liHteitttrti Kitewti 
 

 OF THE 
 
 1Rew l?orK State^tjetennar^ College^ 
 
3 1924 104 226 067 
 
Cornell University 
 Library 
 
 The original of tiiis book is in 
 tine Cornell University Library. 
 
 There are no known copyright restrictions in 
 the United States on the use of the text. 
 
 http://www.archive.org/details/cu31924104226067 
 
A PRACTICAL TEXT- BOOK 
 
 OF 
 
 INFECTION, IMMUNITY 
 AND SPECIFIC THERAPY 
 
 WITH SPECIAL REFERENCE TO IMMUNOLOGIC TECHNIC 
 
 BY 
 
 JOHN A. KpLMER, M.D., Dr.P.H. 
 
 Instructor of Experimental Pathology, University of Pennsylvania ; 
 Professor of Pathology and Bacteriology, Philadelphia Polyclinic, and 
 Pathologist to the Department of Dermatologic > Research ; Pathol- 
 ogist to the Philadelphia Hospital for Contagious Diseases 
 
 WITH AN INTRODUCTION BY 
 
 ALLEN J. SMITH. M.D., Sc.D., LL.D. 
 
 Professor of Pathology, University of Pennsylvania 
 
 WITH 143 ORIGINAL ILLUSTRATIONS, 43 IN COLORS 
 By ERWIN F. FABER 
 
 Instructor of Medical Drawing. University of Pennsylvania 
 
 PHILADELPHIA AND LONDON 
 
 W. B. SAUNDERS COMPANY 
 
 1915 
 
Copyright, 191S, by W. B. Saunders (Jompany 
 'J < 
 
 \ -J I 
 
 <!/ 
 
 PRINTED IN AMERICA 
 
 PRESS OF 
 
 W. B. SAUNDERS COMPANY 
 
 PHILADELPHIA 
 
TO 
 
 RICHARD M. PEARCE, M.D. 
 
 PROFESSOR OF RESEARCH MEDICINE IN THE 
 UNIVERSITY OF PENNSYLVANIA 
 
 IN RECOGNITION OF HIS SERVICES TO THE 
 SCIENCE OF MEDICINE 
 
 BY 
 
 The Author 
 
VETFRlwARV'^0^-^^^ 
 
INTRODUCTION 
 
 The last quarter of a century has witnessed an almost marvelous 
 development of knowledge in the domain o:^ medicine and the allied 
 sciences, only a part, of course, of the extensive progress made in the field 
 of general science. A striking portion of tliis advance has tended to 
 broaden our knowledge of the ijriuciples and <jf the essential details of 
 the processes of infection and immunity, until these branches have today 
 come to form almost a special science in themselves — an imperium in 
 imperio. Aside from the personal factor, the ■writer's immediate inter- 
 est in the present volume, as originally projected, arose from the fact 
 that he was desirous of having appear a series of exercises illustrative of 
 the principles of immunology — a class-book intended to set forth in 
 permanent form the very excellent course of instruction that Dr. Kolmer 
 has been giving during the past few years to selected groups of in- 
 terested students and occasional post-graduatfe" workers in the Medical 
 School of the University of Pennsjdvania. That it should have sur- 
 passed the original simple plan and grofm into a volume of the present 
 proportions is scarcely to be wondered at, if the temptation to elaborate 
 the individual exercises by explanations and cognate considerations was 
 in the slightest to be yielded to. This is due to the fact that in its 
 growth the subject has acquired so much of undoubted importance in 
 the form of isolated observed facts, and itself presents so many analogies 
 and has led to so extensive a terminology, that the author who would 
 attempt to hnk the observed facts into anything hke logical sequence 
 or to add in the least to the bare cook-book-like series of illustrative 
 exercises any explanatory paragraphs, cannot avoid the fullness that 
 Dr. Kolmer has found inevitable in presenting the subject. 
 
 The branch of immunology, including primarily infection, and its 
 ramifications into diagnosis and the actual treatment of disease, has 
 brought to the parent subject of preventive niedicine the greatest offer- 
 ing of the decades of its growth. Itself contributing to world cx-pansion, 
 it has nowhere found a greater stimulusthan in the field of exotic pathol- 
 ogy; and this last, in turn, has enriched internal medicine, even in its 
 most common aspects. The first step in immunology may properly be 
 ascribed to Jenner, with his bovine vaccine for smallpox, a step followed 
 
VI INTRODUCTION 
 
 only after a long lapse of years by Pasteur. On the heels of the latter 
 there appeared at once, and has since then followed, an army of men 
 whose names crowd the history of the subject,, and which many of these 
 are bound permanently to adorn. The old vsEgue theories of infection 
 have taken form, and to observed facts has been added productive 
 theory. The great danger attending this luxurious development is that, 
 temporarily at least, the simpler, and perhaps the more obvious, facts 
 are likely to be neglected; and, also, that sj'mboUzation by theories 
 elaborated to harmonize with discovered facts will be accepted too fully 
 as explanatory when in reaUty it does not explain, and that, as a result, 
 investigation will finally be hampered instead of aided. In the almost 
 universal drift of experimental studies to internal stereochemical factors, 
 are we not in danger of placing too httle stress upon actual and possible 
 physical factors? Is there no danger that, by failing to lay stress upon 
 the obvious importance of the turbinate mechanism in the nose as a 
 natural anatomic factor, our rhinologists may at least feel justified in 
 sacrificing this mechanism too readilj^ for what may be but trivial local 
 reasons? Can we insist that every phenomenon described with facility 
 in terms of the side-chain theory is really a manifestation of chemism, 
 when perhaps, with added investigation along lines of physical absorp- 
 tion and the physical properties of colloids, an equally satisfying con- 
 ception may be had, and possibly new facts be developed? Are we not 
 blundering in rushing madly after matters of specificity as determined 
 by antigen, when perhaps in reahty we are confronted by potential and 
 kinetic modifications due to peculiarities of diet or environmental cir- 
 cumstances? The verity of phagocytosis is open to proof by observa- 
 tion, and its variations are likewise to be demonstrated. Is the explana- 
 tion of opsonins so convincing that merely the word itself is enough to 
 satisfy the investigator? 
 
 Infection and immunity constitute a definite chapter in pathologic 
 science. The processes lack the dignity of a separate science only in 
 that they present variations, and the fact that these are glossed over by 
 brilliant theories and conceptions cannot prevent the deliberate recogni- 
 tion of serious incompleteness. Yet this criticism can be applied to the 
 growth of every branch of scientific knowledge. It in no wise militates 
 against the right and the need for setting forth the subject in the fight 
 that, for the time, is afforded it. The importance of the criticism Ues 
 only in its acknowledgment, lest the subject as at present understood 
 be accepted as fixed. With this danger obviated, and with all theories 
 accepted for the time only as working theories, and their adoption not 
 
INTRODTJCTION VU 
 
 urged to curtail investigations based on other views, their prosecution 
 can be heartily applauded. 
 
 This is the view that the writer believes that Dr. Kolmer has had 
 in mind in his presentation of the subject as here set down. It is cer- 
 tainly true of the chapters that the present writer has had opportunity 
 of examining. In such a sense, therefore, the work is urged on the ap- 
 preciation of the student, whether a laboratory worker or a mere seeker 
 of knowledge. 
 
 I have often been asked to what extent I believe it profitable to pre- 
 sent the subject to the undergraduate studeiit. I do not hesitate to 
 answer that so .far as the roster of the medical curriculum will permit, 
 the laboratory demonstrations and exercises should form a part of the 
 required course; and that, with all due caution to emphasize the fact 
 that our present theory is not known to be final, and is offered merely 
 tentatively, the verbal picture of the subject should be outlined before 
 these beginners. To form some conception is necessary ; and it is better, 
 provided the mind be kept receptive, to follow a certain theory, even if 
 it is unproved, than to do nothing at all or to work in confusion. Our 
 American medical curriculum for undergraduates is so crowded with 
 absolute essentials that the present subject is habitually neglected, save 
 for a rapid lecture outhne; this is an injustice to the student and to 
 American medicine. I have tried to minimize this by providing, 
 through Dr. Kolmer's aid, a reasonable laboratory course in the essentials 
 of the branch to volunteer classes at first, at hours that did not interfere 
 with the regular curriculum— at present during periods open to elec- 
 tion. Nevertheless, the subject, influencing as it does every branch of 
 medical practice, must take its place with other commendable additions 
 to the required schedule. That this can be done only by lengthening 
 the course of study, either in the annual session or by adding a year to 
 our present four-year course, is obvious, and to that end we are rapidly 
 approaching. 
 
 Allen J. Smith. 
 
PREFACE 
 
 Fob the past twenty years the science of inimunity has been one of 
 the most progressive and most active branches in the department of 
 medicine. An enormous hterature has accumillated ; many new terms 
 have been coined, and numerous theories have been adduced; indeed, 
 the subject has acquired an aspect of complexity that is confusing to 
 those not specially interested or engaged in tljis work. 
 
 The purpose of this book is a threefold ona, namclj' : 
 
 1 . To give to "practitioners and students of medicine a connected and con- 
 cise account of our present knowledge regarding the manner in which the 
 body may become infected, and the method, in turn, by which the organism 
 serves to protect itself agairist infection, or strives to overcome the infection 
 if it should occur, and also to present a practical application of this 
 knowledge to the diagnosis, prevention, and treatment of disease. 
 
 2. To give to physicians engaged in laboratory^work and special workers 
 in this field a book to serve as a guide to the various immunologic methods. 
 
 3. To outline a laboratory course in experimental infection and im- 
 munity for students of mediciMe and those especially interested in these 
 branches. 
 
 1. The subject of infection is intimately connected with that of 
 immunity, and this is especially emphasized in those diseases for which 
 a specific therapy exists, for a knowledge of th« nature of the infection 
 is of paramount importance in controlling the dosage and indicating the 
 method of administration of a specific therapeutic agent. By describing 
 principles and technic with considerable detait, a special effort has been 
 made to render Part IV of this book of partici|i|ar value to practitioners 
 of medicine. 
 
 The day is past when the physician and surgeon can relegate the things 
 of immunity entirely to the laboratory. Diagnostic methods and re- 
 actions and the field of specific therapy, — vaccine, serum, and chemo-, — 
 are subjects of such practical importance that it is obvious that the 
 physician and the student of medicine can no longer be merely mildly 
 interested onlookers. The physician who injects salvarsan, a serum, 
 or a vaccine, or who uses a diagnostic reaction,, must be prepared to ex- 
 plain to his patient the nature of the therapy he employs and the sig- 
 
X PREFACE 
 
 nificance of the reaction. This he can do only by equipping himself 
 with the knowledge of the fundamental factors of immunity, or he will 
 be forced into the position of a passive transrnitter of ideas entirely be- 
 yond his own knowledge. 
 
 2. An effort has been made to include data of both practical andi 
 theoretic importance, and in some instances tests are described that are 
 more of theoretic than of practical import, especially in research work. 
 
 It is obviously impossible, in a single volume, to include the very 
 large number of tests and modifications that have been advocated from 
 time to time, and, as a matter of course, most attention has been given 
 those methods that have been shown to be of practical value or that 
 give promise of becoming so. So far as possible original methods are 
 given, these being, in the larger proportion, more or less important modi- 
 fications devised as the result of my own experience in hospital and teach- 
 ing laboratories. 
 
 The technic of the various tests and reactions is described in great 
 detail, thus tending the better to secure accuracy, simplicity, and definite- 
 ness, and to serve as an opening wedge to those about to enter this 
 special field. 
 
 3. The value of the experimental method in the teaching of certain 
 branches of medicine is now well recognized. In no department, how- 
 ever, is this method of greater value than in the study of infection and 
 immunity. A working knowledge of these subjects is so valuable in the 
 practice of medicine and surgery that the student should be well versed 
 in at least their primary principles and practical applications in the 
 prophylaxis, diagnosis, and treatment of diseag 
 
 The laboratory course given in Part V is* based upon the courses 
 given by me in the Laboratory of Experimental Pathology at the Uni- 
 versity of Pennsylvania, and in the laboratories of the Philadelphia 
 Polyclinic and College for Graduates in Medrcine. In including them 
 in this volume I am carrying out my original, plan, for in many of the 
 experiments the exact technic of a given test is described, making a sepa- 
 rate book devoted to this part of the subject imnecessary. Future ex- 
 perience may, however, show the necessity of having this portion of the 
 book form a separate laboratory manual. I shall appreciate the opin- 
 ions of educators who may have occasion to consult the course herein 
 outlined. 
 
 Since the larger portion of our knowledge (5f infection and immunity 
 has been gained from studies upon the lower animals, it is not strange 
 that these were early and directly benefited by a practical application 
 
PREFACE XI 
 
 of this knowledge to the prophylaxis, diagnosis, and treatment of many 
 of the diseases to which these animals are subject. I have, therefore, 
 included in this volume an account of those immunologic diagnostic 
 reactions and applications of specific therapy that have a direct bearing 
 upon veterinary medicine. 
 
 No attempt has been made to cover all literature references on the 
 subject. An effort has been made to state \yell-established facts con- 
 cisely, and, in the case of the more recent subjects, to give the principal 
 references to the literature. I have drawn largely from German, 
 French, and English sources, and have endeavored, wherever possible, 
 to give proper and due credit to each author. In order to keep the work 
 up to the times, I would ask the authors of reprints on immunologic 
 subjects to send me copies. 
 
 The illustrations, all of which have beer^ made by Mr. Erwin F. 
 Faber, will, it is hoped, serve the purpose for'-jvhich they are intended, 
 namely, to elucidate the text and to teach, rather than merely to em- 
 bellish. 
 
 It is with deep and sincere appreciation that I acknowledge the en- 
 couragement and aid given me by Professor Allen J. Smith, who has 
 written the introduction and reviewed severe! chapters. My thanks 
 are also due to Professor Richard M. Pearce fdr reviewing the chapters 
 on Infection; to my assistant, Dr. Anna M. Raiziss, for a number of 
 translations from foreign literature, and to the publishers, whose kind 
 and unvarying courtesy has greatly simplified, the work. 
 
 J. A. K. 
 MoManes Laboratory of Experimental PatholoJst, 
 Univbrsity of Penmsylvania, January, 1915. 
 
CONTENTS 
 
 PAGE 
 InTBODUCTION ; V 
 
 PART I 
 
 GENERAL IMMUNOLOGIC TECHNIC 
 
 Chapter I. — General Technic 17 
 
 Care of centrifuge, 17 — Making a simple capillary pipet, 18 — Making 
 looped pipets, 20 — Graduated pipets, 21 — Making Wright blood-capsules, 23 — 
 Making vaccine ampules, 24 — Preparation of test'tubes for immunologic 
 work, 25 — Selection of a satisfactory syringe, 26 — Solutions, 27. 
 
 Chapter II. — Methods of Obtaining Human and Animal Blood 28 
 
 Obtaioing corpuscles, 28 — Washing er\'throcytes, 28 — Obtaining serum, 30 
 — Obtaining corpuscles and serum, 30 — Obtaining blood plasma, 30 — Obtaining 
 small amounts of human blood, 32 — Obtaining larger amounts of human blood 
 (phlebotomy, wet-cupping, placental blood), 33 — Obtaining cerebrospinal fluid 
 (technic of spinal puncture), 37 — Obtaining small amounts of animal blood 
 (rabbit, guinea-pig, sheep), 41 — Obtaining large amomits of animal blood (rab- 
 bit, guinea-pig, rat, sheep, hog, monkey, dog, and horse), 42. 
 
 Chapter III.— Technic of Animal Inoculation 53 
 
 General rules, 53 — Method of subcutaneous inoculation (fluid and solid 
 inocula), 54 — Method of intramuscular inoculation, 56— Methods of intravenous 
 inoculation (rabbit, guinea-pig, mice and rats, horse) sheep, goat, dog), 56 — 
 Method of iiitiacardial inoculation, 62 — Methods of intraperitoneal inoculation 
 (rabbit, guinea-pig), 64. 
 
 Chapter IV. — Methods for Effecting Active Immunization of Animals . . 65 
 
 Antigens and active immunization, 65 — General technic, 66 — Production of 
 antitoxins, 68 — Production of agglutinins (intravenous knd intraperitoneal inocu- 
 lation), 68 — Production of immune opsonins, 69 — Production of bacteriolysins, 
 69 — Production of precipitins, 70 — Production of herAolysins (intravenous and 
 intraperitoneal inoculations), 71 — Production of cytotoxins, 73. 
 
 Chapter V. — Preservation of Serums — Methods , 75 
 
 Methods for the preservation of normal serums, 75 — Methods for the preser- 
 vation of immune serum in fluid form with antiseptics, 76 — In fluid form by bac- 
 teria-free filtrations, 76 — In fluid form by freezing, 78. — Preservation in powder 
 form, 79 — Preservation in dried paper form, 79. 
 
 PART II 
 
 PRINCIPLES OF INFECTION 
 
 Chapter VI. — Infection 81 
 
 Definition, 81 — Relation of infection to immunity»,*83 — Source of infection, 
 83 — Contagious and infectious diseases, 84 — Exogenous and endogenous in- 
 fection, 85 — Avenues of infection, 8& — Normal defenses against bacterial in- 
 vasion, 90 — Mechanism of bacterial invasion, 91 — Mechanism of infection, 94 — 
 The avenue of infection and tissue susceptibility, 97 — Tlie numeric relationship 
 of bacteria to infection, 99 — General susceptibility in relation to infection, 99 — 
 The defensive mechanism of the microorganism in relation to infection, 102 — 
 Mixed infection, 105 — Summary, 106. 
 
 3 
 
4 CONTENTS 
 
 PAGE 
 
 Chapter VII. — Infection' (continued). PRODucTicp of Di.-^e.^se 107 
 
 Toxins, 108 — Extracellular bacterial toxins, 100— General properties oi' 
 soluble toxins, 109 — Structure of .=:oluble toxins, 110 — Nature of soluble toxin.';, 
 110 — Selective action of soluble toxins, 111. Special Properties of the Principal 
 Soluble Toxins, 112 Diphtheria toxin, 112 — I'he guinea-pig test for virulonee 
 of diphtheria bacilli, 113 — Tetanus toxin, 115 — Botulism toxin, 116 — Dysentery 
 toxin, 116 — Staphylotoxin, 117 — Streptotoxin, 117. Toxins of the' Higher 
 Plants and Animals, 118 — Phytotoxins, lis — Pollen toxin, 119 — Zootoxins, 119 
 — Snake venom, 119. Endotoxins, 120^ — Methods of studying endotoxins, I'-l^ 
 Nature of endotoxins, 121 — Aggressins, 122 — Bail's classification of bacteria, 
 124 — Nature of aggressins, 124 — Anti-aggressins, 126. Bacterial Proteins, 126 — 
 Bacterial split protein, 126 — Nature of bacterial proteins, 127 — Action of bacter- 
 ial proteins, 127 — Theory of Vaughan, 128. Ptoiaains, 129. Mechanical 
 Action of Bacteria, 131 — infection with Animal Parasites, 132 — The Course of 
 Infection, 134 — Stages of infection, 134 — Grades of -infection, 136 — Systemic 
 reaction to infection, 137. 
 
 PART III 
 
 PRINCIPLES OF IMMUNITY AND SPECIAL IMMUNOLOGIC TECHNIC 
 
 Chapter VIII. — Immunity. — Theories of Immunity 138 
 
 Definition, 140 — Historic, 140 — ^E.xhaustion the'ory of immunity, 141 — 
 Retention theory of immunity, 144 — Theory of phag(3cytosis, 144 — Side-chain 
 theory of immunity, 146 — Compatibility of the phagocytic and side-chain 
 theories, 155 — Antigens, 159 — Antibodies, 161. 
 
 Chapter IX. — Various Types op Immunity 165 
 
 Natural Immunity, 165 — Explanation of natural immunity, 166 — l^on- 
 speoiflc immunity, 166 — Local immunity, 167 — Phagocytosis and natural im- 
 njunity, 168 — Natural antitoxic immunity, 169 — Natural bacteriolytic immun- 
 ity, 169 — Natural anti-aggressin immunity, 169 — Athreptic immunity, 170. 
 Acquired Immunity, 170 — Active acquired immunity, 170 — Passive acquired im- 
 munity, 172. 
 
 Chapter X. — Phagocytosis 175 
 
 Historic, 175 — The original theory of phagocytosis, 176 — Kinds of phagocy- 
 tosis, 177 — The relation of the cell types to infectioni 178 — Chemotaxis, 179 — 
 Positive chemotaxis, 179 — Negati\'e chemotaxis, 182 — Results of phagocytosis, 
 182 — The relation of body fluids to phagocytosis, 184 — Revised theory of pha- 
 gocytosis, 186. 
 
 Chapter XI. — Opsonins 187 
 
 Historic, 187 — Definition, IBS — Properties and nature of opsonins, 188 — 
 Susceptibility to opsonification, 189 — Effect of opsonins on bacteria, 189^R61e 
 of opsonins in immunity, 190. 
 
 Chapter XII. — Opsonic Index 191 
 
 Principles involved, 191 — Definition, 191 — Purpo'se of the opsonic index, 
 192 — Limitations of the method, 192 — Precautions in, technic, 193 — Technic of 
 the opsonic index (Wright), 193 — Technic of quantitative estimation of bacterio- 
 tropiiis in immune serum (Neufeld), 200 — Practical value of the opsonic index, 
 203 — Value of the index in diagnosis, 204 — In prognosis, 204 — As a guide to 
 bacterial vaccine therapy, 205. 
 
 Chapter XIII. — Bacterial Vaccines 206 
 
 Definition, 206 — Technic of preparing bacterial vaccines, 206 — Counting 
 a bacterial vaccine (method of Wright), 210 — Counting with the hemocytometer 
 chamber, 211 — Method of KoUe, 212 — Method of Hopkins, 212 — Preparation of 
 "sensitized" bacterial vaccine, 216 — The administration of a bacterial vaccine, 
 217 — ]\Iaking the inoculation, 217 — Effects of inoculation, 218 — Frequency ancl 
 dosage of inoculation, 218 — Ordinary adult doses of flie common vaccines, 218. 
 
CONTENTS b 
 
 PAGE 
 
 Chapter XIV. — Antitoxins » 220 
 
 Definition, 220 — Historic, 220 — Formation of antitoxins, 221 — Structure of 
 antitoxins, 223 — Properties of antitoxins, 22y — Natural antitoxins, 224 — Speci- 
 ficity of antitoxins, 224 — Nature of the toxin-antitoxiij"reaction, 224. Produc- 
 tion of Diphtheria Antitoxin, 227 — Production of diphtiieria toxin, 277 — Testing 
 the toxin, 228 — Immunizing the animals, 228 — Collecting the serum, 230 — Stan- 
 dardizing the serum, 231. Production of Tetanus Antitoxin, 23-1 — Tetanus 
 toxin, 234 — Immunizing the animals, 234 — Collecting the serum, 234 — Standard- 
 izing the serum, 234. Botufinus Antitoxin, 236. Antidysentery Serum, 236 — 
 The culture, 23G — Immunizing the animals, 237 — Collecting and testing the 
 serum, 238. Antistaphylococcus Serum, 238 — Preparation, 239 — Technic of 
 the anti-lysin test, 239. Production of Antivenin, 241. Production of PoUen 
 Antitoxin, 242. The Measure of Antitoxins, 242 — A unit, 242 — Unit of diph- 
 theria antitoxin, 242 — Unit of tetanus antitoxin, 242. 
 
 Chapteh XV. — Ferments and Anti-ferments 244 
 
 Bacterial ferments, 244 — Similarity between toxins and ferments, 244 — Anti- 
 ferments, 246 — Antibodies and antif erments, 247 — Antiferments in disease, 247 — 
 Ferments in pregnancy and disease, 248. Ferment Reactions, 250 — Antitrypsin 
 test, 250 — Abderhalden's sei'odiagnosis of pregnancy, 252 — Practical value of 
 Abderhalden's test, 263 — Sero-enzymes in cancer, 264r-In mental diseases, 265 
 — In syphilis, 265 — In tuberculosis and acute ijifectioii, 265. 
 
 Chapter XVI. — Agglutinins , 266 
 
 Definition, 266 — Historic, 266 -Normal and immune agglutinins, 268 — 
 Formation of agglutinins, 268 — Origin of agglutinin's, 269 — Properties and 
 nature of agglutinins, 270- Mechanism of agglutination, 270 — Specificity of 
 agglutinins, 271 - Absorption methods for differentiating between a mixed and 
 single infection, 272 - Hemagglutinins, 272 — Non-agglutinable races of bacteria, 
 274— Variation in agglutinating strength of a serum, 274 — Role of agglutinins in 
 immunity, 274. Practical Applications, 27.5 — In the diagnosis of typhoid fever, 
 27.5 — Paratyphoid fever, 275 — Dysentery, 27(3 — Cholera, 277 — Cerebrospinal 
 meningitis, 277 — Plague, 277 — Malta fever, 277 — Glanflers, 277 — In the differen- 
 tiation of bacteria, 277 — In the diagnosis of single Etad mixed infection, 278. 
 The Agglutination Reaction, 278 — Microscopic method with serum, 282 — Micro- 
 scopic method with dried blood, 283 — Macroscopic method, 284 — The sat- 
 uration test of Castellani, 288. Tests before Blood Transfusion for Isohem- 
 agglutinins and Isohemolysins, 290. * 
 
 Chapter XVII, — Precipitins 292 
 
 Definition, 292 — Historic, 292 — Nomenclature, 29\ — Structure and propor- 
 tion of precipitins, 294 — Formation of precipitins, 291 — Mechanism of precipita- 
 tion, 296 — Specificity of precipitins, 296 — Role of precipitins in immunity, 297. 
 Practical Applications, 298 — Bacterial precipitins, 298T-pFornet ring test, 299 — 
 Porges-Meier reaction, 299 — Herman-Perutz reaction, 299 — Noguchi globulin re- 
 action, 300 — Differentiation of proteins, 301. Technic of the Precipitin Reac- 
 tions, 303 — Differentiation of human and animal bloodsj 303 — Detection of meat 
 adulteration, 310 — Bacterial precipitins, 313 — Precipitin test in cancer, 314. 
 
 Chapter XVIII. — Cytolysins. Amboceptors and Pomplements 316 
 
 Definition, 317 — Kinds of cytolj'sins, 318 — Nomenclature, 318. Ambocep- 
 tors, 319 — Historic, 319 — Structure of amboceptors, 320 — General properties of 
 amboceptors, 321 — Mechanism of the action of amboceptors, 321 — Formation 
 of amboceptors, 322 — Quantitative estimation of ambeceptors, 325 — Titration 
 of hemolytic amboceptor, 325 — Titration of bacteriolytic amboceptor, 325. 
 Complements, 326 — Historic, 326 — Definition, 326 — Structure and general 
 properties of complement, 326 — .Anticomplements, 327 — r)rigin of complements, 
 328 — Multiplicity of complements, 328 — Endocomplemgnts, 330 — Complement- 
 splitting, 331 — Complement fixation, 332 — Complemetit deviation, 333 — Quan- 
 titative titration of complement, 334. 
 
 Chapter XIX. — Bacteriolysixs 336 
 
 Historic, 330 — Definition, 337 — Origin of bacterioJysins, 338 — Leukins and 
 leukocytic extracts, 338 — Method of preparingleukocytic extracts, 339 — Mechan- 
 ism of bacteriolysis, 340 — General properties of bacterielysins, 341 — Normal bac- 
 
6 CONTENTS 
 
 PAGE 
 
 teriolysins, 341 — Specificity of bacteriolysins, 341. Practical Applications, 341 — 
 Teclinic of tlie Pf eiffer test, 342 — Bacteriolytic test in Vivo for the identification 
 of. bacteria, 343 — Bacteriolytic test in vivo in the diagnosis of disease, 348 — Bac- 
 teriolytic test in vitro (method of Stern and Korte), ^49 — Bacteriolytic test in 
 vitro (method of Wright), 335. 
 
 Chapter XX. — Hemolysins ,= 361 
 
 Historic, 361 — Definition, 362 — Nomenclature, 363 — Nature of hemol3'sins, 
 363 — Analogy between bacteriolysis and hemolysis, 367 — Specificity of hemoly- 
 sins, 368 — Normal hemolysins, 368 — Production of immune hemolysins, 371 — 
 General properties of hemolysins, 371 — Source of hemolysins, 372. Practical 
 Applications, 373 — Quantitative reactions between hemolytic amboceptor and 
 complement, 374 — Method of titration of hemolysin, 375 — Method for removing 
 hemolysin from a serum, 378 — Method of determining natural hemolysins in 
 serum, 378 — The serum diagnosis of paroxysmal hemQglobinuria, 379 — Method 
 ot determining the resistance of red blood-corpuscles, 380. 
 
 Chapter XXI. — Venom Hemolysis 383 
 
 Historic — nature of venom hemolysis, 383 — \'en0m hemolysis in syphilis, 
 385 — Practical value of the venom test in syphilis, 388 — The psycho-reaction of 
 Much in syphilis, 388 — Venom hemolysis in tuberculosis, 390 — Venom hemolysis 
 in cancer, 390. 
 
 Chapter XXII. — Pkinciples of the Phenomenon of Complement Fixation 391 
 
 Historic, 391 — The original complement-fixation method of Bordet, 394 — 
 Mechanism of complement fixation, 395 — Non-specific complement fixation, 
 396 — Quantitative factors in complement-fixation tests, 397 — Practical applica- 
 tions, 399. 
 
 Chapter XXIII. — The Technic op Complement-fixation Reactions 401 
 
 The Wassennann Syphilis Reaction, 401 — Historic, 401 — Principles and 
 theories of the syphilis reaction, 404 — General technic, 404 — Preparation of the 
 fluid to be tested, 408 — Preparation and titration of cojnplement, 411 — Prepara- 
 tion and titration of hemolytic amboceptor, 415 — Preparation of blood-cor- 
 puscles, 416 — Preparation and standardization of antigens, 417 — Technic of the 
 First Method, 435 — Technic of the Second Method, 441 — Technic of the Third 
 Method, 443 — Technic of the Fourth Method, 446. Modifications of the 
 Wassermann Reaction, 449 — Technic of the Noguchi modification, 449 — Technic 
 of the Hecht-\\ einburg modification, 457 — Other modifications, 458. The Was- 
 sermann Reaction in the Various Stages of SyphiUs, 459 — -The specificity of the 
 Wassermann reaction, 465 — The effect of treatment Upon the Wassermann re- 
 action, 466 — The Practical Value of the Wassermann reaction, 469. 
 
 Chapter XXIV. — Complement-fixation Reaottons (continued) 473 
 
 Specific complement fixation in bacterial diseases, 473 — Preparation of 
 bacterial antigens, 473 — Standardizing bacterial antigens, 475 — Principles of 
 complement fixation with bacterial antigens, 476. Complement Fixation in 
 Gonococcus Infections, 477. Complement Fixation ih Glanders, 484. Com- 
 plement Fixation in Contagious Abortion, 486. Complement Fixation in 
 Dourine, 487. Complement Fixation in Typhoid Fever, 489. Complement 
 Fixation in Tuberculosis, 490. Complement Fixation in the Standardization of 
 Immrme Serums, 491. Complement Fixation in Echinococcus Disease, 492. 
 Complement Fixation in the Differentiation of Proteins (blood-stains, meats, 
 bacteria), 494. Complement Fixation in Cancer, 499. 
 
 Chapter XXV. — Cytotoxins 502 
 
 Nomenclature, 502 — Nature and general properties of cytotoxins, 502 — 
 Preparation of cytotoxins, 503 — Methods of studying cytotoxins, 503 — Specificity 
 of cytotoxins, 504— Autocytf)tnxins, 505 — Isocytotoxins, 506 — Anticytotoxic 
 serums, 506 — Kinds of cytotoxins, 506 — Spermatotojin, 506 — Epitheliotoxin, 
 506 — Leukotoxin, 506 — Nephrotoxin, 506 — Hepatotoxin, 507 — Gastrotoxin, 
 507 — Sj'ncytotoxin, 507 — Neurotoxin, 508 — Thyrotoxin, 508 — Role of cyto- 
 toxins in immunity, 508 — Practical applications, 508 — Cytotoxic cancer re- 
 action, 509. 
 
CONTENTS / 
 
 FAGE 
 
 Chapter XXVI. — The Relation of Colloids and ILipoids to Immunity. . . . 511 
 
 Kinds of nolloids, 511 — Nature and properties of colloids, 511 — Analogy be- 
 tween the reactions of immunity and those of colloidal chemistry, 515 — ^Anti- 
 toxins, 516 — Agglutinins and precipitins, 517 — Hemolysins, 518 — Complement 
 fixation as a colloid reaction, 519 — The relation of lipoids to immunity, 520 — 
 The epiphanin reaction, 522 — The miostagmin reaction, 526. 
 
 Chapter XXVII. — Anaphylaxis 531 
 
 Historic, 532 — Definition, 53.5 — Phenomena of anaphylaxis, 536 — Mechan- 
 ism of anaphylaxis, 541 — Anaphylactogens or allergens, 543 — Anaphylatoxin, 
 548 — Anaphylactin (allergin), 552 — Theories of anaphylaxis, 556 — Passive 
 anaphylaxis, 559 — Anti-anaphvlaxis, 561 — Specificity of anaphylaxis, 563. 
 
 PART IV 
 
 APPLIED IMMUNITY IN THE PROPHYLAXIS, "DIAGNOSIS, AND TREAT- 
 MENT OF DISEASE— SPECIFIC THERAPY 
 
 Chaptek XXVIII. — Anaphylaxis in Its Relation to Infection and I.m- 
 
 MUNiTY. Anaphylactic ok Allbkqic Reactions 565 
 
 Relation of anaphylaxis to infectious diseases, 566 — Relation of anaphylaxis 
 to non-infectious diseases, 570 — Serum disease, 571 — Idiosyncrasies, 577 — 
 Relation of anaphylaxis to immunity, 580 — Anaphylactic or allergic reactions, 
 582 — Subcutaneous tuberculin reaction, 592 — Intracutaneous tuberculin re- 
 action, 595 — Cutaneous tuberculin reaction, 596 — ^Conjunctival tuberculin re- 
 action, 598 — Percutaneous tuberculin reaction, 599 — Tuberculin reactions 
 among the lower animals, 600 — The luetin reaction, 601 — The mallein reaction, 
 606 — Allergic reactions in typhoid fever, 607 — Allergic reactions in other dis- 
 eases, 609— Allergic reactions as a measure of immunity, 610. 
 
 Chapter XXIX. — Active I.mmunization. Vacci.xes in the Prophylaxis 
 
 AND Treatment of Disease. Vaccine Therapy 611 
 
 Historic, 611 — Nomenclature, 613 — ilethod of preparing vaccines, 614 
 Mechanism of active immunization, 616 — Living versus dead vaccines, 620 — 
 Sensitized vaccines, 620 — Autogenous versus stock bacterial vaccines, 620 — The 
 negative phase, 621 — Contraindications to active imraunization, 622. Prophy- 
 lactic Immunization or Vaccination, 623 — In small-pox, 623 — In rabies, 636 — In 
 typhoid fever, 643 — In plague, 647 — In cholera, 650-S-In dysentery, 652 — In 
 cerebrospinal meningitis, 652 — In scarlet fc^'cr, 652 — In anthrax, 653 — In 
 blaek-lcg, 655. Therapeutic Immunization. Bacterial Vaccine Therapy, 655 — 
 Principles, 655 — Diseases of the skin, 656 — Diseases of the genito-urinary system, 
 657 — Diseases of the respiratory system, 659 — Acute» general infections, 66*0 — 
 Tuberculin therapy, 661. 
 
 Chapter XXX. — Passive IM^^uNIZATION. Serums tx the Prophylaxis and 
 
 Treatmrnt of Disease. Serum Therapy 681 
 
 Definition, 681- -Purposes of passive immunization, 682 — Kinds of passive 
 immumty, 683 — Indications for passive immunization, 684 — Contraindications 
 to passive immunization, 686 — Technic of subcutafieous inoculation, 688 — 
 Technic of intramuscular inoculation, 690 — Technic of intravenous inoculation, 
 690 — Technic of subdural inoculation, 694. Serum Treatment and Prophylaxis 
 of Diphtheria, 702. Serum Treatment and Prophylaxis of Tetanus, 719. Se- 
 rum Treatment and Prophylaxis of Dysentery, 730. Serum Treatment and 
 Prophylaxis of Hog Cholera, 732. Serum Treatment of Snake Bites, 733. 
 Serum Treatment and Prophylaxis of Hay-fever, 734. Serum Treatment and 
 Prophylaxis of Meningococcus Meningitis, 736. Serum Treatment of Influenzal 
 Meningitis, 749. Serum Treatment of Pneumococcus; Meningitis, 751. Serum 
 Treatment of Other Localized Pneumococcus Infections, 754. Serum Treat- 
 ment of Lobar Pneumonia, 754. Serum Treatment of Streptococcus Infections, 
 760. Serum Treatment of Gonococcus Infections, 765. Serum Treatment of 
 Staphylococcus Infections, 766. Serum Treatment of Anthrax, 767. Serum 
 Treatment of Typhoid Fever, 768. Serum Treatment of Plague, 769. Serum 
 Treatment of Cholera, 770. Serum Treatment of Tuberculosis, 771. 
 
8 CONTENTS 
 
 PAGE 
 
 Chapter XXXI. — SBEtiM Thekapt (continued) 772 
 
 Normal Serum Therapy, 772 — Serum treatment of hemorrhage, 771' — fierum 
 treatment of the toxicoses of pregnancy, 773 — Serum treatment of skin diseases, 
 774. Autoserum Therapy, 775 — Autoserum treatment of skin diseases, 775 — 
 Autoserum treatment of acute infectious diseases, 775 — Autoserum treatment 
 of syphihs (salvarsanized serum), 776 — Autoserum treatment of tuberculosis 
 of serous membranes, 781 — Autoserum treatment of non-tuberculous effusions, 
 782. 
 
 Chapter XXXII. — Chemotherapy 784 
 
 Principles of Chemotherapy, 785 — Organotropism and parasitotropism, 785 
 — Chemoreceptors, 787 — Drug "fastness," 789 — Therapia magna sterilisans, 791. 
 Salvarsan and Neosalvarsan in the Treatment of Syphilis, 792 — Historic, 792 — 
 Properties of salvarsan, 794 — Properties of neosalvarsan, 795 — Methods for pre- 
 paring salvarsan for administration, 795 — Methods for preparing neosalvarsan for 
 administration, 795 — Administration of salvarsan and neosalvarsan by intra- 
 venous injection, 797 — By intramuscular injection, 805 — By intraspinous in- 
 jection, 806 — Contraindications and precautions in salvarsan therapy, 808 — 
 Value of salvarsan and neosalvarsan in the treatment of syphilis, 809. Salvarsan 
 in the Treatment of Non-syphilitic Diseases, 810. Chemotherapy in Bacterial 
 Diseases, 811. Chemotherapy in Malignant Diseases, 812. 
 
 PART V 
 
 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 Introductory, 814— Methods, 814— The Student^, 814— Records, 815— Animal 
 
 Experiments and Autopsies, 815. 
 
 Exercise 1. — Active Immunization of Animals, 815. 
 
 Experiment 1, Production of Agglutinins, Bacteriolysins and Opsonins, 815 — 
 Experiment 2, Production of Precipitins, 816 — Experiment 3, Production of 
 Hemolysins, 816 — Experiment 4, Production of Cytotoxin, 816. 
 
 Exercise 2. — Infection, 816. 
 
 Experiment 5, Experimental Pneumonia, 816. 
 
 Exercise 3. — Toxins, 817. 
 
 Experiment 6, Diphtheria Toxin, 817 — Experiment 7, Method of Testing the 
 Virulence and Toxicity of Diphtheria Bacilli, 818 — Experiment 8, Tetanus Toxin 
 {in vivo), 819 — Experiment 9, Tetanus Toxin {in vitro), 819. 
 
 Exercise 4. — Toxins (continued), 820. 
 
 Experiment 10, Botulism Toxin, 820 — Experiment 11, Dysentery Toxin, 820 — 
 Experiment 12, Staphylotoxin {in vivo), 821 — Experiment 13, Staphylotoxin 
 (in vilro), 821. 
 
 Exercise 5. — Toxins (continued) Plant and Animal Toxins, 822. 
 
 Bx-periment 14, Streptotoxin, 822 — Experiment 15, Phytotoxins (in vivo and 
 in vilro), 822 — Experiment 16, Zootoxin, Cobra venom {in vivo and in vitro), 823. 
 
 Exercise 6. — Endotoxins and Aggi-essins, 823. 
 
 Experiment 17, Endotoxins, 823 — Experiment 18, Natural Aggressins, 824. 
 
 Exercise 7. — Bacterial Protein. Ptomains. Mechanical Action of Bacteria, 824. 
 E-xperiment 19, Bacterial Protein, 824 — Experiment 20, Ptomains, 825 — Ex- 
 periment 21, Mechanical Action of Bacteria, 826. 
 
 Exercise 8. — Kinds of Immunity. Natural Immunity, 826. 
 
 Exi^eriment 22, Phagocytosis in Natural Immunity, 826 — Experiment 23, Nat- 
 ural Antibacterial Immunity, 826 — Experiment 2i, Relative Factors in Natural 
 Immunity, 827 — Experiment 25, Influence of Temperature Upon Natural 
 Immunity, 827. 
 
 Exercise 9. — Acquired Immunity, 827. 
 
 Experiment 26, Acquired Active (Antibacterial) Immunity, 827 — Experiment 
 27, Acquired Passive (Antitoxic) Immunity, 828 — Experiment 28, Acquired 
 Passive (Antitoxic) Immunity, 828 — Experiment 29, Acquired Passive (Anti- 
 bacterial) Immunity, 828. 
 
CONTENTS y 
 
 Exercise 10. — Phagocytosis, 829. 
 
 Experiment 30, Phagocytosis (macrophages), 829 — Experiment 31, Phagocy- 
 tosis (microphages), 829 — Experiment 32, Phagocytosis, 830. 
 
 Exercise 11. — Phagocytosis. Chemotaxis, 830. 
 
 Experiment 33, Positive Chemotaxis, 830 — Experiment 34, Negative Chemo- 
 taxis, 830. 
 
 Exercise 12. — Opsonins, 831. 
 
 E.xperiment 35, Normal Opsonins, 831 — Experiment 36, Immune Opsonin (Bac- 
 teriotropin), 832 — Experiment 37, Hemopsonin, 832. 
 
 Exercise 13. — Opsonins (continued), 832. 
 
 Experiment 3S, Mechanism of Action of Opsonins, S32 — Experiment 39, Specific- 
 ity of Opsonins, 833. 
 
 Exercise 14. — Opsonic Index, 834. 
 
 Experiment 40, Determining the Opsonic Index; 834 — Experiment 41, Quan- 
 titative Estimation of Bactcriotropiiis, 834. 
 
 Exercise 15. — Bacterial Vaccines, 834. 
 
 Experiment 42, Preparation of Typhoid Vaccine, 634 — Experiment 43, Prepara- 
 tion of Staphylococcus Vaccine, 835. 
 
 Exercise 16. — Antitoxins, 835. 
 
 Experiment 44, Standardizing Diphtheria Antitoxin, 835 — Experiment 45, 
 Standardizing Tetanus Antitoxin, 836. 
 
 Exercise 17. — Antitoxins (continued), 836. 
 
 Experiment 46, Specificity of Antitoxins, 836 — Experiment 47, Nature of the 
 Toxin-Antitoxin Reaction. Action of Anti-tetanolysin, 836 — Experiment 4S, 
 Antistaphylolysin, 837. 
 
 Exercise 18. — Ferments and Antiferments, 838. 
 
 Experiment 49, Tryptic Ferment of Leukocytes, 838 — Experiment 50, Testing 
 the Antitryptic Power of Blood-serum, 838. 
 
 Exercise 19. — Ferments (continued), 839. 
 
 Experiment 51, Abderhalden Sero-enzyme Reaction in Pregnancy, 839. 
 
 Exercise 20. — Agglutinins, 840. 
 
 Experiment 52, Gruber-Widal Reaction in Typhoid Fever ("Wet" Method), 
 840 — Experiment 53, Gruber-Widal Reaction in Typhoid Fever ("Dry" 
 Method), 840. 
 
 Exercise 21. — Agglutinins (continued), S41. 
 
 Experiment 54, Macroscopic Agglutination Reaction, 841 — Experiment 55, 
 Macroscopic Agglutination Reaction (Nolle), 842 — Experiment 56, Macro- 
 scopic Agglutination Reaction (Killed Cultures), 842. 
 
 Exercise 22. — Agglutinins (continued), 842. 
 
 Experiment 57, Group Agglutination, 842 — Experiment 58, Pro-agglutination 
 (Agglutinoids), 843 — Experiment 59, The Absorption or Saturation Agglutina- 
 tion Reaction, 843. 
 
 Exercise 23. — Agglutinins (continued), 843. 
 
 Experiment 60, Hemagglutinins, 843 — Experiment 61, Blood Transfusion Tests, 
 844. 
 
 Exercise 24. — Precipitins, 844. 
 
 Experiment 62, Titration of a Precipitin Serum, 844 — Experiment 63, Titration 
 of a Precipitin Serum, 844 — Experiment 64, Specijifcit}' of Precipitins, 844. 
 
 Exercise 25. — Precipitins, 845. 
 
 Experiment 65, Forensic Blood Test, 845. 
 
 Exercise 26. — Precipitins, 846. 
 
 Experiment 66, Lactoserums, 846 — Experiment 67, Bacterial Precipitins, 846 — ■ 
 Experiment 68, Noguehi Globulin Reaction, 846. 
 
 Exercise 27. — Amboceptors and Complements. Hemolysins, 847. 
 
 Experiment 69, Non-specific Hemolysins, 847 — Experiment 70, Serum Hemolysis 
 in vitro, 848 — Experiment 71, Serum Hemolysis in vim, 848. 
 
 Exercise 28. — Ainboceptor and Complements. Hemolysins, 848. 
 
 Experiment 72, Titration of a Hemolytic Amboceptor, 848 — Experiment 73, 
 Quantitative Factors in Serum Hemolysis, 849. 
 
10 CONTENTS 
 
 Exercise 29. — Amboceptors and Complements. Hemolysins, 850. 
 
 Experiment 74, Role of Amboceptor and Complement in Hemolysis, 850 — Ex- 
 periment 75, Specificity of Amboceptors, 850 — Experiment 76, General Proper- 
 ties of Amboceptors, 851. 
 
 Exercise 30. — Amboceptors and Complements. Hemolysins, 851. 
 
 Experiment 77, Mechanism of Amboceptor Action, 851 — Experiment 78, A 
 Further Study of the Mechanism of Amboceptors, 851. 
 
 Exercise 31. — Amboceptors and Complements. Hemolysins, 852. 
 
 Experiment 79, Natural Hemolysins. Removal of Natural Hemolysins, 852. 
 
 Exercise 32. — Amboceptors and Complements, 853. 
 
 Experiment 80. — Hemolytic Complement, 853 — Experiment 81, Inactivation 
 and Reactivation of Complement, 853 — Experiment 82, General Properties of 
 Complement, 854. 
 
 Exercise 33. — Amboceptors and Complements, 854. 
 
 Experiment 83, Titration of Hemolytic Complement, 854 — Experiment 84, 
 Phenomenon of Complement Fixation, 855. 
 
 Exercise 34. — Antigens for the Wassermann Reaction, -856. 
 
 Experiment 85, Preparation of Antigens for the Wassermann Reaction, 856. 
 
 Exercise 35. — Antigens, 856. 
 
 Experiment 86, Method of Titration of Antigens, 856. 
 
 Exercise 36. — Wassermann Reaction, 857. 
 
 Experiment 87, Anticomplementary Action of Serums, 857. 
 
 Exercise 37. — Wassermann Reaction, 857. 
 
 Experiment 88, Wassermann Reaction (First Method), 857. 
 
 Exercise 38. — Wassermann Reaction, 858. 
 
 Experiment 89, Wassermann Reaction (Second Method), 858. 
 
 Exercise 39. — Wassermann Reaction, 85S. 
 
 Experiment 90, Wassermann Reaction (Third Method), 858. 
 
 Exercise 40. — ^Wassermann Reaction, 858. 
 
 Experiment 91, Wassermann Reaction (Fourth Method), 858. 
 
 Exercise 41. — Noguchi Modification of the Wassermann. Reaction, 859. 
 
 Experiment 92, Titration of Antihuman Hemolysin, 859 — Experiment 93, Tech- 
 nic of the Noguchi Modification, 859. 
 
 Exercise 42. — Wassermann and Noguchi Reactions, 859. 
 Experiment 94, Comparison of Methods, 859. 
 
 Exercise 43. — Gonococcus Complement-fixation Reaction, 860. 
 Experiment 95, Titration of Gonococcus Antigen, 860. 
 
 Exercise 44. — Gonococcus Complement-fixation Reaction, 860. 
 Experiment 96, Technic of the Gonococcus Reaction, 860. 
 
 Exercise 45. — Gonococcus Complement-fixation Reaction, 860. 
 Experiment 97, Technic of the Gonococcus Reaction, 860. 
 
 ExjiRCiSE 46. — Complement Fixation in the Differentiiition of Proteins, 861. 
 Experiment 98, Titration of Immune Scrums, 861. 
 
 Exercise 47. — Complement Fixation in the Differentiation of Proteins, 861. 
 Experiment 99, Technic of the Forensic Blood Test, 861. 
 
 Exercise 48. — Venom Hemolysis, 861. 
 
 Experiment 100, Venom Hemolysis in Syphilis, 861.: 
 
 Exercise 49. — Bacteriolysis, 862. 
 
 Experiment 101, Pfeiffer Bacteriolytic Test, 862. 
 
 Exercise 50. — Bacteriolysis, 862. 
 
 Experiment 102, Microscopic Method of Measuring the Bacteriolytic Power of 
 Blood, 862. 
 
 Exercise 51. — Bacteriolysis, 863. 
 
 Experiment 103, Method of Measuring the Bacteriolytic Activity of the Blood 
 in viiro (Method of Stern and Korte), 863. 
 
 Exercise 52. — Cytotoxins, 863. 
 
 Experiment 104, Action of Nephrotoxin, 863. 
 
CONTENTS 1 1 
 
 Exercise 53. — Miostagmin Reaction, 864. 
 
 Experiment 105, Technio of tlie Miostagmin Reaotjon, 864. 
 ExEKCiSE 54. — Anaphylaxis, 864. 
 
 Experiment 106, Anaphylaxis in the Guinea-pig. Specificity of Anaphylaxis, 
 
 864. 
 Exercise 55. — Anaphylaxis, 865. 
 
 Experiment 107, Natmre of the Anaphylatoxin, 865. 
 Exercise 56. — Anaphylaxis, 865. 
 
 Experiment 108, Anaphylaxis in the Dog, 865. 
 Exercise 57. — Anaphylaxis, 866. 
 
 Experiment 109, Passive Anaphylaxis, 866. 
 Exercise 58. — ^Anaphylaxis, 866. 
 
 Experiment 110, Anti-anaphylaxis, 866. 
 Exercise 59. — Anaphylaxis, 867. 
 
 Experiment 111, Local Anaphylactic Reactions, 867. 
 Exercise 60. — Chemotherapy, 867. 
 
 Experiment 112, Salvarsan, 867. 
 
 Index 869 
 
LIST OF ILLUSTRATIONS 
 
 1. An Electric Centrifuge 18 
 
 .2. Method of Making a Simple Capillary Pipet 20 
 
 3. Method of Making a Looped Pipet 21 
 
 4. Graduated Pipets 22 
 
 5. Method of Making a Wright Blood Capsule • 23 
 
 6. Method of Making a Vaccine Ampule of Glass Tubing 24 
 
 7. Method of Making a Large Vaccine Ampule of a Test-tube 25 
 
 8. A Satisfactory Syringe 2(i 
 
 9. A Suction Pump 29 
 
 10. Method of Pricking a Finger ,« 31 
 
 11. Method of Obtaining a Small Amount of Human Blood 32 
 
 12. Collecting Blood in a Wright Capsule 33 
 
 13. Removing Serum from a Wright Capsule 34 
 
 14. Method of Seahng a Wright Capsule , 34 
 
 15. Methods for Securing Blood by Puncture of Vein 3.5 
 
 16. The Keidel Tube for CoUecting Blood 36 
 
 16a. Parts of the Keidel Tube 37 
 
 17. A Wet-cup for Securing Blood from Children (Blat;kfan) 38 
 
 IS. Technic of Spinal Puncture 40 
 
 19. Method of Bleeding a Rabbit from the Ear 43 
 
 20. A Dissection of the Neck of a Rabbit to Show Relation of the Carotid Artery 44 
 
 21. Method of Bleeding a Rabbit from the Carotid Artery 4.5 
 
 22. Method of Bleeding a Rabbit from the Carotid Artery (Pasteur Institute 
 
 Method) 46 
 
 23. Method of Bleeding a Guinea-pig 47 
 
 24. A Dissection of the Neck of a Sheep to show the Relations of the External 
 
 Jugular Vein 48 
 
 25. Method of Bleeding a Sheep from the External Jugular Vein 49 
 
 26. Method of Bleeding a Horse from the Jugular Vein 51 
 
 27. Method of Subcutaneous Inoculation of a Guinea-pig 55 
 
 2S. Method of Intravenous Inoculation of a Rabbit 57 
 
 29. A Dissection of the Neck of a Guinea-pig to show the Relations of the Ex- 
 
 ternal Jugular Vein 58 
 
 30. Method of Intravenous Inoculation of a Guinea-pig 59 
 
 31. Method of Intravenous Inoculation of a Rat 60 
 
 32. Method of Intravenous Inoculation of a Horse 61 
 
 33. Method of Intraperitoneal Inoculation of a Rabbil 63 
 
 34. A Small Berkefeld Filter 77 
 
 35. A Filter 78 
 
 36. Abdominal Wall of Guinea-pig showing Diphtherip Edema Facing 112 
 
 37. Normal Adrenal Gland of a Guinea-pig Facing 114 
 
 13 
 
14 LIST OF ILLUSTRATIONS 
 
 FIG. PAGE 
 
 38. Adrenal Gland of a Guinea-pig after Fatal Diphtheric Intoxication . . .Facing 114 
 
 39. Ehrhch's Side-chain Theory. Formation of Antitoxins 150 
 
 40. Theoretic Structure of a Molecule of Toxin and Ibxoid 151 
 
 41. Ehrhch's Side-chain Theory. Formation of Agglutinins and Precipitins ... . 153 
 
 42. Ehrhch's Side-chain Theory. Formation of Cytolysins (Bacteriolysins, 
 
 Hemolysins, etc.) 155 
 
 43. General Scheme of Antigens and Their Antibodies* 163 
 
 44. Phagocytosis (Macrophages) FaciJig 177 
 
 45. Phagocytosis (Macrophages) Facing 177 
 
 46. Positive Chemotaxis Facing 178 
 
 47. Negative Chemotaxis Facing 182 
 
 48. Capillary Pipet for Opsonic Index Determinations^ - 196 
 
 49. Mixing the Contents of a Capillary Pipet 197 
 
 50. Method of Seahng a Capillary Pipet 198 
 
 51. Method of Preparing a Blood Fihn 198 
 
 52. Blood Films for Phagocytic Counts 199 
 
 53. Tubercle Opsonic Index Facing 200 
 
 54. An Unsatisfactory Fihn for Phagocytic Count Facing 200 
 
 55. A Satisfactory Film for Phagocytic Count Facing 200 
 
 56. An Opsonic Index Chart 204 
 
 57. Preparation of a Bacterial Vaccine 208 
 
 58. A Shaking Apparatus 209 
 
 59. Capillary Pipet for Counting Bacterial Vaccines 210 
 
 GO. A Satisfactory Preparation for Counting a Bacterial Vaccine Facing 211 
 
 61. An Unsatisfactory Preparation for Counting a Bacterial Vaccine Facing 211 
 
 62. Instrument for the Standardization of a Platinum Loop 212 
 
 63. Hopkins' Tube for Standardizing a Bacterial Vaccine 213 
 
 64A. A Stock Ampule of Vaccine (Large) 214 
 
 64B. Stock Bottle of Bacterial Vaccine 214 
 
 65. A Small Vaccine Ampule 215 
 
 66. Comer's Automatic Pipet 215 
 
 67. Theoretic Formation of an Antitoxin 222 
 
 68. A Flask of Diphtheria Culture 228 
 
 69. A Large Toxin Filter 229 
 
 70. Separation of Blood-serum Facing 231 
 
 71. A Kitchens Syringe 233 
 
 72. A Battery of Hitchens' Syringes 233 
 
 73. A Dialysing Cyhnder for the Abderhalden Ferment Test 254 
 
 74. The Xinhydrin Reaction (Abderhalden Ferment Test) Facing 255 
 
 75. Theoretic Formation of Agglutinins 267 
 
 76. Theoretic Structure of Agglutinin and Agglutinoid 268 
 
 77. Diagrammatic Illustration of the Action of Agglutinin and Agglutinoids . . . 269 
 
 78. A Satisfactory Culture for the Microscopic Agglutination Reaction 280 
 
 79. An Unsatisfactory Culture for the Microscopic Agglutination Reaction 280 
 
 80. A Positive Agglutination (Widal) Reaction in Typhoid Fever 283 
 
 81. Microscopic Agglutination Test with Dried Blood. ..,...,. .Facing 284 
 
 82. Macroscopic Agglutination Reaction 285 
 
 83. Macroscopic Agglutination Test. Pro-agglutinatjon 286 
 
 84. Agglutinoscope 287 
 
 85. The Noguchi Butyric Acid Test for Globuhns 301 
 
 86. Hemin Crystals Facing 303 
 
LIST OF ILLUSTRATIONS 15 
 
 FIG. PAGE 
 
 87. Precipitin Test. Preparation of Extract of Blood-stains Facing 304 
 
 88. Uhlenhuth's Filter - 305 
 
 89. A Rack for Precipitin and Agglutination Reactions 306 
 
 90. Titration of a Precipitin (Serum) 307 
 
 91. A Precipitin Reaction. Biologic Blood Test 309 
 
 92. Theoretic Formation of Cytolysins 318 
 
 93. Theoretic Structvu-e of a Polyceptor 321 
 
 94. Scheme showing Mechanism of Complement Fixation 333 
 
 95. Theoretic Structure of a Bacteriolytic Amboceptor 340 
 
 96. Method of Removing Exudate from the Peritonea^, Cavity of a Guinea-pig . . 346 
 
 97. Cultuie of Cholera Undergoing Bacteriolysis. A Positive Pfeifler Reaction 347 
 
 98. Stained Preparation of Cholera Undergoing Bacteriolysis Facing 347 
 
 99. Culture of Cholera Before Bacteriolysis 347 
 
 100. Stained Preparation of Cholera Before Bacterioly^ Facing 347 
 
 101. Bactericidal Test (Looped Pipet Method of Wriglit) Fadng 357 
 
 102. Theoretic Structure of a Hemolytic Amboceptor 364 
 
 103. Titration of Hemolytic Amboceptor .* Facing Zll 
 
 104. Venom Hemolysis Facing 387 
 
 105. A Vial to Contain Blood for the Wassermami Reaction 409 
 
 106. Outfit for Collecting Blood for the "Wassermaim Reaction 410 
 
 107. Titration of Hemolytic Complement Facing 413 
 
 108. Titration of Antigen for Anticomplementary Unit Facing 430 
 
 109. Titration of Antigen for Antigenic Unit ' Facing 430 
 
 110. Wassermarm Reaction (First Method) Facing 438 
 
 111. Reading the Wassermann Reaction t Facing 440 
 
 112. Wassermann Reaction (Second Method) Facing 444 
 
 113. Wassermann Reaction (Third Method) Facin-g 445 
 
 114. Wassermann Reaction (Fourth Method) Facing 449 
 
 115. Titration of Anti-human Hemolytic Amboceptor Facing 454 
 
 116. Moguchi Modification of the Wassermann Reaction Facing 454 
 
 117. Anticomplementary Titration of a Gonococcus Antigen Facing 479 
 
 118. Gonococcus Complement-fixation Reaction Facing 4S1 
 
 119. Urticarial Rash of Serum Sickness Facing 574 
 
 120. Multiform Rash of Serum Sickness Facing 574 
 
 121. Local Serum Anaphylactic Reactions Facing 570 
 
 122. Method of Performing the Cutaneous Tubercuhrj, Test (von Pirquet) .... 597 
 
 123. A Positive Cutaneous Tuberculin Reaction (von Pu-quet) Facing 598 
 
 124. A Positive Conjunctival TubercuUn Reaction (Wolff-Eisner-Calmette) 
 
 Facing 598 
 
 125. A Positive Percutaneous TubercuUn Reaction (Moro) Facing 599 
 
 126. A Positive Luetin Reaction Facing 604 
 
 1,27. Production of Cow-pox Vaccine 628 
 
 128. Method of Vaccination Against Smallpox 631 
 
 129. Vaccinia (7-day lesion) Facing 632 
 
 130. Vaccinia (9-day lesion) Facing 632 
 
 131. Vaccinoid. A Vaccination Scar Facing 632 
 
 132. Preparation of Rabies Vaccine i 641 
 
 133. Preparation of TubercuUn 665 
 
 134. Method of Subcutaneous Injection 689 
 
 135. Method of Intravenous Injection by Means of a Syringe 692 
 
 136. Method of Intravenous Injection by Gravity 693 
 
16 LIST OF ILLUSTRATIONS 
 
 PIG. 
 
 PAGE 
 
 137. Outfit for Intraspinal Injection of Antimeningitis Serum by Cii-a\-it3- 
 
 (Sophian) ' 698 
 
 138. Method of Intraspinal Injection by Gravity 'j^^ 
 
 139. Method of Intraspinal Injection b^,- Means of aSyiingi-- 701 
 
 140. Bloodofliat Infected with S]).recurrentis (dark ground illumination) 791 
 
 141. Same After Administration of Salvarsan (dark ground illumination) 792 
 
 142. Method of Intravenous Injection of Salvarsan 800 
 
 143. Method of Intravenous Injection of Neosalvarsan 801 
 
INFECTION, IMMUNITY, AND SPECIFIC 
 
 THERAPY 
 
 Part I 
 
 CHAPTER I 
 
 GENERAL TECHNIC 
 
 In this chapter simple methods are described for preparing capillary 
 pipets and similar apparatus usually made in the laboratory, and a 
 few general directions are given concerning the preparation of glass- 
 ware and other material employed in the various methods described 
 in succeeding chapters and in experimental werk. 
 
 It may he well here to utter a word of caution to the inexperienced 
 against observing undue haste in performing the manipulations of im- 
 munologic technic. Careful and painstaking work is essential in order to 
 secure reliable and successful results, and should never be sacrificed for 
 speed, the latter being attained only by experience. 
 
 CENTRIFUGE 
 
 1. A good centrifuge is one of the chief requisites of a laboratory 
 equipment. While any good instrument will answer, preference should 
 be given to the larger types, fitted for holding both 15 c.c. and 50 c.c. 
 centrifuge tubes, propelled by electricity, and mounted on a concrete- 
 block in the laboratory (Fig. 1). 
 
 2. The machine must be well oiled. 
 
 3. The counter tubes should be of the same weight — it is our cus- 
 tom to weigh the tubes on a small balance^ and adjust the counter 
 tubes until both are of equal weight. 
 
 4. The centrifuge tubes should rest loosely upon a rubber disc 
 or wad of cotton in the bottom of the metal tube or cup; otherwise 
 centrifuge tubes are quite likely to be broken,: especially if the machine 
 is run at high speed. 
 
 5. The machine should be started and stopped slowly, and un- 
 necessary speed and long running time should be avoided. 
 
 6. Never centrifugalize with cotton plugs in the centrifuge tubes. 
 
 2 17 
 
18 GENERAL TECHNIC 
 
 If the latter must be sealed, as when working aSeptically, rubber stoppers 
 should be used. However, if cotton plugs are large and fit tightly, 
 they may be prevented from becoming displaced by passing through 
 them two cross-pins in such manner that the ends will rest upon the 
 edge of the tube. The plugs are thus prevented from being thrown to 
 the bottom of the tube. 
 
 Fig. 1. — Electric Centrifuge. 
 
 Mounted on a concrete block. The scales are for the purpose of weighing and 
 
 counterbalancing the tubes. 
 
 7. If the centrifuge is out of order, however slightly, it should not 
 be used, but repaired at once, or else it may be ruined. 
 
 PIPETS 
 1. Simple Capillary Pipets. — These are made of soft glass tubing 
 in the following way : 
 
PIPETS 19 
 
 Tubing having a caliber of 6 mm., with thin walls, that does not 
 become opaque, brittle, or "run" on heating; and that does not con- 
 tain lead, may be used. The question of alkalinity is also of importance 
 in connection with the tubing. Many of the cheaper grades undergo 
 disintegrative changes, which are accompanied by the setting free of al- 
 kali, especially when the glass is heated. Glass of this kind shotild be 
 discarded, as it may introduce an element of etror into our experiments 
 and observations. 
 
 2. A convenient length of tubing — about 10 to 12 inches — is chosen; 
 this will make two pipets. If a sufficient length of tubing for both 
 sides is not available, one end may be heated and drawn out with 
 forceps, or a handle may be added by fusing to this short end an odd 
 piece of glass. 
 
 It is convenient to have on hand a supply of tubes cut to correct 
 lengths, plugged at each end with a ball of cotton, and sterilized in a 
 hot-air sterilizer. They are then ready to be drawn out as needed, 
 thus furnishing sterile pipets with cotton plugs that tend to prevent 
 contamination. 
 
 3. The flame must be so regulated as to play upon only so much of 
 the tube as will suffice to furnish the glass required for drawing out the 
 tubing. If a Bunsen flame is used, the tip of the inner greenish flame 
 should be appHed. The margins of the flame are the hottest, and for 
 this reason the tube must be shifted from side to side and be constantly 
 rotated. 
 
 4. In order to secure uniform heating and satisfactory pipets the 
 tube must be kept constantly rotated from the moment it enters 
 until it leaves the flame. The two ends of the? tube are to rest upon the 
 middle finger of each hand while the thumb and forefinger hold the 
 tube in position at either side and impart the rotatory movement. It 
 is also necessary that the tube be displaced laterally from time to time, 
 so as to bring each portion of the middle segment of the tube in turn into 
 the edge of the flame (Fig. 2). If the latter precaution is omitted, we 
 shall obtain a pipet with a central bulb or thicker segment and with 
 thinner segments on each side corresponding to„ the portions of the tube 
 which fie in the edges or hottest portion of the flame. 
 
 5. The tube is heated in this manner untilthe glass is quite plastic. 
 No attempt is made to draw out the tube until it has been entirely 
 withdrawn from the flame, as otherwise a portion becomes unduly thin 
 and plastic and divides, leaving a small, bent, and very poor pipet in 
 each hand. 
 
■20 GENERAL TECHNiC 
 
 The rapidity and force with which the tu|3e is drawn out determine 
 the cahber of the capillary stem. By drawing rapidly a tapering cap- 
 illary tube is obtained; by drawing slowly a larger capillary tube of 
 more uniform caliber is obtained. Of course, the worker cannot take 
 too much time, as the glass hardens quickly. With a httle practice 
 this part of the technic is soon mastered. Thorough and uniform heat- 
 ing and careful, steady pulling when the tube is sufficiently plastic are 
 of primary importance. 
 
 Wlien, owing to an error in judginent in heating the tube, it is with- 
 drawn before it is sufficiently plastic and begins to harden, the situa- 
 tion cannot be remedied by dramng out the* tube quickly with a jerk. 
 Similarly, when a tube has been partially drawn and hardens it cannot, 
 as a rule, be reheated and drawn out to make a satisfactory pipet. 
 
 Fig. 2. — Method of Making a Simple Capillary Pipet. 
 Shows manner of holding tubing in a flame and drawing into capillary tubes, 
 large portion has been removed from the center. 
 
 6. After drawing out the pipets the hands should be held steady 
 for a few seconds, i. e., until the glass has hardened; otherwise the 
 tubes will bend and be less satisfactory. 
 
 7. Capillary pipets are manipulated with rubber teats, which should 
 be of the best soft vulcanized rubber, and should fit snugly upon the 
 pipet, rendering it air-tight. 
 
 2. Looped Pipets. — Looped pipets find their main application in 
 the measurement of the bactericidal power of the blood, after the 
 method of Sir A. Wright. ^ 
 
 The essential features of these pipets are: (a) The capillary stem, 
 which serves for measuring and mixing the b*terial emulsion and serum; 
 (?)) the portion that serves first as a chamber for the sterile nutrient 
 broth and later as a cultivation chamber for determining whether the 
 
 1 "Technique of the Teat and Capillary Glass Tube," 1912. Constable and 
 Company, London. 
 
PIPETS 21 
 
 microbes that have been mixed with the serum have or have not been 
 killed by it; (c) the glass loop, which acts as a trap, preventing ex- 
 traneous contamination; and (d) the handle", upon which the rubber 
 teat can be fitted. With a little practice thesfe pipets are readily made. 
 
 1. Select glass tubing about 6 inches in length. 
 
 2. Heat one portion about two inches from the end, and when 
 sufficiently plastic, draw it out for about two or three inches or until it 
 is long enough to give a spiral loop of the desired dimensions (Fig. 3). 
 While the glass is still plastic hold the left hand steady, and with the 
 right hand lower the tubing and make a spiral loop in such manner 
 that the loop is closely applied, but does not touch the sides of the upper 
 and lower segments of the tube. Actual contact with the sides must be 
 
 E 
 
 Fig. 3. — Method oi' Making a Looped Pipet. 
 
 avoided, for this would produce strain and predispose the tube to 
 fracture. 
 
 3. The longer portion of tubing is now heated and drawn out to a 
 capillary pipet and broken through at the desired point. 
 
 4. Instead of this method, the capillary portion may be drawn be- 
 fore the loop is made. 
 
 3. Graduated Pipets. — 1. In this work 1 c.c. pipets graduated into 
 TTuCC. ; 5 and 10 c.c. pipets graduated into A c.c. mil render satisfactory 
 service. The pipets should be calibrated to the tip, and should pref- 
 erably be long, with a narrow lumen, rather than short, with a wide 
 lumen, as the latter renders the markings too Slose to one another. For 
 pipeting small amounts, as in certain complement fixation tests, a 0.2 6.c. 
 pipet graduated to ttt c.c. will be found quite serviceable, permitting 
 
22 
 
 GENERAL TECHNIC 
 
 00- 
 
 0.10- Si 
 
 0.15- 
 
 0- 
 
 1- 
 
 5- 
 
 ^ 
 
 m 
 
 ci 
 
 ^t 
 
 rl 
 
 ^1 
 
 I 
 
 4 
 
 accurate measureipent of small amounts of 
 fluid. The entire length of these pipets is 
 equal to the ordinary 1 c.c. pipet which 
 renders the subdivisions far apart and quite 
 easy to read (Fig. 4) . These pipets are made 
 by competent dealers upon special request. 
 
 2. These pipets should be perfectly 
 clean and clear, sterilized, and have sharp, 
 easily read marldngs. Pipets with broken 
 tips are difficult to handle, and if calibrated 
 to the tip, are inaccurate. 
 
 3. The worker should practise methods 
 of making accurate measurements. The 
 slightest slip may mean an inaccurate 
 measurement and produce untoward results. 
 The mouth end and the pipeting finger 
 should be dry, otherwise, on measuring 
 small amounts, the delivery will be jerky 
 and usually unsatisfactory. 
 
 4. After pipets = have held infectious ma- 
 terial they should be placed at once in a j ar 
 containing 1 per cent, formalin solution. 
 After pipeting bipod, milk, or serum, the 
 pipets should be rinsed or placed in a jar 
 containing water or a weak lysol solution, as 
 the formalin solution tends to harden these 
 substances and renders cleaning quite diffi- 
 cult. They should be washed thoroughly, 
 the mouth end being plugged neatly and 
 firmly with a bit ol cotton, and then placed 
 in a metal box or wrapped in newspaper and 
 sterilized in the hot-air oven. Unless all 
 the serum, blood, milk, etc., are well washed 
 out, the pipets may become occluded and 
 discolored. If this occurs, they should be 
 soaked for twenty-four hours in strong nitric 
 
 Pig, 4, Ghadtjated Pipets. (50 per cent.) or sulphuric acid, and washed 
 
 These pipets are gen- thoroughly and sterilized, 
 erally used in immunologic 
 
 work. Note the small cotton plugs in the mouth ends. The 1 c.c. pipet (second from 
 the left) is graduated near to, but does not actually include, the tip; this feature is 
 highly desirable, as it permits measuring small amounts of fluid and the pipet is not 
 necessarily ruined, even though a portion of the tip were chipped off. 
 
 '4 
 
BLOOD CAPSULES 23 
 
 5. The jar for holding soiled pipets should contain a layer of cotton, 
 otherwise, the tip may be broken off when the .pipets are dropped in. 
 
 BLOOD CAPSULES 
 
 Blood capsules were devised by Sir A. Wright for collecting small 
 amounts of blood for examination. The essential features of a capsule 
 are: (a) The upper straight limb, which can be drawn out to serve as a 
 needle for puncturing; (b) the recurved limb^ which makes it possible 
 to fill the capsule by gravity, without risk of the inflow being arrested 
 by the blood running down and blocking the straight limb, which pro- 
 vides an outlet for the air. 
 
 These capsules are easily made and prove quite serviceable, es- 
 
 E 
 
 ^,fi//yyyy^yj^y^fy 
 
 I v%%y<^////yvr///^.'^w^////,^ 
 
 Fig. 5. — Method of Making a Wright Blood Capsule. 
 
 pecially for collecting small amounts of blood' [or making agglutination 
 tests, opsonic measurements, etc. 
 
 1. Take a piece of soft glass tubing about 10 or 12 cm. in length, and 
 having an internal diameter of at least 5 mm. 
 
 2. Draw one end out into a capillary stem, and break this at an ap- 
 propriate point (Fig. 5). 
 
 3. Then reinsert the tube into the flame, tod, leaving a portion at 
 least 3 cm. in length to serve as the barrel of the capsule, draw out the 
 tube into a capillary stem about 8 cm. in length, and bend it so as to 
 form a stout recurved limb lying in the horizontal plane; now, before 
 the glass has lost its plasticity, draw the capsule gently upward so that 
 its long axis Mali be at an angle of about 30° hof izontally. Finally, sepa- 
 rate the capsule from the main tube by filing it across the capillary por- 
 tion at the distance indicated in the accompanying illustration (Fig. 5). 
 
24 GENERAL TECHNIC 
 
 4. The straight limb may now be drawn to a sharp point and used 
 as a needle. 
 
 VACCINE BULBS 
 
 These may be made of glass tubing, to hold 1 c.c. or more, al- 
 though when a large number are required, it is cheaper to purchase 
 ready-made ampules, such as are shown on p. 215. 
 
 1. Take a piece of glass tubing at least 10 cm. in length, and having 
 an internal diameter of 6 or 7 mm. 
 
 2. Seal one end by heating it in a hot Bunsen (jr blow-pipe flame. 
 
 3. After cooling, drop in one or two small glass beads, which serve 
 later to aid in mixing the vaccine before it is injected (Fig. 6). 
 
 4. Heat the opposite end and draw out into a wide and stout cap- 
 illary end. 
 
 5. This end may be broken through; the whole ampule is now ster- 
 
 FiG. 6. — Method of Making a Vaccine Ampule of Ordinary Glass Tubing. 
 
 ilized by heat, and after it is cooled, the required amount of vaccine is 
 inserted. 
 
 6. The open end is then sealed in the flame by warming the air in the 
 bulb above the surface of the liquid and finally sealing the tip, other- 
 wise the air may expand after heating and cause an explosion at the 
 tip. Care must be exercised not to heat the glass down to the level 
 of the fluid, as this tends to produce steam and ip crack the bulb. 
 
 Test-tubes may be drawn out and converted into ampules for hold- 
 ing vaccines, serums, or other fluids. 
 
 1. Thin-walled and sterilized test-tubes of appropriate size are 
 chosen. 
 
 2. The tube is heated at a point near the open end in the Bunsen 
 flame, in the same manner as the glass tubing, i.e., by keeping the tube 
 constantly rotating with a lateral movement to insure uniform heating. 
 
 3. When the glass has become plastic, it is drawn out into a stout 
 stem. 
 
TEST-TUBES 
 
 25 
 
 4. After cooling, it is filed through at an = appropriate place, being 
 careful to leave a somewhat long stem (Fig. 7], 
 
 5. The open end may now serve as a funneFfor filling the bulb. 
 
 6. The bulb is now sealed by warming the„air above the level of the 
 fluid and then sealing the tip. With a long stem, in order to secure a 
 portion of the contents, the sealed end may be broken off from time to 
 time; it is readily resealed. 
 
 7. Instead of this procedure the fluid may be placed in the test- 
 tube at once, the upper end being heated in the usual manner and drawn 
 out; the stem is broken through, and the tip sealed. If the tube is 
 small or the contents are such as will almost fill a tube, this method may 
 not be successful, owing to the production of steam on heating the 
 
 Fig. 7. — JIethod of Making a Large Vaccine Ampule of a Test-tube. 
 
 fluid,' which either cracks the bulb or causes the tip to explode at the 
 time of sealing. 
 
 TEST-TUBES 
 
 1. In this work test-tubes of various sizes .are used mainly for mak- 
 ing the agglutination, precipitin, complement' fixation, and other tests. 
 They should be made of good glass, "\idth round bottoms, and be well 
 annealed. 
 
 2. Test-tubes should be thoroughly clean, blear, and sterilized, pref- 
 erably by dry heat. It is not necessary to plug them with cotton unless 
 they are to be used for bacteriologic work, for when a large number of 
 tubes are used, this is a waste of time and of rnaterial. If the tubes arc 
 to be used within from twenty-four to forty-eight hours, merely plac- 
 ing the tube mouth end downward in the wire basket is sufficient. A 
 
26 
 
 GENERAL TECHNIC 
 
 piece of fresh cotton should be placed in the oven at the time of steriliza- 
 tion, and when this has turned a light-brown color, sterilization is com- 
 pleted. 
 
 3. After a tube has been used for holding living and infectious or- 
 ganisms, as in making bactcriologic, agglutination, 
 and opsonic tests, etc., the tube and its plug 
 should be boiled in water or a 1 per cent, solution 
 of caustic soda beforo it is again handled. In 
 making hemolytic experiments, the material is not 
 usually infectious, and it is sufScient to wash the 
 tubes without previous, boiling. In every case all 
 traces of adds or alkalis must he removed by copious 
 washing in plain water j as the slightest trace of 
 either may interfere with the accuracy of some 
 of the tests. Cheaper grades of glass may contain 
 relatively large amounrts of alkali, which would 
 introduce a disturbing element in the reactions. 
 
 SYRINGES 
 
 A good syringe is indispensable in perform- 
 ing bactcriologic and immunologic work. Various 
 sizes should be at hand, but usually a graduated 
 5 c.c. syringe answers most purposes. 
 
 1. Many kinds of syringes are on the market. 
 Those with rubber or feather plungers and pack- 
 ings are unsatisfactory, •& they cannot be sterilized 
 by boiling and soon leak. Nothing is more 
 exasperating than a lealdng syringe, as with the 
 leakage unknown quantities of inoculum are lost, 
 not to mention the possible dangers of con- 
 taminating the fingers, the animal, and the labora- 
 tory. 
 
 Syringes with metal or glass plungers are to 
 be preferred, as are also those upon which the 
 needle may be fitted without screwing (Fig. 8). 
 The old Koch syringe is fitted ^vith a rubber bulb for fdling and ex- 
 pelling the fluid. This arrangement is well adapted for making sub- 
 cutaneous injections, but is somewhat dangerous for purposes of mak- 
 ing intravenous injection, on account of the danger of injecting air. 
 
 Fig. 8. — A Satisfac- 
 tory Syringe (Rec- 
 ord) . 
 
 This syringe has 
 a glass barrel and met- 
 al plunger. It is easily 
 sterilized, durable, and 
 works smoothly and 
 accurately. 
 
SOLUTIONS 27 
 
 3. Syringes may be sterilized by filling them with 1 per cent, formalin 
 solution for a few minutes, followed by several washings with sterile 
 water or salt solution. This method is good for syringes having 
 leather or rubber packings and plungers. It is not safe for blood cul- 
 tures, as spore-forming bacteria may escape the stcriUzing process. 
 
 4. With all glass or metal syringes, it is best to boil the syringe, 
 especially if a careful aseptic technic is to be employed. The plunger 
 should be removed from the barrel, or else, tvhether it be of glass or 
 metal, it will expand more rapidly than the accommodation of the barrel 
 will permit. All parts should be placed in a pan or wrapped in gauze, 
 warm water added, and boiling allowed to take place for a minute or 
 so. After cooling the parts are adjusted. 
 
 Wright's method for sterilizing a hypodermic syringe for the ad- 
 ministration of vaccines is given on p. 217. 
 
 5. If infectious material has been used, the syringe, after using, 
 should be washed out and sterilized. The needles should be dried and 
 wired, and a small amount of vaselin rubbed over to prevent rusting. 
 The plunger may likewise be occasionally rubbed with a small quantity 
 of vaselin. Needles may be kept in oil or in absolute alcohol ; usually 
 thorough drying and wiring preserves them in good condition. 
 
 SOLUTIONS 
 
 1. Salt Solution. — 0.85 per cent, sodium chlorid in distilled water is 
 best adapted for immunologic work. This solution is prepared readily 
 by dissolving 8.5 grams of salt in a liter of water, filtering, and steriUzing 
 in an Arnold sterilizer for at least one hour. 
 
 2. Sodium citrate in 1 or 2 per cent, solution, made with normal salt 
 solution and not with plain or distilled water, is used to prevent the 
 formation of fibrin in drawn blood and exudates. 
 
CHAPTER II 
 METHODS OF OBTAINING HUMAN AND^ ANIMAL BLOOD 
 
 As a general rule, when blood is withdrawn to obtain serum a care- 
 ful aseptic technic should be employed. Similarly, when erythrocytes 
 are to be obtained for purposes of immunization it is necessary to avoid 
 contamination by proper cleansing of the parts and the use of sterile 
 needles, containers, and solutions. In obtaining erythrocytes for mak- 
 ing hemolytic tests it is not necessary that the blood be absolutely 
 sterile, the ordinary precautions against gross contamination being 
 sufficient. 
 
 Blood may be withdrawn to obtain the corpuscles or serum or both. 
 
 OBTAINING CORPUSCLES 
 
 Red blood-corpuscles are usually obtained and washed free of serum 
 for the purpose of making hemolytic reactions ajid experiments. 
 
 Leukocytes are usually obtained for the purpose of estimating the 
 opsonic index. The special technic for obtaining leukocytes for this 
 test is given on p. 195. 
 
 Larger quantities of leukocytes are obtained by injecting sterile 
 irritants, such as sterile aleuronat suspension, into the pleural or ab- 
 dominal cavities of suitable animals. (See p. 2Q1 .) 
 
 (a) Blood may be withdrawn and placed at once in two or three 
 volumes of 1 per cent, sodium citrate in 0.85 per cent, salt solution. 
 Clotting is prevented, and the corpuscles are secured by centrifugaliza- 
 tion or by sedimentation in the refrigerator. 
 
 (b) Blood may be dra'mi into a beaker or flask and defibrinated at 
 once by whipping with a rod or shaking with glass beads. When the 
 fibrin has been removed, the corpuscles and serum are secured by cen- 
 
 ■ trifugalization. 
 
 WASHING ERYTHROCYTES 
 
 For purposes of immunization or in making hemolytic tests red 
 blood-corpuscles are washed free of serum before being used. 
 
 1. Place the citrated blood, which has previously been diluted with 
 sufficient salt solution, in centrifuge tubes. Defibrinated blood may 
 
 28 
 
WASHING ERYTHROCYTES 
 
 29 
 
 be placed in centrifuge tubes and five to ten volumes of sterile normal 
 salt solution added and thoroughly mixed. Tubes are then carefully 
 Balanced and centrifuged at moderate speed for five minutes. 
 
 2. Remove the supernatant fluid down to the corpuscles with a 
 sterile pipet. Add an equal volume of salt solution; mix the corpuscles 
 
 Fig. 9. — A Suction Pump. 
 
 By attaching a capillary pipet to the rubber tubing fluid may be removed without 
 
 disturbing the sediment^ 
 
 and centrifuge. This process should be repeated once more in order to 
 insure thorough washing of the corpuscles to remove all traces of serum. 
 For removing supernatant fluids which are to be discarded, the suction 
 pump shown in Fig. 9 will be found very useful. It is well to fit the 
 
30 METHODS OF OBTAINING HUMAN ANd! ANIMAL BLOOD 
 
 rubber tubing with a capillary pipet, which permits the supernatant 
 fluid flush with the sediment to be removed. 
 
 3. After the last washing the supernatant salt solution should be 
 carefully removed, when the corpuscles are readj^ for use. 
 
 OBTAINING BLOOD-SERUM 
 
 If serum is desired at once, blood should be drawn into sterile cen- 
 trifuge tubes and the tube immersed in cold water for from five to ten 
 minutes; this facilitates clotting. The clot is then broken up with a 
 sterile platinum wire or glass rod, and the serum secured by rapid 
 centrifugalization. Or blood may be drawn into sterile cylinders, 
 Petri dishes, or centrifuge tubes, and allowed to stand at room temper- 
 ature for a few hours, after which they should be placed in a refrigerator 
 until the serum separates. Blood never should- be drawn into Erlen- 
 meyer flasks because of the diflaculty of drawingoff serum without dis- 
 turbing the clot. When drawn into Petri dishes, care should be taken 
 that the layer of blood is not too thin, otherwise drying will occur with 
 poor separation of the serum. As a rule, the best results are secured 
 by placing blood in centrifuge tubes, for if separation is poor or does not 
 occur at all, the clot may be broken up and serum secured by centrif- 
 ugahzation. So far as possible, avoid drawing blood from an animal 
 immediately after feeding, as under these circumstances the serum is 
 likely to be milky or opalescent. 
 
 OBTAINING CORPUSCLES AND SERUM 
 For certain purposes it may be desirable to obtain both serum and 
 corpuscles; these may be secured in the following way : 
 
 1. Place blood in a large centrifuge tube or cylinder, and defibrinate 
 with rods or glass beads. 
 
 2. Centrifuge thoroughly. 
 
 3. Remove the serum, which is slightly discolored on account of 
 defibrination, with capillary tube and rubber teat. 
 
 4. Filter the corpuscles into a centrifuge tube through a wisp of 
 cotton in a funnel to remove small particles of fibrin. 
 
 5. Add normal salt solution, and proceed with the washing process. 
 
 OBTAINING BLOOD PLASMA 
 In obtaining blood plasma it is necessary tb avoid coagulation of 
 blood by securing and handling the blood with the least amount of 
 
OBTAINING BLOOD PLASMA 
 
 31 
 
 trauma to leukocytes (paraffined tubes), and centrifuging rapidly and 
 at once in cold centrifuge tubes. 
 
 1. After warming a centrifuge tube immerse in hot molten paraffin. 
 Remove, drain, and allow the paraffin to harden. This produces a 
 thin coat of paraffin within the tube; if a thicker one is desired, im- 
 merse again until a coat of the desired thickness is obtained; chill the 
 tube thoroughly in cracked ice, but avoid getting water inside the 
 tube. 
 
 2. Blood should be secured quickly by venipuncture, using a dry 
 sterile needle, and passing the blood into a p&affined tube. 
 
 Fig. 10. — Method of Pricking a Finger. 
 The patient's finger is grasped firmly and lanced with a Daland lancet across 
 the folds of skin. When lanced parallel with the skin-folds, the wound is likely to 
 close before sufficient blood is secured. 
 
 3. Centrifuge at once and at high speed. If this is not possible, 
 pack the tube in a large centrifuge cup, and surround with finely cracked 
 ice. This permits of more prolonged centrjfugalization, and at lower 
 speed, and yields a hemoglobin-free plasma. 
 
 4. The clear yellow plasma is removed at once with pipet and nipple. 
 
32 
 
 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 OBTAINING SMALL AMOUNTS OF HUMAN BLOOD 
 For obtaining small amounts of blood — up to 2 or 3 e.c. — for the 
 Widal reaction, complement fixation, and other tests, the following 
 method is satisfactory: 
 
 1. Wash the last joint of the middle finger with alcohol. If the 
 hand is cold, it should be warmed by immersing it in hot water. Before 
 puncturing compress the finger and squeeze in such a manner as to 
 drive the blood toward the end of the finger. 
 
 2. Prick deeply with a broad blood lancet, Hagedorn needle, or 
 scalpel (Fig. 10). 
 
 Fig. 11. — Method or Secukinq a Small Amount of Htohan Blood. 
 By pricking the finger deeply across the lines of the skin with a broad lancet, 
 two or more cubic centimeters of blood are easily collected in a small test-tube. Do 
 not use a large tube, as blood may be lost on the sides of the tube. 
 
 3. Collect the blood in a small test-tube, — about 8 cm. by 1 cm., 
 — such as is used in performing the Noguohi reaction for the serum 
 diagnosis of syphilis (Fig. 11). 
 
 4. By squeezing the finger, sufficient blood can usually be obtained 
 from one puncture practically to fill a tube of the size mentioned. One 
 to two cubic centimeters of serum are easily obtained in this manner, and 
 this is sufficient for condacting the ordinary serum reactions. When 
 
OBTAINING LARGE AMOUNTS OF HUMAN BLOOD 
 
 33 
 
 the treatment of syphilis is being guided by the Wassermann reaction, 
 frequent tests are necessary, and a patient may object to submitting to 
 repeated venipuncture. The method for securing blood just described 
 is so simple and efficient that objections to it are never made. 
 
 5. Blood may also be drawn in a Wright cfipsule, made by drawing 
 ojit ordinary thin glass tubing in the Bunsen burner. (See p. 23.) 
 After sufficient blood has been collected (Fig. 12), the straight empty 
 end is sealed with a flame and then cooled (Fig; 14) . The blood is then 
 shaken into this sealed end, and the bent end, in turn, sealed with 
 the flame. Care should be 
 
 taken not to heat the 
 blood. When the serum 
 has separated, the tube is 
 opened by filing at a point 
 above the clot and breaking, 
 protecting the hands with 
 a towel. The serum is 
 carefully removed with a 
 capillary pipet and nipple 
 (Fig. 13). 
 
 6. To obtain blood from 
 infants and small children 
 the large toe may be punc- 
 tured, but as a rule better 
 results are obtained by wet- 
 cupping or by puncturing a 
 vein. 
 
 Fig. 
 
 12. — CoLL^ci'iNG Blood in a Wright 
 CJapstjlb. 
 
 OBTAINING LARGE AMOUNTS OF HUMAN BLOOD 
 Larger quantities of human blood may be required for making com- 
 plement fixation reactions, the Abderhalden ferment test, etc. 
 
 (a) Phlebotomy. — 1. In adults, a prominent vein at the elbow, such 
 as the median basilic, is usually chosen. In children less than a year 
 old this vein is not suitable, better results being obtained when the ex- 
 ternal jugular or a temporal vein is used. 
 
 2. Place a rubber tourniquet or a few firm turns of a wide muslin 
 bandage above the elbow. 
 
 3. Apply tincture of iodin to the skin over the vein. The vein may 
 be rendered more prominent by directing the patient to open and close 
 the hand several times. 
 
 3 
 
34 METHODS OF OBTAINING HUMAN ANJD ANIMAL BLOOD 
 
 4. Steady the skin over the vein, and insert the needle in the direc- 
 tion of the blood-current (Fig. 15). It is more awkward, and of no 
 practical advantage, to puncture in a downward direction toward the 
 hand. The needle should be sharp, and of a size midway between the 
 ordinary hypodermic and a large antitoxin needle, as the former 
 is too small and the latter is unnecessarily large. The blood is then al- 
 lowed to drop into a sterile tube. It is not necessary to attach 
 
 Fig. 13. — Removing Serum 
 FROM A Wright Capsule. 
 
 Fig. 14. — Method of Sealt 
 iNG A Wright Capsule. 
 
 a syringe, although 5 to 10 c.c. of blood afe obtained more quickly 
 by this means on account of the possible gentle suction. Needle and 
 syringe should be sterilized by boiling. When larger quantities of 
 human serum are required as in auto-serum therapy, a platinum- 
 iridium needle should be used, as coagulation in the needle is less hkely 
 to occur; besides, these needles are readily sterilized by heating in the 
 flame. 
 
OBTAINING LARGE AMOUNTS OF HUMAN BLOOD 
 
 35 
 
 5. Loosen the tourniquet, withdraw the needle quickly, and seal the 
 wound with a touch of flexible collodion. 
 
 Fig. 15. — Methods for Securing Blood by" Puncture of a ^'EIN. 
 The middle figure shows distention of the veins of the arm about the elbow. The 
 needle is entered by a quick upward thrust. Practically any prominent and firm 
 'Vein may be used. The upper right-hand figure shows collection of blood in a test- 
 ;tube. Usually 10 c.c. or more are easily collected before clotting occurs. To secure 
 large amounts, use a larger needle with a smooth bore (preferably a platinum-iridium 
 needle). The lower right-hand figure shows collection of blood in a Keidel tube. 
 
 6. Instead of a syringe, the 5 c.c. vacuum bulb devised by Keidel 
 has proved quite satisfactory (Fig. 16). Thjs apparatus consists of a 
 
36 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 5 c.c. ampule with arm drawn out to a capillary tip and sealed after a 
 vacuum has been created by heating (Fig. 16a, B). A short piece of rubber 
 tubing connects the needle and the capillary portion of the ampule. A 
 needle of No. 25 gage is fitted tightly into the free 
 end of the rubber tubing. A slender glass tube 
 closed at one end and flaring slightly at the otlier 
 serves as a protection for the needle, which it covers 
 when the apparatus is sterilized. The apparatus is 
 sterilized in a hot-air oven at 150° C. for one hour. 
 To obtain a specimen of blood the needle is inserted 
 into a vein, and the capillary end of the ampule 
 crushed with a hemostat through the rubber tubing, 
 blood flowing into the ampule and replacing the vac- 
 uum. The protecting glass tubing is then replaced. 
 Not infrequently, especially in children and in 
 obese adults, one fails to enter a vein. Several 
 attempts to do so may result in ruining one or more 
 of the tubes. My colleague, Dr. Alfred Reginald 
 Allen, has devised a useful modification in the 
 technic of using this handy tube; this consisting 
 in detaching the bulb from the rubber tubing and 
 needle, inserting the latter into the vein, and, when 
 the blood appears, quickly attaching the bulb and 
 breaking the neck with a hemostat, in the usual 
 manner. By this method the bulb is not broken 
 until one is sure he has entered a vein and secured a 
 specimen of blood. 
 
 (&) Wet Cupping. — 1. This method is partic- 
 ularly applicable for securing blood from infants. 
 
 2. Cleanse an area over the back just below the 
 angle of the scapula. 
 
 3. Scarify with a few superficial linear incisions 
 or with a special scarifiert 
 
 4. Apply a cup and e>Jiaust the air with special 
 syringe. The vacuum produces marked congestion 
 of the skin with a ready flaw of blood. 
 
 5. Carefully release the cup and pour blood into a tube. 
 
 6. The apparatus devised by Blackfan, and shown in the accom- 
 panying illustration (Fig. 17), is quite satisfactory and collects blood 
 in a sterile tube. 
 
 =4 
 
 Fig. 16.— The Kei- 
 •DEL Tube for 
 Collecting 
 Blood. (Manu- 
 factured by the 
 Steele Glass Co., 
 of Philadelphia.) 
 
METHOD OF SECURING CEREBROfePTNAL FLUID 
 
 37 
 
 (c) Placental Blood. — For purposes of immunization corpuscles may 
 be obtained by collecting placental blood. 
 
 1. After tying and cutting the cord, the placental end is placed care- 
 fully in a 150 c.c. flask or bottle containing from 25 to 50 c.c. of sterile 
 2 per cent, sodium citrate in physiologic salt solution. To avoid con- 
 tamination, the cord may be Hghtly 
 
 sponged with 1 per cent, formalin 
 solution and severed with sterile 
 scissors. 
 
 2. By exerting pressure on the 
 uterus blood may be squeezed out 
 of the placenta. The flask is then 
 sealed with a sterile cotton plug and 
 gently shaken. 
 
 3. The corpuscles are obtained 
 by centrifugalization or sedimenta- 
 tion. 
 
 ^ 
 
 D. 
 
 full 
 
 METHOD OF SECURING CEREBRO- 
 SPINAL FLUID (RACHICENTESIS) 
 
 The chief purpose in making 
 spinal puncture is to obtain and 
 examine cerebrospinal fluid as an 
 aid to the diagnosis of cerebro- 
 spinal diseases. It is mainly of 
 value in neurologic and psychiatric 
 practice, for the purpose of securing 
 fluid for making the Wassermann 
 reaction, for a study of cytologic 
 changes, alterations in protein con- 
 tent, and the hke. Not infrequently 
 the procedure is required as an aid 
 to establishing a diagnosis of men- 
 ingeal diseases in children, particu- 
 larly tuberculous meningitis, epi- 
 demic cerebrospinal meningitis, meningeal irritation 
 gitis, " etc. 
 
 Contraindications. — Ordinarily, when skilfully performed, spinal 
 puncture is a harmless procedure. Unless thfe necessity for obtaining 
 fluid is very urgent, the operation should not Be done on persons in poor 
 
 Fig. 16a, — Parts ofthb KeidblTube. 
 E is" the vacuum bulb which is at- 
 tached to the needle by a piece of rubber 
 tubing (D) ; the glass tube (B) covers 
 the needle and the whole is sterilized. 
 
 serous memn- 
 
38 
 
 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 physical condition. Kaplan has cautioned against making lumbar 
 puncture in the presence of tumors of the posterior fossa, particularly 
 of the cerebellum. When it is highly desirable to study the fluid of 
 such cases, 2 c.c. may be "\vithdrawn, and immediately replaced with 
 sterile normal salt solution, or if no immediate effects are observed, the 
 patient may be kept in bed for the next twenty-four hours. 
 
 Preparation of Patient. — In bed-fast patients the puncture may be 
 
 Fig. 17. — A Wet Cup for Securing Blood fkom Children. (Devised by 
 
 Blackfan.) 
 The cup is held in this position over a scarified area; air is exhausted by means of 
 a pump attached to the rubber tubing; blood collects in the small test-tube. 
 
 made at any time ; with ambulatory patients,, however, the most suit- 
 able time is late in the afternoon, so as to permit* the patient to rest over- 
 night. 
 
 The ordinary preparations consist in scrubbing the skin of the lumbar 
 region with green soap and hot water, using gauze sponges, followed by 
 washing -ndth alcohol and ether. The area is then covered with sterile 
 gauze, and, just before the puncture is made, an application of 10 per 
 cent, tincture of iodin is made; or the preliminary cleansing may be 
 
METHOD OF SECURING CEREBROSPINAL FLUID 39 
 
 omitted, two or three coats of the iodin being sufficient. After the 
 fluid has been secured, the iodin may be removed with alcohol and 
 gauze. The operator's hands should be cleansed carefully and washed 
 in alcohol and bichlorid solution or weak formalin, or he may put on 
 sterilized rubber gloves before handling the needle and performing the 
 operation itself. 
 
 Anesthesia. — In the majority of instances an anesthetic is not 
 necessary. In tabes dorsalis and general paralysis (two conditions 
 most frequently requiring spinal p\m.cture) the operation is peculiarly 
 painless. Sick children are apparently not greatly chsturbed, but in 
 adults it may be necessary to infiltrate the skin about the site of punc- 
 ture with 1 per cent, eueain (sterile) or cocain solution. Ethyl chlorid is 
 much less satisfactory, except for the mental effect it has upon the 
 patient. Children may receive a few drops; of ether. With nervous 
 patients it is good practice to obviate nervous shock by adopting a few 
 simple precautions against causing unnecessary- pain. 
 
 Technic of Lumbar Puncture. — The patient may either sit in a 
 chair and bend forward, or he on the left side on the edge of a bed or 
 table. In the case of sick persons, particularly children, the latter 
 position is necessary; it is also advisable with nervous patients, as they 
 are likely to bend backward suddenly or jump up when the needle is 
 inserted, and I have known the needle to be broken off at such a time. 
 (See p. 700.) The back should be arched backward, the patient bend- 
 ing forward, and the knees being dra-rni up over the abdomen. 
 
 The needle should be made of flexible, not rigid, material; for adults, 
 a needle 10 cm. long, having a bore of 1 to 1.5 mm. and furnished with a 
 stilet, will be found satisfactory. For children, a shorter needle may be 
 used, but the bore should be about the same as that used for adults. 
 The needle should be sterilized carefully by boihng in water for several 
 minutes. 
 
 The operator now selects a "soft spot" for puncture. By running 
 the finger along the spines of the vertebrae, this will be found to be be- 
 tween the third and fourth lumbar spinous processes, about on a level 
 with the posterior superior spines of the iha. The needle is grasped 
 firmly and inserted with a sudden thrust, exactly in the median hne, 
 and straight forward. The thrust should be sufficient to push the 
 needle through the skin and muscles into the spinous ligaments; it 
 may then be inserted more slowly, a sudden "give way" indicating 
 that the canal has been entered (Fig. 18). This route is better than the 
 lateral route, as there is less danger of striking vertebral processes or 
 
40 
 
 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 otEer obstructions. The stilet is now withdrawn. Usually the first 
 fluid to appear is stained with blood and should be collected in a sep- 
 arate tube. From 5 to 10 c.c. of fluid are then collected in a second 
 sterile tube, the needle is quickly withdrawn, ^nd the puncture wound 
 sealed with collodion and cotton or with adhesive plaster. 
 
 It sometimes happens that, on withdrawing the stilet, no fluid 
 issues forth. In this case the patient is instructed to take a deep 
 , ... _„ 1 
 
 Fio. 18. — Technic of Spinal Pttncture. 
 The patient is sitting on the edge of a chair and is bent forward; the crests of the 
 ilia are indicated by black lines, and are on a level with the spinous process of the 
 fourth lumbar vertebra; the "soft spot" is found just above. The needle is shown 
 in Jigs. 137 and 138. The first tube receives the first few drops of fluid, which are 
 usually blood tinged. 
 
 breath, and if fluid does not appear now, the stilet may be inserted 
 gently to dislodge any material that may be "occluding the needle, or 
 the needle may be withdrawn a trifle if it has, been inserted too far, or 
 may be advanced a little if it has not entered the canal. If, however, 
 the tap proves a dry one, or if only a few drops of blood are obtained, 
 it is not advisable to make another puncture, as the second attempt is 
 likely to prove as unsuccessful as the first. 
 
OBTAINING SMALL AMOUNTS OF ANIMAL BLOOD 41 
 
 After-treatment of the Patient. — Occasionafly the needle may strike a 
 nerve filament, which occurrence is followed b"y more or less pain along 
 the course of its distribution; puncture of the bone is likely to be fol- 
 lowed by pain for several hours. The majority of patients are so little 
 affected by lumbar puncture that no precautions as regards the after- 
 treatment are necessary. As previously stated, it is advisable for the 
 patient to rest overnight. Sudden release of pressure or the nervous 
 shock may give rise to severe headache of one or of several days' dura- 
 tion; persons of hysteric temperament may, in addition, suffer from 
 diarrhea and vomiting. Rest in bed, the application of ice-bags, and 
 the administration of sedatives are usually sufficient to relieve these 
 after effects. 
 
 Disposal of the Fluid. — ^As a general rule, the fluid should be sent 
 at once to a laboratory, as total cell counts and bacteriologic cultures 
 are best made with fresh fluid. For the Wassermann reaction it is not 
 advisable or necessary to add a preservative, as the fluid will keep for 
 several days in a good refrigerator; if, however, the fluid is to be kept 
 for longer periods of time, 0.1 c.c. of a 1 per cent, solution of phenol 
 may be added to each cubic centimeter of fluid. 
 
 OBTAINING SMALL AMOUNTS OF ANIMAL BLOOD 
 Rabbit, — 1. Flip an ear vigorously with the hand, and rub with 
 xylol and alcohol. The xylol produces marked congestion and after- 
 ward should be carefully removed with alcohol and water, as it pro- 
 duces a low-grade inflammatory reaction. 
 
 2. Puncture a marginal vein -with a large needle. The blood will 
 flow quickly in drops and practically any amount up to 10 c.c. or even 
 more may be collected in a centrifuge or test-tube (Fig. 19). For 
 niaking preliminary tests of serum during immunization 2 c.c. of blood 
 is usually sufficient. Bleeding may be checked by making firm pres- 
 sure over the puncture. 
 
 Guinea-pig. — 1. Blood may readily be removed directly from the 
 heart by anesthetizing the animal ■with ether", and inserting a sterile 
 needle into the heart at the point of maximum pulsation. A syringe 
 for aspiration may be attached, but better results are secured by ad- 
 justing a suction apparatus. By means of a short piece of rubber tub- 
 ing the needle may be connected to a test-tube so arranged that a 
 partial vacuum is created by attaching to a water suction pump. As 
 soon as the heart has been entered, blood is ?een to flow into the tube 
 
42 
 
 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 and the constant suction prevents clot formation in the needle. In this 
 manner 5 c.c. of blood may readily be obtained. 
 
 2. Blood may also be secured by aspirating the external jugular vem. 
 The vein is exjDosed by making a small incision', as in giving intravenous 
 injections. 
 
 3. Sufficient blood to make many complement fixation tests may be 
 secured from a large pig by rubbing the ear vigorously with xjdol and 
 making a small incision in the margin. Bleeding is facilitated by at- 
 taching a small test-tube with a side arm to a suction pump. When the 
 
 Fig. 19. — Method op Bleeding A Rabbit from the Ear. 
 
 proper tube is held firmly over the ear, 5 c.c. of blood may be obtained 
 by this method. 
 
 Sheep. — 1. Small amounts of blood may be obtained by pvmctur- 
 ing one of the ear veins. 
 
 OBTAINING LARGE AMOUNTS OF ANIMAL BLOOD 
 
 Rabbit. — After immunization of a rabbit has been completed, the 
 
 animal is usually bled to death, the object being to secure the maximum 
 
 quantity of serum in a sterile condition. Various methods may be 
 
 used. The animal should be anesthetized by ether or high rectal 
 
OBTAINING LARGE AMOUNTS OF jtNIMAL BLOOD 
 
 43 
 
 injection of a gram of chloral hydrate in 10 c.c. of water, deep sleep being 
 induced by the latter in from five to ten minutes, and lasting for several 
 hours, during which time operative procedures produce no pain. 
 
 First Method (Nuttall). — The animal is fastened to an operating 
 board, or, preferably, held by an assistant, and the hair over the neck 
 and thorax is moistened with a 1 per cent, lysol solution. By means 
 
 F 
 
 Fig. 20. — A Dissection of the Neck of a Rabbit to Show the Relations of 
 
 THE Carotid Artery. 
 
 T, trachea; A, carotid artery; V, internal jugular vein; V.N., vagus nerve. 
 
 of a sterile knife the skin is cut longitudinally and the neck muscles ex- 
 posed for a considerable distance. The animal is then held upright by 
 the assistant over a sterile dish or a large sterile funnel, emptying into 
 a cylinder or ,50 c.c. centrifuge tube. The operator stretches the neck 
 .by carrying the head backward, and severs tjie large vessels on one or 
 both sides of the neck with a sharp sterile scalpel or razor, avoiding 
 
44 METHODS OF OBTAINING HUMAN AlSfD ANIMAL BLOOD 
 
 opening the trachea and esophagus. After bleeding, the dish is cov- 
 ered or the tube plugged and set aside for thq serum to separate. This 
 method is quite simple, may be employed by the inexperienced, and 
 usually yields a large amount of sterile serum.. 
 
 Second Method. — The animal is fastened to the operating board and 
 the neck is stretched by placing a roller beneath it. The hair over the 
 neck is clipped close, and the skin moistened with alcohol and 1 per 
 
 Fia. 21. — Method op Bleeding a Rabbit from the Carotid Artery, 
 Second Method. 
 
 cent, lysol solution. The carotid artery of one side is exposed by making 
 a straight incision through the skin over the trachea and skinning well 
 to one side, exposing the sternohyoid muscles and external jugular vein. 
 The carotid artery, internal jugular vein, and pneumogastric nerve are 
 to be found at the outer border of the sternohyoid muscles (Fig. 20). 
 By means of blunt dissection the artery is exposed and carefully isolated. 
 Two small spring clamps or hemostats are then apphed close together 
 
OBTAINING LARGE AMOUNTS OF ANIMAL BLOOD 
 
 45 
 
 at the distal end, and the artery divided betwefen them. The proximal 
 end is then held with forceps within the mouth of a sterile cyUnder or 
 large centrifuge tube. The wall of the artery is. incised with fine scissors 
 proximal to the forceps, and the blood is allowed to flow into the ves- 
 sel. The yield of blood may be increased somewhat by exerting pres- 
 sure on the animal's abdomen and thorax. 
 
 To avoid the risk of contamination in the foregoing method, the 
 apparatus shown in Fig. 21 may be used. The whole apparatus is 
 
 Fig. 22.- 
 
 -Method of Bleeding a Rabbit from the Carotid Artekv, 
 Third Method. 
 
 sterilized in the autoclav before using. After*the artery has been ex- 
 posed and isolated, a temporary clamp is applied to the proximal end. 
 A small incision is made in the wall of the artery, and the cannula in- 
 serted and fastened with a ligature. The clamp is then removed, and 
 blood collected in a large tube. 
 
 Third Method.- — The following method, employed at the Pasteur 
 Institute at Paris, has been found very useful. The animal — a rabbit 
 or a guinea-pig — ^is anesthetized, and secured to an operating-table. 
 The carotid artery is carefully and asepticalfy exposed, and separated 
 
46 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 from the tissues for a distance of at least one inch; a ligature is now 
 tied securely about the artery, at the distal end of exposure; a second 
 
 Fia. 23. — Method of Bleeding a Guinea-pig. 
 
 ligature is placed in position, and looped loosely, ready to tie about the 
 proximal end (Fig. 22). 
 
' OBTAINING LARGE AMOUNTS OF ANIMAL BLOOD 47 
 
 The bottom of a large sterile test-tube is Leated and drawn out to a 
 fine point, as shown in the illustration (Fig. 22), and the tip is broken 
 off. The operator now places his moistened forefinger under the ar- 
 tery, elevating it up and rendering it taut; the tip of the tube is then 
 passed through the wall into the interior of the vessel toward thq heart. 
 The moment the vessel is entered the blood-pressure drives the blood 
 into the tube, so that 20 c.c. are soon secured. An assistant now ties 
 the ligature below the site of puncture; the tube is withdrawn, and the 
 tip sealed in a flame. The ends of the ligatures are cut short and the 
 wound is stitched. Healing usually occurs at once, and if subsequent 
 study of the blood is required, the other carotid and the femorals can 
 be used similarly for securing it. 
 
 Fourth Method. — The animal is fastened to the operating board, and 
 the hair over the neck and thorax moistened with alcohol or lysol solu- 
 tion. The right thorax is then incised and held open by an assistant. 
 The right lung is seized with sterile forceps and quickly severed at the 
 base with sterile scalpel or scissors. The heart is then punctured, and 
 the blood is quickly removed from the thoracic cavity with a sterile 25 
 c.c. pipet with a large opening. Unless the lung is removed, it tends to 
 float and block the end of the pipet. Everything must be in readiness, 
 as otherwise blood will be lost, flooding the thoracic cavity. 
 
 Guinea-pig. — Pig serum is usually secured -to furnish complement in 
 hemolytic tests, and should be used within twenty-four or forty-eight 
 hours after bleeding. Precautions to insure-sterility arenot, therefore, 
 usualljr necessary. 
 
 1. The animal is anesthetized with ether and the large vessels of the 
 neck on one side are exposed h\ a longitudinal incision. These are 
 severed, and the blood is collected in a Petri dish or in a centrifuge tube 
 by means of a funnel (Fig. 23). 
 
 2. By means of a sharp-pointed scissors the vessels on one or both 
 sides of the neck may be incised transversely at one cut, inserting the 
 blade deeply and close to, but avoiding, the trachea and esophagus. 
 
 Rats. — 1. Small quantities of blood may be obtained by snipping 
 off the tip of the tail of the animal and milking blood into an appropriate 
 sterilized tube containing glass beads, or 2; per cent, sodium citrate 
 solution. In this manner one or more culjc centimeters of blood are 
 easily obtained, and at once defibrinated and injected into the perito- 
 neal cavities of other animals, as in inoculating tr^Tjanosomes, etc. 
 
 2. Large quantities of blood are obtained by severing the large vessels 
 :of the neck, under anesthesia. 
 
48 
 
 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 Sheep. — Blood may easily be obtained from a freshly Idlled animal. 
 The first flow of blood is discarded, and a portion of the remainder is 
 collected in a large, sterile, thick-walled flask containing glass beads 
 
 Fig. 24. — A Dissection of the Neck of a Sheep to Show the Relations of 
 THE External Jtjgulak VfciN. 
 T, trachea; O.M., omo-hyoid muscle; E.J.V., external jugular vein; S.C.M., 
 sterno-oleido-mastoid muscle. This dissection was made soon after natural death 
 and shows the position and size of the vein with the head held backward as it is when 
 blood is removed according to the teohnic described in the text. When distended, 
 the vein is even larger than shown ; it is quite superficial and is usually palpable when 
 pressure is made over the vein at the base of the neck. 
 
OBTAINING LARGE AMOUNTS OF ANIMAL BLOOD 
 
 49 
 
 By shaking vigorously the blood is defibrinatcd, if one desires to obtain 
 corpuscles, or the blood may be collected in a cylinder and defibrinated 
 by whipping with glass rods. 
 
 It is usual, however, in large laboratories, to keep a sheep and 
 remove the blood as it may be required. Small amounts may be 
 obtained from the ear vein, larger quantities being secured from an 
 external jugular vein in the following manner :„ 
 
 gflf.,.-^ 
 
 I, 
 
 Fig. 25. — Method of Bleeding a Sheep pbom the External Jdgulah Vein. 
 The operator is distending the vein by pressure over the base of the neck with 
 the left hand. When distended, the vein can usually be felt beneath the skin. The 
 needle here shown is reduced to a little more than hialf the actual size. 
 
 1. One may do the bleeding alone, although the aid of an assistant 
 is usually necessary, especially if the animal is large and vicious. 
 
 2. The sheep is thrown on its back, and the head is held on the knees 
 of an assistant seated on a low box or stool. 
 
 3. The operator may straddle the animal tp*hold down the fore feet, 
 although this is not necessary unless the animal is vicious. 
 
 4 
 
50 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 4. The wool on the left side of the neck is clipped closely with scis- 
 sors and alcohol applied. 
 
 5. The operator then grasps the neck low down with the left hand, 
 and by means of the thumb exerts pressure over the base of the neck. 
 The external jugular vein will be found in a groove between the omo- 
 hyoid and sternomastoid muscles (Fig. 24). Firm pressure over the 
 base of the neck usually distends the vein, which may be seen or easily 
 felt. After locating the vein, the pressure should be released for an 
 instant, when the distention will disappear. In this way the operator 
 may be more certain that he has located the vein. 
 
 6. A sterile stout needle, at least two inches in length and provided 
 with a trocar and special shank for firm grasping, is passed quickly into 
 the distended vein in an upward and inward direction (Fig. 25). It is 
 essential that the needle be sharp, otherwise it will be turned aside by 
 the wall of the vein. The end of the needle must not have too long a 
 bevel, or the point Avill pierce the opposite wall before the body of the 
 needle is well within the vein. The trocar is now removed, and blood 
 collected in a flask or bottle and defibrinated with glass beads and rods. 
 A short piece of rubber tubing may be attached to the needle. A suc- 
 tion apparatus is not needed because the flow of blood is good so long 
 as pressure is preserved over the vein at the base of the neck. 
 
 7. When the required amount of blood has been secured, pressure is 
 released and the needle quickly withdrawn. Bleeding ceases at once, 
 and the neck is then washed with alcohol. 
 
 8. By this method the same vein may be used over and over again 
 for several years. I have never known infection to occur, although the 
 gradual formation of scar tissue about the site of puncture may inter- 
 fere with the operation. 
 
 Hog. — Blood may be secured from hogs by clipping off a small por- 
 tion of the tail with a sharp razor or scissors, beginning at the tip. 
 Bleeding is usually quite free, but is easily controlled by a tourniquet 
 and bandage. The serum of hogs immunized against hog cholera is 
 secured in this manner. 
 
 Monkey. — 1. Small quantities of blood — up to 10 or 20 c.c. — may 
 readily be obtained from a small vein just fceneath the skin which 
 crosses over the inner malleolus at the ankle. When a tourniquet is 
 applied, the vein becomes prominent; the haij- is chpped, and tincture 
 of iodin applied over the skin; a small needle is passed into the vein, 
 and the blood collected in a centrifuge tube. 
 
 2. Large quantities of blood may be obtained from the femoral or 
 external jugular vein under light ether anesthesia. 
 
OBTATXIXG LARGE AMOUNTS OF AXIIIAL BLOOD 51 
 
 Dog. — 1. Small quantities of blood may be obtained in the following 
 manner; Applj'' a tourniquet just above theikiiee; clip the hair over 
 the anterior surface of the leg, and cleanse ^^-ith tincture of iodin and 
 alcohol; make a small incision in the long axis, exactly in the median 
 line; a fairly large vein appears at once just beneath the skin; Ijy in- 
 serting an appropriately sized needle, several iiibic centimeters of blood 
 are quickly and easily secured. The wound should be very small, and 
 usually requires no treatment other than an apfjhcation of collodion and 
 cotton. 
 
 2. Large quantities of blood are obtained from the external jugular 
 vein under ether anesthesia; the neck is shaved and cleansed; the skin 
 is incised over the vein, which is just beneath the skin, and blood re- 
 moved with a sterile needle and sjTinge. Pressure over the base of the 
 neck renders the vein more prominent. In fhe case of large dogs, in- 
 cision is not necessary, as it is easy to enter the vein directly through the 
 skin, as in bleeding the sheep from the external jugular vein or the 
 human from a vein at the elbow. Blood mat also be secured from the 
 femoral vein imder ether anesthesia. 
 
 Horse. — 1. Small quantities of blood for making agglutination and 
 complement fixation tests may readily be secured from a superficial 
 vein about the leg. The hair is chpped over the selected area, and the 
 skin sterilized ■with tincture of iodin. A tourniquet is applied to render 
 the vein prominent, the vessel is steadied between forefinger and thumb, 
 and a needle quickly inserted. 
 
 2. Larger quantities of blood are secured from the external jugular 
 vein. This operation is easily conducted Ltj an aseptic manner and 
 blood collected in sterile jars. The neck about the region of the vein, 
 usually on the left side, is clipped, and a large area washed ■with hot 
 lysol solution. A sterile sheet may be throts-n about the shoulders. 
 The animal is held or placed in specially constructed stalls that pre- 
 vent him from backing away or causing mischief. In large antitoxin 
 laboratories bleeding is conducted in special rooms, where a careful 
 aseptic operating-room technic may be oliserved. 
 
 The external jugular vein is rendered prominent by exerting pres- 
 sure at the base of the neck by the apphcatioti of a special tourniquet 
 or by the thumb and fingers of the left hand.. the thtmib being placed 
 just above the A'ein. A small incision is made through the skin, di- 
 rectly above the vessel. 
 
 A large needle is passed under the skin for a distance of an inch or 
 two and then thrust into the vein. Direct puncture into the vein is 
 
52 METHODS OF OBTAINING HUMAN AND ANIMAL BLOOD 
 
 avoided, as the needle-track under the skin closes after the needle is 
 withdrawn and serves to seal the puncture. The needle is attached to 
 
 Fig. 26. — Bleeding a Horse from the Jugular Vein. 
 
 sterile rubber tubing that conducts the blood into special jars (Fig. 26). 
 In this manner from six to twelve liters of blood are easily obtained. 
 
CHAPTER III 
 
 TECHNIC OF ANIMAL INOCULATION 
 
 This is a highly important part of immunologic work, as both for se- 
 nun diagnosis and for serum therapy the serum must be secured from 
 animals that have been artificially immunized. Successful inoculation 
 requires unremitting care and thoroughness, as the toxic effects of the 
 proteins in general may kill an animal before immumzation has been 
 completed. No hard and fast rules can be laid down — ^the weight of 
 an animal and the reaction to an injection should decide the frequency 
 and the size of subsequent injections. It is better to proceed slowly 
 and gradually, than to give too large a dose at once and at too frequent 
 intervals. 
 
 GENERAL RULES 
 
 1. Select an appropriately sized syringe that does not leak upon 
 being tested with water. As has been stated elsewhere, nothing is 
 more unsatisfactory than a leaking syringe, for not only may the hand 
 become soiled, but an unknown quantity of inoculum is lost. 
 
 2. The inoculmn should be sterile. This is especially desirable 
 when giving intravenous and intraperitoneal injections. When living 
 cultures of bacteria are to be injected, the syriiige and the needle should 
 be sterihzed in order to avoid the introduction of contaminating or- 
 ganisms. 
 
 3. Remove the plunger from the barrel, and sterilize all the parts by 
 boiling for at least one minute. As previously stated, an all-glass 
 syringe or a glass barrel and metal plunger is the most satisfactory. 
 (See Fig. 8.) The old-fashioned syringe with washers and rubber- 
 tipped plunger should find no place in a laboratory. 
 
 4. After cooling, exjiel the water and load the syringe. This may 
 be done by drawing the fluid directly into tlje sjTinge and measuring 
 the dose by its markings or by pipeting the exact dose into a sterile 
 Petri dish or capsule and drawing up in the sjTJnge. 
 
 5. The animal should be fastened or held firmly and in an easy posi- 
 tion. Everything should be in readiness, so that the injections may be 
 given thoroughly and carefully. 
 
 S3 
 
5l TECHNIC OF ANIMAL INOCULATION 
 
 6. In preparing the inoculum care should be exercised that no solid 
 particles enter the syringe. Aside from possibly blocking the needle and 
 interfering with the injection, the subcutaneous injection of small frag- 
 ments may do no particular harm, but in intravenous inoculation they 
 may cause fatal embolism. To obviate this danger the inoculum 
 should, if possible, be filtered through sterile filter-paper before the 
 syringe is filled. 
 
 7. Air-bubbles should be removed. The injection of small bubbles 
 of air into subcutaneous tissues may cause np-harm, but when injected 
 iiito veins they may cause serious disturbances or immediate death. 
 To avoid this the syringe, after being filled, should be held vertically, 
 with the needle uppermost. The needle should be wrapped in cotton 
 soaked in alcohol, and the piston of the syringe pressed upward until 
 all the air is expelled from the barrel and the needle. If a drop of in- 
 oculum is forced out, it will be collected on the cotton, which should 
 immediately be burned. 
 
 8. Injections should be given slowly. 
 
 9. The animal is then tagged or marked, or its coloring recorded. In 
 the case of rabbits, the metal ear tag is best. All data, e. g., the date, 
 size of dose, preparation and kind of inoculum, etc., should be recorded 
 in writing. 
 
 10. Wlien it is necessary to incise the skin in order to reach a vein 
 an anesthetic may be given. With superficial veins, and in subcutaneous 
 inoculations, the injections may be given so readily and easily that no 
 more pain can be felt than that wliich accompanies similar injections in 
 human beings. 
 
 Animals may be actively immunized in a variety of ways and in 
 thfferent locations in tlie animal body. For a particular antibody, a 
 certain method may be found especially efficacious, and this is dealt 
 with in a subsequent chapter. In serologic- work immunization may 
 be performed by subcutaneous, intramnsculai>, intravenous, intracardial, 
 and intraperitoneal injections. 
 
 METHOD OF PERFORMING SUBCXJTANEOUS INOCULATION 
 
 Fluid Inoculiun. — 1. Injections are usuallj^ given in the median line 
 of the abdominal wall. 
 
 2. Have the animal (a rabbit or a guinfea-pig) held firmly by an 
 assistant or secured to the operating-table. 
 
 3. Clip the hair where injection is to be made — it is not always 
 
METHOD OF PERFORMING STJBCUTANEaWS INOCULATION 55 
 
 necessary to shave the area. Apply a 2 per cent, iodin in alcohol solu- 
 tion. 
 
 4. Pinch up a fold of skin between the forefinger and the thumb of 
 the left hand; hold the syringe in the right hand, and insert the needle 
 into the ridge of skin between the finger and thumb, and push it steadily 
 onward until the needle has been inserted about an inch (Fig. 27). 
 Care must be exercised not to enter the peritdiieal cavity. Relax the 
 
 Fig. 27. — Subcutaneous Inoculation op= a Guinea-pig. 
 A fold of skin is pinched up and the needle entered into the fold. The skin is 
 then released, and the injection slowly given. A swelling indicates that the injection 
 is subcutaneous. 
 
 grasp of the left hand and slowly inject the flui;,!. If the skin is raised, 
 this shows that the injection is subcutaneous. If it is not, the needle 
 should be slightly withdrawn and inserted. 
 
 5. Withdraw the needle, and at the same time cover the puncture 
 with a wad of cotton wet with alcohol. A toflch of flexible collodion 
 over the puncture completes the operation. 
 
 Solid Inoculum. — Steps 1 to 3 are the same as" in the preceding. 
 
56 TECHNIC OF ANIMAL INOCTJL'ATION 
 
 4. Raise a small fold of skin mth a pair fif forceps, and make a 
 tiny incision through the sldn with a pair of sharp-pointed scissors. 
 
 5. With a probe, separate the skin from the underlying muscles to 
 form a funnel-shaped pocket. 
 
 6. By means of a fine-pointed forceps or a glass tube syringe in- 
 troduce the inoculum into this pocket and deposit it as far as possible 
 from the point of entrance of the instrument. 
 
 7. Close the wound ^^ith collodion and cotton. A single stitch with 
 fine thread may be necessary. 
 
 METHOD OF MAKING INTRAMUSCULAR INOCULATION 
 
 ^1. These injections are usually made into the posterior muscles of 
 the thigh or into the lateral thoracic or abdominal muscles. 
 
 2. Clip away the hair over the selected area, cleanse, etc., as for 
 subcutaneous injection. 
 
 3. Steady the skin over the selected muscles with the slightly sep- 
 arated left forefinger and thumb. 
 
 4. Thrust the needle of the syringe quickly into the muscular tissue, 
 and slowly inject the fluid. 
 
 METHOD OF MAKING INTRAVENOUS INOCULATION 
 Rabbit. — 1. The posterior auricular vein along the outer margin 
 of the ear is better adapted than a median vein for this purpose. 
 
 2. If a number of injections are to be made, commence as near the 
 tip of the ear as possible, as the vein may become occluded with thrombi, 
 and subsequent inoculations may then be given nearer and nearer the 
 root of the ear. 
 
 3. The animal should be held firmly, as the .slightest movement may 
 result in piercing the vein through and through and require reinsertion 
 of the needle. This is accomplished satisfactorily by placing the rabbit 
 upon the edge of the table and holding it firmiy there by grasping the 
 neck and front quarters, the assistant at thei same time compressing 
 the root of the ear with the thumb and forefinger. 
 
 4. If the hair is long, clip it. 
 
 5. The ear is struck gently with the fingers and washed with alcohol 
 and xylol ; the friction will render the vein more conspicuous. 
 
 6. The ear is grasped at its tip and stretched toward the operator, 
 or the vein may be steadied Ijy rolling the ea¥ gently over the left in- 
 dex-finger and holding it between the finger and thumb. 
 
METHOD OF MAKING INTRAVENOUS" INOCULATION 
 
 57 
 
 7. The inoculum should be free from soli A particles, and all the air 
 excluded from the syringe. As a general rule, the amount injected 
 should be as small as possible, and the. temperature of the inoculum be 
 near that of the body. If the syringe is filled shortly after sterilization, 
 when it has cooled enough to be comfortably hot to the touch the heat 
 will warm the injection fluid and not be hot enough to cause coagulation. 
 
 8. Hold the syringe as one would hold a pen, and thrust the point 
 of the needle through the skin and the wall of the vein until it enters the 
 lumen of the vein (Fig. 28). 
 
 9. Direct the assistant to release the pressure at the root of the ear, 
 and slowly inject the inoculum. If the fluid is being forced into the 
 
 Fig. 28. — Intravenous Inoculation, of a Rabbit. 
 
 subcutaneous tissue, which will be evident at once by the swelling which 
 occurs, the injection must cease and another attempt be made. 
 
 10. The needle is quickly withdrawn, a small piece of cotton moist- 
 ened with alcohol placed upon the puncture wolmd, and firm compression 
 applied. Wash the ear thoroughly with alcohol and water to remove xylol, 
 otherwise a low-grade inflammation which will render subsequent injec- 
 tions more difficult will follow. 
 
 Guinea-pig. — 1. Since the superficial veing are quite small, it is 
 necessary to make the injection into the external jugular vein. 
 
 2. The animal is tied to the operating-table and the hair clipped 
 away about the neck and shoulder on the right side, and 2 per cent, 
 iodin in alcohol applied. 
 
58 TECHNIC OF ANIMAL INOCULATION 
 
 3. A small roll is placed under the neck of the animal, to render the 
 operative area tenser and more easily accessible. 
 
 4. A few drops of ether may be given by an assistant, although one 
 soon learns to expose the vein quickly and there is practicallj' no pain 
 
 Fig. 29. — A Dissection of the Neck op a Guinea-pig to Show the Relations 
 OF THE External Jugular Vein. 
 The skin has been turned aside and the superficial fascia and fat removed; the 
 position of the vein is well shown and is readily exposed by a small and superficial 
 
 after the skin has been incised. If anesthesia is employed, it should be 
 just sufficient to overcome the struggles of the animal. 
 
 5. The assistant is directed to hold the h^ad backward in the med- 
 ian line. 
 
METHOD OF MAKING INTRA VENOUa INOCULATION 
 
 59 
 
 6, Pick up the skin just above and in the middle of the space be- 
 tween the shoulder and the tip of the upper end of the sternum — just 
 above and about in the center of the area where a clavicle in the human 
 would be situated. With sharp small scissors incise the skin for about 
 one-third of an inch. Separate the subcutaneous tissue gently with 
 forceps; a large vein at once comes in view (Fig. 29). Gently dissect 
 it free for about one-fourth of an inch. 
 
 Fig. 30. — Intravenous Inoculation of a Guinea-pig. 
 The vein is steadied by a pair of fine forceps and t}\e injection given through a 
 small needle. The incision here shown is larger than actually required in practice; 
 the vein is also smaller than normal, as the animal was dead for a few hours prior to 
 making the illustration. 
 
 7. Pick up the vein vith a pair of fine forceps, insert the needle 
 of the syringe gently in the long axis of the vein and slowly inject the 
 fluid (Fig. 30). 
 
 8. Withdraw the needle and apply firm pressure ^^dth a wad of clean 
 gauze or cotton. It is not necessary to tie off the vein. A stitch may 
 be inserted to close the skin wound and flexible collodion applied. 
 
60 
 
 TECHNIC OF ANIMAL INOCULATION 
 
 Mice and Rats. — 1. Mice and rats may be injected through a 
 caudal vein of the tail. These veins are quite small, and the injection 
 requires a fine needle and some experience in the manipulations. 
 
 2. Fasten the mouse 
 in a special trap, so 
 that the tail alone will 
 be exposed. Grasp the 
 tip between the left 
 thumb and index- 
 finger, and hold the tail 
 fully extended. 
 
 3. A caudal vein is 
 rendered prominent by 
 the gentle application of 
 heat in the form of hot 
 water, or by vigorous 
 rubbing with xylol or al- 
 cohol. The superficial 
 cells become softened, 
 and may be scraped off 
 with a sharp scalpel, 
 exposing a vein on each 
 side of the middle line 
 of the tail. 
 
 4. It is usually ad- 
 visable to have an as- 
 sistant steady the tail 
 while the inoculation is 
 being given; a fine 
 needle is essential. In- 
 oculation should begin 
 as near the tip of the 
 tail as possible, and in 
 subsequent inoculations 
 gradually approach the 
 root (Fig. 31). 
 
 5. Rats may also be injected through the external jugular vein, in 
 exactly the same manner as a guinea-pig is inoculated. (See Fig. 30.) 
 The animal is fastened to a small operating: board, and an assistant 
 holds the head to the left, which stretches the tissues of the right shoulder 
 
 Fig. 31. — Method of Intravenous Inoculation of 
 a Rat. 
 The hairs and superficial layers of the skin have 
 been scraped away with a scalpel. The vein on each 
 side of the middle line appears as a bluish line in the 
 subcutaneous tissues. 
 
METHOD OF MAKING INTRAVENOUS INOCULATION 61 
 
 and side of the neck. A small incision is made midway between the 
 middle line of the neck and the tip of the fote-shoulder. With super- 
 jicial dissection a prominent vein appears; this vein is jjicked up with 
 ■fine forceps and the injection is readily given through a fine needle. 
 
 Horse. — 1. Horses arc usually injected in the external jugular vein. 
 
 2. The hair of the neck in the region of the site of inoculation should 
 be clipped and thoroughly scrubbed with a hot solution of lysol. 
 
 Fig. 32. — Intravenous Inoctjlation of Horse. 
 The operator causes the vein to distend and become prominent by pressure with 
 the left hand. The needle is entered beneath the skin and is pushed upward for au 
 inch or more before the vein is entered. When withdrawn, this tunneled passage 
 closes and prevents bleeding. Larger injections may be given in the same manner 
 by gravity. 
 
 3. The horse should be held by an assistant; if the animal is vicious, 
 the injections should be given in a specially constructed stall. 
 
 4. The vein is distended by the operator, who grasps the region with 
 his left hand, the thumb being directly over the vein (Fig. 32) . 
 
 5. The needle is inserted beneath the skin, and passed upward for 
 a short distance, and then thrust into the veir^,. 
 
62 TECHNIC OF ANIMAL INOCULATION 
 
 6. After the injection has been given, either with a syringe or, when 
 the inoculum is large in amount by gravity from a large jar, the needle 
 is quickly withdrawn. Bleeding ceases as soop as pressure over the 
 vein is removed. 
 
 Sheep and Goats. — In sheep and goats the intravenous injection is 
 given into the external jugular vein, directly through the skin. The 
 hair is clipped, and the part shaved and disinfe-cted. Compression by 
 the finger at the root of the neck renders the vein more prominent. In- 
 jections are also readily given through a popliteal or a femoral vein. 
 If necessary, a small incision may be made through the skin in order to 
 expose the vein chosen for the injection. 
 
 Dog. — Dogs may be injected through the external jugular or pop- 
 liteal veins. The animal should be fastened to the operating-table. 
 
 2. There is a small vein just beneath the skin, in the median hne, 
 along the anterior surface of the leg, which is readily accessible. Clip 
 away the hair, and disinfect with iodin and alcohol. Direct the as- 
 sistant to grasp the thigh just above the knee, to distend the vein and 
 prevent movement, and make a small incision directly in the median 
 hne. A small vein is seen at once. Dissect free or pick up gently with 
 fine forceps and insert a small sharp needle. The injection can thus 
 be readily given. Withdraw the needle, apply firm pressure, and insert 
 a single stitch. Bind the wound with a few turns of a gauze bandage 
 or seal with collodion and cotton. 
 
 METHOD OF MAKING INTRACARDIAL INOCULATION 
 
 1. Guinea-pigs may be injected by the intracardial roTite instead of 
 intravenously. The techrdc is not, as a rule, more difficult, and no ill 
 effects are noticed. Not infrequently, however, attempts to inject in 
 the teart fail, and frequent trials are not permissible on account of 
 the danger of injuring the organ. p 
 
 2. The animal is tied to the operating board, or held firmly by an 
 assistant; an anesthetic may be given. 
 
 3. Determine the point of maximum pulsation to the left of the 
 sternum by palpation, and quickly insert a thin, sharp needle at the 
 selected area. A flow of blood indicates that the needle has entered the 
 heart. Attach the previously filled syringe and slowly inj ect the contents. 
 
 4. Detach the syringe in order to make sure that the injection was 
 intracardial, as intended, which is indicated by a flow of blood; then 
 quickly withdraw the needle. The puncture wound may be sealed with 
 collodion. 
 
METHOD OF MAKING INTKACARDIAL INOCULATION 63 
 
 FiQ. 33. — Method of Performing Intraperitoneal Inoculation of a Rabbit. 
 The head is held downward; the intestines gravitate toward the diaphragm 
 (note distention); this leaves an area between the umbilicus and pelvis relatively 
 free of intestines and lessens the danger of puncturing the intestines. 
 
64 TECHNIC OP ANIMAL INOCULATION 
 
 METHOD OF MAKING INTRAPERITONEAL INOCULATION 
 
 Rabbit. — 1. Clip the hair and shave an area about two inches in 
 diameter in the median abdominal hne, just below the umbiHcus. 
 Apply 2 per cent, iodin in alcohol. 
 
 2. Direct an assistant to hold the animal firmly, head down. With 
 the animal in this position the loops of intestine tend to sink toward the 
 diaphragm, leaving an area above the bladder which is sometimes free 
 of intestines (Fig. 33) . 
 
 3. The syringe is grasped firmly, and the npedle inserted beneath the 
 skin for a short distance, in the direction of the head in the long axis of 
 the animal when the hand is raised and the needle forced forward 
 through the peritoneum. When the peritoneum has been entered, this 
 is evidenced by a relaxation of the abdominal muscles. The needle 
 is then withdra^Mi slightly and the injection niade. 
 
 Guinea-pig. — 1. Direct an assistant to hold the animal firmly upon 
 its back. This is better than fastening it to an operating-table, for it 
 permits relaxation of the abdominal wall when the injection is to be 
 made. 
 
 2. CUp the hair close to the skin in the m^edian abdominal line. A 
 sm^all area may be shaved although this is not necessary. Disinfect 
 with an application of iodin in alcohol. 
 
 3. With the left forefinger and thumb pinch up the entire thickness 
 of the abdominal parietes in a triangular fold, and slip the peritoneal sur- 
 faces over each other to ascertain that no coils of intestine are included. 
 
 4. Grasp the sj^ringe in the right hand, and insert the needle into 
 the fold near its base. 
 
 5. Release the fold and inject the fluid. If a swelling forms, this 
 shows that the needle is in the subcutaneous tissues, and another at- 
 tempt should be made to enter the peritoneum. 
 
 6. It may be difficult to pinch up the parietes 'ndthout including the 
 intestine. In such case straighten out the animal and stretch the skin 
 between the left forefinger and thumb. Insert the needle obliquely 
 until it is beneath the skin. A slight thrust suffices to pierce the peri- 
 toneum, when the abdominal muscles -n-ill be felt to relax. Withdraw the 
 needle slightly and inject the fluid. 
 
 7. Seal the wound with a touch of collodion. 
 
CHAPTER IV 
 
 METHODS FOR EFFECTING ACTIVE IMMUNIZATION OF 
 
 ANIMALS 
 
 In overcoming an infection, acquired either =naturally or artificially, 
 the macroorganism develops antibodies, or protective substances, 
 against the infecting agent. Possessed of these antibodies, the animal 
 can subsequently withstand a more severe attack of the same infection. 
 Thus, the body-cells of the animal itself are actively concerned in pro- 
 ducing these antibodies, and the resulting protection or immunity is 
 therefore called "active immunity." 
 
 The general term "antigen" has been apphed to any substance that 
 can stimulate the formation of an antibody. The immunity following 
 scarlet fever is an example of active acquired immunity, although the 
 antigen is unknown. In order to acquire inummity it is not always 
 necessary for a person to have had the disease. Thus, a severe infec- 
 tion with the antigen of typhoid fever — Bacillus typhosus — results in a 
 general reaction, exhibiting sjonptoms and course known clinically as 
 typhoid or enteric fever; whereas, if the antigen is attenuated and in- 
 jected artificially in small doses in the form of a vaccine, the body-cells 
 react, producing antibodies and a resulting immunity against tjT)hoid 
 fever, without discomfort or danger to life. This process is known as 
 vaccination, the term being first applied to a similar procedure employed 
 in inducing an immunity against smallpox by the inoculation of a small 
 dose of the antigen attenuated or modified by passage through the cow 
 (cow-pox virus) . 
 
 This process of stimulating the body-cells to produce antibodies is 
 called active immunization, and, while the immunity following disease 
 is an example, the term is generally apphed to= artificial immunization, 
 as in vaccination, which serves in medicine, therefore, the primary pur- 
 pose of effecting prophylaxis. In laboratories active immunization of 
 animals is generally undertaken with a view to obtaining serums to 
 be used for diagnostic or therapeutic purposes. 
 
 Practically, any protein may serve as an antigen. Thus, animals 
 may be immunized not only with various bacteria, but with serum al- 
 bumins and globulins, milk, egg-albumen, epithehal cells, etc. It must 
 5 65 
 
66 METHODS FOR EFFECTING ACTIVE IMMUNIZATION OF ANIMALS 
 
 be remembered that these substances — the antigens — are toxic, and 
 that the process of immunization may evoke, a marked disturbance in 
 the general health of the animal. Special attention should be given to 
 feeding and the general care of the animal, the temperature, weight, the 
 presence of diarrhea, and the occurrence of abscesses, edema, paralysis, 
 etc. 
 
 If the animal dies, a careful postmortem and bacteriologic examina- 
 tion should be made, in order to study the changes produced by the 
 inoculated antigen, and to ascertain if death was induced by the antigen 
 or by contamination and secondary infection. 
 
 In the manufacture of serums on a large scale, especially for thera- 
 peutic use, horses are used almost exclusively. For diagnostic purposes 
 and especially in the study of immunity, smaller animals, such as rab- 
 bits, guinea-pigs, white mice, and rats, and occasionally goats or sheep, 
 are employed. 
 
 So far as their power of producing antibodies is concerned, there are 
 individual differences among the same species of animals; thus, horses 
 immunized against diphtheria differ in the quantity of antitoxin pro- 
 diiced. Similar differences are observed in the smaller animals. 
 
 Immimization with a single antigen usually produces several differ- 
 ent antibodies, although for diagnostic or therapeutic purposes one 
 usually predominates. Thus immunization^ of a rabbit with dead 
 typhoid bacilli produces agglutinin, opsonin, bacteriolysin, and comple- 
 ment-fixing bodies, although the agglutinin is probably the most prom- 
 inent and is used in diagnosis. Immunization with the diphtheria 
 bacillus and its toxin leads to the formation of an antitoxin, opsonin, 
 and complement-fixing body, although antitoxin is by far the most 
 prominent and is used therapeutically. 
 
 We give here methods for making various immune serums from small 
 animals, to be used for the purpose of study and for aiding diagnosis. 
 Curative serums, such as diphtheria and tetanus antitoxin, antimeningo- 
 coccus serum, etc., are made on a larger scale by immunizing horses. 
 
 GENERAL TECHNIC 
 1. The antigens are usually injected subcutaneously, intramuscularly, 
 intraperitoneally, or intravenously. As a rule, the sooner the antigen 
 comes in relation with the body-cells, the more rapid is the immunity 
 gained, and for this reason the intravenous and intraperitoneal routes 
 are frequently chosen. 
 
GENERAL TECHNIC 67 
 
 2. No fixed rules as to the amount to be injected can be given. 
 Experience may show a general method to be the most successful, but 
 as previously mentioned, the general condition and reaction of the ani- 
 mal will be the main guide as to the amount and frequency of the in- 
 jections. Severe reactions may yield unsuccessful results, and doses 
 so small as apparently to give no reaction may lead to a high-grade im- 
 munity. 
 
 3. A single injection seldom yields a highly valent serum. Re- 
 peated inoculations are usually necessary, and may be given in the fol- 
 lowing way: 
 
 (a) A small dose of antigen is injected. If "a reaction sets in, wait 
 until this has subsided, and then, after the fifth day, make a second in- 
 jection of a somewhat larger dose. After another interval of from five 
 to seven days a third injection of a still larger dosage is administered, 
 and so on for two or more injections. 
 
 (6) Same as preceding method, except that the first dose is the max- 
 imum one; subsequent injections arc of decreasing amounts. 
 
 (c) Same as preceding, with a constant dose of antigen at each in- 
 jection which does not produce a severe reaction. 
 
 {d) For several successive days a small cfr medium-sized dose of 
 antigen is injected (Gay). 
 
 The first three methods give excellent results, and the third is es- 
 pecially useful when a serum is needed as soon as possible. 
 
 4. It must be emphasized that good results are largely dependent on 
 the care with which the animals are injected. The operator should work 
 as aseptically as possible, especially when giving intraperitoneal and 
 intravenous injections, and avoid the productipn of embolism by the 
 injection of air or solid particles. Give the injections slowly, and take 
 particular care of the animals. However carefully the injections may 
 be given, unsatisfactory results not infrequently occur. Either the 
 animal refuses to react with the production of antibodies, or dies just 
 when immunization is about completed. It is, therefore, good practice 
 to immunize more than one rabbit at one sitting, in order that immune 
 serum may be had at the time planned. 
 
 Antigens. — ^Animals may be actively immujuzed with the following 
 substances : 
 
 1. With soluble bacterial toxins, as in the manufacture of antitoxins. 
 
 2. With bacteria themselves; whether livihg, attenuated, or dead, 
 as in making bacterial agglutinins, precipitins, immune opsonins, and 
 lysins (bacteriolysins) . 
 
68 METHODS FOR EFFECTING ACTIVE IMMUKIZATION OF ANIMALS 
 
 3. With soluble alien proteins, as scrum,. milk, egg-albumen, etc., 
 as in the manufacture of precipitins. 
 
 4. With various alien cells, as erythrocytes, kidnej' cells, sperma- 
 tozoa, etc., in the manufacture of cytotoxic serums, such as hemolysins, 
 nephrotoxin, spermatotoxin, etc. 
 
 PRODUCTION OF ANTITOXM 
 
 Diphtheria and tetanus antitoxins are prepared on a large scale by 
 the inoculation of horses with increasing doses of the respective toxins. 
 
 The methods for preparing these antitoxins, and also of antimeningo- 
 coccus, antistreptococcus, antipneumococcus,. and other curative se- 
 rums are given in the chapter on Antitoxins and Passive Inmaunization. 
 
 PRODUCTION OF AGGLUTININS 
 
 Agglutinating serums are frequently of iriuch value in making a 
 bacteriologic diagnosis of typhoid fever and cholera. For this purpose 
 unknown microorganisms are mixed with pfoper dilutions of known 
 immune serum, and the presence or the absence of agglutination noted. 
 As a rule, rabbits are used in the preparation of these serums; for the 
 production of larger quantities of serum, goats and horses are occasionally 
 employed. 
 
 The injections may be given intravenously or intraperitoneally; 
 occasionally the first injections are given subcutaneously, to be followed 
 later by intraperitoneal injections. By heating the cultures at a temper- 
 ature not exceeding 60° C. for from one-half to one hour, there is less 
 danger in the subsequent handling of the cultures and agglutinins are 
 readily produced. 
 
 1. Use forty-eight-hour agar cultures of the organism, such as 
 Bacillus typhosus. Spirillum cholerse, etc. Bouillon cultures may be 
 used, but are not recommended on account of the various other con- 
 stituents present in the medium. 
 
 2. With a sterilized three-millimeter platinum loop remove one 
 loopful of culture and rub up in 2 c.c. of sterile salt solution in a small 
 test-tube until a homogeneous emulsion is secured. 
 
 3. Heat the emulsion for thirty minutes at 60° C. in a water-bath. 
 
 4. Inject intravenously. 
 
 5. Give four more injections at intervals of a week as follows: 
 
 Second dose: 2 loopfuls in 2 c.c. NaCl solution, heated. 
 Third dose: 4 loopfuls in 2 c.c. NaCl solution, heated. 
 Fourth dose: 6 loopfuls in 2 c.c. NaCl solution, heated. 
 Fifth dose: 1 agar slant in 4 c.c. NaCl' solution, heated. 
 
PRODUCTION OF BACTERIOLYSINS (BACTERIOLYTIC SERUM) 69 
 
 6. One week after the last injection has bepn made the blood is 
 tested, and if found of satisfactory titer, the animal is killed and the 
 serum secured. 
 
 Intraperitoneal Method (Rabbit). — 1. Same as the preceding, ex- 
 cepting that larger doses are given. 
 
 First dose: 2 loopfuls in 4 c.c. NaCl solution, heated. 
 Second dose; 4 loopfuls in 4 c.c. NaCl solution, heated. 
 Third dose: 6 loopfuls in 4 c.c. NaCl solution, heated. 
 Fourth dose: 1 agar slant in 5 c.c. NaCl solution, heated. 
 Fifth dose: 1 agar slant in 5 c.c. NaCl solution, heated. 
 2. The blood is tested one week after the last injection has been 
 made. 
 
 PRODUCTION OF IMMUNE OPSONINS 
 
 1. These may be produced in the same manner as the agglutinating 
 serums, immune opsonins being readily demonstrated in the same se- 
 rums. For actual diagnostic work, artificial immune opsonins are sel- 
 dom required, but to secure an immune serum for experimental studies 
 on opsonins a culture of Staphylococcus pyogenes aureus may be used in 
 immunizing a guinea-pig as follows: 
 
 First dose: 1 loopful of twenty-four-hour agar culture in 2 c.c. 
 
 NaCl solution heated for one-half hour at 58°C. and given 
 
 subcutaneously. 
 Second dose: 1 loopful in 2 c.c. NaCl, heated; intraperi- 
 
 toneally. 
 Third dose: 2 loopfuls in 2 c.c. NaCl, heated; intraperitoneally. 
 Fourth dose: 3 loopfuls in 2 c.c. NaCl, heated; intraperitoneally. 
 Fifth dose: 6 loopfuls in 2 c.c. NaCl, heated; intraperitoneally. 
 
 2. Bleed the animals one week after the last injection has been made. 
 
 3. Owing to its large size, Bacillus anthracis may be substituted. 
 This is a spore-forming organism, and since it is dangerous unless scru- 
 pulous care in handhng is exercised, it is not usually wise to employ it in 
 experimental work. 
 
 PRODUCTION OF BACTERIOLYSINS (BACTERIOLYTIC SERUM) 
 1. These are prepared in exactly the same manner as agglutinins. 
 In practical diagnostic work the Spirillum cholera) is most frequently 
 used. In experimental studies of bacteriolysis the tjT^hoid bacillus 
 and its immune serum may be employed with equal success. 
 
70 METHODS FOR EFFECTING ACTIVE IMMyNIZATION OF ANIMALS 
 
 PRODUCTION OF PRECIPITINS 
 
 Precipitin immune serums are frequently of value in making a differ- 
 entiation of the proteids, as in the examination of blood-stains, meat, 
 milk, cheese, etc. They are usually prepared by immunizing large 
 rabbits with injections of the sterile antigen» Injections may be given 
 intravenously or intraperitoneally, the former usually yielding the more 
 potent serums. 
 
 Any foreign serum may be used in the preparation of precipitins, 
 such as that of the human, horse, ox, dog, cat, guinea-pig, etc. To pro- 
 duce an antirabbit precipitin a guinea-pig is immunized by intraperi- 
 toneal injections of rabbit serum. An antihuman precipitin serum of 
 high titer is usually obtained with difficulty. It is good practice to 
 immunize a number of rabbits with each antigen, as some animals will 
 produce no precipitin whatever the method used. 
 
 In preparing precipitins for the purpose of identifying blood-stains 
 whole blood may be injected. It is better, however, to use serum only, 
 as the immune serum may be used in diagnosis, according to the method 
 of complement fixation, when the presence of hemolysin is not advisable. 
 
 Serum Precipitins (Intravenous Method). — First Method. — Three 
 injections are given — of 5, 10, and 15 c.c. — on each of three successive 
 days, and the animals are bled twelve days after the last injection has 
 been made. 
 
 Second Method. — One injection of 30 c.c. of serum may be given, and 
 followed twelve days later by bleeding. 
 
 Third Method. — A slower method consists in giving the injections at 
 intervals of a week. After the third dose a few cubic centimeters of blood 
 are withdrawn from the ear, and the serum titrated, as rabbits are most 
 prone to succumb after the third dose, and in many instances the serum 
 is of such strength as to require no further immunization. The ani- 
 mals are bled one week after the last inj ection has been given. 
 
 Doses may be given as follows: 
 
 First dose: 10 c.c. serum intravenously. 
 Second dose: 8 c.c. serum intravenously. 
 Third dose: 5 c.c. serum intravenously. 
 Fourth dose: 5 c.c. serum intravenously. 
 Fifth dose: 3 c.c. serum intravenously. 
 
 Fourth Method. — Rabbits may be immunized by making intra- 
 peritoneal injections after any of the foregoing methods, and with the 
 same or slightly larger doses. 
 
PKODUCTION OF HEMOLYSINS 71 
 
 Milk Precipitins (Lactoserums). — These are prepared by immuniz- 
 ing large rabbits with intravenous or intraperitoneal injections of milk, 
 that of either the human or the lower animals. Rabbits should be im- 
 munized with at least two kinds of milk in order to obtain different 
 lactoserums for the study of specificity. The milk used for the injec- 
 tions should be as sterile as possible, and if heated to 56° C. for one-half 
 hour before the injections are made, the protein remains unchanged and 
 the rabbits are less likely to succumb. Animals may be immunized 
 in the same manner and with the same sized doses as were directed for 
 the preparation of serum precipitins. 
 
 Bacterial Precipitins. — It is usually customary to differentiate be- 
 tween the bacterial and the protein precipitins, but for practical pur- 
 poses this division is superfluous, as the bacterial precipitins are simply 
 antiserums prepared by immunization with bacterial protein. 
 
 PRODUCTION OF HEMOLYSINS 
 
 Because of their use in the serum diagnosis of syphilis and other in- 
 fections, hemolysins possess great practical value. They are best pro- 
 duced by injecting rabbits with washed human or sheep erythrocytes, 
 or with those of some animal of another species. Rabbits differ con- 
 siderably in their power to form hemolysins, and for some unknown 
 reason hemolysins are more readily produced with some erythrocytes 
 than with others. It is not possible, however, to immunize every kind 
 of animal against every type of erythrocyte. As a general rule, an 
 animal produces a better hemolysin the more remote its relationship is 
 to the animal from which the erythrocytes for making the injection are 
 taken. 
 
 Hemolysins may be prepared as the result either of intraperitoneal 
 or of intravenous injections of erythrocytes washed at least three or 
 four times to remove all traces of serum. Unless an antihuman hemoly- 
 sin is required within a short space of time, better results are obtained, 
 as a rule, by using the slower, intraperitoneal naethod. 
 
 In immunizing rabbits it must be remembered that the quantity 
 of amboceptor produced bears no direct relation to the size of the dose$ 
 given. Thus, a highly potent hemolytic serum may be prepared by 
 three intravenous injections of from 3 to 5 c.c. of a 10 per cent, suspen- 
 sion of washed sheep cells. 
 
 It must be emphasized that as far as possible an aseptic technic 
 should be employed, and that corpuscles used for making intravenous 
 
72 METHODS FOR EFFECTING ACTIVE IMMUNIZATION OF ANIMALS 
 
 injections should be filtered to remove small particles of fibrin, and pref- 
 erably washed four times with sterile salt s,qlution. The method of 
 preparing corpuscles for injection has been given in a preceding chapter. 
 After the third or fourth injection the animal should be bled from the 
 ear and the serum tested, as animals not infrequently succumb after 
 the fourth injection, and many possess serum§ of high potency after re- 
 ceiving three injections. By this method success is better assured. 
 
 Intravenous Method.- — For the preparation of most hemolysins 
 the following methods yield very good results. Antihmnan hemolysin 
 is more difficult to prepare, and I have generally found that a slower, 
 intraperitoneal method will yield better resultk 
 
 First Method. — Three injections of 5 c.c. each of a 10 per cent, sus- 
 pension of washed cells at intervals of three days. The animal is bled 
 three or four days after receiving the last injeetion. 
 
 Second Method. — Three injections of 10 c.c. each of a 10 per cent, 
 suspension of washed cells on each of three successive days. The rab- 
 bit is bled four days after receiving the last injection (after the method of 
 Gay). Instead of these large injections, 1 c.c. of corpuscles, removed 
 after thorough centrifugalization and diluted with sufficient sterile 
 salt solution, may be given on each of three successive days. 
 
 Third Method.— K slower method consists in making weekly in- 
 jections of a suspension of corpuscles. The cells must be thoroughly 
 washed to free them from all traces of serunr; if this is not done, the 
 animals may die of anaphylaxis during the course of immunization; 
 aijimals should be tested after the third dose has been injected: 
 
 First dose: 3 c.c. of a 10 per cent, suspension of corpuscles. 
 Second dose: 5 c.c. of a 10 per cent, suspension of corpuscles. 
 Third dose: 10 c.c. of a 10 per cent, suspension of corpuscles. 
 Fourth dose: 15 c.c. of a 10 per cent, suspension of corpuscles. 
 Fifth dose: 20 c.c. of a 10 per cent, suspension of corpuscles. 
 
 Fourth Method. — In the preparation of antihmnan amboceptor, 
 Noguchi advises giving four intravenous injections, — 4 c.c, 3 c.c, 4 c.c, 
 3 c.c, — and possibly another — 4 c.c. — after an interval of four or five 
 days. Rabbits are bled one week after the last injection is given. 
 
 Intraperitoneal Method. — In this method; the most careful aseptic 
 precautions should be observed in washing the cells and in giving the 
 injections, or the animals are likely to succumb from peritonitis just 
 about the time they are fully immunized and ready for bleeding. In- 
 jections may be given every four or five dajfe, and one week after re- 
 ceiving the last injection the rabbits are to be bled. 
 
PRODUCTION OF CYTOTOXINS 73 
 
 This method is especially serviceable in preparing antihuman 
 amboceptor. Whenever possible, blood should be collected aseptically, 
 and the corpuscles washed just before the injections are given. Pla- 
 cental blood is likely to be infected and hemolyzed, and frequently 
 yields unsatisfactory results. In preparing the antihiunan amboceptor 
 several rabbits should be immunized at the same time, the doses being: 
 
 First dose : 5 c.c. of the washed corpuscles. 
 
 Second dose: 8 c.c. of the washed corpuscles. 
 
 Third dose : 12 c.c. of the washed corpuscles. 
 
 Fourth dose: 15 c.c. of the washed corpuscles. 
 
 Fifth dose: 20 c.c. of the washed corpuscles. 
 
 PRODUCTION OF CYTOTOXESTS 
 
 The term "cytotoxins" is usually applied to cell toxins other than 
 hemolysins, such as nephrotoxins, spermatotoxins, etc., and although 
 "lysin" is frequently used, the term "toxin" is better, being descriptive 
 of the changes produced by all cell toxins except the hemolysins and 
 the bacteriolysins. 
 
 Cytotoxic serums can be made theoretically- for any cell, but only 
 the hemolysins possess much practical value. The cytotoxins are 
 prepared with some difficulty by injecting emulsions of cells from one 
 animal into another. Immunization should always be conducted by 
 means of intraperitoneal or subcutaneous injection. 
 
 For the purpose of studying the action of cytotoxic serum, nephro- 
 toxic serum is preferably to be used, as the eftects, e. g., the production 
 of albuminuria, may be observed. A series of two or three animals 
 should be carried along at the same time, as many die after the third 
 injection. So far as possible an aseptic technic should be carried out. 
 
 Practically all cytotoxic serums are hemolytic partly because of the 
 great difficulty of removing all traces of blood from the organ used in 
 preparing the emulsion. This difficulty may be reduced to a minimum 
 by thoroughly washing the organ or tissue prior to preparing an emulsion 
 for injection. 
 
 The following method, after Pearce, illustrates the mode of preparing 
 a nephrotoxic serum by inununizing rabbits with dog kidney. 
 
 1. Anesthetize a dog with ether, open the abdomen, wash the blood 
 out of the kidneys by inserting a cannula high up in the abdominal aorta, 
 and flushing with from six to ten liters of salt solution; open the vena 
 cava. 
 
74 METHODS FOE EFFECTING ACTIVE IMMUNIZATION OF ANIMALS 
 
 2. Remove the kidney as aseptically as possible, and grind it in a 
 meat-grinder; rub through fine-meshed wire gauze, and wash the residue 
 in several changes of sterile salt solution. Tie fat should be rejected 
 and only the cortex used. 
 
 3. Weigh and suspend 10 grams of the substance in 30 c.c. of sterile 
 salt solution. 
 
 4. Inject the rabbit intraperitoneally with 10 c.c. of the emulsion 
 every seven days. 
 
 5. After the third dose has been given test the serum, as subsequent 
 doses increase the danger of losing the animal. 
 
 6. The animal is bled one week after receiving the last injection. 
 
 7. The injection of this serum into dogs is usually followed by al- 
 buminuria and possibly hemoglobinuria. This subject is further con- 
 sidered under the head of Practical Exercises with Cytotoxins. 
 
 Some investigators have asserted that by effecting immunization 
 with the nucleoproteids of an organ more specific cytotoxic serums are 
 secm-ed. These claims have not been confirmed by Pearce, Wells, 
 and others. (See Chapter XXV.) 
 
 Nucleoproteins may be secured as follows: Grind the organ or tissue in a meat- 
 grinder, and finally rub up with sand with a pestle in a mortar; add two volumes of 
 normal salt solution, and pass through a meat press; collect the effluent, place in a 
 refrigerator for twenty-four hours and then filter through gauze and centrifuge the 
 filtrate; to the supernatant fluid add acetic acid to remove the nucleoproteins. Place 
 in the refrigerator for eighteen hours and centrifuge. Collect the sediment and wash 
 several times with normal salt solution. Dissolve the sediment in normal salt 
 solution containing 0.5 per cent, sodium carbonate. Reprecipitate with acetic acid, 
 wash, and redissolve in the alkaline solution. 
 
CHAPTER V 
 THE PRESERVATION OF SERUMS— METHODS 
 
 It is well to remember that serum collected shortly after a meal is 
 likely to be cloudy or opalescent; it is therefore advisable that blood 
 be collected several hours after eating or during a period of fasting. 
 
 After securing a specimen of blood, the container should be set aside 
 and kept at room temperature until the serum separates. If the serum 
 is to be used at once, blood may be collected in centrifuge tubes; allowed 
 to coagulate, and then broken up, as gently as possible, with a sterile 
 glass rod and thoroughly centrifuged. On account of the mechanical 
 rupture of erythrocytes, such serums are usually tinged with hemo- 
 globin. After serum has separated from the clot it should be trans- 
 ferred to another tube, or, if this is not immediately possible, the con- 
 tainer should be placed in the refrigerator, t© retard hemolysis, which 
 may soon occur and render the serum unfit for many purposes. 
 
 Small amounts of serum are best removed from the clot with a cap- 
 illary pipet and teat, or with an ordinary graduated pipet with rubber 
 tubing and mouth-piece, in order that one may see exactly what he is 
 doing and not disturb the clot. As a perfectly clear serum is always to be 
 desired, serums mixed with corpuscles should be centrifuged. 
 
 It may be stated, as a general rule, that allnormal and immune se- 
 rums should be collected as aseptically as possible, and handled in a 
 careful and aseptic manner, so as to insure a clear and sterile product. 
 Notwithstanding the method of preservation all serums should be kept 
 in a refrigerator or ice-chest at a low temperature. 
 
 PRESERVATION OF NORMAL SERUMS 
 Normal serums that are to be used for purposes of immunization are 
 best preserved in small amoimts in separate ampules, or in a large stock 
 bottle holding from 100 to 200 c.c. and well stoppered. In the produc- 
 tion of precipitin-serum, for example, sufficient serum of an animal may 
 be obtained at a single sitting for the whole course of injections, and this 
 serum is best preserved in separate ampules. Each ampule should 
 contain sufficient serum for one injection, and be sealed and marked. 
 
 75 
 
76 THE PRESERVATION OP SERUMS — METHODS 
 
 In this manner the risk of contaminating ^ stock bottle is obviated. 
 In the preservation of normal serum or the serum of luetics to be used 
 as controls for the Wassermann reaction, it is better to store them in 
 small amounts in sterile ampules. 
 
 As a rule, it is best not to add a preservative to serums that are to be 
 used for purposes of immunization, for if the dose of serum is large, 
 enough preservative may be injected to place the health of the animal 
 in jeopardy. However, chloroform may be added in proportion of 1:10 
 or 1:20, provided the serum is .placed in the incubator or heated in the 
 water-bath at 40° C. for fifteen minutes in order to drive off the chlo- 
 roform previous to injection. 
 
 PRESERVATION OF IMMUNE SERUMS 
 
 Immune serums may be preserved either in the fluid or in the dry 
 form. 
 
 Preservation in Fluid Form with Antiseptics. — Practically any 
 immune serum may be preserved in the fluid= state by adding a suit- 
 able preservative in the proper dose without exerting any deleterious 
 influence on the antibody content. The exceptions to this general rule 
 are the precipitin-serums, because these should be crystal clear, and 
 a preservative may render the serum sHghtly cloudy. According to 
 Uhlenhuth, Weidanz, and Wedemann, such serums should be filtered 
 through a sterile Berkefeld filter and then stored without adding an 
 antiseptic. 
 
 Various antiseptics have been advocated for the preservation of 
 serums. Hemolytic serum is well preserved by adding an equal amount 
 of chemically pure glycerin to the serum after it has been inactivated by 
 heating at 55° C. for a half -hour in a water-bath. The addition of 0.1 
 c.c. of a 1 per cent, solution of phenol in salt solution to each cubic 
 centimeter of immune serum usually suffices to keep the fluid free from 
 contamination, and produces only very slight, if any, clouding. Like- 
 wise, the addition of 2 per cent, formalin in a 5 per cent, solution of 
 glycerin in normal salt solution, in the proportion of 1 : 10, makes a very 
 useful antiseptic. Neither lysol nor trikrespl should be used in the 
 preservation of a serum, as they are more likely to produce clouding 
 than does phenol. 
 
 Preservation in Fluid Form, by Bacteria-free Filtration. — If serums 
 are to be preserved in fluid form without the addition of an antiseptic, 
 special precautions in bleeding, collecting, and separating should be 
 
PRESERVATION OF IMMUNE SERUMS 
 
 77 
 
 observed. If contamination has probably ocQurred, the serum should 
 be filtered through a sterile Berkefeld filter (Eig. 34). The apparatus 
 devised by Uhlenhuth (see Fig. 88) is especially useful, as the serum is 
 collected at once in a sterile container, which is =then plugged with sterile 
 cotton and placed in the incubator for twenty-four hours. If the serum 
 
 Fig. 34, — A Small Berkefeld Filter. 
 The fluid to be filtered is poured into the glass cylinjier surrounding the earthen 
 or__porcelain "candle." Negative pressure within the candle is produced by the 
 water-pump, which exhausts the air from the flask. The filtrate is collected in the 
 test-tube within the filter flask. All parts are readily s'terilized in an Arnold sterili- 
 zer or autoclave. The sterile cotton plug prevents air contamination. 
 
 proves to be sterile, it is transferred, with the aid of a sterile pipet, into 
 ampules of 1 c.c. capacity. These are sealed hermetically and kept in 
 the refrigerator. 
 
 Small amounts of serum may be lost in a large filter, and a smaller 
 apparatus should therefore be used. The filter shown in the accom- 
 
78 
 
 THE PRESERVATION OP SERUMS METHODS 
 
 panying illustration (Fig. 35) is quite serviceable, as the flask and 
 earthen candle-filter may be wrapped in a tAwel and sterilized in the 
 autoclave. The apparatus may carefully be attached to a suction 
 pump, and the serum pipeted off into the hollow of the candle and fil- 
 tered, the filtrate being removed, at the completion of the process, by 
 another sterile pipet. 
 
 Fig. 3.5. — A Filter. 
 The cotton plug in the "candle" is removed and the fluid poured within the 
 candle (hollow). The water is then turned on and' the stop-cocks are opened; a 
 vacuum is produced within the flasks, which draw§ the fluid through the candle. 
 The filter is readily cleansed and sterilized (autoclave^ and is quite efficient. 
 
 Preservation in Fluid Form, by Freezing, — Freezing a serum often 
 renders it cloudy or causes a precipitate to be deposited, and interferes 
 with the usefulness of a serum that should be absolutely clear. Freezing 
 is the only practicable method so far devised for the preservation of 
 thermolabile substances, such as complem^ent. A small apparatus. 
 
PRESEBVATION OF IMMUNE SERUMS 79 
 
 named the "Fri'go," has been devised for this„purpose by Morgenroth. 
 A satisfactory apparatus may be made by constructing a wooden box 
 with a smaller sheet-metal-covered iimer compartment, the space be- 
 tween them being well packed with sawdust. This inner box is then 
 filled with crushed ice, and the whole is covered with a lid lined with 
 several layers of felt. 
 
 Preservation in Powder Form.^ — ^When serum is poured out in thin 
 layers and dried, it forms yellowish, amorphous masses, that may be 
 collected and ground into a powder, which keeps well and forms an 
 excellent medium for the preservation of many immune serums, espe- 
 cially those of the agglutinating type. Various toxins, such as tetanus 
 toxin and cobra venom, may also be preserved in this form. 
 
 The serum or toxin may be spread out in thin layers on large glass 
 plates, or placed in shallow dishes and dried in the incubator. After a 
 few hours the dried serum, which adheres only slightly to the dish, can 
 be removed with a spatula and placed in a mortar, and ground and 
 stored in sealed tubes. 
 
 The drying process is better carried out in vacuo, and the large serum 
 institutes are provided wth these special drying apparatus. A simple 
 form may be prepared after the method of Ta=eze, as follows: Place a 
 large glass bell-jar with a ground base and a large opening at the top on 
 a polished iron plate. Set this on a large tripod, as this will facilitate 
 heating with a Bunsen burner. The serum is placed within the jar in 
 a shallow dish, and the jar fastened to the iron plate with hot paraffin 
 or wax. The opening at the top is closed with a three-holed rubber 
 stopper: one hole carries a thermometer; a second is connected with a 
 manometer (not absolutely necessary), and the third carries a bent glass 
 tube which is connected, by means of thick-walled rubber tubing, to a 
 suction pump. A low flame is kept burning so as to keep the tempera- 
 ture at about 35° C. The degree of vacuum sfecured makes little differ- 
 ence, and usually that obtained with an ordinary water-suction pump, 
 allowing for leaks in the tubing, is sufficient, rendering manometric 
 measurements unnecessary. 
 
 The dried serum should be dissolved in sterile normal salt solution 
 before it is used. 
 
 Preservation in Dried Paper Form.— This is a very serviceable 
 method for preserving hemolysins, and to a lesser extent, agglutinins. 
 In the preservation of hemolytic amboceptor Noguchi advises the use 
 of Schleich and SchuU's paper No. 597. The-paper is cut into squares 
 about 10 by 10 cm., and saturated with the serum which, after prelimi- 
 
80 THE PRESERVATION OF SERUM^ METHODS 
 
 nary titration, has been found satisfactory. Sufficient serum is added to 
 wet the sheets evenly, any excess of serum being absorbed with other 
 sheets of paper. Each square is placed separately upon a clean sheet of 
 unbleached muslin and dried at room temperature. When thoroughly 
 dry, the squares are carefully ruled off with a hferd pencil into widths of 
 about 5 mm., and cut into strips. The paper is then standardized and 
 preserved in dark glass vials in a cool, dark place. 
 
 Preservation in the Living Animal. — In the living animal an im- 
 mune serum may be preserved by removing a small amount of blood 
 from time to time as needed, the titer being- preserved or raised by 
 occasional injections. This method, however, may be unsatisfactory 
 and expensive, especially with the smaller aniinals, as they frequently 
 show a marked tendency to sicken and die, or may succumb to anaphy- 
 laxis. After a time, too, they fail to respond to injections with the 
 formation of antibodies, a condition ascribed to atrophy of the cell- 
 receptors (receptoric atrophy). 
 
Part II 
 
 CHAPTER VI 
 
 INFECTION 
 
 Infection is the successful invasion and gtowth of microorganisms in 
 the tissues of the body. 
 
 The skin and adjacent mucous membranes contain numerous micro- 
 organisms, and under normal conditions the|e may invade the tissues, 
 but they are usually quickly destroyed and unable to proliferate, so that 
 mere invasion does not necessarily constitute infection. 
 
 Unfortunately, custom has sanctioned the use of the term infection 
 as synonymous with contamination. The bacteriologist may speak of 
 the air, water, or his culture medium as being infected when they con- 
 tain microorganisms, or, in other- words, are not sterile; similarly the 
 surgeon may speak of a knife or splinter of wood as being infected 
 whereas, while these may be infective or capable of producing infection, 
 it is more accurate to speak of them as being contaminated. In the 
 early days of bacteriology, the mere presence of microorganisms in or 
 on the skin and mucous membranes was regarded as equivalent to in- 
 fection. It is now well known that a person"may harbor various micro- 
 organisms, such as staphylococci, streptococci, and pneumococci, with- 
 out apparent injury to the host, and this surface contamination, or even 
 occasional invasion of the tissues, does not necessarily indicate that the 
 host has been, is, or will be ill. 
 
 Definition. — When, however, microorganisms have passed the normal 
 harriers of the skin or mucous membranes and have invaded and 'prolifer- 
 ated in the deeper tissues, the process is spoken of as an infection. 
 
 By common consent, the term infestation, or infcstment, is being 
 apphed in a similar manner to the presence and growth of animal 
 parasites; thus the intestine may be infected with Bacillus typhosus and 
 infested by Taenia saginata. 
 
 A microorganism may be intimately associated with and have its 
 normal habitat in a certain part of the body and do no harm until special 
 conditions arise, when it may rapidly invade the tissues and produce 
 infection; this condition has been described by Adami as a suhinfection, 
 and is illustrated by the constant presence of staphylococci and strepto- 
 6 81 
 
82 INFECTION 
 
 cocci in the tonsils of most persons, usually harmless, but capable, under 
 special conditions, of producing severe and even fatal infection. 
 
 The abnormal state resulting from the deleterious local and general 
 interaction between a host and an invading bacterium, with consequent 
 tissue changes and symptoms, constitutes an infectious disease. 
 
 ^As has previously been stated, not every invasion of the deeper 
 tissues by microorganisms results in injury or disease. A certain num- 
 ber of bacteria are constantly gaining admissipn to the deeper tissues 
 of the alimentary and the respiratory tracts, ■ndthout producing apparent 
 injury to the host, as they tend to be destroyed very soon after they 
 gain entrance. The terms invasion and infection are not, therefore, 
 synonymous. Every true infection is accompanied by local changes, 
 although these may be so sHght as to escape notice; an infectious disease 
 is practically made up of similar phenomena, but these are of an ex- 
 aggerated or marked degree. 
 
 The hygienist distinguishes between — (1) Sporadic, or isolated, cases 
 of infection; (2) endemic, in which a certain microbic disease affects the 
 inhabitants of a given area year after year; and (3) epidemic, in which a 
 disease appears suddenly and affects a large number of inhabitants, the 
 number of cases rapidly increasing and decreasing. Among the lower 
 animals equivalent terms for the types just described are sporadic, 
 enzootic, and epizootic. A pandemic disease is one that is epidemic over 
 a large territory. 
 
 In all infections there are two inseparable factors to be considered : 
 
 1. The offensive forces of the infecting agent, dependent upon its 
 pathogenic or disease-producing nature and its power of defending itself 
 against the antagonistic forces of the host and of thriving under these 
 conditions. 
 
 2. The resistance offered by the host, and mainly dependent upon 
 certain physical or non-specific local factors or specific antibodies, which 
 constitute the defensive mechanism, or immunologic factors. 
 
 The former is concerned with the general subject of infection, and 
 the latter with that of immunity. 
 
 Microorganisms and host may live together in apparent harmony, 
 owing to the ability of the host to restrain the activity of the micro- 
 organism and neutralize its injurious effects or to an absence of infec- 
 tivity on the part of the microorganism until the vital resistance of the 
 host is diminished or the pathogenicity of the microorganism is increased, 
 when the neutral relations are disturbed and infection occurs. 
 
SOURCES OF INFECTION 83 
 
 RELATION OF INFECTION TO EVIMUNITY 
 From what has been said, it is apparent that the subject of infection 
 forms the basis for the study of immunology, for, paradoxic as it would 
 at first appear to be, infection must usually have occurred in order that 
 immunity may be acquired. This relation is not always apparent; for 
 instance, man and some of the lower animals may possess a natural 
 immunity to a certain parasite because of the presence of various physi- 
 cal or non-specific defensive factors, or to specific antibodies produced 
 as the result of an earlier and unrecognized infection, or even one that 
 has been inherited; under any circumstances, however, natural immu- 
 nity is usually relative and seldom absolute. In passive immunity the 
 same conditions are generally operative, and the antibodies present in 
 the serum used to confer a passive immunity are produced in some other 
 animal as the result of an active infection. 
 
 It may be stated, therefore, that specific antibodies are produced 
 only by stimulation of the body-cells, and thatr this stimulation is fur- 
 nished by the infecting agent either in living, disease-producing form, or 
 in a modified and attenuated state, i. e., in the form of a vaccine; thus 
 it will be seen that infection and immunity are intimately associated, 
 and that, generally speaking, there can be n9 pronounced protection 
 unless infection has taken place. 
 
 SOURCES OF INFECTION 
 
 Bacteria are to be found everywhere. For general purposes they 
 may "he roughly divided into two classes — saprophytes and parasites. 
 The saprophytes are those bacteria which thrive best in dead organic 
 matter, and perform the very important function of reducing, by their 
 physiologic activities, highly organized material into those simple 
 chemical substances that may again be utiUzed by the plants in their 
 constructive processes, and in this manner maintain the important 
 chemical relation between the animal and the plant kingdom. Para- 
 sites, on the other hand, find the most favorable conditions for growth 
 and activity upon the living tissues of higher forms of animal hfe. They 
 include most of the so-called pathogenic or disease-producing bacteria. 
 
 No marked separation between these two divisions can be made, 
 as numerous species occupy a transition point between the two. The 
 terms are merely relative, and bacteria ordinarily saprophytic may 
 develop parasitic and pathogenic powers when the resistance of the host 
 is sufficiently reduced by another infection, fatigue, exposure, or other 
 
84 INFECTION 
 
 deleterious influence. In other words, a jaathogenic microorganism 
 is one that can grow in the living tissues because the immunologic 
 defenses of the host are not sufficiently strong to resist it; in most cases, 
 however, as will be pointed out further on, a higher degree of immunity 
 can be produced artificially, rendering the ba'cterium in question rela- 
 tively harmless for that particular animal. Similarly, under certain 
 circumstances, the resistance of the body or of a part of it may be broken 
 down to such an extent that naicroorganisms ordinarily regarded as 
 saprophytes may gain access to the deeper tissues, flourish, and produce 
 disease. 
 
 Accordingly, no fundamental distinction between pathogenic and 
 non-pathogenic bacteria can be made. Any apparent differences are 
 due not only to various degrees of pathogenicity possessed by the micro- 
 organism, but also to the different degrees of resistance against their 
 attacks, since a microparasite that is highly pathogenic toward one 
 animal, may be quite harmless to another. 
 
 CONTAGIOUS AND INFECTIOU^ DISEASES 
 
 Just as all pathogenic bacteria do not possess the same haljits of 
 growth, so, hkewise, they vary in their vitahty and in their ability to 
 proliferate under various conditions when removed from the animal 
 body. Some are strictly parasitic, and are able to grow only at body 
 temperatiu'e, or, indeed, only in the human body itself; when removed 
 from these conditions, they may retain their vitality for a short period 
 of time, but are unable to prohferate: from this it follows that "com- 
 munication of these bacteria and their disease must be direct or immedi- 
 ate, i. e., from person to person, or almost direct, by the conveyance of 
 the infecting agent in the form oifomites, such as dust, epidermal scales, 
 or discharges, or as the result of bites of suctorial insects. This form 
 of infection, which requires such direct means of transmission, and of 
 which gonorrhea is an example, constitutes what are known as conta- 
 gious diseases. 
 
 Other raicroparasites are not so strictly parasitic; they may be able 
 to preserve their pathogenic powers and proliferate outside of the body 
 at ordinary temperatures, and may even withstand great extremes of 
 heat or cold and various nutritional deficiencies; they may exist thus 
 for weeks, and carry the disease to a second individual through con- 
 taminated material. Infections the result of indirect transmission are 
 known as infectious diseases. 
 
EXOGENOUS AND ENDOGENOUS INFECTIONS 85 
 
 There are no hard-and-fast rules that can be set down in classifying 
 bacterial infections; bacteria that are commonly transmitted by one 
 means may, under slightly altered conditions, be transmitted by another. 
 The usual classification, by which certain diseases are classified as con- 
 tagious and others as infectious, should he abolished, and all should he 
 grouped under the term infectious, there being a definite understanding of 
 those cultural characteristics that render infection more likely to occur 
 by direct and immediate contact, and those that may occur in an in- 
 direct or roundabout manner. It 7nay therefore be stated that all bac- 
 terial diseases are infectious; the term contagious may be reserved for 
 those spread or contracted as the result of direct contact. 
 
 EXOGENOUS AND ENDOGENOUS INFECTIONS 
 
 Infection maj^ occur as the result of the admission of microparasites 
 to the tissues from sources entirely apart from the individual infected 
 {exogenous infection), or from the admission of some of those micro- 
 parasites living normally and harmlessly on the skin and adjacent mucous 
 membranes, and which, under special conditions, have assumed patho- 
 genic properties {endogenous infections) . 
 
 Exogenous infections are the more usual form, and result from con- 
 tact with infective material outside the body. 
 
 1. Microorganisms, such as typhoid and chdlera bacilli, which can 
 live for varying periods of time in water and foods, are particularly 
 likely to gain entrance through the gastro-intestinal tract. Micro- 
 organisms may be present in milk derived directly from diseased animals 
 or tissues, and when ingested, may produce disease. Thus, for example, 
 the germs of tuberculosis may be conveyed in either milk or flesh, young 
 children being particularly exposed to this method of infection. 
 
 2. The atmosphere may be laden ^A-ith microorganisms, which, 
 whether or not capable of prohferating outside of the body, are prone 
 to gain entrance through the respiratory tract, especially through the 
 upper air-passages, the pharynx and tonsils being often the seat of the 
 infection. 
 
 3. Microorganisms capable of existing on the skin may gain entrance 
 to the deeper tissues as the result of wounds. Under these conditions 
 of lowered vitality of the local tissues microorganisms that would 
 otherwise be harmless may become pathogenic and morbidly affect the 
 host, either locally or generally. As the skin is brought so freel}^ in 
 contact -^^ith external objects, various microorganisms, and particularly 
 
86 INFECTION 
 
 the pathogenic cocci, may gain entrance to the dermis. Wounds may 
 be infected by the teeth and secretions of animals, or by various weapons 
 and implements contaminated wth infective material, as, e. g., the virus 
 in the sahva of rabid dogs, or the spores of the tetanus bacillus on rusty 
 nails. 
 
 Contact with unclean objects of various kinds — eating utensils, 
 catheters, syringes, dental instruments, etc.^^may serve to transfer 
 pathogenic bacteria from one person to another. This is especially 
 likely to occur if the skin or mucous membrane is abraded, the infecting 
 parasites thus gaining ready access to the deeper tissues. In some in- 
 fections, however, even this local injury is unnecessary, as the bacterium 
 rriay be able to proliferate and produce lesions= on an intact surface, as, 
 for instance, the diphtheria bacillus in the pharynx, and various fungi, 
 such as Achorion, Trichophj'ton, etc., on the scalp and skin in general. 
 
 Microparasites affecting the genital organs are hkely to be conveyed 
 directly from one sex to the other in conjugation, or to the child' during 
 parturition. 
 
 4. Suctorial insects may serve as the medium by which micro- 
 organisms are transmitted from person to person. In most instances the 
 transmission is a purely mechanical process, as witness the transmissions 
 of the plague bacillus in the intestinal contents of the rat flea; in the 
 case of malaria, on the other hand, the interposition of the mosquito is 
 essential to complete the life cycle of the protozoon. 
 
 5. Microorganisms infecting the placenta may pass to the fetus by 
 way of the umbilical vein. 
 
 Endogenous infections arise as the result- of the activity of micro- 
 organisms having their normal or customary habitat in the body. Such 
 infections do not represent so much an assumption of pathogenic power 
 on the part of the microorganism, as they do a disturbance of the de- 
 fensive mechanism of the host, whereby the -normal relations are dis- 
 turbed, and microorganisms that normally are harmless, become in- 
 fective and disease-producing. While the dis„turbance of the defensive 
 mechanism may be general, it is far more likely to be local; an example 
 is that of appendicitis the result of Bacillus coli infection following 
 passive congestion due to fecal impaction of the colon. 
 
 AVENUES OF INFECTipN 
 
 Local infection m&y occur in any portion of the body, and any part 
 rnay prove the point of entrance of bacteria to the body fluids, the result 
 
AVENUES OF INFECTION 87 
 
 being a general infection. Owing, however, to the peculiar pathogenic 
 properties of different bacteria and their affinity for the cells of certain 
 tissues, coupled with a peculiar tissue susceptibility for certain bacteria 
 or their products, we find that many diseases have regular avenues of in- 
 fection, and, indeed, in a few instances infection (3f the human body may 
 be possible only through a particular and definite route. Infections 
 of the gastro-intestinal, respiratory, and genito-urinary tracts and 
 various sinuses with external openings must be considered as being 
 potentially surface infections. The outer layers do not consist merely 
 of the skin and adjacent mucous membranes, but are made up of all 
 layers covering siu'faces and channels, which, however indirectly, com- 
 municate with the exterior. In the higher aniinals there is only one 
 direct channel of communication between the actual interior and the 
 exterior of the body, this being through the Fallopian tube of the female, 
 which normally has so fine a lumen and is so well protected that to all 
 intents and purposes it may be regarded as closed. In certain inflam- 
 matory conditions of the genital organs, and particularly after parturi- 
 tion, the Fallopian tube may be open, and afford a direct route for the 
 transmission of infection from the external parts to the peritoneal 
 cavity. 
 
 Living in and on the actual and potential external surfaces are count- 
 less microorganisms, which are for the most part, harmless, a few being, 
 however, actually or potentially dangerous. 
 
 1. The skin and adjacent mucous membranes, particularly in those 
 portions where warmth and moisture abound, are well adapted to bac- 
 terial growth, and their contact with surrounding objects causes a large 
 variety of microorganisms to adhere to them. 
 
 As a result, the bacteriology of the skin is quite complex, since it may 
 lodge microorganisms from the air, from water, and from soil. A group 
 of cocci and diplococci, particularly the Staphylococcus epidermidis 
 albus of Welch, and the various pseudodiphtheria bacilli, are habitually 
 present upon the human skin. When local injury occurs, they may 
 produce minor suppurative lesions, and may be concerned in the pro- 
 duction of certain skin diseases, such as eczema, impetigo contagiosa, 
 the pustules of variola, etc. 
 
 Other microorganisms may find temporary lodgment upon the skin, 
 and are in no sense regular inhabitants. For example, the fingers and 
 hands may become contaminated with colon, typhoid, and tubercle 
 bacilli, pneumococci, etc. 
 
 The skin forms a very important barrier against the entrance of 
 
88 INFECTION 
 
 bacteria into the deeper tissues. The greater number of local surgical 
 infections result from the entrance of bacteriii into lesions of the skin, 
 although these lesions may be so small as to escape notice. 
 
 Certain parasites are capable of producing direct action on the skin 
 without previous existing injury, and especially upon the mucous mem- 
 branes, where moisture and higher temperature are more favorable to 
 bacterial growth. For example, a few of the higher fungi, such as 
 Microsporon, Achorion, and Trichophyton, seem able to establish 
 themselves in the superficial cells and invade the deeper tissues through 
 the hair-follicles; staphylococci may reach the roots of hair-follicles 
 and sweat-glands and set up suppurative conditions; diphtheria bacilli 
 may lodge directly on the intact mucosa of the upper air-passages and 
 cause local necrosis and general intoxication; cholera bacilli may have a 
 similar effect upon the intestinal mucosa; the Koch-Weeks' bacillus 
 and the gonococcus may produce severe inflammation of an intact 
 conjunctiva, etc. 
 
 2. The respiratory organs commonly afford admission to certain 
 microorganisms. The nose may be the seat of local infection with 
 Bacillus influenzae, Micrococcus catarrhalis. Bacillus diphtheriae, and 
 other bacteria; it may be the entrance point for meningococci and the 
 virus of anterior poHomyelitis. Similarly, the -entrance of such unknown 
 infectious agents as those of scarlet fever, rneasles, and smallpox can 
 best be accounted for by assuming that they were inhaled and later 
 entered the blood; there is much clinical evidence to support the belief 
 that the contagium of scarlet fever is presenLin the discharges of the 
 upper air-passages of persons suffering from that infection. 
 
 Whether or not tuberculosis of the lungs is the result of the inhalation 
 of tubercle bacilli is a much-disputed point, but it cannot be denied tTiat 
 this theory most readily accounts for the far greater frequency with 
 wTiich tuberculosis affects the lungs than it does other organs of the body. 
 
 Pneumonia, caused by the pneumococcus of Weichselbaum, probably 
 results from the direct inhalation of one of the various types of pneumo- 
 cocci, and bronchopneumonia of children is certainly chiefly inspiratory 
 in origin. 
 
 3. The digestive tract may be the portal of entrance of many in- 
 fections. The mouth usually harbors various fungi and bacteria, which may 
 produce local infections, and either directly or indirectly cause caries 
 of the teeth. The putrefactive changes they may produce is being' 
 generally recognized as having an important bearing on the causation 
 and symptomatology of several infections, and a carious tooth has been 
 
AVENUES OF INFECTION 89 
 
 found the portal of entry of microorganisms causing a general infection. 
 The tonsils are well known to be the breeding- and lodging-place of 
 various microparasites causing many general infections, such as acute 
 rheumatic fever, tuberculosis, and possibly typhoid fever. The pharynx 
 may harbor the microorganisms of diphtheria, pneumococcous angina, 
 etc. 
 
 Normally, except for the presence of a few sarcinse, the stomach is 
 practically sterile. Under special conditions, however, typhoid, dysen- 
 tery, cholera, tubercle, and other infectious bacteria may escape the 
 germicidal effects of the hydrochloric acid, and, reaching the alkaline 
 intestinal contents, which are rich in soluble proteins and carbohydrates, 
 are rendered capable of producing their respective infections. 
 
 Although these conditions are primarily of the nature of local in- 
 fection, there is much experimental evidence to show that bacilU, and 
 particularly tubercle bacilli, may pass throjigh a practically intact 
 intestinal wall and find their way to the Ijmiph-glands or to the blood- 
 stream itself. 
 
 Aside from these direct and specific infections, various other micro- 
 organisms, by fermentative action, may alter the intestinal contents 
 and produce toxic products capable of exciting acute and severe toxemias. 
 Some authorities as, e. g., Mctchnikoff, regard the various types of colon 
 bacilli as producing toxic products responsible for chronic degenerative 
 lesions of the cardiovascular and other organtj.* The digestive tract is 
 therefore regarded by some pathologists as a constant menace to health, 
 in that it permits bacteria to enter the lymphatic and blood-streams, or 
 to produce toxic substances detrimental to heailth and longevity. Adami 
 has drawn particular attention to a condition ^vhich he terms suhin- 
 fection, and which is dependent upon the constant entrance of colon 
 bacilli into the blood, whence they enter the liver, where their final 
 dissolution takes place, appearing as fine, dumb-bell-like granules 
 inclosed in the cells. 
 
 4. The genital organs are the seat of various local infections that may 
 become wide-spread and general. Normally,: the urethra may contain 
 a few cocci which lodge about the meatus; the acid secretions of the 
 vagina are generally inimical to bacterial grQ\\'th, and the uterus and 
 bladder are usually sterile. But three microorganisms — the gonococcus, 
 Treponema pallidum, and the bacillus of Ducrey, here find favorable 
 conditions for growth, and are usually transmitted from person to per- 
 son by means of sexual congress. The local gonococcal lesion may 
 be the portal of entry of gonococci into the blood-stream, resulting in 
 
90 
 
 INFECTION 
 
 wide-spread metastases in the heart valves and joints. The local sjijhi- 
 litic lesion is quickly followed by general infection. Chancroids alone 
 remain locahzed, although the initial lesion frequently spreads quite 
 rapidly by continuity of tissues. In rarer instances other microorgan- 
 isms, such as the ordinary pyogenic cocci, tubercle bacillus, and diph- 
 theria bacillus, may infect these organs and be transmitted by sexual 
 conjugation. 
 
 5. There is considerable contro\'ersy of opinion regarding the suscep- 
 tibihty of the placenta and the filtering properties it possesses for various 
 infectious agents. A study of this subject by Neelow^ would indicate 
 that the non-pathogenic bacteria do not pass from the mother through 
 the placenta to the fetus. Other pathogenic agents may, however, pass 
 through quite readily, for example, pregnant women suffering from 
 smallpox may be delivered of infants showing active lesions of prenatal 
 infection, and syphiUtic infection of the fetus is a well-known condition. 
 Most controversy centers around congenital tuberculosis, and directly 
 opposing views for and against prenatal infections are held by several 
 authorities. Baumgarten is of the opinion that many children are 
 subject to antenatal infection, though the disease infrequently develops 
 in a few of them. 
 
 The general subject of antenatal infection and pathology is a field 
 requiring considerable investigation and research. 
 
 NORMAL DEFENSES AGAINST BACTERIAL INVASION 
 
 When the large area of the body that is suUfect to traumatic injury 
 and accidental infection is considered, it is remarkable that, considering 
 the enormous numbers of various bacteria, infection does not occur more 
 frequently. 
 
 Bacterial invasion of the tissues is of frequent occurrence, but in 
 health they do not usuallj' cause infection, and tend to be destroyed very 
 soon after they enter the tissues. 
 
 It may be well to discuss at this point the factors tending to -prevent 
 invasion, and leave the consideration of the defensive mechanism, 
 whereby the body destroys bacteria after successful invasion and thus 
 prevents infection, for the chapter on Natural Immunity. 
 
 Of the factors preventing bacterial invasion, the following are recog- 
 nized : 
 
 1. The structure of the surface layer of epithelium. The epidermal 
 'Centralb. f. Bakt., August, 1902, 1. Abt., Bd. xxxi, orig., 691. 
 
MECHANISM OF BACTEEIAL INVASION 91 
 
 cells offer a mechanical obstacle to invasion. This resistance is naturally 
 more complete where the cells are thickened and most compact. In the 
 depths of glands and in mucous membranes, where numerous glands are 
 present, and where the layers arc thinner and moisture exists, the 
 barrier is less complete. 
 
 2. Surface discharges are potent factors in preventing bacterial 
 invasions by — (1) Washing away the bacteria mechanically; (2) by 
 germicidal activity through the presence of various chemical agents, 
 such as acids, which they may contain, and (3) by antiseptic and 
 even bactericidal substances that may be present in the form of anti- 
 bodies. 
 
 The sahva, with its antiseptic and germicidal properties, is potent 
 in preventing infections of the mouth and upper air-passages; when 
 this secretion is diminished, as during the course of high fever, bacterial 
 activity is enhanced, which is evidenced by the development of fetid 
 sordes about the teeth and on the lips. 
 
 The acidity of the gastric juice and its germicidal powers are well 
 known and appreciated; similarly the urine, the milk, and to a slight 
 extent, the bile, have been demonstrated by'Adami to exert a distinct 
 antiseptic effect upon certain bacteria, such as the Bacillus coli. 
 
 Surface moisture and discharges about the nose and throat are also 
 potent factors in mechanically removing bacteria from inspired air, and 
 no doubt frequently prevent bacterial invasion of the lower respiratory 
 tract, where more mischief may be done. 
 
 3. The cells of certain excreting glands may possess bactericidal and 
 excretory powers of value in preventing bacterial invasion (Adami) . 
 
 MECHANISM OF BACTERIAL INVASION 
 We will now consider the method by which invasion, the first step 
 of what may be an infection, is brought about. In brief, one or all of 
 the normal defenses just described must be overcome; in some instances 
 the microorganisms, by their inherent diseascrproducing powers, may 
 accompHsh this unaided; in other instances tlie resistance is overcome 
 bjr a general lowering of the vitality of the bodj^ defenses. 
 
 1. Traumatic solution of the surface layers of epithehal cells is a 
 very important factor in the production of infection, as the invading 
 microparasites are thus given easier access to the deeper and less resistant 
 tissues. The pathologist or surgeon may, in the course of his work, 
 contaminate his hands with secretions containing virulent microorgan- 
 
92 INFECTION 
 
 isms, and may yet escape infection, unless a small break in the surface 
 epithelium, in the form of a scratch or a needle-prick, is present. 
 
 2. As has been previously stated, certain bacteria, notably the 
 diphtheria bacillus, by concentrating at one poinSt, may lower the vitality 
 and cause necrosis of superficial cells of the mucosa lining the upper air- 
 passages, and in this maimer induce a local break in the continuity of the 
 epithelial covering. Staphylococci may exert a similar action in the 
 depths of sweat and sebaceous glands, and, indeed, certain fungi, such 
 as the Trichophyton, Microsporon, and Achorion, may attack the 
 intact skin. While, therefore, solution of the surface coverings is a very 
 important source of many infections, it is not essential for the produc- 
 tion of all. 
 
 3. Alterations of the surface discharges, either in quantity or in 
 quality, may permit bacteria to prohferate freely and produce sufficient 
 toxic matter to affect the surface cells, lower their vitality, and destroy 
 them, -nith the result that thej' may gain entrance to the deeper tissues. 
 "When the secretions are diminished or altered, as, for example, the saliva 
 during a fever, im.less the mouth is carefully and frequently cleansed, 
 it becomes putrescent ^^^th bacterial growth. Similarly catarrhal 
 gastritis, or any other factor tending to lower acidity of the gastric juice, 
 favors infection by this route. 
 
 4. Not infrequently bacteria may gain access to the deeper tissues 
 or to an internal organ, and infection may occur ^without any recognizable 
 solution of continuity of the surface epithelium. In these hidden or 
 "cryptogenic infections" the entrance point of the parasites may be 
 healed over, or the infecting microorganisms may have been carried to 
 the circulating body fluids by the wandering cglls. 
 
 Not infrequently, in cases of tuberculosis of the cervical and mesen- 
 teric glands in children, there may be no signs wliatever of local irrita- 
 tion in the fauces or in the intestine to explain the source of infection. 
 The tonsils are now stronglj^ suspected, and indeed Imown to be, the 
 source of entry of bacteria causing several acute and chronic infections. 
 
 The leukocytes, in their phagocytic activities, no doubt, play an 
 important role in the production of crjSTDtogenic infections, especially 
 when an excessive nmnber of pathogenic bacteria have congregated at 
 one point, and congestion, increased leukocytic infiltration, and a 
 lowered natality of the tissues have occurred prior to the invasions of 
 microorganisms. Wandering cells are commonlj^ found on mucous 
 membranes, gathering up various bacterial arfd cellular debris. They 
 may carry a virulent microorganism into the. deeper tissues, and, al- 
 
MECHANISM OF BACTERIAL INVASION 93 
 
 though this may not produce an infection, a large number of bacteria so 
 transported may be able to resist destructipn and prove capable of 
 causing infection. 
 
 5. Aside from the question of local conditions in the process of in- 
 fection, other factors may exert an influence. The temperature of the 
 host may be unsuitable for the growth of a certain parasite, even though 
 it has gained entrance to the deeper tissues; a particular route for the 
 introduction of the infecting agents may be necessary, as in typhoid 
 fever and cholera, which are probably always intestinal infections, and, 
 finally, even after the infecting agent has rekched the deeper tissues, 
 extension is prevented by a local inflammatory reaction. In many such 
 instances the question of natural imnnm.ity is brought into intimate 
 relation with the subject of infection. 
 
 After invasion has occurred, some bacteria can best sustain them- 
 selves against the defenses of the host at the logal point of entry. Such 
 microorganisms may, however, possess unusual vitality, and indirectly, 
 through the lymphatics, find their way to the blood-stream, producing 
 a bacteremia. This is a morbid condition characterized by the presence 
 of microorganisms in the circulating blood. 
 
 Some microorganisms may gain entrance to the general circulation 
 more readily than others, and their mode and route of entry vary in the 
 different infections. It is essential that they possess an unusual degree 
 of invasive power, and be capable of protecting themselves against the 
 manifold defensive factors contained in the blood. Kruse believes that 
 in local infections the high pressure of an inflammatory exudate may 
 force bacteria into the adjacent vessels; that they may sometimes be 
 carried into the deeper tissues, and even into the blood-stream, by 
 leukocytes is not to be denied. 
 
 When bacteria have entered the circulation, they may act as emboli 
 in the finer capillaries, or, being unable to remain in the circulation, may 
 collect in the capillaries of less resistant tissues, proliferating and pro- 
 ducing local metastatic lesions, usually purulent in character. The 
 condition thus produced is known as pyemia. 
 
 Saprophytic bacteria or pathogenic bacteria of feeble invasive powers 
 may be able to grow in diseased tissues, such a.s gangrenous areas, and 
 may assist in effecting morbid changes, producing toxic products of 
 decomposition, which when absorbed into the body, give rise to a series 
 of toxic phenomena, such as fever, rapid pulse, malaise, etc. This con- 
 dition is known as sapremia, a term that has. also been applied to the 
 decomposition of relatively sterile organic material and absorption of 
 
94 
 
 INFECTION 
 
 the toxic products, as when portions of placenta or fetal membranes 
 are retained in the uterus after childbirth. 
 
 The term toxemia is employed rather loosely to mean the presence 
 of any toxic material. Its use should be limited to the condition re- 
 sulting from the absorption of the poisonous Substances produced by the 
 non-invasive bacteria themselves, as in diphtheria and tetanus. Septi- 
 cemia is the term applied to the presence in the bdjdy-fluids of toxic products 
 generated by the pyogenic microorganisms. 
 
 MECHANISM OF INFECTION 
 
 Since bacterial invasion is of frequent occurrence, the question 
 naturally arises. Why are not infections, both local and general, more 
 frequent? Thus abrasions of the surface epithelium are not uncommon 
 in the presence of active microorganisms; tubercle bacilli may be in- 
 spired, and tj'phoid bacilli may be swallowed, the altered local con- 
 ditions affording opportunity for producing infection, and yet the host 
 may escape. 
 
 Bacterial invasion, therefore, does not necessarily mean infection, and 
 it may be stated that infection can only take place when — 
 
 (1) The microorganisms are sufficiently virulent. 
 
 (2) When they invade the body by appropriate avenues and reach 
 susceptible tissues. 
 
 (3) When they are present in sufficient numbers. 
 
 (4) When the host is generally susceptible to their action. 
 
 (5) When the microorganisms are able to resist the defensive forces 
 of the host through special agencies aside from" their offensive forces. 
 
 Not all these factors must necessarily be present before infection may 
 occur. A microorganism may be particularly virulent, so that numbers 
 are relatively xmimportant; a host or a portion of the host may be so 
 susceptible or vulnerable to infection that a microorganism of low 
 virulence, which, under normal conditions, would be totally unable to 
 produce infection, may now prove pathogenic. 
 
 VIRULENCE 
 
 Virulence refers to the disease-producing power of a microorganism, 
 and is dependent upon two variable factors: (1) Toxicity, and (2) aggres- 
 siveness, or the invasive power of the bacteria. In most infections usually 
 both factors are operative. 
 
 Toxicity is the term applied to the kind and amount of poison or toxin 
 produced. This poison may be readily soluble, or exogenous, diffusing 
 
VIRULENCE 95 
 
 into the surrounding tissues and being readily absorbable; or it may be 
 endogenous, and contained chiefly within the microorganisms, and be 
 liberated only upon the dissolution of the bacterial cell. 
 
 Aggressiveness is a term applied to the invasive powers of a micro- 
 organism to enter, live, and multiply in the body"-fluids, or, in other words, 
 to the aggressive or progressive forces of the microorganiam in its new 
 environment. 
 
 Toxicity is generally confused with aggressiveness, a highly toxic 
 microorganism being regarded as an aggressive one. For example, the 
 bacillus of tetanus is highly toxic because of the production of a potent 
 soluble poison which gives rise to the symptoms of tetanus, although 
 it is only slightly aggressive, being almost im.able to multiply in the 
 tissues. The anthrax bacillus, on the other hand, is highly aggressive, 
 owing to the fact that it usually multiplies to such an extent that it can 
 be found in each drop of blood and in every organ of an infected animal ; 
 nevertheless it is but slightly toxic, the animals frequently showing few 
 or no sjrmptoms until shortly before death. The toxicity of a micro- 
 organism should, therefore, be regarded separate from its aggressive- 
 ness, although in many infections both factors are so intimately con- 
 cerned that the term virulence may be used to express the degree of 
 pathogenicity or the total disease-producing power. 
 
 The virulence of a microorganism is more or less specific, i. e., the 
 toxin produced by one species is rhfferent from that produced by another 
 in the kind of disease produced and the spiecies of animal infected. 
 Some toxins are active for certain animals only and not for others. 
 Microorganisms of one group may possess general and common patho- 
 genic properties differing only in degree; those of different morphologic 
 and cultural characters may possess totally different powers. 
 
 The virulence of a given species is subject to great variation. A few 
 bacteria almost constantly retain their virulence, even when kept for 
 years under artificial conditions; as an example may be mentioned the 
 diphtheria baciUus; others quickly lose their virulence as soon as they 
 are grown artificially, as, e. g., the influenza bacillus; in others — and 
 probably the larger class — the virulence may.be raised or lowered ac- 
 cording to the experimental manipulations to which they may be sub- 
 jected. Variations may also be observed among members of the same 
 group of microorganisms, and even among individual microorganisms 
 of the same strain. 
 
 Decrease of virulence of a microorganism may be brought about 
 artificially by repeated growth in or upon culture-media, especially 
 
96 INFECTION 
 
 when transfers to fresh media are made at prolonged intervals. This 
 decrease probably depends upon an actual decrease in virulence, and 
 particularly upon the selection, in artificial growth, of the less virulent 
 or vegetative forms which grow actively anjl soon exceed in number 
 their more pathogenic fellows. Each time the culture is transplanted 
 more of the- vegetative and fewer of the pathogenic microorganisms 
 are carried over, until finally the pathogenic bacteria are entirely elim- 
 inated, or their virulence totally destroyed, and the entire culture is 
 composed only of vegetative or harmless forms of bacteria. 
 
 Various other agencies lead to artificial lessening of virulence, such 
 as exposure, for short periods of time, to a temperature just under the 
 thermal death-point; exposure to sunlight; exjposure to small quantities 
 of antiseptic or germicidal substances; the action of desiccation; sub- 
 jection to increased atmospheric pressure, etc., these methods being com- 
 monly employed in the preparations of vaccines to be used for purposes 
 of active immunization. 
 
 The passage of a microorganism or virus through animals usually 
 iiicreases its virulence, but may modify or attenuate it, as in the case of 
 the passage of smallpox virus through the cali^^ when it loses forever its 
 power of producing smallpox. 
 
 Increase in virulence can best be secured by passing the microorgan- 
 ism through animals. It is practically impossible, by any means, to 
 make a known non-virulent microorganism virulent, although it is 
 comparatively easy to increase the virulence of a culture that has be- 
 come well-nigh non-virulent on account of prolonged artificial cultiva- 
 tion. This fact is worthy of emphasis, and is well illustrated by the 
 large amount of work that has been done in fruitless attempts to render 
 non-virulent, diphtheria-like bacilli virulent b^y i^assage through various 
 animals or growth on special culture-media. 
 
 In cases where the virulence is slight or absent, experimental manip- 
 ulations of the culture are directed toward gradual immunization of the 
 microorganisms to the defensive mechanism of the body of the animal 
 for which the organism is to be made virulent. This is well explained 
 according to the hypothesis of Welch, and will be referred to again in 
 the latter part of this chapter. A number of methods are made use of 
 for this purpose: 
 
 (a) Passage through animals, which enables the microorganisms 
 gradually to immunize themselves or adopt certain morphologic and 
 biologic changes enabling them best to resist the defensive forces of the 
 host. Since these defensive forces vary with different animals, and 
 
THE AVENUE OF INFECTION AND TISSUE SUSCEPTIBILITY 97 
 
 indeed with the various organs of the same animals, it is usual to find 
 that virulence raised by animal passage affects only the animal or the 
 particular organ of a certain animal, and not all animals in general. 
 Thus, in general, the passage of bacteria through rabbits increases their 
 virulence for rabbits and not for mice, dogs, pigeons, etc.; passage 
 through mice may increase their virulence for mice, but not for rabbits, 
 guinea-pigs, etc. 
 
 (b) The use of collodion sacs for increasing virulence has been ad- 
 vocated, especially by French investigators. When microorganisms 
 are inclosed in a collodion capsule of the proper thickness and placed 
 within the abdominal cavity of a suitable animal, the slightly modified 
 body-juices are able to transfuse through the sac, impeding the develop- 
 ment of such microorganisms as are unable to immunize themselves or 
 withstand the injurious influences. In this manner a race of vinilent 
 bacteria are artificially selected which can endure the defensive agencies 
 of those juices with which they have come into contact. 
 
 (c) The addition of animal fluids to the culture-medium may enable 
 the bacteriologist to maintain or even to increase the virulence of a 
 microorganism according to the principles of artificial selection. The 
 fluid, either a serum or whole blood, is secured in a sterile manner and 
 added to the medium in a raw or unheated condition. In this manner 
 the microorganisms are exposed to some of the defensive agencies con- 
 tained in the juices under these conditions, and this tends to destroy 
 the less resistant bacteria, encourage the mote resistant, and at least 
 maintain, for a longer or a shorter time, the virulence of a culture 
 freshly isolated from a lesion or cultivated by-animal passage. 
 
 THE AVENUE OF INFECTION AND TISSUE SUSCEPTIBILITY 
 
 Successful infection of the body by certain bacteria can be accom- 
 plished only when invasion takes place through appropriate avenues. 
 Thus typhoid, cholera, and dysentery infection seems to take place 
 through the gastro-intestinal tract, and doubtfully by inhalation, and 
 not at all through the skin or urogenital system; gonococci usually 
 enter the body through the genital organs or the eye, and not through 
 the respiratory apparatus or through the ski^. The route of injection 
 is less important with microorganisms characterized by great aggres- 
 siveness and producing general, rather than local, infections. For 
 example, in most animals anthrax is a general bacteremia, regardless 
 of the route of invasion; plague rapidly becomes a bacteremia, whether 
 7 
 
98 INFECTION 
 
 the bacilli are inhaled, rubbed into the skin, or reach the lymphatics 
 through superficial abrasions; similarly, local staphylococcus and 
 streptococcus infection may become general, regardless of the route of 
 invasion or the location of the local lesion. 
 
 The avenue of invasion is also of importance in determining the form, 
 nature, and virulence of an infection. Thus virulent pneumococci 
 lodging in the pharynx may produce a pseudomembranous angina; in 
 the eye, a severe conjunctivitis; and in the lungs, a pneumonia. When 
 tubercle bacilli gain admissionthrough the skin, they may produce lupus, 
 or a low-grade inflammatory disease rarely tjbrminating fatally. When 
 inhaled, they may produce tuberculosis of the lungs; in the throat they 
 may reach the tonsils and later the local lymphatic glands, etc. When 
 swallowed, they may produce ulceration of th^intestines, or pass through 
 the intestinal walls and involve the mesenteric glands, and later the 
 Ixmgs or other organs. 
 
 Just as general susceptibility of the host renders infection more 
 likely to occur, so local susceptibility may be induced by injury and 
 fundamental disorders. These changes may not only furnish pabulum 
 for the invading bacteria, but more especially reduce the local resistance 
 of the body defenses. 
 
 Even more important, however, is the predisposition of some patho- 
 genic microorganisms to attack certain tissues or organs, and the fact 
 that these tissues are particularly weak in defensive power, so that the 
 bacteria naturally lodge where conditions are most favorable for their 
 growth. 
 
 While the primary focus of infection is determined largely by the 
 route of invasion, the selective affinity of microorganisms or their toxins 
 for certain tissues and the inherent tissue susceptibility to the toxins 
 are best in evidence in the location of secondary foci or locaHzation of 
 the infection in general bacteremias. Thus the seat of the principal 
 local lesions in pneumonia is the lungs, and in typhoid fever the lym- 
 phoid tissues, especially that of the spleen, and Peyer's patches in the 
 intestine. It is true that mechanical factors may aid in this selection, 
 as, e. g., the occlusion by emboh of microorganisms caught in the capil- 
 laries of organs; but, in general, we must conclude that either — (1) 
 Microorganisms tend to be destroyed in ev^ry tissue or organ except 
 those that are poor in defensive forces and are susceptible, or (2) that 
 microorganisms or their products circulate passively through a tissue 
 and do not lodge because they possess no affinity for these cells. In 
 many infections both processes are probably operative, and at least we 
 
GENERAL SUSCEPTIBILITY IN RELATION TO INFECTION 99 
 
 are led to the very important conclusion, laid down by Adami, that "in 
 infections the body is never involved as a whole. Coincidentally with 
 the growth of the specific germs in individual organs there tends to be a 
 reaction to, and destruction of, the same in other parts." 
 
 The nitmeric relationship of bacteria to infection is very important, 
 and the number alone may determine whether or not it shall occur. 
 Usually the normal defensive factors of the body are sufficient to over- 
 whelm one or a few bacteria unless they are especially virulent. When 
 an intercurrent or chronic disease, malnutritiqn, or injury renders the 
 host more susceptible than normal, fewer bacteria than would other- 
 wise be required may successfully infect the body. Also with true 
 parasites, or those with well-marked aggressiveness, such as the anthrax 
 bacillus, a few may be sufficient, if they reach the circulating fluids, to 
 produce infection. Thus Webb, Williams, and Barbor"^ have found that 
 one anthrax bacillus was sufficient to infect a white mouse, and as few 
 as 20 tubercle bacilU were sufficient in one instance to infect a guinea-pig. 
 
 Park has directed attention to the fact that when bacteria are trans- 
 planted from culture to culture, under supposedly favorable conditions, 
 many of them die; it is highly probable that w'hen they are transplanted 
 to an environment that is hkely to be unfavorable, as are the body 
 tissues with various defensive mechanisms, many more must die. This 
 is an important point to bear in mind in attempting to correlate experi- 
 mental results with the natural cause of an infectious disease. In the 
 laboratory we reproduce disease experimentally by the immediate in- 
 jection of millions of bacteria, whereas in nature there is rarely any such 
 immediate overwhelming of the tissues. For example, pneumonia may 
 be produced experimentally in dogs by the injection of a large munber of 
 virulent pneumococci directly and at once into the bronchi, yielding a 
 positive result with a microorganism which, under natural conditions 
 and in smaller numbers, would be relatively innocuous for the animal 
 under observation. 
 
 GENERAL SUSCEPTIBILITY IN RELATION TO INFECTION 
 Under normal conditions the body-cells of a host will invariably 
 offer some resistance to invasion and infection by pathogenic micro- 
 organisms. When, however, any condition that depresses or diminishes 
 general physiologic activity and vitality exists, the host may be unable 
 to master these defensive forces, and accordingly becomes predisposed 
 or more susceptible to infection. 
 
 'Transactions Sixth International Congress on Tuberculosis, 1908, p. 194. 
 
100 INFECTION 
 
 Predisposition may be inherited or acquired. 
 
 Inherited predisposition may be — -(a) Specific, or species suscep- 
 tibility, as, e. g., dogs to distemper; cattle to contagious pleuropneu- 
 monia; hogs to hog cholera; man to gonorrhea; chancroids, acute 
 exanthemata, typhoid fever, etc. (b) Racial, as Eskimos to measles 
 and syphihs, ordinary sheep to anthrax, whereas Algerian sheep are 
 immune, etc. Racial susceptibility is frequently but a lack of acquired 
 immunity; for instance, measles, syphilis, gonorrhea, and other diseases 
 brought by settlers to foreign peoples among whom these diseases were 
 previously unknown, find them peculiarly suscJeptible and the diseases 
 unusually virulent, (c) Familial, i. e., members of a family may, 
 through generations, be unusually susceptible to scarlet fever, tuber- 
 culosis, rheumatism, rheumatoid arthritis, meta"bolic disturbances, etc. 
 (d) Individual predisposition, which depends principally upon sex, age, 
 and peculiar tissue susceptibility. Thus infants are especially prone to 
 contract certain infections on account of the immature development of 
 the body-cells, and this susceptibiUty to infection is further influenced 
 by acquired factors, chiefly malnutrition. On the other hand, very 
 young children enjoy an immunity to several infections, such as typhoid 
 fever, scarlet fever, and even diphtheria, probably due, as Abbott has 
 suggested, to the fact that pathogenic substances that may set up molec- 
 ular and destructive disturbances in the poorly developed cell have but 
 little effect upon the more inert protoplasm of the immature, cell, and 
 that if certain bacteria gain admission to the* tissues, the cells may 
 destroy them, their toxins not combining with the molecular side-chains, 
 and, as a consequence, not injuring or interfering with the cell functions. 
 
 Acquired susceptibility bears a more important relation to infection, 
 and may be due to various factors, most of which lead to a state of re- 
 duced vitality, normal physiologic processes being impaired to a greater 
 or less degree. 
 
 (a) Overwork or overstrain leads to general or local predisposition 
 to disease. Those engaged in hard labor, mental or physical, which 
 involves late hours and inadequate periods of rest and recreation, fre- 
 quently associated with inadequate nutrition and foul air, are hkely to 
 succumb to tuberculosis, tyi^hoid fever, pneumonia, etc. 
 
 The influence of overstrain on acute infections has been shown ex- 
 perimentally by Charrin and Roger, i who found that white rats natur- 
 ally immune to anthrax became quite susceptible after being compelled 
 to turn a revolving wheel until exhausted before thej^ were inoculated; 
 ' Compt. rend. Soc. de Biol, de Paris, January 24, 1890. 
 
GENERAL SUSCEPTIBILITY IN RELATION TO INFECTION 101 
 
 similarly of four guinea-pigs who were placed in a cage so constructed 
 that they were forced to keep moving for one or two days three died in 
 from two to nine days after the experiment. Sinears and cultures made 
 from the livers, spleens, and blood gave positive results. 
 
 (6) Previous infection with the same or another infectious disease 
 may predispose the individual to renewed infection. Thus some in- 
 fections, such as erysipelas, funmculosis, acute rheumatism, pneumonia, 
 and influenza, not only fail to leave the body-cells immune, but actually 
 predispose to second attacks. Whether the microorganisms of these 
 diseases are not all destroyed, but are retained in the system and become 
 active when the general vitality is lowered, or whether a new infection 
 occurs, is not definitely known, and probably either may occur. 
 
 One attack of an infectious disease may weaken the tissues and 
 render them susceptible to an infection of a different nature. Thus 
 the acute exanthemata may follow one anotljer, and tuberculosis may 
 supervene upon any of them. 
 
 (c) Malnutrition exerts some effect on the resistance to infection. 
 Thus the terrible epidemics of plague, cholera, typhus fever, and typhoid 
 fever which have followed in the wake of faroines in Europe and Asia 
 during the past centuries are examples of the influence of malnutrition 
 as a factor in predisposing to disease. The tendency of marasmatic 
 infants to develop enterocohtis, thrush, bronchopneumonia, and other 
 infections, and of scorbutics to local infections of the mouth, illustrates 
 the influence of insufficient food in decreasing the resistance to disease. 
 Here may also be included local malnutrition; such as loss of nerve or 
 blood supply, prechsposing to local infection, especially with pyogenic 
 microorganisms. 
 
 (d) Diet produces some variation in the resisting powers to infection. 
 For example, the ordinary wild rat is not susceptible to anthrax unless 
 it is fed for a week or more on coarse dry food, when it become sus- 
 ceptible. Here, of course, malnutrition may come in intimate relation- 
 ship with diet, as an inefficient diet may gteatly lower the general 
 resistance. The influence of diet is particularly noticeable from the 
 fact that the diseases of carnivorous animals are not the same as those 
 that affect herbivorous animals, and that each class is frequently im- 
 mune to some of the diseases that attack the other. 
 
 (e) Intoxications of various kinds predispose to infections. Thus it 
 is a common chnical observation that excessive indulgence in alcoholic 
 beverages predisposes to infections, notably pneumonia. Abbott^ has 
 
 I Jour. Exper. Med., 1896, 1, No. 3. 
 
102 INFECTION 
 
 demonstrated experimentally that the daily administration, to rabbits, 
 of 5 to 10 c.c. of alcohol introduced into the stomach by a tube, renders 
 these animals more susceptible to infection with" Streptococcus pyogenes 
 and Bacillus coli. Wagner, Leo, and Platania have also found animals 
 that under the influence of chloral, phloridzin,- alcohol, and curare are 
 more susceptible to infection. 
 
 (/) ExTposure to cold and wet frequently lowers the resistance of 
 man and other warm-blooded animals to infection. The influence of 
 these factors, well illustrated in the etiology of "colds" and pneumonia, 
 is not without experimental foundation. Thus Pasteur found that 
 fowls, which are naturally immune to anthrax, are readily infected if 
 they are inoculated after their body temperature has been reduced by 
 a cold bath. Conversely, Gibieri has shown that frogs, which are also 
 naturally immune to anthrax, are readily infected if their temperature 
 is previously elevated and maintained at 37° G{ 
 
 (g) Trauma and morbid conditions in general may predispose to in- 
 fection. Thus injuries reduce the local resistance and facilitate local 
 infections that vary with the severity and extent of the trauma. The 
 increased susceptibility of injured joints and pileumonic lungs to tuber- 
 culosis; the frequent and oftentimes extensive streptococcus infection 
 accompanying scarlet fever and smallpox; the increased susceptibility 
 of diabetics to furunculosis and local gangrenous lesions of the skin — 
 all show the increased susceptibility of individuals already injured or 
 diseased to infection. 
 
 THE DEFENSIVE MECHANISM OF THE MICROORGANISM IN RELATION 
 
 TO INFECTION 
 
 After bacterial invasion has occurred, the question of whether or 
 not the microorganism can overcome the defensive forces of the host and 
 prove pathogenic may depend to some extent upon the peculiar defensive 
 factors of the invading bacteria against the offensive mechanism of the 
 host, aside from their toxins or other distinctly offensive forces. 
 
 Morphologic and Physiologic Changes of the Microorganisms. — For 
 example, capsule formation or thickening of the ectoplasm of certain 
 bacteria is evidence of their increased powers of resistance against the 
 opposing forces of the host. The capsule may \ye quickly lost when the 
 microorganism is cultivated on artificial media, and its virulence be cor- 
 respondingly lowered, but by repeated animal inoculations a race of 
 
 ' Compt. rend. Acad, de Sci. de Paris, 18$2, xcix, 1605. 
 
DEFENSIVE MECHANISM IN RELATION TO INFECTION 103 
 
 capsulated organisms with increased virulence is produced, explaining 
 in a way the mechanism of animal passage in raising the virulence of a 
 given organism. This, however, is not invariable, and, indeed, may act 
 in a contrary manner, as the passage of smallpox virus through heifers 
 attenuates and modifies instead of increasing' its virulence. 
 
 Aggressins. — The microorganism may actively secrete a material 
 that overwhelms the defensive forces of the host. This phase of the 
 subject has been studied exclusively by Bail, who sought to prove that 
 the question of pathogenicity of a microorganism is dependent upon its 
 ability to secrete substances that are able to paralyze the protective 
 forces of the host, especially the leukocytes. These substances are 
 called "aggressins," and they were distinguished by the fact that they 
 were formed by living bacteria and only in the living body. In support 
 of this theory Bail was able to show that substances are present in the 
 exudates of fatal infections, which, when injected in small quantities 
 into another animal with sublethal doses of the microorganism, would 
 cause a rapidly fatal infection. Later Wassermann and Citron showed 
 that "artificial aggressins" could be prepared by autolyzing bacteria 
 in water or sermn. While the subject of aggjressins is still unsettled, 
 there is strong evidence to show that they are the endotoxins Uberated 
 by the breaking-up of the microorganism. 
 
 The well-known statement of Metchnikoff , that a particular virulent 
 microorganism is not so readily taken up by leukocytes as is an avirulent 
 strain, may be explained by the fact that the microorganism, in its 
 virulent parasitic state, secretes substances that repel the phagocytes, 
 neutraUze the opsonins, or form actual leukocytic toxins. This action 
 may be due to liberated endotoxins, or, as Bail claims, to specific secre- 
 tory substances of the bacterium — the aggressins — specifically formed 
 and liberated by the microorganism for protection against the host. 
 
 Hypothesis of Welch. — Not entirely foreign to this subject is the 
 very interesting hypothesis of Welch. A bacterium may not only pro- 
 duce substances directly inimical to the defeni^ive forces of the host, but 
 it may actually immunize itself against these defensive powers. "Looked 
 at from the point of view of the bacterium, as well as from that of the 
 animal host, according to the hypothesis advanced, the struggle between 
 the bacteria and the body-cells in infections niay be concerned as an 
 immunizing contest in which each participant is stimulated by its op- 
 ponent to the production of cytotoxins hostile to each other, and thereby 
 endeavors to make itself immune against its antagonist." 
 
 It is well known that, when freshly isolated from a patient having 
 
104 INFECTION 
 
 typhoid fever, the typhoid bacillus resists^ agglutination, whereas it 
 becomes easily agglutinable after a period of artificial cultivation. It 
 may be assumed that, when active, the bacillus as an infecting agent 
 gradually became more resistant against the agglutinating properties 
 of the patient's seriim, and that, when grown on artificial media, it loses 
 this resistance by being removed from the stimulating influence of the 
 infected body. 
 
 This hypothesis, however, would go a step further in assuming the 
 possibility of the receptors of the invading bacteria anchoring certain 
 constituents of our body-fluids, and being stimulated to the production 
 of various cytotoxins, which attack the leukocytes, erythrocytes, nerve- 
 cells, liver, kidney, etc. In other words, each bacterium may be con- 
 ceived as being composed of a central atom group with numerous side- 
 chains, just as Ehrhch conceived the hypothetic structure of body-cells, 
 and that these side-chains, primarily present; for the purpose of anchor- 
 ing food material, may likewise anchor various pathogenic animal sub- 
 stances, with the production of substances acting as antibodies to the 
 opposing forces of the host. Welch assumed that these bodies were of 
 the nature of amboceptors, which may become complemented by bac- 
 terial complement or by endocomplements of the tissue-cells; this is of 
 secondary importance, and there is no reason why they may not be of 
 different structxire, and similar to all three orders of antibodies produced 
 by body-cells according to Ehrlich's side-chain theory of immunity. 
 
 This hypothesis may possibly explain certain instances of so-called 
 species and organ virulence, whereby the virulence of an organism arti- 
 ficially increased by repeated passage through animals of the same spe- 
 cies, does not manifest this increased virulence for animals of different 
 species. If, for example, the virulence of the chicken cholera bacillus 
 is increased by repeated passage through the chicken, the increase of 
 virulence affects this animal, but does not affett the guinea-pig. Certain 
 organs may hkewise be subject to a similar selective virulence, if the 
 increase in virulence has been induced by the specific intervention of 
 those organs, and this selective virulence shows itself, irrespective of 
 the manner in which the infection was produced. 
 
 That virulence of this order is playing an important r61e in the pro- 
 cesses of infection is a theory supported by the discovery that different 
 strains of the same species of bacteria are found to produce characteristic 
 lesions,. and while this affinity for a certain organ may be natural and 
 inherent, there can be no doubt that it may also be experimentally 
 induced and acquired. For example, according to Rosenow, a certain 
 
MIXED INFECTION' 105 
 
 strain of streptococcus will produce arthritis;- another, endocarditis; 
 another, gastric ulcer, etc. 
 
 This remarkable species and organ specifici,t*y maj- be due to the fact 
 that the bacteria of a particular culture have been immunized against 
 defensive forces of a particular animal host or a certain organ of the host, 
 so that, when introduced, they thrive as a result of their special and 
 acquired offensive forces. On the other hand, the specificity may be 
 due to the fact that the bacteria have been accustomed to a certain 
 nutriment furnished bj' a particular species or organ, and that thej' 
 cannot thrive unless they receive this special nutriment, and, as a result, 
 the species or organ f ulfillin g this requiremept will become the special 
 seat of infection (Simon) . 
 
 MIXED INFECTION, 
 
 Several different microorganisms may produce infection at the same 
 time, or one may foUow the other or others; and produce secondary 
 infection. The combined effects, upon the tissues of the host, of the 
 products and action of two or more varieties of pathogenic bacteria, 
 and also of the influence of these different forms on each other, are of 
 great importance in the production of disease. The metaboHc products 
 of one bacteria may neutralize or accelerate the action of an associated 
 .species, or combine to form a new substance entirely different from its 
 antecedents. 
 
 Thus pyogenic cocci affect anthrax bacilli in an injurious manner; 
 on the other hand, aerobic bacteria accelerate or make possible the 
 growth of anaerobes by absorbing uncombined osygen. Tetanus 
 bacilli will not grow outside of the body in the* presence of oxj^gen unless 
 aerobic bacteria are associated with them; not infrequenth' tetanus 
 bacilli and their spores would not develop in wotmds were it not for the 
 presence of the aerobic bacteria introduced with them; this factor is of 
 much importance, especially in tetanus produced by cowpox vaccine, 
 where, through careless treatment of the lesion, both tetanus bacilli and 
 pyogenic cocci are admitted to the wound. 
 
 Again, it may be fotmd that one microorganism increases the viru- 
 lence of another; thus the scarlet-fever \-irus ife favorable to the develop- 
 ment of streptococci. 
 
 General!}' all infections of mucous membranes are mixed infections. 
 Numerous bacteria are present upon the mucosa of the air-passages 
 and gastro-intestinal tract ; these are usually harmless, unless the resist- 
 ance of the host is lowered in some manner, in which case not only one 
 
106 INFECTION 
 
 but several varieties of these bacteria invade the tissues and cause 
 infection. When one pathogenic microorganism, such as the typhoid 
 bacillus, has caused the primary infection, becaruse of the local and gen- 
 eral conditions of lowered vitahty of the tissues, these otherwise sa- 
 prophytic baciUi tend to intensify the infection. Blood infections, on 
 the other hand, are usually due to one form of bacteria, and even when 
 two or more varieties are introduced, only one, as a rule, is capable of 
 surviving and developing. The products of certain bacteria, on the 
 other hand, may immunize the host against infection with other bacteria, 
 for, as shown by Pasteur, attenuated chicken-cholera cultures may 
 produce immunity against anthrax. In the intestine harmless varieties 
 of bacteria may be made to crowd out more dangerous ones; this is 
 exemphfied by the ingestion of soured milk which contains lactic-acid 
 bacteria, as advocated by Metchnikoff . 
 
 SUMMARY 
 From what has been said it is clear that infection differs from mere 
 surface contamination, and cannot be said to occur until the invading 
 bacteria have reached the deeper tissues, or a point where they may grow 
 and multiply. The surface epitheUum and various secretions offer the 
 most potent local obstacles to infection, but even when these barriers 
 are broken down, the invaders may not survive the onslaughts of various 
 protective agencies of the host. In order to withstand and overcome 
 these attacks, the bacterium may undergo certain morphologic and 
 physiologic changes, and actively secrete a substance that is inimical 
 to the defensive forces of the host, or immunize itself against these forces. 
 Thus a certain species of bacteria may become selectively fortified or 
 immunized against a certain host or organ of that host, and show a 
 specific affinity for producing infection of a certain animal or a particular 
 organ. When the bacterium has overcome the defensive forces of a host, 
 it may, by the formation and action of exogenous and endogenous toxins, 
 bacterial proteins, mechanical blocking of vessels, or formation of pto- 
 mains, produce disease. These various factors will be considered in 
 greater detail in the following chapter. 
 
CHAPTER VII 
 INFECTION (Continued) 
 
 PRODUCTION OF DISEASE 
 
 When pathogenic microorganisms have reached the deeper tissues and 
 multiplied, infection has occurred, but, as previously stated, tissue 
 changes of sufficient extent to produce definite lesions and symptoms 
 of disease may or may not result, depending upon whether or not the 
 defensive forces of the host are able to overcome the invaders or are 
 overcome by them. If the latter has occurred, and the invading bac- 
 terium is firmly established in its host, the question of how the bacterium 
 and its products cause disease, that is, the viichanism of the production 
 of an infectious disease, arises for consideration. 
 
 The subject is, indeed, quite complex. Although the etiologic 
 relationship of a large number of pathogenic bdcteria to definite patho- 
 logic changes and conditions has been proved by the regularity T\ith 
 which they are found in the diseased tissues, and in many instances has 
 been corroborated by animal experimentation, yet the ways and means 
 by which these bacteria produce disease are quite varied, and are seldom 
 dependent upon one product of bacterial activity. For example, 
 diphtheria and tetanus are apparently simple infections, being caused 
 by soluble toxins secreted by the respective bacilli. There are many 
 factors concerning the action of these toxins, however, which are not as 
 yet understood. Again, the lesions of staphylococcus and other pyo- 
 genic infections are probably due to the activities of soluble toxins, 
 endotoxins, and the protein of the bacterial bodies. All three of these 
 factors are probably concerned in the production of typhoid fever and 
 cholera, whereas the symptoms of sleeping sickness are due in part to 
 blocking of a small but physiologically important vessel in the brain by 
 trypanosomes, with the absorption, at the same time, of toxins and dis- 
 integration products. To these may be added the effects of other 
 biologic activities of the bacteria in living or dead tissues, such as the 
 production of gas from carbohydrates, proteins, etc., in Bacillus aero- 
 genes capsulatus infection. Each infection, therefore, must be regarded 
 as largely a law unto itself, so that all that wll be included within the 
 scope of this book will be the mention and illustration of what are 
 
 107 
 
108 
 
 INFECTION 
 
 generally accepted as the ways and means by which bacteria and proto- 
 zoa produce disease, omitting a description of each disease in detail. 
 
 In the great majority of instances disease is produced as the result 
 of chemical substances generated by the metabolic processes of bacteria. 
 Animal parasites and certain bacteria, such as that of anthrax, may do 
 harm mechanically by forming capillary emboli; but, as stated, bac- 
 teria, as a rule, produce their effects chiefly through chemical means. 
 Accordingly, bacteria may give rise to infection and disease through the 
 following agencies : 
 
 1. Soluble or extracellular toxins, which are poisons generated by 
 bacterial cells and discharged into their surroimding media. 
 
 2. Intracellular toxins or endotoxins, whish are specific poisonous 
 products of bacterial activity, and are contaiifed within the cells. 
 
 3. Aggressins, or substances secreted by bacteria, that neutralize 
 opsonins and prevent phagocytosis. 
 
 4. Bacterial proteins, which are poisonous protein constituents of the 
 bacterial cells and are responsible for certain general and non-specific 
 lesions. 
 
 5. Ptomains, which are the secondary products of decomposition of 
 the media upon which the bacteria are growing; these may be absorbed 
 and produce symptoms of intoxication. 
 
 6. Mechanical action of bacteria, whereby certain symptoms or 
 lesions may be due to the blocking of small but physiologically important 
 vessels with emboli of bacteria, in addition to the effects of mechanical 
 irritation. 
 
 TOXINS 
 
 Nomenclature. — Of all the various means whereby bacteria produce 
 disease, none possesses so much importance as: the poisonous substances, 
 kiiown as toxins, elaborated by the metabolic activities of the micro- 
 organisms. A few classes of bacteria secrete this poisonous principle 
 directly into the tissues or artificial culture-media in which they are 
 growing, and hence are known as soluble, exogenous, extracellular, or 
 true toxins. Other bacteria retain most of their toxins within the 
 bacterial cell, and for this reason are called Endotoxins, or intracellular 
 toxins; these are liberated upon the disintegBation of the bacteria by 
 various mechanical, physical, or chemical meaps. 
 
 By common consent the term "toxin" is applied to the soluble or 
 true toxins, such as those of diphtheria and tetanus, and hence the term, 
 when used without further qualifications, may be considered to refer to 
 toxins of this class. 
 
TOXINS 109 
 
 Aside from bacterial toxins, characteristic poisons are also produced 
 by certain of the higher plants (phytotoxins) and animals (zootoxins), 
 and although few are of medical interest, their studj' has thrown con- 
 siderable light on the phenomena of toxin-antitoxin immunity. 
 
 Extracellular Bacterial Toxins. — Definition-, — Bacterial toxins may be 
 defined as poisonous products produced by bacteria in both living tissues and 
 artificial culture-media. The symptoms resulting from their activity appear 
 after a certain period of incubation, and all are capable of stimulating 
 the production of specific antitoxins. They represent the chief poison- 
 ous product of bacteria, and are mainly responsible for the symptoms 
 of infection caused by the specific bacteria that have produced them. 
 
 The true toxins causing infection in man are chiefly: 
 
 1. Diphtheria toxin. 
 
 2. Tetanus toxin. 
 
 3. Botuhsm toxin (a form of meat poisoning). 
 
 4. Dysentery toxin (Kruse-Shiga) . 
 
 5. Staphylolysin and other bacterial toxins.. 
 
 General Properties of Soluble Toxins. — Many of the true toxins are 
 extremely labile, and susceptible to the action of heat, light, age, etc.; 
 consequently an absolutely pure toxin is practically unknown. Oxygen, 
 even as it occurs in the air, is harmful; all oxidizing agents, including 
 the oxidizing enzymes, quickly destroy them, and Pitini^ has ascribed 
 the harmful effects of toxins to their power of reducing the oxidizing 
 capacity of the tissues. Some substances seem to attack only the 
 toxophore portion of the toxin molecule, e. g., iodin and carbon disulphid 
 (Ehrlich). In the preparation of antitoxin, the first doses of toxin are 
 frequently modified by adding a chemical of this nature. According 
 to Gerhartz^ x-rays tend to weaken the toxins: 
 
 Because of their great lability, the toxins do not lend themselves to 
 accurate chemical analysis. Our knowledge of them has been gained 
 largely through a study of the lesions and sjmiptoms produced by in- 
 jecting the toxins into susceptible animals. 
 
 The toxins are all poisonous, but in order to exert their toxic effect 
 they must enter into chemical combination with cells ; hence there is a 
 necessary period of incubation before symptonis of their activity appear. 
 Most bacterial toxins are not absorbed from the intestine (botulinus 
 toxin excepted), and when introduced into the gastro-intestinal tract, 
 they are usually unable to produce symptoms and are quickly destroyed. 
 
 'Bioohem. Zeit., 1910, 25, 257. ^ Berl. klin. Woch., 1909, 46, 1800, 
 
110 INFECTION 
 
 An essential property of a toxin lies in the fg,ct that we can immunize 
 a ^subject against it, and are able to demonstrate the presence of antitoxin 
 within the serum of the immunized animal. 
 
 Chemical Properties of Soluble Toxins.— ^As has just been stated, 
 the exact chemical nature of toxins is unknown. This is due principally 
 to the fact that pure toxins of bacteria are rarely obtainable, except in 
 conjunction with their associated products, such as lysins, pigments, acids, 
 etc., as well as to the great labiUty of the toxins. A summary of the 
 results of researches into the chemical nature of toxins would indicate 
 that they are toxalbumins, or allied to proteins. Certain investigators 
 have reported that very active toxins obtained by purification processes 
 did not give the protein reactions, yet toxins are digested by proteolytic 
 ferments, and, like proteins, are precipitated by nucleic acid (Kossel). 
 According to Field and Teague,^ the toxins act like electropositive col- 
 loids, but diffuse faster than do proteins. Our present knowledge of 
 the chemistry of the true toxins has been expressed thus by Oppen- 
 heimer: "We must be contented to assume that they are large molecular 
 complexes, probably related to the proteins, eorresponding to them in 
 certain properties, but standing even nearer to the equally mysterious 
 enzymes with whose properties they show the most extended analogies 
 both in their reactions and in their activities." 
 
 Structure of Toxins. — According to Ehrlich, the toxin molecule 
 consists of a main central atom or radical, with^a large number of organic 
 side-chains grouped, as in other organic compounds, about this main 
 radical. Each of the side or lateral arms is composed of two portions — 
 one, the haptophore group, which has a chemical affinity for certain 
 chemical constituents of the tissues of susceptible animals, and the other, 
 the injury-producing portion, called the toxophore group. (See Fig. 40.) 
 An animal is susceptible to a toxin only when its cells contain substances 
 that possess a chemical affinity for the haptophore group of the toxin, 
 and also substances susceptible to the toxic action of the toxophore group. 
 
 The toxophore group is far more unstable and susceptible to dele- 
 terious influences than is the haptophore portion. When the molecule 
 has lost the toxophore radical, it is known as a toxoid, which is still 
 capable of uniting with the side arms of cells but is devoid of toxic action. 
 
 Nature of Toxins. — It has been abundantly demonstrated that 
 
 toxins are colloids, and in many respects bear a close resemblance to 
 
 enzymes. (See p. 244.) The toxins are synthetic products of bacterial 
 
 activity. They are of absolutely specific nature, and in this manner 
 
 ' Jour. Exper. Med., 1907, 9, 86. 
 
TOXINS 111 
 
 differ from ptomains, which are cleavage products from the medium 
 upon which the bacteria have been grown, f'urthermore, ptomains of 
 similar properties may be produced by several different kinds of bacteria, 
 and accordingly are non-specific in nature. Toxins, like ferments, can 
 give rise to antibodies, whereas ptomains cannot produce them. 
 
 The extracellular or soluble toxins differ froiti the intracellular toxins 
 in that they are more easily diffused throughout the animal juices, and 
 that their diffusion occurs independently of the invasiveness of the 
 bacteria, so that comparatively few microorganisms growing at some 
 unimportant focus, and causing but slight local lesions, may be able to 
 give rise to profound general intoxication. This is well illustrated in 
 diphtheria, where the local lesion in the throat may be quite small, 
 and in tetanus, where it may indeed be undiscoverable — yet either, 
 through the action of their toxins on special tissues, may cause profound 
 intoxication and death. 
 
 Selective Action of Toxins. — Extensive studies of the toxins of 
 diphtheria and tetanus and of cobra venom have shown that they are 
 quite complex, and are usually composed of two or more distinct and 
 separate toxins possessing different pathogenic properties, although one 
 T>f these may predominate in producing symptoms. 
 
 All infections with the group of true toxin-^producing bacteria mani- 
 fest certain non-specific sjrmptoms of general intoxication, namely, 
 fever, headache, malaise, prostration, etc.; but the tj^pical symptoms 
 of these diseases are due to the remarkable selective action of the toxins 
 upon certain cells or organs, dependent upon the abihty, chemical, 
 physical, or both, of the toxin to combine with these specific cells. For 
 example, tetanus toxin contains tetanospasmin, that has a special 
 affinity for nervous tissue; and tetanolysin, a poison that has a 
 selective affinity for erythrocytes and is hemotoxic. Ehrhch has shown 
 that these are really different toxins, and not one toxin with a two-fold 
 function, even the antitoxins of the two being different. Similarly, the 
 general sjonptoms and necroses of diphtheria are attributed to the main 
 toxin of the bacillus, and the nerve lesions and paralyses to a secondary 
 but distinct secretory product known as toxon. This latter view of 
 Ehrlich's, however, is much disputed, many ir^vestigators believing that 
 toxon represents a degenerated or modified form of the one toxin. 
 
 The special affinities of toxins for certain tissues have analogies 
 among the poisons of higher plant life, as, for example, strychnin has a 
 similar selective affinity and is said to be specific in its action upon the 
 motor cells. 
 
112 INFECTION 
 
 The venom of various serpents, especially" that of the cobra, has 
 specific action : the erythrocytes of various animals are readily attacked 
 by it, and the cells of the respiratory center are apparently profoundly 
 affected. 
 
 Aside from the special effects of the toxing* upon certain cells and 
 tissues, it must be remembered that toxins may involve the body-cells 
 in general, and particularly those of the parenchymatous organs, such 
 as the kidneys, heart, and liver, causing coagulation of the protoplasm 
 (cloudy swelhng) and final dissolution. The harm brought about by 
 the toxins or toxic products of the pyogenic group of microorganisms, 
 for instance, acts mainly in this manner. 
 
 SPECIAL PROPERTIES OF THE PRINCIPAL TOXINS 
 
 1. Diphtheria Toxin. — Diphtheria bacilli vafy considerably, both in 
 tissues and in artificial culture media, in the quantity of toxin se- 
 creted; thus in bouillon large amounts are seldom found in less than 
 from seven to fourteen days. 
 
 The action of the toxin is dependent upon the dosage, and a certain 
 period of time must always elapse before the symptoms appear, the 
 minimum being about one day. Large doses may shorten this period 
 of incubation, but cannot diminish it below a certain limit. 
 
 The lesion of diphtheria is practically always local, and is usually 
 situated on the mucous membrane of the upper air-passages. It is 
 characterized by the formation of a pearly white membrane that is 
 adherent to the underlying edematous tissues. The toxin produces 
 necrosis of the surface epithelium, and the product, together with fibrin 
 and leukocytes, constitutes the membranous exudate. From this focus 
 toxin is absorbed by the lymphatics and blood'gtream, and distributed 
 throughout the body, the bacilli being rarely found in the blood or in- 
 ternal organs. Later the effects of toxin intoxication are shown by 
 paralyses of certain motor nerves and ganglia, particularly those of the 
 palate and heart. 
 
 When a guinea-pig receives a subcutaneous inoculation with diph- 
 theria toxin, a typical hemorrhagic gelatinous edema develops at the 
 site of inoculation (Fig. 36). Upon opening the abdominal cavity one 
 finds but little peritoneal exudate, but the vessels of the mesentery are 
 injected and the adrenal glands show characteristit; acute hyperemia (Figs. 
 37 and 38). Bloody pericardial and pleural exudates will be found in the 
 thorax, and solidified areas in the lungs. Guinea-pigs survi^-ing a dose of 
 toxin may, after two or four weeks, iDegin to show paralysis of the hind 
 
Fig. 36. — Abdominal \\ all of Guinea-pig Shco^ng Diphthehio Edema. 
 Shows abdominal wall of a guinea-pig forty-eiglil«liouis aftei" subcutaneous in- 
 jection with 2 c.c. of a seventy-two-hour bouillon culture of a diphtheria bacillus 
 isolated from the throat of a diphtheria convalescent. 
 
SPECIAL PROPERTIES OF THE PRINCIPAL TOXINS 113 
 
 and then of the fore extremities, a condition analogous to the post-diph- 
 theric paralysis occurring in man and ascribed to the effects of toxon. 
 
 Method of Testing the Virulence and Toxicity of Diphtheria Bacilli. — ■ 
 Young guinea-pigs weighing from 250 to 300 grams are quite susceptible 
 to diphtheria toxin, and are used in determining the strength of a toxin 
 and in standardizing antitoxin. The test may, be of great value in the 
 management of convalescent and "carrier" ca'ses of diphtheria, harbor- 
 ing bacilh in the upper air-passages, in determining whether the 
 microorganisms are dangerous or merely harmless non-pathogenic sapro- 
 phytes. It is practically impossible, from the morphology of the organ- 
 ism alone, to decide whether or not a given culture is dangerous, and 
 prolonged quarantine may not only be irksome and inconvenient, but, 
 if the organisms are proved to be harmless, it Is unnecessary as well. 
 
 To be rehable, however, such a test must be carried out very care- 
 fully. In the case of a highly virulent culture, the mere introduction 
 of a few organisms beneath the skin will suffice to demonstrate their 
 dangerous character, but with cultures only shghtly virulent, more care 
 is necessary, for although the patient may show no ill effects as a result 
 of the presence of the bacilli, in the throat of another and less immune 
 individual they may be highly dangerous. 
 
 The following method has been used by the author in many hundreds 
 of such tests in the Philadelphia Hospital for Contagious Diseases, and 
 has proved of distinct value : 
 
 1. Make a cuUiire of the part harboring the bacilli on a tube of Loffler serum 
 medium. Incubate at 35° C. for from eighteen to twenty-four hours; prepare a 
 smear and stain with LoflBer's methylene-blue. If diphtheria bacilh are present, 
 they must be isolated in pure culture. Never attempt d guinea-pig test v/ith an impure 
 culture! 
 
 2. Isolate by the "streak" method, on plates of blood-serum. 
 
 3. Inoculate a tube of 1 per cent, glucose bouillon, which is neutral or slightly 
 alkaline, with several different colonies. 
 
 4. Incubate at 35° C. for three days, keeping the tube in a slanted position in 
 order to give the culture as much oxygen as possible: If a good growth does not 
 appear in twenty-four hours, transplant to another tube of bouillon imtU the bacilli 
 have been "educated" to grow on the medium. 
 
 5. Examine for purity. Select a 250- to 300-gram guinea-pig and inject 2 c.c. 
 of the uniiltered culture in the median abdominal line. Animala over the weight 
 specified are more resistant and less reliable for the test. The unfiltered culture is 
 used, since toxin is but one clement of the disease-producing power of diphtheria 
 bacilli, and toxin production in bouillon may not be a true index of the toxin produc- 
 tion in mucous membranes. 
 
 6. Carefully observe the animal for at least four days. Even slight toxemia, 
 especially if accompanied by edema at the site of injefition, should be regarded as a 
 positive result (Fig. 37). 
 
1 14 INFECTION 
 
 7. After death perform a careful autopsy. Make cultures of the edematous 
 area, peritoneum, and heart blood. Diphtheria bacilH may be found in the edema- 
 tous fluid, but will rarely be found in the peritoneum or jn the blood. Observe whether 
 acute hyperemia of the suprarenal glands is present (Figs. 37 and 3S). 
 
 8. Not infrequently animals showing mUd or even an absence of the symptoms 
 of toxemia develop paralysis of the hind quarters two or three weeks later. Accord- 
 ing to Ehrlich, this paralysis is due to the action of "toxon, " a toxic substance se- 
 creted by the bacillus or, as believed by others, a modified form of toxin. 
 
 9. To prove that diphtheria was the cause of the toxemia or death mix 2 c.c. of 
 the culture in a test-tube with 1 c.c. of diphtheria antitoxin (.500 units). After stand- 
 mg aside for an hour at room temperature, inject the mixture subcutaneously in the 
 median abdominal line of a 250 to 300 gram guinea-pig. Symptoms of toxemia do 
 not develop. 
 
 Standardizing Diphtheria Toxin. — The strength of a diphtheria toxin 
 is estimated by injecting subcutaneously a series of guinea-pigs weighing 
 approximately 250 grams, with decreasing amounts of toxin. How 
 many dilutions will be necessary it is impossible to state ; for exact results 
 several pigs of the same weight should be inoculated with the same dose, 
 and the effects should show various gradations, dependent upon the 
 size of the successive doses. In order to obtain a uniform method for 
 estimating the strength of a diphtheria toxin and thus obtain compara- 
 tive values, a standard unit has been adopted, consisting of the smallest 
 amotint of toxin that will Mil a health]] guinea-pig weighing about 250 
 grams in from four to five days. This is known as the minimum lethal 
 dose, or dosis lethalis minimus. The technic used for determining this 
 dose is given in the chapter on Antitoxins, p. 232. 
 
 A quick and accurate method for estimating the amount of diph- 
 theria toxin present in the body-fluids of a diphtheric patient would be 
 of value in controlhng the antitoxin treatment of this infection. At 
 present the amount of antitoxin administered is regulated according to 
 the cUnical condition of the patient. Uffenlieimer has used a method 
 for determining the presence of toxin, con^sting in injecting intra- 
 peritoneally a 250-gram guinea-pig -nith 0.1 to 0.4 c.c. of the patient's 
 serum, diluted \^'ith 2 to 4 c.o. of salt solution. The pi'esence of a dis- 
 tinct doughy edema of the abdominal cavity after seventeen to twenty- 
 four hours indicates the presence of diphtheria toxin, an observation that 
 may be confirmed by making an autopsy at the end of forty-eight hours. 
 The diagnostic value of this method has not been adequately established: 
 it is doubtful if it yields any information other than is more readily 
 gained by making a good cultural examination of the patient, and it 
 does not aid in the estimation of the quantity of toxin, which is the result 
 most desired. 
 
Fig. 37. — Xokmal .\dbe.\.il Gl.\i\d gr a. Giinea-pig. 
 
 Fig. 3S. — Adrexal Gland of a Guixea-i-ig after Fatal Diphthehic 
 
 Intoxication. 
 
SPECIAL PROPERTIES OF THE PRINCIPAL TOXINS 115 
 
 Diphtheria toxins have been classified into three groups, depending 
 upon the degree of avidity for antitoxin they display, viz,, prototoxin, 
 deuterotoxin, and tritotoxin. Each of these toxin groups may, in 
 •whole or in part, be converted into toxoids. The prototoxin has a 
 greater affinity for the antitoxin than has the deuterotoxin, and the 
 deuterotoxin has a greater affinity for the antitoxin than has the trito- 
 toxin. The same relation is apparent with the three toxoids, which are 
 not poisonous, but which have the same power of combining with 
 antitoxin as have the toxins from which they take their origin. 
 
 In standardizing antitoxin, it is found in general that wth a perfectly 
 fresh toxin a certain amount of antitoxin will just neutrafize a definite 
 amount of toxin. If older toxin is used, it is found that the toxin has 
 lost about one-half its toxic power, but retains its initial power for 
 neutralizing antitoxin. Ehrlich explained this by showing that the 
 diphtheria toxin molecule is composed of two groups — one the carrier 
 of the toxic qualities, the toxophore group, which is quite labile; the 
 other uniting the whole molecule with antitoxin, being capable of neu- 
 tralizing it, and characterized bj' its stability. The toxophore group 
 being destroyed as in old toxin, the poison loses its toxic qualities, but 
 retains its power to bind antitoxin. This modified toxin or non-poisonous 
 diphtheria toxin has been designated by Ehrlich "diphtheria toxoid." 
 
 2. Tetanus Toxin. — Of all bacteria classed as true toxin producers, 
 none possesses greater toxicity than does the tetanus bacillus. The 
 number of organisms producing sufficient toxin to cause a fatal infection 
 may be so small that careful anaerobic cultures made from the local 
 lesion of infection, together with injection of the wound secretions into 
 white mice, may fail to disclose the presence .of tetanus bacilli. 
 
 According to Ehrlich, tetanus toxin is composed of two separate and 
 distinct substances — (1) Tetmiospasmin, a neurotoxin, which is very 
 labile and responsible for the severe symptoms of the infection; (2) 
 tetanolysin, a hemotoxin, which is more stable and destructive for ery- 
 throcytes. 
 
 Tetanus toxin is prepared by cultivating- the bacillus in bouillon 
 under strict anaerobic conchtions. Since tetanospasmin is so suscep- 
 tible to the influence of heat, age, and even light, the toxin is best pre- 
 served in a dry form. The standard of tetanus toxin consists of 100 
 minimal lethal doses of a precipitated and dried toxin, preserved at the 
 Hygienic Laboratory of the Public Health and Marine-Hospital Service. 
 
 If susceptible animals, such as mice or guinea-pigs, are injected sub- 
 cutaneously or intravenously with tetanus toxin, they begin to manifest 
 
116 INFECTION 
 
 symptoms after a certain period; tiiese are due to the action of tetano- 
 spasmin upon motor nerve-cells, and are characterized by hyper- 
 sensitiveness, clonic convulsions, and rigidity of the muscles. In man 
 the symptoms of tetanus are similar to those in the animal, the spasm 
 starting quite regularly in the muscles of the lower jaw. 
 
 Experiments by Wassermann and Takaki have demonstrated that 
 an especially close affinity exists between tetanus toxin and certain 
 structures, particularly that of the central nervous system. Most 
 writers agree that the toxin reaches these tissues largely by way of the 
 nerve-paths. 
 
 3. Botulism Toxin. — This poison is generated by the Bacillus bot- 
 ulinus, first isolated, by Van Ermengem in 1896, from a ham during an 
 epidemic of meat poisoning. It is the cause of a type of meat and sau- 
 sage poisoning called botulism, more frequent in those countries where 
 raw meat is eaten, and frequently confused with "ptomain poisoning." 
 
 The bacillus is a motile, spore-forming, apaerobic bacterium, which 
 grows at room temperature and causes marked gas formation in glucose 
 media. 
 
 The toxin is readily produced in anaerobic alkaline bouillon cultures. 
 It is quite labile. 
 
 Symptoms of botulism appear only after a definite period of incuba^ 
 tion, which varies from twenty-four to forty-eight hours. In contra- 
 distinction to the meat poisonings produced by other organisms, those 
 due to Bacillus botulinus may show few or no symptoms directly re- 
 ferable to the intestinal tract, the chief symptoms being due to toxic 
 interference with the cranial nerves: loss of accommodation, ptosis, 
 dilated pupils, aphonia, dysphagia, and hypersecretion of mucus from 
 the mouth and nose. 
 
 Guinea-pigs are quite susceptible, and may be infected by way of the 
 mouth. The symptoms of intoxication usually follow in twenty-four 
 hours, and are characterized by motor paralysis, dyspnea, and hyper- 
 secretion of mucus from the nose and mouth. 
 
 4. Dysentery Toxin. — The distinct types of dysentery bacilli vary 
 exceedingly in their powers to produce toxins, the strongest poisons 
 being produced with bacilli of the Shiga-Kr'use variety, less regularly 
 active ones, with bacilli of the Flexner type. 
 
 Investigations have shown quite conclusively that dysentery itself 
 is a true toxemia, its symptoms being referable to the absorption of the 
 toxins of the bacillus from the intestine. Flexner, who has studied this 
 subject with great care, believes it probable that most of the pathologic 
 
SPECIAL PROPERTIES OF THE PRINCIPAL TOXINS 117 
 
 lesions occurring in the intestinal canal are referable to the excretion of 
 dysentery toxin, rather than to the direct local action of the bacilh. 
 The action of the dysentery toxin upon animals is very characteristic, 
 and throws much light upon the disease in man. Intravenous injection 
 of the toxin in rabbits is followed by marked c^arrhea, rapid fall in tem- 
 perature, respiratory embarrassment, and terminal paralysis. Upon 
 autopsy the intestinal mucosa, especially that' of the cecum and colon, 
 shows marked inflammatory involvement, supporting Flexner's ob- 
 servation of the necrotic action of excreted toxin. 
 
 Dysenter}' bacilU also produce an endotoxin, and poisonous sub- 
 stances are easily obtained by extracting the bacilli themselves or by 
 filtration of properly prepared bouillon cultures. The toxin is fairly 
 stable, and well preserved under toluol in the refrigerator. 
 
 .5. Staphylolysin. — Two definite toxins have been isolated from 
 cultures of Staphylococcus pyogenes aureus And albus, one of which 
 exerts a destructive action on erythrocytes (hemotoxra), and the other 
 on leukocytes (leukocidin) . 
 
 An anti-hemotoxin that counteracts the effects of the toxin may be 
 produced experimentally, and in himian stapliylococcus infections the 
 demonstration of such antihemotoxic substances in the blood-serum 
 may be of aid in making the diagnosis of staphylococcus infections. 
 This antistaphylolysin may be found normally in small amounts in the 
 serum of man and horse, and when anti-hemotoxic tests -ndth hmnan 
 serum are made, a normal control should al-p'ays be included. Anti- 
 leukocidins have also been produced, but p,re not of practical im- 
 portance. 
 
 The hemotoxin is readily formed in cultures of staphylococci; 
 roughly, the amount produced depends upon the virulence of the culture. 
 In human cases of staphylococcus infections this toxin produces hemoly- 
 sis in vivo, and is partly responsible for the grave anemia that is fre- 
 quently present. 
 
 6. Streptolysin. — The grave systemic symptoms that so frequently 
 accompany slight streptococcus lesions arc strong indications that these 
 microorganisms produce a powerful diffusible poison, although extensive 
 researches into the nature of these poisons have not given us any clear 
 understanding of the subject. 
 
 Streptococci may yield soluble toxins that, when administered to 
 guinea-pigs, produce rapid collapse and death. While these toxins are 
 not comparable in potency to the soluble toxins of diphtheria and 
 tetanus, they have, nevertheless, been differentiated from the endo- 
 
118 
 
 INFECTION 
 
 toxins contained within the cell-bodies, and ha've been found to possess 
 less toxicity. 
 
 Beside these toxins, some streptococci produce a hemolysin which 
 may be conveniently observed by cultivatioti of the organisms upon 
 blood-agar plates. This hemotoxin is partlj' responsible for the san- 
 guineous character of a streptococcus exudate. 
 
 TOXINS OF THE mOHER PLANTS AND ANIMALS 
 Phytotoxins 
 
 As has previously been mentioned, the power of forming toxins is not 
 confined to bacteria alone. There is a class of higher plants and ani- 
 mals that produces characteristic poisons against which immunization 
 can be undertaken and an antitoxic serum obtained. Those of most 
 interest medically are pollen toxin and snake poison. 
 
 The most important plant toxins (phytotoxins) are ricin, abrin, 
 cfotin, and pollen. All possess more or less toxic qualities; the first 
 three either agglutinate or hemolyze the corpuscles of certain animals. 
 Antitoxic serums have been prepared that will neutralize the respective 
 toxins, and this factor constitutes the most iniportant evidence of their 
 toxin-like character. 
 
 General Properties. — These toxins resemMe proteins in many re- 
 spects. Jacoby, however, has placed them in the same class as bacterial 
 toxins and enzymes, i. e., large molecular colloids closely resembling the 
 proteins, but still not giving the usual protein reaction. More recent 
 work by Osborne, Mendel, and Harris,^ however, does not support 
 Jacoby's view. These observers found the toxic properties of ricin 
 inseparably associated with the coagulable albumin, and were able to 
 isolate it in such strength that TTnnr milligram was fatal per kilo of rabbit, 
 and solutions of 0.001 per cent, would agglutinate red corpuscles. 
 
 Relation to Immunity. — The phytotoxins, since they obey the same 
 laws as bacterial toxins, have been very serviceable in the study of im- 
 munity; they are more stable than the latter, and can be handled in 
 more exact and definite quantities. They have apparently the same 
 haptophore and toxophore structure as bacterial toxins; antitoxins are 
 readily produced by immunizing animals, and seem to be specific for the 
 toxins; in fact, Ehrlich made his earliest observations on the specificity 
 and quantitative factors in toxin-antitoxin immunity from a study of 
 these plant toxins. 
 
 'Amer. Jour. Physiol., 1905, 14, 259. 
 
TOXINS OF THE HIGHER PLANTS AND ANIMALS 119 
 
 The Toxin of Hay-fever. — Pollen toxin has been described by 
 Dunbar^ as the etiologic factor in the production of hay-fever. In all, 
 the pollen of 25 varieties of grass and seven varieties of plants have been 
 found capable of producing attacks of hay-fever in susceptible persons. 
 This susceptibihty to pollen intoxication is, for-tunately, limited, and the 
 sudden onset of an attack and the characteristic symptoms indicate an 
 anaphylactic reaction due to sensitization with pollen protein. Dunbar 
 has succeeded in producing a pollen antitoxin, which will be described 
 in the chapter on Passive Immunization; the reports of various 
 observers are, however, at variance in regard to its therapeutic value. 
 
 Zootoxins 
 
 The most important animal toxins (zootoxins) are those of the toad, 
 spider, snake, scorpion, and bee. The most striking characteristic of 
 these toxins is that an immunity against thepj can be established; in 
 this respect they resemble true toxins. All ai'e quite complex in struc- 
 ture and properties, and all are more or less heinotoxic. 
 
 Snake Venoms.^ — Medically, these are of particular interest. They 
 were first thoroughly investigated by S. Weir Mitchell (1860) and 
 Mitchell and Reichert (1883), and have aroused considerable attention 
 because of their similarity to bacterial toxins and the aid their study 
 has been in the elucidation of inununologic problems. 
 
 Properties of Venom. — In 1883 Mitchell and Reichert described two 
 poisonous proteins, constituents of venom, one of which seemed to be a 
 globulin and the other a proteose or " peptone. " Faust^ believes that 
 the poisons are not proteins, but glucosids free from nitrogen, and that 
 they belong to the saponin group of hemotoxie agents. It may be that 
 these glucosids are bound to proteins, and can be removed with the 
 globulin in fractional separation, or that they may come down, at least 
 in part, with the albumoses of the venom. 
 
 Various enzymes have been found in venoms; e.g., proteases (Flex- 
 ner and Noguchi) and hpases (Noguchi); thfe latter probably have a 
 definite relation to many of the effects of venom intoxication, especially 
 hemolysis and fatty degeneration of the tissues. 
 
 The poisons, as a rule, produce both local and severe general dis- 
 turbances, the rapidity of the onset of the syraptoms and the prognosis 
 
 'For full review of this subject see Glegg: Jourl Hygiene, 1904, 4; Liefman: 
 Zeit. f. Hygiene, 1904, 47, l.iS; Wolff-Eisner, Deut. med. Woch., 1906, 32, 138. 
 
 ^See Faust: "Die tierischen Gifte," Braunschweig, 1906; Noguchi: Carnegie 
 Institution Publications, 1909, No. Ill; Calmette: "Les venins," etc., Paris, 
 Masson, 1907. 
 
 » Arch. exp. Path. u. Pharm., 1907, 56, 236; 1911,,64, 244. 
 
120 
 
 INFECTION 
 
 in a given case depending largely on the situation of the bite. ^lost of 
 these poisons exert their effect primarily upon' the nervous and vascular 
 systems, besides exhibiting other toxic properties. 
 
 Nature of Vcnams. — All snake ^•enoms possess a hemolytic power, and 
 venom hemoh'sis is one of the most interestiag of biologic phenomena. 
 FLexner and Xoguchi ^ have distinguished and classified the various 
 elements as hemotoxins, hemagglutinins, neurotoxins, leukotoxins, and 
 endothehotoxins (hemorrhagin). The endotheholjtic action of the 
 toxins is sho'mi in the glomerular capillaries, where it causes hemorrhage 
 and hematuria (Pearce'^). 
 
 Cobra hemotoxin is especially characterized by its power of dissolv- 
 ing the corpuscles of certain species (man, dog, guinea-pig, rabbit) ■\nth- 
 out the presence of serum. The explanation of this interesting phenome- 
 non has excited extensive discussion. It is probable that the hemotoxin 
 is in the nature of an amboceptor (Flexner and Xoguchi), which is 
 activated, in the absence of serum, by complementing substances (chiefly 
 lecithin) present in the red cells, and in this manner producing hemolysis 
 of these cells. In s>-philis the quantity' of red-cell lecithin is probably 
 diminished after the primary stage, so that when using definite dilu- 
 tions of venom that are kno'wn to hemolyze a certain quantity of normal 
 erji;hrocytes, an absence of hemolysis of the red corpuscles of a given 
 patient would infer a decrease in complementing lecithin in these cor- 
 puscles and indicate the presence of sj-philis. The technic of this reac- 
 tion and its value as a diagnostic procedure 'will be discussed further on 
 under the head of Venom Hemolysis. 
 
 ENDOTOXINS 
 
 There are a large number of microorganisms, notablj' the cholera 
 spirillum, the typhoid bacillus, the pneumococcus, and other pyogenic 
 cocci, which, when cultivated and separated from the culture-fluid by 
 filtration, are found to be highly poisonous, whereas the filtrate itself 
 is practically devoid of toxicity except for t&e soluble hemotoxic sub- 
 stances. In other words, we are dealing with endotoxins, or poisoiis that 
 are not secreted into the medium in which the bqctcria are groiving, but are 
 contained more or less firmly uuthin the bacterial body, from ivMch they 
 are separable by some method of extraction or by autolysis, only after 
 death. 
 
 1 Jour. Exp. Med., 1903, 9, 257; Univ. of Peana. Med. Bull., 1902, 15, 345, 
 ' Jour. Exp. ;Med., 1909, 11, 532. 
 
ENDOTOXINS 121 
 
 Method of Obtaining Endotoxic Substances. — Endotoxic substances 
 are obtained from bacteria by thorough disintegration of the bodies. 
 This is accomphshed by various methods: (a) The substances may be 
 foimd in old cultures as a result of death and disintegration of numbers 
 of bacteria; (6) they may be obtained by suspe;pding the microorganisms 
 in distilled water and shaking in a machine, much as Wassermann and 
 Citron's artificial "aggressins" are prepared; (c) the bacteria may be 
 dried and ground to a fine powder, as in the preparation of Koch's 
 "bacillen emulsion" of tubercle bacilli; (rf) ]\lacFadyen freezes masses of 
 bacteria With hquid air, and then grinds them into a fine powder; (e) 
 Conradi recommends autolyzing the bacterial cells in non-nutrient 
 fluids; (/) Rosenau has studied tlie endotoxins of pneumococci obtained 
 by alternate freezing and thawing of suspensions in distilled water; (g) 
 Vaughan has demised a method of .growing niassive cultures on solid 
 media several square yards in extent, removing the bacteria -nith sterile 
 salt solution, and digesting the bacterial masses T\'ith an excess of a 2 
 per cent, solution of caustic alkaU in absolute, alcohol. 
 
 In the body, the endotoxins are probably hberated either by autolysis 
 or by disintegration through the bacteriolysins,- complements, or enzymes 
 of the body-cells and fluids. 
 
 Nature of Endotoxins. — Owing to their in^luble nature, endotoxins 
 in pure form and free from other products of bacterial activity, cannot 
 be obtained. As a result, their chemical nature and structure are -un- 
 known. Tuberculin, which was formerly believed to be an albumose, 
 may be produced in a protein-free medium ; it seems probable that this 
 substance is of the nature of a polypeptid, giving no biuret reaction, but 
 being destroyed by pepsin and trj'psin (Laevenstein and Pick ^). Whether 
 or not tubercuhn is an endotoxin hberated upon the disintegration of the 
 bacilli is unknown. Pick regards it as a secretory product closely 
 related to the true toxins. It is probable th'at some toxin is actually 
 secreted into the culture-medium, and that the major portion, which is 
 of a somewhat different nature, is intimately related to the constituents 
 of the bacterial cells. 
 
 There is little doubt but that endotoxic substances are highly poison- 
 pus, and that they are chiefly responsible for the characteristic symptoms 
 of diseases produced by the bacteria that contain them. Whether, 
 however, they are actually preformed definite and specific constituents 
 of bacteria, or merely the poisonous products of disintegration of the 
 bacterial proteins, is still undecided. It would appear that bacteria 
 1 Biochem. Zeit., 1911, 31, 142. 
 
122 INFECTION 
 
 produce and contain toxic substances. When; owing to the peculiar 
 structure of the bacterial protoplasm or natufe of the toxic substance 
 itself, the toxin can diffuse readily into a surrounding medium, the toxic 
 substance is known as a true, soluble, or extracellular toxin; when the 
 toxin enters into combination with the bacterial protoplasm, it becomes 
 known as an endotoxin. This union of toxin and bacterial protoplasm 
 may be so firm as to render the toxic substance inseparable from the 
 bacterial protein. The various toxic substances or toxins differ, there- 
 fore, according to their diffusibility through the membrane of bacterial 
 protoplasm, or their power of combining with these protein substances, 
 or both factors may be operative. 
 
 Satisfactory antitoxins for endotoxins have not been produced, and this 
 is an important point in differentiating between a true toxin or an endotoxin 
 of any particular microorganism. Animals immunized against endotoxin 
 develop substances in their serum that are bactericidal, bacteriotropic, 
 and agglutinative to the bacteria from which the poisons were derived, 
 but the serum itself is not antitoxic for the endotoxins. Therapeutic 
 serums for use against infections caused by the endotoxin class of 
 bacteria are largely bacteriolytic and bacteriotropic in action. The 
 endotoxins of some bacteria, and particularly those of streptococci, 
 seem to repel the leukocytes, or exert a negative chemotactic influence, 
 which may effectually retard or entirely prevent phagocytosis; in this 
 respect they resemble the aggressins of Bail. Immune serums owe a 
 portion, at least, of their therapeutic value to the power they possess of 
 overcoming this influence and facilitating phagocytosis. These serums, 
 however, have not proved of as much value as have the diphtheria and 
 tetanus antitoxins in the treatment of the respective infections men- 
 tioned, and have proved a check to the progress of serum therapy. It 
 is probable that the endotoxins are more specific for the various strains 
 of the same species than are the true toxins, as indicated by the results 
 of Cole in the treatment of pneumonia with an anti-pneumococcus 
 serum corresponding to the type of microorganism responsible for the 
 individual infection, as determined by a rapid method of diagnosis 
 previous to the administration of serum. 
 
 AGGRESSINS 
 
 In an attempt to explain certain observations of Koch to the effect 
 that when a tuberculous animal is injected intraperitoneally with a fresh 
 culture of tubercle bacilli it succumbs quickly to an acute attack of the 
 
AGGRESSINS 123 
 
 disease, the resulting exudate being composed almost exclusively of 
 lymphocytes, BaiH has advanced the hypothesis that bacteria may 
 secrete aggressins, or substances that aim to protect the microorganism 
 by either neutralizing the action of opsonins or directly repelling the 
 body-cells and preventing phagocytosis. Bail foqnd that if he removed a 
 tuberculous exudate, stcrihzed it, and injected it into healthy animals, 
 it had practically no effect. If tubercle bacilli were injected alone, 
 lesions would de'velop in the usual number of weeks; but if sterile 
 exudate and tubercle baciUi were injected together, death would follow 
 in about twenty-four hours, indicating that the exudate contained a 
 substance that acutely paralyzed the defensive forces of the animal, and 
 thus greatly increased the \'irulence of the bacilli. That this effect was 
 not the summation of endotoxins in the exudate plus li^'ing microorgan- 
 isms was sho'mi by Bail, who found that when large quantities of exudate 
 alone were injected no untoward effects resulted, whereas the injection 
 of a small amount of exudate, plus a sublethal dose of bacteria, would 
 regularly produce acute infection and death. Bail therefore concluded 
 that the exudate contained a substance that allowed the bacilU to become 
 more aggressive, and for this reason he called this hypothetic substance 
 "aggressin. " He assumes that in a tuberculous animal the tissues are 
 permeated with the aggressin, and that when fluid collects in the body- 
 ca^'ities after the injection of tubercle bacilh, this fluid contains large 
 quantities of aggressin. This prevents migration and collection of 
 poljTiuclear leukocytes, but not of lymphocjies, and hence allows the 
 bacilh to develop rapidly, producing acute symptoms. On the other 
 hand, when tubercle bacilU are injected into the peritoneal ca\'ity of 
 a healthy guinea-pig, poljTiuclear leukocytes, which engulf the bacilli 
 are attracted, thus inhibiting their rapid development, there being no 
 aggressin to prevent phagocytosis. 
 
 Similar results were obtained with othef microorganisms. Bail 
 inoculated cholera and typhoid bacilli into the pleural and peritoneal 
 canities of animals, and an acute local infection occurred. From the 
 exudates so produced he removed the bacteria by centrifugahzation, 
 and completed the sterihzation -with antiseptics or with heat at 44° C. 
 The clear fluid obtained was found to possess |)ut mild toxic properties, 
 and large amounts could be injected into animals of the same species 
 ^^^thout producing any marked effects; when, however, it was injected 
 into an animal together with a sublethal dos^ of the particular micro- 
 
 ' Wien. klin. Woch., 1905, 8, 14, 16, and 17; Berl. klin. Woch., 1905, 15; Zeit. 
 f. Hyg., 1905, vol. i, 3; Aich. f. Hyg., 1905, 52, 272, and 411. 
 
124 
 
 INFECTION 
 
 organism, an acute and fatal infection followed. Similar results were 
 secured with the bacilli of dysentery, chicken cholera, pneumonia, and 
 other diseases. 
 
 Bail's Classification of Bacteria. — Bail found that bacteria differed 
 in their power of forming aggressins; he therefore used this principle 
 in making a division of bacteria into three classes, according to their 
 disease-producing power, as dependent largely upon whether or not the 
 microorganism can produce an aggressin that is active against the pro- 
 tective forces of the host, particularly against opsonins and leukocytes. 
 
 1. Saprophytes, or those bacteria that, when injected even in large 
 doses, do not produce any characteristic disease. 
 
 2. True parasites, or those bacteria that, when injected even in the 
 smallest amounts, will produce disease and death. These are truly 
 virulent, and the number of bacteria increase so rapidly as to be demon- 
 strable in every drop of blood and in all the organs. Examples of true 
 parasites are the bacilli of anthrax and of chicken cholera, the tubercle 
 bacillus for guinea-pigs, and the bacilli of the group of hemorrhagic 
 septicemia for rabbits. 
 
 3. Half or partial parasites are those bacj^eria the infectious nature 
 of which depends upon the number of bacteria injected. The smaller 
 the number, the milder the symptoms, until a dose is reached below 
 which no disturbances are produced. Organisms of this class possess 
 some virulence and toxicity, examples being the Bacillus typhosus and 
 the Spirillum cholerse. 
 
 It is to be remembered, however, that these effects are but relative, 
 and dependent upon the organism, the species.of animal, and the mode of 
 infection. For example, the bacillus of anthrax is saprophytic for the 
 frog and hen unless the temperature of these animals is brought to the 
 body temperature of the human; a bacillus of the group of hemorrhagic 
 septicemia of rabbits is saprophytic for hunian beings, a half parasite 
 for the guinea-pig if injected subcutaneously, and a true parasite for the 
 same animal if injected intraperitoneally. 
 
 Nature of Aggressins. — The aggressins in inflammatory exudates 
 are presumably substances capable of paralyzing the protective agencies 
 of the body. Bail regards the aggressins as of the nature of endotoxins 
 liberated from the bacteria as a result of bacteriolysis, and believes that 
 they act by paralyzing the polynuclear leukocytes, thereby preventing 
 phagocytosis. In general, the production of these aggressins goes on 
 more actively the greater the resistance to the bacteria; they are pro- 
 duced in greater quantities during the struggle between the bacteria and 
 
AGGRESSINS 125 
 
 the body-cells, although they may be produced artificially in the test- 
 tube with large numbers of bacteria and a non-poisonous agent (serum 
 or distilled water) which can disintegrate the cells. In this manner 
 Wassermaim. and Citron have produced "artificial aggressins, "which act 
 in the same general manner as the "natural aggressins" of Bail. 
 
 By many the aggressins are regarded as enflotoxins, and while they 
 may possess the nature of endotoxic substances, it is to be remembered 
 that there is no definite relation between the poisonous qualities of the 
 aggressins and their power to increase the virijlence of an infection. It 
 is probable, as has been shown by Wassermann and Citron, that patho- 
 genic bacteria contain small amounts of natural aggreasin. This ag- 
 gressin may be regarded as a normal antibody of the bacterium against 
 the defensive forces of the body-cells of a host. During infection these 
 aggressins or antibodies are naturally greatly increased, as the bacteria 
 require more and more protection. Being contained to some extent 
 within the bacterial cells, the antibodies are somewhat similar to endo- 
 toxins: while endotoxins may be regarded as offensive agents of bacte- 
 ria, aggressins may be their defensive agents. This belief is in keeping 
 with the hypothesis of Welch ' and also of Walker ^, according to which 
 it may be presumed that bacteria, as living cells, when so placed that 
 they are exposed to the defensive forces of their host, are, under favor- 
 able conditions stimulated to produce reciprocal antibodies for their 
 protection, and to generate them in increasing amounts as may be neces- 
 sary. I regard aggressins as antibodies of this nature, and consider 
 that they are produced according to the conditions laid down in Pro- 
 fessor Welch's hypothesis. 
 
 Bail regards the aggressins as new substances," as already stated others 
 regard them as simple endotoxins; still others beheve them to be free 
 bacterial receptors, and that these receptors may combine with bacterio- 
 lytic amboceptors, producing, as it were, a deflection of the amboceptors, 
 so that the bacteria themselves are not attacked, and thus continue to 
 proliferate. The action of aggressins is not dependent upon the toxicity 
 9f the endotoxins, for the fluid containing them is devoid of toxic effects; 
 at most, therefore, if they are of the nature of receptors, they possess no 
 toxophorous portion. 
 
 Whatever aggressins may be, and we regard them as antibodies of 
 bacteria, just as bacteriolysins are antibodies of tissue-cells, they appear 
 to be especially directed against opsonins, neut-i^alizing these, paralyzing 
 leukocytes, and thus inhibiting or entirely preventing phagocytosis. 
 
 'Brit. Med. Jour., 1902, 2, 1105. ^joup. of Path., 1902, 8, 34. 
 
126 
 
 INFECTION 
 
 Anti-aggressins may be produced experimentally by gradually 
 immunizing animals with sterile exudates, and this immunity may be 
 transferred passively from one animal to the other by inoculation of its 
 immune serum. These anti-aggressins are quite specific, and neutralize 
 the aggressins in an exudate. 
 
 BACTERIAL PROTEINS 
 
 In practically all bacterial bodies, after removal of toxins and endo- 
 toxins, a certain proteid residue remains, which, when injected into 
 animals, is able to produce various grades of inflammatory reaction 
 leading to tissue necrosis and abscess formation. This substance was 
 first thoroughly studied by Buchner, who named it bacterial protein, and 
 regarded it as identical in all bacteria, and having no specific toxic action, 
 but characterized in general by its power of exerting a positive chemo- 
 tactic influence on leukocytes, and thereby favoring the formation of pus. 
 For example, in the development of an ordinary staphylococcus abscess 
 it is probable that the proteins of the cocci, aside from their toxins, aid 
 in producing tissue necrosis and in attractingJeukocj^es to the infected 
 area. Similarly, an extract of dead tubercle bacilli may produce a 
 tuberculoma or the tissue changes incident to tuberculosis, differing, 
 however, from true tubercle in that they do not contain living bacilli 
 and consequently are not infectious. When cultures of diphtheria 
 bacilli are filtered and the residue washed, it is found that extracts of the 
 bacterial substances or the bodies of the dead bacilh themselves are quite 
 free from the typical toxin; but the bacterial substances or the proteins 
 isolated from them, when injected into the subcutaneous tissues of 
 animals, are found to produce a strong inflammatory reaction and necro- 
 sis of the tissue-Cells. 
 
 Bacterial Split Proteins. — These have been studied extensively by 
 Vaughan and his coworkers, who have ascribed to them the chief role 
 in and a very important relation to the processes of infection and im- 
 munity. 
 
 Massive cultures of colon, typhoid, pneumonia, and diphtheria microorganisms 
 ate grown in special large tanks containing agar; anthrax is grown in Roux flasks, 
 and tubercle bacilli in glycerin beef-tea cultures. After removal of the growths the 
 bacterial cellular substances are washed once or twice with sterile salt solution by 
 decantation, and then repeatedly washed with alcohol; beginning with 50 per cent, 
 and increasing the strength to 95 per cent. The suljstanoe is then placed in large 
 Soxhelet's flasks and extracted first for one or two days with absolute alcohol, and 
 then for three or four days with ether. These extractions should be thorough in 
 order to remove all traces of fats and waxes. 
 
BACTERIAL PROTEI^'^ 127 
 
 After extraction the cellular substance is ground, fifst in porcelain, then in agate 
 mortars, and passed through the finest meshed sieves'to remo\e bits of agar. The 
 person grinding the cellular substance should wear a mask in order to protect him- 
 self against poisoning. Vaughan reports that, despite this precaution, several workers 
 have been acutely poisoned, especially with the typhoid bacillus. Of course, there 
 is no danger of infection, as the bacteria are killed during the treatment. If the 
 finely ground cellular substance, in the form of an impalpable powder, is kept in wide- 
 mouthed bottles in a dark place, it will retain its to.xicity for years. This powder 
 constitutes the bacterial protein substance, which maj! be spUt up by various means. 
 Vaughan found digestion with 2 per cent, caustic soda in absolute alcohol especially 
 satisfactory for extracting the poisonous group from bacterial or any other protein. 
 
 Nature of Bacterial Proteins. — ^^aughan and his coworkers regard 
 bacteria as essentially particulate, specific proteins. He has not been 
 able to demonstrate the presence of cellulose and carbohydrates; fats 
 and waxes that may be present are somewhat secondary and less essen- 
 tial constituents or stored food material. The* sum total of the work of 
 these observers would indicate that the greaterpart of bacteria are made 
 up of true proteins, especially nucleoproteins or glyconucleoproteins, 
 and although they may be simple in structure, they are chemically 
 complex — quite as much so as many of the tissues of the higher plants 
 and animals. 
 
 When bacterial cellular substances are split up mth mineral acids 
 or alkalis they yield ammonia, mono-amino- and diamino-nitrogen, one 
 or more carbohydrate groups, and humin sujDstances. These protein 
 substances are the same as those obtained by the hydrolysis of vegetable 
 and animal proteins. 
 
 By digestion with dilute acids or alkalis, especially the latter, in the 
 form of a 2 per cent, solution of sodium hydroxid in absolute alcohol, a 
 soluble spht product is obtained that resembles in some respects the 
 protamins, although they do not all give a satisfactory biuret reaction. 
 This product is highly toxic, but shows no specificity in its action, being 
 the same whether derived from pathogenic or from non-pathogenic bac- 
 teria, or from egg albumin or other protein substance. All that is defi- 
 nitely known regarding it is that it is toxic, protein in nature, but 
 simpler in structure than the complex proteins of the bacterial cells 
 themselves. 
 
 This soluble toxic portion as obtained in vitro is regarded by Vaughan 
 as the main factor in the production of the general symptoms of infection, 
 the special and distinctive lesions being due to the location of the in- 
 fection. During the infective process the body-cells produce an anti- 
 ferment which, when it reaches a certain concentration or power, begins 
 to spht the protein of the microorganism and new bacterial tissue, with 
 
128 INFECTION 
 
 the liberation of this toxic moiety, in a manner similar to the splitting 
 observed in vitro by dilute alkalis or acids. 
 
 The insoluble and non-poisonous portion of the cellular proteins 
 shows most of the color reactions for protein^ and contains all the car- 
 bohydrate of the unspHt molecule and most of the phosphorus. 
 
 Action of Bacterial Proteins. — ^The effects produced by bacterial 
 proteins are not specific; the protein substance of non-pathogenic 
 bacteria and, indeed, many proteins derived from vegetable and animal 
 sources, have equally marked pyogenic properties. All foreign proteins 
 introduced into the circulation of animals are more or less toxic, and the 
 toxic effects of all bacterial proteins are, in general, quite similar and 
 non-specific. 
 
 Bacterial protein substances may be responsible for certain minor 
 anaphylactic reactions, as has been observed occasionally in the ad- 
 ministration of ordinary bacterial vaccines. They may bear an im- 
 portant relation to the development of the state of hypersensitiveness 
 of a tuberculous person in the course of a series of tuberculin injections. 
 
 Theory of Vaughan. — According to Vaughan and his coworkers, all 
 true proteins contain a common and non-specific poisonous group. 
 This group may be regarded as the central or key-stone portion of every 
 protein molecule, with secondary and possibly tertiary subgroups, in 
 which the specific property of different proteins is inherent. When the 
 main or primary group is detached from its subsidiary group, it mani- 
 fests its poisonous action by the avidity with which it attacks the second- 
 ary group of other proteins. These are detached from their normal 
 positions, and consequently deprive the living protein of its power of 
 functionating normally. When proteins are split, the chemical nucleus 
 or non-specific toxic portion is more or less completely set free, and its 
 toxicity varies according to the thoroughness with which the secondary 
 groups have been removed. 
 
 The pathogenicity of a bacterium is determined not by its capability 
 of forming a poison, but by the ability it possesses to grow and multiply 
 in the animal body. When, during an infection, a pathogenic micro- 
 organism reaches the deeper tissues, it is not immediately killed by the 
 defensive ferments of the host, but continues to grow and multiply, 
 throwing out a ferment that feeds upon the native proteins of the body- 
 cells, tearing them down and building up a specific bacterial protein 
 that may select a certain point of predilection in which it is most prone 
 to accumulate. Thus the typhoid bacillus accumulates in the adenoid 
 tissue of Peyer's patches on the intestine, the spleen, and the mesenteric 
 
PTOMAINS 129 
 
 glands; the pneumococcus tends to lodge in the lungs; the smallpox 
 virus selects the skin, etc. 
 
 The bacterial toxins and -viruses, as, e. g., diphtheria toxin and the 
 virus of smallpox, are regarded as ferments of protein nature, capable 
 of attacking native body protein and building-up a specific foreign pro- 
 tein. This foreign bacterial protein is formed during the period of 
 incubation of disease when there is no effective resistance on the part 
 of the body-cells to its growth and multiplication. During this time 
 the infected person is not ill, so that the foreign protein in itself cannot 
 be toxic, and the body-cells are busy preparing and elaborating a new 
 and specific ferment that will digest and destroy the foreign protein.- 
 When this new ferment becomes active, the first symptoms of disease 
 appear, and the active stage of the disease marks the period over which 
 the parenteral digestion of the foreign protei» extends. These specific 
 ferments split up the foreign protein and liberate the toxic portion or the 
 protein poison; this poison is not a toxin and is not specific, but occurs 
 commonly in all proteins. 
 
 The characteristic symptoms and lesions caused by the various 
 infectious processes are determined largely by the location of the foreign 
 protein. The poison elaborated is the same in all infectious diseases, 
 and it is the location of the infection, rather than the exact nature of the 
 infecting agent, which gives rise to the more or less characteristic symp- 
 toms and lesions of the several infectious diseases. 
 
 Death may be produced by the too rapid breaking-up of the foreign 
 protein, and the consequent liberation of a fatal dose of the protein 
 poison, or it may result from a lesion induced'by the products of this 
 disruption, such as perforation of the intestine and hemorrhage in 
 typhoid fever, or it may follow from chronic intoxication and consequent 
 exhaustion. If recovery takes place, the individual enjoys an immunity 
 of variable duration, owing to the presence of specific ferments capable 
 of destroying the particular substrata if infection should occur. 
 
 It is this power of body-cells, when permeated by a foreign protein, 
 to elaborate a specific antiferment by which the protein is destroyed, 
 that, in the opinion of Vaughan, forms the basis of a correct understand- 
 ing of infection and immunity. 
 
 PTOMAINS 
 It was at one time believed that the symptoms of many diseases 
 were due to the absorption of soluble basic nitrogenous substances pro- 
 
130 INFECTION 
 
 duced by bacterial action upon various albumens, these toxic, alkaloid- 
 like substances being known as ptomains. It was soon found, however, 
 that the ptomains produced by pathogenic bacteria were insufficient of 
 themselves to cause the symptoms and lesions characteristic of the re- 
 spective microorganisms; that they were in general less toxic than the 
 cultures themselves; that the majority of ptomains are not very poison- 
 ous; and that they are not specific, since equally potent ptomains are 
 produced by non-pathogenic bacteria. This lack of specificity is in 
 sharp contrast to the toxins. No matter upon what medium a true 
 toxin producer is grown, the toxin is qualitatively the same, whereas the 
 nature and toxicity of ptomains depend upon the microorganism, the 
 culture-medium used, the duration of growth, and the quantity of oxygen 
 furnished. The same microorganism, when grown on different media 
 or under different conditions, may produce totally different ptomains. 
 
 Ptomains may, however, produce disease, and even death, when they 
 are ingested with food that has undergone bacterial decomposition. In 
 most instances of meat-poisoning, however, which are frequently as- 
 cribed to the presence of ptomains, a specific microorganism, the 
 Bacillus botulinus, or a member of the Bacillus enteritidis group of 
 Gartner, is usually responsible. The commonest sources of ptomain 
 poisoning are improperly preserved meats, fish, sausages, cheese, ice- 
 cream, and milk. This subject received full consideration in Vaughan 
 and Novy's "Cellular Toxins." 
 
 Besides occurring in food-poisoning, ptomains may be formed as the 
 result of putrefactive processes going on in abscesses, gangrenous areas, 
 and within the gastro-intestinal canal, and enough of these may be 
 absorbed to produce symptoms of intoxication. Under these condi- 
 tions it is possible for bacteria to produce ptomains that may be absorbed 
 and produce symptoms of intoxication without the bacteria themselves 
 actually gaining entrance to the tissues, and therefore not constituting, 
 according to our definition, a true infection. Pernicious anemia, 
 chlorosis, and allied conditions have been ascribed to the absorption of 
 such ptomains from the intestinal canal. Obviously it is difficult or im- 
 possible to always differentiate between bacterial toxins and bacterial 
 ptomains, or the products of protein decomposition dependent upon 
 bacterial activity, and we can but admit the possibility of the produc- 
 tion and absorption of both bacterial toxins and ptomains under certain 
 pathologic conditions. Most ptomains probably are produced as the re- 
 sult of decomposition of the dead protein medium upon which the bacteria 
 grow, and to a lesser extent by the destruction of the bacterial cells them- 
 
MECHANICAL ACTION OF BACTERIA 131 
 
 selves. It is extremely doubtful if ptomains are produced in important 
 quantities by pathogenic bacteria infecting living tissues. 
 
 MECHANICAL ACTION OF BACTERIA 
 
 In former years the theory as to the influence of mechanical blocking 
 of vessels with masses of bacteria was regarded with much favor in the 
 etiology of certain infections, particularly anthrax. At the present 
 time this factor has not the same importance, for while it is true that 
 bacterial emboli may occasion harm by blocking important vessels, 
 further researches have shown that it is doubtful if any pathogenic 
 microorganisms are totally devoid of toxic action, and that their toxins 
 are largely responsible for the tissue changes and symptoms of infections. 
 
 It can readily be understood that emboli of microorganisms may 
 produce metastatic lesions; thus when staphylococci are injected into 
 the ear vein of a rabbit they produce abscesses in the kidney and heart, 
 and masses of bacteria from an ulcerative endocarditis, when carried to 
 different portions of the body, will cause abscess formation; but the 
 question in hand, however, deals with the effects of mechanical blocking 
 itself. 
 
 Investigations with anthrax bacilli have shown that they are re- 
 markably free from soluble toxins and endotpxins, although the local 
 lesion develops so rapidly and becomes so quickly necrotic as to suggest 
 very strongly the action of some local toxic substance. Cases of human 
 anthrax seldom prove fatal if the lesion is removed and the blood-stream 
 remains free from the bacilli. Vaughan has sho^vn that anthrax protein 
 possesses toxic qualities, and since the majority of fatal cases of anthrax 
 show enormous numbers of bacilli in the blood-stream and internal 
 organs, it may be that this bacteremia produces an accumulation of 
 toxins which is greatly augmented when the body-cells of the host have 
 produced an antiferment that splits up the protein of the bacilli, the 
 combined toxic substances being responsible for the severe symptoms 
 and death. 
 
 With protozoan disease, the possibility of serious symptoms follow- 
 ing blocking of vessels is far greater, and, indeed, the cerebral symp- 
 toms of malignant malaria and sleeping sickness may be due in part 
 to the blocking of small, but physiologically important, vessels with 
 masses of plasmodia and Trypanosoma gambiensi, together with the ab- 
 sorption of toxic agents and the products of disintegration. Thus Bass, 
 who has successfully cultivated the malarial piasmodium outside of the 
 
132 
 
 INFECTION 
 
 body, believes that the parasites, after attaining sufficient size, lodge in 
 the capillaries of the body, especially where the blood-current is weakest, 
 and where shght obstruction occurs as the result of the protruding in- 
 ward of nuclei of the endotheUal cells. Hero they remain and develop 
 until segmentation takes place. In the meantime other red corpuscles 
 are forced against them, and if the opening'in the infected cell is in a 
 favorable location, one or more merozoites pass directly into another 
 cell; if it is not, the merozoites are discharged into the blood-stream and 
 are speedily killed. 
 
 INFECTION WITH ANIMAL PARASITES 
 
 Infection with animal parasites is simila*r in many respects to in- 
 fection with bacteria. Owing to the difficulty of isolating and culti- 
 vating these parasites in vitro, our knowledge of their toxic properties 
 is somewhat meager. Most attention has been given to a study of 
 their life history and the modes of transmissibn. 
 
 Modes of Infection. — Primary infection with animal parasites is 
 often facilitated by, or in some instances only* rendered possible through, 
 the intervention of special carriers, usually various species of the Ar- 
 thropoda. Thus \ve now know that malaria is transmitted through the 
 bite of infected mosquitos; African relapsipg fever and Texas cattle 
 fever, through the bite of certain infected ticks; trypanosomiasis, 
 through biting flies. The ova of various intestinal parasites may re- 
 quire residence in certain of the lower animals before they can infect 
 man. 
 
 Infection may occur along the same roijtes as bacterial infection, 
 and is governed in general by the same factors of local selection, tissue 
 susceptibility, etc. Biting insects usually deposit the parasite directly 
 in the subcutaneous tissues or in the circulatory fluids. Abrasion of 
 the epithelium may be necessary in order to produce infection with 
 Treponema pallidum, as in the majoritj' of the bacterial infections. 
 The ova or larva of other parasites may he swallowed or find lodgment 
 in the upper or lower air-passages or accessory sinuses. 
 
 It would appear that our natural defenses against infection with 
 aiiimal parasites are much weaker than thosQ against bacteria; this is 
 probably due to the greater resistance offefed by animal parasites to 
 such physical dcstructi\'e influences of the host, as the acidity and ger- 
 micidal activity of the secretions, temperature, etc., as well as to a 
 general lack of natural antibodies in the body-fluids of the host, and 
 inability of leukocytes and other phagocytic cells to deal successfully 
 
INFECTION WITH ANIMAL PARASITES 133 
 
 with the invaders. That natural inxniunitj' against infection with 
 certain animal parasites may exist is shown by the prevalence of certain 
 infections among man, and their absence among lower animals, or vice 
 versA. 
 
 The aggressiveness of animal parasites is in general probably even 
 greater than that of most bacteria, and a more-or less extensive infection 
 apparently occurs in all cases in which the parasite had made successful 
 invasion, some multipljang in the blood-stream (malaria, relapsing 
 fever, trypanosomiasis, Texas fever, filariasis), others in the lymph- 
 stream (filariasis) , and others in the tissues (syphilis, trichiniasis, 
 amebiasis) without much opposition on the part of the host. \\'hether 
 these factors are due to the aggressive forces of ttie parasites which neutra- 
 lize the defenses of the host, or whether thej' are due to the hardiness of 
 the parasites and a lack of defense on the part of the host, is not Icnown, 
 but probably the latter is generally the case. 
 
 As with bacteria, animal parasites show a weU-marked selective 
 affinity for certain tissues, as the malarial Plasmodium for red blood- 
 corpuscles, trypanosomes and spirochetes for blood plasma, trichina 
 for voluntary muscle, various parasites for the" intestinal canal and even 
 for certain portions of the intestinal tract, others for the lung, etc. 
 
 Production of Disease. — Comparatively Mttle is known regarchng 
 the formation of toxic products on the part of the animal parasite. 
 Some, as, e. g., the Treponema pallidum and spirochete of relapsing 
 fever, probably cause disease largely through the production of toxins, 
 especially of the intracellular variety. The chill, fever, and sweat of 
 malaria suggest the liberation of toxic products coincident, or nearly so, 
 with segmentation and rupture of the plasmodjum. The late symptoms 
 of sleeping sickness and the whole course of relapsing fever are strikingly 
 similar to the bacterial toxemias. The metabolic products of all animal 
 parasites are probably injurious in some manner and to some degree. 
 The pathogenicity of others is due, in part at least, to mechanical 
 blocking of vessels, as with the filaria, trypanosomes, and malarial 
 organisms; others (hookworm) abstract blood or consume food material 
 in the intestine, as the intestinal parasites; and others, as migrating 
 foreign particles with irritating secretions, pr^iduce local inflammatory 
 changes. 
 
 Nevertheless, we know comparatively little of the offensive factors, 
 and stiU less of the immunologic defensive factors, operative during the 
 course of infections with animal parasites. With the development of a 
 technic for the cultivation of animal parasites in vitro similar to that 
 
134 INFECTION 
 
 devised for the ameba, certain trypanosomes, spirochetes, and malarial 
 Plasmodia, we will be enabled to study the products of their growth or 
 of disintegration, and the immunologic agencies concerned in infection 
 and recovery; this offers a very important and'fruitful field for research. 
 
 THE COURSE OF INFECTION 
 
 In conclusion, we may briefly consider the results of infection or the 
 general symptoms following bacterial growth and the manner in which 
 these are produced. 
 
 The Stages of Infection. — Practically all infections pass through the 
 following stages : 
 
 1. The 'period of incubation, which begins at the time of infection and 
 ends with the development of the earliest g.eneral symptoms, during 
 which time the invading bacteria are multiplying in the tissues of the 
 host. During this stage no symptoms, or only those of a purely local 
 nature, are present. This period varies considerably in different in- 
 fections, and to a lesser extent in different individuals having the same 
 infection. Some bacteria may be so virulent as to overwhelm the body- 
 cells, thus making the period of incubation very short or entirely un- 
 observable. On the other hand, as, e. g., in rabies, the period may be of 
 several weeks' and, indeed, of several months' duration. In tuberculosis 
 there is usually a primary local growth, which develops so gradually 
 and the toxins are so slowly diffused that it is difficult or, indeed, im- 
 possible, to estimate the length of the period of incubation. 
 
 According to Vaughan, during the period of incubation the bacteria 
 or their toxins or the viruses are actively engaged in changing the natural 
 body proteins into new and specific bacterial proteins, ^nd since this 
 stage is constructive, there are no symptoms and the host is not ill. 
 Even with the experimental administration of the most poisonous of 
 toxins a definite period of incubation is usually to be observed, which 
 cannot be reduced below a certain minimum, independent of the size 
 of the dose injected; in general, however, a large dose of bacteria or of 
 toxin is likely to be followed by a shorter period of incubation than if a 
 smaller dose were administered. 
 
 In a given case the period of incubation, may be determined by 
 several factors: 
 
 (a) The number of bacteria gaining entrance, and especially their 
 toxicity and aggressiveness. The primary factors are the degree of 
 toxicity and the amount of toxic substances produced and absorbed. 
 
THE COURSE OF INFECTION 135 
 
 (6) Upon the site of infection. Thus tbfe introduction of rabies 
 virus or of tetanus bacilli into the tissues of the face or into a deep wound 
 is likely to be followed by a shorter period of incubation than when 
 these are introduced into the foot or in superficial wounds. 
 
 (c) Upon the degree of resistance offered by the host. For instance, 
 one individual may contain more antitoxin or bacteriolysin for a certain 
 bacterium than another, and consequently a longer period of incubation 
 is required, during which these substances are neutralized and an excess 
 of toxic bacterial substance is produced. In fact, these may offer such 
 resistance to the bacterium that the process of infection is inhibited, or 
 but slight and evanescent disturbances appear. 
 
 (d) Upon the general susceptibility of the host and the route of in- 
 vasion. 
 
 2. The period of prodromal symptoms, characterized by systemic 
 disturbances of a relatively mild t3rpe, due to" diffusion of the bacteria 
 and their products into the general circulation and their wide-spread 
 effect upon the body-cells in general. If the bacteria select a special 
 tissue or organ for attack, as the typhoid bacillus for lymphoid tissue, 
 and pneumococci for the lungs, definite symptoms develop later, their 
 nature depending on the special tissue or organ involved. The pro- 
 dromata, however, are more marked, and indicate a wide-spread but 
 mild action upon the body-cells in general. Yaughan believes that these 
 symptoms mark the time when sufficient proteolytic ferments have been 
 generated by the body-cells against the new bacterial protein of the 
 invading bacteria to attack the latter, splitting the molecule and liberat- 
 ing a toxic moiety responsible for the general symptoms of intoxication. 
 
 3. The period of fastigium, or of high /ezier,. during which the disease 
 is at the height of its severity. Special and distinctive symptoms and 
 lesions, according to the organ or organs especi'ally involved, are present; 
 the struggle between the offensive and defensive forces of parasite and 
 host is at its height, with remissions or exacerbations dependent upon 
 the supremacy of any one of these, and the general stability of the body- 
 cells in withstanding the wear and tear. During this time the protect- 
 ive proteolytic ferments of Vaughan are mo^ active in disrupting the 
 newly formed bacterial protein, with the liberation of the toxic portion. 
 This process may be so active as to overwhelm the host with the toxic 
 split product, or lead to grave secondary lesions, such as extensive necro- 
 sis, perforation of a viscus, or hemorrhage. 
 
 4. The period of decline, during which the patient is gradually over- 
 coming the infection, and amelioration of the Symptoms takes place. 
 
136 
 
 INFECTION 
 
 5. The period of convalescence is now ushered in, during which the 
 host gradually overcomes the effects of disease and returns to health. 
 
 During this entire time the emaciation and tissue exhaustion leave 
 the patient quite weak, and undue exertion, errors in diet, or reinfection 
 may lead to a relapse, or a reactivation of the disease. Certain sequelcs 
 ox morbid conditions may follow a disease, and are due to the same orig- 
 inal cause; e. g., in typhoid fever the development of cholecystitis; at 
 any time during the disease complications, or morbid conditions due 
 to some other microparasite, as the development of pneumonia during 
 the course of typhoid fever, may seriously jeopardize the life of the 
 patient. 
 
 Grades of Infection. — ^According to the maimer in which a parasite 
 and its products act upon the cell of a host and the power of the host to 
 neutralize or overcome these the following various grades and types of 
 infection are encountered: 
 
 (a) Malignant or fulminating infection, during which there is no 
 fever, but, on the contrary, a subnormal temperature, with rapid prostra- 
 tion of the patient and death within a brief period. The cells of the body 
 are overwhelmed and paralyzed by the toxic substances; metabolism 
 is arrested, and the heat centers are exhausted with the fall of the tem- 
 perature, an indication of the intense and overwhelming intoxication. 
 
 (&) Acute infection, which is the ordinary type of an infectious disease 
 as previously described, and having a definite incubation period, prodro- 
 mal symptoms, fastigium, defervescence, and convalescence. 
 
 (c) Chronic infection, or a prolonged process characterized by in- 
 sidious onset and symptoms of relatively mild or moderate severity, 
 and terminating either in death, after months or years, or in gradual 
 recovery. A chronic infection may be remittent, as may be observed 
 in the rheumatic group of disorders; during the remission with defer- 
 vescence the infecting bacterium is not totally destroyed, and subse- 
 quently lights up, producing an acute exacerbation of the disease. 
 
 In chronic infections it would appear that the parasites develop and 
 produce their toxins slowly, or that these are slowly and imperfectly 
 absorbed on account of the sluggish local circulation and the presence 
 of scar tissue. The body-cells become accustomed, as it were, to these 
 toxic products, and produce only sufficient antibodies to effect their imme- 
 diate neutralization. The bacteria themselves 'become distinctly resistant 
 to the action of the tissues and the defensive forces, and there is neither 
 the same degree of intoxication nor reaction as are seen in acute infec- 
 tions. Gradually, however, the body-cells become exhausted, and 
 
THE COURSE OF INFECTION 137 
 
 unless the cells are aroused and stimulated, by judicious administration 
 of bacterial vaccines, to produce an oversupply of antibodies, the host 
 shows progressive emaciation and weakness. 
 
 The Systemic Reaction to Infection. — It is not within the scope of 
 this book to discuss the various symptoms of infection, and we will 
 limit ourselves to a brief discussion of the mo$t important, namely, the 
 febrile reaction. 
 
 According to Vaughan, the fever of infection is due mainly to the 
 toxic split protein resulting from the action of the protective proteolytic 
 ferments upon the new bacterial protein. This observer and his asso- 
 ciates were able, by the injection of multiple doses of protein derived 
 not only from the typhoid bacillus but from various vegetable and 
 animal proteins, to reproduce experimentally in rabbits a febrile reaction 
 known as protein fever, and which is not unlike typhoid fever. This 
 induced fever may continue for weeks, and is accompanied by increased 
 nitrogen elimination and gradual wasting; it is followed by immunity, 
 and the serum of immunized animals digests the homologous protein 
 in vitro. As has repeatedly been stated, Vaughan regards the split toxic 
 product as the cause of the general symptoms- of infection, the special 
 and characteristic symptoms and lesions of the different diseases de- 
 pending upon the site where the bacterial proteins have been deposited, 
 and where they are, in large part at least, digested. 
 
 In addition to this toxic action of split protein, fever may be due — 
 (a) to the unusual activity of the cells supplying the proteolytic enzymes; 
 and (6) to the cleavage of the foreign bacterial protein by these ferments. 
 
 The fever of infection, therefore, is caused by the toxic action of 
 pathogenic parasites, both bacterial and animal forms, upon the body- 
 cells and heat-regulating centers. It must be= regarded by itself as a 
 beneficent phenomenon, inasmuch as it marks a reaction of the body- 
 cells to toxic agents, for the purpose of neutralizing these and, by the 
 development of antibodies, ridding the body of foreign substances. 
 
Part III 
 
 CHAPTER VIII 
 
 IMMUNITY.— THEORIES OF IMMUNITY 
 
 In the preceding chapter on the mechanism of infection and the pro- 
 duction of an infectious disease the statement was frequently made that 
 the microparasites of disease are required to overcome the defensive 
 forces of a host which are ever on guard to protect the organism against 
 parasitic invasion and infection. Certain of these defenses are natural 
 to the host, and in a great majority of instances suffice to protect the 
 body against invasion and infection with bacteria, animal parasites, and 
 various inanimate and injurious substances. When, however, these 
 natural defenses are broken down and infection has occurred, the body- 
 cells are not usually rendered powerless and completely overcome, for 
 the products of infection serve as a stimulus to the body-cells, calling 
 forth renewed cellular activity and the production of various specific 
 defensive weapons, termed antibodies, which maintain an incessant 
 struggle against the invading pathogenic agents in an effort to rid the 
 body of them and to neutralize their products. 
 
 Just as microparasites have various offensive weapons, consisting 
 chiefly of their toxins, so, in like manner, the defensive forces of the host 
 are numerous and even more complex. If the toxin of a microorganism 
 is its chief pathogenic weapon, as, e. g., the soluble and extracellular 
 toxin of the diphtheria or the tetanus bacillus, then the body-cells pro- 
 duce an antitoxin as their chief defensive force. If the offensive weapon 
 is largely in the nature of an endotoxin, as, for example, the endotoxins 
 of the typhoid or the cholera bacillus, then a chief antibody is in the 
 nature of a bacteriolysin, which endeavors to dissolve the bacillus in an 
 effort, as it were, to attack the enemy in his stronghold. In other in- 
 fections, especially those due to the pyogenic cocci, certain of the body- 
 cells, and chiefly the polynuclear leukoc3-tes, are observed in the tissues 
 to have engulfed the invaders bodily (phagocytes) in an endeavor to 
 digest them and neutralize their products. In addition to these chief 
 antibodies, there are others that appear to aid them in their work. 
 
 That one attack of many of the infectious diseases may protect the 
 individual against subsequent attacks, or at least render subsequent 
 attacks mild and harmless, is well known. In India and the East for 
 
 138 
 
THEORIES OP IMMTJNIjFY 139 
 
 centuries practical advantage has been taken of this observation in the 
 management of smallpox. In order to protect persons against a severe 
 attack of variola they were deliberately brought in contact with a 
 person suffering with a particularly mild foiMi, in the hope that, by 
 inducing a mild attack of short duration, they would thus obtain pro- 
 tection against the severe, disfiguring, and fatal form of the disease. 
 
 The practice, however, was not without danger to the individual and 
 to the public at large, as the induced disease would at times become 
 malignant, and constitute a focus of infection for an entire community, 
 When Edward Jenner discovered that inoculation with cowpox virus 
 could not produce smallpox, but would, nevertheless, stimulate the pro- 
 duction of specific antibodies and confer immunity against it, an enor- 
 mous forward stride was taken that has since proved a priceless boon in 
 helping to rid the world of the dreadful scourge of smallpox. 
 
 The object of all these procedures has been to secure a resistance or 
 immunity to smallpox, either by inducing a mfid form of the disease or 
 by protecting the individual by means of inoculation with a virus that 
 has been so changed in its passage through a cow as to render it unable 
 to produce smallpox, but yet is capable of stimulating the body-cells to 
 produce antibodies that will neutralize the effect of the true virus. This 
 induced resistance to a given infection constitutes immunity or resist- 
 ance, and since the body was purposely inoculated and the body-cells 
 rendered active in producing the antibodies, this form of resistance is 
 known as active acquired immunity. 
 
 Many persons recover from an infection that may have been unusu- 
 ally severe not because the infecting agent became exhausted or died for 
 want of pabulum, but because it had been gradually worsted in the 
 battle with the defensive forces of the host. In many such instances 
 the host is now immune to this infection for a longer or shorter time, 
 because the body-cells have been so profoundly impressed that they con- 
 tinue generating defensive weapons or antibodies for some time after 
 the last vestige of the infecting agent has disappeared. Or, on the other 
 hand, the quantity of antibodies may be so grgat that they may persist 
 for varying periods of time, even for the remainder of life, ever on guard, 
 and ready to overwhelm their specific enemy should it ever again gain 
 access to the tissues. 
 
 Here, then, arises the question concerning the mechanism of re- 
 covery from an infection, and since this is so intimately concerned with 
 the general subject of resistance to disease, it. is considered under the 
 general head of immunity. 
 
140 IMMUNITY. THEORIES OF IlitMUNITY 
 
 Even superficial observation shows that not all persons are equally 
 susceptible to a given disease, and during the cpurse of epidemics it will 
 be seen that some individuals, although freely exposed, escape infection. 
 Certain species of animals may likewise display a uniform resistance to 
 an infection that will readily enough attack another species of the same 
 general family. It has been demonstrated experimentally that a certain 
 pathogenic bacterium will produce a severe infection in one species of 
 animals and not in another. It may frequently be noticed that even 
 though an infection occurs, it is readily overcome by the natural resources 
 of the host, the latter escaping with slight or no symptoms of disease. 
 In other words, certain persons and animals apparently possess a natural 
 resistance or immunity to disease, which may be general, non-specific, 
 or due to specific antibodies, this type of immunity being frequently 
 relative and seldom absolute. 
 
 Definition. — Immunity, therefore, in a broad sense, is the effective 
 resistance of the organism against any deleterious influence: in the usual 
 and more restricted meaning the term is applied to resistance against in- 
 fection, with vegetable and animal parasites and' their products, which are 
 pathogenic for other animals of the same or of different species. 
 
 It should be remembered that immunity means not only the ability 
 to resist an infection or successful invasion of the tissues by micropara- 
 sites, but also the continual resistance offered' as long as the infection 
 lasts; that is, immunity implies not only resistance to the onset of in- 
 fection, but also to the course and progress of the resulting infectious 
 disease. The science of immunity has, therefore, for its object the study 
 of the mechanism of resistance to and recovery from an infection. 
 
 HISTORIC 
 
 The development of the science of immunity forms one of the most 
 interesting chapters in the history of medicine. Even in ancient history 
 we can trace the conception of our modern ideas on immunization. 
 
 Hippocrates taught that the factor that causes a disease is also 
 capable of curing it — practically the same theory as the more modem 
 homeopathic doctrine of " similia similibus curantur." Pliny the Elder 
 recommended the livers of mad dogs as a cute for hydrophobia, thus 
 coming very near to the basis of the Pasteur discovery. As was pointed 
 out by Elizabeth Eraser, the same idea is expounded in the mythologic 
 tale of Telephus, who cured his wound by applying rust from the sword 
 which inflicted it, and in the story of MithricJLates, King of Pontus (B. 
 
HISTORIC 141 
 
 C. 120), who immunized himself against poisons by drinking the blood 
 of ducks that had been treated with the corresponding toxic substances. 
 
 Immunization against various venoms has been practised by many 
 of the savage tribes of Africa since earliest tinies. Mention has previ- 
 ously been made of the method of preventive Inoculation against small- 
 pox practised in Asia and other Oriental countries for several centuries 
 by exposing the subjects to mild cases of the disease. 
 
 A very definite step in progress must ever be associated with the 
 name of Edward Jenner, who first demonstrated experimentally, and 
 in a scientific manner, that cowpox conveyed to man protected him 
 against smallpox. Jenner was not the first person to make this observa- 
 tion, as many of the Gloucestershire farmers' knew that cowpox pro- 
 tected them against smallpox; nor was he the first deliberately to in- 
 oculate persons with cowpox virus, as this method had been practised 
 sporadically before his time. Jenner was, however, the first medical 
 man to give the matter serious thought and consideration, and to test the 
 method as thoroughly and scientifically as it was possible to do at that 
 period. Thus he inoculated vnth smallpox virus those whom he had 
 previously vaccinated with cowpox virus, and found them immune to 
 smallpox. These experiments were courageously repeated, until a great 
 truth was established, which has resulted in almost completely eradi- 
 cating the disease from those countries or combiunities where vaccina- 
 tion is thoroughly carried out. As was to be expected, Jenner met with 
 considerable opposition, and this is readily understood when it is re- 
 membered that even today — one hundred and eighteen years later — 
 there are those who refuse to accept, or are uAable to realize, the great 
 benefits of this pioneer work. Jenner could not explain his results; he 
 maintained that he was dealing with a modifie^d form of smallpox. We 
 of today have no better means of establishing the truth of the efficiency 
 of cowpox vaccination, nor have we improved any on his method. The 
 specific germ of smallpox is still undiscovered, and we must agree with 
 Jenner that cowpox is probably a modified fqrm of smallpox and prac- 
 tically harmless, the virus of cowpox being the virus of smallpox 
 modified, attenuated, and rendered practicaliy innocuous by passage 
 through a lower animal. 
 
 Nothing further of importance was accomplished during the follow- 
 ing eighty years, until the next and even greater epoch ushered in the 
 discoveries in bacteriology and the first immunization by Pasteur based 
 on scientific reasoning. The chickens around Paris were being destroyed 
 by a virulent intestinal infection, and Pasteur first isolated the causative 
 
142 IMMUNITY. THEOKIES OF IMMUNITY 
 
 microorganism, a minute bacillus, which he fbund was capable of pro- 
 ducing the disease experimentally in healthy chickens. Quite by acci- 
 dent, so it seemed, he discovered that cultures of this bacillus could, by 
 prolonged cultivation be attenuated, for when these cultures were inocu- 
 lated into chickens, the fowls did not die or suffer any ill consequences; 
 further, and what was of the utmost importance, when these same chick- 
 ens were inoculated with virulent cultures, they were found to be im- 
 mune to chicken cholera. Here, then, was the key to active immuniza- 
 tion in the prevention of disease, and Pasteur possessed the genius to 
 realize the full significance of his discovery. 
 
 Armed with this knowledge Pasteur, and his assistants, Roux and 
 Chamberland, next studied anthrax, an infectious disease of cattle that 
 was causing a great annual loss to the farmers of France, and the bacillus 
 of which was among the first pathogenic micro&rganisms to be discovered. 
 It was found that prolonged cultivation of this bacillus was insufficient 
 to attenuate the cultures, as the spores were highly resistant and re- 
 tained their pathogenicity under extreme circumstances and over pro- 
 longed periods of time. 
 
 In 1880 Touissant published a method of attenuating the bacilli by 
 heating the blood of an infected animal to 55° C. for a few minutes; 
 later, Chauveau secured similar results by heating fresh cultures for a 
 few minutes at 80° C. Both methods were uncertain, and neither safe 
 nor practical. After prolonged experimentation Pasteur found that 
 cultivating the bacilli at the relatively high temperature of from 42° to 
 43° C. resulted in gradual attentuation, and if this cultivation was con- 
 tinued, the cultures were robbed entirely of their disease-producing 
 power. Further, subcultures of these growths when kept at 37° C. 
 did not regain their original virulence, but maintained for generations 
 the grade of attenuation reached in the original culture the result of 
 cultivation for a certain number of days at the higher temperature. In 
 this manner Pasteur was able to control to some extent the degree of 
 attenuation, and by inoculating first a highly attenuated and later a 
 less markedly attenuated culture he could immunize animals against 
 anthrax. This discovery was soon amply verified. The original method 
 is practically the one employed today, and is proving of considerable 
 economic value. 
 
 Pasteur's next great experiment was undertaken for the relief of 
 rabies, a condition in which, for the first time, he came to deal with a 
 disease that not infrequently affects man. His success and the results 
 of his discovery of an effective means of immunization against hydro- 
 
HISTORIC 143 
 
 phobia were even greater than in previous experiments. Here he was 
 deahng with a disease of unknown etiology, the causative agent of which 
 he could not cultivate artificially, but which he sought to attenuate by 
 a new process — that of drying. 
 
 Pasteur first established that the virus of rabies is contained within 
 the tissues of the brain and spinal cords of infected animals. 
 
 He then invented a method of inoculating animals by making sub- 
 dural injections of an emulsion of these tissues. By repeated passage 
 of a virus through a number of rabbits a virus of fixed pathogenic power 
 {virus fixe) was obtained. By inoculating rabbits with this virus and 
 removing their spinal cords immediately after death and drying these 
 over a desiccating agent at room temperature, he found that he could 
 modify the virulence of the virus at will, depending on the length of the 
 period of drying. By emulsifying small portions of attenuated spinal 
 cord in salt solutions and injecting these he was able gradually to im- 
 munize animals against rabies, and finally he applied the treatment 
 successfully to the prevention of rabies in the human being. 
 
 Antirabic vaccination is largely responsible for extending our knowl- 
 edge of the possibility of securing immunization. Pasteur has taught 
 us at least three different methods for modifying a virus in the prep- 
 aration of a vaccine, and that each disease, being itself a special 
 entity, having its own characteristics, must be dealt with along special 
 lines. 
 
 These discoveries were largely empirical, and the explanations of 
 their mechanism are now only of historic interest. It was not until 1883, 
 when Metchnikoff shed light upon the problems of immunity by 
 making a scries of remarkable studies on the r£le played by certain of 
 the body-cells in overcoming infection, and the part they played in the 
 processes of immunity in general, that the world was given a glimpse 
 into the dark problems of immunity. These observations were soon 
 followed by investigations showing the importance of the body fluids, and 
 since that time a great deal of work has been done upon these subjects. 
 As a consequence, a large amount of data of a wholly new order has 
 accumulated, accompanied by the introduction of a host of new terms 
 expressing diverse views and theories advanced by individual workers. 
 Of the many theories advanced from time to time to explain the phenom- 
 enon of immunity, two have claimed the most attention: one ascribes 
 protection and cure to the activity of certain body-cells; this is known 
 as the cellular theory; and the other attributes these qualities to the body- 
 fluids — the humoral theory. The chief exponent of the former is Metch- 
 
144 IMMUNITY. ^THEORIES OF fMMUNITY 
 
 nikoff, with his theory of phagocytosis, whereas Ehrlich is the father and 
 leader of the latter, with that marvelous invention of human ingenuity, 
 the side-chain theory. 
 
 THEORIES OF IMMUNITY 
 
 The earlier hypotheses advanced by various investigators are now 
 only of historic interest, as in the light of subsequent discoveries and 
 observations they have failed to offer adequate explanations. 
 
 Pasteur's own theory and explanation of the mechanism of acquired 
 immunity sought to show that the microorganisms living in the infected 
 animal used up some substance essential to their existence, so that, for 
 lack of proper nourishment, the microorganisms were eventually forced 
 to retire, the soil being unfit for further occupation. This was known 
 as the "exhaustion theory." 
 
 Chauveau considered it more probable that the microorganisms, 
 after having lived in the body of an infected animal, produced sub- 
 stances that, accumulating in the blood, had an inhibitory action on the 
 bacteria and were inimical to their further existence. This was known 
 as the "retention theory," and in some particulars was just the opposite 
 of the exhaustion theory. 
 
 The Theory of Phagocytosis 
 
 In 1883, when Metchnikoff showed that certain of the body-cells, 
 and, particularly, the polynuclear leukocytes, were active in the defense 
 of the human body against invasion by microparasites, real light was 
 thrown upon the unknown problems of immunity. Although he has 
 since amplified his theory, as new theories were adduced to describe the 
 part played by the body-fluids and the organisms themselves, yet his 
 theory of phagocytosis remains a demonstrable fact, and establishes the 
 important role of cells in the processes of immunity. 
 
 According to this theory, certain of the body-cells are able to ingest 
 ah infecting parasite, a red corpuscle, or other cell in the same maimer 
 as an ameba ingests a food-particle, and to dispose of it by intracellular 
 digestion through the agency of ferments known as "cytases. " To 
 such cells Metchnikoff applied the name phagocyte, as he likened them 
 to scavengers, i. e., they were concerned in picking up and disposing of 
 offensive material, both living and dead. 
 
 Various body-cells are capable of becoming phagocytes. The poly- 
 nuclear leukocj'tes are particularly active in acute infections, and have 
 been called microphages. Endothelial cells, mononuclear leukocytes, 
 
THEORIES OF IMMUNITY 145 
 
 and embryonic connective-tissue cells are more active in chronic infec- 
 tions and have been designated macrophages. 
 
 Although the original explanation of phagocytosis was quite plain 
 and far reaching, subsequent discoveries by the adherents of the "hu- 
 moral theory" showed the potent influence of the body-fluids upon the 
 process. It was demonstrated that without the aid of these fluids 
 phagocytosis is almost negligible. Metchnikqff early recognized this 
 fact, and sought to explain the influence of the body-fluids by assuming 
 that they contained "stimulins, " or substances" that stimulated phagocy- 
 tosis. It was likewise shown that the body-fliiids contained antibodies 
 and were antibacterial, independent of cells. Metchnikoff recognizes 
 the existence of these conditions, but claims that they are due to the 
 soluble products of phagocjiiic cells, and thusfn a broader sense would 
 jnaintain the importance of his phagocytes. 
 
 It was soon observed that in certain infections leukocytes or other 
 cells, instead of being attracted toward the seat of infection by some 
 unknown chemical stimulus, (positive chemotaxis), were repelled, or at 
 least the attraction was counterbalanced or did not exist {negative 
 chemotaxis). While a satisfactory explanation of these phenomena is 
 still lacldng, it may be stated that, in general", the degree of negative 
 chemotaxis is in proportion to the virulence of the microparasite. 
 
 In 1903 Wright and Douglas, and Neufeld» and Rimpau threw con- 
 siderable light upon this subject. They demonstrated experimentally 
 that one action of the body-fluids was directed against the microorgan- 
 isms, lowering their resistance, and, maldng' them, as it were, more 
 attractive to the phagocytes, the process of phagocytosis was facilitated. 
 To these substances the name of "opsonins" (from opsono, I prepare for) 
 was applied by Wright, while Neufeld called them "bacteriotropins." 
 
 The leukocytes are not, however, entirely passive and willing to wait 
 until their prey is weakened and fully prepared for their attack. In the 
 presence of an infection they are found to increase, and this leukocj^tosis 
 is known to be a valuable addition to the defensive forces. They prob- 
 ably also undergo qualitative changes, which increase their antibacterial 
 power. It has been shown that opsonized bacteria attach themselves 
 to the protoplasm of the leukocytes, a physiochemical phenomenon 
 that occurs regardless of whether the leukocyte is dead or alive, although, 
 of course, only the living leukocyte is able to ingest them. 
 
 That phagocytosis is a potent and very important factor in the 
 mechanism of recovery from certain infections is generally admitted, 
 
 and although it probably has not the far-reaching significance originally 
 10 
 
146 IMMUNITY. THEORIES OF IMMUNITY 
 
 attributed to it, yet, throughout the discussion of immunity the im- 
 portance of the phagocyte itself is emphasized. This theory of Metch- 
 nikoff is treated more fully in the chapter on Phagocytosis. 
 
 SmE-CHAiN Theory 
 
 The humoral theory of immunity, which would ascribe the power to 
 resist infection to the body-fluids, may be said to have had its origin in 
 1896, when Fodor discovered that the blood of the rabbit will kill an- 
 thrax bacilli in the test-tube, independent of cells and phagocytosis. 
 Later Buchncr adopted this theory, and sought to explain the bacteri- 
 cidal action of blood-serum as dependent uppn a special constituent 
 which he called alexin. 
 
 With the discovery, in 1890, of antitoxins by von Behring and Kit- 
 asato, the theory received fresh support, and while an effort was made 
 to demonstrate that antitoxins were of paranjount importance in ac- 
 quired immunity, evidence soon accumulated to show that this anti- 
 toxic power is operative oiJy in a few diseases, chiefly in diphtheria and 
 tetanus. 
 
 Fresh support to the "humoral" as against the "cellular" explana- 
 tion of immunity was given by Pfeiffer in 1894, with the discovery that 
 cholera vibrios introduced into the peritoneal cavity of a guinea-pig 
 previously immunized against cholera became transformed into granules, 
 and ultimately passed into complete solution (bacteriolysis), apparently 
 without the aid of cells. Bordet then showed that this phenomenon 
 was due to two distinct substances — one, the "sensitizing substance," 
 which is specific and exists only in the immune serum, acting only on the 
 bacteria against which the animal was immunized, and the other a non- 
 specific substance, found in the fresh serum of practically all animals, 
 and to which he gave the name "alexin, " and which was later renamed 
 by Ehrlich and called "complement." 
 
 Of the various theories offered in explanation of these observations, 
 the suggestive, fascinating, though highly hypothetic theory of Ehrlich, 
 known as the side-chain theory, has been mo^t widely accepted and 
 adopted to explain new discoveries as they were made. The theory 
 has, indeed, aided investigators in making new discoveries. Nevertheless 
 the contention of Bordet, that its too ready acceptance without sufficient 
 convincing proof has retarded investigation, should" not be ignored. 
 
 The basis of this theory, as originally proposed, bore no relation to 
 the subject of immunity, but was advanced in 1885 to explain the pro- 
 cesses of nutrition. 
 
THEORIES OF IMMUNITY 147 
 
 Ehrlich asserts that a cell has two important functions: The first is 
 the special physiologic function, as that of a nerve-cell to conduct; of a 
 gland-cell, to secrete, etc. The second function is that of nutrition, 
 and presides over the processes of waste and repair. Furthermore, 
 each of the molecules composing the complex cell is believed to possess 
 these two functions, i. e., one is concerned with the special function of 
 the molecule, and the other, the more important functional portion, is 
 concerned in the nourishment of the molecule. 
 
 The second portion, or that concerned with nutrition, is of more 
 importance in relation to the problems of immunity. Ehrlich con- 
 ceives this as consisting of a special executive, center or main portion 
 ("Leistenkern"), in connection with which there are nutritive side- 
 chains, receptors, or haptines (" Leitenketter ")^ which " seize, " or rather 
 enter into chemical combination with, suitable food .atoms, which is 
 followed by a sort of digestive or absorptive process, whereby the food 
 material is incorporated in the molecule; 
 
 The function of "seizing" molecules of food from the surrounding 
 tissues implies a selective action or chemical affinity between food 
 atoms and the portion of a cell or side-arm for which it has a chemical 
 affinity, for we cannot conceive that all atoms that circulate in the blood 
 and lymph are suitable for all cells at all times: 
 
 The food molecule in the fluid surrounding the cell is conceived as 
 possessing a special or haptophore portion for union with the side-arm 
 of a cell molecule, and when brought into relation with one of the side- 
 arms or receptors of the cell molecule, the two are "anchored, " or unite, 
 just as a key fits a lock. The second stage involves a process that may 
 be compared to digestion, by which the food material is prepared and 
 absorbed, in whole or in part, into the molecule of protoplasm. 
 
 These processes, therefore, are conceived as being chemical rather 
 than physical, and our diagrammatic representations of them have no 
 necessary or actual morphologic basis. One is quite likely to regard the 
 main central portion as the nucleus of a cell, alid the side-arms as small 
 morphologic projections resembling the pricliles of certain epidermal 
 cells. These processes are concerned with each molecule of a cell, the 
 main portion, or " Leistungskern, " being conceived as diffusing through 
 the nutritive part of the molecule, and the side-arm receptors, or "Lei- 
 tenketter," as numerous atoms or groups of atoms, each of which has 
 achemical affinity for some particular food-substance circulating in the 
 body-fluids, and necessary for the life of the molecule in question. 
 
 Later this theory was amplified by Ehrlich to explain the action of 
 
148 IMMUNITY. THEORIES OF IMMUNITY 
 
 toxins and the production of antitoxins. It assumes that the side-arms 
 to a cell molecule are exceedingly numerous, not only because nutritive 
 substances are varied, but because special cells also possess different 
 and special side-chains, which anchor pathologic material. When 
 infection occurs, and in addition to toxins the- physiologically normal 
 substances are brought to the cells, they likewise find suitable receptors 
 in practically all or certain cell groups, and become anchored, causing 
 more or less damage to the cells. 
 
 Having combined with the side-arms or receptors of a cell, the toxin 
 may be sufficiently potent to kill the cell, and if d large number of cells are 
 so injured, symptoms of disease present themselves and death of the 
 infected host may follow. On the other hand, although the cell has lost 
 one or more of its side-arms, it may not be dead, and it proceeds at once 
 to repair the damage done. According to Weigert's "overproduction 
 theory, " nature is lavish in its processes of repair, and the cell not only 
 replaces the lost receptors, but produces them in numbers; the excess 
 receptors, having no space for attachment to the cell, are thrown off 
 into the blood-stream. Each of these cast-off receptors or haptines 
 possesses the same structure as the original receptor. These free re- 
 ceptors, then, are capable of combining chemically with their antigen, 
 neutralizing the antigen and rendering it innocuous. In diphtheria 
 and tetanus the antigen is largely the soluble toxin of the bacilli, and the 
 antitoxins are these cast-off receptors produced: as a result of the stimu- 
 lating action of the toxins upon the cells. This excess of receptors is 
 made by repeatedly injecting a horse with increasing doses of diphtheria 
 toxin. By injecting this receptor-laden (antitoxin) serum into one 
 suffering from diphtheria, the receptors unite wi,th free diphtheria toxin 
 and thus protect the body-cells. 
 
 For the production of these receptors, or antibodies, as they are now 
 called, it is necessary, as previously stated, that the antigen enter into 
 chemical combination with the cell, so that the usual illustrations 
 showing the theoretic union of antigen and side-arm by physical contact 
 alone probably do not correctly portray what actually occurs. As 
 Adami points out, the antigen probably enterp into intimate relation- 
 ship with the cell, and the continued stimulation of its presence is re- 
 sponsible for the production of an excess of receptors, in addition to 
 the overproductive tendencies of nature's repair. 
 
 It is also necessary that the antigen possess, sufficient toxic power at 
 least to stimulate the cell, for otherwise antibodies may not be produced. 
 Food material, for instance, being physiologic, is assimilated by the 
 
THEORIES OF IMMUNITY 149 
 
 cells without stimulating the production of antibodies, as otherwise 
 the food would be attacked by cast-off receptors and rendered useless 
 before it reaches cells, the process ending in slkrvation and death. 
 
 The host in whom certain cells with special receptors for a given 
 poison are present will make use of these, no matter how the pathologic 
 agent is introduced. This affinity is well illus'trated in tetanus, where 
 the effects produced are dependent to a large extent upon the selective 
 affinity of the toxin for nerve-cells. 
 
 The Thhbe Orders of Receptors and Corresponding Antibodies 
 
 First Order: Antitoxin and Simple Antiferments. — The simplest 
 receptor of the cell molecule is composed of a single arm or haptophore, 
 for union with the haptophore portion of a food molecule. As previously 
 stated, a molecule of toxin is conceived as being composed of two por- 
 tions — one, the haptophore, for union with the side-arm or receptor of a 
 cell molecule, and the second, the toxophore, in which its toxic action 
 resides. 
 
 The first stage of intoxication of a cell produced by a true toxin 
 consists in the union of the haptophore portion of the toxin molecule 
 to a receptor or side-arm of the ceU molecule, this receptor being one 
 that fits the toxin molecule "like a key fits a l9ck. " Each molecule of 
 the body-cell has innumerable receptors, of which only a certain number 
 are suitable for a particular toxin. The toxin molecule is now anchored 
 to the living cell, and, as animal experiments with a great number of 
 toxins show, this union is a firm and enduring pne (Fig. 39). 
 
 So long as the union lasts the side-chain involved cannot exercise its 
 normal nutritive physiologic function — the taking up of food-stuifs. 
 Furthermore, the toxophore group of the toxin molecule maj' now exert 
 an injurious, enzyme-like action on the protoplasm of the cell, with the 
 result that the protoplasm is poisoned. If only a few of the cell receptors 
 are united with toxin molecules, or if the to^jin is of low toxicity, the 
 effects on the cell may be slight. If more are joined to the molecule 
 or the toxin is highly poisonous, the whole molecule, and finally the ceU 
 itself, may be greatly disturbed, and produce marked symptoms, or the 
 host may be destroyed. 
 
 Since the receptors joined to the toxin molecules are incapacitated 
 or destroyed, the damage is repaired by the regeneration of new recep- 
 tors. According to the reparative principles worked out by Weigert, 
 the repair is not a simple replacement of the defect — the compensation 
 proceeds far beyond the necessary hmit; indped, overcompensation is 
 
150 
 
 IMMUNITY.- 
 
 rHEORIES OF IMMUNITY 
 
 the rule, and this forms the basis of Ehrlich's theory. If, after repair 
 has taken place, new quantities of toxin are administered at proper 
 intervals and in suitable quantities, the side-dhains that have been pro- 
 duced by the regenerative process are taken up anew in combination 
 with the toxin, and so again the process of regeneration gives rise to the 
 formation of fresh side-chains. "The lasting and ever-increasing 
 regeneration must finally reach a stage at which such an excess of side- 
 
 FiG. 39. — Formation op Antitoxins. 
 
 The central white area represents a molecule of a "cell; the shaded portion repre- 
 sents the cell itself; the surrounding area represents the body-fluids about the cell. 
 
 r, A receptor of the molecule (first order) ; A, overproduction of receptors, which 
 are being cast off; A^, a cast-off receptor free in the body-fluids — now an antitoxin; 
 A', a molecule of antitoxin combination with a toxin molecule T*. A*, a cast-off 
 receptor still within the parent cell ; T, a toxin molecule in combination with the re- 
 ceptor of a cell molecule; "P, a toxin molecule free in the body-fluids; T', a toxin 
 molecule in combination with antitoxin; T*, a molecule of toxoid (toxophore group 
 lost). 
 
 chains is produced that, to use a trivial expression, the side-chains are 
 present in too great a quantity for the cell to carry, and are, after the 
 manner of a secretion, handed over as a needless ballast to the blood. 
 Regarded in accordance with this conception, the antitoxins represent 
 nothing more than side-chains reproduced in excess during regeneration, 
 and therefore pushed off front the protoplasm and so coming to exist in the 
 free state" (Ehrlich). 
 
THEORIES OF IMMUNITY 151 
 
 This theory explains the specificity of the antitoxins for a given 
 toxin; thus the latter causes specific chemical stimulation of the cell, 
 which induces the formation of specific side*-thains, — the cast-off re- 
 ceptors, — which are capable of uniting with the toxin molecules free in 
 the body-fluids and thus neutralizing them; they are, therefore, called 
 antitoxins. 
 
 This theory also explains why a minute quantity of toxin is capable 
 
 of stimulating the production of a large amount of antitoxin, and why 
 
 the production of antitoxin persists for some time. The toxin molecule 
 
 rnust be conceived as entering into the protoplasm of a body molecule 
 
 and residing there for some time, acting as a stimulus to the cell, with 
 
 consequent production of antitoxin. Diagrammatic 
 
 representations of this process would seem to show 
 
 that a physical union exists between toxin and cell 
 
 receptors, resulting in the destruction of receptor, 
 
 which drops off and is replaced by a number of 'Tecep- 
 
 tors that, for lack of space for attachment to the 
 
 cell, are thrown off into the blood-stream. In ^'■'^- 40.— -Theo- 
 retic BTHUC- 
 
 reality, by the act of immunization certain cells of xukeofaMole- 
 
 the body become converted into cells that secrete amd^Toxoid"'^''* 
 
 specific antitoxin, and, as shown by Salmonson and 1, Toxin: H, 
 
 Madsen, the administration of pilocarpin, which for^S°''^itf°the 
 
 augments the secretion of most glands, also pro- receptors of cells or 
 , . . . , . , . , . . , antitoxin: T, toxo- 
 
 duces m immunized ammals a rapid increase in the phore group. 
 
 antitoxin content of the serum. The formation of ^ ■^' Toxoid: Same 
 
 structure as toxin 
 antitoxin is constantly going on, and so throughout molecule except 
 
 a long period the antitoxin content of the serum group is lo°st°^ 
 remains nearly constant. 
 
 In the production of antitoxin the haptophore group is the essential 
 and important portion of the toxin molecule.* Even though the toxo- 
 phore group is lost, — and when this occurs the toxin is called toxoid 
 (Fig. 40), — the haptophore group is capable of uniting with receptors 
 and stimulates the production of antitoxin. In fact, in effecting immu- 
 nization with powerful toxins it may be necessary, in the first few in- 
 jections that are given, to convert artificially all or a portion of the 
 toxin into toxoid, so that antitoxins will be produced that will protect 
 the animal against subsequent overdoses of toxin. 
 
 The production of antitoxins must, in keeping with this theory, be 
 regarded as a function of the haptophore group of the toxin. It is easy, 
 therefore, to understand why, out of the great number of alkaloids, 
 
152 IMMUNITY. TIIEOKIES OF IMMUNITY 
 
 none is in a position to cause the production of antitoxins, for alkaloids 
 possess no haptophore group ttiat anchors them to the cells of organs. 
 Ai3 has been stated, in the formation of antitoxin the haptophore 
 group of the toxin molecule is the essential portion; the toxophore 
 group is much less important, and during immunization the symptoms 
 of illness due to the action of the latter group are not essential to and 
 play no part in the production of antitoxin. It must be said, however, 
 that a toxin molecule with an intact toxophore group is more stimulating 
 than a toxoid in which this group is absent; therefore, in artificially 
 immunizing horses for the production of antitoxin, after the first few 
 injections increasing amounts of toxin are administered. 
 
 Antibodies of the Second Order (Agglutinins and Precipitins). — 
 As new cUscoveries were made, Ehrlich amplified his theory of the for- 
 mation of antibodies, but always upon the original and basic conceptions 
 as just set forth. 
 
 We have seen that the simplest molecules of food substances, toxins, 
 and ferments, substances really in solution, are anchored to molecules 
 of cell protoplasm by means of the simple side-arms of the latter. When 
 this chemical union has taken place, the food or toxin may be assimi- 
 lated without undergoing any further change. With more complex 
 food substances, however, some preparatory treatment is necessary be- 
 fore they become available for final assimilation. The large molecule 
 may readily enough be anchored tO the molecule of the cell, but it 
 probably requires some preparation before it becomes available for the 
 nutrition of the cell. 
 
 Accordingly, Ehrlich assumed that the body-cells are furnished with 
 another order of side-chains or receptors composed of two portions; 
 one part or group for union with the food substance, and called the hapto- 
 phore group; and the second portion, called the toxophore or zymophore 
 group, in which the special function of the receptor resides. 
 
 Similarly, certain pathogenic agents that are more complex than 
 soluble toxins or ferments combine with receptors of this kind. One 
 ami, the haptophore group of the receptor, combines with the hapto- 
 phore portion of the pathogenic molecule, and then the second or toxo- 
 phore portion of the receptor exerts some special action upon the at- 
 tached molecule. Receptors or haptines of this nature are known as 
 receptors of the second order; antibodies of the same structure, pro- 
 duced and cast off into the blood-stream as the result of toxic injury 
 and stimulation of body-cells, are known as antibodies of the second 
 order. 
 
THEOBIES OF IMMUNITY 
 
 153 
 
 Two such antibodies are well kiio\\Ti. In one we find that the toxo- 
 phore group of the antibody causes clumping or agglutination of its 
 antigen, or the agent that caused its productioij, and hence this antibody 
 is called an agglutinin. In typhoid fever, for example, the bacillus or 
 one of its more complex products causes the production of an antibody 
 of this nature, so that when the serum of a- typhoid fever patient is 
 mixed with the bacilli, the latter lose their motility and form clumps or 
 agglutinated masses. This phenomenon was first observed by Gruber 
 and Durham, and was applied in a practical way to the diagnosis of 
 
 Fig. 41. — Formation of Agglutinins and Precipitins. 
 
 The central white area represents a molecule of a cell; the shaded portion repre- 
 sents the cell itself; the surrounding area represents tHe body-fiuids about the cell. 
 
 R, Receptor of the molecule {second order); B?, overproduction of receptors, 
 which are being oast off; A, a cast-off receptor which now constitutes the antibody; 
 A, A'', agglutinins in combination with the antigen (bacilli). 
 
 typhoid fever by Widal and Griinbaum. The second antibody of this 
 class, the precipitins, resemble the agglutinins quite closely (Fig. 41). 
 Kraus discovered that if a bouillon culture of the typhoid bacillus 
 is filtered through porcelain, and a few drops of serum from a typhoid 
 fever patient or from an animal immunized by injections of typhoid 
 bacilli are added to a small quantity of the baoilli-free filtrate, a faint 
 cloud will appear resembling in some respects that observed at the line 
 of contact between nitric acid and urine that contains a trace of albumin. 
 
154 IMMXJNITY. — THEORIES OP IMMUNITY 
 
 The toxophore portion of this antibody, therefore, appears to coagulate 
 or precipitate soluble substances, and, accordingly, the antibody is 
 known as a precipitin. As will be pointed 6ut later, various protein 
 substances, such as blood-serum, milk, egg-albumin, etc., may cause the 
 production of specific precipitins. 
 
 Antibodies of the Third Order (Hemolysins, Bacteriolysins, Cyto- 
 toxins). — Still more complex molecules of food material require con- 
 version into simpler substances before they may be assimilated by the 
 molecules of the cell. It is essential that they undergo a sort of digestion, 
 and accordingly Ehrlich has conceived that special side-arms or re- 
 ceptors exist for this purpose, these being cpmposed of two grasping 
 portions, or haptophore groups, one for union with the complex food 
 molecule, the second for union with a special, ferment-like substance 
 present in the blood and called complement. The receptor, therefore, 
 acts simply as a connecting link or interbody between food molecule 
 and complement, bringing the two into relation with each other when 
 the food molecule is rendered soluble, i. e., undergoes lysis. 
 
 With highly organized cell material, such as red blood-corpuscles or 
 bacteria, it is found that receptors of this nature bring about their de- 
 struction by lysis by attaching them to a suitable complement. During 
 infections with various bacteria, therefore, we«find that numerous anti- 
 bodies are produced. If the bacteria produce soluble toxins, specific 
 antitoxins are produced to coimteract the effects of these; other prod- 
 ucts stimulate the production of agglutinins and precipitins ; still other 
 products or the whole cell cause the production of antibodies, which 
 are not in themselves destructive, but which have the specific power of 
 combining with the cell and bringing about its lysis or destruction by 
 bringing it into relation with the ferment-like complement. It is only 
 by means of a special antibody of this nature that a complement may be 
 united with the pathogenic agent, i. e., the complement itself cannot 
 act directly upon the cell, but must be united by means of the antibody. 
 
 Ehrlich has termed an antibody of this nature an amboceptor, or 
 interbody. In structure, amboceptors are believed to possess two com- 
 bining or grasping portions: one, the haptophore or antigenophore 
 group, for union with the cell; the other the complementophile group, 
 for union with a complement (Fig. 42). 
 
 The lysins (bacteriolysins, hemolysins, and other cytolysins) are 
 antibodies of this order. If, for example, the erythrocjrtes of one animal 
 are injected into an animal of a different species, hemolysins will be pro- 
 duced, the hemolysin being a specific hemolytic amboceptor that will 
 
PHAGOCYTIC AND SIDE-CHAIN. THEORIES 
 
 155 
 
 unite corpuscles of the animal used in the injeetion and only these cells, 
 ■nith a complement, and thus bring about t^eir solution or lysis. If 
 certain bacteria (e. g., the cholera bacillus) are injected into an animal, 
 specific bacteriolysins (bacteriolj'tic amboceptors) will be produced. 
 Similarly, specific amboceptors are produced during the course of in- 
 fections with typhoid bacilli, and are largely instrumental in combating 
 and overcoming this infection. It is important to remember, however, 
 that although these amboceptors probably prepare their antigens for 
 
 Fig. 42. — Formation of Cttoltsins (Hemolysins, BsiCTEHioLYSiNS, Cttotoxins). 
 
 The central white area represents a molecule of a cell; the shaded portion repre- 
 sents the cell itself; the surrounding area represents the body-fluids about the cell. 
 
 R, Receptor of the molecule {third order) ; R', overproduction of receptors, which 
 are being cast off; A, a cast-off receptor wliich now ccCofetitutes the antibod>y or am- 
 boceptor; C, molecule of complement free in the body-cells and body-fluids; A-A^, 
 amboceptors in combination with molecules of a cell (antigen) and a complement; 
 A', an amboceptor in combination with a molecule of a cell. The cell (antigen) is 
 now said to be sensitized. Lysis does not occur because a complement is not united. 
 
 lysis, or, in the meaning of Bordet, "sensitize" them, thej' are not in 
 themselves lytic, final solution of the antigen being accomplished by the 
 ferment-hke substance — ^the complement. 
 
 COMPATIBILITY OF THE PHAGOCYTIC AND SIDE-CHAIN THEORIES 
 
 When we seek to compare the theory of Metchnikoff -n-ith that of 
 Ehrlich, we find that they differ only in minor details, the fundamental 
 
156 IMMUNITY. THEORIES OF IMMUNITY 
 
 principles not being contradictory; they may, rather, be regarded as 
 one set of phenomena viewed from different .aspects. 
 
 Since its original announcement, Metchnikofi has, on different oc- 
 pasions, enlarged upon his theory to meel? certain discoveries, made 
 chiefly by adherents of Ehrlich's theory, showing the presence of sub- 
 stances in the blood-serum and other body-fluids that are potent in the 
 processes of immunity independent of cells. Metchnikoff claims, 
 however, that these antibodies are derived from the group of cells 
 classified as phagocytes, and thus would preserve the primary impor- 
 tance of his theory. Ehrlich, on the other hand, while not denying that 
 these cells may be a source of their formation, points out that they are 
 not necessarily the sole or supreme source, but may be formed by the 
 general body-cells or by special groups of cells possessing a selective 
 affinity for the pathogenic agent. 
 
 The theory of Ehrlich is essentially a chemical one, and maintains 
 that the union of food or pathologic material with cells is a chemical 
 union; his views, therefore, possess that degree of definiteness necessary 
 to constitute a plausible chemical theory. The theory of Metchnikoff 
 would explain processes of nutrition and immunity as largely founded 
 on a physical basis, and is therefore, necessarily more general, being 
 largely biologic and vitalistic. 
 
 The two theories differ in two more or less hypothetic points: (1) 
 In the manner by which material enters into relation with cells, and 
 (2) the relative importance of certain cells in the formation of anti- 
 bodies. Otherwise both are intimately related, in that phagocytosis is 
 unimportant if removed from the influence of antibodies in the body- 
 fluids, and these same antibodies, although probably formed accord- 
 ing to Ehrhch's theory, are derived in part from Metchnikoff's phago- 
 cytes. 
 
 Phagocytosis, whether by leukocytes, endothehal cells, or by newly 
 developed connective-tissue cells, is very common, and is obviously a 
 most important factor in the destruction of pathogenic bacteria and in 
 the cure of infectious disease. In virulent infections, however, phagocy- 
 tosis may not be apparent; the leukocytes are not attracted, and those 
 in the vicinity undergo dissolution. Later ih these infections, however, 
 phagocytosis may become apparent, due, according to Metchnikoff, to 
 the "adaptation" of the cells to the products of the invading micro- 
 organism, whereby the weak or negative chemotaxis is converted into an 
 active positive chemotaxis with vigorous digestion. This, however, is 
 not primarily due to increased digestive capacity of the phagocytes, 
 
PHAGOCYTIC AND SIDE-CHAIN THEORIES 157 
 
 but to an increase of opsonins in the body-fluidH; these opsonins prepare 
 the bacteria for digestion. 
 
 The original phagocytic theory did not explain the destruction of 
 bacteria within the living tissues without the intervention of leukocytes, 
 and, what is even more striking, a similar destruction occurring in vitro 
 by serum and other body-fluids totally devoid of cells. Bacteriolysis 
 has been shown to be due to two different substances — one, a thermo- 
 labile, ferment-like body called "cytase" by Metchnikoff and "comple- 
 ment" by Ehrlich, and the other a more specific thermostabile body 
 called "fixateur" by Metchnikoff and "amboceptor" by Ehrlich. 
 These substances appear to play an important role in certain infections, 
 as, for example, in typhoid fever and cholera, , and were studied mainly 
 by the adherents of the side-chain theory. Metchnikoff recognized 
 their existence and significance, but endeavored to preserve the primary 
 importance of the phagocytic theory by claiming that they are products 
 of the group of cells classified as phagocytes. Ehrhch, however, while 
 not denying that these cells may be one source-, holds that they are not 
 necessarily the sole source, but that they are products of general cellular 
 activity or of special groups of cells that have shown a combining affinity 
 for the antigens. 
 
 For example, Metchnikoff holds that there are but two comple- 
 ments, — macrocytase and microcytase, — and that these are formed by 
 destruction or solution of macrophages and rnicrophages. Ehrlich has 
 shown quite conclusively that there are many complements, and that 
 these are the excretory products of leukocytes, and probably of other 
 cells as well. Ehrlich teaches also that specific amboceptors or fixateurs 
 may be products of various body-cells other than those classified as 
 phagocytes, and Metchnikoff recognizes their existence, but holds that 
 they are formed and discharged solely by the leukocytes or other pha- 
 gocytic cells. Ehrlich has shown the manner in which complement and 
 amboceptor produce bacteriol3^sis, and Metclinikoff has amplified his 
 theory to meet these observations, to the extent that destruction of 
 bacteria is recognized as being brought about either intracellularly, by 
 the digestive action of the leukocytes, or extracellularly, by the enzyme- 
 like action of the cytase, or complement, working through the inter- 
 mediation of the fixateur or amboceptor, and that cells that are poten- 
 tially phagocytic give origin to these antibodies. 
 
 Regarding the structure of toxins and the action of antitoxins the 
 two theories are divergent, and whereas Metchnikoff is inconclusive, 
 Ehrlich presents definite conceptions that are well supported by experi- 
 
158 IMMUNITY. THEORIES OF IMMUNITY 
 
 mental data. Metchnikoff maintains that il is the cells that absorb 
 the "toxin" that furnish the antitoxin. In other words, the enzymes, 
 as microcytase and macrocytase, exert their action not only upon the 
 more complex molecules of microorganisms, but also upon their simpler 
 toxins, fixing or otherwise altering them until they can finally be de- 
 stroyed. This explanation would lead us to conclude that the nerve- 
 cells which bind the tetanotoxin are capable of furnishing antitoxin, 
 whereas experimental observations are absolutely opposed to this 
 narrower view. Metchnikoff also maintains that antitoxin acts by 
 stimulating the leukocytes to absorb and destroy toxin, whereas EhrUch 
 has clearly shown that antitoxin, by combining chemically with the 
 toxin, neutralizes it, a process that may be shown in vitro entirely in- 
 dependent of cells. 
 
 From what has been said it will be seen that the two theories are not 
 essentially divergent, and that we are unwarranted in clinging to one 
 view to the absolute exclusion of the other. The question rests largely 
 on which of the body-cells are most active in° forming antibodies, and 
 also on a recognition of the role played by phagocytosis in certain in- 
 fections, such as staphylococcus, streptococcus, and pneumococcus in- 
 fections. Ehrlich has attempted an explanation of the method by which 
 body-cells form antibodies, and the manner in which these antibodies 
 overcome their antigens; he has placed both processes upon a chemical 
 basis, involving no one particular group or class of cells. Metchnikoff, 
 on the other hand, has shown the important role played by phagocy- 
 tosis in many infections, and claims that the antibodies in the cir- 
 culating fluids are the products of these phagoeytes ; he places immunity 
 more largely upon a physical basis. 
 
 The various phenomena of immunity cannot be ascribed either to 
 the activity of the body-cells or to the body, fluids alone, to the total 
 exclusion of the other — both are intimately concerned in the various 
 phases of immunity. 
 
 It is, moreover, becoming more obvious that too httle attention has 
 been paid to the influence of the microorganism in the phenomena of 
 immunity reactions. It is important to recognize that some bacteria 
 are apparently able to immunize themselves against the combative 
 forces of their hosts, as is demonstrated by thQ manner in which strepto- 
 cocci and pneumococci protect themselves with a capsule and resist 
 phagocytosis. Virulent strains and "resistant races" may be evolved 
 in this manner. This has been demonstrated by Ehrlich with regard 
 to the action of various arsenical compounds on protozoa, work that 
 
ANTIGENS 159 
 
 finally culminated in the brilliant discovery of salvarsan. Thus atoxyl 
 may not kill all the trypanosomes in an infected animal, those escaping 
 acquiring a new power of resistance to the poison and become atoxyl- 
 resistant. The production of "resistant races," not only among the 
 protozoa, but also in the class of bacteria, complicates enormously the 
 practical problems of immunity. 
 
 ANTIGENS 
 
 Briefly defined, antigens are substances that can cause the formation 
 and appearance of antibodies in the body-fluids. 
 
 So far as is now known, antigens are colloids, and are usually protein 
 in nature. Every known soluble protein may. in some degree act as an 
 antigen, and recent investigations would seem to show, although they 
 do not definitely' prove, that toxic glucosids and various lipoids may to 
 some extent act in this same capacity The "protein antigens may be 
 quite varied: thus antibodies are produced not only by the injection 
 of bacteria or their toxins, but also by erythrocytes, serums of different 
 animals, egg-albmnen, milk, etc. 
 
 Of the cleavage products of proteins, it isS certain that none of the 
 amiao-acids and simple poh-peptids can act as. antigens; there is, how- 
 ever, some evidence to show that the proteose^ possess antigenic proper- 
 ties. It has been shown by Gay and Robertson^ that if the antigenic 
 cleavage products of casein are resynthesized' by the reverse action of 
 pepsin into a protein resembling paranuclein, this synthetic protein is 
 capable of acting as an antigen. Protamins and globin were found to 
 be non-antigenic, although globin combined with casein formed a com- 
 pound of antigenic power in that it produced an antibody yielding 
 complement -fixation reactions with globin. 
 
 Whether the entire protein molecule, or only groups thereof, deter- 
 mine the characteristics of the antigen and thcantibody is not definitely 
 known. Wells and Osborne^ have recently submitted evidence showing 
 that a single protein molecule can act as an antigen and produce more 
 than one antibody. 
 
 Non-protein Antigens. — Ford' was able t*o immimize rabbits by 
 
 injecting a toxic glucosid contained in extracts of Amanita phalloides, 
 
 producing an antibody antihemolytic for the hemolysin of Amanita 
 
 when diluted 1 :1000. Abderhalden and others have found that specific 
 
 enzjTnotic substances appear in the blood of Animals injected with car- 
 
 1 Jour. Biol. Chem., 1912, 12, 233; Jour. Exp. Med„ 1912, 16, 479; 1913, 17, 535. 
 ^ Jour. Infect. Dis., 1913, 12, 341. ' j^ur. Infect. Dis., 1907, 4, 541. 
 
1.60 IMMUNITY. — THEORIES OF JMMUNITY 
 
 bohydrates and fats. Recent developments in iimnxinologic research 
 would indicate, therefore, that a complex toxic glucosid that can be 
 hydrolyzed by enzymes may act as an antigen. 
 
 The intimate relationship of hpoids to complement fixation reactions, 
 especially in syphilis, has natm-ally led to investigations regarding the 
 possibility of lipoids acting as true antigens. In testing for the Wasser- 
 mann reaction the use of lipoids in the form of tissue extracts to serve 
 as an antigen does not mean that it is a true antigen; in fact, experi- 
 mental work indicates quite strongly that these lipoidal substances are 
 incapable of producing antibodies when injected into animals. 
 
 Much and others have worked with lipoids secured from a strepto- 
 thrix, and which is called "nastin, " and they assert that this substance 
 may be used in immunizing animals with the production of an antibody 
 yielding complement fixation, with nastin as the antigen. Similar 
 results have been described for the fatty substances from tubercle bacilli 
 ("tuberculonastin"). Kleinschmidt "■ accepts the antigenic nature of 
 nastin in reactions, but was imable to secure antibodies by immunizing 
 rabbits with this substance. 
 
 Ritchie and Miller ^ could demonstrate no antigenic activity in the 
 lipoids of serum or corpuscles. Thiele ' calls attention to the fact that 
 Upoids possess no specificity, and that they cannot act as antigens with 
 the production of antibodies. On the other hand, Meyer * has reported 
 the production of specific complement-fixation antibodies by immuniz- 
 ing rabbits with acetone-insoluble lipoidal substances obtained from 
 various teniae. He has also found the acetone-insoluble fraction of 
 tubercle bacilli to serve as antigens in complement-fixation reactions 
 ■With antibodies of the tubercle bacillus, and much more effectively than 
 with the protein residue of the bacilli. Beigel^ has observed that after 
 injecting lecithin in rabbits an increase occurs in the lipase content of 
 the blood and tissues, with the presence of complement-fixing anti- 
 bodies, and Jobling and Bull^ have found an increase in serum lipase 
 after immunizing with red corpuscles. 
 
 It will be noticed, therefore, that the results of various investigations 
 regarding the true antigenic properties of lipoids are not in accord. 
 It should be emphasized that the complement-fixation reaction does not 
 constitute a reliable index to the study of this problem, as so little is 
 
 ' Berl. klin. Wochschr,, 1910, 47, 57. « Jour. Path, and Bact., 1913, 17, 427. 
 
 3 Zeit. f. Immunitat., 1913, 16, 160. 
 
 *Zeit. f. Immunitat., 1910, 7, 732; 1911, 9, 530; 1912, 16, 355. 
 
 6 Deut. Arch. f. Hin. Med., 1912, 106, 47. 
 
 « Jour. Exper. Med., 1912, 16, 483. 
 
ANTIBODIES 161 
 
 understood of the actual nature of this reaction itself. That lipoids 
 serve a very important purpose in the absorption or fixation of comple- 
 ment in vitro, as is so well demonstrated in Wassermann's reaction for 
 syphilis, is undoubtedly true, but this does not inthcate that the anti- 
 body in the blood-sermn of syphilitics is in the nature of a true hpoid 
 antibody, and, indeed, investigation on this' subject would seem to 
 inchcate that it is not.-"- 
 
 It will be understood, therefore, that the question of substances other 
 than proteins acting as true antigens must be regarded as an open one, 
 requiring further investigation. The relation of proteins, however, to 
 the production of antibodies has been fully established, and is at present 
 receiving renewed attention through the researches of ^'aughan and his 
 coworkers and Abderhalden. As has been sta±ed in a previous chapter, 
 Vaughan regards the protein constituents of bacterial and other cells as 
 the main antigenic principle capable of causing the production of specific 
 proteolytic ferments, which spUt the new bact-erial protein, releasing a 
 toxic product responsible for the symptoms and lesions of the infection. 
 Abderhalden has also demonstrated the presence of proteolytic ferments 
 in the blood-serum after experimental immunization with proteins, and 
 in the serum of pregnant women, due to the antigenic stimulation of 
 syncytial cells, capable of sphtting their substrata in vitro into amino- 
 ■acids and other simple cleavage-products. These investigations serve 
 .to show the intimate relation that proteins bear to the problems of 
 infection and i mm unity, and demonstrate that^antibodies may be largely 
 in the nature of ferments, and that immunologic reactions, both in the 
 hving tissues and in the test-tube, are largely in the nature of disin- 
 tegrative enzymic processes. 
 
 ANTIBODIES 
 
 The term antibody is used to designate the specific bodies produced by 
 the cells of a host in reaction against an antigen, as an infecting micropara- 
 site and its products or other foreign protein. 
 
 Various kinds of antibodies may be produced by the same antigen 
 
 and by different antigens. Some neutrahze the soluble toxin of the 
 
 antigen (antitoxin); others agglutinate or precipitate their antigens 
 
 (agglutinins and precipitins) ; still others cause complete dissolution of 
 
 the antigen (hemolysins, bacteriolysins, etc.),, and others again may so 
 
 alter the antigen and lower its resistance as to render it more easily 
 
 phagocjiiable by the body-cells (opsonins or bacteriotiopins). 
 
 ' Further discussion on the question of lipoids arting as antigens will be found 
 in Chapter XXVII in a consideration of anaphylactogens. 
 11 
 
162 IMMUNITY. THEORIES OF IMMUNITY 
 
 Specificity of Antibodies. — Antibodies are usually specific for their 
 antigen, and it is upon this general law that the-reactions of immunity are 
 based. It should be remembered, however, that not all antibodies are 
 protective; the agglutinins, for instance, apparently do not injure their 
 antigen. On the other hand, an animal may enjoy an immimity with- 
 out demonstrating the presence of any antibody in the body-fluids, and 
 another animal may show antibodies generally considered as possessing 
 protective powers, as, for example, the bacteriolysins, without neces- 
 sarily being immune. 
 
 Upon what does the specificity of antibodies and immunologic 
 reactions depend? Specificity was at first believed to depend solely 
 upon some peculiar biologic relationship of the antigens, for it was found 
 comparatively easy to diiferentiate the serum of animals of dissimilar 
 nature by means of the precipitin and other reactions, and, as serum pro- 
 teins, which seemed to be quite similar chemically, but which were 
 obtained from unrelated species, were sharply differentiated by the 
 biologic reactions, it was considered that the specificity must be depend- 
 ent upon some principle quite apart from the ordinary chemical sub- 
 stances. 
 
 With the use of proteins other than serums, and especially when more 
 or less purified proteins were employed, it has been quite firmly estab- 
 lished that specificity depends upon chemical composition, and that 
 differences in species, as exhibited by their biologic reactions, depend upon 
 distinct differences in the chemistry of their proteins (Wells) . 
 
 Pick and his coworkers have shown that two kinds of specificity 
 exist in each protein molecule: (1) One of these is easily changed by 
 various physical agents, such as heat, cold, and partial coagulation. 
 When an antigen is altered by heat, it produces an antibody that reacts 
 best with the heated antigen; heating does not, however, destroy the 
 characteristics of the antigen of this species, as its antibody will not react 
 with the heated antigen of another species. (2) The second alteration 
 involves a profound chemical change of the antigen, whereby it is so 
 altered that it loses the characteristics peculiar to the species, and pro- 
 duces an antibody that will react with the altered antigen, but not with 
 the unaltered antigen, even from the same animal. For example, it is 
 possible so to alter the serum protein of a rabbit by treatment with 
 nitric acid that the nitroprotein injected back into the same rabbit will 
 produce an antibody specific for the nitroprotein, but which does not 
 react with the unchanged serum protein. These changes are apparently 
 closely related to the aromatic radicals of the protein antigen, for they 
 
ANTIGENS 
 
 163 
 
 are effected by introducing into the protein molecules substances that 
 are known to combine with the benzine ring, e. g., iodin, diazo- and nitro- 
 groups. Pick, appreciating the fact that the number of different aromatic 
 radicals in the protein molecule are limited, interprets the significance 
 of these radicals as depending upon their arrangement, rather than upon 
 their number, in the protein molecule. Granting the number of possible 
 variations in the arrangement of the aminoacids in a protein molecule 
 which the great number of these radicals provides, there is no difficulty 
 in understanding the possibility of an almost limitless number of specific 
 distinctions between proteins. 
 
 .#-'•'' 
 
 
 t^ 
 
 AKTfTOXINS 
 
 a and 
 4nTI FERMENTS 
 
 •A"P 
 i), PREnjPITINS 
 
 Bacteria, S9ra,JnilK^anrt elhf 
 Saltiile ^r'oIifn% 
 
 Hemolysins 
 bacteriolyjins 
 (;Cbacteriotropins 
 aHJ3;,CYT0TOMN5 
 
 Fig. 43. — General Scheme of Antigens and Antibodies. 
 
 Antitoxins and antiferments: R, Receptor of a molecule of a cell; T, a toxin 
 molecule; (, toxophore group of the toxin molecule; h, haptophore group of the toxin 
 molecule; A, cast-off receptor and constitutes antitoxin. 
 
 Agglutinins and precipitins: A. R, Receptor of cell with antigen attached; B, 
 a bacterial molecule (antigen) attached to a receptor; A or P, an agglutimn or pre- 
 cipitin; h, haptophore group of the antibody; a, aggliitinophore group of an agglu- 
 tinin. 
 
 Hemolysins, etc.: A, Cast-off amboceptor (hemolysins, bacteriolysiu, etc.); 
 h, haptophore group of amboceptor; c, complementophile group; C, molecule of 
 complement. 
 
 It may be stated, however, in general, that immunologic reactions, 
 such as that of anaphylaxis, are as delicate in distinguishing between 
 proteins as are chemical analyses. Distinctions may be m^de by these 
 reactions with quantities too small for making accurate chemical de- 
 terminations. 
 
 In succeeding chapters we shall consider, first, the different kinds of 
 immimity, as dependent upon the presence of various factors, and then 
 the role played by the phagocytes and the body-fluids in immunity, 
 with a more detailed consideration of the various antibodies. 
 
164 
 
 IMMUNITY. — THEORIES OP IMMUNITY 
 
 It may be useful here to draw up in tabular form a list of the varioua 
 antigens and antibodies with which we are mainly interested in that 
 portion of immunity invohing infection with vegetable or animal para- 
 sites, and the products of their metabolism'or. degeneration (Fig. 43). 
 
 Antigens 
 Toxins: 
 
 1. Soluble bacterial toxins (diph- 
 
 theria and tetanus toxins, etc.). 
 
 2. Phyto- (vegetable) toxins (ricin, 
 
 abriii, etc.), 
 
 3. Simple zoo- (animal) toxins; 
 
 (snake, spider, toad venoms). 
 
 4. Complex zo6toxin.s, as snake 
 
 venom, requiring complement 
 for action. 
 
 Enzymes or ferments (rennin, lipase, 
 etc.). 
 
 Precipitogenous substances (soluble ani- 
 mal and vegetable proteins). 
 
 Agglutinogenous substances (bacteria, 
 erythrocytes, etc.). 
 
 Opsonigenous substances (bacterial en- 
 dotoxins or aggressins?). 
 
 Cytoligneous substances: 
 
 1. Vegetable cells (bacteria). 
 
 2. Animal cells (erythrocytes, sper- 
 
 matozoa, kidney tissue, etc.). 
 
 Antibodies 
 Antitoxins : 
 
 1. Antitoxins (diphtheria and 
 
 tetanus antitoxins, etc.). 
 
 2. Ai}ti- (phyto-) toxins (antiricin, 
 
 antiabrin, etc.). 
 
 3. Anti- (zoo) toxins (antivenins). 
 
 4. Antihemolysins, etc. 
 
 Anti-enzymes (antirennin, antilipase, 
 
 etc.). 
 Precipitins. 
 
 Agglutinins. 
 
 Opsonins (acting singly or with comple- 
 ment) . 
 Cy tolysins : 
 
 1. Bactcriolysins. 
 
 2. Hefnolysms^ spermatolysins, 
 
 nephrolysms, etc. 
 
CHAPTER IX 
 
 THE VARIOUS TYPES OF IMMUNITY 
 
 As has been stated in the preceding chapter, it is generally agreed 
 that various antibodies and other protective, agencies exist, although 
 opinions differ as to the source and relative importance of these to re- 
 sistance to and recovery from various infections. Whether or not a 
 particular antibody is derived from a certain group of cells is largely a 
 matter of individual opinion, because of the difficulty of deciding the 
 point by actual experimental evidence. Of far more importance is a 
 knowledge of the properties of antibodies and of the role they may play 
 in the processes of immunity. It is seldom that resistance to, or re- 
 covery from, an infection is dependent upon one defensive factor; 
 usually several agencies are operative, although one factor may pre- 
 dominate. For example, antitoxins arc known to neutralize their 
 respective toxins, and are of most value in combating the toxemias, 
 such as diphtheria and tetanus; bacteriolysins cause the death of and 
 may totally destroy their antigens, and play £Cn important part in the 
 recovery from infections with bacilli of the typhoid-colon and cholera 
 groups; phagocytosis in itself is of importance in staphylococcus in- 
 fections, and is of primary importance, in conjunction with the opsonins, 
 in recovery from pyogenic infections in general; agglutinins and pre- 
 cipitins do not appear to have a direct iniinical influence on their 
 antigens, but are probably secondary factors, and contribute in some 
 manner toward their destruction. Along with important non-specific 
 factors, these various antibodies are responsible for the different forms 
 of immunity, which may now be considered in their more general as- 
 pects. 
 
 There are two forms of immunity — natural^ and acquired. 
 
 NATURAL IMMUNITY 
 
 Natural immunity is the resistance to infection normally possessed, 
 lisually as the result of inheritance, by certain individuals or species under 
 natural conditions. 
 
 The mechanism of this type of immunity is very complex, and bears 
 an intimate relation to the subject of infection, both local and general, 
 
 165 
 
166 THE VARIOUS TYPES OF I]\#UNITY 
 
 the nature of the infecting parasite, and the presence or absence of 
 specific antibodies in the body-fluids. In many instances this type of 
 immunity is dependent upon non-specific cai^ses — is frequently relative 
 and seldom absolute. For example, fowls are immune to what may be 
 called an ordinary dose of tetanus toxin, but succumb readily to larger 
 doses ; rats are highly immune to diphtheria to:Sin, and readily withstand 
 the effects of an amount equaling 1000 lethal doses for a guinea-pig, but 
 still larger doses may prove fatal; hedgehogs possess complete or almost 
 complete immunity for the amount of snake venom deposited in an 
 ordinary strike, but if the venoms of several snakes are collected and 
 injected at one time, the result is fatal. 
 
 Species ijnmunity is a type of natural immunity, best illustrated by 
 the immunity of man to certain diseases of the lower animals, such as 
 fowl cholera, swine-plague, distemper, Texas cattle fever, mouse septi- 
 cemia, etc.; and, conversely, by the immunity of animals to diseases 
 common to man, such as measles, cholera, typhoid fever, scarlet fever, 
 chicken-pox, etc. Although the close relation of man to the domestic 
 animals furnishes ample opportunity for infection, yet a complete 
 immunity is frequentlj' observed. 
 
 Racial immunity is that type of natural immimity existing among 
 members of the same species. For example, negroes are believed to 
 enjoy immunity to yellow fever and Mongolians to scarlet fever. As 
 a matter of fact, well-marked examples of raciai immunity are extremely 
 rare, as not infrequentlj' the disease in question may have been acquired 
 in early infancy in a clinically unrecognized form. 
 
 Similarly, close biologic relationship is no guarantee that animals 
 will behave alike toward infection. For example, the white mouse is 
 inamune to glanders, the house mouse is somewhat susceptible, and the 
 field mouse is highly susceptible. The rabbit, guinea-pig, and rat are 
 rodents, but though the rabbit and the guinea-pig are susceptible to 
 anthrax, the rat is largely immune. Mosquitos, though closely re- 
 lated, behave differently toward, the malarial parasite. The Cvlex 
 does not carry the parasite at all, and of the Anopheles, one species. 
 Anopheles maculipennis, is quite susceptible and well recognized as a 
 carrier of the parasite, whereas Anopheles punctipennis, though closely 
 related, is not susceptible to it. 
 
 Examples of individual immunity, while not infrequent, are not con- 
 stant and seldom absolute. Certain persons appear to possess a defi- 
 nite immunity to scarlet fever and diphtheria, although they may be 
 freely exposed; others may pass through various epidemics of other 
 
NATURAL IMMUNITY 167 
 
 infectious diseases, such as measles, pertussis, etc., without becoming 
 infected. I have noticed, on several occasions, that resident physicians, 
 on service in scarlet-fever wards for many months or years, having 
 escaped infection though brought in intimate contact with severe forms 
 of the disease, finally contracted the disease uppn returning from a short 
 vacation. 
 
 Natural immunity may be due to the following causes; 
 
 1. Various non-specific factors may prevent infection; among these 
 may be mentioned — (a) The integrity of the epithelium of the skin and 
 mucous membranes, and (b) the chemical and physical action of various 
 secretions, such as the gastric fluid, the intestinal juices, and the saliva. 
 
 2. A particular route for the introduction of infecting microparasites 
 may be necessary. For example, intestinal diseases, such as typhoid 
 fever and cholera, are usually due directly to swallowing of the infecting 
 microorganisms, infection in this type of disease seldom, Lf ever, occurring 
 through the skin. This is probably due in part to the lowered vitality 
 of the intestinal mucosa, together with a peeuliar selective afEnity of 
 the bacteria for the cells of these tissues, aided by — (a) the biologic 
 nature of the invading bacterium, which grows best under the more 
 favorable cultural conditions of the intestinal canal. This selective 
 action is further illustrated by the tendency of dysentery toxin to attack 
 the intestinal mucosa when the bacilli or tdxin is administered intra- 
 venously. 
 
 3. Certain tissues appear to possess a marked local immunity to certain 
 bacteria. In considering examples of local immunity, various factors, 
 such as the question of exposure, the thickness "of the epidermis, and the 
 kind and quantity of the local secretions must be borne in mind. 
 For example, Trichina spiralis affects the rnuscles, never the bones, 
 and but rarely any other tissue. Likewise, although diphtheria in the 
 throat may spread in many directions, it seldom passes down the esopha- 
 gus. 
 
 Some differences are known to exist in regard to local immunity as 
 observed in the child and in the adult. For example, ring-worm of the 
 scalp is practically unknown among adults,, whereas children under 
 seven years of age are quite susceptible to the disease. These differences 
 may be due to the greater susceptibility in general of young tissues to 
 infection, and the local immunity constitutes but an index to the gen- 
 eral rise in resisting power accompanying improvement in strength and 
 vitality. In some cases this may be due perhaps to an actual strengthen- 
 ing of local tissues, as in the case of the adult.vaginal mucosa, which is 
 
168 THE VARIOUS TYPES OF IMMUNITY 
 
 immune to the gonococcus, whereas the thin and immature infantile 
 membrane is peculiarly susceptible. 
 
 In general, our knowledge of local immunity is quite incomplete. 
 The subject is a difficult one, hence most attention has been given to the 
 study of general immunity. A striking example of acquired local im- 
 munity may be seen in a patch of psoriasis, where the center is observed 
 to be largely free from scales, whereas the margins are quite active. 
 
 The question of local immunity may be largely determined by vari- 
 ous local non-specific factors, such as loss of blood supply due to trau- 
 matism, thrombosis, tight bandaging, etc., and the action of severe 
 irritants, tending to produce necrosis of the tissues. 
 
 4. The importance of phagocytosis in natural immunity must be em- 
 phasized. Microorganisms are constantly gaining entrance to the 
 tissues through numerous small abrasions of the skin and along the in- 
 testinal and respiratory tracts, and investigations have shown how im- 
 portant the wandering cells are in preventing infection, being ever on 
 guard and ready to pick up and dispose of any irij'urious material. Even 
 after mild infection has occurred, the local iiiflammatory reaction in 
 which the phagocyte is a prominent factor may be so prompt in over- 
 coming the invaders that the host will escape serious infection. 
 
 The natural immunity of the frog to anthrax has been shown to be 
 partly dependent upon the activity of the leukocytes in engulfing and 
 disposing the bacilli. 
 
 Similarly a mild irritant may produce hyperemia and exudation or 
 local accumulation of leukocytes, which aid in establishing a local im- 
 munity largely dependent upon phagocytosis. In this manner the 
 intraperitoneal injection of sterile bouillon or even of salt solution may 
 produce exudation and increase the immunity to infection. 
 
 5. It may be that even after the introduction of a microorganism or its 
 toxin no harm results because of a lack of suitable receptors on the part 
 of the body-cells of the host for union vnth the pathogenic agent. For 
 example, tetanus toxin, being unbound by the cells, produces no 
 effect on the turtle, and antitoxin is not produced. On the other hand, 
 suitable receptors may be present that will bind the toxin, but produce 
 no harmful effects because the body-cells are not susceptible to the 
 action of the microparasite or its products. Thus it is asserted that 
 tetanus toxin has no effect upon the alhgator, although the toxin is 
 bound and antitoxin is produced by its body-cells. 
 
 In other instances, a host may escape infection owing to the fact 
 that there is a lowered affinity between a pathogenic agent and the 
 
NATURAL IMMUNITTl 169 
 
 body-cells, so that but a small amount of harmful substances are bound 
 to the liody-cells, and no particular harm results, whereas a larger dose, 
 uniting with a greater number of cells, is capable of producing some dis- 
 turbance. 
 
 6. A natural antitoxin immunity may exist, as the immunity of the 
 alligator to tetanus toxin, just mentioned. Similarly natural diph- 
 theria antitoxin may prevent infection, especially in those persons 
 known to harbor virulent bacilli in the fauces. In such instances, how- 
 ever, it is difficult to exclude entirely the possibility that a previous 
 minor infection has occurred, as natural antitoxin immunity persists 
 much longer than the passive immunity resulting from the administra- 
 tion of an antitoxin serum. 
 
 Otto, who has recently investigated the content of diphtheria anti- 
 toxin in the blood of normal persons, found mpre than ywii o^ ^ ^^^^ o^ 
 antitoxin in each cubic centimeter of the blood of those who had been 
 in close contact with cases of diphtheria without having been sick them- 
 selves; others usually had much less. Observations would tend to 
 show that this quantity of antitoxin is generally sufficient to confer 
 immunity to diphtheria, and the object of von Behring's method of 
 active immunization is to induce the production of at least that much 
 antitoxin by the body itself. Otto found that diphtheria carriers, both 
 those who had had the disease and those who" had not, contained more 
 antitoxin in their blood than did patients who had just recovered from 
 an attack. This shows that the mere presence of bacilli in the throat 
 is sufficient to stimulate the production of antitoxin, on which the im- 
 munity of the carrier himself would seem to depend. Undoubtedly 
 physicians and nurses who are freely exposed to diphtheria and yet 
 escape infection owe their safety rather to aft acquired immunity the 
 result of repeated contact with the bacilli, th^n to a natural antitoxin 
 immunity. 
 
 7. In some instances a natural immunity may he dependent, at least 
 in part, upon antibacterial activity, due to the presence of bacteriolysins and 
 bacteriotropins in the body-fluids, as, for example, that shown by the dog 
 and the rat to anthrax. In other instances, however, a similar immunity 
 may be observed that cannot be ascribed to the presence of antitoxins or 
 bacteriolysins. In this type of immunity micfoparasites are apparently 
 unable to sustain themselves, and proliferate, in one animal, although 
 aggressive enough in another of the same species. 
 
 8. An immunity to infection, especially with such microorganisms as 
 the anthrax bacillus, which is markedly aggressive and but slightly toxic, 
 
1-70 THE VARIOUS TYPES OF IMMUNITY 
 
 may he due to the presence or production of dnti-aggressins. This im- 
 munity would seem to depend not upon the bactericidal properties of 
 the serum or leukocytes nor upon the antitoxins, but on the presence of 
 substances that prevent the microorganisms from exercising their special 
 aggressive forces. 
 
 9. Finally, an immunity may exist because the parasite or other for- 
 eign cell does not obtain suitable nutrition in a host and thus fails to grow. 
 This condition of athrepsia, is responsible for what has been called athreptic 
 immunity. It has been more recently studied by Ehrlich, who found 
 that upon transferring mouse cancer to the rp,t, the tumor grew for a 
 short time only, or presumably until the special nutriment carried over 
 with the tumor was consumed. While there' is no experimental basis 
 for assuming that a similar condition may be present in bacterial life, 
 yet such a cause may be operative and should be kept in mind. 
 
 ACQUIRED IMMUNIXY 
 
 Acquired immu7iity occurs in two distinct forms: (1) Active and (2) 
 Passive. A mixed form may exist, brought about by a combination 
 of factors necessary for the development of the other two. 
 
 Active Acqtiired Immunity. — Active acquirM immunity is that form of 
 resistance to infection brought about by the activity of the cells of a person 
 or animal as a result of having had the actual ifisease in question, or as a 
 result of artificial inoculation with a modified or attenuated form of the 
 causitive microparasite. 
 
 The essential feature of this immunity is that the cells and tissues 
 of persons or animals should, by their own efforts, and as a result of 
 their own active struggle against the action of a microparasite or its 
 products, overcome these and become less susceptible to them than they 
 were before. 
 
 This form of immunity is gained, therefore, only as the result of an 
 active struggle between body-cells and infecting agent, and this battle 
 may be of any degree of severity, ranging from an attack of the disease 
 itself that may threaten life, down to the most transitory and trivial re- 
 action due to the injection of a minute dose of,a mild vaccine. 
 
 Active acquired immunity may be gained — (1) By accidental infection, 
 Which is the most famiUar form of acquired immunity, and follows an 
 attack of an infectious disease, such as scarlet fever, measles, varicella, 
 variola, or typhus fever; (2) by inducing an attack of the disease by arti- 
 ficial inoculation. This latter method of producing an active acquired 
 
ACQUIBED IMMUNITY 171 
 
 immunity was illustrated by the ancient, obsolete, and discarded prac- 
 tice of smallpox inoculation, by which healthy persons were inoculated 
 with the virus of a mild case of smallpox, at a time when no epidemics 
 existed and the person was in good general health and able to secure 
 proper attention from the outset. 
 
 This process of immunization is used much more extensively in 
 veterinary practice, where an occasional untoward or fatal result is of 
 comparatively little importance if by its means an outbreak can be con- 
 trolled or the great majority of the animals saved. As a rule, an at- 
 tempt is made to render the induced disease as mild as possible by — (a) 
 Using a small amount of infective material; (b) By inoculating it through 
 an unusual avenue; or (c) by inoculating it at^a time when the animals 
 are naturally less susceptible, or (d) by a combination of these methods. 
 For example, Texas cattle fever, which is due to a protozoan (Piro- 
 plasma bigeminum) conveyed by the bites of infected ticks, may be 
 combated by exposing calves while still milk fed to the bites of a few 
 infected ticks. Another method consists in injecting a small amount of 
 blood from an infected animal directly into the jugular vein. The ob- 
 ject is to induce a mild attack of the disease. Occasionally a severe or 
 fatal reaction occurs, but the number of these untoward results is much 
 lower than the mortahty among untreated animals. 
 
 (3) Active immunity may also he gained hy. vaccination, i. e., by in- 
 oculation with a virus or microparasite or its products, modified and 
 attenuated by passage through a lower animaj (Jennerian vaccination) 
 or by various other means, as age, unfavorable cultural conditions, heat, 
 germicides, etc. (Pasteurian vaccination or bacterination). These subjects 
 are considered more fully in the chapter on Active Immunization. 
 
 Active immunity, whether induced accideiltally or artificially, may 
 be antitoxic, as after recovery from diphtheria b*r as the result of active 
 immunization with diphtheria toxin, as by von Behring's method; or 
 antibacterial, as the immunity following typhoid fever or induced by 
 typhoid vaccination, and largely dependent u|ion the presence of bac- 
 teriolysins in the circulating fluids. 
 
 During the process of active immunization an animal not infre- 
 quently fails to react to relatively large doses of toxin, and at the same 
 time the quantity of antibody in the body-flb'id may decrease. This 
 phenomenon has been explained as being due to atrophy of the receptors 
 of the body-cells (receptoric atrophy), wherebjj the toxin fails to exert 
 its deleterious influence because it fails to unite with the bodj^-cells. 
 It is curious, however, that the toxin is innocUous when present in a 
 
172 THE VAEIOTJS TYPES OF IMMUNITY 
 
 free state within the body-fluids, even thougll unbound to the body- 
 cells; this condition is not well understood, and may be dependent upon 
 other factors. A rest may restore the activity of the receptors and cells, 
 a fact that is well recognized in the immunizationof horses for the prepara- 
 tion of antitoxin. Not infrequently a rabbit fa;ils to produce hemolytic 
 amboceptor if the injections of erythrocytes are too frequent. After 
 a rest, however, the animal may react promptly with much smaller 
 doses. 
 
 Passive Acquired Immunity. — As the name indicates, this is a form 
 of immunity that depends upon defensive factors not originating in the 
 -person or animal protected, hut is passively acquired by the injection of 
 serum from one that has acquired an active immunity to the disease in 
 question. 
 
 This is a sort of secondary immunity, acquired by virtue of haying 
 received antibodies actively formed by another animal that has had to 
 resist the infecting agent in order to produce them. Two well-known 
 examples of this type of serums are the diphtheria and tetanus anti- 
 toxins. These are produced by injecting horses with successive doses 
 of the respective toxins. The horses are requited to combat the effects 
 of the toxins, and acquire an active innnunity of increasing grade, due to 
 the production of antitoxins. When the animals are bled, the antitoxin- 
 laden serum, separated from the corpuscular elements, may be used for 
 conferring an immunity in a person or another toimal simply by injecting 
 the serum, the latter receiving and enjoying an immunity in a passive 
 manner. 
 
 Passive immunity is specific, that is, the , serum of an animal im- 
 munized against one microorganism will protect a second animal against 
 that and against no other. This type of irmnunity is acquired just as 
 soon as the immune serum has become mixed with the blood of the per- 
 son or animal injected, and there is no negative phase. Hence in 
 severe infections our hopes of specific therapy rest on the production of 
 passive immunity. No matter how sick the -recipient may be, under 
 ordinary circumstances the immune serum produces no further dis- 
 turbance than would be expected from the injection of a normal serum. 
 The recipient's body-cells have no additional burdens, or very slight 
 ones only, to bear and these are more than counterbalanced by the re- 
 lease from combat with toxic substances neutralized bj- the antibodies 
 in the immune serum. Unfortunately, this fi,eld of therapy is limited, 
 although recent discoveries are indicating the reasons for failure, and 
 when these are eliminated, the field of usefulness will be much extended. 
 
ACQUIRED IMMUNITY 173 
 
 Passive immunity is of shorter duration than active immunity, and 
 the former is especially indicated in prophylaxis for warding off an 
 acute infection that has a relatively short incubation period. The de- 
 gree of passive immunity is also seldom equal to that of an active im- 
 munity. The antibodies produced by our own cells are more lasting 
 and possess higher protective value. This is an important factor in 
 von Behring's method of immunization in diphtheria, when a small 
 amount of toxin loosely bound to antitoxin is injected in the~belief that 
 the toxin becomes dissociated and sei-ves to stimulate our body-cells into 
 producing our own antitoxin. 
 
 Passive acquired immunity is usually antitoxic, as, for example, 
 that induced by the administration to man of diphtheria antitoxin 
 prepared by the body-cells of the horse. Antibacterial serums may 
 likewise induce a passive immunity, as, for instance, that used in im- 
 munization against plague. 
 
 It is evident, therefore, that the processes whereby infections are 
 overcome and immunity is conferred, and the general reactions that 
 follow the introduction into the body of modified antigens in the prac- 
 tice of immunization, are complex processes, and in none is one anti- 
 body produced or solely responsible for the resulting immunity. The 
 properties and action of the known antibodies are considered in sub- 
 sequent chapters, particular attention being given to methods for de- 
 termining their presence in the body-fluids, which serve as an aid to the 
 diagnosis of infection as based upon the general law that the antibody 
 is specific for its antigen, and so, when the presence of an antibody 
 is demonstrated, it may be assumed that the antigen is or has been 
 present. 
 
 Nothing is known concerning the nature of the immunity that is ac- 
 quired against several infections, such as scarlet fever, measles, small- 
 pox, etc., nor will much be known until the causes that give rise to these 
 conditions have been discovered. 
 
 Theory of Vaughan. — According to Vaughan, the inability of a bac- 
 terial cell to grow in the animal body either because it cannot feed upon 
 the protein of the body or because it is itself destroyed by the ferments 
 elaborated by the body-cells explains all forms of bacterial immunity, 
 either natural or acquired. Thus in antitoxin immunity the toxin is 
 regarded as a ferment that splits up the proteins of the body-cells, setting 
 the protein poison free. The body-cells react with the formation of an 
 antiferment or antitoxin, which neutralizes the toxin and prevents 
 cleavage. The toxin itself is regarded as harrfiful only in so far as it is 
 
H4 
 
 THE VARIOUS TYPES OF Il^MUNTTY 
 
 able to set free the protein poison responsible for the symptoms of the 
 infection. 
 
 Natural immunity to any infection, according to Vaughan's theory, is 
 explained as being due to an inability of the infecting agent to grow in 
 the animal body. 
 
 Acquired immunity, due to recovery from dA infection or occurring as 
 a result of vaccination, is regarded as the outcome of the developrrent 
 in the body, during the course of the infectivfe process, of a specific fer- 
 ment that, on renewed exposure, immediately destroys the infection. 
 The vaccine is the same protein that causes tHe disease, so modified that 
 it will not produce the disease, but yet so little altered that it will stim- 
 ulate the body-cells to form a specific ferment that will promptly and 
 quickly destroy the infecting agent on exposure. 
 
 The various kinds of immunity and the factors probably concerned 
 in their production, may be summarized as follows : 
 
 1. Due to non-specific factors: 
 
 1. Barrier of epithelium. 
 
 2. Various secretiojis. 
 
 3. A particular route of infection may be nec- 
 essary, aided, by the biologic nature of 
 the invading bacterium. 
 
 Natural Immunity 
 
 Acquired Immunity 
 
 2. Due to local tissue immunity and selective ac- 
 tion of micro-parasites for certain tissues. 
 .3. Due to phagocytosis., 
 
 4. Due to lack of suitable receptors of body-cells 
 
 for a particular bacterium. 
 
 5. Due to natural antitoxins. 
 
 6. Due to natural bacteriolysins. 
 
 7. Due to anti-aggressins. 
 
 8. Due to lack of suitable food matf-rial — athrep- 
 
 sia. 
 
 Active (accidentally or 'I 1, Antitoxic, 
 artificially acquired) J 2. Antibacterial. 
 
 1. Antitoxic.' 
 
 2. Antibacterial. 
 
 Passive 
 
CHAPTER X 
 PHAGOCYTOSIS 
 
 Historic. — As Lord Lister stated in 189fi, "If ever there was a 
 romantic chapter in pathology, it has surely been that of the story of 
 phagocytosis." The author of this "story" is Elie Metchnikoff. His 
 early researches on phagocytosis in the lowly* organized forms of life 
 constituted the starting-point for an entirely new series of researches on 
 the subject of Immunity, and his treatise on the "Comparative Pathology 
 of Inflammation" must ever remain a most entertaining work and a 
 medical classic. 
 
 Several observers before Metchnikoff had considered that leuko- 
 cytes might assist in bringing about the destruction of microparasites. 
 Panum (1874) suggested that the bacilli of putrefaction might, by mak- 
 ing their way into the blood-corpuscles and being carried off to the 
 lymph-glands, spleen, etc., thus disappear from the body-fluids. Carl 
 Roser (1881) had also observed the ability of certain "contractile cells" 
 to ingest the enemy that enters the animal Ijody. These statements, 
 however, were poorly supported by scientific data, and the subject was 
 not followed in subsequent research. As the result of zoologic studies, 
 Metchnikoff was led to discover the part pljjyed by body-cells in the 
 processes of immunity. He observed that when a food-particle arrives in 
 the vicinity of a simple unicellular organism as an ameba, the latter, 
 by reason of its "irritability, " moves forward and sends out processes of 
 its protoplasm (pseudopodia) that flow around the particle and finaUy 
 gather it into the interior of the cell. The particle then undergoes a 
 process of intracellular digestion, losing its sharp outUne and clear ap- 
 pearance, becoming granular, and disappearing within the protoplasm 
 of its host. Similar studies were made of the processes of nutrition in 
 many unicellular animals, then through actininas, sponges, and similar 
 animals of transparent and simple organization. 
 
 Metchnikoff was primarily a biologist, and up to this time was 
 mainly interested in the processes of nutrition in the simple forms of 
 animal Ufe. At this stage, however, he became greatly impressed by 
 Cohnheim's description of the phenomena of inflammation. The dia- 
 pedesis of leukocytes through the walls of the blood-vessels in an 
 
 175 
 
176 
 
 PHAGOCYTOSIS 
 
 inflammatory reaction impressed him as significant and similar to the 
 movements of the ameba in its process of feeding, and led to com- 
 parative studies in inflammation in the lower forms of life, where 
 processes were much simpler to watch and may indicate what occurs 
 in the higher vertebrates. 
 
 He found that when daphnias are invaded by the spores of a yeast, — 
 the monospora, — they may multiply in the bedy of the host and bring 
 about its destruction. When, however, a few spores gained access, he 
 found that the daphnia's leukocytes approached them, formed a wall 
 around them, and finally brought about their destruction by a process 
 of digestion. He also observed that if rose prickles were stuck into 
 large bipinnaria larvse of star fish, these were soon enveloped in a mass 
 of ameboid cells. From this he concluded that, in inflammation, the 
 exudation of leukocytes may be regarded as a reaction against any sort 
 of injury, whether mechanical or due to bacterial invasion. 
 
 Metchnikoff traced this defensive reaction against an invading 
 microorganism from small invertebrates up to man, and showed that 
 instead of bacteria attacking leukocytes and forcing a passage into them, 
 as was then believed, they were indeed pursued and engulfed by the 
 leukocytes. Connecting his various discoveries, he was able to formu- 
 late the idea that "the intracellular digestion of unicellular organisms 
 and of many invertebrates had been hereditarily transmitted to the 
 higher animals, and retained in them by the arneboid cells of mesodermic 
 origin. These cells, being capable of ingesting and digesting all kinds 
 of histologic elements, may apply the same power to the destruction of 
 microorganisms." To a cell, and especially to a leukocjrte, possessing 
 this activity and power he applied the name phagocyte, because of its 
 ability to act as a scavenger, gathering up the living and dead material. 
 
 THE ORIGINAL THEORY OF PHAGOCYTOSIS 
 
 The theory as originally adduced by Metchnikoff regarded the leu- 
 kocytes and certain other cells as specific fighting cells, able to engulf 
 and consume living as well as dead bacteria and cellular debris. The 
 outcome of any infection would depend upon the success or failure of the 
 phagocytes to overcome the invaders: if they were successful in in- 
 gesting all the bacteria, their victory meant recovery; if, on the other 
 hand, they were destroyed by the invaders, the host was overcome by 
 the infection. 
 
 Phagocj'tes may ingest not only living and dead bacteria, but also 
 
Fi(i. 14, — Fir,Afjo< 1 joMH — AlAcjioriiAtjKs. 
 A snjciir of |i(^iif,oncal cxiidalo from a ffiiiiicii-pin twejify-foiir houi'H after inji'r- 
 tion witli '■', c.e. of a 5 per i:cut. sUHpciiKioii of pinoori'H fllooil. Note that the corpUBcles 
 (nucleated) are l>oiii;^ iii^cslcd by the Ihtk'' endothelial cclla of the pi-ritoni'iim. 
 
 ¥10. 45. — PuAOorvTOSis — MAfiiopiiAfir.s. . 
 A smear of peritoneal exiid.'i.te froro tfje same Kijine:i-|)jff forty-ei(;ht lioiirH after 
 injection with piju'on's Mood. Note fhe lfir<:e numbefK of corpusdcH ir]p;ested by the 
 endothelial cells. In most, in.starifo.s the ''orpiit^rle.-; have unrlerKone dlKestion, the 
 nuclei beiJ]^ more re.-:i.sl!irit. These nuclei are .shrunken, and in .some inHtance,-; are 
 broken up. The extracellular red blooil-corpu.scle,-, are swollen and stain lightly. 
 
TARTETIES OF PHAGOCYTES 1 / 7 
 
 red corpuscles, cellular debris, inorganic particts. such as coal-dust, and 
 even soluble substances, such as bacterial toxins. 
 
 Subsequent discoveries have shown that many other factors are 
 present that considerably modify the workings of so simple a process. 
 Metchnikoff has, therefore, modified his theorj- from time to time as 
 new discoveries were made, but has always preserved the primarj" im- 
 portance of the phagocj"te itself. 
 
 Before stating the revised theory as it stan^ todaj', we wiU describe 
 the kind of cells that may act as phagoc>i:es, and consider the methods 
 and reasons whj" these cells assume the functions of phagocji:es. 
 
 VARIETIES OF PHAGOCtTES 
 Xot only leukocytes, but other bodj'-cells ■ have been found active 
 in the processes of phagocj-tosis. Metchnikoff has divided the phago- 
 £^1:65 into two great classes : 
 
 1. ^ficTophage.s — ^principallj' the polynuclear neutrophilic leukoc}"tes. 
 ''See Fig. 46.) The eosinophiUc leukocytes are also included in this group, 
 but are of doubtful importance and weak in phagocytic powers. The 
 small lymphocj'te may be included in this class, although it is usuaUj' con- 
 sidered with the second group. 
 
 2. Macrophages, principally the large mononuclear leukocj-tes; 
 ameboid cells of the spleen and hinphatic glajids; alveolar cells of the 
 lung; endotheUal cells of the serous ca^'ities &nd lymph-spaces: bone- 
 corpuscles and giant-ceUs of bone-marrow and embrj'onic connective- 
 tissue cells Tigs. 44 and 4.5). 
 
 The most important are the leukocytes, especially the poljTiuclear 
 leukocj-tes and the large hTnphocj-tes of the blood. AH the leukocji:es, 
 however, have phagocjiiic powers, as is weU seen in opsonic determina- 
 tions. Eosinophiles are seldom known to ingest bacteria, but in in- 
 fections with animal parasites, or after the injection of extracts of ani- 
 mal parasites, both a local and a general increetse in eosinophilous forms 
 inay be observed. 
 
 Small hTnphoc}-tes are much less active thgji the large, presumably 
 because they contain less of the mobile cT,i:opl!asm, and consist chiefly 
 of the structuralh' fixed nuclear substance, and while they take up but 
 a. small number of bacteria, they may be obser\'ed to contain various 
 other cells, such as red corpuscles and cellular jdebris. 
 
 Besides the leukocj-tes, some of the tissue-cells which are free or 
 have the power of becoming so are actively phagocytic. Endothelial 
 12 
 
178 PHAGOCYTOSIS 
 
 cells of the lymph-spaces and serous cavities are especially active, not 
 only in the phagocytosis of other cells and Cellular debris, but also of 
 various bacteria. In exceptional instances epithelial cells may act as 
 phagocytes. In the presence of an irritant these cells may become de- 
 tached and act as phagocytes; this is exempHfied in the case of chronic 
 passive congestion of the lungs, in which the alveolar cells of the lung 
 ingest the hemosiderin formed and deposited (heart failure cells). 
 
 Relation of the Cell Types to Infection.— The kind of cells that take 
 piart in phagocytosis is determined to some extent by the nature of the 
 irritant. Thus in acute pyogenic infections the polynuclear cell is 
 found to be most active. (See Fig. 46.) It is extremely rare to find these 
 cells containing bacilli in the tissues, although they will take them up 
 readily enough under the artificial conditions of an opsonic determina- 
 tion. (See Fig. 53.) In chronic bacterial infection, such as tubercu- 
 losis and syphihs, and in infections with various fungi, the small lympho- 
 cyte and macrophages are the types most concerned. 
 
 Experimental evidence regarding lymphocytic activity is quite con- 
 tradictory. Undoubtedly many of the cells in the lymphocytic accumu- 
 lations seen in such conditions as tuberculosis.and syphilis arc not really 
 lymphocytes from the blood, but are newly formed cells of the tissues. 
 There is no direct means of inducing experimentally a local accumula- 
 tion of lymphocytes similar to that induced by most any irritant, 
 resulting in an outpouring of polynuclear cells. Long-continued in- 
 toxication of animals may result in increasing the numbers of lympho- 
 cytes, but the local introduction of the toxin leads to an accumulation 
 of polynuclear cells, rather than lymphocytes. Reckzeli ' found that 
 in lymphatic leukemia, in which the lymphocytes greatly exceed the 
 polynuclears, the pus from an acute lesion or the fiuid from the vesicles 
 produced by cantharides, contained practically no IsTnphocytes, but 
 was composed of the usual polynuclear cell f oyms. Wlassow and Sepp ^ 
 state that lymphocjrtes are not capable of ameboid movement or 
 phagoc3rtosis at ordinary body temperature; Wolff, ^ on the other hand, 
 claims that tetanus and diphtheria toxins produce lymphocytosis in 
 experimental animals, and Zieler^ claims that in the skin of rabbits 
 exposed to the Finsen light active migration of lymphocji;es takes place 
 during the reaction. General lymphocytosis may be produced experi- 
 mentally by the injection of pilocarpin and muscarin, but these bear no 
 relation to the vital process of phagocytosis, as they are apparently ex- 
 
 ' Zeit. f. klin. Med., 1903, 50, 51. = Virchow's Archives, 1904, 176, 185. 
 
 ' Berl. klin. Woch., 1904, 41, 1273. * Central.f. Pathol., 1907, 18, 289. 
 
Fig. 46. — Positive Chemotaxis. Phabocytosis of Staphylococci. 
 
CHEMOTAXIS 179 
 
 trudcd from the lymphoid organs by contraction of the smooth muscles 
 (Harvey^). 
 
 As previously stated, the eosinophiles undergo a marked increase 
 during infections with various animal parasites?. 
 
 The typical macrophages, such as the endothelial cells of serous 
 cavities (Figs. 44 and 45) and the lymph-spaces, are mostly concerned in 
 the phagocytosis of other cells and inorganic material. Brodie ^ con- 
 siders the phagocytosis of leukocytes and red corpuscles by the endo- 
 thelial cells of the lymph-glands and the spleen as the normal end of 
 these cells. It is a mistake to believe, however, that they do not in- 
 gest bacteria, since endothelial cells are extremely active phagocytically 
 for bacteria, and, on the other hand, polynuclear leukocytes may be 
 observed to contain red corpuscles, especially when aided by a suitable 
 opsonin, 
 
 CHEMOTAXIS 
 
 An important question in the study of the phenomena of phago- 
 cytosis is the manner in which the various leukocytes and other body- 
 cells are attracted to a focus of infection and brought into contact with 
 the microparasites or other foreign substances. It must be assumed 
 that some means of communication must exist between this point and 
 the leukocytes in the circulating blood. Since there is no direct com- 
 munication by way of the nervous system or other structural route, it 
 would appear that the only mode of communication is through the body- 
 fluids. Chemical agencies, 'produced either directly by the bacteria or other 
 foreign substance, or indirectly by their action upon cells at the site of resi- 
 dence in the tissues, are regarded as furnishing the attractive forces that are 
 transmitted through the body-fluids and exert what has been called chemo- 
 taxis. 
 
 The movement of a cell in response to a chemical stimulus is a phe- 
 nomenon that is displayed by almost all motile and unicellular organ- 
 isms, whether animal or vegetable, and by the leukocytes and other un- 
 fixed cells of the higher animals. As a rule, chemical stimuH serve to 
 attract cells to the site of infection, thus constituting what is known as 
 positive chemotaxis; on the other hand, the stimuli may fail to attract 
 or actually repel the cells, or be so powerful as to paralyze tham en 
 route, this constituting negative chemotaxis. 
 
 Positive Chemotaxis. — That leukocytes reach the site of an infec- 
 tion because of chemical substances produced by bacteria at this point 
 
 1 Jour. Physiol., 190e, 35, 115. » Jour. Anat. and Physiol., 1901, 35, 142. 
 
180 PHAGOCYTOSIS 
 
 was first clearly demonstrated by Leber ^ in 1879. This writer ob- 
 served that in keratitis leukocytes invadeU the avascular cornea 
 from the distant vessels, not in an irregular manner, but direct to the 
 point of infection, where they accumulated. As dead cultures of 
 staphylococci produced a similar although a- less pronounced accumu- 
 lation of leukocytes, Leber sought the chemjotactic substance in their 
 bodies, and isolated a crystalline, heat-resisting substance — phogosin — 
 which attracted leukocj^tes in the tissues. 
 
 Since these fundamental studies were made many other investiga- 
 tions, with various chemical substances of many different origins, have 
 been undertaken upon leukocytes, amebse, ciliata, and plasmodial forms, 
 indicating that chemical substances are mainly concerned in exerting 
 either a positive or a negative chemotactic influence. 
 
 Experimental evidence tends to show that cells respond to stimuli 
 of various kinds chiefly through the effect of these stimuli upon surface 
 tension: if they decrease the surface tension, the cell goes forward; if 
 t^iey increase the tension, the cell recedes. 
 
 The behavior of leukocytes in inflammation may be explained on 
 these purely physical grounds. At the site of cell injury or infection 
 chemical substances are produced that tend to -lower the surface tension 
 of leukocytes and thus exert a positive chemotactic influence. These 
 chemical stimuli are transmitted by the body-fluids to the nearest cap- 
 illaries, where they enter through the vessel-wall and come in relation 
 with the slowly moving peripheral leukocytes. The leukocytes will be 
 brought into touch by the chemotactic substances most largely on the 
 side from which the substances diffuse ; accordingly, the surface tension 
 being least nearest the stomata in the capillary wall, this results in the 
 formation of pseudopodia, and motion in this direction, dragging the 
 nucleus along in an apparently passive manner. Those cells, therefore, 
 containing most of the mobile cytoplasm, sucl^ as the polynuclear leuko- 
 cyi;es, are chiefly affected in these processes; those containing little 
 cytoplasm and a relatively large and dense nucleus, such as the lympho- 
 cytes, are affected primarily to a much less extent. 
 
 Once outside of the vessel-wall, the leukocytes tend to move toward 
 the focus from which the chemotactic substance comes. If the leuko- 
 cyte meets a substance that greatly lowers its surface tension, it will flow 
 around the object and inclose it, this constituting phagocytosis. The 
 toxins of the ingested bacteria may kill the oell, or so equalize surface 
 tension that movement ceases. Otherwise thfe leukocytes tend to move 
 » Fortschritte der Med., ISSS, 0, 460. 
 
CHBMOTAXIS 181 
 
 forward until checked by any one of several influences, as pointed out 
 by Wells, — as (1) Until the chemotactic substance has been used up or 
 removed, or from any of the causes that terminate inflammation; (2) 
 the leukocytes may reach a point where the chemical stimulant is so 
 generally diffused that surface tension is decreased equally in all di- 
 rections and motility stops; (3) the leukocytes' may reach a place where 
 toxins or other chemical substances coagulate their cytoplasm or fer- 
 ments cause their solution ; (4) they may be Mocked by a dense wall of 
 leukocytes and other cells while being held fixed by the chemical attrac- 
 tion that diffuses through this wall. These factors would explain the 
 formation of the wall of leukocytes about an area of infection. When, 
 for example, the abscess has ruptured or has been incised, with removal 
 of the chemotactic substances, there may be less chemotactic substances 
 in the center of the inflamed area than there is further out; hence the 
 leukocytes will move away from the center toward the periphery, follow- 
 ing the chemotactic substances back into the, blood-vessel and lymph- 
 stream. This would explain the dispersion of living leukocytes at the 
 close of an inflammatory process. 
 
 General leukocytosis can be explained equally well by assuming that 
 the chemotactic substances from the area of inflammation, reaching the 
 blood-stream, pass through the bone-marrow, lowering surface tension 
 and attracting leukocytes into the blood-stream as long as it contains 
 more chemotactic substances than the marrow. 
 
 The exact chemical nature of chemotactie substances is unknown. 
 In bacterial infection the toxins, and especially the protein of dead micro- 
 organisms, are regarded as mainly responsible for the occurrence of posi- 
 tive chemotaxis. Chemotaxis and phagocytosis of chemically inert 
 particles, such as coal-dust, stone-dust, and pigments, are more difficult 
 to explain on this physical basis of alteration in surface tension. It is 
 probable that the death of tissue-cells, brought about by these materials, 
 may produce the chemical stimulant responsible for a mild but defuiite 
 chemotactic influence. Although the movemeat of amebse and similar 
 higher animals cannot be fully explained on- this physical basis, the 
 surface tension theory best explains leukocytic movement. Although 
 the ameba may possess some special property that endows it with the 
 power of selecting and engulfing a food particle, it would appear to be 
 entirely unreasonable to assume that a simple, undifferentiated, and 
 naked leukocyte possesses similar powers. The physical theory, there- 
 fore, appears to be the most reasonable offered in explanation of the 
 ameboid movements of these simple cells. 
 
182 PHAGOCYTOSIS 
 
 Negative Chemotaxis. — In nearly all infections we find that leuko- 
 cytes are attracted in large numbers into the involved area, i. e., nearly 
 all bacteria give off substances that are positively chemotactic. In cer- 
 tain infections, however, we may find the tissues poor in leukocytes, as 
 exemplified in infections due to the presence of virulent streptococci. 
 This negative chemotaxis is more difficult of explanation. Kantlack 
 doubts the existence of really negative chemotactic action upon leuko- 
 cytes. Verigo^ also considers that as yet no actual negative chemo- 
 tactic substances have been satisfactorily danonstrated; certainly no 
 marked example of negative chemotaxis has been shown since methods 
 involving the study of phagocytosis in vitro have been devised. It is 
 true that virulent bacteria appear to repel the leukocytes, but, as 
 Kantlack has pointed out, these are not necessarily examples of nega- 
 tive chemotaxis, and it is probable that the paucity in mmibers of the 
 leukocj^es about such an area of inflammation is due to their over- 
 stimulation or paralysis and destruction of the powerful ferments that 
 are given off by the bacteria. Thus Metchnikoff has asserted that 
 leukocytes might, after a time, be attracted toward substances that 
 would kill them. Therefore, while leukocytes will migrate freely 
 toward substances that would kill them, they may be destroyed before 
 they reach the inflammatory area, or, having rSached there, are promptly 
 destroyed and pass into solution (Fig. 47). 
 
 While it is doubtful, therefore, whether substances are produced by 
 bacteria that actually repel leukocytes, the point has not been definitely 
 settled. If such substances exist, it would appear that they are closely 
 identified with either the endotoxins or the aggressins, the latter being 
 definite secretory products of bacteria that neutralize opsonins and 
 retard phagocytosis. In many instances it is probable that the same 
 substances that exert a positive chemotaxis are, when concentrated, 
 negatively chemotactic, through overstimulation and paralysis of the 
 leukocjrtes. With diminution in the numbers or vitality of bacteria 
 and dilution of their chemotactic substances, this inhibiting influence is 
 removed and the leukocytes are attracted to the focus of infection, thus 
 explaining in a way those instances in which positive chemotaxis is 
 observed to follow a primary period of negative chemotaxis. 
 
 RESULTS OF PHAGOCYTOSIS 
 
 After phagocytosis has been accomplished, the fate of the engulfed 
 objects depends upon their nature. In general they undergo a process 
 ' Arch. d. med. Exper., 1901, 13, 585. 
 
Fig. 47. — Negative Chemotaxis. 
 A smear of exudate from the peritoneal cavity of- a guinea-pig twenty-four 
 hours after injection with virulent streptococci. The exudate was thin, serous, and 
 tinged with hemoglobin. Note the large numbers of streptococci and relatively few 
 leukocytes. ' 
 
RESULTS OF PHAGOCYTOSIS 183 
 
 of digestion. The ameba, for example, is aljle to kill and digest en- 
 gulfed material through an intracellular ferment regarded as a form of 
 trypsin, demonstrated by Alouton^ and cajled amebadiastase. Ac- 
 cording to MetchnikofE, the digestion of erytJhrocj'tes and tissue frag- 
 ments is accompUshed through an enzyme of the macrophages called 
 macrocytase; that of bacteria or other substances engulfed by micro- 
 phages by a similar enzyme called microqjtase. 
 
 Following the general law that hving protoplasm cannot be digested, 
 we are confronted with the very important question as to whether living 
 bacteria are engulfed by phagocj^tes or whether they are first destroyed 
 by extracellular agencies before they undergo phagocj-tosis. 
 
 Endolysins. — It seems positively established at the present time 
 that leukocj-tes do take up hving bacteria, which may either grow in- 
 side the leukocjiie or be destroyed by intracfeUular substances called 
 endolysins. On the other hand, leukoc3i;es do not take up extremely 
 ■virulent bacteria, hence the question arises as to the importance of 
 substances in the body-fluids which neutraUze the repeUing substances 
 of bacteria and facUitate phagocj'tosis. This subject, which has con- 
 siderably modified Metchnikoff's -sdews of phagocytosis, v,i.]l be con- 
 sidered in a succeeding chapter. It wiU suffice here to state that leuko- 
 cytes may engulf hving bacteria possessing, some virulence, for not 
 infrequently an infection may be spread by bacteria transported into 
 deeper tissues by phagocj-tes, when the}' resist the germicidal acti^-it}- 
 of the endolysins, bring about the death of the phagocj-te, and are thus 
 liberated into new tissues. 
 
 Death of the engulfed bacteria is, therefore, brought about by en- 
 dolysins ^ that are probably different from the digestive enzjTnes. These 
 substances are strongly bactericidal, and have a complex structure re- 
 sembling bacteriolysins. According to Weil,^ they are not specific. 
 They are resistant to 65° C. or even higher, do not readily pass through 
 porcelain filters, are precipitated by saturation with ammonium sul- 
 phate, and resemble the enzymes in many respects (]\Ianwaring*). It 
 is probable that the endolysins act not only upon bacteria that have 
 been phagocytosed, but also upon free bacteria when hberated through 
 disintegration of the leukocytes. In this manner the endolysins would 
 elosely resemble the normal opsonins or bacteriolysins, and support the 
 contention of Metchnikoff that these important substances contained 
 
 ' Compt. rend, de I'Acad. Sci. de Paris, 1901, cxxxiii, 244 
 
 ' For general review, see Kling: Zeit. Immunitate, 1910, 7, 1. 
 
 ' Arch. f. Hyg., 1911, 74, 289. ■> Joi|r. Exper. Jled., 1912, 16, 250 
 
184 PHAGOCYTOSIS 
 
 ill the body-fluids are derived primarily from the cells which he has 
 classed as phagocytes. According to Schneider,^ lymphocytes and 
 macrophages seem to contain little or no endolysin, and these cells are 
 not so active in the phagocytosis of virulent bacteria as are the micro- 
 phages. 
 
 Indigestible substances, if chemically inert, may remain in cells, 
 particularly in fixed tissucTcells, for variable periods of time. The leu- 
 kocytes seem to transfer such particles to other tissues, particularly to 
 the lymph-glands. It is probable that these phagocytes are in turn 
 engulfed by the endothelial cells. Macrophages of the lymph-sinuses or 
 the leukocytes may be destroyed in the glands, and their contents re- 
 phagocyted by these cells. In just what manner these insoluble par- 
 ticles reach the gland stroma or perilymphajigeal tissues is unknown; 
 it is probable that they are liberated from the lining endothelial cells, 
 and are again seized by the young connective-tissue cells. 
 
 THE RELATION OF THE BODY-FLUIDS TO PHAGOCYTOSIS 
 
 Important and far-reaching as were Metchnikoff's researches and 
 conclusions, they were not allowed to pass unchallenged, especially by 
 the adherents of the humoral school, who were able to show the potent 
 influences of the body-fluids in the mechanism of recovery from in- 
 fections where phagocytosis was little in evidence, or, indeed, where 
 phagocytosis was impossible. It was shown that Metchnikoff's original 
 theory was untenable, and that the leukocyte is almost impotent if re- 
 moved from the influence of the body-fluids. 
 
 As demonstrated by Denys, Leclef, Fliigge, Nuttall, Pfeiffer, and 
 others, bacteria may be killed, i. e., may undergo a process of lysis or 
 disintegration, by means of substances in thje blood-serum entirely in- 
 dependent of phagocytosis Later researches by Wright, Neufeld, and 
 their coworkers demonstrated most clearly that even in those infections 
 in which phagocytosis was observed and known to be of great importance, 
 the bacteria are first prepared for phagocytosis by substances in the 
 body-fluids, and that without this preliminary preparation of the bac- 
 teria phagocytosis was slight and of little consequence. 
 
 Metchnikoff corroborated most of these discoveries, and modified 
 his theory from time to time to meet the developments and keep them 
 within the limits of the phagocytic theory. For example, when bac- 
 teriolysis was shown by Bordet to be due to two separate substances ia 
 
 ' Arch. f. Hyg., 1909, 70, 40. 
 
RELATION OF THE BODY-FLUIDS TO PHAGOCYTOSIS 185 
 
 the body-fluids, which he called substance sensibilisatrice and alexin 
 (later renamed by Ehrlich amboceptor and camplement respectively), 
 Metchnikoff claimed that this phenomenon was extracellular digestion, 
 similar to the intracellular digestion that occurs within the phagocyte, 
 and brought about by ferments secreted and hberated from leukocytes 
 or other cells classed as phagocj^tes. He regards alexin as a cytase se- 
 creted by leukocytes, or liberated upon their disintegration; similarly 
 the substance sensibilisatrice is regarded as ft free ferment (fixateur), 
 derived principally from leukocytes, and concerned in preparing the 
 bacterial or other cell for the digestive-hke action of the cytase. 
 
 Aside from these free ferments that are capable of producing extra- 
 cellular lysis, Metchnikoff has long known that other substances that 
 aid phagocytosis itself may be present in the> body-fluids; he regards 
 these as of the nature of stimulins, or substanjces that stimulate leuko- 
 cytes to become more actively phagocytic. On the other hand, Leish- 
 man, Wright and Douglas, Neufeld, and Rimpau, Hektoen, and others 
 have clearly demonstrated that they facilitate phagocytosis, not by 
 stimulating the leukocytes, but rather by lowering the resistance of 
 bacteria or in some way rendering them more vulnerable to phagocytosis 
 (opsonins, bacteriotropins) . 
 
 Thus the gap between the original cellular theory, which ascribed 
 protection and cure to phagocytosis pure and'simple, and the humoral 
 theory (finally summed up by Ehrlich in his side-chain theory), which 
 ascribed the chief and primary roles to substances in the body-fluids, 
 and relegated phagocytes to a position of secondary importance, re- 
 garding them only as scavengers that remove" dead or disabled micro- 
 organisms, has been filled with discoveries correlating both processes. 
 
 The vitality of the leukocyte is to be regarded as important in the 
 consideration of phagocytosis as a means of defense. While the body- 
 fluids are acting upon the invaders, the leukocytes themselves are prob- 
 ably undergoing quantitative and qualitative changes. They are in- 
 creasing in numbers, and, as Rosenow has shown, are undergoing more 
 specific changes. Thus, for instance, the leukocytes from a pneumonia 
 patient were found more vigorous against invasion of the pneumococcus 
 than are those from a normal person, regardless-of the influence of serum. 
 
 When a microparasite is ingested, the process has only begun. Un- 
 less suitable endolysins are present and the endotoxin is absorbed or 
 otherwise dealt with, and unless suitable digestive enzymes are secreted 
 and the bacterium is dissolved, the process is useless, or, indeed, if viable 
 bacteria are transported to other parts of the bbdy, it may be dangerous. 
 
186 PHAGOCYTOSIS 
 
 THE REVISED THEORY OF PHAGOCYTOSIS 
 
 As previously stated, Metchnikoff has revised his theory from time 
 to time, as these discoveries were made on the influence of substances in 
 the body-fluids, not only upon phagocytosis itself, but also upon the 
 processes of immunity in general. He would regard extracellular cy- 
 tolysis (bacteriolysis, hemolysis, etc.) as due to the same ferments that 
 bring about the destruction and solution of the ingested bacterium or 
 other cell within the phagocjd^e, and, further, these extracellular fer- 
 ments are derived from the cells that are classed as phagocytes. By this 
 method of reasoning he would preserve the importance of the phago- 
 cytic theory. 
 
 In local infections phagocytosis is usually well marked, and no 
 doubt plays an important part in resistance to and recovery from these 
 conditions. In general infections, however, as in bacteremias due to 
 staphylococci, streptococci, and particularly the tjrphoid and allied 
 bacilli, it is rare indeed to find evidence of a leukocyte in the blood- 
 stream becoming phagoc3^ic. Here extracellular substances or anti- 
 bodies are chiefly operative in affording protection or in overcoming in- 
 fection. Even in local infections, where phagocytosis along the more 
 simple lines originally described by Metchnikoff is well marked, the 
 resistance of the bacteria is first attenuated or modified by the opsonins 
 of the body-fluids before the phagocytic process' becomes well marked. 
 
 The question, then, of the relative importance of the cellular and 
 humoral theories of immunity resolves itself to a consideration of the 
 origin of the substances in the body-fluids so potent in both processes. 
 If they are derived solely from the cells known to act as phagocytes, then 
 the cellular theory of phagocytosis, in its broader meaning, is the one 
 explanation of the processes of immunity as they are now understood. 
 This, however, has never been proved, and it is entirely Ukely that these 
 substances are products of a general, rather than of a more restricted, 
 cellular activity, so that ultimately all immunologic processes are cellular 
 in origin. For this reason we prefer to speak of the phagocytic cell in its 
 relation to immunity when deahng with the relation and activity of mi- 
 crophages and macrophages in a hmited sense in the process of phago- 
 cytosis. 
 
CHAPTER XI 
 
 OPSONINS 
 
 Historic. — Although there can be no doubt as to the importance of 
 phagocytosis in the mechanism of recovery from infection, yet it was 
 shown by Metchnikoff, as early as 1893, that the body-fluids contained 
 substances that greatly facilitated the phagocytic process, and that 
 leukocytes removed from this influence were practically powerless to en- 
 gulf and destroy the invading bacterium. In other words, if leukocytes 
 and bacteria are washed free from all traces of serum and then mixed, 
 very few of the leukocytes will be found capable of phagocytizing the 
 bacteria, which means that spontaneous phagokytosis is feeble and hence 
 of slight importance. When, however, fresh serum is added, especially 
 the serum of an animal immunized against the microorganism used in 
 the experiment, phagoc}d;osis is marked, and,. indeed, most impressive. 
 Metchnikoff attributed this difference to the influence of a substance 
 in the serum that stimulated (stimulins) the leukocytes to become phag- 
 ocytes, but later researches have shown that this is probably erroneous, atid 
 that the serum facilitates phagocytosis not by exerting a stimulating influ- 
 ence upon the leukocytes, but by preparing the bacteria for the process by 
 nialing them, as it were, more attractive to the leukocytes. 
 
 Denys and Leclef, in 1895, were among the first to demonstrate 
 the effect of scrum on bacteria in the process of phagocytosis, and the 
 fact that the active substance was not bactericidal in action, but in the 
 nature of a new antibody. Since Metchnikotf had shown that freshly 
 isolated or virulent strains of bacteria were not readily phagocytized, 
 but seemed to resist or repel the leukocytes, it was natural for these 
 observers to suggest that the action of this substance in serum was to 
 neutraUze the exotoxins and endotoxins of microorganisms that were 
 regarded as responsible for negative chemotactic influences, and thus, 
 by robbing them of at least two defensive weapons, prepare them for 
 phagocytosis. 
 
 The subject remained in an uncertain state until 1903, when Wright, 
 and later Wright and Douglas, demonstrated more clearly this action of 
 serum upon bacteria in aiding phagocytosis. Using their own modifica- 
 tion of the technic devised by Leishman for measuring the phagocytic 
 
 187 
 
188 
 
 OPSONINS 
 
 power of the blood, these observers first deternlined the direct depend- 
 ence of phagocytosis upon some ingredient of the blood-serum, and fur- 
 ther proved that this substance acts directly upon bacteria, is bound by 
 the bacteria, and renders them more easily ingested by the leukocytes, 
 i. e:, more readily phagocytable. To this substance they gave the name 
 opsonin (from opsone, I prepare food for). At the same time, and in- 
 dependently of Wright, Neufeld and Rimpau conducted similar inves- 
 tigations with immune serum and reached similar conclusions, but called 
 the substance bacteriotropin. Since then botlj terms have been used, — 
 the former more frequently in English literature,^-and this is permissible, 
 providing that it is understood that both are pra.ctically the same anti- 
 body, and not distinct and separate from each other. 
 
 As will readily be understood, the bacterial opsonins have been 
 studied most extensively, but opsonins may be. present in normal and 
 immune serums for other cells, such as erythrocytes, and these hemopso- 
 nins are regarded as separate antibodies, distinct from hemagglutinins 
 and hemolysins. 
 
 Definition. — Opsonins are substances in normal and immune serums 
 which act upon bacteria and other cells in such a manner as to prepare them 
 for more ready ingestion by the phagocytes. 
 
 Properties and Natiire of Opsonins. — There is considerable differ- 
 ence of opinion regarding the identity and probable structure of opsonins 
 in normal and immune serums. Just as agglytinin for a bacterium, 
 such as Bacillus tj^hosus, may be found in varying amounts in normal 
 serum, so various opsonins for different bacteria may be found in normal 
 serums. These normal opsonins appear more or less specific for a given 
 bacterium, and in immune serum the specific opsonic substance for the 
 particular bacterium or cell with which immunization has been pro- 
 duced is developed to a high degree. Both owe their full effect to the 
 interaction of two substances. One of these, the common substance, 
 is thermolabile, and destroyed by heating the serUm to from 56° to 58° C. 
 for half an hour, whereas the other more specific substance remains un- 
 affected. The latter, in both normal and immruie serums, is opsonic 
 by itself, although in the absence of the common thermolabile substance, 
 to a less degree, and is produced anew and specifically by artificial im- 
 munization or as the outcome of spontaneous infe'ctions. 
 
 The true nature of opsonins is, therefore, difficult to understand. 
 They have been compared to receptors of the second order, with a hap- 
 tophore and a toxophore or opsoniferous group. Receptors of this 
 order, however, are active and independent of the presence or absence 
 
SUSCEPTIBILITY TO OPSONiriCATION 189 
 
 of complement, whereas the opsonins, although active to some e;xtent 
 in the absence of complement, are far more so if a complement is present. 
 In this respect they resemble amboceptors, or receptors of the third 
 order, opsonins in normal and inunune serums representing respectively 
 normal and immune bacterial amboceptors. One objection tothis 
 view of their structure is their activity, however slight, when the ther- 
 molabile substance, is removed by heating, unless the amboceptors are 
 complemented by an endocomplement, as from the bacteria themselves. 
 
 At the present time, therefore, not a few observers doubt that opso- 
 nins exist as true and separate antibodies, and are inclined to regard 
 thermolabile opsonin (largely the opsonin in fresh normal serum) as a 
 complement, and thermostabile opsonin (largely immime opsonin or 
 bacteriotropin) as an amboceptor; it would appear that either alone, 
 but more especially the latter, may facilitate phagocytosis to some ex- 
 tent. This process is, however, much more marked when both sub- 
 stances are acting in unison. While it is true that the bacteriolysin 
 and opsonin content of a serum do not run parallel, our methods for 
 measuring these are not entirely satisfactory; both intracellular and 
 extracellular lysis may be mere differences in degree, depending upon 
 the nature of the bacterium or the concentration of the antibodies, 
 rather than upon separate and distinct antibodies. 
 
 Source of Opsonins. — Little is definitely known regarding the source 
 of opsonin. Thermostabile opsonin — ^that which is increased by arti- 
 ficial immunization or during disease, and is largely in the nature of an 
 amboceptor — is probably a product of general cellular activity, and 
 especially of the local cells at the site of infection. Thermolabile opsonin 
 — largely the opsonin occurring in normal serum, and in the nature of a 
 complement — is probably a product of the leukocytes and other cells as 
 well, as it has never been proved that the leukocytes are the sole source 
 of the complements, as Metchnikoff would have us believe. 
 
 Susceptibility to Opsonification. — As previously stated in the chap- 
 ter on infection, not all bacteria are equally ^susceptible to opsonifica- 
 tion. As a general rule, recently isolated and virulent microorganisms 
 resist the influence of opsonins until they have undergone culture sev- 
 eral times. This resistance may be due to capsule formation, thicken- 
 ing of the ectoplasm, actual self-immunization of the bacterium, or the 
 influence of endotoxins as a protective means against the antibodies of a 
 host, all of these being weakened or lost upon artificial culture-media. 
 
 Effect of Opsonins on Bacteria. — ^We know= nothing definite regard- 
 ing the manner in which opsonins prepare bacteria for phagocytosis 
 
190 OPSONINS 
 
 except that opsonification in itself apparently does not impair the vital- 
 ity of the bacterium, in so far, at least, its viability is concerned. 
 
 Role of Opsonins in Immunity. — Although the exact identity of 
 normal and immune opsonins and their relation to other antibodies is as 
 yet unsettled, the important relation they bear to processes of immunity 
 is generally recognized, especially their ability in aiding resistance to 
 infection by facilitating phagocytosis. They are operative in some in- 
 fections more than in others, and they are especially active in those con- 
 ditions in which phagocytosis is recognized as the chief defensive force, 
 as, for example, in pyogenic infections. In these conditions their pres- 
 ence has been taken as a measure {opsonic index of the resistance of the 
 host) and, largely through the researches of Wright and Douglas, a 
 technic for detecting their presence, kind, and. quantity in the body- 
 fluids has been devised, the method and information it yields being of 
 value imder certain limitations and in some infections. (See next 
 chapter.) 
 
 If experiments in vitro may be taken as an example of what occurs 
 in vivo, it must be true that leukocytes are capable of consuming an 
 enormous number of bacteria. Experiments with washed leukocytes 
 ■ — those removed from the influence of serum — show that spontaneous 
 phagocytosis is very slight. Metchnikoff declared these experiments to 
 be untrustworthy for the reason that the various manipulations of wash- 
 ing injures the vitality of the leukocytes. When, however, bacteria are 
 opsonized, that is, are exposed to a serum containing opsonins, and then 
 are thoroughly washed, it is found that the washed leukocytes engulf 
 enormous numbers of bacteria, showing that Metchnikoff' s objection to 
 these experiments is unwarranted. Granting, then, that what we call 
 opsonins are substances that facilitate phagocytosis, and that phago- 
 cytosis is a process of great importance, especially in certain infections, 
 we must conclude that opsonins play a very important role in immunity; 
 in fact, they constitute the very basis of the phenomenon of phagocytosis 
 in the broader meaning of the term. 
 
CHAPTER XII 
 OPSONIC INDEX 
 
 Whether opsonins are regarded as separate antibodies or as being 
 identical with complements and amboceptors, a measure of their quan- 
 tity and power may be of aid in formulating a* diagnosis, as a guide to 
 active immunization, and as one means of determining the potency of 
 various immune serums used for therapeutic purposes, such as antimen- 
 ingococcus and antipneumococcus serums. We are mainly indebted 
 to Leishman, Wright and Douglas, Neufeld and Rimpau, and their 
 coworkers for devising a technic that, however imperfect it may be ac- 
 cording to the results obtained, has opened a" new and important field 
 for the study of imrnunologic processes. 
 
 Principle. — This is based upon the method devised by Wright and 
 Douglas, whereby it was sought to determine the amount and kind of 
 opsonin in a patient's serum by comparing the degree of phagocytosis 
 with that occurring when normal serum was used. 
 
 Definition. — The opsonic index is the ratio of the number of bacteria 
 ingested by a given number of phagocytes in the presence of a patient's serum, 
 to the number ingested by the same number of phagocytes in the presence of 
 normal serum. 
 
 "An equal volume of the patient's serum; measured in a capillaiy 
 pipet, is mixed with an equal volume of a suspension of washed leuko- 
 cytes derived from a normal blood. After this 'phagocytic mixture' 
 has been digested for a suitable period at 37° C, film preparations are 
 made and stained. 
 
 "A 'phagocytic count' is then undertaken, i: e., the average bacterial 
 ingest of the leukocytes in the phagocytic mixture is determined, and 
 this is compared with the average ingest of the leukocytes in a phago- 
 cytic mixture made with normal blood. 
 
 "The expression thus obtained, 
 
 Average ingest of the individual phagocyte in the mixture containing the 
 
 patient's serum. 
 
 Average ingest of the individual phagocyte in the mixture containing nor- 
 mal serum is denoted the opsonic index" (Wright). 
 
 191 
 
192 OPSONIC INDEX 
 
 Purpose of the Method. — The opsonic index aims to serve as a guide : 
 
 1. In diagnosing the presence of bacterial infection, or rather in dis- 
 covering whether the natural protective powers of the patient's blood 
 have been diminished or increased as the result of the immunizing in- 
 fluence of the infection. 
 
 2. In connection with vaccine therapy, to guard against diminishing 
 the opsonin content of the patient's blood; to assure ourselves that 
 our efforts to increase them have been successful, and occasionally to 
 ascertain how long the store of opsonin that has been obtained for the 
 patient remains in the blood. 
 
 Limitation of the Method. — In ascertaining the opsonic index of a 
 patient's serum, we must take it for granted — although it has not been 
 proved : 
 
 1. That the bacteria act the same in the body as they do in the test- 
 tube. This is known not to be the case, for virulent organisms resist 
 phagocytosis, whereas a non-virulent strain of the same bacterium is 
 easily phagocyted. If, therefore, a laboratory culture of attenuated 
 organisms is used in making the opsonic index, the result can hardly 
 be accepted as a criterion of the power of the patient to overcome the 
 "resistant" or more virulent organism as it occurs in the body. This 
 source of error can be overcome in a manner if the microorganism is 
 isolated and used at once before attenuation occurs. 
 
 2. That the leukocytes are a constant factor, and need not be taken 
 into account. Investigation has shown that, as a result of infection, 
 the leukocytes probably undergo qualitative changes and it is hardly 
 fair to accept phagocytosis by normal leukocytes as a criterion of pha- 
 gocytosis with the patient's own leukocytes, as it occurs in the body dur- 
 ing the infection. 
 
 3. The method assumes that phagocytosis by the polynuclear leuko- 
 cyte plays a large part in overcoming the infection. In many cases, 
 however, this is by no means proved. For example, in tuberculosis 
 it is not this form of leukocyte, but the mononuclear form or the lympho- 
 cyte, which seems to be more important, and hence it is difficult to un- 
 derstand how the index with the polynuclean leukocyte can aid the 
 question of diagnosis or treatment. 
 
 4. The chances for error are considerable. To be of any value, the 
 work requires experience and painstaking care. The results obtained 
 by competent workers with the same blood itiay show variation, but 
 it must be said that, with strict attention to technic and insistence upon 
 perfect preparations, the worker may usually obtain valuable results. 
 
TECHNIC 193 
 
 An index taken at one time by one person and later by another, and so 
 on, will not be of as much value as when all are taken by the same worker, 
 who bruigs practice, skill, and conscientious care to his aid. 
 
 Precautions in Technic.^ — 1. Proper controls should be used. When 
 dealing with the tubercle bacillus, the staphylococcus, or any other 
 saprophyte of the external surfaces, or with any pathogenic organism 
 with which we have normally no relations, the serum of a normal in- 
 dividual or the mixed serum of a number of normal persons will furnish 
 the standard of comparison. When, on the other hand, we are deahng 
 with intestinal bacteria or with a saprophyte of the mucous membrane, 
 where, as a rule, any relation with them will be denied, it is difficult to 
 establish a standard of health. Pooled serum is, therefore, necessary, 
 and will furnish a standard for comparison for the purpose of measuring 
 the fluctuations that may occur in the patient's blood. 
 
 2. A reasonable degree of phagocytosis should occur in the control 
 serum. This is one of the main drawbacks to the value of the method 
 for certain pathogenic organisms, as the pneumococcus, meningococcus, 
 streptococcus, etc., may resist phagocytosis in normal serum, and thereby 
 show abnormally high indices with immune serum. 
 
 3. Efforts at spontaneous phagocytosis should be suppressed in 
 order to measure more accurately the opsonin,, as shown by the degree 
 of phagocytosis independent of the inherent activities of the cell itself. 
 Spontaneous phagocytosis can largely be overcome by using 1 per cent, 
 solution of sodium citrate such as is used for the collection of leukocytes. 
 
 4. The ingest of a sufficiently large number "of phagocytes should be 
 counted. As a general rule, 100 cells should represent the minimum. 
 
 TECHNIC 
 
 The necessary constituents for making the test are as follows: 
 
 1. The patient's serum and normal serums to serve for the control. 
 
 2. A bacterial emulsion. 
 
 3. A suspension of washed human leukocytes in normal salt solution. 
 Collection of Patient's and Control Serum. — 1. The blood is col- 
 lected in a Wright capsule, as described in Chapter II. 
 
 2. Care must be taken not to heat the blood when sealing the tube. 
 In drawing off the serum, avoid an admixture of corpuscles, as these may 
 lower the opsonic index. 
 
 3. If coagulation is incomplete or the serum has not been well sep- 
 arated, the clot may be broken up gently with= a platinum wire and the 
 
 13 
 
194 OPSONIC INDEX 
 
 tubes centrif uged. Slight discoloration of the* serum from mechanically 
 breaking-up a few erythrocytes will not interfere with the test. 
 
 4. If gross contamination is avoided, blood may be kept for one or 
 two days in a dark place without much deterioration of its opsonin 
 content. 
 
 5. It is always well to collect the control bloods at the same time the 
 patient's blood is taken, or, if this carmot be done, to place them in an 
 ice-chest as soon after collection as possible. When conducting the 
 test, the control serums are pooled and mixed in a clean watch-glass. 
 
 The Bacterial EmxJsion. — 1. This must be perfectly uniform, free 
 from clumps, and must not undergo agglutination, either spontaneous 
 or with the serum to be tested. 
 
 With many bacteria, especially the motile ones, such as Bacillus 
 coli and Bacillus tj^hosus, it is comparatively easy to secure a uniform 
 emulsion. Staphylococci, streptococci, and pneumococci, as a rule, 
 present no diiSculties. After growing the culture for from eighteen to 
 twenty-four hours on slants of a suitable medium, add three cubic centi- 
 meters of sterile 1 per cent, salt solution, and gently remove the bac- 
 terial growth with a platinum loop. The mixture is then pipeted into a 
 separate flask or thick glass test-tube containing glass beads, and shaken 
 by machine or by hand until it is thoroughly- emulsified. If necessary, 
 the emulsion may be ccntrifugalized to remove clumps and is then ready 
 for use. 
 
 2. It must consist of bacteria that stain evenly and well. 
 
 Only young cultures should be used; for example, an eighteen to 
 twenty-four-hour culture of freely growing organisms and a seven days' 
 growth of tubercle bacillus. 
 
 3. It must be of such strength as to give a leukocytic ingest that will 
 enable adequate differentiation to be made of the opsonin content of the 
 various serums. 
 
 An emulsion that does not yield a count of at least 250 bacteria 
 within 100 leukocytes is too weak to yield a satisfactory differential 
 count. If the emulsion is too thick, bacteria overlie the leukocytes 
 and introduce error. As a general rule, a suspension containing 500,- 
 000,000 bacteria per cubic centimeter is satisfactory. Experience will 
 teach the right density to be used, and frequent trials may be necessary 
 before the right one is secured. 
 
 4. The bacteria must be such as will not prove seriously dangerous. 
 In order to obviate any danger attending wofk with such organisms as 
 the glanders and the tubercle bacillus, first kill the culture by pouring 
 
TECHNIC 195 
 
 on a 10 to 40 per cent, solution of formalin, mix the culture, shake, 
 transfer to a centrifuge tube, and centrifugalize until the bacteria have 
 been carried to the bottom of the tube. Pipet off the supernatant 
 formalin, wash in normal salt solution, centrifuge, pipet off again, and 
 finally mix the sediment in sufficient salt solution to make a satisfactory 
 suspension. 
 
 The "Washed Leukocytes.' — These should consist mainly of the poly- 
 nuclear leukocytes of a healthy person washed-free from any admixture 
 with serum. As usually obtained, the leukocytes are mixed with red 
 corpuscles. It is necessary to collect blood for the leukocytic mixture 
 from a person whose corpuscles are known to be insensitive to agglutina- 
 tion, as otherwise there is an undue lowering of the opsonic effect. 
 
 1. Place 4 c.c. of sterile 2 per cent, solution of sodium citrate in dis- 
 tilled water in sterile 10 c.c. centrifuge tubes. 
 
 2. Prick the finger, and add 1 c.c. of blood. Agitate well to insure 
 thorough mixing. 
 
 3. Centrifuge at a sufficiently high speed to mix the red corpuscles 
 and leukocytes at the bottom of the tube an'd avoid clumping of the 
 leukocjiies. 
 
 4. Draw off the supernatant fluid, add 5 c.c. of 1 per cent, salt solu- 
 tion, mix, and centrifuge. 
 
 5. Wash once more. Draw off the supernatant fluid. 
 
 6. Add sufficient salt solution to bring the total volume up to that of 
 the blood originally taken — i. e., 1 c.c. Mix toeU. 
 
 The Test. — 1. Prepare capillary pipets of approximately the same 
 caliber. These are made by taking 6-inch lengths of soft clean glass 
 tubing having an external diameter of t^ inch, heating them in the 
 middle in the tip of the blow-pipe or the Bunsen flame until about }/2 
 inch length of tubing is quite soft. Remove from the flame, and by rap- 
 idly separating the two hands draw out the molten glass to a length of 
 from 18 to 20 inches. After cooling, the capillary thread is cut across 
 with a small file, so that from 6 to 8 inches is kft attached to each piece 
 of tubing. The ends must be cut square, as < ragged and uneven ends 
 are difficult to handle. By means of a wax-pencil make a fine mark at a 
 point about an inch from the free end of each capillary thread. This 
 indicates the unit volume (Fig. 48). 
 
 2. Adjust a well-fitting rubber teat, and draw up the unit volume of 
 blood-cells. A tiny bubble of air is now allowed to enter the thread, and 
 then one volume of the bacterial emulsion is a(Jded; another air-bubble 
 is allowed to enter, and finally one volume of serum, so that we have 
 
196 
 
 OPSONIC INDEX 
 
 il 
 
 .1, 
 
 
 5 
 
 Fig. 48. — Capil- 
 
 LART PiPET FOR 
 
 Opsonic Index De- 
 termination. 
 
 named in their order in the capillary tube from above 
 downward, one volume of bioocl-cells, an air-bubble, 
 one volume of bacterial emulsion, an air-bubble, and 
 one volume of serum (Fig. -t^ . 
 
 3. By making gentle pressure on the teat these 
 are then blown out on the surface of a clean glass 
 slide, and perfect mixture effected by making alter- 
 nate aspiration and expulsion from the capillary tube 
 at least six times (Fig. 49). 
 
 4. Carefully reaspirate into the capillary thread, 
 so that the mixture occupies about the middle, and 
 seal the tip in a low Bunsen flame (Fig. 50). 
 
 5. Remove the teat, and with the wax-pencil mark 
 the tube with the name or number of the serum. 
 
 6. A similar preparation is prepared with the 
 pooled serum (control) . 
 
 7. The phagocytic mixtures are then placed in 
 an incubator at 37° C. for fifteen minutes, except in 
 the case of such bacteria as the Bacillus tjrphosus 
 and the Bacillus coli, as lysin and agglutinin may 
 be present in the serums of such bacteria when the 
 period is reduced to ten minutes. 
 
 8. The tubes are then removed from the incuba- 
 tor, the teats readjusted, the tip of the capillary 
 threads scratched with a file, and evenly broken off. 
 The phagocytic mixture is carefully expelled on a 
 clean glass slide, and a perfect mixture made by 
 alternate aspiration and expulsion. Avoid air- 
 bubbles. The whole is then reaspirated, and a 
 small drop of the mixture plated on each of two clean 
 slides that have been roughened with emerj^ paper 
 about M inch from one extremity. With the edge 
 of a spreader slide held at! an angle of about 30 
 degrees, and with moderate pressure, the drop is 
 distributed evenly along about IJ^ inches of the 
 surface of the slide (Fig. 51). _ The smears are made 
 in duplicate, because one may be more nearly 
 perfect (Fig. 52) than the other, or one may be 
 spoiled in the staining, when the second may be 
 utilized. Each slide is then labeled at one end. 
 
TECHNIC 
 
 197 
 
 9. After drying in the air, the slides must be fixed and stained. 
 
 (a) For Ordinary Bacteria. — 1. Fix by covering the shde with a sat- 
 urated aqueous solution of mercuric chlorid for one minute. Wash in 
 water. 
 
 2. Cover with carbolthionin and stain for one or two minutes. 
 Wash in water. 
 
 Fig. 49. — Mixing the Contents of a Capillary Pipet. 
 Due precautions must be exercised to avoid thtf formation of bubbles. 
 
 3. Dry thoroughly. 
 
 (b) For Acid-fast Bacilli.— 1. Fix by inverting the films for thirty 
 seconds over a watch-crystal or jar containing formalin, being careful 
 that there are no drops of formalin on the edge' of the vessel that might 
 come in contact with the preparation. The films may be fixed also 
 
198 
 
 OPSONIC INDEX 
 
 with a saturated solution of mercuric chlorid or with pure methyl 
 alcohol for two minutes. Wash in water and dry. 
 
 2. Heat a portion of carbolfuchsin almost to boiling in a test-tube, 
 and pour the hot stain over the films. Allow to remain for at least 
 fifteen minutes. Wash under the tap and dry. 
 
 Fig. 50. — Method of Sealing a Capillary Pipet. 
 The tip of the pipet is placed in the edge of a flame. The teat is held in the 
 same position until the tip has been sealed, when it may be removed without disturb- 
 ing the contents of the pipet. 
 
 3. Cover with a 5 per cent, solution of nitric or sulphuric acid for 
 half a minute or longer if necessary, until decolorization is complete. 
 Wash thoroughly under the tap. > 
 
 Fig. 51. — Method of Preparing a Blood Film. 
 The slide is laid on a flat surface; the drop of blood is placed near one end; the 
 spreader is held between the thumb and middle finger of the left hand, at an angle of 
 about 30 degrees, and quickly -pusihed to the opposite end of the slide. 
 
 4. Cover with 4 per cent, aqueous solution of acetic acid for one to 
 two minutes to remove the hemoglobin from the red cells. Wash and 
 blot lightly. 
 
 5. Cover with LofHer's methylene-blue for two minutes. Wash in 
 water and dry thoroughly (Fig. 53). 
 
TECHNIC 199 
 
 10. Examination of the stained films with the oil-immersion ob- 
 jective of the microscope will show that polynuclear leukocytes have 
 collected more toward the edges and the end at which the spreading was 
 completed. The individual leukocytes, however, should be separated 
 from one another (Figs. 54 and 55). 
 
 11. The edge of the film is examined, and the number of bacteria 
 found in each series of five consecutive phagocytes is noted. If the 
 technic has been satisfactory, no great divergence should be found in 
 the count of each set of five cells. 
 
 Fig. 52. — Blood Films for Phagocytic Codnts. 
 The first slide (on the extreme left) is too thick and honeycombed, due to a 
 greasy slide and large drop of blood; the second is likewise thick and vmeven; the 
 third is too thin, and was spread with too small an amount of blood and with the 
 spreader held too upright; the fourth (extreme right) is a satisfactory film; it was 
 spread on a dean shde, is even, smooth, and of the proper thickness. 
 
 12. If the films are satisfactory, divide 100 phagocytes into groups 
 of 20. The average ingest of each group should not show a difference of 
 over 10 per cent., otherwise the technic has been faulty and it is neces- 
 sary to count 250 phagocytes or to repeat the test. If divergence is 
 due to the fact that every now and then onp cell has a considerable 
 higher ingest than others and the bacteria are well separated, hyper- 
 activity of the cell is probably the cause. If the bacteria are all clumped 
 together, it must be assumed that there has been a lack of care in prepar- 
 ing the bacterial emulsion or that agglutinin is present in the serum, 
 and the test must be repeated with fresh precautions. 
 
200 OPSONIC INDEX 
 
 13. In opsonic work the question as to how a certain element ought 
 to be counted becomes quite evident and important. The proper 
 method of procedure is to determine definitely how they may best be 
 dealt with, and then to follow the rule adbpted consistently. If an 
 organism rests on the border of a cell, it will be better to consider it as 
 within the cell and count it. Diplococci dr division forms may be 
 counted as one or as two, provided we are consistent in our method. In- 
 dividual organisms, as distinguished from zoogleic masses, which may 
 be lying on the top of the cell, are counted as if they were within the 
 cell; for we have no means of determining definitely whether or not our 
 suspicions are well grounded. In the case of a beaded or vacuolated 
 bacillus, it is always better to count the whole «element as a unit. 
 
 14. The 'phagocytic index is the average number of bacteria or other 
 cells ingested per leukocyte after counting at least from 50 to 100 cells. 
 The total number of bacteria ingested is divicJed by the total number of 
 phagocytes, the result being the average number of bacteria ingested 
 per leukocjfte — i. e., the phagocytic index. 
 
 15. The opsonic index is then given by the" ratio — 
 
 Patient's phagocytic index 
 Control phagocytic index 
 
 For example, with patient's serum, 100 phagocytes contain 300 bac- 
 teria, the phagocytic index being -f-f~|- = 3. With the control serum, 100 
 phagocytes contain 500 bacteria, the phagocytic index being -f-j-j-^S. 
 The opsonic index is : 
 
 3 Patient's phagocytic index _„ . 
 5 Control phagocytic index 
 
 16. Simon and Lamar have suggested a modification of Wright's 
 method that has been adopted by many laboratories. It consists in 
 diluting the patient's and control serums up to 1 : 10 or 1 : 100, and pre- 
 paring mixtures of various dilutions with leukocytes and thinner bac- 
 terial emulsions. The percentage of phagocytic cells in the mixtures 
 containing the serum to be tested is compared with the mixtures con- 
 taining normal serum. It is, therefore, a method of comparative phago- 
 cjrtic indices. 
 
 QUANTITATIVE ESTIMATION OF BACTERIOTROPINS (NEUFELD) 
 Of the various methods for standardizing an immune serum, par- 
 ticularly antimeningococcus serum, and of obtaining some idea of its 
 
Fig. 53. — Tdbekcle Opsonic Index. 
 A smear, stained after the method given in the text. Case IX, C. M., aged twenty- 
 two years; active pulmonary tulierculosis; opsonic in(Icx, +1.6. 
 
 Fig. 54. — An Un.sati,sf.\ctory Film for a Pil^lGOCYTIC Count. 
 
 No(.e that the leukocytes are collected in ma.sses of erythrocytes. The slide was 
 
 greasy and the smear too thick. 
 
 Fig. 55. — A Satisfactory Film for a Phagocytic Count. 
 
QUANTITATIVE ESTIMATION OF BACTERIOTROPINS 201 
 
 potency, that of determining the bactcriotropic or opsonic index of the 
 serum is in most general use. 
 
 Neufeld's technic is that generally employed, and is similar to the 
 serum dilution method employed by Simon. It varies from the 
 technic of Wright in several particulars : 
 
 1. The immune serum is free from complement (thermolabile op- 
 sonin). 
 
 2. The actual number of bacteria within the leukocytes are not 
 counted. Various dilutions of serum are used, and the highest dilution 
 in which the bacteria are ingested in great numbers is compared with a 
 normal serum in similar dilution as a control. The highest dilution that 
 still favors phagocytosis is then taken as the hacteHotropic titer of the serum. 
 
 Serum. — The serum is inactivated by heating it to 55° C. for one- 
 half hour. In old or carbolized serums this may be omitted, as they 
 are usually free from complement. Tuberculous serums also should 
 not be heated, as their bacteriotropins are very= susceptible to heat. 
 
 Normal serum from an animal of the same species as was used in the 
 preparation of the immune serum should be used as the normal control. 
 
 An exactly parallel series of dilutions with normal salt solution are 
 made of the immime serum and pooled normal senuns in a series of 
 small test-tubes. At least 0.5 c.c. of each dilution should be available 
 for the test; the following dilutions may be used: 1:10, 1:20, 1:50, 
 1:100, 1:200, 1:400, 1:600, 1:800, 1:1000, 1:2000, etc. After working 
 for some time with normal serums one soon learns the dilution in which 
 the normal bacteriotropins are attenuated. It may not be necessary, 
 therefore, to use the whole series of dilutions with the normal serum. 
 
 Leukocsrtes. — Leukocytes may be obtained in several different ways : 
 (1) By injecting a guinea-pig intraperitoneally sixteen to twenty-four 
 hours previously with from 5 to 10 c.c. of sterile aleuronat solution (for 
 method of preparation see p. 339). Pipet the peritoneal exudate into 
 about 20 c.c. of sterile 1 per cent, sodium citrate in normal salt solution 
 in centrifuge tubes. Centrifugalize, and wash the leukocytes again 
 three times with sterile normal salt solution. The sodium citrate solu- 
 tion prevents the coagulation and formation of clumps of leukocjrtes. 
 
 2. Instead of aleuronat, a sterile saturated, solution of peptone may 
 be injected. 
 
 3. If rabbit's leukocytes are preferred, 10 c.c. of aleuronat should be 
 injected into each pleural sac or 20 c.c. intraperitoneally. For mice, 
 an injection of 1 c.c. of aleuronat intraperitoneally is sufficient; human 
 leukocytes may be obtained after the method of Wright. 
 
202 OPSONIC INDEX 
 
 4. After the final washing, the leukocytes are suspended in sufficient 
 normal salt solution until an opacity equal to a 0.3 per cent, lecithin 
 emulsion in salt solution is attained. 
 
 Culture. — Cultures should be selected witjl great care, in order to 
 avoid using one that displays a well-marked tendency to undergo 
 "spontaneous phagocytosis," or, on the other hand, one unduly re- 
 sistant to phagocytosis. Usually an old strain of meningococci is 
 serviceable; it is generally necessary to try out a number of strains 
 and select the best. 
 
 Meningococci are grown for twenty-four hours on slants of glucose 
 agar. To each slant add 0.5 c.c. each of bouillon and of normal salt 
 solution, and emulsify the growth. Or the bacteria may be employed 
 in the form of a sixteen to twenty-four hour homogeneous broth culture. 
 Tubercle bacilli may either be triturated in an agate mortar with 1.5 
 per cent, salt solution added slowly drop by drop, or the tubercle powder 
 of Koch may be employed in an emulsion prepared in the same manner. 
 The resultant emulsion should be freed from coarser clumps by brief 
 centrifugalization, but, as a general rule, it is very difficult to secure a 
 uniform emulsion of tubercle bacilli by any method. 
 
 The Test. — 1. The mixtures are made preferably in a series of small 
 test-tubes about 5 cm. long and 1 cm. wide. 
 
 2. Mix 0.1 c.c. of each dilution of immune serum with 0.1 c.c. of bac- 
 terial emulsion. Plug each tube with cotton. 
 
 3. Mix and incubate for one hour. 
 
 4. Add 0.1 c.c. of leukocjdic eiiuilsion to each tube. Double this 
 quantity may be used if the emulsion is weak. 
 
 5. Mix and incubate for from one-quarter to two hours, depending 
 upon the variety of nucro5rganisin. 
 
 6. iVt the end of the second incubation the leukocytes will have 
 settled to the bottom of the tubes. Carefullj^ remove the supernatant 
 fluid from each tube ; mix the sediment well with a loop, and make smears 
 on slides. Label each slide carefully to correspond to its serum dilution. 
 
 7. Dry the smears in the air, fix with methyl alcohol, and stain with 
 carbolthionin, as previously described. 
 
 The Controls. — 1. A series of the lower dilutions of pooled normal 
 serums are set up in exactly the same maimer. 
 
 2. A tube containing bacteria and leukecytes without serum — ^to 
 detect the extent of spontaneous phagocytosis. 
 
 Readings. — A great number of fields are examined microscopically, 
 and a note is made of the weakest dilution that still favors phagocyiiosis. 
 
QUANTITATIVE ESTIMATION OF BACTERIOTROPINS 203 
 
 No attempt is made to count the phagocyted bacteria. The relative 
 amount of phagocytosis with the immune serum in various dilutions is 
 compared with the normal controls, and the result is expressed as the 
 bacteriotropic titer. 
 
 Simon's Method. — This method of counting the number of empty 
 leukocytes with a given dilution of serum is followed ; a similar count is 
 made of the normal serum control in the same dilution; thus, if in the 
 control film 25 per cent, of leukocytes were empty, and in the patient's 
 film, 50 per cent., the index would be |-f- = 0.5. A study of the results 
 obtained by this method, and by careful counting after Wright's method, 
 shows that they are fairly comparable, and the method may be used 
 where it is only necessary to determine whether the index is high or low. 
 
 Precautions.^ — 1. If phagocytosis is entirely absent, one should not 
 conclude that bacteriotropins are not present. The leukocytes may 
 have been injured, especially if heterologous leukocytes have been 
 present; control examinations with homologous leukocytes (i. e., from 
 the same animal) as the serum should result in phagocytosis. 
 
 2. The time during which the tubes were ifi the incubator may have 
 been too short or too long. Most microorganisms require from one- 
 half to two hours — meningococci require about one-half hour; pneumo- 
 cocci usually need at least two hours; typhoid and cholera bacilli about 
 fifteen minutes to thirty minutes, as they undergo extracellular or even 
 intracellular lysis quite readily. 
 
 3. If the control of bacteria and leukocytes alone shows well-marked 
 phagocytosis the test should be repeated with another strain. 
 
 Practical Value of the Opsonic Index 
 
 1 . In competent hands, the opsonic index of normal persons to most 
 pathogenic organisms has been found to vary from 0.8 to 1.2. As 
 previously mentioned, it is difficult to find a perfectly normal serum for 
 such microorganisms as the Bacillus coli, tlje staphylococci, Bacillus 
 tuberculosis, etc., as it is unlikely that any individual can altogether 
 escape active infection at some period of his life. As menstruation ap- 
 proaches, even wider fluctuations occur, the* normal index being re- 
 established by the second or third day. During the first year of life the 
 opsonic index varies to such a degree that it has little or no practical value. 
 
 2. Although a large amount of work haa been done with the op- 
 sonins in disease, it is the consensus of opinion that the determination of 
 the opsonic index has less practical significanoQ than was originally be- 
 lieved. One point is clear, however, that the work of Wright and others 
 has broadened the field of vaccine therapy and placed it upon a firmer 
 
204 
 
 OPSONIC INDEX 
 
 foundation. Aside from the 10 per cent, of chances of technical error 
 in making an opsonin measurement, other factors may be present that 
 are entirely beyond control and cannot be measured by the immunisator, 
 and that may seriously affect the value of the opsonic index. 
 
 3. As a diagnostic procedure, Wright has shown that the opsonins 
 possess a certain specificity, and that in a given infection a low index 
 
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 Fig. 56. — An Opsonic Index Chaht. 
 
 for a certain microorganism indicates that this organism is probably the 
 etiologic factor. This possibility is strengthened if the opsonic index 
 for this microorganism is increased by careful rUanipulation or exercise 
 of the diseased part, when auto-inoculation occurs, with consequent 
 increase of opsonin. 
 
 4. In prognosis the opsonic index may have some value in deciding 
 
QUANTITATIVE ESTIMATION OP BACTERIOTEOPINS 205 
 
 whether an infection has been entirely overcome or is still active. An 
 attempt is made to induce auto-inoculation, as" by gentle massage of a 
 knee-joint or a hip; active exercise; deep breathing, etc., and the in- 
 dex is made before, and at frequent intervals after, such attempts. If 
 the index remains unchanged within the normal limits, the assumption 
 is that the infection has been overcome; if, on the other hand, an in- 
 crease in opsonin occurs, this indicates that an active focus remains. 
 
 5. Most value was placed by Wright upon" the opsonic index as a 
 guide to the size and frequency of doses of bacterial vaccines in the treatment 
 of disease. A large number of careful determinations showed that an 
 injection of vaccine is followed by a decrease of the opsonins (negative 
 phase), which is of variable degree and duration, according to the amount 
 injected (Fig. 55). This is followed by an increase (positive phase), 
 and coincidentally there is a corresponding improvement in the patient's 
 condition. This subject is discussed more fully in the chapter on Active 
 Immunization. 
 
 The purpose of proper vaccination, therefore, is so to gage and 
 time the different doses that a pronounced or prolonged negative phase 
 is prevented, as far as possible, and a high positive phase secured and 
 maintained. It is obvious that the technic of opsonic measurement con- 
 sumes much time, and that the immunisator cannot mark the index at 
 the time a dose of vaccine is given. However, the determination of the 
 opsonic index at proper intervals after the firstidose of vaccine may give 
 valuable information as regards the reaction of the patient, and serve 
 as a guide to the size and frequency of subsequent doses. 
 
 As a routine measure, the opsonic index has fallen into disuse, vac- 
 cine therapy being largely guided by the clinical evidences of reaction 
 and the condition of the patient. That it has distinct value, particularly 
 in scientific investigation, is generally admitted, and it is well to re- 
 member that in the early years following Wright's investigations the 
 practice of vaccine therapy was limited to those skilled in determining 
 the- index, preparing the vaccine, and carefully guiding and guarding 
 its administration. It is to be regretted that, the wholesale and indis- 
 criminate manufacture and use of vaccines have brought this valuable 
 field of therapy inevitably into disrepute. This is being realized more 
 and more, and the effort is being made to restore the value of this form 
 of therapy. This effort consists in recognizing the possibilities and 
 hmitations of the method, and confining its practice to those who possess, 
 at least, sufficient knowledge of bacteriology to prepare a vaccine and 
 make an opsonic measurement, the best results being secured by co- 
 operation between bacteriologist and clinician.. 
 
CHAPTER XIII 
 BACTERIAL VACCINES 
 
 In this chapter a method for preparing bacterial vaccines will be 
 described, the general discussion of vaccine therapy, with the special 
 technic for preparuig cow-pox vaccine, rabies vaccine, tuberculin, and 
 other special vaccuies, being taken up in Chapter XXIX. 
 
 Definition. — Bacterial vaccines are "sterilized and enumerated sus- 
 'pensions of bacteria which furnish, when they dissolve in the body, sub- 
 stances which stimulate the healthy tissues to a production of specific 
 hacteriotropic substances which fasten upon and directly or indirectly con- 
 tribute to the destruction of the corresponding bacteria" (Wright). 
 
 TECHNIC FOR PREPARING BACTERIAL VACCINES 
 
 Bacterial vaccines are made — (1) By procuring the infected ma- 
 terial; (2) by preparing pure cultures of the bacteria that are to be 
 attacked; (3) by making suspensions of these, in saline solution, adding 
 a preservative, and placing in proper containers. 
 
 1. Procxmng Infected Material. — Varioas precautions, according 
 to existing circumstances, should be taken to avoid contamination and 
 to secure material that is truly representative of the focal secretions. 
 For instance, pus should be collected from an abscess cavity or sinus 
 after the surrounding tissues have been cleansed with dilute tincture of 
 iodin, for if we secured a culture of the relatively harmless Staphy- 
 lococcus epidermidis albus from the skin instead of the Staphylococcus 
 aureus, which may be the cause of infection, o„ur vaccine will have little 
 or no value. 
 
 Nasal secretion may be secured after cleansing the nasal orifice with 
 soap and warm water, passing a sterile cotton swab through a nasal 
 speculum, and rubbing the surfaces of the lower turbinates and septum 
 lightly. 
 
 An ear should be cleansed, the excess of secretions removed with 
 sterile swabs, and the culture be made of pus from the infected tissues. 
 Various saprophytes quickly gain admission and grow in the necrotic pus, 
 whereas the infecting bacterium is more likely to be found in the tissues. 
 
 206 
 
TECHNIC FOR PRBPAKING BACTERIAL VACCINES 207 
 
 In the collection of sputum special care is required: the patient 
 should be instructed to brush the teeth with a sterilized brush, rinse the 
 mouth several times with boiled water, and after swallowing several 
 mouthfuls of water, to cough and expectorate into a wide-mouthed 
 sterilized bottle. The sputum may be plated at once, or washed sev- 
 eral times in sterile Petri dishes with sterile water and then cultured. 
 
 Lung Puncture may occasionally be required in infective lung con- 
 ditions in which sputum is not obtainable or is too badly contaminated. 
 A 5 to 10 CO. all-glass syringe with a strong needle is sterihzed by boiling, 
 and 2 or 3 c.c. of peptone broth introduced. The skin of the chest-wall 
 over the site of infection, as shown by clinical evidence, is sterilized with 
 tincture of iodin, and puncture made into the pulmonary tissues. When 
 the desired depth has been reached, 1 c.c. of thfe broth is injected gently 
 into the tissues, and after the lapse of a few seconds reaspirated as far 
 as possible into the syringe. During the operation the patient should 
 refrain from respiratory movements, in order" to minimize any risk of 
 lacerating the pulmonaiy tissues (Allen). 
 
 Urine should always be withdrawn with a sterile catheter after 
 thoroughly cleansing the meatus. This last is especially important, for 
 the infection may be due to a certain strain of Bacillus coli, and unless 
 we are successful in obtaining a culture of this particular strain, the 
 vaccine will have little value. 
 
 Blood specimens are taken with a sterile syringe from a prominent 
 vein at the elbow after the skin has been cleansed and sterilized with 
 tincture of iodin, and cultured in large amounts on proper culture-media. 
 
 2. Preparing Pure Cultures. — This is frequently the most difficult 
 step in the whole technic, for some microorganisms, as, for example, the 
 gonococcus and Bacillus influenzae, grow slowly, require special culture- 
 media, and their colonies may easily be overlooked. To secure pure 
 cultures, and especially to select one or at most two organisms that may 
 be the chief offenders, considerable bacteriologlc knowledge is necessary, 
 and no simple rules or directions can be given in the limited space of this 
 volume, 
 
 1. Stained smears of the secretions of a lesion may indicate the nature 
 of the infection and the best culture-medium and technic to use for pur- 
 poses of isolation. 
 
 2. Cultures of the lesion may be made upon solid media, and isola- 
 tion carried out after a primary growth has been secured. With proper 
 care, primary cultures may be grown, such as staphylococci from the 
 pus of a freshly incised abscess, or the microorganism of a case of cystitis 
 
208 
 
 BACTERIAL VACCINES 
 
 or pyelitis by securing urine with the aid of a sterilized catheter. If 
 slowly growing organisms, such as Bacillus influenza, gonococcus, 
 pneumococcus, etc., are to be cultured, "streak" plates are usually sat- 
 isfactory, and as a routine the best culture-media are, as a rule, those 
 containing serum or blood. 
 
 3. The cultures that are to be worked up into a vaccine are usually 
 best made on solid media. 
 
 4. In making a bacterial vaccine of a freely growing microorganism 
 for an individual patient, it ^vill suffice to plant two agar slant tubes; 
 when dealing with bacteria that grow much less luxuriantly, such as 
 streptococci and pneumococci, four to six tubes should be used. 
 
 5. Cultures are usually grown for twenty^-four hours at 37° C, but 
 
 Fig. 57. — Preparation op a Bacterial Vaccine. 
 
 Removing the culture of bacteria by gently rubbing over the medium (agar-agar) 
 
 with a sterilized platinum loop. 
 
 in the case of rapidly growing organisms a shorter period in the in- 
 cubator will suffice. 
 
 6. When the cultures are ready, a smear of each growth is made and 
 stained in order to see that pure cultures of th§ right microorganism were 
 made. 
 
 7. Inasmuch as the immunizing power of a vaccine is in most cases 
 a factor of the virulence of the organism, thi^ being especially true of 
 such organisms as the pneumococcus, streptococcus, and influenza 
 bacillus, it is well, whenever possible, to employ the first pure subculture 
 for the preparation of the vaccine. 
 
 3. Preparation of the Emulsion. — Carefully observing aseptic pre- 
 caution throughout, pour a portion of a test-tube of a sterile xiormal salt 
 solution over the surface of the first culture, shaking the fluid in such a 
 
TBCHNIC FOR PREPARING BACTERIAL VACCINES 209 
 
 way as to bring the microorganisms into suspension. If the culture is 
 not easily washed from the medium, a sterile platinum loop may be used 
 to remove the growth, care being taken not to cut into the medium and 
 mix the fragments with the bacterial suspension (Fig. 57). 
 
 The bacterial suspension thus obtained is poured on the surface of 
 the second culture, bringing this into suspension, and repeating the 
 process until the whole series of cultures have been suspended, adding 
 more salt solution if necessary. 
 
 The final suspension is transferred to a sterile, thick-walled flask 
 containing glass beads, and shaken by hand or in a mechanical shaker 
 until the bacterial masses are broken up (Fig. 58). This may be es- 
 pecially difficult with diphtheria bacilli and streptococci. Unless the 
 
 Fig. 58, — A Shaking Apparatus (Electric, 110 volts, direct). 
 A satisfactory machine for sliaking vaccines, preparing antigens, malcing emulsions, 
 
 etc. 
 
 elnulsion is perfectly homogeneous, the larger particles may be removed 
 by brief centrifugalization or, better, by filtering through a sterile filter. 
 There is evidence to show that bacteria grown on culture-media con- 
 taining peptone may produce objectionable toxic substances capable 
 of producing anaphylactic phenomena (Reichel and Harkins^). In addi- 
 tion, when, in the preparation of a vaccine, bacteria grown on a serum 
 medium are washed off with normal salt solution, a portion of the serum 
 may be removed and this may be capable of producing disagreeable 
 local and general reactions. For these reasons.it is advisable to wash all 
 suspensions by repeated centrifugalization until the supernatant fluid 
 reacts negatively to the biuret or ninhydrin reaction (Willard Stone). 
 
 ^Centralb. f. Bakteriolog., etc., 1913, 69. 
 14 
 
210 
 
 BACTERIAL VACCINAS 
 
 4. Coimting of the Bacterial Suspension.^Standardization, best ac- 
 complished by counting the bacterial elements con- 
 tained in a unit volume of Ihe suspension, is necessary 
 in order to adjust our initial dose as experience will 
 dictate and for guidance in making subsequent in- 
 jections. 
 
 In dealing with a vaccine we have to count both 
 the dead and the living bacteria, making no dis- 
 tinction, for both furnish the chemical agent that 
 calls forth the elaboration of bacteriotropic sub- 
 stances. Inasmuch as sharp definition and the 
 staining properties of bacteria may be lost in the 
 process of sterilization by heat, the specimen of 
 vaccine to be examined should be secured before 
 sterilization is undertaken. 
 
 The counting or standardization may be done 
 in several ways: (a) By mixing equal portions of 
 normal blood and bacterial emulsion and counting 
 the proportion of corpuscles to bacteria in our mix- 
 ture (Wright) ; (6) by diluting, staining, and counting 
 with the hemo cytometqr, as in the enumeration 
 of red blood-corpuscles; (c) for standardizing large 
 quantities of bacterial vaccine the method of KoUe 
 or (d) that of Hopkins may be used. 
 
 (o) Method of Wright. — Prepare a simple capil- 
 lary pipet, making a mark on its stem about an 
 inch from the tip, and fit a teat to its barrel (Fig. 
 59). Cleanse and prick the finger, press out a drop 
 of blood, take up the pipet and draw up into it first 
 one volume of sodium citrate solution, one of blood 
 and then either one volume of bacterial suspension 
 or two or more volumes, if it appears on inspection 
 to contain much fewer than 500,000,000 of bacteria 
 to the cubic centimeter. To guard against crimping 
 of the corpuscles in drying the films, Wright advo- 
 cates aspirating one or two volumes of distilled water 
 after the blood and bacterial suspension. 
 
 Now expel from the pipet first only the distilled 
 water and bacterial emulsion, and mix these, so that 
 there may be no danger of the red corpuscles becoming hemolyzed, and 
 
 •a 
 
 c 
 
 H ■§ 
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 o 3 
 
 < a 
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 m s 
 
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 « £ 
 
 fit ^ 
 
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 05 -^ 
 
 o 
 
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 ■3 
 
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 Og i3 
 
 u 
 
 Q O . ,-^C\is 
 
 ,0° 9.0 _^o oc^fol 
 
 ■„Goo . -ho 
 
 ^0/ 
 
 CJ- 
 
 CX: 
 
 00 
 
 Fig. 60. — ^A Satisfactory Fkefaration for CouNTiNn a Raotertal Vaccine, 
 The microorganisms are well separated and evenly distributed among the cor- 
 puscles; the spread is even and regular, and the corpuscles are not gathered in 
 rouleau formation or irregular clumps. 
 
 Fi.G. 61. — An Unsatisfactory Preparation for Counting a Bacterial Vaccine. 
 The microorganisms are mostly gathered in irregular masseB instead of being 
 separated and evenly distributed; the corpuscles ar« not separated and evenly dis- 
 tributed, but show a tendency to gather in clumps; both factors render a count 
 difficult, inaccurate, and unsati.#'actory. 
 
TECHNIC FOR PREPARING BACTERIAL VACCINES 211 
 
 then proceed to mix together the whole contents of the pipet, aspirating 
 and recxpelling these a dozen times. Then make two or three micro- 
 scopic films from the mixture, spreading these out on slides that have 
 been roughened with emery. 
 
 The film s are dried in the air, fixed by immersing them for two min- 
 utes in a saturated solution of corrosive subUmate, washed thoroughly, 
 and stained for a minute with carbolfuchsin diluted IVIO or carbolthionin 
 for two to five minutes, and then washed and dried. 
 
 The films are now given a preliminary examination. If red cor- 
 puscles and bacteria are found in approximately the same numbers and 
 the suspension is free from bacterial aggregates, the count may be made 
 (Fig. 60). If either the bacteria or the corpuscles are largely in excess, 
 new mixtures and new films must be made. In case the bacteria are 
 gathered in clumps, the suspension should be shaken again and new 
 films prepared (Fig. 61). 
 
 When satisfactory films have been obtained, the actual counting 
 may be done. This is carried out with an oil-immersion lens, and in 
 order to secure accuracy, it is necessary to restrict or divide the field by 
 a small square diaphragm made of paper or cardboard, or by inscribing 
 cross lines on a small clean cover-glass and dropping them on the dia- 
 phragm of the eye-piece. 
 
 A field is now chosen at random, and the corpuscles and bacteria 
 are counted, the results being jotted down on a sheet of paper, keeping 
 each enumeration separate and writing the numbers in two columns. 
 Proceed at random from field to field, traversing every part of the 
 slide. Establish a rule for counting corpuscles that transgress or touch 
 the edge of the field. Eliminate from consideration any parts of the 
 films in which the preparation is unsatisfactory as regards the staining, 
 or with respect to the integrity of the red corpuscles. The examination 
 is continued until at least 500 corpuscles have been counted, half of the 
 count being made from the second slide. The number of microorganisms 
 counted is now totaled, and the approximate number per cubic centi- 
 meter estimated. Let us assume, for example, that 600 red cells and 
 1200 bacteria have been counted. Now, a cubic niillimeter of blood 
 contains 5,500,000 red coipuscles, and equal volumes of blood and emul- 
 sion were taken. A cubic millimeter of the emulsion, therefore, contains 
 
 5,500,000X1200 ,i„„„„^^ . ,- •„■ . 
 
 = 11,000,000 organisms per cubic millimeter, or 
 
 600 
 
 11,000,000,000 per cubic centimeter. 
 
 (6) The second method of counting is precisely gimilar to that em- 
 
212 
 
 BACTERIAL VACCINES 
 
 P 
 
 ployed for the enumeration of blood-corpuscles", the diluting and staining 
 fluid being made by adding to a 1 per cent, solution of sodium chlorid 
 in distilled water sufficient formalin to make 2 per cent., and alcoholic 
 gentian-violet, 5 per cent. The emulsion is drawn up in a white cor- 
 puscle pipet to the mark 0.5, and with diluting fluid to the mark 11. 
 The contents are then mixed thoroughly for several minutes, several 
 drops expelled, a drop placed in the counting chamber and properly 
 covered with a special thin cover-glass. The , bacteria are allowed to 
 
 remain in the counting cell for 
 at least half an hour prior to 
 enumeration. A large number 
 of small squares are counted, 
 and the average of one square 
 obtained. By multiplying this 
 figure" by 4000 and then by 20, 
 the number of bacteria per 
 cubic millimeter is obtained, 
 and 1000 times this figure 
 gives the number of bacteria 
 contained in one cubic centi- 
 meter of the vaccine. If the 
 enmljsion is highly concen- 
 trated, the red cell pipet may 
 be used and the calculations 
 madg accordingly. 
 
 (c) Method of Kolle.—k 
 platJform loop adjusted to fit 
 No. 2 of a Lautenschlager wire 
 gage (Fig. 62) measures about 
 4 mm., and holds approxi- 
 mately 2 mg., or about 2,500,000,000 orga=iiisms. By growing an 
 organism on slants of agar and emulsifying a certain number of loopfuls 
 in a measured quantity of saline solution, an approximate method 
 of standardization is obtained. According to KoUe, ordinary slants of 
 agar will hold about 15 loopfuls of staphylococci, Bacillus typhosus, 
 or Bacillus coli, and about 5 loopfuls of streptococci and gonococci. 
 
 (d) Method of Hopkins.^— This is based upon concentrating a 
 bacterial suspension by centrif ugalization and, .the preparation of stand- 
 ard emulsions from the sediment. The emulsion is filtered through a 
 1 Jour. Amer. Med. Assoc, 1913, xl, 1615. 
 
 Fig. 62. — Instbtiment fob the Stamjaediza- 
 
 TION OF PlATINTTM LoOFS. 
 
 The 2 mm. loop holds approximately 2,500,000,- 
 000 bacteria (as, Bacillus typhosus). 
 
TECHNIC FOR PREPARING BACTERIAL VACCINES 
 
 213 
 
 small cotton filter to remove larger clump of bacteria and particles of 
 agar, and is then placed in specially constructed centrifuge tubes (Inter- 
 national Centrifuge Company, see Fig. 63), covered with rubber caps, 
 and centrifugalized for half an hour at a speed of approximately 2,800 
 revolutions a minute. The salt solution and bacteria above the 0.05 
 mark are then removed, and 5 c.c. saline solution is measured into the 
 tube, so as to make a 1 per cent, emulsion. If the sediment does not 
 reach the 0.05 mark, its volume is read on the scale, and a corresponding 
 quantity of saline is added to make the emulsion 1 per cent, in strength. 
 The bacteria are forced into suspension, the 
 vaccine transferred to a sterile tube, and the 
 microorganisms killed in the usual manner. 
 
 Estimations of carefully counted suspensions 
 obtained by centrifugalization in the foregoing 
 manner gave the following results: 
 
 Staphylococcus aureus and albus . 
 
 Streptococcus lisemolyticus 
 
 Gouococcus , 
 
 Pireuniococcus . . 
 
 Bacillus typhosus . 
 
 Bacillus coli 
 
 Per 
 
 Cent 
 
 ..1 
 ..I 
 ..1 
 ..1 
 . .1 
 . .1 
 
 Billion 
 Per C.c. 
 
 10.0 
 8.0 
 8.0 
 2.5 
 8.0 
 4.0 
 
 5. Sterilizing the Vaccine and Testing its 
 Sterility. — When, after the preliminary examina- 
 tion, the films for counting have been found satis^. 
 factory, a pause is made to start the process of 
 sterilization, which may continue while the count 
 is being made. Either heat or a germicide may be 
 used for sterilizing vaccine, preferably the former. 
 
 The vaccine may be placed in a test-tube, 
 which is then sealed (Fig. 64), and the whole im- 
 mersed in the water-bath; a simpler method, and 
 one just as good, is to place the flask or tube of 
 vaccine in the bath, observing special care to see that the water is above 
 the level of the vaccine. 
 
 Efficient sterilization is dependent upon permitting the process to con- 
 tinue at the minimum temperature for the minimum length of time. With 
 the water-bath at 56° to 60° C. sterilization is nearly always complete in 
 an hour. 
 
 The vaccine should be now cultured to test its sterility. At least a 
 dozen platinum loopfuls are transferred, under strict aseptic precautions, 
 
 Fig. 63. — Hopkins' 
 Tube for Stand- 
 ardizing A Bacte- 
 rial Vaccine. 
 
214 
 
 BACTERIAL VACCINES 
 
 to a slant of a suitable culture-medium, such as Loffler's blood-serum or 
 blood-agar; this is incubated at least twenty-four hours, or longer if the 
 organism is a slowly growing one. It is then examined, and if found 
 sterile, the preparation of the vaccine may be completed. If not, the 
 vaccine is heated for another hour, or, preferably, a new vaccine is 
 prepared. 
 
 6. Diluting the Vaccine and Adding a Preservative. — Having made 
 the count and sterilized the vaccine, it is next diluted with sterile saline 
 
 solution so that each cubic centimeter 
 
 contains the dose decided upon. If 
 
 (5|~ 
 
 A 
 
 "^^y 
 
 Fig. 64A. — -A Stock Ampule op 
 Vaccine. 
 
 Fig. 64B. — Stock Bottle of Bacterial 
 Vaccine. 
 
 the treatment is likely to be prolonged, a sufficient number of doses 
 should be provided for. It is a good plan not to dilute all the vaccine, 
 but to preserve the remainder undiluted in case larger doses are subse- 
 quently needed. If, for instance, a vaccine of Staphylococcus aureus 
 contains 1,500,000,000 organisms per cubic centimeter and the dose 
 decided upon is 500,000,000 per cubic centimeter, sufficient vaccine for 
 .30 doses is prepared by withdrawing 10 c.c. of vaccine in a sterile con- 
 tainer and adding 20 c.c. of sterile salt solution. This mixture is agitated. 
 
TECHNIC FOR PREPARING BACTERIAL VACCINES 
 
 215 
 
 dilution of phenol is 
 
 f 
 
 to insure thorough mixing, and 01 c.c. of a 1:-.100 
 added to each cubic centimeter of vaccine aS a 
 preservative against chance contamination. Thus, 
 in the foregoing example, 3 c.c. of the diluted 
 phenol would be added. The amount of stock 
 vaccine is estimated or measured, 0.5 per cent, 
 phenol is added, and the vaccine stored in a 
 sterile container in the refrigerator, being first 
 properly labeled with the patient's name, the 
 date, and the number of bacteria per cubic centi- 
 meter. 
 
 The vaccine may now be placed in a sterile 
 vaccine bottle, fitted with a sterile rubber cap, 
 and properly labeled (Fig. 64). When it is to be 
 - administered, the cap is 
 
 touched with tincture of iodin, 
 the needle plunged through the 
 cap, and a dose withdrawn with 
 a sterile syringe. The punc- 
 ture in the cap is then scaled 
 with a drop of flexible collo- 
 dion. This method is inex- 
 pensive, and with proper care 
 is quite satisfactory, especially 
 for stock vaccines. 
 
 Another and probaBly 
 better method, especially for 
 autogenous vaccines, consists 
 in tubing each dose in separate 
 sterile ampules (Fig. 65), which 
 are then sealed in the flame. When the vaccine 
 is to be administered, the ampule is well shaken, 
 the neck broken in a towel, and the contents 
 aspirated into a sterile syringe. These ampules 
 may be purchased ready for use or be made in 
 the laboratory, using 6 mm. soft glass tubing 
 (Fig. 6). For pipeting a vaccine into ampules tiie 
 special automatic pipet shown in the illustration 
 (Fig. 66) is quite convenient. As a rule, vac- 
 cines should be preserved in a cool place, such as a refrigerator. 
 
 Fig. 65.— a Small 
 
 Vaccine Ampule. 
 
 Capacity 1 c.c. 
 
 Fig. 66.— Comee's 
 Automatic Pipet. 
 (Steele Glass Co., 
 Philadelphia.) 
 
 The inner tube to 
 the tip of the pipet 
 holds exactly 1 c.c. If 
 the rubber teat raises 
 too much fluid, the 
 excess is received in tlie 
 gla^s reservoir; when 
 too much fluid accumu- 
 lates in this, it may be 
 emptied by turning the 
 poiiit of the inner tube 
 downward and ejecting 
 the fluid by pressure on 
 the teat. 
 
216 BACTERIAL VACCINES 
 
 PREPARATION OF SENSITIZED BACT.ERIAL VACCINES 
 
 A highly immune serum is prepared by imflniunizing a series of rabbits 
 or a goat with the microorganism to be used in preparing the vaccine. 
 The first injections consist of heat-killed emulsions, administered 
 subcutaneously. After the first or second dose the period of heating is 
 gradually reduced, and the dose increased, tmtil finally the injections 
 may be given intravenously and with living microorganisms. From time 
 to time a small amount of serum should be examined for immune 
 bodies: with the typhoid-cholera group, by testing for bacteriolysin and 
 agglutinins; with staphylococci and streplJococci, by agglutination, 
 bacteriotropic, and complement- fixation tests; with pncumococci, gon- 
 ococci, and meningococci, by bacteriolj^tic, agglutination, and bacterio- 
 tropic tests. When a highly immune serum is secured, the animal is 
 bled, the serum isolated, heated to 56° C. for half an hour, and stored 
 in a strictly aseptic manner. 
 
 To "sensitize" the bacteria, thick, even emulsions of young cultures 
 in normal salt solution are treated with onQ-half to an equal bulk of 
 inactivated immune serum, and the mixture gently agitated at room 
 temperature for from six to twelve hours. The emulsion is then thor- 
 oughly centrifuged, and the residue of bacteria washed three times mth 
 sterile salt solution, after the manner in which the red corpuscles are 
 washed. After the final washing the bacteria are resuspended in salt 
 solution, shaken for a time to insure breaking Jip of agglutinated clumps, 
 counted, heated at 60° C. for an hour, cultured as a test for sterility, and 
 then diluted so that the emulsion will contain slightly larger doses than 
 a corresponding dose of ordinary vaccine prepared for administration. 
 
 "Sensitization" probably consists in the union of bacteriolytic 
 amboceptor with its antigen, and when injected, serves, with the 
 patient's complement, to hasten solution or lysis of the bacteria (antigen), 
 thereby liberating quickly the chemical substances required for the 
 stimulation of antibodies. 
 
 Metchnikoff and Besredka are using sensitized living bacteria, and 
 their work is being followed with much interest. In this country strict 
 legal restrictions and regulations exist regarding the sending of living 
 cultures through the mails. If, therefore, the method should fulfil the 
 high claims and expectations made for it, there may be considerable 
 difficulty in bringing it into general use. 
 
THE ADMINISTRATION OF A BACT^JIIAL VACCINE 217 
 
 THE ADMINISTRATION OF A BACTERIAL VACCINE 
 
 Syringe. — Vaccines are best administered with the aid of a 1 c.c. 
 
 all-glass syringe, furnished with a sharp platinum iridium or steel needle. 
 
 These may be sterilized in boiling water for a minute or longer. After 
 
 sterilization, the parts should be carefully adj usted and the syringe loaded. 
 
 According to Wright, time and trouble may=be saved by sterilizing in 
 oil at a temperature of 125° to 140° C. If this temperature is not ex- 
 ceeded, the oil can be drawn into the syringe without danger of breaking 
 the glass. The procedure is as follows: Partly fill a tablespoon with 
 any vegetable oil, and into this place a bread-crumb about the size of 
 a large hemp-seed. Then heat over a spirit-lamp until bubbles of steam 
 begin to appear about the bread-crumb. The temperature of the oil 
 is now about that of boiling water. After bubbles have ceased to form 
 about the bread-crumb — ^meanwhile drawing up the oil once or twice 
 into the syringe — reapply the heat very cautiously until the bread- 
 crumb shows the first sign of turning brown (at about 140° C). Then, 
 without allowing time for the oil to cool, dra-s^ it up two or three times 
 into the syringe, being careful to see that it -comes into contact with 
 every part of the interior. 
 
 If it is desired, so as to improve the appearance, to get rid of all re- 
 maining traces of oil from the syringe, this pan be easily effected by 
 drawing up into the syringe a very weak (0.25 to 0.50 per cent.) solution 
 of sterilized sodium carbonate. 
 
 Method of Making the Inoculation. — As the administration of a 
 vaccine is frequently followed by a temporary depression of the resist- 
 ing powers of the individual and a feeling of lassitude, the injections are, 
 as a rule, best given during the afternoon and evening, the night's rest 
 aiding in overcoming the depression. Since the determination of proper 
 dosage rests mainly on the observation of such clinical signs and symp- 
 toms as temperature, pulse, and local reaction at the site of the lesion, 
 the patient should be watched during the following twenty-four to 
 forty-eight hours. 
 
 The injections should be given at a point where the tissues are loose, 
 where muscular action is not much in evidence, and where pressure by 
 clothing or weight is not made. The most suitable localities are in 
 front of the shoulder, at a site about IJ^ inches below the center of the 
 clavicle; high up in the buttock, or in the side of the abdomen, about 
 two or three inches inside the anterior-superior spine of the ilium. The 
 skin at the point of injection should be touched with tincture of iodin, 
 
218 BACTERIAL VACCINES 
 
 which is removed after the injection has been given by washing with 
 alcohol. 
 
 Both convenience and experimental woi;k to test the comparative 
 efhcacy of inoculation into different tissues point to the subcutaneous 
 tissues as the most suitable site for inoculation. The best method of 
 procedure is to pick up a fold of skin between the finger and thumb, and 
 then to push the needle well down into the middle of the fold, and slowly 
 inject the fluid. 
 
 Since it is known that the power of response of the tissues to the 
 stimulus of a vaccine is somewhat limited, it would seem advisable to 
 choose a new site for each successive inoculation. 
 
 The Effects of Inoculation. — The local effects produced at the site 
 of inoculation vary considerably, being influenced by the nature of the 
 individual, the variety and amount of the inoculum, and the sensitive- 
 ness of the patient's tissues to stimulation.. In the majority of cases 
 the local reaction is Hmitcd to a very shght reddening of the skin around 
 the puncture for an area of about one incb. In some instances, oc- 
 casionally encountered where a large number of typhoid inoculations 
 have been made, the reaction after the flrst dose is more severe than 
 after subsequent doses, and is accompanied by considerable edema, 
 hyperemia, and pain. 
 
 The focal effects about the lesion are exceedingly important in de- 
 termining the reaction of the patient, and sei-ve as a guide to the 
 adjustment of dosage and intervals. Where, in a case of furuncle, an 
 appropriate dose of staphylococcus vaccine is administered, within a 
 few hours increased hyperemia is seen around the focus, and there is a 
 slight increase in the swelling. When very small doses are given, these 
 focal symptoms may practically be absent, but, as a rule, a slight re- 
 action does no harm, but serves rather to show that the vaccine possesses 
 some degree of potency and may aid in the curative process. 
 
 The constitutional effects may also vary wjthin wide limits. An ade- 
 quate, but not excessive, dose may, within a few hours, produce a feeling 
 of lassitude, headache, slight rise in temperature, and acceleration of the 
 pulse-rate. Severe constitutional reactions are generally due to ex- 
 cessive dosage, but may occur in some persons after doses that were 
 previously well borne. 
 
 Frequency and Dosage of Inoculation. — No definite rules can be 
 laid down, each patient being a law unto himself. The opsonic index 
 has been largely abandoned as a guide to the administration of vaccine, 
 the reaction and condition of the patient now governing the dosage. 
 
THE ADMINISTRATION OF A BACTERIAL VACCINE 219 
 
 In more acute infections, and in delicate pesrsons, smaller doses are 
 usually indicated. It is well to make the first dpse small, and if no reac- 
 tion occurs within forty-eight hours, a second and a larger dose may be 
 given. If, however, the patient presents other symptoms of a general 
 reaction, the dose given was large enough, and may be repeated, as 
 necessary, at intervals of from five to seven days. It should be care- 
 fully borne in mind that an increase in dosage is contraindicated so long 
 as any sign of general or focal reaction is produced and steady progress 
 is maintained. One should always be on guard to detect any signs of 
 fresh infection by some other organism, and if a given vaccine is failing 
 to exert a beneficial effect, additional cultures should be made, instead 
 of continuing to administer dose after dose of the same vaccine. 
 
 The intervals at which injections are to be- made are of some im- 
 portance. It is certainly better to wait too long than to inoculate 
 prematurely, but the ghost of the "negative phase" is always too prom- 
 inent in the minds of the inexperienced. The inoculations may be 
 given while improvement is still in progress or convalescence well es- 
 tablished, in the endeavor to secure a summation of positive phases of 
 clinical improvement; or one may wait for the first signs of retrogression 
 before administering another dose. The formea: method is the preferable 
 procedure, but is difficult to accomphsh; the latter is less ideal, but 
 is easier to perform and more devoid of risk. 
 
 The dosage varies according to whether the infection is acute or 
 chronic, the nature of the microorganism, and the age of the patient. 
 No fixed rules can be given. In acute infections the dose should be 
 small and may frequently be repeated; in chronic infections larger doses 
 may be given at longer intervals. If in doubt- as to the size of the dose 
 to be given, it is better to give a small dose, and carefully observe the 
 effect on the patient, letting this serve as an index to subsequent doses. 
 Children tolerate relatively large doses of bacterial vaccines, but the 
 dosage should depend on the weight and not on the age of the child. 
 
 The following is a list of the ordinary dosesifor adults of various bac- 
 terins : 
 
 Staphylococcus aureus 100,000,000 to 1,000,000,000 
 
 Staphylococcus albus and citreus 200,000,000 to 1,000,000,000 
 
 Streptococcus pyogenes 2.5,000,000 to 200,000,000 
 
 Gonococcus 25,000,'000 to 200,000,000 
 
 Typhoid bacillus 2o0,000;000 to 1,000,000,000 
 
 Colon bacillus 100,000;000 to 1,000,000,000 
 
CHAPTER XIV 
 ANTITOXINS 
 
 For general purposes the antibodies produced during infection may 
 be divided into two groups, the first consisting of those antibodies that 
 are truly antagonistic to the bacterium or its products responsible for 
 their production, and the second those that are not in themselves de- 
 structive, but that probably prepare the bacterium for the action of a 
 more powerful antibody of the first group. 
 
 To the first group belong the antitoxins, which neutralize the toxins 
 of a bacterium without being directly destructive to the microorganism 
 itself; and the bacteriolysins, which are truly destructive, causing the 
 bacterium to break up and finally disappear. 
 
 To the second group belong the opsonins, which, as we have seen, 
 prepare the bacterium for phagocytosis; and the agglutinins and pre- 
 cipitins, which, while not in themselves des"tructive, probably in some 
 manner prepare their antigen for the action of bacteriolysins, just as 
 opsofiins prepare them for phagocytosis. 
 
 Definition. — Antitoxins are antibodies in the blood that are capable of 
 directly and specifically neutralizing the dissolved toxins that caused their 
 production. 
 
 Historic. — Bacteriolysins were discovered before antitoxins. Their 
 discovery is due to the researches of Nuttall, Fodor, Buchner, and others, 
 who showed that normal serum, and especially the serum of animals 
 artificially immunized against a certain bacterium, was able to exert a 
 destructive action on the microorganism, causing its dissolution and 
 final disappearance. This property of the blood-serum was found to 
 diminish with age, and to disappear completely when the serum was 
 heated to 56° C Buchner laid greatest stress upon the importance of 
 the thermolabile substance which he called alexin, but later researches 
 have shown that the main factors are the specific bacteriolysins, which, 
 however, are practically powerless to destroy their antigen without the 
 cooperation of alexin (later renamed "complement" by Ehrlich). 
 
 While these studies were being made, in the hope of thus explaining 
 all phases of immunity, Behring discovered that in diphtheria infections 
 
 220 
 
FORMATION OF ANTITOklNS 221 
 
 induced experimentally, while the animals became more and more im- 
 mune, virulent bacilli may, nevertheless, be present at the site of in- 
 jection. Here, then, was an example of immunity that could not be 
 explained on the basis of bacteriolysis. Later, in 1890 and 1892, Behr- 
 ing, in collaboration with Kitasato and Wernicke, made further im- 
 portant discoveries, showing that the blood-serum of animals actively 
 immunized against diphtheria and tetanus would protect normal animals 
 against these diseases, and, furthermore, that the blood-serum of the 
 immune animals did not possess bactericidal properties. These ob- 
 servers also demonstrated that such serum could be used therapeutically 
 for the cure of an infection already in progress. 
 
 Soon after these discoveries Ehrlich showed that specific antitoxins 
 (antiricin, antiabrin, etc.) could also be produced against the poisons 
 of some plants, and Calmette produced a similar antitoxin (antivenin) 
 against snake poison. Other observers since then have increased the list 
 of poisons against which antitoxins can be produced; as, for example, 
 Kempner has produced an antitoxin against the;poisonof Bacillus botu- 
 linus, and Wassermarm one against that of Bacillus pyoeyaneus. 
 
 Formation of Antitoxins. — It was formerly believed that there was a 
 direct conversion of toxin into antitoxin, but this certainly is not the 
 case, for the amount of antitoxin produced is altogether out of propor- 
 tion to the amount of toxin injected. 
 
 Antitoxins are formed by those cells that anchor the toxins.' In 
 order to produce them it is necessary that the toxin enter into direct 
 union with the cells and exert a stimulating influence on them, for where 
 a loose union occurs, as between cells and alkaloids, antibodies are not 
 formed. 
 
 Having entered into chemical union with the side-arms of cells, 
 a toxin may destroy the entire cell, and if' a sufficient number of 
 these are destroyed, the host will show symptoms of infection and may 
 succumb. If, however, the cell itself is not destroyed, but only one or 
 more of the side-arms injured, the damage is r-epaired by the cell form- 
 ing new side-arms that have a specific affinity' for the toxin responsible 
 for their production. According to Weigert's overproduction theory, 
 a cell once stimulated to produce these side-arpis or receptors continues 
 to produce them for some time, even after the stimulus has been re- 
 moved. In this manner the specific receptors arc produced in excess, 
 and since all cannot remain attached to the parent cell, the excess is 
 discharged into the blood-stream. Each of these cast-off receptors is 
 capable of uniting with toxin, thus neutralizing the poisonous principles 
 
222 
 
 ANTITOXINS 
 
 the toxin, and rendering it practically harmless. Antitoxins, therefore, 
 are nothing more than these cast-off receptors, vfhich have a specific affinity 
 for their toxins (Fig. 67). 
 
 As Adami has pointed out, it is probable that the toxins exist for 
 some time within the cell, not as part and parcel of the cell, but as a 
 stimulating agent that causes the cell to develop the habit of producing 
 the specific receptors. The mere union of toxin with a receptor, causing 
 it to fall off, and being followed by nature's mode of repair, with the 
 formation of an excess of receptors and no further stimulation, is hardly 
 sufficient to explain the enormous activity of the cells. 
 
 (? 
 
 ^ 
 
 Fig. 67. — Theoretic Formation of Antitoxins. 
 
 That antitoxins may be produced locally was illustrated by the ex- 
 periment of Romer with abrin. This substance has a peculiarly power- 
 ful effect upon the conjunctiva. By gradually immunizing the right 
 conjunctiva of a rabbit with increasing doses, it was shown that, after 
 killing the animal and triturating the conjunctiva with a fatal dose of 
 abrin, an injection of the emulsion of the right or immunized conjunctiva 
 was without effect, whereas the emulsion from the left proved fatal. 
 Thus it will clearly be seen that the cells that had absorbed the abrin 
 had developed and contained antiabrin in sufficient amounts to neu- 
 tralize the poison. 
 
PROPERTIES OF ANTITOXINS 223 
 
 While leukocytes, such as Metchnikoff 's macrophages, arc likewise 
 active in the formation of antitoxins, it is certain that they are not the 
 only cells involved. Metchnikoff claims that antitoxins are merely 
 toxins altered by leukocytic activity, rather than constituents of tissue- 
 cells; this explanation is, however, inadequate, and it has been shown 
 experimentally that the quantity of antitoxin produced is so far in ex- 
 cess of the amount of toxin injected as to render this view untenable. 
 
 Structure of Antitoxins. — According to the side-chain theorj^, anti- 
 toxins are the simplest of antibodies, being composed of a single arm or 
 haptophore group for union with the toxin, and called receptors of the 
 first order. While illustrations of this theoretic structure will convey 
 the impression of mere physical contact or union with toxin, it is to be 
 remembered that experimental data indicate that the union and con- 
 sequent neutralization of the toxin are chemical processes. 
 
 Properties of Antitoxins. — ^While chemical analyses to determine 
 the nature of antitoxin serums were made as early as 1897, little is 
 known regarding it because it is impossible td secure the antitoxic ele- 
 ment free from serum and serum constituent^. Belfanti and Carbone 
 found that most of the antitoxin in a serum is precipitated with the 
 globulins by saturation with magnesium sulphate. This work, which 
 has been verified by Atkinson and Pick, shows that the antitoxin is 
 carried down with the globulin precipitates, but does not necessarily 
 prove that it is itself a globulin. Later Gibson and Banzhaf showed that 
 the portions of the globulin precipitate soluble in saturated sodium 
 chlorid solution carried most of the antitoxin, and with this discovery 
 a practical method of eliminating much of the non-antitoxic portion of 
 the serum was perfected. 
 
 The relation of antitoxins to proteids has also been studied, digestive 
 ferments being permitted to act on antitoxic serum. It has been shown 
 that antitoxin resists tryptic digestion to a well-marked degree; in this 
 respect it resembles the serum globulin. All the evidence obtained in- 
 dicates that a closer relation of antitoxins to, proteids exists than has 
 been shown for the toxins, although all attempts to separate antitoxins 
 from proteids have thus far failed. 
 
 Antitoxins are fairly resistant bodies, and a properly prepared anti- 
 toxic serum, when kept in a cool place and protected from light and air, 
 may be preserved for a year or more with very little deterioration in 
 strength. At times, however, for unknown reasons, antitoxins gradu- 
 ally deteriorate, losing about 2 per cent, in strength a month. Manu- 
 facturers have endeavored to calculate this loss in strength, and have 
 
224 ANTITOXINS 
 
 pjaced a label on each package of antitoxin, bearing a date beyond which 
 the serum is not guaranteed to contain the amount of antitoxin present 
 at the time it was put up. 
 
 The antitoxins, with few exceptions, are far more stable than the 
 toxins, resisting heating up to 62° C, but gradually deteriorating with 
 higher temperatures. Boiling destroys thefli completely. They are 
 readily preserved with small amounts of chlbroform, phenol, tricresol, 
 etc., although strong solutions of these produce destructive changes. 
 Putrefaction of the serum destroys the antitoxin content. Ehrlich has 
 devised the best method for their preservation, which consists in drying the 
 serum in vacuo and preserving it in the dark, at a low temperature, in the 
 presence of anhydrous phosphoric acid. So preserved, antitoxin retains 
 its strength for prolonged periods and is used in standardizing toxins. 
 
 Natural Antitoxins. — The appearance of so-called natural antitoxins 
 can be explained on the basis of Ehrlich's theory. Since the antitoxin 
 is composed of receptors that are not new bodies, but simply normal 
 receptors produced in excess, it is reasonable to assume that a few may 
 be thrown off occasionally, constituting the ng,tural antitoxin. 
 
 Small amounts of natural diphtheria antitpxin may be found in cer- 
 tain individuals and lower animals. Since the diphtheria bacillus is 
 so wide-spread in its distribution, it is possible that minor subinfections 
 may be responsible for antitoxin production, and this is probably always 
 the case when large amounts arc found. 
 
 Information regarding natural antitoxins for other members of the 
 toxin-producing group of microorganisms is less complete, although it 
 is highly probable that natural antitoxins for these exist. 
 
 Specificity of Antitoxins. — ^Antitoxins well illustrate the law of 
 specificity that exists between antigen and antibody, since they are 
 strictly specific for their toxins. Diphtheria antitoxin will neutralize 
 only diphtheria toxin; tetanus antitoxin, only tetanus toxin, and so on 
 through the list. This specificity is not confined to the particular 
 toxin-producing organism that generates ths antitoxin; for example, 
 there are various kinds of diphtheria bacilli, differing as regards 
 morphology and toxicity, although one antitoxin appears to act the same 
 with their various toxins. 
 
 Nature of the Toxin-Antitoxin Reaction. — While the injection of toxin- 
 antitoxin mixtures into the lower animals is the only practical method 
 of testing and standardizing the curative an4 prophylactic powers of 
 their serums, this method does not throw much light upon the nature of 
 the toxin-antitoxin reaction, or show how antitoxin overcomes the toxin. 
 
NATURE OF THE TOXIN-ANTITOXIN REACTION 225 
 
 Antitoxin is protective and curative, in that it actually destroys the 
 toxin, in a manner similar to the dissolution of a bacterium caused by a 
 specific bacteriolysin; or it may influence the tifesue-cells in some way and 
 render them more resistant to the toxins, a view that was held by 
 Roux, and particularly by Buchner; or the antitoxin may form a di- 
 rect chemical union with the toxin, similar to the chemical neutraliza- 
 tion of an acid by a base — an opinion early held.by Behring and elabora- 
 ted later by Ehrlich. 
 
 Experimental data support the view of chemical union with the 
 toxin. In the test-tube some time is required for the union of toxin and 
 antitoxin to occur; this union is hastened by heat and retarded by cold; 
 it is more rapid in concentrated than in dilute? solutions, and in general 
 takes place in accordance with the law of multiple proportions — all 
 of which tends to show the close similarity of tlfe toxin-antitoxin reaction 
 to a chemical process. 
 
 It is generally conceded that antitoxin does not directly destroy the 
 toxin, for when neutral mixtures of toxin and antitoxin are injected into 
 animals, portions of toxin maj' become dissociated and unite with tissue- 
 cells possessing greater affinity for the toxin, and symptoms of infection 
 may result. It is probable that toxin and antitoxin form a distinct 
 compound, and this action requires time for its consummation. For 
 example, Martin and Cherry, by filtering mixtures of toxin and anti- 
 toxin through fine filters that would permit the toxin molecule to pass 
 through but I'estrain the larger antitoxin molec=ule, found that, if filtered 
 immediately, all the toxin in the mixtures was extruded, but that, as the 
 interval between mixing and filtration was prolonged, less and less toxin 
 appeared in the filtrate, until finally, two houTS after mixing, no toxin 
 whatever passed through the filtei'. 
 
 This element of time in support of the chemical nature of the reaction 
 is further strengthened by the experiments of Calmette with snake venom 
 and antivenin, and likewise serves to demonstrate that the antitoxin 
 apparently does not directly destroy the toxin. Although most toxins 
 are thermolabile, Calmette found that snake* venom is rendered inert 
 by heating to 68° C, whereas the antivenin remains uninfluenced by a 
 temperature of 80° C. When neutral mixtures, of venom-antivenin were 
 heated to 70° C, they were found to become- foxic again, presumably 
 on account of the destruction of the antiverifn, the venom itself not 
 being destroyed. Similar experiments were carried out by Wassermann 
 with mixtures of pyocyaneus toxin-antitoxin, with similar results. In 
 both instances, however, as developed later, if the mixt^ures had been 
 
226 ANTITOXINS 
 
 allowed to stand longer, these results would not have been secured. Al- 
 though performed originally to show that an antitoxin does not act by 
 actually destroying its toxin, these experiments simply demonstrate the 
 importance of the element of time in the reaction, without throwing any 
 real light upon the nature of the new toxin-antitoxin compound, if such 
 exists. 
 
 That toxin is counteracted by antitoxin, ind^endent of the partici- 
 pation of living tissue-cells, has been quite conclusively proved by ex- 
 periments in vitro. Ehrlich showed that the agglutinating qualities of 
 ricin — a vegetable toxin — may be overcome in the test-tube by adding 
 antiricin, the corresponding antitoxin. Similar results were obtained 
 by Ehrlich with tetanolysin and tetanus antitoxin, and by Stephens 
 and Myers with cobra venom and its antivenin. 
 
 It is probable that antitoxin has a similar action when injected for 
 therapeutic purposes, as for curing an infection. The longer the in- 
 terval that has elapsed between the time of infection and the administra- 
 tion of antitoxin, the less satisfactory will be the result, as antitoxin 
 becomes less powerful when toxins have formed a firm union with the 
 body-cells. This is especially true in tetanus, where even very large 
 doses of antitoxin may be incapable of dissociating the toxin molecule 
 from the nerve-cells, the serum, therefore, being of greatest value in 
 prophylaxis. In diphtheria, however, the union between toxin and cells 
 is less firm, and the antitoxin is probably capable of neutralizing the 
 toxin already present in the cells, and especially any toxin that may 
 become dissociated from the cell or is freshly prepared by the diphtheria 
 bacillus at the site of infection. The indication, therefore, in giving 
 antitoxin, is to give a dose large enough to neutralize all free and loosely 
 bound toxin, with an excess to neutralize dissociated toxin and that pre- 
 pared by the bacillus during the course of the infection. 
 
 The introduction of the test-tube experiment into the investigation 
 of these reactions permitted more exact observations to be made, and 
 the evidence secured by this means, as well as by carefully graded quan- 
 titative animal experiments, would seem to indicate that we should ac- 
 cept, for the present at least, the conception of the chemical nature of 
 the process. 
 
 PRODUCTION OF ANTITOXINS FOR THERAPEUTIC PURPOSES 
 
 Diphtheria and tetanus antitoxins are manufactured on a large 
 scale, and are used extensively in the prevention and cure of these in- 
 
PRODUCTION OF ANTITOXINS FOR THERAPEUTIC PURPOSES 227 
 
 fections. They are prepared by immunizing horses with carefully 
 graded and increasing doses of the respective toxins until the serum of 
 the animals shows a sufficiently high antitoxin content, after prelim- 
 inary trials, to warrant more extensive bleeding. Large quantities of 
 blood arc then collected aseptically by puncturing the jugular vein. 
 The serum is carefully separated and standardized according to an ac- 
 cepted technic, in order to determine the antitoxin content in units. 
 A small amount of preservative is added, and the serum is finally dis- 
 pensed in special containers or syringes ready for administration. In 
 some laboratories it is customary to precipitate the globulin fraction of 
 the serum with magnesium or ammonium sulphate, and redissolve the 
 portion containing most of the antitoxin in satmrated sodium chlorid 
 solution. The bulk of the serum is thus grbatly decreased, and ob- 
 jectionable constituents largely eliminated, to the obvious advantage 
 of the preparation for therapeutic purposes. 
 
 Antitoxins have also been prepared for other bacterial toxins, as 
 those of the dysentery bacillus (Kruse-Shiga) and Bacillus botulinus, 
 for the vegetable toxins in pollen, and for the animal toxins in snake ven- 
 oms. 
 
 There are other serums for the treatment of certain infections, which 
 depend for their effects chiefly upon the presence of bacteriolysins and 
 immune opsonins, and these are described in a subsequent chapter. 
 
 The Production of Dn>HTHERiA Antitoxin 
 
 The following, taken largely from Park, is a widely used and ac- 
 cepted technic for the production of diphtheria antitoxin: 
 
 Production of the Diphtheria Toxin. — ^A strong diphtheria toxin 
 should be obtained by growing a virulent culture in a 2 per cent, nutri- 
 ent peptone bouillon made from "bob" veal, of an alkalinity that should 
 be about 9 c.c. of normal soda solution per liter above the neutral point 
 to litmus, and prepared from a suitable peptone (Witte). The broth 
 should be poured into large-necked Erlenmeyer flasks in comparatively 
 shallow layers, so as to allow of the free access of air, and maintained at a 
 temperature of about 35° to 36° C. (Fig. 68). 
 
 In the Hygienic Laboratory of the Public Health and Marine-Hos- 
 pital Service "Smith's bouillon" is used for preparing the toxin. This 
 is made of fresh lean beef, after the muscle sugar and all other sugars 
 have been removed by fermentation with a good culture of Bacillus coli. 
 The reaction is adjusted until 0.5 per cent, acid to phenolphthalein, that 
 IS still distinctly alkaline to litmus, and 1 per cent, peptone, 0.5 per cent. 
 
228 
 
 ANTITOXINS 
 
 sodium chlorid, and 0.1 per cent, dextrose are added. The reaction is 
 again noted, and adjusted to + 0.5 per cent. Tie broth is then filtered 
 through filter-paper into fiasks and test-tubes and steriUzed in the auto- 
 clave at a temperature of 120° C. for twentj' minutes. 
 
 After incubating for from seven to ten days the culture is removed, 
 and its purity having been tested by microscopic and cultural methods, 
 it is rendered sterile by the addition of 10 per cent, of a 5 per cent, solu- 
 tion of carbolic acid. After forty-eight hours the dead bacilli have 
 settled on the bottom of the jar, and the clear fluid above is siphoned 
 off, filtered, and stored in full bottles in a cold place until needed (Fig. 
 69). 
 
 Fig. 68. — A Flask of Diphtheria Culture. 
 The bacilli grow on the surface and form a scum. As the culture grows older, 
 the bacUh die and sink to the bottom of Ihe flask, A flask of this shape affords a 
 large surface of culture-medium in contact with oxygei) and facilitates toxin pro- 
 duction. 
 
 Testing the Toxin. — The strength of the toxin is then tested by 
 injecting a series of guinea-pigs with carefully measured amounts. 
 When injected hypodermically, less than 0.005 c.c. should kill a 250- 
 gram guinea-pig, and a toxin requiring more than 0.01 c.c. to kill a pig 
 of this weight is too weak for present purposes. This preliminary titra- 
 tion of the toxin will suffice for determining the dosage for horses, but 
 in standardizing antitoxin, the technic must necessarily be more ac- 
 curate. 
 
 Immunizing the Animals. — The horses used' should be young, vig- 
 orous, of fair size, and absolutely healthy. They should be severally 
 
PRODUCTION OF ANTITOXINS FOR THERAPEUTIC PURPOSES 229 
 
 injected with 10,000 units of antitoxin and with 5000 units of toxin, an 
 amount sufficient to kill 5000 guinea-pigs each weighing 250 grams. If 
 
 Fig. 69. — A Large Toxin Filter. 
 The culture is contained in the large bottle on the shelf, and drains mto the flask, 
 which in turn empties into the earthen " candle." By me|ins of a vacuum the culture 
 IS faltered through the "candle" and collects in the large bottle at the base of the stand. 
 
 antitoxin is not given with the first doses of toxii^, only one-tenth of the 
 dose advised is to be given. After from three to four days, or as soon as 
 
230 ANTITOXINS 
 
 the temperature reaction has subsided, a second subcutaneous injection 
 of a slightly larger dose is given, the amount" of toxin increasing about 
 10 to 15 c.c. per dose, until, six weeks later, the animal is receiving from 
 20 to 30 times the amount originally given. At the end of this time a 
 trial bleeding is made and the serum tested. 
 
 There is absolutely no way of judging which horses will produce the 
 highest grades of antitoxin. Roughly estimated, those horses that are 
 extremely sensitive and those that react feebly are the poorest, but 
 there are exceptions even in these cases. The only reliable method, 
 therefore, is to bleed the horses at the end of six weeks or two months 
 and test their serum. If only high-grade serum is wanted, all horses 
 that give less than 150 units per cubic centinieter should be discarded. 
 The remaining horses should receive steadily increasing doses, the 
 rapidity of the increase and the interval of time between the doses 
 (three days to one week) depending somewhat on the reaction following 
 the injection, an elevation of temperature of more than 3° F. being un- 
 desirable. 
 
 For example, according to Park, a horse that yielded an unusually 
 high grade of serum was started on 12 c.c. of toxin {^ko ^-c fatal dose), 
 together with 10,000 units of antitoxin. Sixty days later a dose of 675 
 c.c. was given, and the serum contained IQOO units of antitoxin per 
 cubic centimeter. Regular bleedings were made weekly for the next 
 four months, at the end of which time the serum had fallen to 500 units 
 in spite of weekly gradually increasing doses of toxin. At the end of 
 three months the antitoxic serum of all the horses should contain over 
 300 units, and in about 10 per cent, as much as 800 units in each cubic 
 centimeter. Not more than 1 per cent, give above 1000 units, and, 
 according to Park, so far none has given him as much as 2000 units per 
 cubic centimeter. The very best horses, if pushed to their limit, con- 
 tinue to furnish blood containing the maximum amount of antitoxin 
 for several months, and then, in spite of increasing injections of toxin, 
 begin to furnish blood of gradually decreasing strength. If an interval 
 of three months' freedom from inoculation is allowed once every nine 
 months, the best horses will furnish high-grade serum for from two to 
 four years. 
 
 Collecting the Serum. — In order to obtain the serum, the neck of 
 the horse should be cleansed thoroughly as for an aseptic operation, and 
 a special tourniquet applied to distend the jugular vein. A small slit 
 is made through the skin over the vein, and a special sharp-pointed 
 cannula is passed upward under the skin for two inches or more and 
 
Fig. 70. — Prepauation of Diphthekia Awtitoxi.n. Sefakation of Blood- 
 
 SEKTJJI. 
 
 The bottle on the left shows blood after standing about an hour; the bottle on the 
 right shows the separation of serum about twelve hours after bleeding. 
 
PKODTJCTION OF ANTITOXINS FOE THERAPEXTTIC PURPOSES 231 
 
 then plunged into the vein. From 6 to 12 Hters of blood are collected 
 by a rubber tube into cylindric jars provided with special tops, facilitat- 
 ing filling with blood and subsequent withdrawal of the serum. The 
 cannula, tubing, jars, and everything used in. collecting the blood and 
 serum should be carefully sterihzed, and the whole operation should 
 be conducted with scrupulous aseptic care in order to avoid contamina- 
 tion. (See Fig. 26.) 
 
 The jars are set aside (Fig. 70) for three or* four days, and the serum 
 is drawn off by means of sterile glass and rubber tubing and stored in 
 large sterile bottles. When the globulins are to be separated, the blood 
 may be added directly to one-tenth of its volume of a 10 per cent, solu- 
 tion of sodium citrate, which prevents clotting of the blood. 
 
 The serum should be clear and free from blood, and its sterility 
 should be proved by culture tests. An antiseptic, such as 0.4 per cent, 
 tricresol, 0.5 per cent, phenol, or chloroform, may be added, but this is 
 not necessary unless it is desired to keep the serum for some time. The 
 serum is poured into small bottles fitted with jubber stoppers, or placed 
 in special syringes labeled with the number of units contained. The 
 whole process should be conducted with scrupulous aseptic technic. 
 Diphtheria toxin varies too much to be used as a standard in determin- 
 ing the antitoxin content of a serum; hence a dried antitoxin is pre- 
 pared by the Hygienic Laboratory and is distributed for this purpose. 
 The serum is evaporated and dried in vacuo by passing dry sterile air 
 heated to 35° C. through it, and when perfectly dry, is preserved in 
 special containers over anhydrous phosphoric acid at a constant temper- 
 ature of 5° C. Preserved in this manner, the? antitoxin is quite stable. 
 Just before use it is dissolved in the required amount of sterile normal 
 salt solution. 
 
 Standardizing the Sertun. — During the earlier investigations it was 
 tieheved that toxin was quite stable, and that it possessed a definite 
 toxicity with a constant value in neutralizing antitoxin. Upon these 
 suppositions the original Behring-Ehrlich antitoxin unit was based, 
 consisting of 10 times the amount of antitoxin that neutralized 10 fatal 
 doses of toxin. For example, if the minimal lethal dose (M. L. D.) of 
 toxin was 0.001 c.c, and 0.01 c.c. was neutralised by 0.01 c.c. of serum, 
 then 0.1 c.c. of serum equaled one unit, or 10 units in a cubic centimeter. 
 Later stronger serums were found, and von Behring and Ehrlich modi- 
 fied the unit, which they now call the immunity umt, to be that quantity 
 of antitoxin which will neutralize 100 times thi minimal fatal dose for a 
 250-gram guinea-pig. 
 
232 
 
 ANTITOXINS 
 
 It was soon discovered that toxins are unstable compounds, and that, 
 almost immediately after their production, they begin to change into 
 toxoids, which are not acutely poisonous, but which retain their power 
 to neutralize antitoxin. 
 
 In order to standardize a serum it is necessary that the strength of 
 the toxin be known, and since this is so variable, a standard antitoxin is 
 supplied by the Hygienic Laboratory, by which the various antitoxin 
 plants may measure the strength of their toxins. By mixing varying 
 quantities of toxin with one uiiit of this standard antitoxin and injecting 
 these into 250-gram guinea-pigs, the L_i_ {limes death) dose is obtained, 
 which is the dose of toxin required to kill a pig in four days with the one 
 unit of antitoxin. In order accurately to determine this dose many 
 pigs may be required, but this method of titration is the key-note to 
 successful standardization. 
 
 Such a titration for instance, has shown a toxin to react as follows : 
 
 TABLE 1.- 
 
 -METHOD 
 
 One antitoxin umt+0.2 
 
 
 
 ' +0.22 
 
 
 
 ' +0.24 
 ' +0.2.5 
 ' +0.2(1 
 
 ' +0.28 
 
 
 
 ' +0.3 
 
 
 
 ' +0.32 
 ' +0.34 
 ' +0.35 
 
 METHOD OF DETERMINING THE L+ DOSE OF DIPH- 
 THERIA TOXIN 
 
 c.c. toxin = No visible symptom.'s. 
 = No symptoms. 
 
 = Uijually no symptoms or a very slight reaction. 
 = Very slight congestion and edema. 
 = Slight edema at site. 
 = Edema; sometimes late paralysis. 
 = Acute edema and' sometimes death. 
 = Always acute death about the fourth day. 
 = Death from second to third day. 
 = Death about the second day. 
 
 Here the L+ dose is 0.32 c.c. The dose of toxin that just neu- 
 tralizes the antitoxin \vithout causing symptoms has been called by 
 Ehrlich the L6 {limes zero) dose, and in this instance it is about 0.24 c.c. 
 This determination, however, has not the same practical value as the 
 L_|_ dose. 
 
 Having determined the L+ dose of the toxin, a series of six to eight 
 guinea-pigs are injected with this constant dose of toxin and increasing 
 amounts of the corresponding antitoxin serupi; for instance. No. 1 
 would receive 0.001 c.c. of serum; No. 2, 0.002 c.c; No. 3, 0.003 c.c; 
 No. 4, 0.004 cc; No. 6, 0.005 c.c; No. 6, O.OOB c.c, etc If at the end 
 of the fourth day Nos. 1, 2, 3, and 4 were dead and Nos. 5 and 6 were 
 alive, the serum would contain 200 units of antitoxin in a cubic centi- 
 meter. These injections are best given with ptecision syringes, the one 
 devised by Hitchens being particularly servicealile (Fig. 71). The syr- 
 inges are sterilized, and the needles are dipped in sterile vaselin to 
 
PRODUCTION OF ANTITOXINS FOR THBRAPETJTIO PURPOSES 233 
 
 plug them. The mixtures are made in the barrel of the syringe, and 
 sufficient sterile salt solution is placed in the side-arm to bring the total 
 volume of the injection up to 4 c.c, and to wash in all traces of toxin and 
 
 Fig. 71. — A Kitchens Syringe. 
 The needle is plugged by dipping the tip in carbelized va.selm. The aide arm 
 holds sterile salt solution; when the needle has been entered, the injeotion is given by 
 pressure on the bulb; the side arm is then turned upward, when the contents flow 
 into the main barrel, and injected in this manner insares accuracy in dosage and 
 uniform bulk of inoculum. 
 
 antitoxin. The mixtures are allowed to stand, for at least fifteen min- 
 utes (Park) before being injected (Fig. 72). Tlie pigs must be of proper 
 weight— i. e., about 250 to 300 grams; the abdominal wall is shaved, 
 
 Fig. 72. — ^A Battery of Kitchens" Syringes. 
 
 and the injection given directly in the median abdominal line. The 
 animals are placed two in a cage, and carefully observed for four or 
 five days for symptoms of toxemia and edema about the site of injection. 
 
234 ANTITOXINS 
 
 PRODUCTION OF TETANUS ANTITOXIN 
 
 The method used in the production of tetanus antitoxin is similar to 
 that employed in producing diphtheria-antitoxin, the horses being 
 inoculated with increasing doses of a strong tetanus toxin. 
 
 Tetanus Toxin. — The toxin is secured by inoculating large flasks 
 or tubes of neutral veal broth containing 1 per cent, of sodium chlorid 
 and peptone with abundant tetanus culture,: and growing these anae- 
 robically at 37° C. for two weeks. The cult.ures are then filtered rap- 
 idly through Berkefeld filters, and the toxin preserved in fluid form with 
 the addition of 0.5 per cent, phenol. As previously mentioned, the 
 toxin rapidly deteriorates — especially tetanospasmin — and for pur- 
 poses of antitoxin standardization it is usually preserved in a dry state 
 after being precipitated with ammonium sulphate. The yellowish, 
 crystalline masses are readily soluble in water or salt solution, and should 
 be used immediately after solution takes place. The strength of the 
 toxin is determined by injecting increasing aihounts into white mice or 
 350-gram guinea-pigs. 
 
 Immunizing the Animals. — According to Park, the "horses receive 
 5 c.c. as the initial dose of a toxin, of which 1 c.c. kills 250,000 grams of 
 guinea-pig, and along with this twice the amount of antitoxin required 
 to neutralize it. In five days this dose is doubled, and then every five 
 to seven days larger amounts are given. After the third injection the 
 antitoxin is omitted. The dose is increased at first slowly until appre- 
 ciable amounts of antitoxin are found to be present, and then as rapidly 
 as the horses can stand it, until they support 700 to 800 c.c. or more at a 
 time. This amount should not be injected in a single place, or severe 
 local and perhaps fatal tetanus may develop." 
 
 Collecting the Senim. — The horses are bled, and the serum is col- 
 lected under strict aseptic precautions, in a manner similar to the col- 
 lection of antidiphtheric serum. The serum should be clear and free 
 from blood, and should be proved sterile by cultural tests. It may be 
 preserved in the liquid state by adding 0.5 per cent, of phenol or 0.4 
 per cent, of tricresol. 
 
 Standardizing the Servim. — The official immunity unit of tetanus 
 antitoxin of the United States Government is based largely upon the 
 work of Rosenau and Anderson. These investigators, together with a 
 Committee of the Society of American Bacteriologists, have defined the 
 unit of tetanus antitoxin to be "ten times the l6ast amount of serum neces- 
 sary to save the life of a SBO-gram guinea-pig for ninety-six hours against 
 
PRODUCTION OF TETANUS ANTITOXIN 
 
 235 
 
 the official test dose of a standard toxin. This test dose consists of 100 
 minimal lethal doses of a precipitated and dried toxin, tested out 
 against 350-gram pigs, and preserved in the Hygienic Laboratory, 
 from where it is sent to various antitoxin plants for the purpose of secur- 
 ing a uniform method and unit of standardization. 
 
 In standardizing tetanus antitoxin, the Lq- dose of toxin is em- 
 ployed. A standard toxin and an antitoxin^ arbitrary in their first 
 establishment, are preserved in the Hygienic Laboratory, and arc kept 
 constant by making frequent tests one against the other. In determin- 
 ing the L+ dose, increasing amounts of toxin i|re mixed with a constant 
 amount of antitoxin equal to one-tenth of ari immunity unit, and in- 
 jected into 360-gram pigs. The L+ dose must contain just enough 
 toxin to neutralize this amount of antitoxin and kill a pig in four days. 
 This L_)_ dose of toxin is sent out by the Hygienic Laboratory to those 
 interested, commercially or otherwise, in the manufacture of antitoxin 
 for purposes of standardization. 
 
 For determining the strength of an unknown serum a large number 
 of mixtures are made, each containing the L+ doses of the toxin and 
 increasing quantities of antitoxin. The measurements are made with 
 accurate volumetric pipets, and the total volume brought up to 4 c.c. 
 with sterile salt solution in order to equalize concentration and pressure. 
 The mixtures are allowed to stand at room temperature for an hour, and 
 are then injected subcutaneously into 350-gram pigs. This method 
 of titrating the antitoxin is shown in the following example from Rosenau 
 and Anderson; 
 
 TABLE 2.— METHOD OF TITRATING TETANUS ANTITOXIN 
 
 No. or 
 
 Weioht of 
 Pig in Ghams 
 
 SUBCUTANEOTJS INJECTION OF 
 
 A Mixture of — 
 
 Time op Death 
 
 Pig 
 
 Toxin (Test 
 Dose) 
 
 Arititoxin 
 
 1 
 
 360 
 350 
 350 
 360 
 350 
 
 Gram 
 
 0.0006 
 0.0006 
 0.0006 
 0.0006 
 0.0006 
 
 C.c. 
 
 0.001 
 0.0015 
 0.002 
 0.0025 
 
 o.dtis 
 
 Two days four hours 
 Four days one hour 
 Symptoms 
 Slight symptoms 
 No symptoms 
 
 2 
 
 3 
 
 4 
 
 s 
 
 
 In this series the animal receiving 0.0015 c.c. of antitoxin died in 
 approximately four days; this amount of serum, therefore, represents 
 TTT of one unit. 
 
236 ANTITOXINS 
 
 BOTULINUS ANTITOXIN 
 
 The nature of the botuhnus poison has previously been described. 
 Wassermann has recently immunized horses against this toxin, and the 
 antitoxin shows unmistakable value in animal experiments, although 
 it has not been employed frequently enough in this form of poisoning 
 in human beings to prove its value. 
 
 ANTIDYSENTERIC SERUM 
 
 The Kruse-Shiga type of dysentery bacillus has been shown to pro- 
 duce varying amounts of a soluble toxin; aind antiserums, which are 
 partly antitoxic and partly bactericidal in nature, have been prepared, 
 and have apparently yielded good therapeutic results in the hands of 
 several observers. Potent antiserums for the Flexner type of bacillus 
 and for various strains isolated from the feces of cases of infantile ileo- 
 colitis have not been produced. Even a virulent strain of the dysentery 
 bacillus does not produce true soluble toxiris in a manner comparable 
 to those produced by tetanus and diphtheria. Potent toxins are sel- 
 dom secured with less than two to three weeks' incubation, and fresh 
 cultures of whole or autolyzed bacilli are likewise quite too toxic, in- 
 dicating that although a soluble toxin may be produced, considerable 
 endotoxin is also present in the bacilli. 
 
 Antidysenteric serum has very little prophylactic value, but in in- 
 dividual cases it frequently exerts a curative action, and should be 
 available for use in institutions and armies: when dysenteric infection 
 is prevalent. 
 
 The older investigators, such as Kruse and Shiga, produced anti- 
 serums by immunization with whole bacilli. Later Kraus and Doerr 
 prepared antitoxic serums with the toxin alone. At the present time 
 the evidence would seem to indicate that the best serums are prepared 
 by injecting both toxins and bacilli, producing a serum that is essen- 
 tially antitoxic and bactericidal in action. 
 
 Culture. — Young and healthy horses are best adapted for immuniza- 
 tion. Two methods may be followed: (1)- Immunization with toxin 
 or (2) with young cultures of whole bacilli. As previously mentioned, 
 investigations have tended to show that the most potent serums are 
 secured by using mixtures of both toxin and microorganisms. 
 
 Several strains of dysentery bacilli should be used, in order that a 
 polyvalent serum may be prepared. Cultures should be grown for two 
 
ANTIDYSENTERIC SERUM 
 
 237 
 
 weeks at 37° C, in alkaline broth similar to that' used for preparing diph- 
 theria toxin; this should be neutralized to phenolphthalein, and 7 c.c. 
 normal soda solution to a liter added. The minimal lethal dose of the 
 mixed unfiUered cultures is determined by giving young rabbits increas- 
 ing doses intravenously, in order to obtain a guide as to the proper dose 
 for immunization. Fatal doses produce severe diarrhea and paralysis 
 of the extremities, with rapid loss in wejght. Rabbits and horses are 
 quite susceptible to the toxin; guinea-pigs and mice are more resistant. 
 
 TABLE 3.- 
 
 -METHOD OF DETERMINING THE MINIMAL LETHAL DOSE 
 OF DYSENTERY CULTURE 
 
 No. 
 
 Weight, Grams 
 
 Dose in Co. 
 
 Result 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 710 
 690 
 695 
 690 
 700 
 
 0,025 
 
 0.05 
 
 0.1 
 
 0.2 
 
 0.3 
 
 No symptoms 
 
 No symptoms 
 
 Diarrhea. Recovered 
 
 Death third day 
 
 Death second to third day 
 
 In this instance the minimal lethal dose was 0.2 c.c. and subsequent 
 cultures of the same strains, grown under similar conditions, showed this 
 dose to remain quite constant. 
 
 It is good practice to keep the cultures growing during the entire 
 time of immtmizatibn. Cultures may, however, be grown for three 
 weeks, filtered through porcelain, and with the addition of 0.5 per cent, 
 phenol, the toxin preserved for long periods of time. The minimal 
 lethal dose of such a toxin is determined in the manner directed above. 
 
 Immunizing the Animals. — Since horses are quite susceptible, the 
 initial dose of unfiltered and unheated culture should not be. larger 
 than the minimal lethal dose for a young rabbit. The dosage is grad- 
 ually increased, and the injections are given subcutaneously for from 
 four to six months, after which several injections of from 300 to 350 c.c. 
 may be given intravenously at one time. If at any time diarrhea and 
 other symptoms of toxemia are well marked, subsequent doses should 
 be smaller and should be given at longer intervals until a higher immu- 
 iiity is produced. 
 
 Instead of using bouillon cultures, young agar cultures may be used, 
 the bacilli being grown for seventy-two hours, and one-tenth of an ordi- 
 nary slant being given as the first dose. The early doses are heated to 
 60° C. for an hour and injected subcutaneously; the later doses consist 
 of cultures washed from 30 to 40 tubes, and are given intravenously. 
 
238 
 
 ANTITOXINS 
 
 Collecting and Testing the Serum.— Aftor three or four months a 
 trial bleeding should be made and the serum tested as follows: the mini- 
 mal lethal dose of a culture is determined and ten times this amount 
 placed in a series of tubes or syringes with increasing doses of serum; 
 the total quantity of injection is made up to 4c.c. with sterile salt solu- 
 tion. The mixtures are set aside for one hour at 35° C. and injected in- 
 travenously in young rabbits. The animals are to be observed for at 
 least five days for diarrhea, paralysis and loss in weight. 
 
 TABLE 4. 
 
 -METHOD OF TESTING ANTIDYSENTERIC SERUM (KRUSE- 
 SHIGA) 
 
 No. 
 
 Weight, 
 Grams 
 
 CULTUKK, 
 
 0.2C.C. M. L.D. 
 C.c. 
 
 Serum, C.c. 
 
 Result 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 G 
 
 600 
 610 
 615 
 590 
 600 
 590 
 
 2.0 
 2.0 
 2.0 
 2.0 
 2.0 
 2.0 
 
 0.00035 
 
 0.0005 
 
 0.001 
 
 0.002 
 
 0.004 
 
 0.006 
 
 Died second day 
 Died third day 
 Diarrhea, recovered 
 Diarrhea, paralysis 
 No symptoms 
 No symptoms 
 
 In this instance 0.004 c.c. of serum was sufficient to protect young 
 rabbits against 10 fatal doses of culture, and demonstrated that it is 
 possible to secure a fairly potent serum against the toxins of the Kruse- 
 Shiga microorganism. 
 
 According to Todd, if the antiserum is given at least one-half hour 
 after administering the culture, it will protect the rabbit. If given 
 twenty-four hours later, it affords no protection. Similarly, the mix- 
 tures of culture and scrum must not be injected immediately after mix- 
 ing, as the results are more irregular than if they are allowed to stand for 
 one-half to one hour before injecting. 
 
 If the trial bleeding shows a satisfactory serum, the horse is bled 
 aseptically, as was previously described, and the serum is separated and 
 preserved with 0.5 per cent, phenol in quantities of 10 c.c. in sterile 
 containers. As there is no official immunity unit, the serum is admin- 
 istered in doses of from 5 to 10 c.c. until a therapeutic effect is secured. 
 
 ANTISTAPHYLOCOCCUS SERUM 
 
 Both Staphylococcus pyogenes aureus and S. pyogenes albus have 
 
 been shown to produce certain soluble toxins, such as a leukocidin and 
 
 a hemolysin, which are partly responsible for the tissue destruction and 
 
 symptoms that accompany these infections. Severe staphylococcus 
 
ANTISTAPHYLOCOCCUS SERTTM 239 
 
 infection is probably due in part to the paralyzing effect and actual de- 
 structive action of the leukocidin upon the leukocytes, preventing, for 
 the time being, the walling-off of the lesion and effectual phagocytosis. 
 Antistaphylococcus serums have been shown to counteract the action 
 of the leukocidin and the hemolysin, and may be useful in the treatment 
 of severe, spreading, or metastatic staphylococcus infections. 
 
 According to Neisser and Wechsberg, during staphylococcus dis- 
 ease an antihemotoxin is produced against the hemotoxin of the cocci; 
 later Bruck, Michaelis, and Schulze attempted to show that a demon- 
 stration of this antistaphylolysin in the seruni may be regarded as evi- 
 dence of a staphylococcus infection. 
 
 Preparation of Antistaphylococcus Serum.: — For immunization pur- 
 poses several different cultures of the Staphylococcus aureus should be 
 used, in order that the antiserums may be polyvalent. Goats or horses 
 may be employed. Cultures may be grown on neutral agar for forty- 
 eight hours, and an emulsion, equivalent to half an agar slant, heated to 
 60° C. for one hour and injected subcutaneously in an adult goat. If 
 10 different strains are used, a four-millimeter loopful from each culture, 
 emulsified in 5 c.c. of sterile salt solution, will be about the proper dose 
 for the first injection. Subsequent doses are given at intervals of a 
 week, and are rapidly increased in size until full, living, unheated cul- 
 tures are injected intravenously without harm to the animal. The 
 serum may be tested by determining its content of antilysin or of bac- 
 teriotropin. Complement-fixation tests are occasionally useful for 
 obtaining an insight into the quantity of bacteriolysin present. 
 
 Technic of the Antilysin Test. — The object of this test is to determine 
 the amount of antihemolysin present in a serunj, which is dependent on 
 the amount of serum necessary to protect the red blood-cells of rabbits 
 against a solution of the staphylolysin. 
 
 (a) Staphylolysin. — This is prepared by growing a known hemolysin- 
 producing staphylococcus in slightly alkaline broth for three weeks, 
 filtering through a Berkefeld filter, and preserving the filtrate with 0.5 
 per cent, phenol in the refrigerator. 
 
 (&) Rabbit Blood. — Remove 2 or 3 c.c. of blood from the ear of a rab- 
 bit and place in 5 c.c. of a 1 per cent, sodium citrate in normal salt solu- 
 tion. Wash the corpuscles three times, and make up in a 1 per cent, 
 suspension (dose 1 c.c.) or up to the original volume of blood (dose, 1 
 drop). 
 
 (c) Patient's Serum. — The serum is inactivated by heating to 56° C. 
 for half an hour. 
 
240 
 
 ANTITOXINS 
 
 (d) Control Serum. — As every normal serum contains a certain 
 amount of antilysin, it is necessary to use a normal control serum. 
 Normal horse serum, dried mi vacuo to prevent deterioration, and freshly 
 dissolved for each test in 10 volumes of sterile distilled water or salt 
 solution, has been advocated by Bruck, Michaelis, and Schulze. 
 
 (e) The Test. — It is first necessary to titrate the staphylococcus fil- 
 trate to ascertain the amount of lysin present. This is accomplished 
 according to the following scheme: 
 
 TABLE 5.- 
 
 -METHOD OF TITRATING STAPHYLOLYSIN 
 
 Amount of 
 Staphti-olysin 
 
 FlWRATE 
 
 Rabbit 
 
 Bt.ood 
 
 One Pee Cent. 
 
 Normal 
 Salt Solution 
 
 Result of Hf.moltsis After Two 
 Hours at 37° C. and Twenty- 
 Four Hours in Refrigerator 
 
 0.005 c.c. 
 
 1 C.C. 
 1 C.C. 
 1 C.C. 
 1 C.C. 
 1 C.C. 
 
 1 c.c. 
 1 c.c. 
 
 1 0.0. 
 
 q. s. 2 CO. 
 q. s. 2 c.c. 
 q. s. 2 c.c. 
 q. s. 2 c.c. 
 q. s. 2 c.c. 
 q. s. 2 c.c. 
 q. s. 2 c.c. 
 
 No hemolysis 
 No hemolysis 
 SUght hemolysis 
 Marked hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 
 0.01 c.c 
 
 0.02 c.c. . . 
 
 0.05 c.c 
 
 0.1 c.c. ... 
 
 0.2 c.c. 
 
 0.5 c.c 
 
 1.0 CO... . 
 
 
 In this test 0.1 c.c. is the smallest amount of ly'sin that can completely 
 hemolyze the given quantity of erjrthrocytes, acid is taken as the unit 
 for the second part of the test. 
 
 The lytic dose of filtrate just determined is hew placed in a series of 
 small test-tubes, with increasing doses of serum to be tested and a con- 
 stant dose of corpuscles. 
 
 TABLE 6.— METHOD OF TITRATING ANTISTAPHYLOLYSIN IN A SERUM 
 
 Amount of 
 Filtrate 
 
 Inactivated 
 
 Serum 
 
 1 Per Cent. 
 
 Rabbit 
 Corpuscles 
 
 0.001 c.c. 
 
 1 CO. 
 
 0.005 0.0. 
 
 1 c.c. 
 
 0.01 c.c. 
 
 1 CO. 
 
 0.05 c.c. 
 
 1 c.c 
 
 0.1 c.c. 
 
 1 c.c 
 
 0.2 c.c. 
 
 1 cc. 
 
 Normal Salt 
 Solution 
 
 Readings After Incubation at 37° C. 
 
 FOR Two Hours and Twentv-foub 
 
 Hf^URS IN THE Refrigerator 
 
 0.1 CO, 
 0.1 CO, 
 0.1 CO 
 0.1 CO 
 0.1 CO 
 0.1 CO 
 
 s. 2 c.c. 
 s. 2 c.c. 
 s. 2 CO. 
 s. 2 CO. 
 s. 2 CO. 
 s. 2 c.c. 
 
 Complete hemolysis 
 Slight inhibition of hemolysis 
 Marked inhibition of hemolysis 
 Complete inhibition of hemolysis 
 Complete inhibition of hemolysis 
 Comfjlete inhibition of hemolysis 
 
 In this instance 0.05 c.c. of the patient's se»um was sufEcient com- 
 pletely to neutralize the lysin. 
 
 A similar test is carried out with normal horse serum. The antilytic 
 dose of this serum is taken as 1, and the patiept's serum is compared 
 
PRODUCTION OF ANTIVENIN 241 
 
 with this unit. For example, if 0.1 c.c. of norcfial horse serum was suf- 
 ficient to neutraUze the lysin in this experiment, then the antilysin value 
 of the patient's serum is 2. 
 
 According to Arndt and others, a high antilysin content of a serum 
 is to be regarded as indicating a staphylococcic infection, even if it is 
 impossible to establish fixed limits for the values. 
 
 PRODUCTION OF ANTIVENIN 
 
 Snake venom contains two toxins, one being largely neurotoxic and 
 producing paralysis of the respiratory centers, and the other being hemo- 
 toxic and irritant, and producing local necrosis of tissues, hemolysis, 
 etc. In venom poisoning the neurotoxic effect is jnost dangerous. Largely 
 as the result of the work of Calmette and Fraser an antivenin has been 
 prepared that is capable of counteracting the neurotoxic action not only 
 of cobra venom, but to a lesser extent of other venoms as well. These 
 serums, however, appear to have no effect or but very little upon the 
 irritant toxins. In the poisonous American snakes, such as the rattler, 
 inoccasin, and copperhead, the effects of the irritant toxins are largely 
 in evidence, a:nd satisfactory antiserums for these venoms have not 
 been prepared (McFarland). 
 
 In preparing antivenins the toxins, since they are thermolabile, 
 must be used unheated; subcutaneous injections are usually followed by 
 extensive sloughing, and although a certain amount of immunity may 
 be induced in the horse by intravenous injection, there is apparently no 
 protection against the local action of the toxins. 
 
 Preparation of Antivenin. — According to Calmette, horses maj- be 
 immunized by giving them weekly subcutaneous injections of gradually 
 increasing doses of cobra venom, heated to TQ" C, for an hour, which 
 precipitates the irritant toxins without injuring the neurotoxin. The 
 initial dose is usually 0.01 gram, gradually increased until, by the end of 
 four months, 4 grams may be given at a single jiose. The serum is then 
 tested by mbdng increasing doses with the minimal lethal dose for a 
 young rabbit, and injecting the mixtures intravenously into a series of 
 rabbits. 
 
 Since tlie neurotoxin may prove dangerous in any case of snake-bite, 
 antivenin may be given to advantage, although the local pain and ne- 
 crosis are not relieved by the serum. 
 
 16 
 
242 ANTITOXINS 
 
 PRODUCTION OF POLLEN AJSITITOXIN 
 
 The pollen of certain plants is markedlj; toxic for susceptible in- 
 dividuals. In America the pollen of the golden-rod and of rag weed 
 frequently produce a syndrome of distressing symptoms known as 
 "autumnal catarrh." The onset and chara'cter of the symptoms of 
 pollen intoxication are strongly suggestive of- an anaphylactic reaction. 
 Dunbar has studied pollen toxins quite extensively, and considers them 
 the etiologic factor in the production of hay-fever. 
 
 Pollen antitoxin has been prepared by immunizing susceptible horses, 
 the toxin being isolated by mixing the ground pollen with 5 per cent, 
 sodium chlorid solution and 0.5 per cent, phenol at 37° C for ten hours. 
 In the form of a proteid, it is then precipitated by adding eight to ten 
 volumes of 96 per cent, alcohol, dissolving the resultant white precipi- 
 tate in physiologic salt solution (Citron). 
 
 THE MEASURE OF ANTITOXINS 
 
 Antitoxin Unit. — A unit is the definite measure of antitoxin in any 
 serum or solution that will neutralize a certain amount of toxin. As pre- 
 viously stated, the United States Goveriunenf has estabhshed a definite 
 unit for the standardization of diphtheria and tetanus antitoxins, and 
 frequently examines the serums made by various licensed manufacturers. 
 Officers of the Public Health and Marine-Hospital Service purchase 
 from reliable pharmacists several grades of antitoxins made by each 
 manufacturer, which are then sent to the Hygienic Laboratory at Wash- 
 ington, where they are tested for potency, freedom from contamination 
 by bacteria, chemical poisons, especially tetamis toxin, and for excessive 
 amounts of preservative. Delinquencies are reported immediately, and 
 steps are taken to withdraw that particular lot=of serum from the market. 
 
 A unit of diphtheria antitoxin may he defined as the "amount of anti- 
 toxin that will just neutralize 100 minimal fatal doses of toxin for a 250- 
 gram guinea-pig." 
 
 A unit of tetanus antitoxin may be defined as the "amount of antitoxin 
 which will just neutralize 1000 minimal fatal doses of toxin for a 350- 
 gram guinea-pig." 
 
 The standardization of these serums is useful as a guide to their ad- 
 ministration, especially when given for prophylactic purposes, where 
 experience has taught that so many units usually confer protection; it 
 also serves for purposes of record. In the treatment of diphtheria and 
 
PRACTICAL applicati6n 243 
 
 tetanus, however, the serums are usually given until a therapeutic effect 
 is noted, regardless of the number of units administered. If it were 
 possible to determine quickly and accurately the amount of toxin in a 
 given patient, then neutralization could be accomplished along the same 
 lines that make this possible in the test-tube. The indications are to 
 administer at once sufficient antitoxin to neutralize all the toxin, giving 
 subsequent doses large enough to overcome the toxin as it is produced 
 until the focus of infection is removed. 
 
 Antitoxin should be kept in a cold place and protected from air and 
 light. When this is done, they usually do not deteriorate more than 30 
 per cent, of their original strength, and often much less, within a year. 
 All manufacturers place a larger number of units in the container than 
 the label calls for, in this way allowing for the gradual loss in strength up 
 to the date specified on the label. According*lo Park, the antitoxin in 
 old serum is just as effective as that in fresh serum, except that there is 
 less of it. 
 
 PRACTICAL APPLICATION 
 
 The employment of antitoxic serums both in prophylaxis and in the 
 treatment of infection, is considered in greater detail in the chapter on 
 Passive Immunization and Serum Therapy. 
 
 Romer's Method of Determining Small Amomits of Diphtheria Antitoxin. — The 
 
 principle of this method is based upon the observation that, when very small amomits 
 of diphtheria toxin are injected inlracutaneously into the abdominal skin of guinea- 
 pigs, small areas of edema and necrosis result in about forty-eight hours. When 
 such injections are made with mixtures of to.xin and antitoxin, the presence of free 
 toxin is indicated by such tissue changes. It is chiefly used in determining the anti- 
 toxin content of human serums after active immunization with the toxin-antitoxin 
 mixtures of von Behring. (See p. 718.) 
 
 Technic. — I conduct this test in the following ma,nner; The "limes-necrosis" 
 (Li) dose of a toxin is first determined, which is the amount of toxin which, together 
 with 1 jVb of a unit of standard antitoxin, will still produce a mininial amount of ne- 
 crosis in forty-eight hours after intracutaneous injection into .guinea-pigs. A series of 
 dilutions of the L+ dose of a toxin is made, ranging from 1 ; 5 to 1 : 100, and 0.2 
 c.c. of each mixed with 0.2 c.c. of antitoxin so diluted that each 0.1 c.c. contains 
 TuVis of a unit. These mixtures are made in small test-tubes, the cotton stoppers 
 paraffined, and the tubes incubated for three hours and placed in the refrigerator for 
 twenty-one hours, after which 0.2 c.c. of each is injected into guinea-pigs (prepared 
 by pulling out the hairs); several injections may be maUe in each pig. 
 
 When the Ln dose of the toxin has been determined this amount is mixed in a 
 similar manner with varying amounts of the patient's serum being tested. The 
 amount of serum just neutralizing the toxin contains ysVj of a unit of antitoxin from 
 which the amount of antitoxin per cubic centimeter of sfirum may be computed. For 
 example I have found that 0.003 c.c. of serum of a person reacting negatively to the 
 Schick test (p. 719) neutralized this amount of toxin; therefore each cubic centimeter 
 of this person's serum contained 0.33 unit of antitoxin. 
 
CHAPTER XV 
 FERMENTS AND ANTIFERMENTS 
 
 Bacterial Ferments. — In addition to the toxins, most bacteria possess 
 certain ferments or enzymes tliat may play an important role in the pro- 
 cesses of disease. After toxins have destroyed body-cells, proteolytic 
 ferments, partly derived from the bacteria, ai4 in their digestion and 
 produce a homogeneous puriform substance. A similar condition may 
 be demonstrated m vitro in cultures of Bacillus subtilis on coagulated 
 blood-serum media when the entire tube of medium is quickly liquefied 
 into a creamy fluid resembling pus. The action of the proteolytic fer- 
 ment is also demonstrated in the liquefaction of gelatin. 
 
 Pyocyanase is a ferment that is capable of digesting other bacteria, 
 such as Bacillus anthrax, Bacillus diphtherise, staphylococci, and strep- 
 tococci. It has been asserted that the injection of this ferment is 
 followed by an increased resistance to infection. Some years ago pyo- 
 cyanase was manufactured extensively and its use advocated in the 
 treatment of local infections, the purpose being to effect digestion of 
 the disease-producing microorganisms. These claims have not, how- 
 ever, been substantiated, and the treatment hg-s been generally aban- 
 doned. 
 
 In addition to this proteolytic ferment, bacteria may possess dia- 
 static ferments capable of converting starches into sugars; invertiag 
 ferments, which may change polysaccharids into monosaccharids; 
 rennin-like ferments capable of coagulating milk, etc. Not all bacteria 
 possess all these ferments, but a study of theni: may aid greatly in the 
 identification of the various bacterial species. 
 
 S imil arity Between Toxins and Ferments. — Aside from the definite 
 ferments that are in the nature of secretory products of the bacteria, 
 there are many points of resemblance between the toxins, both exo- 
 toxins and endotoxins, and the ferments or enzymes. It would seem that 
 the whole subject of infection and immunity is becoming more and 
 more closely identified with physiologic chemisjry, especially with the 
 lipoids and lipolytic ferments. This subject constitutes today a most 
 important field for investigation. 
 
 244 
 
SIMILARITY BETWEEN TOXINS AND FERMENTS 245 
 
 1. Both toxins and ferments are products of the metabolism of living 
 animal and vegetable cells, and may be extracellular (free enzymes and 
 soluble toxias) or intracellular (intracellular errzymes and endotoxins). 
 
 2. Both exhibit a latent period before manifesting their individual 
 activities; in general, the effect of each is rnore rapid the larger the 
 amount present. 
 
 3. Both substances represent a method or means by which the or- 
 ganism attempts to modify its environment and render the surroundings 
 suitable for its nutrition and growth. 
 
 4. Both show a strong affinity for their substratum, and first manifest 
 their activity by combining with it. For example, fibrin placed in 
 gastric juice at 0° C. and then repeatedly washed in cold water to remove 
 all traces of pepsin will undergo digestion when raised to body tempera- 
 ture. Similarly, if red corpuscles are placed in fresh tetanus toxin at 
 0° C. for an hour, washed repeatedly with cold normal saline solution, 
 and then raised to 37° C, hemolysis will take place, indicating the pri- 
 mary union of the bacterial hemolysin or tetanolysin with the corpuscles. 
 In a similar manner toxins probably iinite chemically with tissue-cells, 
 as the toxia quickly disappears from the blood following its iajection 
 and but a small fraction can be recovered from the excretions. Further- 
 more, the injection of an emulsion of these cetifi into other animals may 
 be followed by specific symptoms of intoxication. 
 
 5. The activities of both toxins and ferments seem to depend largely 
 upon the temperature to which they are exposed. For instance, in the 
 example previously cited, tetanus toxin is harmless for the frog until 
 the temperature of the animal is raised to about 37° C. 
 
 6. Both are usually affected by temperatures above 70° C. 
 
 7. The one great difference, however, between toxins and enzymes 
 is the greater activity of the latter, even very minute amounts of an 
 enzyme having the power to split up or decompose large quantities of 
 complex organic compounds. An enzyme attaches itself to a substance 
 and absorbs water; the molecule breaks down^ the enzyme is liberated, 
 and then attacks another molecule, this process being repeated until 
 large amounts of fermentable substances have been attacked. When, 
 however, a toxin has united with a substance it loses its identity, and in 
 this maimer it follows the law of multiple proportions. This has been 
 discussed as it relates to the soluble toxins of diphtheria and tetanus, 
 and is likewise easily demonstrable in the action of tetanolysin upon 
 erythrocytes of the rabbit. It is true that a toxin may become dissoci- 
 ated and attack another molecule, but this action is different from that 
 
246 FERMENTS AND ANTIFERI\fENTS 
 
 of an enzyme, because the molecule first attacked is not injured. How- 
 ever, as Adami points out, the toxins may be equally active in the body 
 until arrested by antitoxins, although experiments in vitro clearly demon- 
 strate the greater activity of the ferments. 
 
 8. Even in serimi hemolysis it would appear that a lipolytic ferment 
 is vitally concerned. According to Jobling and BuU,"^ the end-piece of 
 split complement contains a lipolytic ferment. These observers be- 
 lieve that a parallelism exists between lipase content and complement 
 activity of the serums of various animals. As will be pointed out further 
 on, the complements possess many properties resembling those of the 
 ferments, and many factors are being established to show the important 
 relation that lipoids and lipases bear to immunologic processes. 
 
 ANTIFERMENTS 
 
 According to some investigators, antiferments are to be found in large 
 amounts in all normal serums, and are probably vitally concerned in the 
 processes of life in preventing autodigestion. That they may be in- 
 creased in nimaber artificially by immunization up to a certain limit 
 has been disputed; it is certain that they never attain the extreme 
 amounts possible with the injection of toxins. This may be due to the 
 formation of anti-anti-enzymes, produced by a regulating mechanism 
 that prevents anti-enzymes from accumulating beyond a certain point 
 and interfering with nutrition. It is possible that the body mechanism 
 exerts a strict regulating effect between the formation of enzymes and 
 anti-enzjrmes. Furthermore, when free receptors, such as normal 
 anti-enzymes, are present in the body-fluids, the body-cells are not 
 stimulated to produce these anti-enzymes in excess, nor does the pres- 
 ence of the free receptors stimulate the cells to produce anti-bodies 
 against their normal side-chains. 
 
 Many investigators claim to have prodmced antiferments experi- 
 mentally. Morgenroth ^ believed that he obtained a specific antireimin 
 by inoculating goats with rennin. Sachs ' and Achaline * assert that they 
 have produced specific antipepsin or antitrypsin by inoculating animals 
 with these ferments. Antisteapsin and antilactase have been prepared 
 by Schutze,' antityrosinase by Gessard,'' and antiurease by Moll.' 
 
 On the other hand, these observations have not been generally con- 
 
 1 Jour. Exper. Med., 1913, xvii, 61. '' Centralljl. f. Bakteriol., 1899, xxvi, 349. 
 3 Fortschr. d. Med., 1902, 20, 425. ^ Ann. deJ'Inst. Pasteur, 1901, xv, 737. 
 
 6 Zeitschr. f. Hygiene, 1904, 48, 457; Deutsch. mod. Wochen., 1904, 30, 308. 
 6 Aim. de I'Inst. Pasteur, 1901, 15. ' Hofmeister's Beitr., 1902, 2. 
 
ANTIFBRMENTS 247 
 
 firmed. The inhibitory substances on trypsin in blood-serum, for in- 
 stance, have been ascribed by Jobling and Petersen ^ to the presence of 
 compounds of the unsaturated fatty acids. 
 
 Antibodies and Antifennents. — Elsewhere has been discussed more 
 fully the intimate parallelism that exists between bacterial antibodies 
 and antiferments. One is impressed with the similarity of the 
 processes concerned in the breaking-down of food-stuffs into simple sub- 
 stances for assimilation by ferments and the destruction of bacteria and 
 their products by antibodies. The processes that occur when a cell 
 digests an ingested microbe by a cytase must be similar to that which 
 occurs in the digestion of any other foreign matter, and it is but a short 
 step to conceive that bacteriolysis in the body-fluids is similar to the 
 processes concerned in the digestion of fibrin in the gastric juice. The 
 close similarity that exists between the toxins and the ferments, the anti- 
 toxins and the antiferments, complements and kinases, and the quantita- 
 tive relations existing between a toxin and its antitoxin, between a fer- 
 ment and its antiferment — all these indicate, as Adami has pointed out, 
 the close parallelism that exists between toxins and cytolysins and fer- 
 ments of different orders and grades. They would seem to indicate, 
 moreover, that we are probably dealing with one common group of en- 
 zymic substances that act not by physical contact, but by chemical 
 combination. 
 
 Antiferments in Disease. — In this coimection a subject of consider- 
 able interest is the probable nature of the syphilitic antibody so vitally 
 concerned in the Wassermann reaction. Although the true nature of 
 this reaction is unknown, there can be no doubt as to the intimate re- 
 lation of lipoidal substances to the processes concerned. It is probable 
 that the Treponema pallidum produces a true antibody, and a second 
 body, in the nature of a cellular reactionary substance, which has a 
 marked affinity for lipoids. When the two substances arc mixed and 
 complement added, the latter is adsorbed or inactivated to a greater or 
 less degree, so that upon the subsequent addition of washed erythrocytes 
 and its corresponding hemolytic amboceptor hemolysis either does not 
 occur at all or is more or less incomplete. A similar "rcagin" is present 
 in the blood-serum of persons suffering with yaws and tuberculous lep- 
 rosy, and although its exact nature is unknown, its peculiar behavior 
 toward the lipoids suggests strongly that it is a product of the toxic 
 action of the causal parasite upon the lipoids of the body-cells and is in 
 the nature of an antilipoid. (See Wassermann reaction.) 
 1 Jour. Exper. Med., 1914, xix, 459. 
 
248 FERMENTS AND ANTIFERMENTS 
 
 Jochmaim and Muller have demonstrated the presence of an anti- 
 ferment in the serum used against leukocytic ferments in diseases as- 
 sociated with great destruction of the leukocj^tes. Following these 
 observers, Marcus Brieger and Trebing ^ found that 90 per cent, of the 
 patients suffering from carcinoma or sarcoma ex3,mined by them showed 
 an increase of antitrypsin in the blood. Von Bergmann and Meyer ^ 
 confirmed this observation, although they found that a similar increase 
 also occmred in 24 per cent, of non-cancerous patients. More recent 
 work would indicate that the antitrypsin may be present in acute in- 
 fections, such as pneumonia, typhoid fever, etc., in chronic infections, 
 such as tuberculosis and syphilis, in exophthalmic goiter, and in severe 
 anemias. As previously mentioned, Schwartz,' Suginoto,^ and Job- 
 ling and Petersen'^ believe that the antitrj-ptic influence of blood-serum 
 is due to the lipoids, and especially to the compounds of the unsaturated 
 fatty acids. 
 
 The tryptic ferment liberated by disintegrating leukocytes and con- 
 nective-tissue cells is largely responsible for the liquefaction of these 
 cells and the formation of pus, as in abscess fornjation and autodigestion 
 of infected surface wounds. On the other hand, an antitrypsin-like 
 substance tends to limit the activities of the ferment and protect the 
 surrounding tissues from progressive destruction. A deficiency of this 
 substance may account for the rapid breaking-down of infected glands 
 and of a walled-off tuberculous lesion, the development of carbuncles, 
 etc. A study of the antitryptic power of the blood may, therefore, prove 
 of value in suppiu'ative processes and in malignant disease, and consider- 
 ably influence a prognosis. 
 
 Ferments in Pregnancy and Disease. — It is largely to the researches 
 of Abderhalden and his associates that we owe our knowledge of the 
 fact that when food-stuffs are introduced into the body parenterally, i. e., 
 by subcutaneous or intravenous injection, fermenfe are produced that, by 
 process of cleavage and reduction, deprive them of their individuality. 
 
 For example, normal dog serum cannot reduce cane-sugar, whereas 
 the serum of a dog immunized by several injectitjns of this sugar is able 
 to reduce it in vitro. Similarly, normal serum is unable to cleave edestin 
 (vegetable albumin), whereas the serum of an immunized dog will split 
 this protein into simpler substances. 
 
 1 Berl. klin. Woohschr., 1908, xlv, 1349. 2 Ihid., 1908, xlv, 1673. 
 
 'Wien. klin. Wochschr., 1909, xxii, 1151. 
 
 ^Arch. f. exper. Path. u. Pharmakol., 1913, Ixxii, 374. 
 
 s Jour, ex-per. Med., 1914, xix, 239 and 459. 
 
ANTIPERMENTS 249 
 
 After he had proved experimentally that the animal organism is able 
 lo defend itself against foreign substances by mobilization of ferments, 
 Abderhalden next took up the question whether protective ferments are 
 produced when substances native to the body but foreign to the blood are 
 introduced into the circulation. Having learned from the researches 
 of A'eit, Schmorl, Weichard, and others, that during pregnancy sjaicytial 
 cells frequently enter the maternal circulation, Abderhalden used the 
 serums of pregnant animals, and found that they contaiaed a fennent 
 capable of splitting placental peptone into amino-acids and coagulated 
 placenta into peptones, polypeptids, and amiao-acids. 
 
 It was thus established that the body-cells are harmonically attuned 
 to one another, and if new or modified cells or tjieir products are brought 
 into relation with other cells, they are received as foreign invaders, and 
 their entrance is followed by the production of protective ferments 
 ("Abwehrfermente") capable of bringing about their cleavage into 
 simpler products. In this manner the presence- in the circulation of some 
 of the body-cells may give rise to the production of these ferments if the 
 cells in question are really foreign to the blood-plasma and other cells. 
 
 Abderhalden is careful to note that although he was led to make 
 these investigations on the supposition that syncytial elements were 
 present in the blood of pregnant women, it is not necessary that they be 
 constantly in the blood, for every case of pregnancy has a complicated 
 protein metabolism and there is a general exchange of substances between 
 the placenta and the maternal blood that permits the entrance into the 
 latter of protein products that have not been broken down completely 
 into amino-acids, and that cause the organism to produce defensi^-e 
 proteolji;ic ferments. 
 
 In cancer, where the production of new cells is so marked, some of 
 these cells or their products may easily be swept into the general circula- 
 tion, where they act as foreign invaders and ca,use the formation of pro- 
 tective proteoh-tic ferments. It is a noteworthy fact, moreover, that 
 the serum of carcinoma cases reacts best with carcmoma cells and that 
 of sarcoma with sarcoma cells. 
 
 Sunilar ferments have been described in other conditions. Fauser 
 has demonstrated that the blood-serum of dementia prsecox patients 
 contaios ferments that act on the reproduction glands, so that the serum 
 of males reacts with testicular extracts and that of females with ovarian 
 extracts. These scrums'were, however, also found to react v,-ith thyroid 
 tissue and brain cortex. In general paresis reactions were obtained with 
 braiti cortex and liver, also at times with th^Toid gland, reproductive 
 glands, and more rarely with kidney. 
 
250 FERMENTS AND ANTIFERMENTS 
 
 Abderhalden has found ferments for the tubercle bacillus in the blood- 
 serum of tuberculous persons, and they have also been found in the blood- 
 serum of syphilitics for the Treponema pallidum, either in pure culture 
 or in organs containing large numbers of the parasites. 
 
 Although these protective ferments are in general similar to the cyto- 
 lysins or antibodies produced during bacterial and protozoan infections 
 capable of lysing or digesting their antigens, their exact nature and rela- 
 tion to the cytolysins have not been determined. From the evidence 
 at hand it would appear that the ferment-like cytolysin is specifically 
 directed against the toxic portion of a bacterial cell, and the proteolytic 
 ferment against the bacterial cell itself. Recent work by Pearce and 
 Williams-' would seem to indicate that the cytolysins and protective 
 ferments are separate substances, but considerable additional experi- 
 mental investigation is required to clear up the point. 
 
 Although recent investigations would tend to show that these pro- 
 teolytic ferments are not so specific as Abderhalden believes, the subject 
 is one of the greatest importance, and its elucidation may possibly throw 
 much light upon the nature of immune bodies in general. The technic, 
 specificity, and practical value of the methods devised by Abderhalden 
 for detecting the proteolytic ferments in the serum of cases of pregnancy, 
 cancer, etc., are considered in a later portion of this chapter. 
 
 Ferment Reactions 
 
 ANTITRYPSIN TEST 
 
 Bergmann and Meyer ^ have devised a test for estimating the titer 
 of antitrypsin that possesses value in determining the presence of tryp- 
 tic ferment in the blood-serum and in the intestinal and stomach con- 
 tents. The test may also prove of value iii making a functional study 
 of the pancreas. At present it is regarded by some as an aid to the 
 diagnosis of cancer, and it may also be of service in establishing the 
 diagnosis and prognosis of suppurative processes. 
 
 Solution of Trypsin. — This is made by dissolving 0.5 gm. of pure 
 trypsin (Griibler) in 50 c.c. of NaCl solution and adding 0.5 c.c. of normal 
 soda solution; make up to 500 c.c. with physiologic salt solution. 
 
 Casein Solution. — Dissolve one gram of pure casein in 100 c.c. of 
 sodium hydroxid solution with the aid of gentle heat. Neutralize to 
 litmus with -^ hydrochloric acid solution and dilute with physiologic 
 
 1 Jour. Infect. Dis., 1914, xiv, 351. = Berl. klin. Wochenschr., 1908, No. 37. 
 
ANTITRYPSIN TEST 251 
 
 salt solution up to 500 o.c. Filter and sterilize in an Arnold sterilizer. 
 Preserve in the refrigerator. 
 
 Acetic Acid Solution. — To 5 o.c. of acetic acid (c. p.), add 45 c.c. 
 of absolute alcohol and 50 c.c. of distilled water. 
 
 The patient's serum must be fresh, and should be diluted 20 times 
 with salt solution. Dose, 0.2 c.c. 
 
 Technic. — A titration of the trypsin solution must precede the test 
 proper. Into each of several small test-tubes place increasing amounts 
 of trypsin solution, as, for example, 0.1, 0.2, U.4, 0.6, 0.8, and 1.0 c.c. 
 Add 2 c.c. of the casein solution to each tube; shake carefully and place 
 in an incubator or water-bath for half an hofur at 50° C. Then add 
 three or four drops of the acetic acid solution to each tube, and observe 
 which tube first shows cloudiness after a few minutes. The tube con- 
 taining the smallest amount of trypsin and which remains perfectly 
 clear contains enough trypsin fully to digest the 2 c.c. of casein solution. 
 
 Into each of six small test-tubes now place 0.2 c.c. of the 1 : 20 dilu- 
 tion of the patient's serum, and increasing ampunts of the trypsin solu- 
 tion, beginning with the completely digesting dose, as determined above, 
 and LQcreasing by 0.1 c.c. Add 2 c.c. of casein solution to each tube, 
 and bring all tubes to a like volume by the addition of normal salt solu- 
 tion. Shake gently, and incubate at 50° C. for half an hour. Add 
 several drops of acetic acid solution to each tube, and again observe the 
 tiibc containing the smallest anaount of trypsift in which cloudiness can 
 be seen. In this way the amount of trypsin*neutralized by the anti- 
 trypsia of the serum is determined. 
 
 For example, in an experiment the preliminary titration showed that 
 0.5 c.c. of trypsin completely digested the casein. In the second part 
 of the test the lower limit of trypsin was this 0.5 c.c. increased by 0.1 
 c.c. in successive tubes up to 1 c.c. It is now found that 1 c.c. of the 
 trypsin solution is required to bring about the complete digestion of the 
 casein in the presence of the serum, or 1 c.c — 0.5 c.c. =0.5 c.c, which is 
 the amount of trypsin neutralized by 0.01 c.c.^ of undiluted serum. 
 
 A control experiment is conducted with the pooled serum of several 
 normal persons, and a comparison of the vaJlue thus obtained shows 
 whether the antitryptic power of the serum tested is altered. 
 
 The method of Marcus,' which is a modification of the method of 
 Miiller and Jochmann,^ is described in the laboratory exercises on 
 Experimental Infection and Immunity. 
 
 1 Berl. klin. Wochschr., 1908, No. 4; 1909. 
 
 2 Mlineh. med. Wochschr., 1909, Nos.'?9 and 31. 
 
252 FERMENTS AND ANTIFERMJiNTS 
 
 ABDERHALDEN'S SERODIAGNOSIS OF PREGNANCY' 
 Principles. — ^As previously stated, this test aims to discover ia the 
 blood-serum of pregnant women, the presence of a proteol3rtic ferment 
 capable of splitting coagulated placenta or placental peptone into simple 
 substances, such as amino-acids. The presence of this ferment indi- 
 cates that its antigen is present, i. e., the woman is pregnant, and syn- 
 cytial cells or their products have gained access to the maternal circu- 
 lation with the result that a protective ferment has been produced. 
 According to Abderhalden, while this ferment is not absolutely specific 
 for the placenta of the same species, it is incapable of cleaving any other 
 protein. For example, the serum of a pregnant cow contains a ferment 
 capable of splitting the protein of human placenta, but does this less 
 satisfactorily than does the ferment in human sermn. The ferment of 
 pregnancy, however, will not split the protein of cancer-cells or of nor- 
 mal tissues. 
 
 Although all investigators are in accord regarding the prssence of a 
 proteolytic ferment in the blood-serum of pregnancy, the specificity of 
 the ferment has been questioned. • To all such queries Abderhalden 
 has usually replied by calling attention to errors in technic, and, indeed, 
 while the dialysis method is easy of manipulation, opportunities for 
 technical error are so numerous that the test is far from simple. 
 
 Methods. — Two methods have been devised by Abderhalden for 
 the demonstration of the protective ferments in the blood-sermn of 
 pregnancy : 
 
 1. The Dialyzation Method. — Specially prepared and coagulated 
 placenta and fresh serum are placed in a dialyzing capsule so prepared 
 that it will permit the passage of peptones and amino-acids only. The 
 filled capsule is placed in sterile distilled water, and incubated for from 
 sixteen to twenty-four hours, when the dialysate is tested by the biuret 
 or ninhydrin test for peptones and amino-acids. Under proper condi- 
 tions the presence of these substances indicates that the placental tis- 
 sue has been digested by a ferment in the serum, and, if this ferment 
 is specific for placental cells, this indicates that th-e serum tested is from a 
 pregnant woman. This is the method usually employed. 
 
 2. The Optical Method. — This method is based upon the same prin- 
 ciple as the dialyzation method. Into the tube of a polariscope place a 
 solution of placental peptone and the serum to be tested. Warm the 
 
 ' Abderhalden : Abwehrfermente des tierischen Organisims, Julius Springer, 
 third edition, 1913. 
 
abdekhalden's sekodiagnosis of pregnancy 253 
 
 mixture to 37° C, and after an hour note the degree of rotation and 
 record it; repeat this at intervals during the following twenty-four to 
 forty-eight hours. If the serum contains a proteolytic ferment, the 
 peptone is split into amino-acids and the degree of rotation increased 
 from 0.05° to 0.5° and higher. This method requires an expensive polar- 
 iscope, considerable practice in making the observations and readings, 
 and is only reliable in skilful hands. 
 
 The Dialyzation Method 
 Testing the Dialyzing Shell. — The quality of the dialyzing shell 
 largely determines the success of this method. It must fulfil two re- 
 quirements : 
 
 1. It must be absolutely non-permeable for albumin. 
 
 2. It must be evenly permeable for the protein cleavage products, 
 such as peptones, polypeptids, and amino-acids. 
 
 Special shells arc made by Schleichter and Schull, No. 579a being 
 recommended at the present time. The shells must be of correct size, 
 and every one must be tested before being used. If a shell allows un- 
 cleaved protein to pass through, then all reactions would react posi- 
 tively regardless of the presence or the absence of the specific ferment. 
 If the shell is too thick and too tight, and prevents the passage of pep- 
 tones and amino-acids, then all reactions would be negative, even though 
 the ferment were present in the serum and had digested the placental 
 protein. Accordingly, each shell must he tested and standardized, and only 
 those employed that have proved satisfactory. 
 
 Glassware. — It is highly important that all glassware should be 
 free from clinging particles or traces of albamin, acids, and alkalis. 
 Pipets and dialyzing cylinders should be washed in water, alcohol, 
 ether, and finally in distilled water, and sterilized by dry heat. Boiling 
 rods of solid glass (10 cm. by 0.5 cm.) should be washed in alcohol, 
 ether, and distilled water, wrapped in bundles- of six in newspaper, and 
 sterihzed by dry heat. 
 
 A very convenient dialyzing cylinder is shown in the accompanying 
 illustration (Fig. 73). This cylinder measurep 8 by 3 cm. It should 
 be plugged with cotton and sterilized. When the shell is loaded with 
 coagulated placenta and scrum and covered with toluol, it will rest well 
 beneath the surface of the outside distilled water. The wide mouth of 
 the cylinder facilitates all manipulations and the shell cannot upset. 
 The apparatus is easily sterilized, and the cotton plug prevents bac- 
 terial contamination and undue evaporation of-the contents. 
 
254 
 
 FERMENTS AND ANTIFERMENTS 
 
 /• 
 
 General Precautions. — According to Abderhalden, the work should 
 be conducted in a special room, where there is no dust or fumes of acids, 
 and where no bacteriologic work is in progress. This observer also 
 recommends that a special incubator be used for this work. If, however, 
 
 the working table is scrupulously 
 
 clean and the glassware is clean and 
 
 - ~ sterile, and if the shells are handled 
 
 / with sterile forceps and the dialyzing 
 
 cylinder is stoppered with a plug of 
 sterile cotton, all requirements are 
 practically fulfilled. 
 
 Reagents. — The presence of albu- 
 min or its split products may be 
 detected by two color reactions: (1) 
 the biuret reaction, and (2) the nin- 
 hydrin reaction. The first is espe- 
 cially delicate for uncleaved albumin, 
 and the latter for peptones and amino- 
 acids. The technic of the biuret test 
 is described, with the technic of testing 
 shells for permeability to albumins. 
 
 Ninhydrin. — This is the trade 
 name for triketohydrindenhydrate. 
 It is a whitish yellow, readily soluble 
 powder, dfepensed in brown glass 
 vials containing 0.1 gram of the drug. 
 A circular describing its method of 
 use accompanies each package. As 
 0.2 c.c. of a 1 per cent, watery solu- 
 tion is the amount necessary for a 
 test, the contents of the vial are dis- 
 solved in 10 c.c. of distilled water, 
 and the vral rinsed with a portion 
 of the solvent. This solution should 
 be preserved in a brown bottle in a cold place, and precautions taken 
 to prevent infection. Triketohydrindenhydrate has been described by 
 Ruheman,! who gives its formula as follows: 
 
 Fig. 73. — A Dialyzing Ctmndek 
 
 FOR THE AbDEKHALDEN FeKMENT 
 
 Test. 
 
 The shell contains placental 
 tissue and fresh serum; it is sur- 
 rounded with 20 c.c. of distilled water 
 and covered with toluol. The cotton 
 plug prevents contamination. The 
 cylinder is readily sterilized in a hot- 
 air oven and affords a simple and 
 efficient means for conducting the 
 test by the dialyzation method. 
 
 CeHi 
 
 / 
 
 ^CO 
 
 C(0H)2 
 
 1 Jour. Chem. Soc, London, 1910, xgvii, 2025. 
 
Fig. 74. — NiNHTDRiN Reaction (.Abderhalden Ferment Test). 
 The tube on the left shows a positive reaction with the serum of a pregnant 
 woman; the tube on the right is the beruin control and shows a faint violet color, due, 
 presumably, to the passage of dialyzabk' substances in thi.s serum. 
 
abderhalden's serodiagnosis of pregnancy 255 
 
 Owing to the fact that it gives a blue color in the presence of any 
 compound that possesses an amino-group in the alpha position of the 
 carboxyl group, it is of great value as an aid in recognizing the products 
 of protein digestion (Fig. 74). 
 
 Testing the Shell for Non-permeability to "Albumin. — 1. New shells 
 should be softened by soaking them for half an hour in sterUe distilled 
 water. A dozen or more may be tested at one time. 
 
 2. The albumin solution is prepared by placing 5 c.c. of the albu- 
 min of fresh eggs in a mixing cylinder, and adding distilled water to 
 make 100 c.c. Mix well. There must be no flakes. Instead, a clear, 
 hemoglobin-free serum may be used in doses of 2 c.c. for each shell. 
 
 3. Carefully pipet 2.5 c.c. of the albumin solution into each shell. 
 Great care should be exercised that none of the solution contaminates 
 the outside of the shell. The preferable method is to hold the shell with 
 a pair of broad-toothed sterilized forceps and carry the pipet to the 
 bottom, in order that none of the albumin should contaminate the upper 
 portion of the inside of the shell. The pipet may easily touch the edge 
 of the shell and thus contaminate the dialysate. If in doubt, cover the 
 upper end of the shell with the forceps or with a clean thumb and fore- 
 finger and wash the outside with running water. 
 
 4. The loaded shell is now placed in a sterile dialyzing cylinder con- 
 taining 20 c.c. of sterile distilled water. Never load the shell in this 
 cyUnder, for some of the albumin may fall into the distilled water. 
 
 5. Cover the contents of the shell and the surrounding distilled water 
 with a layer of toluol about j^ inch in depth. Replace the cotton plug 
 in the cyhnder. 
 
 6. Incubate at 37° C. for sixteen hours. 
 
 7. Pass a sterile pipet quickly through the layer of toluol and remove 
 10 c.c. of the dialysate to a clean sterile test-tube, and test for albumin 
 by the biuret reaction. Add 2.5 c.c. of a 33 per cent, solution of sodium 
 hydroxid; shake gently, but remove the thxunb from the top of the tube. 
 The solution may become slightly cloudy. Carefully overlaj^ with 1 c.c. 
 of a 0.2 per cent, solution of copper sulphate in such manner that a sharp 
 line of demarcation separates the alkaline dialysate from the copper 
 sulphate solution. A delicate violet tint at this line indicates that al- 
 biunin is present and that the shell is useless. If one cannot see this 
 color or is in doubt, it is well to make the ninhydrin test. To do this 
 dialysis should be continued for twenty-four hours; ninhydrin reacts 
 With albumin in addition to peptones and an\ino-acids, but, according 
 to Abderhalden, this test is less sensitive than the biuret test. 
 
256 FERMENTS AND ANTIFE^MENTS 
 
 8. All shells should react negatively, — i. ex, they should not permit 
 the passage of unchanged albumin. If the ninhydrin test is used, the 
 tubes should be inspected one-half hour after 'boiling, and the contents 
 should be as clear as water or show but the faintest blue tint. If this is 
 not the case, shells should be discarded as being permeable to albumm. 
 Those that are satisfactory in this respect should be tested further as 
 follows: 
 
 Testing the Shell for Permeability to Peptone. — 1. The shells should 
 now be thoroughly cleansed, but not with a stiff brush, washed in 
 running water, and boiled for thirty seconds. 
 
 2. Prepare a 1 per cent, solution of silk peptone (Hochst) in dis- 
 tilled water, and carefully pipet 2.6 c.c. into each shell, using every pre- 
 caution against contaminating the upper portion on the inside, and 
 especially of the outside, of the shell. 
 
 3. Place the loaded shell in a sterile dialyzing cylinder containing 
 20 c.c. of sterile distilled water, and cover the contents of the shell and 
 water with toluol. Replace the cotton plug apd incubate at 37° C. for 
 twenty-four hours. 
 
 4. Remove 10 c.c. of the dialysate (avoid removing toluol) to a 
 clean, sterile, thin-walled test-tube, and add D.2 c.c. of the 1 per cent, 
 ninhydrin solution. Insert a sterile boiling rod and hoil for exactly one 
 minute. 
 
 5. The boiling process is quite an important feature of this test. 
 Always boil in precisely the same maimer. A high Bunsen flame should 
 be used, and about one minute after air-bubblps first appear on the sides 
 of the tube lively boiling commences. The flame should then be turned 
 down and the boiling continued for exactly oiie minute. 
 
 6. Place the tube in a rack. With a fresh sterile pipet remove 10 
 c.c. of dialysate from the next cylinder and test in the same manner, 
 and repeat until the entire scries have been finished. 
 
 7. After half an hour inspect all the tubes; they should show a 
 deep blue color; if they do not do so they 4re impermeable or partly 
 permeable to peptone and should be discarded. There is usually a 
 difference in the degree of color reaction amdng a number of shells, as 
 their permeability varies. 
 
 8. Those shells that have withstood both tests are now thoroughly 
 washed in running water, boiled for from thir.ty seconds to one minute, 
 placed in a jar of sterile distilled water contaifling a few drops of chloro- 
 form, and covered with toluol. From this tinie on they should not be 
 handled with the fingers, but only with forceps- that have been sterilized 
 
abderhalden's serodiagnosis of pregnancy 257 
 
 by boiling. Of the entire number of shells, usually from 20 to 30 per 
 cent, or more are found to be unsatisfactory. 
 
 Preparation of the Placental Tissue. — Thfer is the substratum, and 
 should consist of coagulated placental protein free from dialyzable sub- 
 stances that react with ninhydrin. 
 
 1. A fresh normal placenta should be prepared soon after delivery. 
 It is highly important to wash it free from all blood, Abderhalden having 
 laid considerable stress upon this point. He explains that in the blood 
 of all animals there is always a specific ferment for the red blood-cor- 
 puscles, as even the smallesL hemorrhage into the tissue calls forth a 
 protective ferment. For this reason all organs that contain blood may 
 contain the substratum and ferment, and yield false positive reactions. 
 
 2. The placenta should be placed in warm water and freed as far 
 as possible of clots. The membranes and cord are removed, and the 
 placental tissue cut into pieces about the size of a dime. These are 
 placed in a sieve under running water, and each piece squeezed with the 
 hand. From time to time the entire mass is thoroughly squeezed out 
 in a towel. Tissues that cannot be freed from clots should be discarded. 
 The tissues are now crushed in a mortar, connective-tissue strands re- 
 moved, and the washing continued until the tissue is snow white. De- 
 colorizing substances, such as H2O2, should not be used. If the tissue is 
 not white and free from blood it should not be employed. Liver, spleen, 
 and kidney tissue cannot be made perfectly white, although all traces of 
 blood have been removed. 
 
 3. Add 100 times as much distilled water as there is tissue, and to 
 each liter add five drops of glacial acetic acid and boil for ten minutes. 
 
 4. Wash the coagulated tissue with distilled water, and boil again 
 without the addition of acid. This should be repeated six times in 
 succession. If an interruption occurs, cover the tissue and water with 
 a layer of toluol. 
 
 5. After the sixth boiling add a small quantity of water to the tissues 
 — just sufficient to enable it to boil for about five minutes mthout burn- 
 ing, for the water is now to be tested with the ninhydrin reaction and 
 it is important that this be as concentrated as possible. Filter the water, 
 and to 5 c.c. in a sterile test-tube add 1 c.c. of the ninhydrin solution. 
 Boil vigorously for one minute. If there is th'e slightest discoloration 
 within half an hour, the tissues must be boiled again, but with only five 
 volumes of water and no longer than five minutes each time. These 
 boilings should be repeated as often as is necessary until the ninhydrin 
 reaction remains water clear for at least one-half hour, 
 
 17 
 
258 FERMENTS AND ANTIFBI|MENTS 
 
 6. The tissues are again gone over with a sterile forceps, and a search 
 made for brown masses resembhng blood-clots. These are to be dis- 
 carded. 
 
 7. The tissue is now preserved in a sterile jar containing sufficient 
 sterile water and chloroform and covered with toluol. All tissue should 
 be handled with sterile forceps, and when once removed from the jar, 
 they should never be returned. The whole operation requires several 
 hours and it should be conducted without interruption. If the process 
 is interrupted, the tissue should be covered with a layer of toluol. 
 
 8. It is well to try out the tissue with a known serum of pregnancy 
 to make certain that it is a suitable substratum. 
 
 9. Only normal placenta should be used, as in certain instances a 
 normal organ may be satisfactory, whereas ^ diseased organ would be 
 unsuitable. 
 
 10. Animal placenta may be substituted for human placenta and 
 vice versa, but Abderhalden cautions against this substitution until 
 further work has been done. 
 
 The Blood-serum. — 'The serum to be tested must fulfil three condi- 
 tions : 
 
 (1) It must contain the smallest amount of dialyzable substances 
 that would react with ninhydrin. Blood is best drawn in the morning 
 before breakfast. In all diseases accompanied by marked protein dis- 
 integration, such as cancer, the blood-serum may contain large amounts 
 of dialyzable substances. 
 
 (2) It must be absolutely free from hemoglobin and clear. 
 
 (3) It must be /ree/roTOceWs. Even an apparently clear serum may 
 contain millions of erythrocytes. 
 
 1. From 10 to 20 c.c. of blood are withdrawn from a vein at the 
 elbow with a dry sterile needle into a sterile centrifuge tube. This is 
 placed aside at room temperature for several hours, when sufficient 
 serum has usually separated out; if this has not occurred, centrifuge for 
 several minutes. The sermn is removed to a second sterile centrifuge 
 tube, and centrifuged at high speed for several minutes imtil all cor- 
 puscles have been precipitated to the bottom of the tube. 
 
 2. The sermn should be used within twelve hours after the blood has 
 been withdrawn. Abderhalden claims that Jieating a serum to 60° C. 
 robs it of its digesting powers. Pearce and Williams have found that 
 inactivation considerably weakens the reactioji, but does not abolish it 
 altogether. 
 
 3. Specimens of blood sent through the mails are really unsatisfac- 
 
abderhalden's serodiagnosis of pregnancy 259 
 
 tory, for even if they are delivered within twelve hours after bleeding, 
 the amount of handling has usually resulted in the breaking up of a 
 number of corpuscles and the tinging of the, serum with hemoglobin. 
 The Test. — 1. Absolute cleanliness should be employed. The glass- 
 ware should be sterile and dry, and everything should be in readiness. 
 The technic should be aseptic and thoroughly understood. 
 
 2. Remove a portion of the prepared placenta with sterile forceps 
 and wash in a dish of sterile distilled water to remove toluol and chloro- 
 form. Place on sterile filter-paper, and squeeze to remove any excess 
 of water. Weigh and place 0.5 gram in each of two shells (one for a 
 control). 
 
 3. Holding each shell with a second pair of boiled forceps, pipet 1.5 
 c.c. of the patient's serum into one shell containing placenta, and the 
 same amount into a third shell which is to serve as a control on the 
 serum. Place 1.5 c.c. of sterile distilled water in the placental tissue 
 control shell. 
 
 4. Unless one is absolutely sure that neither the tissue nor the serum 
 has touched the outside of the shells, they should be held shut and washed 
 with sterile distilled water. 
 
 5. Each of the three shells is now placed in cylinders containing 
 20 c.c. of sterile distilled water. Under no circumstances are the shells 
 to be loaded while they are in the dialyzing cylinders. 
 
 6. The contents of each shell and the water surrounding them are 
 covered with a layer of toluol about J^ inch in depth, and the cylinders 
 plugged with cotton to prevent evaporation and contamination. The 
 shell should be at least 34 to H inch above the level of the outside fluids, 
 and due care must be exercised in carrying the cylinder back and forth 
 from the incubator that the contents of the aiiell and the surroimding 
 water do not become mixed. 
 
 7. If it is at all possible, it is well to set up t'^o more shells as controls, 
 each containing placenta and normal serum and the serum of pregnancy 
 respectively. 
 
 8. All the cylinders are incubated at 37° C. for twenty-four hours. 
 Ten c.c. of the dialysate are then removed from each tube with a sepa- 
 rate sterile pipet and placed in sterile test-tubes of the same size and 
 boiled with 0,2 c.c. of the 1 per cent, ninhydrin solution for exactly one 
 minute. After standing for half an hour, the readings are made. 
 
 Reading the Reaction. — The dialysate of the serum alone should bo 
 clear as water or show but the faintest blue tinge. The dialysate of the 
 placenta alone should be clear; the dialysate of the patient's serum 
 
2B0 FERMENTS AND ANTIFERMENTS 
 
 plus that of the placenta may show a deep yiolet-blue color when the 
 reaction is strongly positive, or a fainter blue \then it is weakly positive. 
 If this dialysate is water clear or has a faint blue color, comparable to 
 the controls, the result is negative. If there is any doubt, the test 
 should be repeated. The negative control should be water clear or have 
 a faint tinge comparable to its control. The positive control should 
 show a deep violet-blue color. 
 
 I generally control the result given by the shell containing tissue and 
 patient's serum by cleansing it thoroughly, boiling for a minute, and 
 testing it with egg-albumen solution or a serum, in case the reaction 
 was positive, to make sure that the shell has not allowed the passage of 
 serum, or with peptone solution, in case the reaction was negative, to 
 make sure that it was not thick enough to block the passage of peptones 
 and amino-acids. This procedure delays the neport on a serum for an- 
 other twenty-four hours, but the greater accuiracy obtained warrants the 
 delay. 
 
 Readings should never' be made by artificial light. Tubes should be 
 held against a white background the better to appreciate the color 
 changes. 
 
 A pinkish or brownish yellow discoloration has nothing to do with 
 the ninhydrin reaction. 
 
 Sources of Error in the Dialyzation Method. — There are many 
 sources of error, and until the technic has been improved sufficiently to 
 eliminate these, Abderhalden's directions should be followed minutely. 
 
 1. The shells may become spoiled in tiipe. They should not be 
 cleansed with rough brushes or boiled too long. They should be 
 cleansed at once after using, and tested everj-four weeks. If a wrong 
 diagnosis results, the shell should be retested at once. 
 
 2. The placental tissue is an important sburcc of error, due to the 
 fact that it contains blood. 
 
 3. The serum should be fresh and free from hemoglobin and cor- 
 puscles. 
 
 4. The controls on placenta alone and eq,Qh serum alone are abso- 
 lutely necessary, as both may contain various substances capable of 
 reacting with ninhydrin and thus yielding false positive reactions. 
 
 5. The water used should be distilled and sterile. The glassware 
 should be chemically clean and sterile, and the laboratory free from the 
 fumes of acids and alkalis. It is very important that absolutely the 
 same conditions should exist for the control tests as for the main test 
 itself. 
 
abderhalden's seeodiagnosis of pregnancy 261 
 
 The Optical Method 
 
 In the dialyzation method, we establish the transformation of a 
 colloid into a diffusible crystalloid; in the optical method we start, 
 for purely technical reasons, not with the whole protein molecule, but 
 with a peptone prepared of placental protein. The unsplit protein it- 
 self cannot be used, as this will interfere with the determination of the 
 rotation of the mixture of substratum plus serum. Further, in such 
 mixtures precipitation may occur and render the readings difficult. 
 Instead, we transform the protein into peptone, and observe the final 
 changes in the tube of the polariscope. 
 
 Placental Peptone. — This requires considerable care in its prepara- 
 tion. Placental peptone may be purchased of the Hochst Farhwerke, 
 and is expensive. Each specimen should be tested and its rotation de- 
 termined, as otherwise uncertain and unreliable results may be secured. 
 According to Abderhalden, a peptone may be prepared as follows: 
 
 The tissues are first cut into small pieces and thoroughly washed until they are 
 white, as in the preparation of tissues for the dialyzation method. 
 
 The tissue is freed of any excess of water by pressing it through several layers of 
 filter-paper, and the process of hydrolysis is begun. If»a larger quantity of the same 
 tissue is to be collected, the pieces are prepared as the/ are secured by washing them 
 free from blood, boiling in water for ten minutes, and jSreserving in a stock jar r-on- 
 taining sterile water and chloroform, and covered with toluol. The tissues are boiled 
 in order to destroy the cell ferments and to prevent autolysis. 
 
 The tissues are now weighed, placed in a large flask, and treated with three 
 volumes of cold 70 per cent, sulphuric acid. The flask Js well shaken and carefully 
 stoppered, and placed aside at room temperature (not higher than 20° C.) until-the 
 tissues have gone into solution. The flask is shaken occasionally. Solution is 
 usually completed within three days. The flask is nbw placed in iced water and 
 treated with 10 volumes of distilled water added slowly so that the temperature 
 does not rise above 20° C. 
 
 The sulphuric acid is now precipitated with pure crystallized ba.rium hydroxid 
 until the solution gives no precipitate with either the hydroxid or sulphuric acid. 
 A precipitate of a barium salt of peptones may appear* in spite of the fact that no 
 sulphuric acid is present. This precipitate is soluble fry nitric acid, whereas barium 
 sulphate is not soluble. The neutralization point is controlled with litmus paper. 
 Small portions are then filtered through filter-paper and tested, first with barium 
 hydroxid and then with sulphuric acid. If a turbidity develops on testing with the 
 barium hydroxid. nitric acid is added and the whole gently warmed. If the pre- 
 cipitate persists, it is evident that more barium suljShate should be added. The 
 final neutralization is effected with dilute sulphuric acid and barium hydroxid. 
 When the solution is free from sulphuric acid, the precipitate of barium sulphate is 
 secured by filtering through filter-paper or by centrifug&lization. It is then worked 
 up in a mortar with distilled water and again filtered. It is well to repeat this wash- 
 ing once more. 
 
 The peptone solution is now concentrated in a special apparatus that permits 
 
2Q2 FERMENTS AND ANTIFERMENTS 
 
 e\faporation of the solution under diminished pressure; and at 40° C. A special drop 
 funnel delivers the peptone solution drop by drop and thus prevents foaming. 
 
 The yellowish, syrupy residue that remains is covered with 100 volumes of 
 methyl alcohol and boiled. The boiling hot solution is then filtered through five 
 thicknesses of filter-paper into five volumes of cold ethyl alcohol, which is kept in 
 ice water. Precipitation may be accomplished by the addition of ether. Just as 
 soon as the precipitate has formed it is filtered out, preferably through a porcelain 
 filter. The filter is then placed in a vacuum desiccator, and after one or two days 
 the peptone is dry and easily removed and weighed. 
 
 A 10 per cent, solution in 0.9 per cent, salt solution is prepared, and the rotation 
 determined. If the rotation is more than 1°, the solution is diluted until the rota- 
 tion is 0.75°. 
 
 Testing the Peptone.-^One cubic centimeter of fresh, clear, hemo- 
 globin- and corpuscle-free serum from a man and an equal amount of a 
 5 to 10 per cent, solution of the peptone are placed in a sterile polaris- 
 cope tube, warmed to 37° C, and the rotation read. If marked changes 
 occur, the peptone is not free from sulphuric' acid or barium hydroxid. 
 With the serum of pregnancy, readings should be taken each hour for 
 six hours, and then at intervals of from thirty-six to forty-eight hours. 
 It is well to test a number of pregnancy serums until a normal curve 
 can be charted. 
 
 Peptones of other tissues and bacteria may also be prepared. 
 
 The Polariscope. — A perfect and delicate: instrument is necessary, 
 that of Schmidt and Hansch, Berlin, being recommended by Abder- 
 halden. The instrument must be delicate enough to record differences 
 in rotation of 0.01°, and be furnished with an electric incubator attach- 
 ment for keeping the tube at a constant terfiperature of 37° C. The 
 tubes may, however, be kept in a bacteriological incubator, removed, 
 quickly read, and then returned. 
 
 The Test. — One cubic centimeter of fresh and absolutely hemoglo- 
 bin- and corpuscle-free serum is placed in the polarization tube with 
 1 c.c. of a 10 per cent, solution of standardized placental peptone; suf- 
 ficient sterile saline solution is added to fill the tube. The tube is then 
 placed in an incubator at 37° C. for an hour, and a reading made. An- 
 other reading is taken an hour later, and the two readings should not 
 show more than a minute difference in rotation. Another reading is 
 made at the end of six hours, and others at intervals during the next 
 thirty-six or forty-eight hours. 
 
 Reading the Reaction. — In serums of pregnancy cleavage is usually 
 apparent at the end of six hours, and rotation may amount to 0.05° to 
 0.2° in thirty-six hours. With non-pregnant serums the rotation is 
 seldom more than 0.03°. 
 
abderhalden's serodiagnosis o'f pregnancy 263 
 
 Considerable experience is required in making these readings, and 
 the novice should practise well before conducting diagnostic tests. 
 
 Individual readings by different persons may vary 0.02°, and in 
 order to secure absolute certainty Abderhalden gives 0.04° as the limit 
 
 for error. 
 
 A large margin for error will result if the primary reading is taken 
 when the tube is cold, as it is immediately after being filled. Only that 
 reading taken after the tube has been in the incubator for at least one or 
 two hours is to be taken as the guide for determining the amount of 
 digestion according to the degree of rotation. 
 
 The greatest source of error lies in the observer himself. One must 
 be trained to make the readings in about thirty seconds; the eye soon 
 grows tired, and the readings are then unreliable. 
 
 Practical Value of Abderhalden-s Pregnancy Test 
 
 1. It is too soon to express a definite opinion of the specificity and 
 diagnostic value of this reaction. Most reports have been based upon 
 the dialyzation method. According to Abderhalden, ^ Veit,^ Frank 
 and Hermann,' Franz and Jarisch,* Petriji^" Judd,* Schwartz,'' and 
 others the ferment is highly specific, and the test is of considerable value 
 in the diagnosis of pregnancy. 
 
 2. The reaction appears in the middle of the second month, and dis- 
 appears in from ten to fifteen days after pregnancy has been interrupted, 
 regardless of whether the fetus is born before, at, or after the normal 
 period of gestation. Nursing has no effect upqn the reaction. 
 
 3. The reaction has been recommended in making an early diagnosis 
 of pregnancy, when the symptoms and physical signs are indefinite; 
 also in making the differential diagnosis between pregnancy and tumors 
 in the pelvis. 
 
 4. The reaction is likely to be positive in hydatidiform disease and 
 in chorio-epithelioma. 
 
 5. In acute febrile and cachectic diseases the serum may contain 
 relatively large amounts of dialyzable compounds; positive reactions 
 have occurred in tuberculosis of the female generative organs. 
 
 1 Miinch. med. Wochenschr., 1912, lix, 1305; 1912, lix, 1939 and 2172. Berl. 
 tierarzt. Wochenschr,, 1912, xxvii, 446; 1912, xxviii, 685 and 774. Zeitschr. f. 
 physiol. Chem., 1912, Ixxvii, 249. Deutsch. med. Wochenschr., 1912, xxxviii, 2160 
 and 2252. Prakt. Ergebn. d. Geburtsh. u. Gynak., 1910, ii, 367. 
 
 ' Zeitschr. f. Geburt. u. Gyn. 1913, 77, 463. 
 
 2 Berl. klin. Wochenschr., 1912, No. 36. 
 
 ' Wien. klin. Wochenschr., 1914, 44, 144. 
 
 ' Centralbl. f . Gyn., 1913, No. 7. ' Jour. Amer. Med. Assoc, 1913, Ix, 1947. 
 
 ' Interstate Med. Jour., St. Louis, 1913, xx, 195. 
 
264 FERMENTS AND ANTIFERIVJENTS 
 
 6. All investigators in this field are in general accord regarding the 
 constant presence of the reaction in the serums of pregnancy, but there 
 is a growing tendency to regard the ferment asjnon-specific and capable 
 of splitting the coagulated protein of other organs, and, indeed, of or- 
 gans from the lower animals. For example, Pearce and Williams' 
 have found positive reactions with pregnancy serums, liver, and kidney; 
 normal male serums and those of various diseases have reacted with 
 coagulated placenta. Williams and Ingrahanl^ have had positive re- 
 actions with definitely non-pregnant persons. Michaelis and Luger- 
 mark^ have likewise found non-specific reactions. Pearce is inclined 
 to believe that there is a general proteolytic ferment that is not specific 
 for any one protein. He is careful to add that this opinion is based en- 
 tirely upon the results of the dialyzation method. Abderhalden has 
 usually replied to these adverse criticisms by calling attention to possible 
 errors in technic, and at the present time all that one can do is to follow 
 his directions closely and preserve a mind receptive to results until the 
 question of specificity is settled definitely. 
 
 SERO-ENZYMES IN DISEASE 
 Cancer. — Freund and Abderhalden^ claim to have found protective 
 ferments in the serum of cancer that will digest coagulated cancer pro- 
 tein in the same manner as the ferments in pregnancy digest placental 
 protein. Frank and Heiman ^ reported positive results in 53 of 54 cases 
 of cancer; Markins and Munze,^ Epstein,^ Gambaroff,* Erpicum,' 
 Brockman,!" Lampe," Lowy,'^ Ball," and others have reported highly 
 favorable results. Frankle'^ and Lindig'^ have found the reactions 
 generally non-specific in character. 
 
 ' Surg., Gyn., and Obst., April, 1913, 411. 
 
 2 Colorado Med., Denver, 1913, x, 367". 
 
 ' Deut. med. Wochenschr., 1914, No. 7, 316. 
 
 * Munch, med. Wochenschr., 1913, 14, 763. 
 
 5 Berl. Win. Wochenschr., 1913, 1, No. 14. 
 
 « Berl. klin. Wochenschr., 1913, 1, No. 17. 
 
 ' Wien. klin. Wochenschr., 1913, x.xvi, No. 17. 
 
 8 Berl. klin. Wochenschr., 1913, 1, No, 17. 
 
 9 Bull, de I'Acad. Roy. de Bclg., 1913, xxvii, 624. 
 •» Lancet, London, November 15, 1913. 
 
 11 Miinch. med. Wochenschr., 1914, lxi,oNo. 9. 
 
 12 Jour. Amer. Med. Assoc, 1914, Ixii, 437. 
 " Jour. Amer. Med. Assoc, 1914, Ixii, 599. 
 " Deutsch. med. Wochenschr., xl, No. 12. 
 
 IS Munch, med. Wochenschr., 1913, 60, 288. 
 
SERO-BNZYMES IN DISEASE 265 
 
 Either the dialyzation or the optical method may be used in conduct- 
 ing these tests, precisely the same technic being followed as in making 
 the pregnancy test, except that several different" cancer tissues should be 
 used with each serum. It is well to make the experiment with a number 
 of cancer tissues taken from various parts, also with sarcoma tissue and 
 that of various benign tumors. 
 
 Mental Diseases. — Fauser^ has studied the serums of 88 cases of 
 dementia praecox and other mental diseases with various antigens com- 
 posed of the ductless glands, testicles, ovaries, etc., and attained in- 
 teresting results, tending to show that in many of these brain affections 
 there may be associated lesions in other organs, and that the symptoms 
 may be due to perverted functions of certain ductless glands. Munzer,^ 
 Bundschue and Roener,^ and Fisher ^ have also found in the serums of 
 mental and nervous diseases ferments for the protein of the ductless and 
 generative glands, tending to show that lesions of these organs may be 
 operative in the symptomatology of these conditions. 
 
 Syphilis. — Baeslack^ has reported having" had exceptionally good 
 results with the serums of syphilitics and a Substratxun composed of 
 coagulated syphilitic lesions of rabbit's testicle. Using the dialyzation 
 method, he found the sero-enzyme test more constant and earlier than 
 the Wassermann reaction. 
 
 Tuberculosis and Acute Infections. — Abderhalden and Andryewsky ^ 
 have suggested the use of the dialyzation or t*he optic method in the 
 diagnosis of acute infections. The peptone may either be prepared of 
 the bacilU, or the boiled organisms used in the dialyzing shell. In pre- 
 paring a bacterial substratum, the material iriust be carefully centri- 
 fuged in order to facilitate washing. The tubercle bacilli are degreased 
 by extraction in fat solvents. According to Abderhalden, the presence 
 of ferments in acute infections indicates that the animal is defending 
 itself. Abderhalden and Andryewsky found ferments present in the 
 serum of cattle receiving injections of suspensions of dead tubercle 
 bacilli and in experimental infections, and suggest that the test may 
 prove efficacious in testing cattle. This work should receive further 
 study in human infections. 
 
 ' Deutsch. med. Woohenschr., 1913, xxxix. No. 7. 
 
 = Berl. klin. Woohenschr., 1913, 1, No. .'i. 
 
 ' Deutsch. med. Wochenschr., 1913, No. 42^2069. 
 
 <• Deutsch. med. Wochen.schr., 1913, No. 44, 2138. 
 
 = Jour. Amer. Med. Assoc, 1914, Ixii, 1002;' ibid, Ixiii, 559. 
 
 ^ Munch, med. Wochenschr., 1913, Ixi, 1641-. 
 
CHAPTER XVI 
 AGGLUTININS 
 
 As previously stated, given any infectton, several antibodies of 
 different properties may be produced. If the infecting microorganism 
 produces characteristically an exogenous toxin, as, for example, that 
 produced by the diphtheria bacillus, an antitoxin is produced as the most 
 prominent of several antibodies. With other pathogenic bacteria that 
 produce mainly an endogenous toxin various antibodies are formed, and 
 one or more may play a prominent role in protecting the host, such as 
 opsonins, agglutinins, precipitins, bacteriolysins, etc. 
 
 If typhoid immune serum from an immunized animal or a patient 
 suffering from typhoid fever is added to an emulsion of typhoid bacilli 
 in a test-tube and the mixture placed in an incubator, the following 
 phenomenon will be observed: the bacteria, which previously formed a 
 uniform emulsion, clump together into little masses, settle at the sides 
 of the test-tube, and gradually fall to the bottom, the fluid becoming 
 almost clear. In a control test to which no active serum is added, the 
 fluid remains uniformly cloudy. If the reaction is observed microscopic- 
 ally in a hanging drop, it is noted that with the addition of the serum the 
 bacilli move nearer and nearer one another, this process being followed 
 by a gradual loss in motility and the formation of clumps. The sub- 
 stance in the serum causing this phenomenon* is called agglutinin, and 
 the reaction is known as agglutination. 
 
 Definition. — Agglutinins are antibodies that possess the power of caus- 
 ing bacteria, red blood-corpuscles, and some protozoa (trypanosomes) sus- 
 pended in a fluid to adhere and form clumps. 
 
 Historic. — Although Metchnikoff, Charrin, and Roger had noticed 
 peculiarities in the growth of Bacillus pyocyaneus when cultivated in 
 immune serum which we now believe were due to agglutinins, Gruber 
 and Durham and Bordet (1894-1896) were the first to recognize that 
 the agglutination reaction was a separate function of immune serum. 
 While investigating the Pfeiffer phenomenon of bacteriolysis with Bacil- 
 lus coli and the cholera vibrio, these investigators found that if the 
 respective immune serums were added to bouillon cultures of these two 
 
 266 
 
HISTOKIC 
 
 267 
 
 species, the cultures would lose their turbiditjc, flake-like clumps would 
 form and sink to the bottom of the tube, and the supernatant fluid 
 would become clear. Gruber at the same time called attention to the 
 fact that agglutinins were not absolutely specific for their own antigen, 
 but would agglutinate, to a lesser extent, closely allied species of bacteria. 
 In 1896 Pfaundler drew attention to a peculiar phenomenon ob- 
 served when bacteria were grown in an immune serum. Long and more 
 or less interlaced threads of bacteria developed, which were regarded as 
 due to agglutinins. At that time considerable* emphasis was laid upon 
 the importance of Pfaundler's reaction, but at present the ordinary 
 
 Ftg. 75. — Theorettc Formation of Aooltttinins. 
 
 agglutination tests have superseded this reaQtion as a practical diag- 
 nostic procedure. 
 
 In 1896 Widal and Griinbaum first turned these facts to practical 
 use in the diagnosis of typhoid fever. These investigators found that 
 the serum of patients suffering from typhoid fever acquires a high agglu- 
 tinating power for Bacillus typhosus, and since this phenomenon gen- 
 erally manifests itself comparatively early in the disease, its recognition 
 has considerable diagnostic importance. It is purely accidental that 
 we speak of the "Widal reaction" in typhoid, fever, rather than of the 
 "Griinbaum reaction," for the latter observer conducted similar studies 
 
268 
 
 AGGLUTININS 
 
 independent of Widal, but, owing to a lack of patients, Widal preceded 
 him in the publication of a more extensive work. 
 
 At the present time this diagnostic reaction is known as the Grubler- 
 Widal reaction. It has proved of great value to a large number of dif- 
 ferent investigators, not only in making the serum diagnosis of typhoid 
 fever, but in other infections as well. 
 
 Normal and Immime Agglutinins. — Normal serums are frequently 
 capable of agglutinating bacteria, such as the typhoid, colon, pyocy- 
 aneus, and dysentery bacilli. In some cases the typhoid bacillus may be 
 agglutinated in dilutions as high as 1 : 30, a point of practical impor- 
 tance in the clinical use of the test. When a normal serum is found to 
 have a high agglutinating power, it is probable that 
 a previous infection by the microorganism has oc- 
 curred. Since the serum of a new-born child is 
 largely devoid of agglutinins that are found in later 
 life, the so-called normal agglutinins may, after all, 
 be acquired properties. 
 Fig. 76.— -Theo- 'p]-,g -^gj-jQ immune aqqlutinin is applied to the 
 
 RETIC StRUC- '"' -"^^^ 
 
 TUBE OF Agglu- agglutinating substance in! a seriun developed as the 
 
 TININ AND Ag- 1i f ■ i- i- c ± ±- ■ ■ J.- 
 
 GLUTiNoiD, result 01 miection or oi systematic immunization 
 
 1, Agglutinin: with the microorganism. 
 
 Sou^''^fo°^L°ion Formation of Agglutinins.— According to Ehr- 
 
 with antigen;^, the lich's side-chain theory, agglutinins are antibodies of 
 agglutmopnore or rr n rr-vi . . 
 
 zymophore group. the second order (Fig. 7o). They resemble antitoxins 
 
 -, Agglutmoid. Qj. receptors of the first order in possessing an afiinity- 
 
 aggUitinin, except bearing or haptophore group that unites with the 
 
 phore or zymophore antigen, but they differ from them in having also a 
 
 group is lost. functional or agglutinophofe group that agglutinates 
 
 the antigen when this union has occurred (Fig. 76). 
 
 Agglutinins that have lost their zymophore or agglutinophore group 
 through the action of heat, age, acids, etc., but that still possess their 
 haptophore group, are called agglutinoids, just as toxins that have lost 
 their toxopliore group are called toxoids. Such agglutinoids, then, may 
 still combine with the bacteria or blood-cells without being able, how- 
 ever, to produce agglutination (Fig. 77). 
 
 It is found, at times, that even a fresh serum, when concentrated, 
 will cause less agglutination than when it is diluted. This is ascribed to 
 the presence of agglutinoids, which have a stronger affinity for agglutin- 
 ogen than has the agglutinin. When producing a reaction of this char- 
 acter they are called pro-agglutinoids. When" the serum is diluted, the 
 
OniGIN OF AGGLL'TIXIXS 
 
 269 
 
 pro-agglutinoids become less concentrated, aid finally, when they are 
 diluted as to have no influence on the reactioil, the agglutinins are still 
 present in sufficient quantity to bring about agglutination. As a practi- 
 cal fact, in agglutination reactions the action' of pro-agglutinoids is of 
 much importance, for the inexperi- 
 enced may be misled by the absence 
 of or by poor agglutination in lower 
 dilutions to neglect the use of higher 
 dilutions (see Fig. 83). 
 
 The substance in bacteria or 
 other cells that produces agglutinin 
 is called agglutinogen. It appears 
 to be formed in the cell, and in 
 some cases may be excreted into 
 the surrounding medium. Certainly 
 when bacteria die and become dis- 
 integrated, agglutinogen is Uberated 
 and the filtrates (entirely free from 
 bacterial cells), when injected into 
 animals, will cause the formation of 
 agglutinins. 
 
 Agglutinogen must be con- 
 sidered as ha%'ing a simple hapto- 
 phorous group, through which it 
 may unite with the receptors of the 
 tissue-cells. This haptophore 
 comes into plaj' again in the union 
 between agglutinogen and agglu- 
 tinin, which precedes agglutination. 
 It is a passive bodj', similar to 
 
 the haptophore of antitoxin, and has no other function than that of 
 uniting either mth cell or -nith agglutinin. 
 
 Origin of Agglutinins. — The investigation^ that have been carried 
 out for the purpose of determining the site ofi formation of agglutinins 
 have not thus far ;\-ielded conclusive T-esulta The IjTuphoid tissues 
 appear especially concerned, agglutinins being found earh' in the bone- 
 marrow and the spleen (Pfeiffer and JNIai-x). INIetchnikoff believes that 
 agglutinins maj- be derived from leukocj^:es arid endothelial cells. It is 
 more probable, however, that the formation is general, and is the result 
 of wide-spread cellular acti^-itv. 
 
 Fig. 77.-*-A Diagrammatic Illvstha- 
 TiOi\- OF THE Action of Aggluti- 
 xixs AXD Aggltttixoids. 
 
 In t^e first tube Geft) most of the 
 bacilli {B) have been agglutinated and 
 massed iji the bottom of the tube by 
 the agglutinins (a). 
 
 In ishe second tube (right) the 
 bacilli (B) are in combination with the 
 agglutinoids (A), but agglutination 
 does not occur because the agglutino- 
 phore groups are lost. A few bacilli 
 ha\e been- agglutinated by the agglu- 
 tinins (a). 
 
270 
 
 AGGLUTININS 
 
 In accordance with the side-chain theory, the ability of an animal to 
 form agglutinins for a certain microorganism would depend on its pos- 
 session of receptors of the second order, which are able to unite with the 
 agglutinogenic receptors of the microorganism. It has been well es- 
 tablished that the number of such suitable receptors vary in animals, and 
 that different animals may not produce serums with equal agglutinating 
 powers. 
 
 Agglutinins do not appear in the serum immediately after inocula- 
 tion, but require an incubation period of from two to four days for their 
 production. 
 
 Properties and Nature of Agglutinins. — (1) Agglutinins are fairly 
 resistant substances that withstand heatrug to 60° C. and lose their 
 power only when heated to higher temperatures. It is possible, there- , 
 fore, to make a serum bacteriolytically inactive by destroying comple- 
 ment at 55° C, and still retain its agglutinating power. 
 
 (2) They resist drying, and their activity is best preserved in this 
 state. 
 
 (3) They are precipitated from a serum by magnesium or ammonium 
 sulphates, when these salts are used in proper concentration, and are 
 thus closely associated with the globulin fraction of serum. 
 
 (4) They are separate and distinct antibodies, and arc not associated 
 with bacteriolysins. Thus the agglutinins of an immune serum may be 
 lost, destroyed, or absorbed and the bacteriylysins retained. As pre- 
 viously mentioned, the bacteriolytic power of *a serum may be inhibited 
 by heating it to 55° C. for one-half hour without influencing the agglu- 
 tinin content, and during disease processes the formation of agglutinins 
 and that of bacteriolysins arc apparently not parallel processes. 
 
 Mechanism of Agglutination. — The true nature of the phenomenon 
 of agglutination is unknown, as is shown by the number of theories ad- 
 vanced. Thus — 
 
 1. Gruber's idea of the mechanism of this phenomenon was that the 
 agglutinin so changed the bacterial membrane as to render it more 
 viscous, and that this increased viscosity caused the bacteria to adhere 
 and form clumps. No visible changes in the organisms or red corpuscles 
 can, however, be seen. 
 
 2. Paltauf's theory is somewhat similar, he believing that the agglu- 
 tinogen is precipitated on the surface of the bacteria by union with the 
 agglutinin, with the formation of a sticky substance. He cites evidence 
 that tends to show that such substances are actually thrown out from 
 the bacteria during agglutination, as may be seen in a properly stained 
 
SPECIFICITY OF AGGLUTININS 271 
 
 preparation in the form of a precipitate stirrounding the bacterial 
 cells. 
 
 3. The presence of some salt is necessary far the occurrence of agglu- 
 tination. Bordet found that if the salts were. removed from the serum 
 and from the suspension of bacteria by dialysis and that the two were 
 then mixed, agglutination did not occur, but that if a small amount 
 of sodium chlorid was added, agglutination promptly took place. Ac- 
 cording to this view, therefore, agglutination is a phenomenon of molec- 
 ular physics — the agglutinin acts on the bacteria or other cells and 
 prepares them for agglutination by altering the relations of molecular 
 attraction between them and the surrounding fluid, the second phase, 
 the loss of motility, clumping, etc., being brought about by the presence 
 of salt. This second phase, therefore, would be a purely physical 
 phenomenon, the salts altering the electric conditions of the colloidal- 
 like agglutinin-bacterium combination, so that their surface tension is 
 increased. To overcome this the particles adhere together, presenting 
 in a clump less surface tension than if they remained as individual par- 
 ticles. Bordet cites the precipitation of clay as an analogous case: if 
 a little salt is added to a fine emulsion of potters' clay in distilled water, 
 the clay immediately clumps and falls to the bottom, the resemblance 
 between these flakes and the clump of agglutinated bacteria being very 
 striking. 
 
 Specificity of Agglutinins. — For a time after their discovery the ag- 
 glutinins were regarded as strictly specific, i. e., a typhoid-immune serum 
 would agglutinate only typhoid bacilli and no others. Gruber early 
 pointed out that an immune serum will frequently agglutinate other 
 closely related organisms, although not usually to so high a degree. 
 
 Group or partial agglutinins, therefore, refer to the presence in a serum 
 of certain agglutinins that agglutinate certain other microorganisms 
 that are morphologically, biologically, and often pathogenetically 
 closely related to the homologous microorganism (the bacterium causing 
 the infection or used in artificial immunization). For example, a ty- 
 phoid-immune serum possesses, besides its greatly increased aggluti- 
 nating power for Bacillus typhosus, some agglutinin for Bacillus para- 
 typhosus, notably above that of normal serum. This is explained by the 
 very close biologic relationship of these bacteria, together with the fact 
 that the agglutinin-producing substance (agglutinogen) is a complex 
 and not a single chemical substance. This has been explained by Dur- 
 ham in the following example : If the typhoid agglutinogen is composed 
 of various elements. A, B, C, D, it is conceivable that the closely related 
 
272 
 
 AGGLUTININS 
 
 paratyphoids might contain one or more of these four agglutinogens, 
 and, therefore, the agglutinating power of the- typhoid serum for a para- 
 typhoid bacillus, though not so great as on the typhoid bacillus, is still 
 considerable. Accordingly, in an infection with one microorganism 
 a specific agglutinin will be formed for that microorganism, and group 
 agglutinins for other more or less allied microorganisms, and conse- 
 quently the specificity of the agglutinating rfeaction depends upon the 
 principle of dilution, the specific agglutinin^ being present in largest 
 amount and operative in dilutions above the range of the group agglu- 
 tinins. 
 
 Absorption Methods for Differentiating Between a Mixed and a 
 Single Infection. — In 1902 Castellani discovered that if the serum of an 
 animal immunized against a certain microorganism contains that 
 microorganism in large numbers, the serum will lose its agglutinating 
 power not only for that microorganism, but also for all other varieties 
 on which it formerly acted. If, however, the serum contains the organ- 
 ism corresponding to the group agglutinins, the agglutinating power of 
 the serum for the homologous organism is reduced but little or not at all. 
 
 In a mixed infection due to two or more varieties of bacteria there 
 iwill be specific agglutinins for each of the microorganisms, and group 
 agglutinins for each of them as well. If the immune serum is saturated 
 with one of these varieties its chief or major agglutinins and some or all 
 of the group agglutinins will be removed, but the major agglutinin of the 
 second species will remain. On the addition of the second bacterium to 
 the immune serum agglutination occurs and its agglutinin is absorbed. 
 Park, who has carefully investigated this subject, finds that the absorp- 
 tion method proves that when one variety of bacteria removes all agglu- 
 tinins for a second, the agglutinins in question were not produced by the 
 second variety. 
 
 Hemagglutinins. — Agglutination, like other immunity reactions, is a 
 manifestation of broad biologic laws and is not limited to bacteria. As 
 hemolysins are produced by the injection of an animal with red blood- 
 corpuscles from another species, so agglutinins that agglutinate the red 
 blood-corpuscles may be developed at the same time. When a serum 
 containing hemagglutinin is added to a suspension of the corresponding 
 red blood-corpuscles contained in a test-tube,, it causes these to collect 
 into clumps and flakes and sink to the bottom; just as a typhoid immune 
 serum agglutinates typhoid bacilli. These clumps are broken up with 
 some difficulty, and may interfere with hemolytic reactions. They are 
 especially to be observed in antihuman hemolytic serums when agglu- 
 
HEMAGGLUTININS 273 
 
 tination may be so marked as to prevent hemolysis unless the tubes are 
 frequently and vigorously shaken. 
 
 Small amounts of normal hemagglutinins may be found. Of par- 
 ticular practical importance are those for animals of the same species, 
 the so-called isohemagglutinins. 
 
 Isohemagglutinins were discovered independently by Landsteiner 
 and Shattuck in 1900, and were studied quite extensively by Hektoen 
 and Gay. At first the occurrence of isoagglutination was regarded as 
 of pathologic significance, but later researches showed that they may be 
 found in a large percentage of normal bloods. According to Land- 
 steiner and Hektoen, human bloods may be divided into four groups, as 
 follows : 
 
 Group 1: Here the corpuscles are not agglutinated by any human 
 serum, whereas the serums agglutinate the corpuscles of the other groups. 
 This group includes about 50 per cent, of all persons. 
 
 Group 2 : In this group the corpuscles are agglutinated by the serums 
 of other groups, whereas the serums agglutinate the corpuscles of Group 
 3 but not of Group 1. 
 
 Group 3: The corpuscles are agglutinated by all other serums, and 
 the serums agglutinate the corpuscles of Group 2 but not of Group 1. 
 
 Group 4: The corpuscles are agglutinated by all other groups, but 
 the serums are unable to agglutinate any human corpuscles. These are 
 quite rare. 
 
 The group characteristics arc hereditary, and permanent throughout 
 life. 
 
 With the increasing number of blood transfusions, the phenomena of 
 isoagglutination and isohemolysis — the two b„eing closely related — are 
 of considerable practical importance, especially if the patient is suffering 
 with cancer, when the serum is likely to be actively hemolytic for the 
 donor's corpuscles. 
 
 In selecting the donor for a transfusion, agglutination and hemolysis 
 
 tests should always be made before operation if time permits. The 
 
 tests made in vitro are usually safe guides as to conditions existing in 
 
 vivo, and such preliminary tests may prevent the occurrence of untoward 
 
 symptoms associated with intravascular hemolysis or agglutination, 
 
 such as fever, dyspnea, edema, and hemoglobinuria. As a rule, the 
 
 donor selected should be a near relative, and whenever time permits, a 
 
 Wassermann reaction and the isoagglutination and isohemolysin tests 
 
 should be made. That donor should be chosfin whose blood shows no 
 
 mter-agglutination or hemolysis with the patient's serum and corpuscles. 
 18 
 
274 AGGLUTININS 
 
 If such a donor cannot be obtained, it is safer ts'use a person whose serum 
 is agglutinative toward the patient's cells than one whose cells are 
 agglutinated by the patient's serum. 
 
 The technic of these transfusion tests is! given at the end of this 
 chapter. 
 
 Non-agglutinable Species of Bacteria. — Cjertain species of bacteria, 
 especially when freshly isolated from the animal body, may prove them- 
 selves immune to the action of agglutinins; this is true especially of 
 the bacillus of Friedlander. As a rule, this resistance is lost when the 
 microorganism is grown for some time in artificial media. In some in- 
 stances the typhoid bacillus, when freshly isolated from a patient, may 
 resist agglutination until after it has passec^;'a period of existence on 
 artificial media. This variability is probably due to some change 
 taking place in the agglutinable substance of the agglutinins during the 
 sojourn of the bacilli in the animal body, and possess such an excess of 
 agglutinogcnic receptors as to require a much* larger amount of aggluti- 
 nin to cause agglutination. 
 
 It should be remembered that agglutinins act on dead as well as on 
 living bacteria, those killed by heat, formaliil, phenol, etc., being simi- 
 larly agglutinable. In making the microscopic test the use of dead 
 bacteria is not so satisfactory as when the test.is made with living motile 
 bacteria, for the influence of the serum on motility alone is of value in 
 interpreting a reaction. 
 
 Variation in Agglutinating Strength of a Serum. — In a given infection, 
 such as typhoid fever, there is usually a continued increase in the amount 
 of agglutinin in the blood from the fourth day until convalescence is 
 established, and then a decrease occurs. It is a fact of practical impor- 
 tance that the agglutinating power of a seru'tai may vary from day to 
 day, so that it is very strong one day and may become weak or disap- 
 pear entirely on the next day or two. Hence the importance of making 
 more than one test in a suspicious case when the first trial has been 
 doubtful or negative. There is no satisfactory explanation for this 
 variation, mixed infection, intestinal hemorrhage, etc., being regarded 
 by some as responsible for it. 
 
 Role of Agglutinins in Immunity. — The agglutinins were formerly 
 regarded as possessing a true protective and curative power. It has 
 previously been mentioned that bacteria may be grown in a specific 
 agglutinating serum, and cultures made of agglutinated bacteria show 
 them to be fully alive and as virulent as before agglutination took place. 
 In certain cases agglutinins for a microorganism may be entirely absent, 
 
PRACTICAL APPLICATIONS 275 
 
 and yet the animal enjoy an immunity. Bacteria that have been acted 
 upon by an agglutinin are apparently not altered in appearance, viabil- 
 ity, or virulence. 
 
 Many observations tend to show that the agglutinating power of a 
 serum gives no indication of the degree of imlnunity that exists. For 
 instance, relapses may occur in typhoid fever at a time when the agglu- 
 tiuating power of the patient's blood is at its highest. 
 
 At present agglutinins are regarded as playing a subsidiary role in 
 immunity, their presence being of diagnostic v^lue, and an' indication of 
 the presence of more important factors, and as an aid to bacteriolysis, 
 and phagocytosis. 
 
 PRACTICAL APPLICATIONS 
 
 The agglutination reaction is used for the following purposes: 
 
 1. For the diagnosis of disease, by identifying the bacterial infection 
 from which the patient is suffering. To do this satisfactorily we must 
 have on hand stock cultures of bacteria, and test the patient's serum for 
 agglutinins for these bacteria. For instance, if a patient presents symp- 
 toms of typhoid fever, the serum is tested for typhoid agglutinins; if 
 the reaction is very weak or negative and continues so, the serum is 
 further tested for agglutinins for Bacillus paratyphosus A and B. 
 
 In typhoid fever the Gruber-Widal reaction may be positive as early 
 as the third day; usually, however, the positive reaction is obtained 
 somewhat later — about the seventh or the eighth day. A day or so 
 earlier the bacilli used in making the test may be seen to lose their mo- 
 tility, and two or three may form a loose clump. This is the doubtful 
 reaction, and it is well to test every day or every other day until a de- 
 cisive reaction is obtained. 
 
 According to Park, "about 20 per cent, of typhoid infections give 
 positive reactions in the first week; about 60 per cent, in the second 
 week; about 80 per cent, in the third week; about 90 per cent, in the 
 fourth week, and about 75 per cent, in the second month of the disease." 
 In about 90 to 95 per cent, of cases in which repeated examinations are 
 made a positive reaction is to be found at some*time during the patient's 
 illness. 
 
 Occasionally the reaction appears first during the stage of convales- 
 cence, and at times it may even be absent, the diagnosis being confirmed 
 by cultivating typhoid bacilli from the blood. The possibility of a given 
 case reacting strongly one day and weakly or entirely negative a day or 
 so later has been emphasized elsewhere. 
 
276 
 
 AGGLUTININS 
 
 Usually the reaction is strongest during convalescence, remains posi- 
 tive for several weeks, and then gradually returns to the normal. Occa- 
 sionally the reaction remains positive for months or even years after the 
 attack of typhoid fever — many such cases are "carriers" and harbor the 
 bacilli in the gall-bladder, although the persoa appears to be quite well. 
 
 Only very rarely does normal serum immediately agglutinate typhoid 
 bacilli in a dilution higher than 1:10; where a time limit of one to 
 two hours is given, a few may show some agglutination in dilutions up 
 to 1 : 30. 
 
 If the typhoid bacillus is agglutinated by 'the patient's serum in a 
 dilution of 1:100, or at least 1:40, the Widal'reaction may be regarded 
 as positive. It is not safe to use lower dilutions, as occasionally the 
 serum of healthy persons may agglutinate Bacillus typhosus in dilutions 
 up to 1:30. Due care must be exercised not to mistake a pseudo- 
 reaction about detritus for true agglutination. 
 
 Positive reactions are occasionally obtained in other diseases — acute 
 miliary tuberculosis, malaria, malignant endocarditis, and pneumonia. 
 It is also well to bear in mind the possibility of a patient having been 
 vaccinated against typhoid at some early dates, with resulting agglutinin 
 formation. 
 
 Owing to the similarity of symptoms an infection with Bacillus para- 
 typhosus A and B may be difficult to distinguish from typhoid fever. 
 This difficulty is increased by the confusion of the Widal reaction 
 owing to the presence of group agglutinins in the serum if proper dilu- 
 tion is not practised. Bacillus A and Bacillus B are not identical in their 
 agglutinable properties, the latter being more closely related to the 
 typhoid bacillus than the former. In this country Bacillus paratyphosus 
 is usually held responsible for pajatyphoid fever. Conclusions should 
 not be drawn until tests have been made with both strains of the para- 
 tjrphoid bacillus and with the typhoid bacillus. In case of mixed in- 
 fection the absorption method of Castellani will serve to clear up the 
 diagnosis. 
 
 In dysentery the agglutination reaction wfth the serum of patients 
 shows great variability. In spite of the presence of bacilli in the feces 
 ithe reaction is sometimes absent, often disappears rapidly during con- 
 valescence, and rarely is as high as in typhoid fever. The tests should 
 always be performed with both the "Flexner" and "Shiga" types of 
 bacilli, as the two do not possess identical agglutinable properties, and 
 either may be the cause of infection in a given case. The absence of the 
 reaction does not exclude a dysenteric infection. 
 
PRACTICAL APPLICATldNS 277 
 
 In cholera the agglutination test has so far proved of doubtful aid in 
 establishing a diagnosis of the disease. However, for the purpose of 
 recognizing bacilli isolated from the feces of suspicious cases the reaction 
 with known immune serum is of great value. 
 
 In cerebrospinal meningitis the agglutination occurs within an hour 
 in dilutions of 1 : 10. It is seldom that the patient's serum agglutinates 
 in a dilution higher than 1 : 50. 
 
 In tuberculosis the agglutination reaction has no value as a diagnostic 
 procedure. Koch recommended the agglutination test for the estima- 
 tion of the degree of immunity conferred by tdbercuhn treatment. As 
 pointed out elsewhere, agglutinins have apparently no antimicrobic 
 influence, but, as with typhoid vaccination, may indicate the degree of 
 reaction and the presence of other antibodies. 
 
 Many strains of tubercle bacilli are almost non-agglutinable. The 
 preparation of a homogeneous emulsion is not easily made, and the results 
 are Ukely to be confusing and contradictory. 
 
 In plague the agglutination reaction becom'es quite marked about 
 the ninth day of the disease — too late, however,, to be of much practical 
 value in diagnosis. It is occasionally useful, however, for deciding 
 whether a patient in the convalescent stage has really suffered from the 
 disease. 
 
 In Malta fever the agglutination reaction is of considerable value in 
 making the diagnosis. 
 
 In pneumonia the reaction is of value in rapidly differentiating pneu- 
 mococci and as an aid in specific serum therapy,* and it may also be of aid 
 in making the diagnosis of infection with Bacillus enteritidis and Bacil- 
 lus botulinus. 
 
 In veterinary practice agglutination reactions are of value in the 
 diagnosis of glanders, infected horses reacting hi some instances to dilu- 
 tions as high as 1:2000. For diagnostic purposes the agglutination 
 test in glanders must be in dilutions higher than 1:800. A positive 
 reaction in dilutions of 1:1000 is regarded as suggestive, anc! is con- 
 trolled by a complement-fixation test; agglutination in dilutions of 
 1 : 1500 practically always indicates an infectiorS. The complement-fixa- 
 tion test, however, is a better diagnostic reaction. 
 
 2. Agglutination reactions are also of valuQ as an aid to the identi- 
 fication of a microorganism that has been cultivated from a patient. 
 For this purpose we must have on hand various standard immune 
 serums. For example, if a bacillus resembling the typhoid bacillus is 
 isolated from the feces of a patient, the diagn'osis may be aided by a 
 positive agglutination reaction with typhoid immune serum. 
 
278 AGGLUTININS 
 
 The "group agglutinins" constitute a source of difficulty in making 
 the differentiation among the numerous menjbers of a group of micro- 
 organisms, but if a highly potent agglutinati-ng serum is used and the 
 test is carried to the point of determining the highest dilution that will 
 agglutinate the bacteria, it will in most cases be possible to differentiate 
 the variously allied microorganisms by this test. 
 
 In conducting these reactions it is best to ilse a macroscopic method, 
 and the agglutinating serum used must have been previously titrated 
 against an easily agglutinable and known strain of the microorganism 
 in question 
 
 In this connection it is well to remember that freshly isolated cultures 
 of a microorganism may be not at all or but yery slightly agglutinable. 
 Thus colonies of t5T3hoid bacilli found in feces or in an abscess may, if 
 picked from a plate, resist agglutination until subcultured several times 
 in artificial media. 
 
 The agglutination test has great value as"a mode of differentiating 
 a.mong the members of the typhoid-colon group of bacilli. In the diag- 
 nosis of cholera, suspicious bacilli isolated from the feces may be tested 
 with a known cholera immune serum, and the bacteriologic diagnosis 
 thus be greatly facilitated. The test also has some value in making a 
 biologic differentiation between meningococci and gonococci, and also 
 between other groups of bacteria. 
 
 3. Agglutination tests are of value in determining whether, in a case 
 in which more than one microorganism has been cultivated, the condi- 
 tion at hand is a single or a mixed infection. The absorption agglu- 
 tinin test is made with the patient's serum, and the cultures are isolated 
 from the patient. 
 
 4. Agglutination tests are also of value for measuring the immuniz- 
 ing response that a patient is making to his infection or to artificial 
 immunization. Thus the test is of some value in determining the re- 
 sponse to inoculation with typhoid vaccine, although it is probable 
 that the agglutinin itself does not possess true antimicrobic properties. 
 
 THE AGGLUTINATION REACTION 
 
 The value of the agglutination test in the diagnosis of disease is 
 limited chiefly to typhoid and paratyphoid fevers, and, in a less degree, 
 to cerebrospinal meningitis and bacillary dysentery. It is especially 
 useful as a practical test in the diagnosis of obscure and atypical cases of 
 typhoid fever and also in the diagnosis of "typhoid carriers." 
 
THE AGGLtJTINATIOX REACTION 279 
 
 Two methods may be employed : 
 
 1. The microscopic method which is generally employed where the 
 Gruber-Widal reaction for tj-phoid fever has been employed, as the re- 
 action is quickly done and requires but a small amount of blood. 
 
 This test is usually performed with serum separated from the clot 
 and in various dilutions (\\et method). The tfet may also be performed 
 with dried blood (dry method), the agglutinins* being preserved and re- 
 dissolved with a diluent. The technic of ttfe latter method is very 
 simple. The blood is easily collected and m*ay be sent for long dis- 
 tances, and for these reasons the method has been adopted by many 
 boards of health. 
 
 2. The macroscopic method is that generally preferred if sufficient 
 blood is on hand, and is the method of choice in scientific research. Ab- 
 sorption tests must be performed with the macroscopic technic. 
 
 Requisites for Conducting Agglutination Reactions. — (o) Bacterial 
 Emulsion. — This may be prepared by growing the microorganism in 
 broth. Solid media may be used, and the culture washed off ■with sterile 
 salt solution and emulsified. 
 
 (1) The bacterial emulsion should be prepared of young cultures, 
 should be homogeneous and free from clumps, and of such density as to 
 furnish a sufficient number of microorganisms to give the reaction (Fig. 
 78). 
 
 (2) For the ordinary microscopic Widal test, eighteen to twenty- 
 four hour bouillon cultures of Bacillus typhosus, Bacillus coli, and Bacil- 
 lus paratyphosus jield uniform and satisfactory results. An old lab- 
 oratory culture — one that is kno-rni to be agglnlinable — should be used. 
 Broth cultures should be cultivated at a temperature lower than body 
 heat, in order that long motile forms may be secured. During the sum- 
 iner and early autumn months the culture can be grown at room tem- 
 perature; during the winter, on top of the incubator. 
 
 Thick cultures are unsatisfactory for making the microscopic test, 
 as there is always some false cliunping and motility is not well marked 
 (Fig. 79). 
 
 ^\ hen these tests are done routinely, it is gqod practice to subculture 
 in broth every day in order that a satisfactorj- cultiu-e may always be 
 on hand. When performed at irregular intervals, a broth culture can 
 be prepared from a stock agar culture and the, test performed twenty- 
 four hours later. 
 
 (3) Emulsions may be prepared of young cultures on solid media by 
 removing portions of the growth with a platinum loop and emulsifying 
 
280 
 
 AGGLUTININS 
 
 in a diluent, such as normal salt solution or broth. This may be per- 
 formed by placing the diluent in a test-tube and rubbing the loop over 
 the glass just at the margin of the fluid, thg bacteria being gradually 
 emulsified and floated into the diluent. When larger quantities of emul- 
 sion are desired, as for making the macroscopic test, 5 c.c. of diluent may 
 be poured upon the culture and the growth washed off with the aid of 
 the platinum loop. The emulsion is gently shaken and removed to a 
 second tube, when unresolved bacterial clumps will sink to the bottom. 
 In other cases the emulsion may be centrifuged for a short time or fil- 
 tered through sterile filter-paper. Sufficient salt solution is added to 
 
 Fig. 78. — A Satisfactoky CuurxfRE for 
 THE MiuRoscoric Agglutination 
 Reaction. X 430. 
 
 Thi.s shows a satisfactory culture of 
 the proper density ;iiiil free of clumps of 
 bacilli. (Twenty-four hour culture of 
 Bacillus typhosus grown at room tem- 
 perature.) 
 
 Fig. 79. — An Un.'satisfactort Culture 
 for the michoscofic agglutina- 
 TION RpACTioN. X 430. 
 
 The culture is rather too dense and 
 shows considerable spontaneous or false 
 agglutination of the bacilli. (Twenty- 
 four hour, culture of Bacillus typhosu.s 
 grown at 37° C.) 
 
 give the emulsion a density equal to that of a rich twenty-four-hour 
 bouillon culture. 
 
 (4) To emulsify a culture of the plague bacillus or any other micro- 
 organism that displays a strong tendency to undergo "spontaneous" 
 agglutination, distilled water or 1:1000 salt solution should be used. 
 
 (5) In the case of a culture of tubercle bacillus, the growth can be re- 
 solved into its elements by prolonged trituratibn in normal salt solution, 
 and any residue or unresolved clumps removed by centrifugalization. 
 A less laborious and dangerous method is to use the tubercle powder of 
 Koch, which is obtained by reducing dried tubercle cultures to a fine 
 
THE AGGLUTINATION REACTION 281 
 
 powder by machinery. The powder may be made up into a suitable 
 suspension by rubbing it in a mortar with normal salt solution. 
 
 (6) When it is necessary to work with highjly dangerous microorgan- 
 isms, or to operate from day to day ^ath the same bacterial suspension, 
 one may employ suspensions that have been heated for one hour to 60° 
 C, or suspensions in salt solution to which 1 per cent, formahn has been 
 added. These will keep well in the refrigerator, but the sediment of 
 dead bacteria must be well shaken before it is used. 
 
 (&) Serum. — Serum should preferably be fr^sh, clear, and free from 
 corpuscles. 
 
 (1) For the microscopic "wet " method, ample seriun is furnished from 
 the blood contained in the ordinary Wright capsule (see p. 33). 
 
 (2) For the microscopic "dry method," bloftd is secured by pricking 
 the finger or lobe of the ear and collecting a few drops of blood upon 
 aluminum foil, on a clean glass slide, or on partially glazed paper. The 
 blood must not he heated to hasten drying, or agglutinins may be destroyed. 
 Smears on aluminum foil and on glass slides are to be preferred to those 
 on paper, as the blood can be moistened and portions removed without 
 the likelihood of transferring extraneous material, such as paper fiber. 
 While there are certain objections to this methdd to be pointed out later, 
 yet practical experience has demonstrated its value, as the serum does 
 not readily deteriorate or become contaminated with bacteria, and the 
 ease with which blood may be collected and mailed recommends the 
 process for board of health laboratories. 
 
 (3) For the macroscopic test larger amounts of serum are needed. 
 Sufficient blood is easily obtained by pricking the finger deeply and 
 collecting several cubic centimeters in a small test-tube. 
 
 (4) Because normal agglutinins may be active in dilutions as high as 
 1;30, for diagnostic tests in typhoid fever the serum should not be di- 
 luted lower than 1 : 40. For routine work dilutions of 1 : -50 and 1 : 100 
 are well adapted for the microscopic test. Dilutions of 1 : 40 and 1 : 80 
 are readily made with the white corpuscle pipSt and are equally useful. 
 
 With the macroscopic technic, dilutions of from 1:20 up to any 
 dilution are readily made in appropriate test-tubes. 
 
 Precautions. — In bacteriologic technic due care should be observed 
 to avoid contamination and possible infection when working with living 
 cultures. 
 
 (a) Agglutinated bacteria are not necessarily dead, and hanging-drop 
 preparations, test-tubes, etc., should be immersed in 1 per cent, formalin 
 before cleansing. 
 
282 AGGLUTININS 
 
 (b) The working table or desk and the hands should be washed with 
 a solution of lysol or 1 per cent, formalin after the reactions have been 
 made and the work completed. 
 
 (c) Early in typhoid fever the bacillus may be present in the blood, 
 and consequently due care should be exercised in handling it, in diluting 
 the serum, and in the disposal of the clot. 
 
 Technic of the Microscopic Agglutination Test (Wet Method).— The 
 WiDAL Reaction in Typhoid Fever 
 
 (1) Dilute the patient's serum by placing one drop from a capillary 
 pipet in a small watch-glass and adding 19 drops of normal salt solution. 
 This gives a dilution of 1 : 20. Mix thoroughly. 
 
 (2) With a 3 or 4 mm. platinum loop place a drop on a clean cover- 
 glass that is sufficiently thin to permit the use of an oil-immersion lens. 
 The loop is better than a capillary pipet because the drop it gives is 
 smaller, and when it is later diluted with an equal quantity of bacterial 
 emulsion, it is not too large and is easily manipulated. 
 
 (3) With the same sterilized platinum loop add one loopful of a 
 twenty-four-hour broth culture of Bacillus typhosus to the drop of 
 diluted serum on the cover-glass. Mix gently and without spreading the 
 drop. This gives a final dilution of 1 : 40. 
 
 (4) Edge a hanging-drop slide with vaselin, and invert the cover- 
 glass slide over the hollow portion in such a manner that the drop will 
 be suspended in its center. Care must be exercised not to spread the 
 drop, for if this occurs and the fluid flows arDund the margins of the 
 chamber a new preparation must be made. Inspect the slide, and add 
 vaselin, if necessary, until it is sealed tightly. By means of a grease 
 pencil label the slide with the name of the patient, the dilution, and the 
 time when the preparation was made. 
 
 (5) Place 2 to 5 drops of serum dilution; 1 : 20 in a second watch- 
 glass, and add an equal quantity of normal salt solution. Mix well. 
 This gives a dilution of 1 : 40. 
 
 (6) Prepare a second slide by mixmg a loopful of this dilution with 
 an equal sized loopful of culture. Mix gently. This gives a final dilu- 
 tion of 1 : 80. Mark the slide with the name, dilution, and the time. 
 
 (7) Prepare a third slide by placing a loopful of culture on a cover- 
 glass and invert over a concave slide to which vaselin has been apphed 
 in the usual manner. This is the culture control. Label the slide. 
 
 (8) Place the sUdes in a dark place at room temperature and examine 
 at the end of an hour with the ^/q or oil-immefsion lens. 
 
THE AGGLUTINATION REACTION 
 
 283 
 
 (a) First inspect the control. The bacilh should not be clumped, 
 but should be motile, and preferably in the form of long slender rods. 
 (See Fig. 78.) 
 
 (6) Examine the 1 : 40 and 1 : 80 dilution preparations : a positive 
 reaction is indicated by loss of motility and definite clumping (Fig. 80). 
 A few free motile bacilli may be seen, or a clump may be seen to move, 
 owing to the efforts of the bacilli to break away. A doubtful reaction 
 is indicated by a partial loss of motility and a few indefinite clumps. 
 A negative reaction is indicated when there is no loss in motility or no 
 clumping, or when the reactions resemble the control to which no serum 
 has been added. In reporting 
 upon agglutination tests always 
 state the time at which the test 
 was made and the dilution used. 
 
 A 1 : 20 and a 1 : 40 dilution 
 may be prepared and examined at 
 the end of half an hour. Prompt 
 agglutination is found practically 
 only in t3rphoid fever. 
 
 Dilutions may be conveniently 
 prepared by drawing the serum up 
 to the mark 0.5 in the white cor- 
 puscle pipet, and the distilled water 
 up to the mark 11. Mix well. 
 This gives a dilution of 1 : 20. 
 Gne loopful of this diluted serum 
 and one loopful of bouillon culture 
 of the microorganism to be tested 
 
 give a dilution of 1 : 40. One loopful of the 1 : 20 diluted serum and 
 three loopfuls of the culture give a dilution of 1 : 80. Having mixed the 
 diluted serum and the bacterial suspension on a cover-glass, prepare the 
 cultures on the vaselined concave slides in the usual manner. 
 
 Fig. 80. — A Positive Agglutination 
 (Widal) Reaction in Typhoid Fe- 
 ver. X 430. 
 
 Serum from a patient ill about 
 
 twenty-two days; a 1 : 100 dilution at 
 
 the end of one hour. 
 
 The Microscopic Agglutination Test^ (Dry Method) 
 
 (1) Place a loopful of a twenty-four-hour bouillon culture of Bacillus 
 typhosus in the center of a clean cover-glass. 
 
 (2) Moisten the dried blood which has beeii collected on aluminum 
 foil, glass slide, or paper with a loopful of normal salt solution. (A 
 second and smaller loop may be used for this purpose.) Gently rub up 
 the dried blood and transfer a sufficient amount to the drop of culture 
 
284 AGGLUTININS 
 
 on the cover-glass until, when thoroughly mixed, it presents a delicate 
 orange tint (Fig. 81). Avoid transferring tdo much debris with the 
 solution of blood, especially if the blood has been collected on paper. 
 It is good practice to mix the culture and solution of blood with the 
 cover-glass held over a white surface, in order that the color may readily 
 be observed. 
 
 (3) Having made the mixture on the cover-glass, invert it over a 
 vaselined concave slide, label, and stand aside ior an hour. 
 
 (4) Prepare the culture control in the usufal manner and label. 
 
 (5) Examine at the end of an hour with the 3^ or oil-immersion lens. 
 If minute fragments of fiber, etc., have been transferred, due allow- 
 ance for false agglutination for these should be made. Otherwise the 
 readings are made in exactly the same manner as in the "wet" method. 
 
 (6) Accurate dilutions are not possible with this technic. Satisfac- 
 tory results are dependent largely upon the color; a faint orange tint 
 of the suspension is desirable, and probably represents a dilution of 
 about 1 : 40. This method, however, is very simple, and when care- 
 fully performed, yields results in the practical serum diagnosis of typhoid 
 fever almost as satisfactory as the serum-dilution method. 
 
 It is possible, however, to work with known approximate dilutions 
 by the dried blood method if a good chemical balance is available. 
 Blood must be collected on aluminum foil or glass, and is then scraped 
 off and weighed. To each five milligrams of dried blood 0.5 c.c. of salt 
 solution is added which equals a dilution of' 1 : 25 of whole blood or 
 1 : 100 of dried blood (Wesbrook). After permitting the mixture to 
 stand for half an hour it is centrifuged for a short time. To one drop of 
 the dilution thus obtained one drop of culture is added, which gives a 
 final dilution of about 1 : 50. At the end of an hour it is examined. 
 Higher dilutions can be prepared from this stDck dilution at the will of 
 the operator. 
 
 Macroscopic Agglutination Test 
 
 This is frequently the method of choice in? conducting agglutination 
 tests, especially in investigation and research work, where a high degree 
 of accuracy is desirable. All dilutions and measurements are to be made 
 with accurate volumetric pipcts. 
 
 1 . Place a row of seven small test-tubes (lO x 1 cm.) in a test-tube 
 rack and add 1 c.c. of normal salt solution to each. 
 
 2. Dilute the serum 1 : 5 in the first tube, ^s follows: 0.2 c.c. serum 
 plus 0.8 c.c. salt solution. This now gives in this tube 2 c.c. of a dilution 
 of 1 : 10. Mix well with the pipet. 
 
^' /9 
 
 Fig. 81. — Microscopic Agglutination Test -svith Dried Blood. 
 Shows the proper color of the suspended drop of typhoid culture when the 
 solution of dried blood has been added. The tinge sholild be light nrutigc or yellow, 
 and a shade lighter than ordinary vaselin used in sealing the preparation. 
 
THE AGGLUTINATION REACTION 
 
 285 
 
 ■3. Place 1 c.c. of the serum from tube 1 into tube 2. Mix well, and 
 place 1 c.c. of the mixture from tube 2 into tube 3, and so on. When the 
 sixth tube has been reached, discard 1 c.c, as no serum is to be added to 
 the seventh tube, which is the culture control; i. e., it will contain salt 
 solution plus bacterial emulsion. 
 
 4. Add 1 c.c. of bacterial emulsion to each tube, which doubles the 
 serum dilution in each. Tube 1 now contains a serum in a dilution of 
 
 Fig. 82. — Macroscopic Agglutination Reaction. 
 Serum of a person who had received three injections of typhoid vaccine. This 
 drawing was made twenty-four hours after the test was set up. The dilutions are 
 marked on the tubes. 
 
 1 :20, acting on the bacteria; tube 2, one of 1 :40; tube 3, one of 
 1:80; tube 4, one of 1:160; tube 5, one of 1:320; tube 5, one of 
 1 :640. Tube 7, as just stated, contains the bacterial emulsion in salt 
 solution and is the culture control. 
 
 In determining the agglutination titer of a highly immune serum, 
 these dilutions may be continued to any degree. 
 
 5. On each tube the final dilution is marked with a wax pencil. 
 
|i..-4, 
 
 ^^ 
 
 
 '^~y 
 
 "W 
 
 "^ 
 
 Fig. 83. — MACROSconc Agglutination Reaction, Shows Action of Aggltjtin- 
 oiDS (Pho-aggltjtination). 
 Note absence of agglutmation in dilution 1 : 50; agglutination beginning in 1: 100, 
 and fairly well marked to 1:4000 inclusive. Note uniform cloudiness of control. 
 This reaction was set up with a typhoid immune serum over six months of age; the 
 drawing was made after the tubes had been incubated two hours and placed in a 
 refrigerator overnight. 
 
 286 
 
THE AGGLUTINATION REACTION 
 
 287 
 
 The tubes are then shaken gently, stoppered with cotton plugs, and 
 placed in the incubator at 37° C, for two hours. The tubes are then 
 allowed to remain at room temperature for six hours, or in the re- 
 frigerator for twenty-four hours, after which readings are made. 
 
 6. The culture control should show a uniform cloudiness, with no 
 sediment or flakes, or at most a very slight precipitate that is readily 
 
 Fig. S4. — Agglutinoscope (Altman). 
 The test-tubes are arranged in the rack and vie»ed from below in the mirror. 
 In this manner the smallest deposits are easily seen and compared with the control. 
 
 broken up by gentle agitation, A positive reaction shows masses and 
 clumps of bacteria adhering to the sides and bottom of the tube, which 
 are broken up with some difficulty (Fig. 82): The supernatant fluid 
 should be clear. As dilutions become higher and the amount of con- 
 tained agglutinin correspondingly less, agglutination becomes less and 
 less complete. There is less sediment, and the turbidity of the super- 
 natant fluid is greater, until the negative tube closely resembles the cul- 
 
288 AGGLUTININS 
 
 ture control. A microscopic examination of a deposit will sliow that 
 tiie bacilli point in all directions, whereas in a deposit of unagglutinated 
 bacilli they lie horizontally side by side. 
 
 When agglutinoids are present, agglutination is absent or incomplete 
 in the lower dilutions of serum, and complete iji the tubes containing the 
 higher dilutions. This is called pro-agglutinafion (Fig. 83). 
 
 Readings are facilitated by the use of a special instrument known as 
 the agglutinoscope (Fig. 84). The tubes are: placed in a rack havipg 
 numbered holes, and are viewed from beneath with the aid of a mirror. 
 In this way one looks upward through the column of fluid, and secures a 
 combined view of sediment and turbidity, and when examined with the 
 culture control, fine and accurate readings may be made. 
 
 The method of Kolle and Pfeiffer is very convenient, and may be 
 safer than that of adding live cultures with a pipet. It is conducted as 
 follows : 
 
 1. Make dilutions of serum as described. 
 
 2. Emulsify thoroughly a loopful (2 mg.) of culture from an eighteen- 
 to twenty-four-hours-old agar culture in the, first test-tube, repeating 
 the process in the second tube, and so on through the series. In this 
 method the serum dilutions are not doubled ; thus in the foregoing series 
 the dilutions would be 1 : 10, 1 : 20, 1 : 40, 1 : 80, 1 : 160, 1 : 320. 
 
 3. The tubes are gently shaken, labeled, plugged, and incubated as 
 directed in the preceding method. 
 
 Technic of the Absorption Agglutination Test in Mixed Infection 
 (The Saturation Test of Castellani) 
 
 The practical importance of partial agglutinins is recognized in the 
 diagnosis of mixed infections. Thus the serum of a patient may agglu- 
 tinate typhoid as well as paratyphoid bacilli in dilutions up to 1 : 100. 
 This may indicate one of three possibilities : 
 
 1. The patient may be infected with typhoid, but has formed an 
 exceptionally large quantity of group agglutinins for paratyphoid bacilli. 
 Saturation of this serum with typhoid bacilli will remove all the typhoid 
 and a portion, if not all, of the group agglutinins. Saturation with para- 
 typhoid bacilli will remove the group agglutiiiins, but not the main or 
 typhoid agglutinin. 
 
 2. The patient may be infected with paratyphoid bacilli, but has 
 formed, at the same time, many partial agglutinins for typhoid baciUi. 
 Saturation of the serum with paratyphoid bacilli will remove all the 
 paratyphoid and a large portion of the typhoid agglutinin. 
 
THE AGGLUTINATION EEAOTION 289 
 
 3. The patient may have a mixed infectioh of typhoid and para- 
 typhoid, and therefore agglutinin for both may be present. Saturation 
 of the serum with typhoid bacilli will remove the typhoid and probably 
 a small portion of the paratyphoid agglutinin; After this reaction the 
 serum will still show the presence of a decided quantity of paratyphoid 
 agglutinin. 
 
 In selecting the most likely one of these hj^Dotheses a decision may be 
 reached by adopting the method of Castellani (Citron), which is as fol- 
 lows: 
 
 1. Four rows of test-tubes are arranged, each row being made up of 
 four small tubes each containing 1 c.c. of serum dilutions 1 : 20, 1 : 40, 
 1 : 80, and 1 : 160 respectively. 
 
 2. In each of the tubes of the first and second rows five loopfuls of 
 typhoid bacilli are emulsified. An extra tube containing 1 c.c. of nor- 
 mal salt solution receives a similar amount of bacteria, and serves as the 
 typhoid control. 
 
 3. In each tube of the third and fourth rows five loopfuls of para- 
 typhoid bacilli are emulsified. Arrange the paratyphoid culture control. 
 
 4. Mix gently and incubate for four hours. Carefully record the 
 presence or absence of agglutination in each test-tube. Centrifuge all 
 the tubes excepting the two controls, and transfer the supernatant fluid 
 of each to other test-tubes arranged in the same order. 
 
 5. To each tube of the first and third rowa add five loopfuls of ty- 
 phoid bacilli; to each of the second and fourth rows, five loopfuls of 
 paratyphoid bacilli. Mix well and incubate for four hours. 
 
 (o) If typhoid is present, the agglutination titer in the first part of 
 the test will be strong in the tubes of the first aiid second rows, and weak 
 in those of the second and third rows. In the second part of the test the 
 titer for typhoid will be weak or nil in the first; second, and fourth rows, 
 whereas in the third row it will remain practically the same. 
 
 (b) If paratyphoid exists, the agglutination titer in the first part of 
 the test will be strong in the tubes of the third and fourth rows, and weak 
 in those of the first and second rows. In the second part of the test the 
 titer for paratyphoid will be less or nil in the fourth row, and strong or 
 unchanged in the second row. 
 
 (c) If a mixed infection exists, the agglutination titer in the first part 
 of the test will be strong in the tubes of all four rows. In the second 
 part of the test the titer in the first and fourth rows is much weaker or 
 nil, and in the second and third rows it will remain the same. 
 
 19 
 
290 AGGLUTININS 
 
 TESTS BEFORE TRANSFUSION FOR ISOHEMAGGLUTININS 
 AND ISOHEMOLYSINS 
 
 1. Two or three c.c. of blood are obtains^ from each donor from a 
 vein at the elbow and 0.5 c.c. is placed at once* in a centrifuge tube con- 
 taining 5 c.c. of a 1 per cent, sodium citrate in normal salt solution. 
 The remainder is placed in a small, dry test-tube until coagulation has 
 occurred and the serum has separated. 
 
 2. From the recipient, 3 to 4 c.c. of blood are necessary; 0.5 c.c. is 
 placed in sodium citrate solution, and the remainder is allowed to coagu- 
 late and the serum collected. 
 
 3. The sodium citrate tubes are centrifuged; the supernatant fluid 
 is pipeted off, and the cells are washed again with normal salt solution. 
 After the final washing enough normal salt solution is added to the sedi- 
 nient of cells to bring the total volume up to 5 c.c. 
 
 4. The serum tubes are also centrifuged, so that clear serums are 
 obtained. These should preferably be free from hemoglobin stain. 
 
 5. The following mixtures should be set up within twenty-four hours 
 of the time of collecting blood, in order that native complements may 
 not have undergone deterioration. Measurement may be made ac- 
 cording to a drop from an ordinary 1 c.c. graduated pipet held vertically. 
 Small sterile test-tubes (8 by 1 cm.) are to be used. 
 
 Tube 1 : 4 drops of donor's serum + 1 cirop of recipient's red-cell 
 emulsion. 
 
 Tube 2 : 4 drops of recipient's serum -|- I drop of donor's red-cell 
 emulsion. 
 
 Tube 3: Control: 4 drops of donor's serum -|- 1 drop of donor's 
 red-cell emulsion. Should show no p,gglutination and no hem- 
 olysis. 
 
 Tube 4: Control: 4 drops of recipient's serum -|- 1 drop of re- 
 cipient's red-cell emulsion. Should show no agglutination or 
 hemolj'sis. 
 
 Tube 5 : Control : 1 drop of donor's red-cell emulsion -|- 4 drops 
 of normal salt solution. This serves as a control on the toxicity 
 of the corpuscles and isotonicity of the salt solution. 
 
 Tube 6: Control: 1 drop of recipient's red-cell emulsion -|- 4 
 
 drops of saline solution. 
 
 One cubic centimeter of salt solution is added to each tube, and the 
 
 tubes are gently shaken and placed in the incubator for two hours. 
 
 They are to be inspected every half hour. Agglutination is recognized 
 
TESTS FOR ISOHEMAGGLUTININS AND ISOHEMOLYSINS 291 
 
 macroscopically by the clumping of the red blood-cells into small masses 
 that later sink to the bottom of the tube as a small clot. Hemolysis 
 is likewise easily detected, as corpuscles tencj to become precipitated 
 within two hours. If doubt exists, the finer grades of hemolysis may be 
 detected after the tubes have been allowed to stand overnight in an ice- 
 chest, or at once by thorough centrifugalizatiqn. 
 
 In cases where, for any reason, the quantities of blood previously 
 named cannot be secured, the whole operatioii may be conducted with 
 smaller amounts, using Wright's capillary pipets (Epstein and Otten- 
 barg). This method is as follows: 
 
 Blood is secured from both recipient and donor by pricking the finger 
 of each and allowing five or six drops of blood to flow into 5 c.c. of sodium 
 citrate solution, and collecting a good-sized Wright capsule full for se- 
 curing the serum. The corpuscles are washed twice and enough normal 
 salt solution added to make up approximately a 10 per cent, suspension. 
 The capsules are centrif uged if necessary, filed, opened, and the serum 
 pipeted off. 
 
 The mixtures are prepared in Wright's capillary pipets (see Fig. 48) 
 fitted with rubber nipples. The unit volume= is marked off by a blue 
 pencil on the stem about an inch from the tip. Four volumes of serum, 
 one volume of cell emulsion, and four or five volumes of salt solution 
 are drawn up into the pipet and then mixed gently, running them out on 
 a watch-glass. The entire mixture is then drawn into the barrel of the 
 pipet and the tip sealed. The pipets are carefully labeled, incubated for 
 two hours, and examined for agglutination and hemolysis. 
 
 The results are somewhat more difficult to' read, but the method is 
 of value when many donors are to be examined or when the supplies of 
 serum and corpuscles are limited. 
 
CHAPTER XVII 
 PRECIPITINS 
 
 Closely allied to the agglutinins are antibodies known as precipitins. 
 They act on dissolved albuminous bodies in a manner quite similar to 
 the action of agglutinins upon formed cellular elements. For example: 
 (1) If tjrphoid immune serum is added to a bouillon culture of typhoid 
 bacilli, agglutination occurs; (2) if the culture is filtered and the im- 
 mune serum is added to the clear sterile filtrate, cloudiness appears and 
 finally a precipitate forms. The first is an example of the action of 
 agglutinins upon the formed bacilli, and the "second illustrates the ac- 
 tion of precipitins upon the albumins of dead and dissolved bacilli. 
 
 Precipitins are formed not only for bacterial albumins, but for most 
 any soluble animal (zooprecipitin) and vegetable protein (phytopredpi- 
 tin) as well. If the serum of a rabbit immunized with horse serum is 
 added to horse serum, a precipitate forms, owing to the presence of a 
 specific precipitin in the immune serum. Normal rabbit serum does not 
 possess this power. 
 
 Definition. — The precipitins are specific antibodies that develop in ike 
 serum of animals inoculated vrith bacteria or with solutions of animal or 
 vegetable albumins, which possess the power of producing a precipitate in a 
 clear solution of the particular albumin or culture filtrate against which the 
 animal has been immunized. 
 
 Historic. — Kraus (1897) was the first to study and describe the bac- 
 terial precipitins. He observed that when the serums of animals that 
 have been immunized against cholera, typhoid, or plague are added to a 
 clear filtrate of the respective bouillon cultures of their bacteria, instead 
 of to the bacteria themselves, the clear solutipn becomes turbid and a 
 precipitate forms. 
 
 This reaction was found to be quite specific, i. e., it occurs best with 
 the filtrate of the homologous bacteria, and to a much less extent with 
 closely allied species. For example, the typhoid immune serum does 
 not produce a precipitate with a filtrate of Spirillum cholerae, and simi- 
 larly a cholera immune serum does not produce a precipitate with the 
 filtrate of Bacillus typhosus. Kraus advocated the precipitin reaction 
 
 292 
 
HISTORIC 293 
 
 as a means of identifying and differentiating cei-tain species of bacteria, 
 but the test possesses no advantage for these purposes over agglutina- 
 tion reactions and is not generally employed. 
 
 Tchistovitch was the first to call attention to the non-bacterial pre- 
 cipitins. This observer found that the serum of rabbits inoculated with 
 eel serum, when mixed with a small quantity of the eel serum, caused a 
 precipitate to form. 
 
 About the same time (1899) Bordet found that the serum of rabbits 
 inoculated with the serum of chickens, when mixed with the chicken 
 serum, gave a specific precipitate. A little l&ter Bordet produced an 
 anti-milk immune serum {ladoserum) by inoculating rabbits intraperi- 
 toneally with milk partially sterilized by heating to 6.5° C. When this 
 immune serum was mixed with the homologQUS milk, small particles 
 appeared, which gradually formed larger flakes and sank to the bottom 
 of the fluid. It was found that the lactoserums were specific — i. e., 
 cow lactoserum would precipitate only cow casein, human serum only 
 human casein, etc. 
 
 Wladimiroff was the first to use the bacterial precipitin reaction as a 
 practical diagnostic test. He showed that the serum of a horse suffering 
 from glanders would, when added to a clear filtrate of a culture of Bacil- 
 lus mallei, produce a precipitate. The technic of these reactions is, 
 however, more diflacult than with the agglutination tests, and as the 
 reactions are usually not more delicate or more advantageous than the 
 latter, they are seldom employed. 
 
 Pollowing Wladimiroff, Uhlenhuth and Wassermann made a very 
 important practical demonstration of the value of serum precipitins in 
 differentiating the blood and secretions of man and animals. For ex- 
 ample, the serum of rabbits immunized with various bloods would react 
 with, solutions of old and dried specimens of tlftjir respective bloods, and 
 although "group" precipitins were found present in the tests with the 
 blood of closely allied species, yet the value of the reaction was not 
 impaired to any extent when a proper technic, with correct dilutions, was 
 employed. These discoveries were found to possess considerable value 
 in forensic medicine, particularly in the recognition of the source of 
 blood-stains. 
 
 Nuttall, in a thorough and painstaking research with the blood from 
 500 animals, was able to study the "blood relationship" of various ani- 
 mals as based upon group precipitins. For example, the serum of a 
 rabbit immunized with human blood will react best with human serum, 
 then with the serums of the higher apes, and finally with the lower orders 
 
294 PRECIPITINS 
 
 of monkeys. Similar reactions were found to occur among the lower 
 animals. 
 
 Nomenclattire. — The antibody in an immune serum responsible for 
 the phenomenon of precipitation is called precipitin; the substance or 
 antigen responsible for the production of this antibody is known as the 
 precipitinogen; the precipitate is the end-product of the reaction between 
 precipitinogen and precipitin. Just as toxoids and agglutinoids may be 
 formed, so precipitin may be modified to precipiioid. 
 
 Although, since bacterial precipitins are produced by the protein 
 constituents of bacteria, the custom of differentiating between bacterial 
 and protein precipitins is superfluous, nevertheless, when the mean- 
 ing is clearly understood, the term bacterial precipitin is convenient 
 and may be employed. 
 
 The precipitins derive their names from their precipitinogens, as, 
 for example, a precipitin produced by injecting rabbits with ox serum 
 is designated anti-ox precipitin. 
 
 Normal Precipitins. — Although agglutinins may be found in normal 
 serum, it is decidedly uncommon to find normal precipitins. Extracts 
 of organs have been known to contain norrpal precipitins for certain 
 albumins, although at the same time they were absent from the serum 
 of the animal. In this case the active bodies, exist in the cells as "ses- 
 sile receptors," and by the process of extraction they are brought into 
 solution. During immunization these same reteptors are stimulated to 
 overproduction and are thrown into the circulation as free precipitin 
 receptors. 
 
 Immime precipitins are antibodies produced by immunization with 
 a foreign albumin, either during the course of a bacterial infection or as 
 the result of artificial inoculation. 
 
 Structure and Properties of Precipitins. — According to the side-chain 
 theory, precipitins are antibodies or receptors of the second order, com- 
 posed of a combining arm or haptophore group for the precipitinogen, 
 and a zymophore or precipitinophore group that precipitates the antigen. 
 Their structure is, therefore, seen to be quite similar to that of agglu- 
 tinin, the difference being largely due to the different functions of the 
 zymophore group. 
 
 The properties of precipitins are quite similar to those of agglutinins. 
 They are fairly resistant bodies, resist the effect of drying for prolonged 
 periods, but are gradually destroyed by heating to 60° to 70° C. When 
 inactivated by exposure or heat, they cannot be reactivated by the addi- 
 tion of fresh normal serum, and therefore they bear no relation to the 
 
FORMATION OF PRECIPITINS 295 
 
 complements. As with agglutinins, the presence of a salt is necessary 
 to secure the reaction. 
 
 The haptophore or combining arm is quite stable; the precipito- 
 phore group is more labile, and is affected by heat, and when this less 
 resistant arm is lost, the receptor is called a precipitoid. Like agghitin- 
 oids, the precipitoids are of practical interest from the fact that their 
 haptophore arm will not only combine with precipitinogen, but displays 
 a greater activity in this direction than the whole receptor or precipi- 
 tin itself, and when union between precipitinogen and precipitoid has 
 occurred, precipitation does not result. Hence in low dilutions of a 
 precipitin serum the phenomenon of precipitation is slight or altogether 
 absent, whereas in higher dilutions the reaction becomes evident. 
 
 Group precipitins are not so prominent as group agglutinins, yet they 
 are formed to a certain degree and are of much practical importance in 
 attempting to differentiate bacteria and serums by the precipitation 
 method. Although precipitins are highly specific, the principle of serum 
 dilution, as emphasized imder Agglutination, must be closely observed 
 in order to dilute the group precipitins to such small amounts as to pre- 
 vent them from interfering with the chief precipitin. This principle is of 
 particular importance in differentiating the bloods of various animals, 
 and especially in medicolegal cases, where the precipitin reactions are 
 employed for the diagnosis of blood-stains. 
 
 Formation of Precipitins. — Immune serums for diagnostic purposes 
 are produced by injecting the precipitinogenous fluid into the veins, 
 peritoneal cavity, or subcutaneous tissues of animals, usually rabbits. 
 The power of forming precipitins is probably disseminated among the 
 organs and general body tissues. Kraus and Levaditi assign the leu- 
 kocytes as the chief source of precipitin formation. 
 
 As in the case of agglutinin formation, not ail animals possess equally 
 the power of forming a precipitin for a given albumin. While this point 
 is of general interest with the bacterioprecipitins, it becomes of panticular 
 importance in relation to serum precipitins. For example, an animal 
 will not form a precipitin active against its own serum. If formed, it 
 would be an autoprecipitin, or isoprecipitin, and, as a rule, animals do 
 not form antibodies for their own tissue constituents. Furthermore, 
 animals are unlikely to form precipitins for the proteins of other members 
 of the same species, or if precipitins are produced, they are usually the 
 result of prolonged immunization of a number of animals. Precipitin 
 formation is also slight for the proteins of other animals that are closely 
 related either zoologically or biologically. For example, attempts at 
 
296 PRECIPITINS 
 
 immunization of a guinea-pig with the serum of a rabbit, a pigeon with 
 that of a hen, or a monkey with human serum, are procedures that do not 
 usually yield good precipitating serums. 
 
 Attempts have been made to produce qntipreci'pitin by effecting 
 immunization with immune precipitating serums. Such attempts have 
 been reported as partially successful with serum and milk, but not with 
 bacterial precipitins. Antiprecipitins possess* no practical value. 
 
 Mechanism of Precipitation. — Of the various theories advanced to 
 explain the phenomenon of precipitation, none has received so much 
 support experimentally as that advanced by Bordet in explanation of 
 agglutination. 
 
 Colloids may be precipitated by salts, and probably the salts so alter 
 the electric state of colloidal particles that their surface tension is de- 
 creased, and, as a result of this change, neighboring particles coalesce 
 in such quantities as to produce a visible precipitate. Salts are likewise 
 necessary for serum precipitation, and there is a close analogy between 
 serum and colloidal precipitation. 
 
 The origin of the precipitate formed during the reaction is of interest. 
 When a very potent inunune serum is employed, the precipitinogen is so 
 highly diluted that it no longer gives any of the chemical reactions for 
 proteins, but when the precipitating serum is added, it may yield, never- 
 theless, a heavy precipitate. The precipitate can, therefore, hardly be 
 regarded as due to the slight trace of albumin in the precipitinogen, and, 
 furthermore, if the precipitating serum is diluted, the precipitate be- 
 comes smaller and smaller, and, if the dilution is increased, it finally 
 disappears altogether. For this reason the precipitate is generally 
 considered as originating in the immune serum. 
 
 Specificity of Precipitins. — Precipitins react but feebly on closely 
 related albumins of the same species, but are specific against those of 
 unrelated species. In other words, the precipitation test merely deter- 
 mines the animal species from which the proteid originates, but cannot 
 demonstrate positively whether it comes from the blood, the semen, 
 milk, or other albuminous body. For medic9legal purposes, therefore, 
 a diagnosis of "human blood-stain" cannot be made without chemical 
 evidence to prove that the stain actually consists of blood. 
 
 An immune serum prepared by the injection of the serum of a certain 
 animal gives a precipitate also with the juices of the various organs of 
 that animal. The only exception to this rule is the protein of the crystal- 
 line lens of the eye, which gives no precipitate with the antiblood im- 
 mune serum. The same albumin exists in the crystalline lenses of all 
 
EOLE OF PRECIPITINS IN IMMUNITY 297 
 
 animals, — from fishes to man, — and a serum produced by immunization 
 with lens substance will react with the protein derived from the lens of 
 any animal, but with no other animal proteid. This theory of species 
 specificity may, however, be carried a little further, for by carefully 
 freeing the organs from all blood and then using organic extracts for 
 inoculation, it is possible to produce serums that will yield a precipitate 
 best with the particular variety of organic extract (liver, kidney, etc.), 
 and a weak or no precipitate with extracts of other organs of the same 
 animal. 
 
 Besides this animal specificity, precipitin reactions also demonstrate 
 the "constitutional specificity" of proteins. If, instead of using a pure 
 animal or plant albumin for immunization, variously altered albumins 
 are used (heated albumins, acid albumin, formaldehyd albumin, and the 
 like), the organism reacts by producing antibodies of a characteristic 
 nature, differing from those developed after inoculation with pure al- 
 bumin. For example, if a rabbit is immunized with normal horse serum, 
 the resulting immune serum will produce a precipitate when added to 
 pure horse serum, but not when added to horse serum that has been 
 heated. On the other hand, if a rabbit is inoculated with horse serum 
 that has been diluted and boiled for a short time, the resulting immune 
 serum will react not only with normal horse serum, but also with heated 
 serum and a group of its decomposition products with which the normal 
 immune serum ordinarily never produces a precipitate. 
 
 This observation is of practical importance in detecting meat sub- 
 stitution by precipitin reactions. In order to render the detection dif- 
 ficult, the meat is commonly boiled; with the aid of precipitins produced 
 by immunization with heated proteins, this fraud is more easily detected 
 than if a normal immune serum were used. 
 
 Obermeyer and Pick have demonstrated that while animal specificity 
 is not destroyed when the albumins are modified by heat, tryptic di- 
 gestion, or oxidation, their specificity is lost when an iodin, nitro- or 
 diazo-group is inserted into the protein molecule. Immunization with 
 such transformed proteins, e. g., xanthoprotein, can produce a precipi- 
 tating serum that will react with every xanthoprotein, even that of 
 different animals. These investigators conclude that species specificity 
 is probably dependent upon a certain aromatic group of the protein 
 molecule. 
 
 Role of Precipitins in Immunity. — Precipitins are probably not truly 
 protective antibodies, like antitoxin and the lysins, but they are quite 
 similar to the agglutinins in being secondaiy products of cellular activ- 
 
298 PRECIPITINS 
 
 ity, and they are of value chiefly as indicators of this general antibody 
 formation. They may, however, he concerned in preparing their anti- 
 gens for destruction and solution, just as opsonins prepare cells for pha- 
 gocytosis, but precipitins themselves possess no appreciable curative or 
 protective virtues, and are of value chiefly in diagnostic procedures. 
 
 PRACTICAL APPLICATi6nS 
 Bacterial Precipitins 
 
 Bacterial precipitins have no clinical diagnostic value. Their re- 
 actions have no advantage over the agglutination test, they are more 
 difficult of execution than the latter, the sources of error are greater. 
 
 In scientific research they may be of value in differentiating micro- 
 organisms from closely allied species, but even here agglutination 
 reactions serve the purpose equally well and are less difficult of 
 execution. 
 
 Occasionally bacterial precipitins are of service in demonstrating 
 the presence of soluble bacterial substances within exudates or organic 
 fluids. For example, Vincent and Bellot recommend the reaction as 
 being of considerable value in the diagnosis of cerebrospinal meningitis. 
 The cerebrospinal fluid is centrifugalized until it is clear; 2 c.c. of this 
 clear fluid is then placed in one tube and 4 c.c. into another. One-tenth 
 cubic centimeter of a standard antimeningococcic serum is added to 
 each tube. The tubes are kept for from twelve to fifteen hours at 37° C. 
 In the presence of cerebrospinal meningitis a precipitate forms. If the 
 fluid is normal or if it was derived from some= other form of meningitis, 
 no cloudiness results. This reaction is said to occur within the first 
 twenty-four hours of the illness and to persist until the twelfth to the 
 twentieth day. 
 
 Similar reactions have been advocated in the diagnosis of other in- 
 fections, particularly syphilis. Fornet believed that the presence of 
 typhoid antigen (precipitinogen) ought to be capable of demonstration 
 in the blood-serum of typhoid fever patients long before antibodies 
 themselves are in evidence. One cubic centimeter of potent immune 
 serum in concentrated and diluted form (1 : 5 and 1 : 10 with normal salt 
 solution) is placed into small test-tubes, anc| an equal amount of the 
 serum for examination, also in concentrated and similar dilutions, is 
 carefully floated on top of the immune serum. Control tests with nor- 
 mal and immune serum and normal with unknown serum are made. 
 The mixtures are allowed to stand undisturbed at room temperature for 
 
PRACTICAL APPLICATIONS 299 
 
 two hours, and if the reaction is positive, a whitish ring makes its appear- 
 ance at the point of contact of the two serunJs, the controls remaining 
 negative. According to Citron, this ring test is, also evident In the pres- 
 ence of scarlet fever, measles, and syphilis. 
 
 Floccule-forming Reactions 
 
 Fomet Ring Test. — Owing to the wonderful activity that has marked 
 the research work of syphilis several precipitation tests for diagnostic 
 purposes were devised. These have all been oi^ershadowed and forsaken 
 for the Wassermann complement-fixation test. Fornet applied his ring 
 test, using the serum of patients with manifest luetic symptoms as the 
 precipitinogen, and the serum of paretics as the precipitating or immune 
 serum. Klausner advocated a simple test consisting of mixing in a small 
 test-tube 0.2 c.c. of fresh, active, and absolutely clear serum, with 0.6 
 c.c. of distilled water. This serum and the control mixtures are allowed 
 to stand at room temperature for from seven, to fifteen hours, when a 
 thick, floceulent precipitate of fibrin globulin will appear at the bottom 
 of the tube. 
 
 Porges-Meier Reaction. — Porges and Meier observed that luetic 
 serums are capable of producing floceulent precipitates from solutions 
 of lecithin and similar salts. Two-tenths of a cubic centimeter of a 1 
 per cent, solution of Merck's sodium glycochplate in distilled water is 
 placed in narrow test-tubes, and an equal amount of the patient's 
 serum, which must be absolutely clear and inactivated by heating at 
 56° C. for thirty minutes, is added. This miAure and the known nor- 
 mal and luetic controls are kept at room temperature for from eighteen 
 to twenty-four hours. A positive reaction is marked by the appearance 
 of distinct coarse floccuh, mere turbidity or faint precipitation being 
 regarded as negative. 
 
 Herman-Perutz Reaction. — More recently Herman and Perutz have 
 devised a similar test requiring the following two solutions: Solution 1 
 (stock solution, diluted 1:20 with distilled water before use) consists of: 
 Sodium glycocholate, 2 gm., cholesterol, 0.4 gm.; 95 per cent, alcohol, 
 100 c.c. Solution 2 (freshly prepared before use) is a 2 per cent, solu- 
 tion of sodium glycocholate in distilled water. The test is performed by 
 adding to 0.4 c.c. of clear inactive serum (heated at 56° C. for half an 
 hour) in a small test-tube 0.2 c.c. of solution 1 and 0.2 c.c. of solution 2. 
 The tubes are tightly plugged with cotton and* set aside at room tem- 
 perature for twenty-four hours, after which the presence or absence of 
 precipitation is noted. It is well in this test, a^ in all inmiunologic re- 
 
300 PRECIPITINS 
 
 actions," to prepare controls with known normal and luetic serums and 
 with distilled water. 
 
 None of these reactions has been found specific, and none has been 
 generally adopted, the far greater accuracy of the Wassermann reaction 
 having made this method more valuable. 
 
 Noguchi Butyric-acid Test. — Noguchi has devised a very useful 
 test for the detection of an increased amount of protein, particularly 
 globulin, in cerebrospinal and other body-fluids. In my experience this 
 test has proved of particular value in establishing the differential diag- 
 nosis between serous and tuberculous meningitis, being negative in the 
 former and positive in the latter, whereas in both the fluid may be clear, 
 the cytology may be indefinite, and tubercle bacilli may escape detection. 
 Serous meningitis is not a true infection, but a reflex vasomotor disturb- 
 ance of the cerebral vessels, causing an outpouring of serum that leads 
 to various pressure symptoms closely resembling those of a true meningi- 
 tis. This condition is particularly common during childhood, and the 
 general symptoms, the increased pressure of the cerebrospinal fluid, and 
 its clear, watery character, are features that resemble those of tubercu- 
 lous meningitis. It is just in such cases — and they are frequent — that 
 I have found this protein reaction of considerable value. A positive 
 reaction practically always means a true meningitis; a negative reaction 
 usually means "serous meningitis," with a milch better prognosis if the 
 underlying cause is corrected. 
 
 Noguchi has found the test positive in about 90 per cent, of cases of 
 general paralysis and in 60 per cent, of cases of locomotor ataxia or cere- 
 bral or spinal syphilis. In the diagnosis of syphilis the' Wassermann 
 reaction with cerebrospinal fluid has greater value than the protein re- 
 action. However, the best results in diagnosis arc usually secured by a 
 Wassermann test, but3Tic-acid test, and total and differential cell-counts. 
 In a case where the diagnosis rests between tpberculous meningitis and 
 syphilis, a positive butyric-acid test and a negative Wassermann reaction 
 would decide in favor of the former. 
 
 The test is extremely simple. Into a small, thin-walled test-tube 
 place 0.2 c.c. of cerebrospinal fluid (which must be clear and free from 
 blood) ; add 1 c.c. of a 10 per cent, solution of butyric acid in normal salt 
 solution; heat over a low flame and boil for a short period. Then add 
 quickly 0.2 c.c. of a normal solution of sodium hydroxid and boil once 
 more for a few seconds. The presence of an increased content of protein 
 is indicated by the appearance of a granular or fiocculent precipitate, 
 
PRACTICAL APPLICATIONS 
 
 301 
 
 which gradually settles to the bottom of the tube, under a clear super- 
 natant fluid (Fig. 85). 
 
 The velocity and intensity of the reaction vary with the quantity of 
 the protein contained in a given specimen. The granular precipitate 
 appears within a few minutes in a specimen containing a considerable 
 increase in protein, whereas one hour may be required to obtain a dis- 
 tinct reaction in specimens weaker in protein. In obtaining the reaction, 
 the time period should not be greater than two hours. A faint opales- 
 cence without the formation of a distinct -precipitate is to he regarded as 
 mthin the limits of the normal. 
 
 Fig. 85. — The Nocuchi Butyric-acid Test for Globulins. 
 The tube on the extreme left shows the formation of flocculi within a few minutes 
 after adding NaOH; the middle tube shows a strongly positive reaction after stand- 
 ing several hours (supernatant fluid quite clear) ; the tube on the extreme right shows 
 a very slight opalescence, but no flocculi (withui the limits of normal). 
 
 Proteusi Precipitins 
 
 The protein precipitins have a larger range of value and represent 
 one of the most important practical aids in forensic medicine. As men- 
 tioned elsewhere, a precipitating immune serum reacts only, or at least 
 best, with its homologous serum. The precipitin reaction is, therefore, 
 highly specific, and offers a method whereby proteins can be easily and 
 definitely determined — a problem that could not be solved by chemistry. 
 
 By means of lactoserums various milks and cheeses may be recognized 
 and their source determined. 
 
302 
 
 PRECIPITINS 
 
 By using appropriate immune KeruniK seminal fluids and stains may 
 be detected, and in medicolfgal cases, where the question is one of rape, 
 this reaction possesses considerable value in differentiating seminal from 
 leukorrheal stains. 
 
 The precipitin reaction is likewise of great value in medicolegal cases 
 in determining the source of blood-stains, the original application and 
 technic having been largely worked out by Wassei-rjiann, Hchutze, Uh- 
 lenhuth, and Weidanz. Thus in a case whe're, for example, a bloody 
 towel is found in the possession of a man charged with murder, tiie prose- 
 cution may see in this a proof of crime, whereag the defendant may claim 
 that the stains are those of dog's blood. Microsco^jic and chemical 
 t(;sts may show that the stains are blood-stains, but they cannot deter- 
 mine their source. The blood-stained towel is placed in water or salt 
 solution, and a portion of the extract is mixed with the serum of a raVjbit 
 immunized against human serum, and another: portion with the serum of , 
 a^abbit immunized against dog's serum. If the first mixture shows a 
 precijMtate, thr; stain was made by blood frorp a human bfdng; if, on 
 the other hand, this mixture remains clear and the second shows a pre- 
 cipitate, this is strongly indicative of the presence of dog's blood. 
 
 This method has also cleared up a number of scientific problems, 
 especially that of showing the blood relatiorifikip of man and thr; lower 
 animals. Just as a group agglutination demonstrates the close relation- 
 ship existing between various bacteria, so, als«j, serum preT^ipitins prove 
 that a distinct relationship exists iK-tween the different speeies of animals. 
 
 For example, an antihuman serum in low dilution will precipitate the 
 serum of monkeys. The differentiation ?jetween iiuman and monkey 
 serum can be accomplished, however, Vjy imnjunizing the monkey with 
 human serum, when a precipitin is formed that* reacts with human serum 
 alone, an isoprecipitin, or one active against the monkey's own serum, 
 not being developed as a general rule. 
 
 The precipitin tests are likewise of value in food inspection, as, for 
 instance, to determine the nature of meats. For example, in order to 
 detect the presence of dog or horse flesh in sausage, extracts of the sausage 
 are made and tested with anti-dog and anti-horse serum, the presence of 
 precipitates indicating strongly the presence of the meat of thr^se animals. 
 With an appropriate technic even salted and cOoked fiesi] may ije rt^eog- 
 nized, although when the meat has been cooked it is necessary to prepare 
 immune serums by immunizing rabbits with extracts of cooker! meats. 
 
 In this connection it may be staterl that specific organic reactions 
 have been secured \>y various investigators \>y, prolongf-d immunization 
 
Fig. S6. — Hbmin Crystals. 
 
 Prepared after the method described m the text. From a stain (over two months 
 
 nlrli of sheep blood on ;i towel. 
 
TECHNIC OF PRECIPITIN EE\CTI0NS 303 
 
 of rabbits with certain organ extracts. Thus it is possible to differen- 
 tiate between the Hver and kidney of the same animals; such tests have, 
 however, but limited practical value. Maragliano attempted to apply 
 this test of organic specificity to the serodiagnosis of malignant tumors 
 by preparing immune serums by the injection of tumor juices, securing a 
 serum that yielded a precipitate with the albumins of a cancerous tumor. 
 The test is not absolutely specific, and its practical value in diagnosis 
 requires confirmation. 
 
 TECHNIC OF PRECIPITIN REACTIONS 
 Differentiation of Human and Animal Blood — Biologic Blood Test 
 
 Unless a stain is definitely known to be a blood-stain it is necessary 
 to establish its identity by making a chemical teSt before proceeding with 
 the precipitin reactions. For example, old stains upon clothing may be 
 due to substances other than blood, such as coffee and fruit-juices. 
 Blood-stains upon clothing, metal, wood, or glass may be used for making 
 these reactions and their source determined. 
 
 To identify the stain as one of blood, a portion may be taken into 
 solution in distilled water, rendered slightly acid with dilute acetic acid, 
 filtered until clear, and examined spectroscopically. Or the Teichmann 
 hemin crystal test may be applied to the stain by transferring to a clean 
 slide a small amount of material scraped from tKe stain; add a few small 
 crystals of sodium chlorid, crush the crystals, apd mix the powder with 
 the dry material. Place a clean cover-glass over the stained material 
 and run a small amount of glacial acetic acid under the cover-glass. 
 Heat the preparation to just about the boiling-point for a minute, re- 
 plenishing the acid as may be necessary. The fluid turns brown. 
 The specimen is allowed to cool a few minutes, and is then examined 
 microscopically for the presence of brown rhombic crystals of hemin 
 (Fig. 86). It may be necessary to reheat the specimen several times 
 before the crystals are obtained. 
 
 Having shown that a given stain is actually a blood-stain, the source 
 of the blood may be determined as the result of the precipitin reaction, 
 which consists in extracting the stain in normal salt solution and mixing 
 with antiserums prepared by immunizing rabbits with human and various 
 animal serums. Since the antiserums are known, a precipitate with any 
 one of the extracts indicates that the blood in the stain was derived from 
 the same species of animal. The reaction is based upon the principle of 
 the specificity of antigen and its antibody. Here the antibody is known, 
 and is used in the test to detect the antigen. 
 
304 PEECIPITINS 
 
 As mentioned elsewhere, because of the presence of group precipitins 
 the reaction is fraught with certain technical difficulties of importance, 
 especially in medicolegal cases. In most instances it may suffice to show 
 that a stain is of human blood, as will be indipated by a strong reaction 
 with human blood and negative reactions with the bloods of lower ani- 
 mals. If the reaction is negative with antihunian serum, the antiserums 
 of the domestic animals, such as that of the dqg, cat, hog, ox, horse, etc., 
 are tried. Although the blood of the higher apes, and even of the lower 
 orders of monkeys, may react slightly with human blood, this factor may 
 be determined by observing a proper technic, of dilution, or the possi- 
 bility of a given stain being one of monkey blood being definitely ruled 
 out. 
 
 The reaction can be obtained from blood in an advanced state of 
 putrefaction, or from a stain that has been dried for a year or more. 
 Tests with mummies, however, have reacted negatively, and stains or 
 clots altered by heat may not react unless the antiserum has been pre- 
 pared with a similar antigen. 
 
 This same technic may be employed for the recognition of seminal 
 stains, especially in cases of rape. The staiij is taken into solution in 
 exactly the same maimer as the blood-stain, and tested with an anti- 
 human semen serum prepared by immunizing rabbits with human semen. 
 
 Preparation of the Extract of Stain (the Precipitinogen). — If the 
 stain is on clothing, a portion three inches square should be carefully 
 torn into shreds with forceps and scissors, and not with the fingers, 
 and placed in 40 c.c. of normal salt solution. If the stain is upon wood, 
 glass, or metal, the staining substance should be carefully scraped off 
 with a knife and placed in the salt solution. As a further control on the 
 technic an unstained portion of the clothing should be extracted in the 
 same manner, in order to show that the latter alone does not give the 
 reaction. The mixtures must not be shaken, but should be stood aside 
 for from two to twenty-four hours, dependmg ; upon the rapidity of ex- 
 traction. The extract should preferably not be stronger than 1 : 1000. 
 The strength may be estimated by removing; 1 c.c. of the extract into 
 another tube, diluting with from 10 to 20 c.c. of normal salt solution, and 
 gently shaking. If a persistent froth appears upon the surface of the 
 fluid, sufficient extraction has occurred. Place 2 c.c. in a test-tube, heat 
 to boiling, and add a drop of a 25 per cent, solution of nitric acid. A 
 faint opalescence indicates that the strength of the extract is about 1 : 1000 
 (Fig. 87). If a heavy precipitate forms, the amount of normal salt solu- 
 tion that must be added to a portion of the extract to reduce it to a 
 
■ I 
 
 t 'i 
 
 Fio. 87.— Precipitin Reaction. Prepahation of Extract of Blood-stain. 
 The tube on the left shows the color of an extract of a blood-stain; the middle 
 tube shows tliis extract so diluted as to vicli 1 a faint albumin reaction with nitric acid ; 
 the tube on the right shows the foam tes^t with the same diluted extract (about 1 ;1000). 
 
TECHNIC OF PRECIPITIN REACTIONS 
 
 305 
 
 dilution of 1 : 1000 should be determined. The extract should be almost 
 colorless by transmitted light, and must be crystal clear ; this may be ac- 
 complished by filtering it through a clean sterile Berkefeld filter. It is 
 highly desirable that the extract be sterile, as- the reaction may require 
 
 -■^■- 
 
 Fig. 88. — An Uhlenhuth Filter. 
 Serum is poured into the glass cylinder surrounding the "candle"; a vacuum is 
 produced by the water-pump; the filtrate is collected in the small bottle attached by 
 rubber tubing to the graduated cylinder at the lower portion of the apparatus. This 
 filter is especially adapted for filtering small amounts of serum or other fluid. 
 
 several hours, and cloudiness due to bacterial growth may interfere with 
 the result. 
 
 The Immxme Serum. — A highly potent, sterile, and absolutely crys- 
 tal-clear serum immune against the protein to be recognized must be 
 prepared. The method of preparation is given in the chapter on Active 
 Immunization of Animals. For the recognition of blood-stains it is not 
 20 
 
306 
 
 PRECIPITINS 
 
 necessary that whole blood be injected, as the serum alone will suffice. 
 It is better to inject a number of rabbits with each serum and to give all 
 injections intravenously. From the third injection on, preliminary 
 titrations are made according to the technic to be described later, as 
 many animals succumb after a large number of injections have been 
 given them. 
 
 The serum must be absolutely clear. Animals should be bled after 
 a period of fasting, as the opalescence of the serum following feeding 
 cannot be removed by filtration and will interfere with the reaction. 
 Precipitin immune serum should be collected with a scrupulous aseptic 
 technic, and stored in ampules holding 1 c.c. Although it is best not to 
 add a preservative, the addition of 0.1 c.c. of a 1 per cent, solution of 
 
 Fig. 89. — Test-tube Rack for Precipitin and Agglutination Reactions. 
 The strips of black material in the rear of the tubes faoiUtate reading the reactions. 
 
 phenol to each cubic centimeter of serum does not render the fluid cloudy 
 and aids greatly in its preservation. 
 
 If, after long standing, a precipitate has become deposited in an anti- 
 serum, this should not be shaken up, but the ampule should be carefully 
 opened and the clear supernatant serum drawn off with a capillary pipet. 
 A serum that has become cloudy may be cleared partially or entirely by 
 filtering it through a small candle filter, although even an infected and 
 offensive sermn will give the reaction (Fig. 88). 
 
 Apparatus. — Long and narrow test-tubes, 10 cm. by 0.8 cm., are 
 used. These must be absolutely clean, and preferably sterile. 
 
 The test-tube rack devised by Uhlenhuth^ in which the tubes hang 
 suspended in beveled holes, is quite satisfactory*. Where a test is carried 
 out with many controls, a rack similar to the <Jne shown in the illustra- 
 tion (Fig. 89) is quite serviceable. A strip of black material placed be- 
 
TECHNIC or PRECIPITIN REACTIONS 
 
 307 
 
 hind the tubes aids greatly in the detection of the finer degrees of opal- 
 escence or precipitation. 
 
 Preliminary Titrations. — The precipitin content of an immune serum 
 is titrated frequently during the process of immunization and after the 
 animal has been bled. For medicolegal purposes, Uhlenhuth advises 
 the use of only highly valent serums. He considers an antiserum ef- 
 
 FiQ. 90. — Titration of a Precipitin (Sebum). 
 Not all tubes of the series are here shown. Note the well-marked precipitate in 
 the bottom of the first two tubes (1 : 100 and 1 : 500); the third tube (1 : 1000) 
 shows less precipitate; the fourth tube (1 : 2000) is negative (clear and no precipi- 
 tate). The titer of this serum was recorded as 1 : 1000. 
 
 ficient if 0.1 c.c. of it, when added to its respective serum-antigen 
 diluted 1 : 1000, produces a distinct turbidity, either at once or in from 
 one to five minutes at the latest. 
 
 Into a series of six test-tubes place 2.0 c.c. of the following dilutions 
 of serum-antigen, prepared with normal salt solution: 1:100, 1:500, 
 1:1000, 1:2000, 1:5000, and 1:10,000. To each tube add 0.1 c.c. of 
 
308 PRECIPITINS 
 
 clear iinmune serum. The tubes must not be shaken. Within from one 
 to five minutes a faint, misty cloud appears a^t the bottom of the tubes 
 reacting positively, and this becomes a distinct precipitate within one- 
 half to one hour (Fig. 90). 
 
 Before performing the actual test with tt|,e unknown blood-stain it 
 is advisable to test the entire reaction with a similar known blood-stain 
 in order to make sure that all ingredients are in good working order. 
 In laboratories equipped for medicolegal examinations stains upon linen, 
 as by the blood of man, dog, cat, ox, horse, etc., and their respective 
 antiserums are always kept in readiness for making the preliminary and 
 actual tests. 
 
 Technic of the Test. — The following mixtures are set up in a series of 
 
 test-tubes. Fresh sterile pipets should be used in handling the various 
 
 solutions. The immune serum should be added slowly, and in such a 
 
 way that it will flow down the side of the tube and collect at the bottom. 
 
 Tube 1 : 2 c.c. of extract of unknown substance in dilution of 
 
 1:1000+0.1 c.c. of immune serum. 
 Tube 2: 2 c.c. of the unknown extract in dilution of 1 : 5000-|-0.1 
 
 c.c. of immune serum. 
 Tube 3: 2 c.c. of the unknown extract in dilution of 1 : 10,000-|-0.1 
 
 c.c. of immune serum. 
 Tube 4: 2 c.c. of the unknown extract in (Silution of 1 : 100-|-0.1 c.c. 
 
 of normal rabbit serum (control). 
 Tube 5: 2 c.c. of a 1: 1000 dilution of the=serum of that species of 
 animal whose blood is suspected to be present in the unknown 
 solution+0.1 c.c. of immune serum (control). 
 Tube 6: 2 c.c. of the extract of unknown substance alone (control). 
 Tube 7: 0.1 c.c. of the immune serum -1-21 c.c. of normal salt solu- 
 tion (control). 
 Tube 8: 2 c.c. of the extract of the unstained portion of clothing -|- 
 0.1 c.c. of immune serum (control). 
 The tubes are not shaken, are kept at ropm temperature, and the 
 results are read after from ten to twenty minutes. Exposure to incu- 
 bator temperature facilitates the reaction. Wi.th proper immune serums, 
 and especially in medicolegal work, a positiye reaction should appear 
 within two to five minutes as a faint, misty cloud at the bottom of the 
 test-tube. Within five minutes this becomes more definite, and in from 
 ten to twenty minutes the precipitate is seeB (Fig. 91). Any cloudi- 
 ness that develops later than twenty minutes after the beginning of the 
 reaction has no significance. 
 
TECHNIC OF PBECIPITIN REA<:;TI0NS 
 
 309 
 
 In tests other than those employed in medicolegal work, especially 
 if the antiserums are weaker than desired, the reaction may be read after 
 one to two hours. 
 
 In the foregoing test, if positive results are obtained in tubes 1 , 2, or 
 
 Fig. 91. — A Precipitin Reaction — Biologic Blood Test. 
 
 This reaction was set up with an extract of a stain of sheep blood on a towel, 
 over three months of age, with an anti-sheep immune serum. 
 
 The first tube, containing a 1 : 1000 dilution of the blood (extreme left), shows a 
 well-marked precipitate; in the second (1 : 2000) the reaction is also well marked; 
 the third (1 : .5000) shows no precipitate; the fourth contains a 1 : 1000 dilution of 
 blood-stain, with normal rabbit serum as a control, and shows no precipitate; the 
 fifth is the positive control with known sheep blood extract and immune serum ; the 
 last tube is No. 8 of the series, and contains an extract of the non-bloody portion of 
 the towel with immune serum; no precipitate. 
 
 3 and tube 5, and all the others react negatively, the presence of the blood 
 or protein of the species suspected in the unknown extract is established. 
 If the entire test proves negative, the species" to which the unknown 
 specimen belongs must be determined with new' antiserums prepared for 
 each species, and the tests conducted in the manner described. 
 
310 PKECIPITINS 
 
 Partial reactions between closely related species due to group pre- 
 cipitins seldom occur, and are easily detected when the technic described 
 is employed. The precipitin test, as determined by the extensive ex- 
 perience of Nuttall, is highly specific, and it is;only between very closely 
 related animals, such as the hare and the rabbit, the horse and the 
 mule, the sheep and the goat, etc., that any doubt can arise. 
 
 When only very limited amounts of the unknown stain are available, 
 the test, according to Hauser, can be carried out in slender, clean, and 
 sterile capillary tubes. The piece of stained clothing is torn into shreda, 
 extracted, and filtered until clear. The tests are performed by drawing 
 a small amount of the unknown solution into aleapillary tube, and under- 
 lying this with a small amount of immune serum. As many controls 
 as possible are put up in the same manner. A distinct whitish ring will 
 form in the positive tubes at the line of contapt between the two fluids; 
 this is best seen by holding the tube against a black background. 
 
 Detection of Meat Adulteration 
 
 The principle of this method is the same as that in the foregoing test. 
 An extract of a meat will yield a precipitate when mixed with its anti- 
 serum, prepared by immunizing rabbits with, an extract of the flesh or 
 with the blood-serum of some other animal. 
 
 The method is especially serviceable in food inspection for the de- 
 tection of horse, dog, or other foreign flesh in meat mixtures, such as 
 sausage and the like. Even salted and cooked meats may be used in the 
 test, although the latter may require the use of antiserums prepared by 
 immunizing with heated or cooked antigen. 
 
 Preparation of the Meat Extract. — To prepare this, about 50 grams 
 of flesh are removed from the deeper parts of the specimen by means of a 
 sterile knife, and through a fresh opening, as this portion has been least 
 exposed to the methods of preservation, especially at the high tempera- 
 tures to which the meat may have been exposed. It should contain as 
 little fat as possible. It is then placed on a clean sterile tile and cut into 
 smaller pieces, and finally minced by passing it through a perfectly clean 
 meat-grinder or chopping it with a sterile chopping knife. After being 
 finely minced the meat is placed in a sterile Erlenmeyer flask, and cov- 
 ered with 100 c.c. of sterile normal salt solution. The mixture of meat 
 and salt solution is kept for about six hoiirs: at room temperature, or 
 overnight in the refrigerator, the flask being gently shaken from time to 
 time. 
 
 Salted meat should be ground and freshened by placing it in a large 
 
TECHNIC OF PRECIPITIN REACTIONS 311 
 
 sterile Erlenmeyer flask and covering it with sterile distilled water, re- 
 newed several times in the course of fifteen minutes without shaking the 
 flask. 
 
 Since the presence of a great deal of fat interferes with the reaction, 
 it is advisable to remove it beforehand by extracting it with ether and 
 chloroform for from twelve to twenty-four hours (Miessner and Herbst) . 
 Pork sausages are usually quite fatty, and may require this preliminary 
 treatment. To make the extraction, take about 75 to 100 grams of the 
 minced meat or sausage and place it in a large Erlenmeyer flask and cover 
 with equal parts of ether and chloroform. After twenty hours the ether 
 and chloroform are poured off, the meat is washed once or twice with 
 sterile normal salt solution, and then extracted in 100 c.c. of salt solu- 
 tion, as described elsewhere. 
 
 To determine whether a sufficient quantity of protein substances has 
 passed into solution place 2 c.c. in a test-tube and shake vigorously. If 
 a foam develops and persists for some time, the extraction may be said 
 to be complete. It must then be filtered until it becomes perfectly clear. 
 With extracts of fresh lean meat this is usually accomplished by filtering 
 through a hard filter-paper moistened with salt solution. If it is not 
 crystal clear, and especially if the meat to be examined is fat or salt, it 
 may be necessary to filter through a sterile Berkefeld filter. 
 
 To make the test the extract should contain about one part of pro- 
 tein in 500 parts of salt solution. To determine this, 2 c.c. of the clear 
 filtrate are placed in a test-tube and heated, &= drop of dilute nitric acid 
 being added. If a marked cloudiness and a flocculent precipitate de- 
 velops, the extract is too concentrated and must be diluted with clear 
 normal salt solution until the heat and acid test causes only a diffuse, 
 opalescent cloudiness that settles at the bottom of the tube after five 
 minutes as a slight precipitate. 
 
 Before proceeding with the experiment the reaction of the solution 
 should be tested with litmus paper, and if it is found to be acid, it should 
 be neutralized very carefully with ^ sodium hydroxid. 
 
 Extracts of the meats that are known or likejy to be present, such as 
 extracts of pork and beef, should be prepared as controls. 
 
 Preparation of Immxme Senim. — An imnjune serum against that 
 variety of flesh that is to be determined in the unknown specimen is 
 prepared by injecting rabbits intravenously with the sermn of an ani- 
 mal of that species. For example, if the object is to test for dog meat, 
 an anti-dog serum is prepared by immunizing rabbits with sterile dog 
 serum. 
 
312 PRECIPITINS 
 
 As has repeatedly been mentioned, it is advisable to immunize a 
 number of rabbits at the same time, for only a small number will yield a 
 satisfactory serum after the third injection. 
 
 Immunization may be performed with extracts of flesh that have been 
 filtered and heated at 56° C. for an hour to secure partial sterilization. 
 Such injections, when given subcutaneously, are likely to produce ex- 
 tensive sloughing, and with any method of immunization the mortality 
 is high. 
 
 After the third inoculation it is well to remove a small amount of 
 blood from the ear and make a preliminary titration. This is performed 
 in exactly the same manner as in making the forensic blood test pre- 
 viously described. An antiserum is satisfactory if 0.1 c.c. produces a 
 well-marked cloudiness and a precipitate in ten minutes with 2 c.c. of a 
 1 :-1000 dilution of the serum or extract of flesh. 
 
 In addition to being highly potent, the immune serum must be crystal 
 clear and sterile. To avoid opalescence the animal should be bled only 
 after a period of fasting. 
 
 Technic. — If, for example, the object is to determine whether a 
 piece of meat is horse flesh or, if sausage, contains the meat of this ani- 
 mal the test is conducted as follows: 
 
 Tube 1: 2 c.c. of unknown extract, 1 : 5004-0.1 c.c. of antihorse 
 
 serum. 
 Tube 2: 2 c.c. of unknown extract, 1 : lOOO+O.l c.c of antihorse 
 
 serum. 
 Tube 3: 2 c.c. of unknown extract, 1 :5000-[-0.1 c.c. of antihorse 
 
 serum. „ 
 
 Tube 4: 2 c.c. of horse flesh extract, 1 : 1000-|-0.1 c.c. of antihorse 
 
 serum (positive control). 
 Tube 5: 2 c.c. of unknown extract, 1:500+0.1 c.c. of normal 
 
 rabbit serum. 
 Tube 6: 2 c.c. of pork extract, 1 : 500+0.1 c.c. of antihorse serum. 
 Tube 7: 2 c.c. of beef extract, 1 : 500+0.1 c.c. of antihorse serum.. 
 Tube 8: 2 c.c. of unknown extract. 
 
 Tube 9: 2 c.c. of sterile salt solution+0,1 c.c. of antihorse serum. 
 The immune serum is added to each tube very carefuUy and run 
 down the sides of the tube, rather than dropped into them. The tubes 
 should not be shaken. 
 
 If the preliminary titration of the immune serum fulfils the ideal 
 requirement of yielding a well-marked cloudiness within five to ten 
 minutes with a 1 : 1000 extract, the foregoing test should be recorded at 
 
TECHNIC OF PRECIPITIN REACTION 313 
 
 the end of half an hour at room temperature. If in tubes 1, 2, 4, and 
 possibly 3 a misty cloudiness should appear within five minutes, the 
 extract is very probably one of horse flesh. If a definite precipitate 
 forms within thirty minutes, the other tubes remaining clear, horse 
 flesh or the flesh of some other single-toed animal is present. 
 
 If the preliminary titration does not show a precipitate with the 
 immune serum until at the end of one or two hours, this interval may be 
 utiUzed for conducting the test. 
 
 In a similar manner tests may be made for the meat of dogs, cats, 
 or any other animals if the respective immune .serums are used with the 
 extract. 
 
 Bacterial PREcrpixnMS 
 
 As has previously been stated, these precipitins have slight diagnostic 
 significance, as the information they yield in the diagnosis of an infec- 
 tion or in the differentiation of bacterial species may be gained much 
 more easily with the agglutinin test. 
 
 Bacterial precipitinogens are prepared by filtering ten to twenty-one 
 day bouillon cultures through Berkefeld filters. The filtrates must be 
 absolutely clear and sterile, for the reaction frequently requires a num- 
 ber of hours, and if bacteria arc present, they may grow quickly, produce 
 turbidity, and mask a reaction. 
 
 Immune Serum. — This is prepared according to the technic described 
 under Active Immunization. Rabbits are given intravenous injections 
 of increasing doses of cultures of the bacteria themselves or of filtrates, 
 the inoculum being heated at 60° C. for an hour previous to making the 
 injection. After the third dose the serum is titrated and the injections 
 continued unless the serum is satisfactoiy. 
 
 Technic. — ^A known quantity of precipitinogen and varying amounts 
 of immune serum are employed. If too much precipitinogen is fur- 
 nished, the precipitate will not form, and one that has already formed 
 niay dissolve on the addition of more precipitinogen. 
 
 If, for example, one desires to determine if typhoid precipitin is 
 present in a given serum, the test is conducted" as follows: 
 
 Tube 1: 2 c.c. of typhoid bouillon filtrate+0.05 c.c. of unknown 
 
 serum-|-0.9 c.c. of normal salt solution 
 Tube 2: 2 c.c. of tj^pfioid bouillon filtrate+0.1 c.c. of unknown 
 
 serum-f 0.9 c.c. of normal salt solution. 
 Tube 3: 2 c.c. of typhoid bouillon filtrate+0.5 c.c. of unknown 
 serum-f-0.5 c.c. of normal salt solution. 
 
314 PRECIPITINS 
 
 Tube 4: 2 c.c. of typhoid bouillon filtrate 4-1 c.c. of unknown serum. 
 
 Tube 5: 2 c.c. of typhoid bouillon filtrate+1 c.c. of typhoid im- 
 mune serum (positive control). 
 
 Tube 6: 2 c.c. of typhoid bouillon filtrate+1 c.c. of normal salt 
 solution. 
 
 Tube 7: 2 c.c. of typhoid bouillon filtrate+1 c.c. of normal serum. 
 
 Tube 8: 1 c.c. of serum+1 c.c. of normal salt solution. 
 The tubes are not shaken, and are kept at room temperature for 
 from one to six hours. If the unknown serum contains considerable 
 typhoid precipitins, a positive reaction will be noticed in the first four 
 tubes in a short time — often within from ten to fifteen minutes. Tube 
 5 should show a strong reaction and the other tubes should remain clear. 
 In studying the biologic relationship of an organism to others of the 
 same group its immune serum may be used in amounts of 1 c.c. of varj^- 
 ing dilutions, as 1:50, 1:100, 1:500, 1:1000, 1:2000, 1:4000, and 
 so on, with a constant dose of 1 or 2 c.c. of the bouillon filtrates of the 
 various organisms studied. A comparison of the precipitates in the 
 re.spective dilutions of the different filtrates indicates the relationship, 
 according to the amount of group precipitins present in the immune 
 serum. 
 
 PRECIPITIN TEST IN CANCER 
 
 Freund and Kaminer have devised a pretapitin test to be used in 
 conjunction with their cytolytic reaction in the diagnosis of cancer. 
 The extract of cancer-cells is prepared by treating fresh tissue or tissues 
 preserved in alcohol, finely minced with 10 volumes of 0.6 per cent, acid 
 sodium phosphate solution. After agitating the mixture the cells are 
 allowed to settle, and the extract is decanted off and preserved in an ice- 
 chest, a few crystals of thymol being added as a preservative. For use 
 add 5 c.c. of a 5 per cent, acetic acid solutioli to 100 c.c. of the fluid; 
 heat the mixture in a water-bath for fifteen minutes at 80° C, and filter. 
 Cool, and neutralize to litmus with sodium carbonate. Heat again 
 as previously directed, cool, and filter. The extract may now be tested 
 undiluted, and diluted 10, 50, and 100 times with 10 drops of known 
 normal and cancerous serums, the latter yielding a precipitate that is 
 plainly visible in test-tubes held against the light. The extract keeps 
 for only a few days. 
 
 The dilution of extract that yields a precipitate with known can- 
 cerous serum, but not with a normal serum, is used in testing unknown 
 serums. Place 10 drops of the patient's serum in a small test-tube and 
 
PRECIPITIN TEST IN CANCER 315 
 
 add 2 c.c. of the properly diluted extract. Controls should be made up 
 with normal serums, and also the extract with a fluid corresponding to 
 that with which the tissue extract was prepared; that is, by adding to 
 100 c.c. of a 1 per cent, solution of acid sodium phosphate 5 c.c. of 5 per 
 cent, acetic acid and neutralizing with sodium' Carbonate. 
 
 The precipitate forms at once, and must be viewed by transmitted 
 and not by reflected light. The practical value of the test, however, 
 has not been established. 
 
CHAPTER XVIII" 
 CYTOLYSINS 
 
 Amboceptors and Complements 
 
 The cytolysins include a number of antibodies of considerable diag- 
 nostic and therapeutic importance, for example, the hemolysins and the 
 bacteriolysins. It will be remembered that the various antibodies act 
 differently upon their antigens, and that, according to the side-chain 
 theory, as their antigens become more highly organized, their structure 
 becomes more complicated. For example, the molecule of a soluble 
 toxin may be considered as simple in structure, and accordingly its 
 antibody has been conceived as being likewise pimple, and composed of a 
 plain cast-off receptor or side-arm that unites directly with the toxin 
 and neutraUzes it without further aid. Antitoxins and antiferments 
 are antibodies of this nature. For more highly organized antigens, 
 however, so simple an antibody will not suffice; and we now find a more 
 complicated antibody, composed of a portion that unites with the anti- 
 gen and another portion, an integral part of the antibody, that exerts a 
 special selective action upon the antigen, and either neutralizes its activ- 
 ity or prepares it for ultimate destruction. To this class of antibodies 
 belong the agglutinins and precipitins, which agglutinate or precipitate 
 their antigens preparatory, in a sense, to their* final disintegration. For 
 still more complex antigens nature has provided special ferments, al- 
 ways present in varying proportions in the blood, which, when united 
 with the antigen, cause its disintegration and solution in a manner simi- 
 lar to the process of digestion as it takes place in the intestinal canal. 
 These ferments are, however, powerless unless, united with the antigens, 
 and here we find that the antibody serves as the connecting link, bind- 
 ing antigen with ferment, which results in a fb'rm of digestion and final 
 lysis or solution. The antibody is, thereforej simple in structure, and 
 is composed of two binding or grasping arms — one for the antigen and 
 one for a ferment. This interbody, or amboceptor, is specific for the anti- 
 gen, and will act only and specifically with this antigen. It is important 
 to remember that the ferment or complement is not an integral part of 
 the antibody, but is free in the blood-stream; that the antibody is only 
 a connecting link, but preserves its importance by being specific for its 
 
 316 
 
AMBOCEPTORS AND COMPLEMENTS 317 
 
 antigen; that the primary function of this antibody is to unite antigen 
 and complement, and that the latter then causes the lysis or solution of 
 the antigen. The connecting link or antibody of this nature is known 
 as an antibody or receptor of the third order. 
 
 Different cells produce their own and specific interbodies or ambo- 
 ceptors. Thus bacteria or vegetable cells, blood-corpusoles, and va- 
 rious other cells, such as ciliated epithelium, spermatozoa, renal epi- 
 thelium, etc., when present in the form of an infection or when injected 
 into an animal, generate different and specific amboceptors, which bring- 
 about their solution by binding them with a- ferment or complement. 
 One ferment or complement does not serve for all; there are various 
 ferments, which act with the different amboceptors, but all ha,ve proper- 
 ties so nearly alike that many believe, with Bordet, that but a single 
 complement exists. 
 
 Definition. — This special digestive and lytic process is known to 
 occur with cells, and hence the antibodies capable of bringing about this 
 action are called cytolysins, or substances that cause lysis or solution of 
 the various cells that may be their antigens. 
 
 It is well to remember that, according to Ehrlich, the three orders of 
 antibodies each have their counterpart, both ill structure and in effect, 
 in the receptors serving for the normal nutrition of cells. For the sim- 
 plest molecule of food that is in solution the celL is provided with a simple 
 receptor for union with the molecule, which is then directly assimilated. 
 This receptor is similar to an antitoxin, or an antibody of the first order, 
 which destroys its toxui directly and without further ado. More 
 complex food material must first undergo some preparation by the cell 
 before it can be assimilated, and accordingly we find receptors provided 
 with a more complex structure which have their counterpart in the anti- 
 bodies of the second order, or those possessing a special toxic portion 
 that agglutinates or precipitates their antigen or prepares it for phagocy- 
 tosis. It is possible that with physiologic substances this is all that the 
 cell requires of its receptor, but so far as is known, it would appear that 
 for antibodies this action does not in itself injure the antigen, but is 
 rather one step toward preparation for its further destruction. Or- 
 ganized and complex food substances must be^ digested before assimila- 
 tion can occur, and here we find that the receptor acts as a link in binding 
 the food molecule to a ferment, with resultingldissolution and assimila- 
 tion of the products of solution. These are called receptors of the third 
 order, and have their countei-part in similar antibodies, — the cytoly- 
 sms, — ^which act as links or interbodies between antigen and a comple- 
 
318 
 
 CYTOLYSINS 
 
 ment, the latter being entirely free and separate, and independent of 
 the receptor or antibody (interbody) itself (Fig. 92). 
 
 Varieties of Cytolysins. — The cytolysins produced by bacteria are 
 known as baderiolysins, i. e., antibodies producing disintegration and 
 lysis of bacteria. The cytolysins known as hemolysins cause lysis or 
 hemolysis of the erythrocjrtes. Similar cj^olysins may be formed for 
 practically all cells, such as leukocytes, epithelium, liver, kidney, spleen, 
 etc., and to these the general name cytotoxin/h&s been given; thus we 
 have leukotoxin, hepatotoxin, nephrotoxin, neurotoxin, etc., these 
 
 Fig. 92. — Theoretic Formation of Cytolysins. 
 
 terms being more nearly correct and expressive* of the actual mechanism 
 by which their action is produced. 
 
 Nomenclature. — In no other field of immunity have so many differ- 
 ent names been applied to the same substances as have been applied to 
 this order of antibodies. This confusion of terms, added to the various 
 interpretations placed upon their significance,, has rendered the subject 
 incomprehensible to those not specially interested. 
 
 The ferment-like and thermolabile substances present in all serums 
 and actively concerned in lytic processes have been given the name of 
 alexin by Bordet; Mctchnikoff called it cytasc; and Ehrlich designated it 
 as complement because in the conception of- the side-chain theory it 
 
AMBOCEPTORS 319 
 
 completes the reaction after being linlied witlj the antigen. The term 
 "alexin" was first applied by Buchner to the germicidal substance found 
 in normal serum. We now know that Buchner was working with both 
 amboceptor and complement, although Bordet was the first to discover 
 the former, Buchner having been unconsciously most interested in the 
 thermolabile complement. 
 
 To the antibody itself the term substance sensibilisatrice has been 
 applied by Bordet, for he believes that this antibody sensitizes or pre- 
 pares the cell for the action of the alexin or complement. The following 
 names have been applied to the antibody by various observers: fixateur, 
 by Metchnikoff; preparator, by Miiller; and amboceptor, interbody, and 
 immune body by Ehrlich. Of these, the term "amboceptor" is in most 
 general use, signifying a two-armed body that unites antigen on the one 
 hand, with a complement on the other. 
 
 When using the term amboceptor, care should be used to designate 
 its specific character; thus, for example, a hemolytic amboceptor and a 
 bacteriolytic amboceptor mean respectively a hemolysin and a bacterio- 
 lysin. 
 
 It is common practice to designate an amboceptor according to the 
 cell for which it has a special affinity; thus antisheep amboceptor or 
 hemolysin means an amboceptor for sheep cells, the prefix "anti" being 
 affixed because it is destructive for those cells. 
 
 AMBOCEPTORS 
 
 Although antitoxins have received considerable study from a thera- 
 peutic standpoint, probably no order of antibodies has been given more 
 attention than the cytolysins have received, not only because of their 
 vast therapeutic possibilities, but also from their value as an aid to diag- 
 nosis. The hemolysins especially have been utilized in making the 
 Wassermann test for syphiHs and similar reactions, the very nature of the 
 phenomenon offering a visible and fascinating method of study. 
 
 Since the general structure, formation, and Action of the various am- 
 boceptors, such as the bacteriolysins, hemolysins, and other cytolysuis, 
 are essentially similar, the general character of amboceptors may be here 
 considered, a study of the special characteristics of each being reserved 
 for subsequent chapters on the more important members of the group. 
 
 Historic. — The alexins or complements werfe first discovered through 
 the researches of Nuttall and Buchner in 1889. The amboceptors were, 
 of course, present in the various serums with which these observers were 
 
320 CYTOLYSINS 
 
 working, but it was not until 1895 that Bordet showed quite clearly that 
 two substances were concerned in the phenomena of bacteriolysis and 
 hemolysis. At this time he demonstrated that the alexin or comple- 
 ment may be removed from a serum by heatirig it to 55° to 66° C, and 
 that it may be reactivated by the addition of fresh serum from another 
 animal; that an old bacteriolytic serum cannot produce bacteriolysis 
 unless it is reactivated by a fresh normal serum or is placed in the peri- 
 toneal cavity of a living animal, from which it may derive the thermola- 
 bile alexin. In other words, the amboceptors in these serums with- 
 stood the effects of heating and age, but were unable to produce lysis 
 without the aid of an alexin furnished by a fresh normal serum. 
 
 Structure of Amboceptors. — ^According to ,the theory of Ehrlich, an 
 amboceptor is but a simple interbody furnished with two haptophore or 
 grasping portions. One haptophore group attaches the antibody to its 
 antigen, whatever that may happen to be-^bacterium, erythrocyte, 
 epithelial cell, etc., while the other attaches a suitable complement 
 (Fig. 92) . The first is called the cytophile or antigentophile group, and 
 the second, the complementophile group, ifie amboceptor is specific 
 in the sense that it will unite only with its antigen or other very closely 
 related body. For example, when a rabbit is injected with sheep cor- 
 puscles an amboceptor is formed that will unite only with sheep, and 
 not with human, dog, ox, or other cells. 
 
 As will be shown further on, Ehrlich believes that many different 
 complements may be present in a serum, whereas Bordet believes 
 that one complement exists that will act with the amboceptor, whether 
 this is bacteriolysin or hemolysin. This view-is based mainly upon the 
 observation that the complement in a serum may be absorbed out by 
 furnishing an excess of either bacteriolytic or hemolytic amboceptors, 
 the one variety of amboceptor removing all the complement for the 
 other. Although the results of experimental work would seem to indi- 
 cate that Ehrlich's belief in the plurality of complements is correct, and 
 while this view is quite generally held, conclusive proof regarding this 
 has not as yet been furnished. An amboceptor may have more than 
 one complementophile group, and may bind a. number of different com- 
 plements simultaneously {polyceptor) (Fig. 93). Ehrlich and Morgen- 
 roth called attention to this possibility when they stated: "Finally, it 
 is possible that an immune body, besides one particular cytophile group, 
 contains two, three, or more complementophile groups." Later Ehrlich 
 and Marshall showed that, in order to get a specific lytic effect, it was not 
 necessary for all complements to become active, but that only a few 
 
AMBOCEPTORS 321 
 
 are necessary in any single instance to bring alsout effect. These com- 
 plements are termed "dominant complements," the remainder being 
 known as "non-dominant complements." 
 
 Whether amboceptors can undergo degenerative changes and lose 
 their cytophile or complementophile groups and become arnhoceptoids, 
 just as toxoids and agglutinoids are formed, is still doubtful. Reasoning 
 from analogy to the toxins and agglutinins, it is probable that ambocep- 
 toids may be produced by a loss of the complementophile group, the 
 cytophile portion of all antibodies being more- stable; such ambocep- 
 toids, by uniting with their antigens, may effectually block the action 
 of an amboceptor, just as agglutinoids prevent agglutination. 
 
 General Properties of Amboceptors. — Amboceptors are fairly re- 
 sistant bodies, withstanding to a well-marked degree the effects of heat, 
 acids, alkalis, exposure, and drying. A 
 hemolytic serum, for instance, may be pre- 
 served in a sterile condition for many 
 months and show but slight deterioration 
 in its activity. Such a serum may be dried 
 in vacuo or on suitable filter-paper, and 
 preserve its activity for remarkable inter- Fig. 9.3.— Theoretic Struc- 
 
 .,,,,,.,, , , , TURE OF A POLTCEPTOR. 
 
 vals of time, with but slight and gradual ^^ ^.^^^ ^^^-^^ ^^ ^^. 
 
 deterioration. While a temperature of 55° boceptor in combination with 
 
 ^ .,,.,. . , ... a cell; C, dominant comple- 
 
 L. will inactivate complement m from ment; c, lesser complements, 
 fifteen to thirty minutes, amboceptors can 
 
 tolerate from 60° to 65° C. for an hour and show but slight depreciation 
 in activity. 
 
 Mechanism of the Action of Amboceptors. — An amboceptor or anti- 
 body of the third order is believed to act as a simple chemical interbody 
 between antigen and complement — i. e., it is a connecting link between 
 the two. Complement is, therefore, the active agent in lytic processes, 
 but is practically powerless to act upon the aptigen until brought into 
 chemical union through the intervention of an amboceptor. Comple- 
 ment is present in normal serums, and its quafitity cannot be increased 
 by immunization. Amboceptors are specific for their antigen, may be 
 present in small amounts in a normal serum,, but may be greatly in- 
 creased during infection or as the result of artificial immunization. A 
 hemolytic amboceptor for sheep coi-puscles is specific for these, and will 
 not unite with the corpuscles of any other animal. A bacterioljd;ic 
 amboceptor as for Bacillus typhosus will unite only with those bacilli, 
 
 and to a lesser extent with closely related bacilli, such as the paratyphoids 
 21 
 
322 CYTOLYSINS 
 
 but not with other bacterial species or other cells. As to the specificity 
 of complement, opinions differ. Ehrlich believes that there are many 
 complements, as, e. g., one for a particular hemolytic amboceptor, an- 
 other for a bacteriolytic amboceptor, and so, on through the extensive 
 list of known amboceptors. Bordet, on the other hand, believes that 
 there is but one complement, which will act with any amboceptor. 
 
 Bordet's conception of the mechanism of this order of antibodies is 
 also different from that of Ehrlich. Both agree that the antibody pre- 
 pares the cell for the lytic action of a complement, but Ehrlich holds 
 that the antibody acts as a simple link between antigen and comple- 
 ment, whereas Bordet believes that the antibody acts as a mordant or 
 dye, penetrating its antigen, and sensitizing or preparing it for the action 
 of the complement. For this reason Bordet calls these antibodies 
 "sensitizers," or substances sensibilisatrice. The term "sensitizing" is 
 now in general use, and is employed especially in hemolytic tests when 
 corpuscles or bacteria are mixed with their ahaboceptors to effect their 
 union. It is expressive and satisfactory, and may be used without 
 necessarily subscribing to Bordet's view. 
 
 Metchnikoff believes that amboceptor or his "fixator" is found in 
 the leukocytes. He asserts that the amount of fixator or amboceptor 
 produced is proportional to the amount of phagocytosis and phagolysis 
 that occur during the absorption of the antigen. Metchnikoff considers 
 the fixators as analogous to enterokinase, and he believes that, like the 
 latter, the fixator acts as an accessory digestive ferment, having for its 
 object the linking of the more potent ferment, as a cytase or comple- 
 naent, to the cell or molecule to be digested. 
 
 Formation of Amboceptors. — ^While experimental data are at hand 
 to show that amboceptors may be produced by local tissues, it is entirely 
 probable that in wide-spread infection or as the result of artificial im- 
 munization there is general cellular activity with extensive antibody 
 formation. The spleen and hematopoietic tissues in general and the 
 mononuclear leukocytes are regarded by mdny as being particularly 
 active in the formation of hemolysins and bacteriolysins (Pfeiffer and 
 Marx, Deutsch, Wassermann) . 
 
 As has been stated, Metchnikoff believes that antibodies of the class 
 under consideration are the products of the le.ukocjrtes, thus tending to 
 preserve the importance of the phagocytic theory. While there is little 
 doubt that the various leukocytes, endothelial cells, and other phag- 
 ocytic cells are sources of amboceptor production, there is no reason for 
 accepting the belief that their formation is confined strictly to these cells. 
 
AMBOCEPTORS 323 
 
 Specifitity of Amboceptors. — It has been stated elsewhere in this 
 volume that amboceptors are highly specific bpdies. This specificity is 
 not, however, absolute, for just as group agglutinins are produced by 
 one bacterium for closely allied species, so in like mamier experimental 
 investigation by Ehrlich and Morgenroth, von.Dungern, and others has 
 shown that immunization of an animal with the erythrocytes of another 
 animal would produce one chief hemolysin for- these cells and a second- 
 ary hemolysin for the cells of another animal. For example, on innxiuniz- 
 ing a rabbit with ox blood a hemolytic serum was obtained that was 
 hemolytic not only for goat blood, but also for ox blood. These second- 
 ary amboceptors are known as group or partial immune bodies. Their 
 production may readily be understood when it' is remembered that the 
 body-cells are conceived as being provided with various side-arms for 
 many different blood-ceUs, bacteria, etc. Now, if the erythrocytes of 
 the goat possess receptors not only for the particular goat-blood side- 
 arms of the body-cells, but also for the ox-blood side-arms, both sets of 
 side-arms will be attacked and consequently two amboceptors are 
 formed — one, the main one, for goat corpuscles, and a secondary one 
 for ox corpuscles. Ehrhch and Morgenroth, therefore, claim that the 
 immune body of a hemol3i;ic serum is composed of the sum of the partial 
 immune bodies, which correspond to the individual receptors used to 
 confer the immunity. Since the cells of various animals of the same and 
 of different species vary in the number and variety of side-arms or re- 
 ceptors, which are not present in another, the different combining group 
 possessed by a blood-cell or a bacterium will not, therefore, find fitting 
 receptors in every animal, and thus there may be a different varietj' of 
 partial immune bodies in two animals. This! would lead to the possi- 
 bility of the occurrence of antibodies for the same blood-cell or bacterium, 
 differing from one another in the partial immjme bodies of which they 
 are composed, depending on the variety of the animals used in preparing 
 the serum. 
 
 This view is directly opposed to that of Metchnikoff and Besredka, 
 who beheve that a certain immune body is al^aj's the same, no matter 
 what species of animal was used in preparing, the serum. As will be 
 pointed out further on, in addition to theoiietic interest, the subject 
 possesses great practical importance, for, as is well known, most cura- 
 tive serums are best prepared with many different strains of a particu- 
 lar microorganism because of certain differences in their antigenic prop- 
 erties, and if, in addition, the value of a bacteriolytic serum depends 
 upon the sum-total of the immune bodies, it m,ay be advisable to secure 
 
324 CYTOLYSINS 
 
 as many of these as possible by preparing the serum from various animals 
 of the same and of different species. 
 
 It will be understood, therefore, that the specific action of antibodies 
 of this order is not limited to the cells used in the immunizing process, 
 but extends to other cells that have receptors in common with these, 
 a condition that is analogous to group agglutinins and precipitins for 
 closely allied cells and bacteria or dissolved albumins. 
 
 Natural or Native Amboceptors 
 
 Difference Between a Normal and a Specific Immune Serum. — Just 
 as small and varying amounts of native agglutinins and antitoxins may 
 be found in normal serums, so, in like manner, various native bacterio- 
 lytic and hemolytic amboceptors may be found. According to the side- 
 chain theory, these various amboceptors are normally attached to body- 
 cells, hence it is probable that a few are being continually swept off into 
 the blood-stream. In some instances the amo-imt of a natural ambocep- 
 tor may be quite high; thus, for example, many human serums contain 
 relatively large amounts of antisheep hemolytic amboceptor. These 
 natural amboceptors will be considered mor^ fully in the chapters on 
 Hemolysins and Bacteriolysins. 
 
 The difference between a normal and an immune serum lies in the 
 fact that the normal serum contains a number of amboceptors in small 
 amounts, whereas the immune serum contains a greatly increased 
 amount of at least one amboceptor for a particular cell. As has been 
 shown by numerous investigators, this difference is not due to the 
 complements, as these are not increased during the process of immuni- 
 zation. Since the presence of an amboceptor cannot be demonstrated 
 unless complement is present, in testing a serum for an amboceptor we 
 must furnish sufficient complement to bring out the maximum activity 
 of the amboceptor. If the serum of a rabbit before and after immuniza- 
 tion is titrated with sheep erythrocytes, it may be found that the im- 
 mune serum contains from a hundred to many thousand times the 
 normal quantity of antisheep amboceptor. 
 
 These facts bear a further practical relation to the treatment of 
 infectious diseases with bacteriolytic serums. Ordinarily, when we in- 
 ject an immune serum we furnish but one bactericidal substance, namely, 
 the bacteriolytic amboceptor, and no complement at all. If the pa- 
 tient's complement is decreased or at least insufficient to activate the 
 amboceptor furnished, lysis will not occur, andaccordingly an increased 
 therapeutic effect may be secured by the injection simultaneously of 
 
AMBOCEPTOHS 325 
 
 an immune serum and a fresh normal serum. This procedure presents 
 certain diiEcultics, and the subject is considerefd more fully in the chap- 
 ter on Passive Immunization. 
 
 Anti-amboceptors. — Just as anti-agglutinins and antiprecipitinfg 
 may be formed, so anti-amboceptors may be produced experimentally 
 by immunizing an animal with an amboceptor-laden serum. An anti- 
 amboceptor is specific for the amboceptor that, caused its production, 
 and when these are mixed, the activity of the amboceptor is impeded by 
 the anti-amboceptor, which unites with its cytophilic group. It is 
 possible that old erythrocytes are destroyed by an autohemolysin present 
 in the blood-stream under normal conditions^ and that a physiologic 
 equilibrium is maintained through the production of an anti-amboceptor. 
 
 QuANTrrATrvE Estimation of Amboceptors 
 Titration of a Hemolytic Amboceptor. — The quantity of amboceptor 
 in a given amount of serum may be determined by titration. If, for 
 instance, a rabbit is injected with sheep corpuscles, the amount of hemo- 
 lytic amboceptor for these cells in the rabbit sferum may be determined 
 by the following method of titration: To a series of test-tubes increasing 
 amounts of the rabbit immune serum (heated to remove complement) 
 are added, mth a constant dose of sheep-cells and a constant dose of 
 fresh guinea-pig serum to furnish complement. After a suitable period 
 of incubation the tube showing complete hemolysis would contain suf- 
 ficient hemolytic amboceptor, i. e., just one unit. The value of the 
 immune serum may then be expressed by stating that so much serum, 
 as, e. g., 0.001 c.c, contains one unit of amboceptor. Of course, if the 
 amounts of complement or corpuscles are varied the unit will likewise 
 vary. In order to establish or measure the content of hemolytic am- 
 boceptor a constant amount of corpuscles ar),d complement must be 
 used. The details of this amboceptor titration, are given in the chapter 
 on Hemolysins. 
 
 Titration of a Bacteriol3rtic Amboceptor. — A bacteriolytic amboceptor 
 may be measured in much the same manner as a hemolytic amboceptor, 
 although less accurately, because of technical ddflaculties. If a standard 
 and fixed dose of an emulsion of living bacteria and a fixed dose of com- 
 plement are mixed with varying amounts of inactivated immune serum 
 containing amboceptors for these bacteria, th^ amount of amboceptors 
 present may be determined by plating the mixture and estimating the 
 number of bacteria that have been killed. Or we may use fixed doses of 
 immune serum and complement with varying amounts of bacteria and 
 
326 CYTOLYSINS 
 
 obtain an approximate estimate of the content in bacteriolytic ambo- 
 ceptors after the same manner. 
 
 The technic of these tests is given in the chapter on Bacteriolysins. 
 
 COMPLEMENTS 
 
 Historic. — ^As early as 1876 Landois described the hemolytic action 
 of fresh blood-serum upon the blood-corpusples of animals of certain 
 species. Traube and others observed that animals could withstand the 
 injections of relatively large amounts of septic material, but it was not 
 until 1886-90 that Fodor, Nuttall, Buchner, and others fully established 
 the bactericidal properties of fresh blood-serum. Buchner demon- 
 strated the fact that the active principle causing bacteriolysis or hem- 
 olysis is very labile, and can be inactivated by a temperature of 55° C, 
 by dialysis, or by dilution with distilled water. He designated the 
 active principle "alexin." 
 
 Subsequently, in 1899, Bordet found that the alexin of Buchner was 
 composed of two distinct substances — one a sensitizing substance, which 
 is thermostabile, and a second, the thermolabile substance. Somewhat 
 later (1899) Ehrlich and Morgenroth confirmed these observations, but 
 applied the name "amboceptor" to the sensitizing substance and 
 "complement" to the alexin. These terms are most widely employed 
 at the present time. Bordet adheres to the term alexin, meaning thereby 
 the thermolabile principle, and does not use it in the original sense of 
 Buchner, which included both the sensitizing substance and the alexin. 
 Metchnikoff's cj^tases are practically the same as Ehrlich's complement 
 and Bordet's alexin. 
 
 Definition. — Complement [Lat., complementum, that which completes] 
 is the substance, present alike in normal and in immune serum, which is 
 destroyed by heating to 55° C, and which acts with an amboceptor to produce 
 lysis. 
 
 As mentioned in the discussion on amboceptors, the complement is 
 the active lytic substance concerned in the phenomenon of cytolysis, 
 but is powerless until united with the cell, corpuscle, or bacterium by 
 means of the interbody or amboceptor. 
 
 Structure and General Properties of Complement. — Complement is 
 ordinarily not attached to the body-cells and is free in the blood-serum. 
 According to Ehrlich, complement is simple in structure, and is composed 
 of a haptophore portion for union with the complementophile hapto- 
 phore of an amboceptor, and a second toxic or lytic portion, called the 
 
COMPLEMENTS 327 
 
 cytophile group. In other words, the theoretic structure is similar to 
 that of a toxin, although the function and action of the two are quite 
 different. 
 
 By heating a complement serum to 55° C. for half an hour the cyto- 
 phile portion is destroyed, and the serum is now said to be inactivated, 
 as the complement is no longer active. If the complement serum is 
 allowed to stand at ordinary room temperature for forty-eight hours or 
 longer, the same change will take place. The- cytophihc or active por- 
 tion of the complement is, therefore, quite unstable. When this portion 
 is altered or lost, the substance is called complementoid, and this is anal- 
 ogous to toxoids and agglutinoids. 
 
 Since complementoids have their haptophore groups intact, they will 
 unite with amboceptors and to some extent prevent lysis by blocking the 
 active complement, just as toxoids unite with antitoxin and agglutin- 
 oids with their antigens. 
 
 Anticomplements may be obtained by imniunizing suitable animals 
 with serums that contain complement or coiAplementoid. A\Tien an 
 inactivated anticomplement serum is mixed with the homologous com- 
 plement, the haptophores of the latter are bound by means of the 
 haptophores of the anticomplements. A proof of this union lies in the 
 fact that a complement serum that has been treated with its specific 
 anticomplement is no longer able to activate an appropriate amboceptor. 
 
 According to Gay, the production of anticomplements is only ap- 
 parent; he explains the loss of complement activity when a fresh serum 
 and its antiserum are mixed as due to the absorption of complement in 
 the precipitate which forms, although the latter may be invisible. 
 
 Anticomplements may be of practical importance owing to the forma- 
 tion of auto-anticomplements. The complements must exercise an im- 
 portant function, not only in the destruction of bacteria, but also in the 
 digestion and solution of all kinds of foreign albuminous bodies that 
 enter the organism. As was shown by Wassermarm, anticomplements 
 may so bind up their complements as to render their host much less 
 resistant to certain infectious diseases. The spontaneous development 
 of auto-anticomplement in an animal has never been demonstrated, as 
 there are no receptors in an organism of the complements of the same 
 organism. The injection of the serum of another animal containing 
 complements that are almost identical may, hojvever, lead to the forma- 
 tion of an auto-anticomplement in the serum of the immunized animal. 
 
 The extreme lability or sensitivity of complements to heat, exposure, 
 adds, alkalis, etc., is their most prominent general characteristic. An 
 
328 CYTOLYSINS 
 
 active serum is one containing complement, and this must, under ordi- 
 nary circumstances, be a fresh serum. On heating or exposure the 
 serum becomes inactive. An inactivated serum may be reactivated by 
 the addition of fresh complement serum. These terms are in general 
 use, especially in testing for hemolytic and bacteriolytic reactions. 
 
 Origin of Complement. — Buchner regarded complement as a true 
 secretory product of the leukocytes, and IVtetchnikoff also maintains 
 that leukocytes are the main source, the complement being liberated 
 upon disintegration of the white cells. There is considerable experi- 
 mental evidence both for and against these views, but at the present 
 time the consensus of opinion would tend to regard the leukocytes as 
 an important, but not necessarily the sole, source of complement forma- 
 tion. 
 
 Complement has been demonstrated in plasma, where its presence 
 is probably due to continual disintegration of = leukocytes and liberation 
 of complement during life. It is probably increased to a slight extent 
 as serum is left in contact with the blood-clot, indicating that disintegra- 
 tion of leukocytes may augment the complement supply. 
 
 The liver (MiiUer and Dick), pancreas, and other organs have been 
 regarded as sources of complement formation, but in general the evi- 
 dence points to the leukocytes as the chief source of supply, the liver 
 being probably concerned through its activity; in blood destruction. 
 
 Multiplicity of Complements. — Ordinarily^ a fresh serum, such as 
 that of the guinea-pig, will furnish complement for either bacteriolytic 
 or hemolytic amboceptors, and the question arises as to whether one 
 complement unites equally well with all amboceptors, or whether several 
 complements are present in one serum that a6t more or less specifically 
 with different amboceptors. 
 
 Bordet believes that only one complement* is present, and bases this 
 opinion mainly on the fact that a complement that can be shown to 
 activate either a liemoljrtjc or a bacteriolj^ic amboceptor may be 
 absorbed out of a serum by furnishing an excess of either amboceptor. 
 
 Metchnikoff maintains that there are twa cytases or complements, 
 one being derived from macrophages and mainly hemolytic, and the 
 second derived from microphages and chiefly bacteriolytic. 
 
 Ehrlich and Morgenroth, Sachs, Was8erm=ann, Wechsbcrg, and the 
 German school in general believe that many different complements are 
 present in amounts varying with the different serums. These observers 
 have sought to prove this experimentally, and while the evidence is not 
 absolutely convincing, because of the difficulty of working with sub- 
 
COMPLEMENTS 329 
 
 stances that are so labile, yet the doctrine of the multiplicity of comple- 
 ments is quite generally accepted. 
 
 (a) By digesting 20 c.c. of fresh goat serum that was found to activate 
 different hemolytic amboceptors with 3 c.c. of a 10 per cent, solution of 
 papain in the incubator for from thirty to forty-five minutes, it was 
 found that the complement for one amboceptor was destroyed, whereas 
 those remaining were left intact or but slightly impaired. 
 
 (b) By treating 10 c.c. of ^is goat serum with 1 c.c. of a 7 per cent, 
 solution of soda for an hour it was found that some complements were 
 destroyed and others were weakened. 
 
 (c) By sensitizing different blood-cells with homologous amboceptors 
 and adding these to a fresh serum for short and varying periods of time, 
 some complements could be destroyed, whereas others would be left 
 behind with undiminished or but slightly decreased activity. Pro- 
 longed exposure would remove all complements. 
 
 (d) As was previously stated, anticomplements may be produced by 
 immunizing an animal with the complement of an animal of a different 
 species. The anticomplements appear to be specific for the comple- 
 ments responsible for their production, and by means of these anticom- 
 plements different complements may be demonstrated in one serum. 
 Since the formation of anticomplements would depend upon whether or 
 not the body-cells of the immunized animal possess suitable receptors for 
 the various complements, in a series of animals it may be found that one 
 does not produce anticomplements for all the complements injected, 
 a finding that would tend to support the theory of the multiplicity of 
 complements. In addition, Marshall and M6rgenroth actually found 
 in ascitic fluid an anticomplement for at least=one of two complements 
 present in guinea-pig serum. 
 
 These experiments go to show that complements differ in this respect 
 at least: that not all have identical haptophores. Whatever differences 
 between complements exist must be slight; probably the cytophilic 
 group of all are alike. At present the subject has more theoretic than 
 practical importance. In the various diagnostic reactions guinea-pig 
 serum ordinarily furnishes the complement for "hemolysin, bacteriolysin, 
 or other cytolysins, and in the therapeutic administration of bacterioly- 
 tic serums we are compelled in any case to depend for activation of the 
 amboceptor upon the natural complement in the patient's serum. 
 
 The Nature and Action of Complement. — The true nature of the 
 complements is unknown. In many respects they bear a resemblance 
 to ferments, and certainly the part they play in the processes of cytolysis 
 
330 
 
 CYTOLYSINS 
 
 suggests a ferment-like activity. They differ'from true proteolytic fer- 
 ments, such as trypsin, in not digesting the stroma of corpuscles, al- 
 though recent work by Dick would seem to indicate that proteolysis 
 actually occurs, a process that increases the permeability of the cell and 
 permits the escape of hemoglobin. 
 
 On the other hand, it is possible that the nature and action of com- 
 plements may be placed upon a chemical basis. Following the dis- 
 covery of the hemolytic power of cobra venom by Flexner and Noguchi, 
 a power they ascribed to the presence of an amboceptor in the venom 
 acting with serum complement, Kyes found that the amboceptor may 
 be activated not only by a complement in the blood-serum, but also by 
 some constituent of the red blood-corpuscles themselves. This last 
 observer speaks of the latter as endocomplement, i. e., endocellular com- 
 plement. 
 
 In attempting to discover the nature of this endocomplement various 
 substances existing normally in the erythrocytes, such as cholesterin and 
 lecithin, were obtained in a pure state and their activating powers for 
 cobra amboceptors tested. These investigations showed that lecithin 
 has an activating power, whereas cholesterin is antihemolytic. Al- 
 though all erythrocytes contain lecithin, yet all are not equally suscep- 
 tible to the action of venom amboceptors, which is probably due to the 
 fact that the lecithin in the cells of some animals is bound to other cell 
 constituents in a loose way and is thus available as complement. In 
 syphilitic infection the lecithin content of the erythrocytes is actually 
 diminished or in some manner rendered less available, so that the in- 
 hibition or absence of venom hemolysis is diagnostic of this infection. 
 
 Kyes was able to obtain the union of cobra amboceptor and lecithin, 
 forming what is known as cohra lecithid. Although lecithin is an unstable 
 substance and is difficult to obtain free from fatty acids and soap, there 
 is little doubt but that Kyes' lecithid is a phosphatid compound and is 
 actively hemolytic after all traces of fatty acids have been removed. 
 
 The next important observations were made by Noguchi,^ who found 
 that soap isolated from blood and various tissues possessed active he- 
 molytic properties. The salts of the fatty acids, and particularly of oleic 
 acid, were found to possess similar hemolytic properties. Pure soluble 
 olcates mixed with serum were found to produce compounds possessing 
 many of the characteristics of true complements: (1) They are inacti- 
 vated by heating to 66° C. for half an hour; (2) they are inactive at 0° 
 C. ; (3) the addition of acids, alkalis, and yeast renders them inactive. 
 1 Proe. Soc. Exper. Biol, and Med., 1907, 4, 107; Biochem. Zeitscbr., 1907, 6. 
 
COMPLEMENTS 331 
 
 Von Liebermann/ as the result of his own experiments, came to practi- 
 cally the same conclusions, and advanced the hypothesis that the com- 
 plements of the blood are to be sought for in the soaps of the serum; 
 that these soaps are united with serum albuniin, and are inactive until 
 liberated by the amboceptors, when they become actively hemolytic. 
 
 Further than this, it was shown that oleic! acid may act as an am- 
 boceptor, and when added to an inactive soap-albumin combination, it 
 would render this actively hemolytic. Von Liebermann and Fenyvessy^ 
 have shown that a mixture of soap, serum, and oleic acid possesses a 
 striking resemblance to complements and amboceptors, and that the 
 amboceptor-complement action is much more than a mere linkage of 
 complement to antigen by means of an amboceptor. These observers 
 suggest that an amboceptor may have an affinity for certain constituents 
 of the cell or bacterial body, and, on the other hand, act upon the com- 
 plement and separate one of its constituents, -^vhich then breaks up the 
 cell. These artificial hemolysins, however, completely dissolve the 
 stroma of the corpuscles, whereas the immujie hemolysins appear to 
 dissolve out the hemoglobin, leaving the stroritia undissolved. As has 
 been mentioned elsewhere, recent work would tend to show that the 
 stroma is also dissolved, at least in part, in speoific hemolysis, so that the 
 difference in action between the two is not quite so apparent. 
 
 While the simplicity of the substances concerned in these observations 
 does not harmonize with the great variety and complexity of the im- 
 mune bodies, nevertheless, as Adami has pointed out, the points of re- 
 semblance between artificial and natural complements and amboceptor 
 are so striking that material advances in our knowledge of their nature 
 and action may be gained by further researches into the chemistry of 
 immunity. 
 
 Complement Splitting. — During recent years considerable attention 
 has been directed toward a phenomenon known as the splitting of com- 
 plement. It was generally conceded that when treated with hydro- 
 chloric acid (Sachs), carbon dioxid gas (Liefmann), or acid and alkaline 
 phosphates (Michaehs and Skwissky), or when diatyzed against dis- 
 tilled water (Ferrata), complement may be split into two parts, known as 
 amid-piece and an end-piece. According to certain investigators, these 
 two components of the complement differ in their behavior in hemolytic 
 processes: one, the mid-piece, is bound by the sensitized cells, while 
 the other, or end-piece, possesses the lytic action. Speaking in terms 
 of the side-chain theory, it is just as if the haptophore and cytophilic 
 
 ' Biochem. Zeitschr., 1907, 4. ^ Bioohem. Zeitschr., 1907, 5. 
 
332 CYTOLYSINS 
 
 portions of the complement were separated: the haptophore portion 
 corresponding to the mid-piece (the cell or bacterium being the first 
 piece), and the lytic cytophilic portion corresponding to the end-piece. 
 In successful complement splitting the mid-piece is believed to be in the 
 globulin fraction and the end-piece in the albumin fraction. Noguchi 
 and Bronfenbrenneri have cast considerable* doubt upon these views, 
 and have shown that what is known as complement splitting is really 
 nothing more than an inactivation of the active principle of complement, 
 since both globulin and albumin fractions contain a part of the comple- 
 ment, a fact that can be demonstrated by the removal of the inhibiting 
 action of the acid or alkali used in the process. 
 
 The Bordet-Gengou Phenomenon of Complement Fixation 
 
 In the endeavor to demonstrate the unity of complement Bordet 
 and Gengou devised an experiment that has proved of great practical 
 viilue in the serum diagnosis of syphilis and other infectious diseases. 
 By mixing bacteria and their amboceptors wi£h a little fresh serum con- 
 taining complement and letting the mixture stand aside for an hour or 
 so it was found that, upon the addition of corpuscles and their ambo- 
 ceptor, hemolysis did not occur, although the serum that had been used 
 as complement was capable, in its original condition, of producing hem- 
 olysis of these corpuscles. Bordet advanced this experiment to show 
 that the complement concerned in bacteriolysis is the same as that at 
 work in hemolysis, and consequently concluded that there is but one 
 single complement. 
 
 This experiment of Bordet is usually spoken of as the "Bordet- 
 Gengou phenomenon," and is now used extensively in determining 
 whether or not a given serum contains certain amboceptors. The serum 
 to be tested is first inactivated, treated with the antigen composed of an 
 emulsion of the bacterium whose amboceptor it is desired to discover, 
 and then mixed with a small quantity of a fresh normal complement 
 serum. The mixture is placed in the incubator for an hour, during which 
 time the bacterial antigen unites with its amboceptor and the comple- 
 ment, i. e., fixes the complement, so that when red blood-cells previously 
 sensitized with heated hemolytic serum are added, hemolysis does not 
 occur because the complement in the fresh serum, which was suitable 
 for lysis of the sensitized corpuscles, has been "fixed" by the bacteria 
 by reason of the presence of specific amboceptors in the serum tested 
 
 1 Jour, Exper. Med., 1912, 15, 598 and 625. 
 
COMPLEMENTS 
 
 333 
 
 (Fig. 94). If these amboceptors were not present, then the complement 
 would remain unfixed and be free to hemolyzc the sensitized corpuscles, 
 a negative reaction being indicated, therefore, by hemolysis, whereas 
 the absence or inhibition of hemolysis indicates a positive reaction. 
 
 CA.C 
 
 PQ! 
 
 -C.I 
 
 Fig. 94. — Mechanism of Complement Fixation. 
 
 Tube 1 shows the hemolytic system; C, a red blood-corpuscle; A, a hemolytic 
 amboceptor; Cp, complement; C.A.C., complement uniffed to a corpuscle by means 
 of the specific amboceptor. Hemolysis results. 
 
 Tube 2 shows complement fixation by bacterial antigen and amboceptor; Ai 
 antigen; C, complement united to the antigen ^i by the amboceptor A. When 
 hemolytic amboceptors are added hemolysis does not occur because the complement 
 has been previously fixed by the bacterial antigen and amboceptor. 
 
 Tube 3 shows absence of complement fixation because the bacterial amboceptor 
 Ai is not specific for the bacterial antigen Ai and hence complement is not fixed; 
 when hemolytic amboceptor and the corresponding corpuscles are added comple- 
 ment unites with these, C.A.C. and hemolysis occurs. 
 
 Wassermann, Neisser, and Bruch have applied this test to the serum 
 diagnosis of syphilis, the technic of the test being considered in a subse- 
 quent chapter. 
 
 Deviation or Deflection of Complement. — ^While large doses of anti- 
 toxin are indicated in the treatment of diphtheria and tetanus, theoretic- 
 ally the administration of too large an amount of a bacteriolytic serum 
 
334 CYTOLYSINS 
 
 may result in more harm than good. As has been pointed out by Neisser 
 and Wechsberg, more amboceptors may be introduced than can be taken 
 up by the bacteria causing the infection, and those that remain free are 
 capable of combining with some of the complement that is present, and 
 thus prevent a portion of the complement from acting with the ambo- 
 ceptors attached to the bacteria — i. e., the complement has been de- 
 viated or deflected from its natural course. This would really mean a 
 decrease in bacteriolysis, but while deviation of complement has been 
 demonstrated as a fact, its importance in serum therapy is probably 
 overrated and has certainly not been definitely proved. 
 
 Quantitative Titration of Complements 
 Titration of Hemolytic Complement.— The quantity of a hemolytic 
 complement present in a fresh serum for certain corpuscles may be 
 measured in the same manner as hemolytic amboceptors are measured, 
 namely, by adding to a series of test-tubes increasing amounts of the 
 fresh serum with a constant dose of an emulsion of corpuscles and a 
 constant and sufficient dose of the corresponding hemolytic amboceptor 
 for these corpuscles. After a suitable period of incubation, that tube 
 that shows complete hemolysis contains just sufiicient complement, or 
 one unit. Since we know how much complement serum was placed in 
 this tube, we now know that this quantity contains one unit of hemolytic 
 complement. Of course, the amount of corpuscles and amboceptor must 
 be constant in all tubes; if the quantity or quality of one or both of these 
 is altered, the unit of complement will vary. 
 
 Titration of a Bacteriolytic Complement. — The quantity of bacterio- 
 lytic complement in a given amount of fresh serum may be determined 
 in a similar manner, although far less accurately, for instead of viewing 
 the results of lysis in the test-tube as we can do in hemolysis, the degree 
 of lysis must be determined by plating out the "mixtures to determine the 
 relative numbers of living and dead bacteria.. The degree of bacterio- 
 lysis may, however, be viewed after a manner with the aid of the micro- 
 scope. 
 
 Gay and Ayer employ a direct method, which consists in adding 
 varying amounts of the serum to be tested to a definite volume (0,5 c.c.) 
 of a suspension of cholera vibrios, prepared by emulsifying a twenty- 
 four-hour agar culture in 10 c.c. of normal salt solution and adding a 
 constant and sufficient dose of serum from a rabbit immunized against 
 cholera. The mixtures are placed in small test-tubes and incubated for 
 one and one-half hours at 37° C. Films are then prepared, stained, and 
 
COMPLEMENTS 335 
 
 examined to ascertain the degree of clianges undergone by the vibrios. 
 Or the changes may be observed in hanging-drop preparations. In 
 either case a control is prepared of the culture which has also been 
 incubated along with the mixtures. Gay and Ayer found that 0.02 c.c. 
 of normal human serum contained sufficient complement to effect com- 
 plete lysis of this dose of vibrios — even 0.001 c.c. produced distinct 
 changes. 
 
CHAPTER XIX 
 BACTERIOLYSINS 
 
 Having considered the general nature and properties of amboceptors 
 and complements and the mechanism of their action in producing solu- 
 tion or lysis of cells, we will now study more closely the bacteriolysins, 
 which are antibodies belonging to this group and possessing diagnostic 
 and considerable therapeutic importance. 
 
 Historic. — The early liistory of the discov.ery of the bacteriolysins 
 is closely associated with the history of immunity in general, for with 
 the discoveries in bacteriology and the establishment of the relation of 
 bacteria to disease, it followed as a matter of course that investigations 
 should be undertaken to ascertain the mechanifem of resistance to and 
 of recovery from an infection. 
 
 In 1874 Traube showed that septic material may be destroyed in the 
 blood of living animals, and in 1881 Lister demonstrated the same phe- 
 nomenon in extravascular blood. These experiments were naturally 
 somewhat crude, as they antedated the period in which the pyogenic 
 microorganisms were isolated and studied in pure culture, but they 
 served, nevertheless, to demonstrate the germicidal powers of the blood. 
 
 In 1886 Fodor demonstrated the germicidal action of blood-serum 
 upon anthrax bacilli. This work was followed shortly after by that of 
 Fliigge and Nuttall, who showed the germicidal powers of the body-fluids 
 in general independent of cells. The controversy between the adherents 
 of the cellular and humoral theories of immunity now began, as Metchni- 
 koff was actively engaged in studying phagocytes and in formulating 
 his phagocytic theory. 
 
 Buchner and others took up the subject, emphasizing the important 
 germicidal powers of the body-fluids and ascribing this function to the 
 presence of "alexins" (substances that ward off disease). Buchner 
 showed that if the serum was heated this gfeirmicidal power was lost; 
 hence it followed that the active bacteriolytic agent was considered very 
 unstable and was quickly destroyed outside of the body. 
 
 In 1894 Pfciffer demonstrated most clearly the phenomenon of bac- 
 teriolysis, which gave great encouragement ^nd impetus to studies in 
 
 336 
 
DEFINITION 337 
 
 immunity, and incidentally strengthened the claims of the humoral 
 theory. He showed that cholera vibrios introduced into the peritoneal 
 cavity of a guinea-pig that had been immunized against cholera lost 
 their motility and finally became disintegrated and passed into solution 
 regardless of the presence of cells. 
 
 It remained for Bordet, however, to show the mechanism of this in- 
 teresting phenomenon. This observer demonstrated the fact that the 
 thermolabile body was but one substance concerned in the reaction, and 
 that the specific substance was thermostabile and the actual product 
 of immunization, results that were later corroborated and elucidated by 
 his researches upon hemolysis, and by those of Ehrlich and his pupils on 
 cytolytic phenomena in general. As previously mentioned, Bordet 
 retained the name "alexin" for the thermolabile substance and appUed 
 the new term, "substance sensibilisatrice," to the specific thermostabile 
 antibody. Later, both substances were renamed by Ehrlich, and called 
 "complement" and "amboceptor" respectively. 
 
 As will be pointed out further on, as the result' of these observations 
 Metchnikoff modified his phagocytic theory, and recognized the exist- 
 ence of both substances, which he named "cytases" and "fixateurs," 
 believing that both were derived from cells clasfeed as phagocytes. 
 
 All are agreed as to the presence of two difl'erent bodies in the body- 
 fluids concerned in bacteriolysis, although opinions vary as regards their 
 origin and mechanism of action. The side-chain theory has been widely 
 accepted in explanation of their action, and the, terms applied by Ehrlich 
 to the two substances concerned, namely, complements and ambocep- 
 tors, are in general use. 
 
 Definition. — Bacieriolysins are substances present in the serum and 
 other body-fluids that kill bacteria with or without lysis. 
 
 The term itself would infer that solution or lysis of the bacterium is 
 an essential property of an antibody of this order. Bactericidins are 
 substances that kill bacteria without lysis, and, strictly speaking, an 
 effort should be made to differentiate between the terms, although from 
 a practical standpoint this is not important. Certain microorganisms 
 may be killed and resist solution or digestion fpr a comparatively long 
 time, whereas, on the other hand, the same bacteria, under different 
 circumstances, may readily be lysed. 
 
 Although the endotoxins liberated from the lysed bacteria may pro- 
 duce symptoms of disease, followed by death, yet the bacterium itself 
 is usually destroyed and unable to proliferate. A bacteriolysin is, there- 
 fore, always bactericidal, although the converse is not necessarily true. 
 
338 BACTERIOLYSINS 
 
 Custom, however, has never strictly differentiated between the two 
 terms, and bacteriolysis appears to be but a continuation of and a more 
 nearly complete bactericidal process. Hence the definition just given 
 covers both terms — bacteriolysins and bactepcidins. 
 
 Origin of Bacteriolysins. — Our knowledge regarding the origin of the 
 bacteriolysins is quite fragmentary. The investigations of Pfeiffer and 
 Marx in cholera, and Wassermann in typhoid, have shown that the 
 spleen and hematopoietic organs in general miy be especially concerned. 
 
 According to Metchnikoff, the "bacterial fixateurs," which are 
 practically the bacteriolysins, are secretory or excretory products of 
 phagocytic cells, especially the polynuclear leukocytes or microphages. 
 It is commonly believed that during infection the bacteria cause phag- 
 olysis or disintegration of these cells, with liberation of both comple- 
 ments (cytases) and amboceptors (fixateurs), which produce extracellu- 
 lar lysis of the invading bacterium (bacteriolysis). If, on the other 
 hand, the phagocytes are fortified and phagolysis is prevented, the bac- 
 teria are phagocytized and undergo intracellular lysis, a condition that, 
 according to Metchnikoff, may be induced experimentally by giving an 
 animal an intraperitoneal injection of sterile bouillon twenty-four hours 
 before bacteria or other cells are injected. 
 
 That leukocytes afford a bacteriolytic substance is supported by ob- 
 servations showing that leukocytic exudates, secured by the injection 
 of a sterile aleuronat suspension, possess a well-marked germicidal ac- 
 tivity. Issseff found that the intraperitoneal injection of sterile bouillon 
 and other mild irritants, by producing a leukocs/tic exudate that supplied 
 certain bactericidal substances and facilitated phagocytosis, increased 
 the resistance of animals to bacterial infection. At one time surgeons 
 made practical use of this observation by injecting nucleinie acid and 
 other substances into the peritoneal cavity before performing laparot- 
 omy, in order to induce a local resistance to a possible infection. 
 
 LEUKINS AND LEUKOCYTIC EXTRACTS 
 
 The bactericidal substance contained within leukocytes has been ex- 
 tensively studied by Schattenfroh, Schneider, Peterson, Hiss and 
 Zinsser, Manwaring, and others. It has been observed that when leu- 
 kocytes are suspended in diluted blood-serum, the bactericidal proper- 
 ties of the serum are increased without coincident destruction of the 
 cells, showing that the leukocytes may secrete germicidal substances into 
 the fluid. The same observation has been made with Bier's congestive 
 
LEUKINS AND LEUKOCYTIC EXTRACTS 339 
 
 lymph, indicating that this activity takes place both in the test-tube 
 and in living tissues. 
 
 Hiss, and later Hiss and Zinsser, found that autolyzed leukocytic 
 exudates possess some bactericidal activity, and that they may pro- 
 foundly modify experimentally induced infection of rabbits and guinea- 
 pigs with the pneumococcus, staphylococcus, streptococcus, and other 
 bacteria. In applying this method of treatment to man by means of 
 subcutaneous injections, these investigators qbserved distinctly bene- 
 ficial results in cases of epidemic cerebrospinal meningitis, lobar pneu- 
 monia, staphylococcus infections, and erysipelas. 
 
 Preparation of Leukocytic Extracts. — Hiss and Zinsser have pre- 
 pared leukocytic extract by giving rabbits intrapleural injections of 
 aleuronat suspension. Manwaring has secured much larger quantities 
 by making his injections into the horse. 
 
 The aleuronat is prepared by making a 3 per cent, solution of starch 
 in bouillon without heating, and adding 5 per cent, of powdered aleuronat 
 to this emulsion. The starch helps to keep the"aleuronat in suspension. 
 The mixture is boiled for five minutes and placed in large sterile test- 
 tubes, 20 c.c. being placed in each tube. Fin^l sterilization is done in 
 an autoclave. 
 
 For making the injections large rabbits are* selected. The hair over 
 both sides of the thorax is removed, the skin is sterilized with tincture 
 of iodin, and 10 c.c. of the aleuronat suspension are injected into each 
 pleural cavity at a point in the anterior axillary line, at the level of the 
 sternum, great care being taken to avoid puncturing the lungs. After 
 twenty-four hours the animals are chloroformed and the pleural cavities 
 carefully and aseptically opened. The cellulaf exudate is pipeted into 
 centrifuge tubes containing at least 10 c.c. of sterile 1 per cent, sodium 
 citrate in normal salt solution. One or more cubic centimeters of exu- 
 date may be obtained from each cavity. The exudate is usually tinged 
 with blood. It is then centrifuged and the supernatant fluid removed. 
 The sediment is broken up with a platinum spatula, and 20 volumes of 
 sterile distilled water are added. The tubes are set aside in the incuba- 
 tor for twenty-four hours, after which cultures.are made to insure steril- 
 ity. A small amount of preservative may be added, and the extract 
 placed in bottles or ampules ready for administration. It is given sub- 
 cutaneously in doses of from 5 to 10 c.c. every four to six hours. 
 
 The endocellular bactericidal substances, or endolysins, mentioned 
 in a previous chapter, which can be extracted. from leukocytes, are not 
 in the nature of complements, as they are not rendered inactive by tem- 
 
340 BACTEEIOLYSINS 
 
 peratures below 80° C. They cannot apparently be increased by im- 
 munization, the quantity present in each leukocyte being probably at 
 all times just sufficient for the digestion of a limited number of bacteria, 
 which can be ingested at one time by the individual leukocyte. It 
 is probable that the excess of bactericidal substance is thrown off into 
 the blood-stream, representing the serum bacteriolysins, and at least 
 indicating that the leukocytes are one source for the production of bac- 
 teriolytic amboceptors. 
 
 Mechanism of Bacteriolysis. — ^According to the side-chain theory 
 of Ehrlich a bacteriolysin is an antibody of the third order, or an ambo- 
 ceptor furnished with two haptophore or grasping arms for uniting the 
 bacterium on one side with a suitable complement on the other. The 
 antibody, therefore, acts simply as an interbody or connecting link; 
 it is specific for the bacterium causing its production, but is unable itself 
 directly to injure the bacterium, lysis being brought about by an at- 
 tached complement. Bacteriolysis is, 
 therefore, an interaction of ambocep- 
 tor and comi3lement upon the bacterial 
 
 Fig. 95. — Theoretic Structure 
 
 OF A Bacteriolytic Ajmbocep- While bacteriolytic amboceptors 
 
 ™^'. , , „ , will unite with their antigens under 
 
 A, Amboceptor; C, comple- . 
 
 ment; r, receptor of bacillus; h, practically all conditions, nevertheless 
 
 tTTa^'cor^lLlSlT'g^^^^^^^^^ ^ ^'^it-ble complement may not be 
 of the amboceptor. present, and hence a bacteriolytic 
 
 serum may not be active in all animals. 
 
 The influence of bacteriolysins upon endotoxins is a question of con- 
 siderable interest and importance. As the result of convincing experi- 
 ments performed, especially by Pfeiffer, it is evident that a bacteriolytic 
 serum does not neutralize the endotoxin at the time the bacterium xmder- 
 goes disintegration. Highly immune serums appear to be unable to pro- 
 tect an animal against the endotoxins, and, indeed, may even increase the 
 intoxication and, by liberating an excess of endotoxin, kill the animal. 
 
 The bactericidal substances derived from leukoc5rtes are, however, 
 apparently capable of neutralizing endotoxins,: to some extent at least, 
 as Hiss and Zinsser were unable to ascribe the beneficial effects of leu- 
 kocytic extracts to the bacteriolytic action alone. 
 
 As mentioned elsewhere, both Metchnikoff and Bordet maintain 
 that the bacteriolysin is in the nature of a "sensitizer," preparing the 
 bacterium for the action of the alexin or cytase, just as a mordant aids 
 in the penetration of a dye-stuff. 
 
LEUKINS AND LEUKOCYTIC EXTRACTS 341 
 
 General Properties of Bacteriolysins. — As with other cytolysins, 
 the bacteriolysins are thermostabile and resist heating to 60° C, being 
 gradually destroyed at temperatures ranging from 70° to 80° C. They 
 are likewise highly resistant to acids and alkalis, and when preserved in 
 a sterile condition with the addition of small quantities of a preservative 
 bacteriolytic serums for therapeutic, and diagnostic purposes may re- 
 main active for long periods of time. Cholera immune serum as a diag- 
 nostic aid in making the agglutination and Pfeiffer bacteriolytic reac- 
 tions is best preserved in dry form, the serum retaining its activity under 
 these conditions for considerable periods of time. 
 
 Normal Bacteriolysins. — As a result of the contention of Metchnikoff 
 that bacteriolysins (fixateurs) are produced only upon the disintegration 
 of leukocytes, and that the plasma is accordingly free from these anti- 
 bodies, much experimental work has been done. The weight of evi- 
 dence is against this view, as both amboceptors and complement.s have 
 been demonstrated in plasma, although the quantity of bacterial ambo- 
 ceptors normally present in the body-fluids is> quite small. It is quite 
 natural to expect that under normal conditions small amounts 
 will be present, as receptors are being constantly thrown off into the 
 blood, and leukocytes are, of course, being= constantly formed and 
 destroyed. 
 
 Specificity of Bacteriolysins. — The bacteriolysins are highly specific 
 antibodies, and are useful in making the differentiation of bacterial 
 species. Group bacterioljrtic reactions are leSs common as compared 
 to group agglutination, as was shown by Kolrrier, Williams, and Raiziss 
 with the typhoid-colon group of bacilh. As a practical procedure, 
 however, the agglutination reaction is so easily secured as to be 
 the test of choice in making a differentiation between closely allied 
 organisms. As with the agglutinins, the influence of partial bac- 
 teriolysins may be removed by using highly immune serums in high 
 dilutions . 
 
 P*racticELl Applications. — The bacteriolysins have considerable value 
 in the following procedures : 
 
 1. In making a differentiation of bacteria, especially when the pres- 
 ence of cholera vibrios is suspected. 
 
 2. In the diagnosis of certain infections, such as cholera and typhoid 
 fever. 
 
 3. In the treatment of some infections with specific bacteriolytic 
 serums. 
 
342 BACTERIOLYSINS 
 
 TECHNIC OF BACTERIOLYXie TESTS 
 The Pfeiffer ExperlheWts 
 
 The essentials of this important test have been described at the be- 
 ginning of this chapter. Briefly, it consists in making intraperitoneal 
 injections of a bacteriolytic serum mixed with living bacteria into a 
 nonnal guinea-pig. The resulting bacteriolysis is studied microscopically 
 by withdrawing small amounts of peritoneal exudate at varying inter- 
 vals. By performing the experiment with varying dilutions of serum, 
 the bacteriolytic titer may be determined by noting the dilution in which 
 bacteriolysis just fails to occur in a specified time. 
 
 Pfeiffer also showed that the phenomenon could be produced by in- 
 jecting a mixture of serum from an immunized animal and the culture 
 of cholera into the peritoneum of a normal guinea-pig. This phenome- 
 non appeared when an old specimen of serum w^s used, as well as when a 
 fresh specimen that had been heated to 60° C. was employed. Later, 
 this observer found that if an old immune serum was injected into the 
 peritoneal cavity and allowed to remain for a time, it regained its bac- 
 tericidal powers. 
 
 Pfeiffer believed that the bacteriolytic substance may exist in the 
 serum of an immunized animal either in an active or in an inert state. 
 In the blood-serum or peritoneal fluid of the living animal it occurs as 
 an active substance, but when kept for a few days or when heated 
 rapidly to 60° C. it becomes inert; it may be rendered active again 
 by coming in contact with the lining endothelial cells of the perito- 
 neum. 
 
 The foregoing constitutes the classic Pfeiffer experiment. The bac- 
 teriolytic amboceptor present in the immune serum is activated by the 
 complement furnished by the guinea-pig. The same serum will not 
 produce bacteriolysis in the test-tube in case it has been heated or the 
 complement is lost through age unless fresh normal serum or peritoneal 
 exudate is added. By immunizing guinea-pigs with gradually increasing 
 doses of cholera serum and then introducing fatal doses of cholera cul- 
 ture intraperitoneally, the samfe phenomenon qf bacteriolysis is observed. 
 In this instance the guinea-pig furnishes both amboceptor and comple- 
 ment. 
 
 By these and similar experiments and observations Bordet was able 
 to show the role played by the two bodies concerned in cytolysis in 
 general, namely, the thermolabile alexin or complement and the specific 
 sensitizer or bacteriolytic amboceptor. 
 
TBCHNIC OF BACTERIOLYTIf: TESTS 343 
 
 Bacteriolytic Test in vivo for the Identification of Bacteria Recovered 
 from Feces, Water-supplies, etc. — This method is employed chiefly ia 
 the identification of suspected cholera cultures.. According to Citron, 
 in Germany, the Pfeiffer test, made with miefoorganisms obtained in 
 pure culture from ■suspected patients, is required for the official diagno- 
 sis of the first cases of cholera. 
 
 As a rule, the agglutination test is first applied in making the diag- 
 nosis of a suspected microorganism, as the technic of this test is more 
 easily carried out. 
 
 This bacteriolytic test may also be employed in the study of typhoid 
 and paratyphoid bacilli, although bacteriolysis of these microorganisms 
 is less complete than that observed with cholera, and agglutination 
 tests answer all practical requirements. 
 
 The test consists in mixing varying dilutions of a known and highly 
 immune serum with a constant dose of unknown microorganisms, and 
 injecting the mixtures intraperitoneally into guinea-pigs. After twenty 
 minutes small amounts of exudate are withdrawn by nieans of fine capil- 
 lary pipets, and studied in hanging-drop preparations. In the presence 
 of a positive reaction the bacilli are observed; to lose motility, become 
 swollen and coccoid in shape, and gradually form granules, ultimately 
 disappearing in complete solution. 
 
 Preparing the Immune Serum. — A highly immune serum is required. 
 This may readily be prepared by giving rabbits a series of intravenous 
 injections of a known culture, according to the technic described in the 
 chapter on Active Immunization of Animals. The official test in Ger- 
 many demands that the serum be of such strength that 0.0002 gram of 
 dried serum will suffice to disintegrate completely within one hour one 
 loopful (2 mg.) of an eighteen-hour-old culture of virulent cholera in 
 1 c.c. of nutrient bouillon when injected into the peritoneal cavity of a 
 guinea-pig. The Hygienic Laboratory^ in Washington is prepared to 
 furnish board of health laboratories with a dried scrum of high titer for 
 diagnostic purposes. 
 
 In testing an immune serum to determine its bacteriolytic titer the 
 'dose of microorganisms should not be larger than one loopful, so that if 
 any particular strain of cholera or typhoid is not sufficiently virulent, 
 necessitating the use of larger doses, the virulence should be increased 
 by passing the organism through guinea-pigs. 
 
 Method of Testing the Virulence of a Culture. — The unit of measure- 
 ment is a 2 millimeter platinum loop, which, when loaded, will usually 
 ' Personal communication from Dr. John F. Anderson, Director of the Laboratory. 
 
344 BACTERIOLYSINS 
 
 hold about 2 milligrams of microorganisms. (See p. 212.) All dilu- 
 tions are made with sterile neutral broth, and not with salt solution. 
 Mixtures are injected intraperitoneally into, 250-gram guinea-pigs to 
 determine the dose that will be fatal within twenty-four hours. 
 
 Great care should be exercised in all manipulations to avoid acci- 
 dental infection. The mouth-ends of pipets should be plugged with 
 cotton. Sufficient assistants should be on hand to facilitate the making 
 of injections and the examination of peritoneal exudates with ease, 
 caution, and certainty. All pipets, measuring glasses, test-tubes, and 
 hanging-drop preparations should be immersed after using in 1 per cent, 
 formalin solution before cleaning. In other* words, every precaution 
 should be taken to carry out a thorough and Conscientious bacteriologic 
 technic. 
 
 Guinea-pig No. 1 : Four loopfuls of agar culture emulsified in 4 c.c. 
 
 of bouillon; inject 1 c.c. intraperitoneally (=1 loopful). 
 Guinea-pig No. 2: 1 c.c. of foregoing emulsion -}- 1 c.c. of bouillon; 
 
 inject 1 c.c. intraperitoneally ( = }^ loopful). 
 Guinea-pig No. 3: 1 c.c. of first emulsion + 4 c.c. of bouillon; inject 
 
 1 c.c. intraperitoneally ( = ^ loopful). 
 Guinea-pig No. 4: 1 c.c. of emulsion No. 3 -|- 1 c.c. of bouillon; 
 inject 1 c.c. intraperitoneally { — yij loopful). 
 A satisfactory culture is one in which a dose of |- loopful will prove 
 fatal within twenty-four hours. The immune §erum is then titrated with 
 five times this amount of culture, or one loopful. 
 
 Method of Titrating a Bacteriolytic Serum. — The serum is inacti- 
 vated by heating to 56° C. for half an hour, and dilutions are made with 
 bouillon in sterile shallow glasses or test-tubes.* One loopful of an eigh- 
 teen-hour agar culture of the microorganism is thoroughly einulsified 
 in the diluted serum, and the mixtures are injected intraperitoneally 
 in 250-gram guinea-pigs. Higher dilutions than those given here may 
 be employed until the limit of bacteriolytic activity is reached. 
 
 1. Mix 0.5 c.c. of inactivated serum with 4.5 c.c. of bouillon (1 : 10). 
 Place 2 c.c. of this mixture in a separate test-tube, and add 2 loopfuls of 
 culture. Inject 1 c.c. ( = 0.1 c.c. immune serum). 
 
 2. Mix 2 c.c. of the first dilution (1 : 10) with 18 c.c. of bouillon ( = 
 1 : 100). Place 2 c.c. in a separate tube and add 2 loopfuls of culture. 
 Inject 1 c.c. ( = 0.01 c.c. immune serum). 
 
 3. Mix 1 c.c. of the second dilution (1 : 100) with 4 c.c. of bouillon 
 ( = 1 : 500). Place 2 c.c. in a separate tube and add 2 loopfuls of culture. 
 Inject 1 c.c. ( = 0.05 c.c. immune serum). 
 
TECHNIC OF BACTERIOLYTIC TESTS 345 
 
 4. Mix 2 c.c. of the third dilution (1 : 500) with 2 c.c. of bouillon 
 (= 1 : 1000). Place 2 c.c. in a separate tube and add 2 loopfuls of cul- 
 ture. Inject 1 c.c. ( = 0.001 c.c. immune serum). 
 
 5. To 1 c.c. of the fourth dilution (1 : 1000) add 9 c.c. of bouillon 
 (=1 : 10,000). Place 2 c.c. in a separate tube, and add 2 loopfuls of 
 culture. Inject 1 c.c. ( = 0.0001 c.c. immune serum). 
 
 6. Control: Emulsify 2 loopfuls of culture in 2 c.c. of bouillon. In- 
 ject 1 c.c. This animal will probably succumb within twenty-four hours. 
 
 7. Control: To 2 c.c. of a 1 : 10 dilution of inactivated normal rabbit 
 serum add 2 loopfuls of culture and inject 1 e.c. intraperitoneally. If 
 goats or horses are used in preparing the immune serum, this control 
 should be conducted with normal goat or horse serum. According to 
 KoUe, one loopful of virulent cholera culture is destroyed in the peri- 
 toneal cavity of a guinea-pig by 0.1 to 0.3 c.c. of normal rabbit's serum; 
 0.02 to 0.03 c.c. of normal goat's serum; 0.005 to 0.01 c.c. of normal 
 horse's serum. 
 
 8. Control: A pig may be injected with 1 cfi. of a 1 : 100 dilution of 
 the immune serum, and with a loopful of some other microorganism, 
 such as Bacillus coli. This control, however, is not absolutely neces- 
 sary. 
 
 An area of the abdominal wall of the guinea-pig about one inch in 
 diameter is shaved and cleansed with alcohol. After the injections have 
 been made the bacteriolytic phenomena are observed. 
 
 In making this test fine capillary pipets are prepared and used for 
 withdrawing the peritoneal exudate. 
 
 After permitting the animal to inhale a f ews drops of ether, to make 
 sure that it will not suffer, a small incision is made through the skin of 
 the abdomen. The capillary pipet, the large end of which is kept closed 
 with the index finger, is then quickly passed into the abdominal cavity. 
 The index-finger is released, and the tube is gently moved about and 
 withdrawn. As the result of capillary attraction sufficient exudate 
 usually passes into the tube without the aid of suction (Fig. 96). The 
 tube may be fitted with a rubber teat in case suction should be necessary. 
 Hanging-drop and smear preparations are made and studied microscop- 
 ically (Fig. 97). Stained smears are, however, less reliable and not so 
 useful in determining the degree of bacteriolysis (Fig. 99). 
 
 It is best to withdraw the exudate immediately after injection, and 
 then at intervals of ten, twenty, thirty, forty, and sixty minutes. 
 
 A hanging-drop preparation, made from the culture by emulsifying 
 a minute quantity in bouillon, should be on hand as a control in studying 
 
346 
 
 BACTERIOLYSINS 
 
 bacteriolysis in the exudate (Fig, 98). A smear of the culture stained 
 with dilute carbolfuchsin should also be on hand for making comparison 
 with smears of the exudate (Fig. 100) . 
 
 Bacteriolysis is first manifested by loss df motility. As the process 
 progresses many of the bacilli become swollen and distorted, and later 
 irregular and broken fragments or granules become apparent. Finally, 
 at the end of an hour, the exudate is practically sterile. In those cases 
 in which bacteriolysis is complete in an hour the animal generally sur- 
 
 **^w*^«efi?r''' 
 
 <-^"^ 
 
 Fig. 96. — Removing Exudate from the Pebitoneal Cavity op a G0inea-piq 
 
 (Pfeifper Bacteriolytic Test). 
 
 A small incision is made through the skin, and the exudate is withdrawn by 
 
 means of a pipet. 
 
 vives. In the higher dilutions of serum bacteriolysis is incomplete, and 
 the animal becomes toxic and may succumb after twenty-four hours, 
 double the virulent dose being used in all infections. 
 
 In the foregoing titration a serum that ife bacteriolytic in dilutions 
 of 1 : 1000 or over will be satisfactory for purposes of diagnosis. When 
 preserved in a sterile condition in separate ampules in a dark cold place 
 the titer usually remains unaltered for several months. 
 
 Dried Serum. — If the serum is dried in vacuo, it will be necessary to 
 titrate with dilutions of the dried products in bouillon, as during the 
 
Fig, 99. — A Smear of Peritoneal Exudate Removed Twenty Minutes after 
 
 Injection of Culture of Cholera in Fin. 100, 
 
 Stained with 1:10 carbolfuchsin. 
 
 Fig. 100. — Stained Preparation of Cholera before Bacterioltsis. 
 
TECHNIC OF BACTERIOLYTICf TESTS 
 
 347 
 
 process of drying the amboceptor content may be decreased. Dried 
 serum is to be preferred, however, as it keeps much longer and there is 
 no danger of rendering it worthless by contamination. After drying, 
 the product is ground in a mortar and stored in amounts of 0.1 or 0.2 
 gram in separate ampules. 
 
 In determining the bacteriolytic titer of dried serum 0.1 gram is 
 carefully weighed out and dissolved in 9.9 c.c. of sterile bouillon. From 
 this stock dilution other dilutions may be prepared, in the manner pre- 
 viously described, and injected with a loopful of the culture. 
 
 ^^--:r-^ 
 
 
 
 ^ 
 
 
 
 / 
 
 I -^x 
 
 / 
 
 / , 
 
 \ ^ ^ \ 
 
 A . ■•■ J^ - \ 
 
 / 
 
 ^ 
 
 ■^ "^ \ 
 
 - .. ^ .^ \ 
 
 1 
 
 
 v_^ \ 
 
 ■ ^■^'-\- oO;: 
 
 \ 
 
 > 1 
 
 (' 
 
 ' "", -' '^ " / 
 
 \ 
 
 V. 
 
 1 ^ i 1 
 
 X -'^ ' X 
 
 \ 
 
 V 
 
 
 Fig. 97. — Culture of Cholera Under- 
 going Bacteriolysis. A Positive 
 Pfeiffer Reaction. 
 A hanging drop of peritoneal exudate 
 removed from a guinea-pig one-half hour 
 after injection with ] c.c. of the suspension 
 shown in Fig. 99 with 1 c.c. of 1 : 1000 
 dilution of cholera immune serum. Note 
 that the bacilli are now quite short and 
 coccoid in shape. At the end of seventy 
 minutes the exudate was sterile. What 
 appears to be two or three coccoid forms 
 in apposition is really one bacillus under- 
 going lysis. 
 
 Fio. 98. — Culture of Cholera Before 
 Bacteriolysis. X720. 
 A hanging drop of cholera in normal 
 salt solution prepared from a twenty- 
 four-hour culture of cholera on agar- 
 agar. 
 
 After securing an immune 
 
 serum of satisfactory potency the 
 
 main test may be conducted. 
 
 While the technic is more difficult 
 
 than that of agglutination tests, 
 
 the results, under proper conditions, are more conclusive, for there is 
 
 less likeUhood of group or partial amboceptor activity for closely alUed 
 
 bacteria taking place. 
 
 Technic of the Pfeiffer Test. — The suspected culture to be tested 
 should be grown on agar for from eighteen to twenty-four hours, and 
 used in doses of one loopful (2 mg.). 
 
 If cholera is suspected, cholera immune serum of known titer should 
 be used. Dilutions of serum are made with sterile bouillon, and 2 c.c. 
 
348 BACTERIOLYSINS 
 
 of each dilution, representing 2, 5, and 10 tim^ the titer dose, are placed 
 in separate glasses or small test-tubes. A foifrth tube should contain 2- 
 c.c. of a 1 : 100 dilution of normal serum, according to the animal used 
 in producing the immune serum; a fifth tube should contain 2 c.c, of 
 sterile bouillon (culture control). 
 
 Two loopfuls of suspected culture are thoroughly emulsified in each 
 of the five tubes of the series, and 1 c.c. of each is injected intraperi- 
 toneally in five guinea-pigs weighing about 250 grams each. 
 
 At intervals of ten, twenty, forty, and sij{ty minutes the peritoneal 
 exudate should be removed with capillary pipfets and examined in hang- 
 ing-drop preparations. Smears may be prepared and stained with dilute 
 carbolfuchsin, although they give less information than hanging-drop 
 preparations. 
 
 If guinea-pigs Nos. 1, 2, and 3 show granule formation at the latest 
 after an hour, while in the fourth and fifth animals the bacteria remain 
 whole, motile, and well preserved, the reaction may be regarded as 
 positive and the diagnosis as established. 
 
 Pfeiffer Bacteriolytic Test in vivo in the Diagnosis of Disease. — Bac- 
 teriolysins are usually produced somewhat later than agglutinins, and 
 reach their highest point of production during convalescence. Bac- 
 teriolytic tests are used only for diagnostic purposes, when agglutination 
 reactions are negative or doubtful. The most satisfactory results from 
 these tests are obtained in cholera. Bacteriqlysis with typhoid bacilli 
 is less typical and more incomplete; with the paratjrphoid and dysen- 
 tery bacilli it is even more unsatisfactory, and in anthrax, pest, and the 
 various diseases due to cocci it does not occur. 
 
 The test is conducted in a manner similar to the preceding test, ex- 
 cept that instead of an immune serum the patient's serum is used, with 
 a known culture of typhoid, cholera, paratyphoid, etc., according to the 
 infection suspected to be present. 
 
 One cubic centimeter of the patient's serum is secured in a sterile 
 manner, inactivated by heating to 55° C. for one-half hour, and dilutions 
 of 1 : 20, 1 : 100, 1 : 250, and 1 : 500 prepared with sterile bouillon. Two 
 cubic centimeters of these dilutions are placed in separate tubes, and 
 two loopfuls of an eighteen-hour culture of the test organism emulsified 
 in each. A fifth tube contains 2 c.c. of bouillon with two loopfuls of 
 culture, and serves as the culture control. 
 
 One cubic centimeter of each dilution and of the culture control is in- 
 jected intraperitoneally into five guinea-pigs, and the exudate examined 
 after twenty, forty, and sixty minutes. 
 
TECHfnC OF BACTERIOLYTIC TESTS 349 
 
 If granule formation occurs in the first two or three animals, but is 
 absent in the culture control, the reaction may be regarded as positive, 
 and the diagnosis of typhoid, cholera, etc., considered as established. 
 
 If granule formation occurs to any extent in^the fifth animal (culture 
 control), the culture is to be regarded as unsuitable and the test repeated. 
 
 Measuring the Bactericidal Power of the Blood in vitro by the Plate 
 Culture Method (Stern and Korte). — Since the earliest days of bac- 
 teriology the aim and purpose have been to de^^ise some means by which 
 the bactericidal power of the blood could be measured, just as is done in 
 testing an ordinary germicidal substance, namely, by adding a bacterial 
 suspension to the serum and observing the effect on the bacterial con- 
 tent. This is sho-OTi by counting the bacteria in a loopful immediately 
 after adding the bacterial suspension, and repeating this at regular in- 
 tervals, the counting being done by the method of plate culture. 
 
 The use of the platinum loop in these tests is objectionable, since the 
 dose is quite variable, depending upon whether the loopful is removed 
 from the fluid edgewise, or with the loop held, flat, hke a spoon in use. 
 If the serum contains agglutinins, the results \fith any form of technic 
 are likely to be irregular, and the number of colonies upon the plate stand 
 in no relation to the actual number of microorganisms present. The 
 results may be masked by a rapid multiplication of the survivors, and 
 accordingly several controls are necessarj' witk any technic. 
 
 Neisser and Wechsberg have recommended the so-called bactericidal 
 plate culture method. By this method the patient's serum is inactivated, 
 and varying amounts mixed with definite and constant quantities of 
 bacteria. To this a constant quantity of active normal serum is added 
 as complement, to reactivate the bacteriolytic amboceptors. These 
 mixtures are then incubated for several hours. To determine whether 
 and in what proportion death of bacteria has r.esulted from the effect of 
 the bacteriolysins, agar is added, and the mixtures are then plated and 
 incubated for twenty-four hours or longer. The number of colonies are 
 then counted and the results compared with control plates of the culture 
 alone. 
 
 Stern and Korte have modified this technics slightly, and recommend 
 the procedure as a substitute for the Pfeiffer test in the clinical diagnosis 
 of typhoid fever. The method spares a certain number of animals, but 
 it is somewhat more compHcated than the Pfeiffer reaction, and its re- 
 sults are less trustworthy. As a clinical test, therefore, it is to be recom- 
 mended only in cases in which the agglutination reactions have yielded 
 uncertain results, although with practice and. care the test oftentimes 
 
350 
 
 BACTERIOLYSINS 
 
 yields uniform and satisfactory results, and may be employed in making 
 special investigations for determining the bactericidal power of the blood, 
 as after typhoid immunization. 
 
 Technic of the Test. — The requisites for success are that all vessels 
 and diluting fluids, as well as the serums employed, should be absolutely 
 sterile. To secure uniform and reliable results it is necessary to famil- 
 iarize oneself with the technic by repeated practice. In order to carry 
 out the steps in the technic according to strict aseptic bacteriologic 
 principles the services of an assistant are required. 
 
 Sterile bouillon is largely used throughout, to maintain proper os- 
 motic conditions. 
 
 With so many tubes and controls, it is important that all tubes and 
 Petri dishes be properly labeled with a wax-pencil to avoid confusion. 
 
 One cubic centimeter of sterile patient's sepum and an equal amount 
 from a normal and healthy person to serve as a control are inactivated 
 by heating to 55° or 56° C. for half an hour. These serums are then 
 diluted 1 : 50 by adding 49 c.c. of sterile salt solution to each and mixing 
 thoroughly. 
 
 Complement is prepared by securing 4 to 5 c.c. of sterile rabbit's 
 blood and separating the serum. This serum is chosen because it should 
 be from the same species of animal as that used in producing the immune 
 serum to be tested. In determining the bactericidal titer of human 
 serum either guinea-pig or rabbit complement serum may be used. 
 Dilute 2 c.c. of this fresh serum (not over eighteen hours old) with 18 
 c.c. of sterile normal salt solution (1 : 10). Dose, 0.5 c.c. 
 
 Secure a good twenty-four-hour bouillon culture of typhoid bacilli 
 (grown in the incubator) and dilute 1 : 500 by thoroughly mixing 0.1 
 c.c. of tlie culture in a flask containing 50 c.c. ^f sterile normal salt solu- 
 tion. This dilution of culture, when used in constant doses of 0.5 c.c, 
 generally yields satisfactory results, but it may be necessary to try it 
 out beforehand, for the control plates must regularly show a uniformly 
 good growth and contain many thousands of colonies before uniform 
 results can be expected. The bactericidal effect will then be distinctly 
 shown by the reduction, in the proper plates, of this large number of 
 colonies to zero or near it. 
 
 Place 1 c.c. of sterile neutral bouillon in each of 12 test-tubes which 
 have been plugged with cotton, sterilized, and large enough to hold at 
 least from 12 to 15 c.c. Place in the first of these tubes 1 c.c. of the 
 diluted patient's serum and mix thoroughly by alternate sucking up and 
 forcing out of the fluid; then, with the same pipet, draw up 1 c.c. and 
 
TECHNIC OF BACTERIOLYTIC TESTS 351 
 
 transfer it to the second tube of the series; mix as before, and transfer 
 1 c.c. to the third tube; continue in this manner to the last tube, 
 from which, finally, 1 c.c. is discarded. 
 
 Each tube now contains 1 c.c. of fluid, representing dilutions of 1 : 100, 
 1 : 200, 1 : 400, 1 : 800, 1 : 1600, 1 : 3200, and so on up to 1 : 204,800 of 
 the patient's serum. Higher or lower dilutions than these may be em- 
 ployed. It is very desirable that the dilutions^be high enough to secxrre 
 the limit of bactericidal activity, so that the la,st plates will show an in- 
 crease in the number of colonies. 
 
 Arrange four tubes with the normal control serum, each containing 
 1 c.c. of fluid and representing the first four dilutions, viz., 1 : 100, 
 1 : 200, 1 : 400, and 1 : 800. 
 
 Label each tube carefully with the initials of the patient and the dilu- 
 tion it contains. Add 0.5 c.c. of the diluted- bacterial emulsion, and 
 finally 0.5 c.c. of the diluted complement serum, to each tube. 
 
 All manipulations should be made with strict bacteriologic care and 
 with the aid of an assistant, as the introduction of contaminating micro- 
 organisms that may cause spore-formation will considerably vitiate the 
 value of the test. 
 
 The following controls are necessary: 
 
 1. 0.5 c.c. culture directly in a sterile Petri dish -t- 5 to 10 c.c. of 
 
 neutral agar cooled to 42° C. and thoroughly mixed. This 
 control will show the original number of bacteria contained in 
 the enmlsion. Mark as control No. 1. 
 
 2. 0.5 c.c. culture -1- 1.5 c.c. bouillon. Mark as control No. 2. 
 
 After three hours' incubation this tube receives the usual 
 amount of agar and is plated. It will show to what degree the 
 culture has multiplied in the incubator. 
 
 3. Since the complement serum frequently contains bacteriolysin, 
 
 it is necessary to control this element carefully. To a series of 
 four tubes add the following doses of the serum, diluted 1 : 10: 
 1 c.c, 0.5 c.c, 0.2 c.c, 0.1 c.c. Add 0.5 c.c culture to each 
 tube, and sufficient bouillon to make the total volume in each 
 tube 2 c.c. 
 
 4. 0.5 c.c. complement -|- 1.5 c.c. bouillon. To control the sterility 
 
 of the complement serum. 
 
 5. 1 c.c. of patient's serum (1 : 100) + 1 cc bouillon. To control 
 
 the sterility of this serum. 
 
 6. 1 c.c. of the control serum (1 : 100) + 1* c.c. of salt solution. To 
 
 control the sterility of this serum. 
 
HACI'l'.HIOIiVSINS 
 
 7. 1 c.(^ of Irlu' piilioiii's sonim in diluliuii of 1 : 100 |- 0.r> c.c. cul- 
 Uirc -\- O.f) (■.!■. bouillon. .\ coiiij-ol on the possililr ijI'chcmcc of 
 coiiiplcincnl, in t.iiin scriini. 
 S. 1 c.r. of Ulc couliol f^cruin in diiulion of I : KIO H ().,S v.r. cnlliii-c 
 + ().;") cc. l)ouillou. \ conli'ol on Uir powHililc pirscncc of 
 coniplcnicnl,. 
 
 All l.lilicH wilili IIk^ cxccpliion of \hv liruli coii-li-ol wliirli Ii.mm hccn |il.'ilc(l 
 nrr pl.'iccd in ;in inciili.'iior ni '.i7" < '. for lin-cc Jionis. A(, I, lie end of I Imm 
 tinic tJic conlrcnls of ('.■icli I.mIic ■aw piiiJ.cd in iW'iilr.'d ;itj;:i,i'. Slri'ii<' I'dri 
 diislics ulioidd li(^ jjiopi'iiy ;nid ])liunly Itdu'lcd willi ;i, \v:i\ pencil luid 
 ;uT,'inn'(Ml ill onku'. A lliisk of ])lii,in ncnii'.'d .M^'iir is incll.cd in lioilinfi; 
 Vv;d,i'r ;mi<I (^ool('<i tol'J"('.. Tiic Iriihcs .'ire rcn'iciNrd from llir inrnlud^nr 
 li.nd wli.'ikrn j^rrd-iy .'ind wifii IJif !l-iil of ;i.n .'insinf.'inlr from i") |.o S i-.r. of 
 :\ti:i\' ur(^ iiddcd cin'cfnlly lo cnrli tulic willi n, slcrilc 10 c.i'. pipclr. 'I'lic 
 conlcnfs :i,ri' mixi'd by frcnIJc roddion of ili(> l(d>r;ind iJii-n ponrcd in Uic 
 (^orrcspondinfi; l'i'l,ri dinii, followed liy !i,n inMiLionn,! niixinn' .■irrordinjj; in 
 tlu! nsnid l),'iclr('riolof!;ii'. )ir-oci'dnr('. WilJi or'dfmiiry Hpci'd Ilir vvlioli' Hi'l. 
 of lubes may be ixmred in h sidisfii.eiory rTiiUiiier befor'i^ l.iie IIuhK uf ,'i,(i;iu' 
 }r;iM luul IJme to lliuden. 
 
 AnoUier mid-liod of pl.'iiin^; conHiHtH in ))ipi'l'inn \,\]i' eonfenlii of ench 
 inbe info il,n <',()i-ren[)ondin)); <lisli, iuid IJien wiiiiliini.'; IJie fube vvilJi ini 
 addiiion.'d 1 e.e. of HJ.erile huH/ Holnfion, l.o remove all fra,een of Herinn luid 
 cull.nre. Or ilu! end of e.M.eli lube miiy be llamed mid ilie conlcnl.H 
 poured direi'lly itlfo a diHii. If IJliM Irielliod Ih a,dopl.ed^ HmaJI le;il, Inben 
 Bliould be uned. Wliile IJie inelJiod is mole con Venietll,, if \H llHUally iinl. 
 HO ii,ccui'.'d,e an tluf firHl fwo meiliod:i. 
 
 NeisK.er phifeH Ind- 5 or 10 drops from eacli tube. TIk; dose dccidi^d 
 Upon is tlie one employed iliroiiKlionf. I'or example, ii. would be erro- 
 ireoiis l.o fake 5 (lro[)H from one tube and lit fnun anol.lier. NeinMcr, 
 liowever, uses miicli Htriulli:r jitnourd'.H of tli<' H<'rnm, as 1, U.'.',, 0.1, O.O.'l, 
 and 0.01 c-.c,., instead of the nincli liij^lier dilutions t'^iven in this tecliiiic. 
 '"i'liesediCfereiiceH inusi/ bi: remembered and tlie pr(jperdilutioiiMeinployeil, 
 and but 5 to |0 drops should be |)lided in performiiip: the ba.ctericidiil 
 plali' test a.ccordiiifr to Neisser's lechnic. 
 
 Tiipfer a,nd .lalT<> jioiu' a, tJiili layer of !i,f.far jnto .'i, I'etri diiJi a,ud liJIoW 
 it to lia,rdeii. Ujiou i/liis they pour the culture iiernm a,(';ar mixture, 
 which, after H(M.tlin(»;, is covered with a.nother" thin hi.yer of ii,)z;ar. In 
 this way a, culturi' in the wid-ei' of eonilen:;aliiii;i is avoided. In the iiHiinJ 
 ti;clinic, 1,his MKiy lie a,void<'d by I urriiriK i,lie plnJe:^ oyer soon after hiU'dcn- 
 ing occurs so tlial- the w;d-ei- of cdiidens.'ition (^olleclji on the cover. 
 
TECHNIC OF BACTERIOLYTIC TESTS 
 
 353 
 
 All plates are incubated at 37° C, for from twenty-four to thirty-six 
 hours and the colonies then counted. In some plates the number may 
 b(; so large that counting will be inaccurate" and unnecessary. Sig- 
 nificance can be attached only to marked and easily recognizable dif- 
 ferences. According to Neisser, the growth is best and most rapidly 
 described by means of approximate cstirrjatfs, nsing a scheme somewhat 
 like the following: or almost none; about! 100; several hundreds; 
 thousands; very many thousands; infinite numbers. A distinct Vtac- 
 tericidal action is then present only if the controls react normally, and 
 if a reduction of colonies from an infinite numbel- or many thousands to 
 or very few has occurred. As previously state4, the test can then be re- 
 garded as satisfactory only if the lower limits of Ijactericidal activity have 
 been reached and the last plates show an increase- in the numberof colonies. 
 Examination of thes<; plates is very much facilitated by using a colony 
 counter, that of Stewart being particularly serviceable with agar plates. 
 
 The controls are first examined and the results recorded, and finally 
 those of the patient's serum are set down. 
 
 As a practical illustration, the results of a test with a rabbit typhoid 
 immune serum are given, because this is in general an average example 
 of those usually obtained: 
 
 1 c.c. DrbUTroNS 
 
 OF Immune 
 
 Seuuu 
 
 Ij 100 ... . 
 
 1 : 200 
 
 1 : 400 
 
 1 : 800 
 
 .1:1600 
 
 1:3200 
 
 1:6400 . . . . 
 1:12,800 . . . 
 
 1:25,600 . . 
 
 1:51,200 . .. 
 
 1:102,400 . . 
 
 1:204,800 .. 
 
 2r 
 
 Twenty-four- 
 
 HOUH CULTUriK 
 OF li.VCILLUB 
 
 Typhobuh (Dilu- 
 tion, 1:600), Co. 
 
 0.5 
 
 0..5 
 
 0.5 
 0.5 
 0.5 
 0.5 
 
 0.5 
 0.5 
 
 0.5 
 
 0.5 
 
 0.5 
 
 0.5 
 
 Rabbit Comple- 
 ment SiCKUM, 
 1 ; 10, C.c. 
 
 0.5 
 
 0.5 
 
 0.5 
 0.5 
 0.5 
 0.5 
 
 0.5 
 0.5 
 
 0.5 
 
 0.5 
 
 0.5 
 
 0.5 
 
 Places Poured after Three Hours 
 
 AT .37^ C- Counted after 
 
 TwK-NTr-FOUR Hours 
 
 Immune Serum 
 (Inactivated; 
 
 About, .500 colo- 
 nSes 
 
 AhsDut 125 colo- 
 nies 
 
 Sterile 
 
 Sterile 
 
 Sterile 
 
 Two colonies; 
 practically 
 s'terile 
 
 lOOcolonics 
 
 Ab'out 800 colo- 
 iiie.s 
 
 About 2000 col- 
 onics 
 
 M u!iy thous- 
 and 
 
 Many thous- 
 4nd 
 
 Many thous- 
 and 
 
 Normal Rabbit 
 Serum (In- 
 activated) 
 
 Many thousand 
 
 ?\lany thousand 
 
 Many thousand 
 Many thousand 
 
354 bacteriolysins 
 
 Controls 
 Control 1: 0.5 c.c. culture plated immefiiately : many thousands 
 
 of colonies. 
 Control 2; 0.5 c.c. culture plated after being incubated three hours: 
 
 plate very crowded; number of bacteria increased. 
 Control 3: Varying amounts of normal rabbit serum used as com- 
 plement. The first plate containing 0.1 c.c. serum showed a 
 slight bactericidal action; the remaining plates showed many 
 thousand of colonies and were comparable to control No. 1. 
 Control 4: 0,5 c.c. complement serum: sterile. 
 Control 5: 0.01 c.c. inactivated immune serum: sterile. 
 Control 6: 0.01 c.c. inactivated normal control serum: plate shows 
 
 several colonies of contaminating bacteria. 
 Control 7: 0.01 c.c. inactivated immune serum plus culture: plate 
 
 shows many thousands of colonies. 
 Control 8: 0.01 c.c. of inactivated normal serum plus culture: 
 many thousands of colonies. 
 An examination of this experiment shows that the dose of culture 
 was satisfactory, that the culture increased ^uring the three hours of 
 primary incubation, that the complement serum was very slightly 
 bactericidal in a dose lower than that used in making the test, and that 
 the technic was fairly satisfactory, slight contamination being present 
 in but one plate — ^that of the inactivated normal control serum. 
 
 The most striking result observed is the absence of bactericidal ac- 
 tivity in the first two plates, which contained the largest amount of 
 immune serum and where one would naturally expect to find complete 
 sterility. The titer of this serum was betwe.en 1 : 3200 and 1 : 6400. 
 According to Neisser and Wechsberg, these paradoxic results are caused 
 by deviation or "deflection of complement," fis was explained in a pre- 
 vious chapter. In bactericidal experiments, according to Neisser, the 
 deflection is caused by an excess of amboceptors in the immune serum. 
 In a mixture of bacteria, complements, and large amounts of amboceptor 
 the complement is bound not only by the amboceptors anchored to the 
 bacteria, but also in large measure by "free" amboceptors that are not 
 anchored to bacteria. A portion of the anchored amboceptor, therefore, 
 finds no complement at its disposal, and is, therefore, unable to exert any 
 bactericidal action, which gives rise to a relative lack of complement. 
 
 This phenomenon resembles the action of agglutinoids in the agglu- 
 tination reaction, where, in the lowest dilutions, agglutination is feeble 
 or absent, but becomes manifest in the higher dilutions. 
 
TECHNIC OF BACTERIOLYTIC TESTS 355 
 
 In the foregoing experiment the immune serum used was several 
 weeks old; perfectly fresh serums are not so liKely to show this so-called 
 "deflection of complement." In many instances, and especially if a 
 fresh serum is used, one caimot help thinking that agglutinins may be 
 responsible for the absence or diminution of bactericidal activity in the 
 lower dilutions. It is certainly true that hemagglutinins considerably 
 inhibit hemolysis, and this is especially the case with serums of low hemo- 
 lytic activity. With more potent serums the agglutinins are diluted 
 until activity ceases and hemolysis is ready and complete. Reasoning 
 from analogy, therefore, the absence or dinaiaution of bactericidal ac- 
 tivity may be due to agglutinins, and the theory of "deflection of com- 
 plements" may be emphasized a little too strongly. 
 
 According to Hahn, normal human serums show bactericidal activity 
 in only about one-third of the cases, and the titer is only very exception- 
 ally demonstrable in dilutions higher than 1 : 500. The serums of ad- 
 vanced cases of typhoid fever or of those but recently recovered are 
 bactericidal in dilutions greater than 1 : 1000, and may reach 1 : 50,000 
 or higher. Similarly after typhoid immunization the patient's sermii 
 may show a high bactericidal titer. Weston has found such sermns 
 active in dilutions of 1 : 20,000, higher dilutions not being used. 
 
 Besides being used in typhoid, the plate culture method has been 
 employed for experimental purposes in cholera, dysentery, paratyphoid, 
 and other infections with bacilli of the typhoid-colon group. With 
 these, however, the test possesses but little diagnostic significance. 
 
 The bactericidal titer does not run strictly parallel with the agglu- 
 tinins or complement-fixing bodies. 
 
 Measuring the Bactericidal Power of the Blood by Capillary Pipet 
 Method (After Wright). ^ — ^By this technic it is= sought to overcome the 
 fallacies of the "loopful" method of measurement and those due to 
 agglutination of the test organism. 
 
 The native complements of the patient's own serum are used; hence 
 the serums used in this technic must be fresh. Quantitative titration is 
 accompHshed by furnishing varying dilutions of culture, with a constant 
 quantity of serum. A series of volumes of serum is taken, and to these 
 are added equal quantities of progressively increasing dilutions of a 
 counted bacterial culture. The mixtures are kept at 37° C, for twenty- 
 four hours, after which each is introduced intoi nutrient broth and culti- 
 vated to see whether a complete bactericidal effect has been exerted. 
 
 'Wright, A. E.: Technique of the Teat and Capillary Glass Tube, 1912, Lou- 
 don, Constable & Co. 
 
356 BACTERIOLYSINS 
 
 The largest number of bacteria that a constant quantity of serum has been 
 able to kill furnishes a measure as to its bacteriddal power. 
 
 After a few technical details have been mastered, this method is 
 quickly performed and yields fairly constant and reliable results. It is 
 not adapted for the titration of old immune serums, but is a ready clin- 
 ical test, finding a special field of usefulness in- determining the bacteri- 
 cidal powers of the blood after typhoid immunization. 
 
 Requisites for Carrying out the Test. — 1. A. specimen of the patient's 
 blood is collected aseptically, a process that may be accomplished by 
 thoroughly cleansing the finger with alcohol and collecting blood in a 
 sterile Wright capsule, or better, perhaps, by means of venipuncture, 
 when from 2 to 5 c.c. may be collected aseptically in a sterile centrifuge 
 tube. The control blood from a healthy individual may be collected 
 in a capsule. The serums are carefully separated and pipeted into small 
 sterile test-tubes; 1 c.c. of each is ample for the test. 
 
 2. A twenty-four-hour-old broth culture of the test organism (a 
 young culture is required, because such a culture contains a few dead 
 microorganisms and the absorption of bactericidal elements by dead 
 organisms is thus avoided). 
 
 3. About two dozen "looped pipets," made according to the direc- 
 tions given in Chapter I. 
 
 4. Sterile neutral broth for making dilutions and cultivations. This 
 is prepared and sterilized in the usual manner, from 5 to 10 c.c. being 
 placed in test-tubes. When working with the typhoid bacillus a special 
 broth containing 1 per cent, of mannite and suSicient litmus to color 
 it a deep blue (Smallman) should be on hand. 
 
 5. Two dozen small test-tubes, plugged and sterilized, for making 
 dilutions of the culture. 
 
 Preparation and Enumeration of the Bacterinl Culture. — As the prin- 
 ciple of the test depends upon measuring the bactericidal activity of the 
 blood according to the number of organisms that are killed, it is neces- 
 sary to prepare somewhat high dilutions and count the number of 
 organisms in a unit volume in each dilution. 
 
 For this purpose place 10 sterile test-tubesr in a rack, and arrange 10 
 sterile Petri dishes on the table to correspond to these. To the first 
 tube add 4.9 c.c. of plain sterile broth; to the second, fourth, sixth, 
 eighth, and tenth tubes add 1 c.c, to the third, fifth, seventh, and ninth 
 tubes, add 4 c.c. 
 
 With a sterile graduated 1 c.c. pipet add 0.^1 c.c. culture to the first 
 tube and then discard the pipet by placing it in a cylinder of disinfecting 
 
i 
 
 f 
 
 ^j^^ 
 
 Fig. 101. — Bactericidal Test (Looped Pipet Method of Wright). 
 The pipet shown on the extreme left contains th'e mannite-litmus broth and an 
 equal volume of patient's serum and emulsion of typhoid bacilli; the second pipet 
 shows the serum and biicillary emulsion mixed; the middle pipet shows the serum- 
 bacillary mixture cultured in the broth; the fourth pipet shows acid production in 
 the brotli by hving bacilli which escaped destruction afier twenty-four hours' incuba- 
 tion; the fifth pipet (extreme right) is the seriuii control. The broth, being clear 
 untl unchanged, sliows that the serum was sterile. 
 
TECHNIC OF BACTERIOLYTIC TESTS 357 
 
 solution or hot watpr. With a second sterile pipet mix the contents of 
 tube No. 1 by carefully sucking it in and forciiig it out of the pipet sev- 
 eral times, and place 0.1 c.c. in Petri dish No; 1. Then, with the same 
 pipet, transfer 1 c.c. to the second tube, mix well, and place 0.1 c.c. in 
 Petri dish No. 2; next transfer 1 c.c. to the third tube, mix, and place 
 0.1 c.c. in the third Petri dish. Continue in tljis way until the last tube 
 is reached, when 1 c.c. is discarded into a germicidal solution and 0.1 
 c.<!. placed in the tenth Petri dish. To each Petri dish now add from 8 
 to 10 c.c. of neutral agar at 41° C, and mix thoroughly. After the 
 plates have hardened they are turned over in order that the water of 
 condensation may collect on the cover. They are then incubated for 
 twenty-four hours, and the colonies carefully counted. 
 
 We now have the following dilutions of culture: 1 : 50, 1 : 100, 
 1 : 500, 1 : 1000, 1 : 5000, 1 ; 10,000, 1 : 50,000, 1 : 100,000, 1 : 500,000, 
 and 1 : 1,000,000. Since but 0.1 c.c. of these dilutions were plated, the 
 total number of colonies in each plate must be multiplied by 10 in order 
 to obtain the approximate number per cubic centimeter of the various 
 dilutions. 
 
 To secure fairly uniform results, the various dilutions must be 
 thoroughly mixed and the pipeting be accurately performed. We have 
 found that this method usually gives better results than are obtamed 
 by plating but one or two of the higher dilutions, which are used as a 
 basis for calculating the number of bacteria in the other dilutions. 
 
 Filling the Pipets. — ^With a wax pencil make a mark upon the capillary 
 stem of a sterile looped pipet at a point 2 cm. from the lower end, and 
 fit a rubber teat to the barrel. The point of the capillary stem is now 
 broken off between the finger and thumb, the lower portion is sterilized 
 ia the flame, and the air is expelled from the teat. 
 
 Mannite broth is then aspirated into the pipet until the bulb is about 
 two-thirds full and the capillary portion contains an an- column rismg 
 to at least 5 cm. from the end (Fig. 101). 
 
 The end of the capillary stem is now inserted into the tube contain- 
 ing the patient's serum, and the serum is allowed to flow in until it 
 reaches the pencil mark. 
 
 The orifice of the pipet is now raised above the surface of the serum, 
 and a small bubble of air is admitted into the tube, to serve as an index 
 for the measurement. The end of the capillary stem is now carried 
 into the tube containing the highest dilution of the organism, and the 
 culture is allowed to flow in until the bubble of = air has been carried just 
 past the pencil mark. 
 
358 BACTEEIOLYSINS 
 
 The serum and culture must now be mixed, being careful not to con- 
 taminate the broth. This is effected by blowing these two volumes out 
 into a sterile mixing tube and drawing up and blowing out the fluid 
 several times in succession. By starting witt the highest dilution, the 
 same mixing tube may be used throughout the series. With an air- 
 bubble of at least 5 cm. between serum and broth, this can be quite easily 
 achieved without driving the sterile broth down from the bulb of the 
 pipet into the lower part of the capillary stem and contaminating it there. 
 
 The column of mixed serum and culture is now drawn up into the 
 middle region of the capillary stem and the lower end of the tube is 
 sealed. The teat is then removed, and the dilution that has been used 
 is written on the barrel of the pipet. 
 
 The series of pipets are now filled with the nine measuring dilutions 
 of the culture. 
 
 Controls. — The serum from a healthy individual, or, better still, the 
 pooled serums of several persons, may be used in precisely the same way, 
 with at least the second, fourth, sixth, and eighth dilutions of culture. 
 
 Several culture controls are advisable. The pipets are filled with 
 mannite broth in the usual manner, and then with one volume of at least 
 four different dilutions, usually 1 : 50, 1 : 5000, 1 : 100,000, and 1 : 1,000,- 
 000. The tubes are sealed, labeled, and incubated together with those 
 concerned in the test proper. 
 
 One pipet is to contain the usual quantity of broth and one volume 
 of the patient's serum. This is a control on the sterility of the patient's 
 serum. A similar preparation is made with the control serum to serve 
 as a control on its sterility. 
 
 When the whole series of tubes have been filled, these are placed 
 upright in a large test-tube or cylinder labeled with the date and the 
 source of the serum, and incubated at 37° C. for from eighteen to twenty- 
 four hours. 
 
 Test for the Germicidal Activity of the Serunt. — ^The serum and culture 
 in the capillary portion of the tube must now be mixed with the broth in 
 the barrel. This is accomplished by taking each pipet in hand singly, 
 and heating the lower portion of the capillary stem in a "peep-flame" 
 and drawing out into a small thread with a p^ir of forceps. 
 
 A collapsed teat is now fitted over the barrel, and the negative pres- 
 sure carefully regulated by keeping the finger and thumb in position on 
 the teat, and the finely drawn out end of fie capillary stem gently 
 snapped across. The colunm of scrum and culture will then be carried 
 up into the bulb of the pipet. The end of the tube is now sealed. 
 
BACTERIOLYTIC SERUMS IN THE TEEATHENT OF DISEASE 359 
 
 When the whole series of pipets has been dealt with in similar fashion 
 they are returned to the incubator for another twenty-four hours. 
 
 Reading the Result. — The continued sterility of the broth may be de- 
 termined from direct naked-eye inspection. 
 
 When a complete bactericidal effect has been secured, the broth will 
 have undergone no color change, but will still be clear. 
 
 When a growth of the typhoid bacillus has occurred, a perceptible 
 cloudiness will be apparent in the broth, and the color will have changed 
 from blue to reddish-blue, indicating that acid has been formed from the 
 mannite. 
 
 Turbidity of the broth without a change of color would indicate the 
 admission of contaminating microbes that were" unable to split mannite 
 with the formation of acid. 
 
 The controls are first examined and recorded. All the culture con- 
 trols should show a growth and change of color. The serum controls 
 should be sterile ; the normal serum controls may or may not be sterile, 
 depending upon the amount of natural bactericidal amboceptors which 
 it contains for the test organism. 
 
 The pipets containing the patient's serum are now examined, and a 
 simple numeric expression for the result is obtained by referring to the 
 result of the enumeration of the various dilutions, as determined bj^ the 
 counting of the plates. In this manner the number of bacteria contained 
 in 1 c.c. of the lowest dilution of the bacterial culture that has been com- 
 pletely sterilized is calculated. This will give the number of micro- 
 organisms which 1 c.c. of serum would be capable of killing. 
 
 Although this test affords a convenient basis for the comparison of 
 serums, it must be understood that the expression is entirely arbitrary, 
 and will vary according to the culture emplqyed; this is true of any 
 technic for determining the bactericidal titer of a scrum. 
 
 BACTERIOLYTIC SERUMS IN THE TREATMENT OF DISEASE 
 
 While bacteriolysis is readily demonstrated with some bacteria both 
 in vivo and in vitro, nevertheless, when such- serums are used thera- 
 peutically, beneficial results are not dependent solely upon lysis of the 
 infecting bacterium, but are usually the result of a combination of lysis 
 with increased phagocytosis, due to the simultaneous presence of bac- 
 teriotropins (immune opsonins). When one recalls the close similarity 
 in general properties that exists between bacteriolysins and bacterio- 
 tropins, the difference between extracellular and intracellular lysis is 
 
360 BACTEKIOLYSINS 
 
 relatively slight, and tends to strengthen Metiohnikoff's views regarding 
 the close relationship of the bacteriolysins to leukocytic products and 
 phagocytosis. While extracellular lysis can occur in the absence of 
 cells, especially in vitro, yet in the administration of antibacterial serums 
 the activities of the leukocytes are much in evidence, extracellular lysis 
 or bacteriolysis proper being rather subsidiary to intracellular lysis or 
 phagocytosis. 
 
 The preparation and methods of standardization of antibacterial 
 serums are given in the chapter on Passive Immunization. Most effort 
 in this direction has been expended on the preparation of antiserums for 
 the treatment of meningococcic, streptococcic, pneumococcic, and 
 gonococcic infections, and the greatest success has been attained with 
 the various antimeningococcic serums. 
 
CHAPTER XX 
 HEMOLYSINS 
 
 Hemolysis is the term applied to the solution or lysis of red blood- 
 ccyrpuscles. Strictly speaking, it would include the disintegration and 
 solution of the stroma, although in practice the term is applied to any- 
 process in which the cells are so injured as to liberate hemoglobin into 
 the surrounding fluids, with or without solution of the stroma. 
 
 Hemolysis may be caused by various physical, chemical, and specific 
 agencies. The prolonged agitation of blood with glass beads, for in- 
 stance, may result in the mechanical rupture of erythrocytes. The 
 addition of blood to hypotonic solutions of sodium chlorid or to plain 
 water, results in ready and complete hemolysis, the fluid being trans- 
 formed from an opaque red suspension of erythrocytes to a clear, trans- 
 parent red fluid. Various chemicals, such as acids and alkalis, may also 
 produce hemolysis. As previously stated, a few bacterial toxins, such 
 as tetanolysin and staphylolysin, are known tq be hemolytic; the same 
 is true of certain vegetable toxins, such as abrin and ricin, and of some 
 animal toxins, such as cobra, toad, and scorpion venoms. 
 
 Just as bacteria may be killed and possibjy broken up by specific 
 bacteriolytic amboceptors and complements, so, in like manner, hemol- 
 ysis may be caused by specific hemolytic antibodies known as hemolysins. 
 Working in unison with complements, the mechanism of both bacterio- 
 lysis and serum hemolysis is probably identical. The simplicity of 
 hemolytic experiments, the rapidity with which they may be performed 
 and terminated, and the ease with which hemolysis maj^ be observed 
 by the naked eye have rendered the specific serum hemolysins particu- 
 larly useful in the study of amboceptors and of complements, and of 
 cytolytic phenomena in general. In fact, bacteriolysis was not thor- 
 oughly understood until Bordct discovered the hemolysins, and demon- 
 strated the analogy that exists between bacteriolysis and hemolysis, a 
 discovery that led to a vast amount of research work and controversy, 
 to many important discoveries, and to the final evolvement of diagnostic 
 reactions of great value. 
 
 Historic. — For many years physiologists were aware that the bloods 
 of various animals transfused into man or animals of a different species 
 
 361 
 
362 HEMOLYSINS 
 
 were more or less directly injurious, and incapable of replacing human 
 blood. In 1875 Landois demonstrated experimentally that while trans- 
 fusion of blood from one animal to another of a different species may 
 prove injurious and even fatal, transfusion to an animal of the same or of 
 very closely related species produced no ill effects. The explanations 
 offered were inadequate, until later researches^ on the hemolysins showed 
 that the normal blood-serum of one animal may contain hemolysins for 
 the er5rthrocytes of other animals, and, consequently, upon transfusing 
 this blood to another animal the hemolysin acting with the complement 
 present produced hemolysis in vitro, thereby explaining in part the tox- 
 icity of the ahen blood. 
 
 In 1898 Belfanti and Carbone made the observation that the serum 
 of a horse receiving several injections of rabbit blood was toxic for rab- 
 bits, whereas normal horse serum was without injurious effects. 
 
 At about the same time Bordet published his epoch-making discov- 
 eries. He observed that while normal guinea-pig serum had little or 
 no hemolytic action on rabbit erythrocytes, the serum of a pig that had 
 received a few intraperitoneal injections of rabbit blood was able quickly 
 and completely to hemolyze rabbit blood, just as an animal may acquire, 
 through immunization with cholera, the property of dissolving cholera 
 vibrios. He demonstrated further that this acquired hemolytic activity 
 was highly specific, for when animal A was immunized with the corpuscles 
 of animal B the serum of A acquired the power of hemolyzing only the 
 erythrocytes of B, and possibly of other animals closely related zoologic- 
 ally. It was found also that the hemolytic activity of an immune serum 
 was lost by age or could be removed by heating; that in either case the 
 serum could be reactivated by the addition of a little normal serum or 
 peritoneal exudate — phenomena closely resembling that observed in 
 bacteriolysis, and due to the action of a thermolabile body or alexin and 
 a second and specific thermostabile antibody named by Bordet the 
 "substance sensibihsatrice." 
 
 These observations were soon confirmed' by Landsteiner and von 
 Dungern, and were followed by very extensive studies by Ehrlich and 
 Morgenroth, who likewise confirmed Bordet's experiments, but offered a 
 different explanation for the mechanism of the phenomenon, according 
 to the side-chain theory, and renamed the alexin and sensitizing sub- 
 stance concerned in the process "complement" and "hemolytic ambo- 
 ceptor" or "inunune body," respectively. 
 
 Definition. — Hemolysins [Gr., aliJia — blood + \vav = to dissolve] 
 are antibodies in a serum that, when acting with complement, have the 
 
NATURE OF HEMOLYSINS 363 
 
 -power of "lysing" or breaking up red blood-corjiusdes, or so altering their 
 envelop as to allow the hemoglobin to escape. 
 
 Nomenclature. — Normal hemolysins are those found in normal se- 
 rums; specific or immune hemolysins are those produced as the result of 
 the injection of blood-corpuscles from an animal of a different species. 
 Heterolysins are the hemolysins formed by immunization with corpuscles 
 of a different species (the immune hemolysins). Isolysins are hemoly- 
 sins for the corpuscles of animals of the same, species. Autolysins are 
 hemolysins that act upon the corpuscles of the same animal and are quite 
 rare. It should be remarked that isolysin and autolysin are not strictly 
 synonymous terms, as the former does not act upon the corpuscles of the 
 animal producing the hemolysin, but may hemolyze the corpuscles of 
 other animals of the same species. For example, Ehrlich has shown that 
 the serum of a goat that had received several injections of blood from 
 other goats, although actively hemolytic for the corpuscles of goats 1, 2, 
 4, 5, 6, 9, and less so for goats 3 and 8, was not able to hemolyze those of 
 goat 7 or of itself at all. This immunity of the corpuscles of an animal 
 to its own isolysin was subsequently shown to be due to a complete 
 absence of suitable receptors in its corpuscles. Therefore in cases in 
 which a large internal hemorrhage occurs and the blood is absorbed an 
 autohemolysiu is not produced, or produced only in small amounts, and 
 likely to be followed by the formation of an anti-autolysin which 
 regulates the process of blood destruction within physiologic limits. 
 Although little is known concerning the processes of normal blood destruc- 
 tion, as in the disposal of old erythrocytes, it is possible that an auto- 
 hemolysiu is being produced, and that its activity is held within normal 
 limits by an anti-autolysin. A disturbance of this physiologic equilib- 
 rium may then be the basis of certain types of primary anemia character- 
 ized by excessive blood destruction. 
 
 Nature of Hemolysins. — According to the side-chain theory, he- 
 molysins are amboceptors or antibodies of the=third order, requiring the 
 action of a complement before hemolysis can be produced (Fig. 102). 
 Bordet showed that two substances were concerned in the phenomenon 
 of serum hemolysis, although his views on the mechanism of the process 
 differ from those advanced by EhrUch and Morgenroth. 
 
 Ehrlich argued that the hemolytic amboceptor or hemolysin must 
 be an antibody to the receptors of the red blood-corpuscles used in the 
 process of immunization, and if this is true, it ought to unite with the 
 corpuscles. Taking the serum of a goat that had been injected with and 
 was hemolytic for the erythrocytes of a sheep, he destroyed the comple- 
 
364 HEMOLYSINS 
 
 ment by heating the serum to 56° C. To this he added some sheep's 
 corpuscles, and allowed the mixture to stand for a short time at room 
 temperature, after which it was centrifuged and the supernatant fluid 
 pipeted off in another test-tube. No hemoljfsis had occurred, and the 
 corpuscles were to all appearances imaltered, but it was now found that 
 if a small amount of normal goat serum, as complement, was added to 
 the corpuscles and the mixture placed in the incubator, hemolysis oc- 
 curred. By adding sheep's corpuscles and normal goat serum (comple- 
 ment) to the supernatant fluid that had been removed to a separate test- 
 tube, hemolysis did not occur. This experiment indicated, therefore, 
 that the red blood-corpuscles had combined with all the antibody. 
 That the action was specific was shown by the fact that the corpuscles 
 of other animals, such as rabbits or goats, f or, example, exerted no com- 
 bining power when used instead of the sheep's.<;ells. The union between 
 
 cell and antibody was considered as 
 being in the nature of a chemical com- 
 bination and quite firm, as repeated 
 washing of the cells with normal salt 
 
 ^ ^ „ solution did hot break it up. 
 
 Fig. 102. — Theoretic Structuke . ^ 
 
 OF A Hemolytic Amboceptor Havmg shown that the antibody 
 
 (Hemolysin). , had a combining affinity for the cells, 
 
 ^4, Amboceptor; C, comple- 
 ment; r, receptor of the corpuscle; it was important to Solve the question 
 
 t^XthZ Slementopa «f ^he relatioh. of alexin or complement 
 
 group of the amboceptor. to the process. In other words, does 
 
 this substance, unite directly with the 
 
 cell or does it unite with the antibody and thus indirectly with the cell? 
 
 EhrUch and Morgenroth studied this by means of a similar experi- 
 ment. Sheep's corpuscles were mixed with normal goat serum (com- 
 plement), and after a time the mixture was centrifuged and the two por- 
 tions tested separately. To the corijuscles heated immune serum was 
 added, but hemolysis did not result. To the supernatant fluid cor- 
 puscles and heated immune serum were added and hemolysis occurred. 
 This indicated that the alexin or complement did not unite with the 
 corpuscles, as did the antibody in the first experiment, but remained 
 free in the supernatant fluid. 
 
 By mixing corpuscles, immune serum, and= complement and keeping 
 the mixture at 0°-3° C. for several hours, hemolysis did not take place. 
 By centrifuging the mixture and separating the supernatant fluid from 
 the corpuscles, a similar test showed that the red cells had combined 
 with all the antibody, but had left the alexin practically undisturbed. 
 
NATURE OF HEMOLYSINS 365 
 
 At higher temperatures these relations were more difficult to demon- 
 strate, as hemolysis occurs rapidly, but by leaving the cells and serum in 
 contact for short periods of time, centrifuging rapidly, and testing the 
 corpuscles and supernatant fluid in the same manner, similar relations 
 were found to exist. 
 
 These experiments led Ehrlich to formulate the theory that comple- 
 ment will unite only with the antibody and not with the red corpuscles, 
 but that it acts upon the corpuscles when united indirectly by means of 
 the antibody. As will be pointed out further on, this view is in direct 
 opposition to that of Bordet, who does not accept this interpretation, 
 but believes that the complement acts directly upon the corpuscles. 
 
 Ehrhch, therefore, conceived the antibody as being in the nature of 
 an amboceptor or of an interbody between an antigen and complement, 
 with two combining arms — one the cytophile haptophore for union with 
 the cell, and the second, the complementophile haptophore for union with 
 complement. The amboceptor is unable in itself to injure the cell, but 
 preserves its importance in being the only and specific means by which 
 the ferment or complement can attack the cell and cause its destruction. 
 
 The process of specific sermn hemolysis is therefore supposed to be 
 as follows: In fresh immune serum containing both amboceptors and 
 complement, or in a mixture of old or heated immune scrum and fresh 
 normal serum {i. e., of amboceptor and complement), the two substances 
 occur independently of each other. When the* corpuscles corresponding 
 to the amboceptor are added, the amboceptor unites with these and the 
 complement unites with the amboceptor, the amboceptor, therefore, 
 standing midway between corpuscles and complement. When these 
 unions have taken place, hemolysis will result: The amboceptor has a 
 greater affinity for the corpuscles than the complement has for the am- 
 boceptor, and will unite with the cells at a low temperature, whereas the 
 complement unites with the amboceptor only very slowly at low tem- 
 peratures. Body temperature favors a quicker union of both, and 
 especially that of complement with amboceptor. Hemolysis, therefore, 
 may occur at low temperatiores, but is hastened by higher temperatures, 
 and occurs best at 37° C. 
 
 As stated in a previous chapter, Ehrlich believes that a great many 
 complements exist in normal serums, which view is in direct opposition 
 to the "unitarian theory" of Bordet, which hoids that the one alexin or 
 complement will act with any sensitizer or airlboceptor. Of the large 
 number of complements, each is especially adapted for the solution of 
 one or more varieties of cells, which it can dissolve in conjunction with a 
 
366 
 
 HEMOLYSINS 
 
 suitable amboceptor. This is known as the main or dominant comple- 
 ment; other complements that may aid in the process are termed non- 
 dominant. In general, it is held that tho^e complements that are 
 especially active in hemolysis are but shghtly active in bacteriolysis, and 
 vice vers4. Although the amboceptor is depicted as having but one 
 haptophore arm for the dominant complement, it is really a polyceptor, 
 and is so constituted that it combines with the.cell to be dissolved, on the 
 one hand, and with a number of complements, on the other. 
 
 Bordet has never accepted these views. He holds that the antibody 
 is not an amboceptor for uniting cell and compjement, but that it sensi- 
 tizes the cell and renders it susceptible to the direct lytic action of the 
 alexin or complement. According to his views, both antibody and 
 complement may unite directly with the cell, and he has borne out this 
 belief by making experiments almost exactly similar to those made by 
 Ehrhch. 
 
 In accepting Ehrlich's view, it is a question of considerable practical 
 importance whether the complement may ur^te directly with free am- 
 boceptor. Ehrlich maintains that the two may enter into a loose and 
 easily dissociated chemical combination, which is hastened by heat and 
 retarded by cold. The imion of hemolytic amboceptor in cobra venom 
 with the lecithin of corpuscles (Kyes' cobra lecithid), which acts as com- 
 plement, while it tends to strengthen this view can hardly be accepted 
 as direct proof, as lecithin differs markedly from the ordinary comple- 
 ments found free in serum. Likewise the thebyy of Neisser and Wechs- 
 berg regarding complement deviation, whereby, it appears that an excess 
 of amboceptor may combine directly with complement and in this 
 manner rob those amboceptors that are attached to cells of the comple- 
 ment necessary to produce lysis, is quite complicated, and is not uni- 
 versally accepted, the evidence of direct union of complement and am- 
 boceptor having not been proved beyond the peradventure of a doubt. 
 It follows, then, as Emery has stated, that we must either assume that 
 the complementophile haptophore of an amboceptor united with its 
 antigen has an increased affinity for complement over and above that of 
 free amboceptors, or we must agree with Bordet that cell and comple- 
 ment unite directly after the former has been sensitized by the action of 
 the antibody. This latter view of the separate union of cell with anti- 
 body and complement is supported by the observations of Muir, who 
 found, upon saturating red blood-corpuscles with antibody and then with 
 complement, that some of the former but none of the latter may be dis- 
 sociated from the combination and become free in the fluid. However, 
 
NATURE OF HEM0LYSI!NS 367 
 
 the dissociated amboceptors found free of compJement may be those that 
 did not have time to unite with complement, and there does not appear 
 to be any direct and positive experimental evidence to permit one to 
 decide between Ehrlich's and Bordet's views. 
 
 While Ehrhch believes that the union between cell and amboceptor 
 is a chemical one and follows ordinary chemical laws, obeying the law 
 of multiple proportions, Bordet holds that the antibody acts as a mor- 
 dant and sensitizes the cell, comparing the process to the staining of 
 filter-paper when immersed in a dye, or to the use of mordants prepara- 
 tory to the staining of flagella of certain bacteria. For example, 0.4 
 c.c. of a hemolyiic serum, if added at once, was found to dissolve 0.5 
 c.c. of corpuscles. If 0.2 c.c. of corpuscles were first added, and amounts 
 of 0.1 c.c. were added subsequently, no lysis taok place after that of the 
 first portion added. Bordet cited this as an example of a physical pro- 
 cess of the nature of absorption, just as filter-paper when added at once 
 to a dye will be stained a uniform color, whereas if it be added a little at 
 a time, the first pieces inserted will be stained deeply, the subsequent 
 ones less and less so, until the dye is completely absorbed. Recent re- 
 searches on the colloidal theory of antibodies, would indicate that the 
 hemolysins are governed by very complex chemicophysical laws, not as 
 yet fully understood, which regulate the action ^of colloids on one another 
 and are probably intimately concerned in the processes under discussion. 
 
 As was stated in a previous chapter, Metchnikoff maintains that both 
 substances concerned in hemolysis are ferments, and that both are 
 adapted for intracellular digestion. He regards complement or his cy- 
 tase as a digestive ferment derived from leukocytes, and believes that it 
 is set free only when leukocytes are dissolved (phagolysis) , either as 
 the result of the injection of a foreign substance or during the process of 
 coagulation. Amboceptor or his "fixateur" is= likened to enterokinase, 
 and like it acts as an accessory ferment that unites the more potent fer- 
 ment (cytase) to the particle to be digested. He also regards it as being 
 derived from leukocytes, and considers that the amount formed depends 
 upon the degree of phagocytosis that occurs during the absorption of the 
 antigen. In the conception of immunity as being fundamentally a pro- 
 cess of nutrition, and in the belief of the existence of more than one com- 
 plement, the similarity between the views of Ehrhch and those of Metch- 
 nikoff is indeed striking. 
 
 Analogy between Bacteriolysis and Hemolysis. — Studies in hemol- 
 ysis aided greatly in a correct understanding of the mechanism of bac- 
 teriolysis. It became apparent that two substances were concerned in 
 
368 HEMOLYSINS 
 
 bacteriolysis — one, the thermostabile amboceptor present in tlie immune 
 serum, and the second, thermolabile alexin or complement, furnish(;<l 
 by the peritoneal exudate in the Pfeiffer test- or by any fresh normal 
 serum in the test-tube bacteriolytic reaction. The discovery and study 
 of the specific serum hemolysins aided greatly, in a better understanding 
 of bacteriolysis and cytolysis in general. 
 
 Specificity of Hemolysins.— The hemolysips are highly specific anti- 
 bodies, and although partial or group hemolysins may be formed and 
 act upon the corpuscles of closely related species, as antihuman hemolysin 
 on the corpuscles of the higher apes, yet the main hemolysin may be so 
 potent that high dilution of the immune serurn practically rules out the 
 activities of group hemolysins, the hemolytic amboceptor proving highly 
 specific for the alien corpuscles responsible fo^ its production. 
 
 NORMAL HEMOLYSINS 
 
 Just as small amounts of antitoxins, agglutinins, and opsonins may 
 be found in normal serums, so, also, normal hemolysins may be present. 
 Their most important practical significance is concerned with comple- 
 ment-fixation reactions, where a close and intimate quantitative rela- 
 tion exists between the complement and hemolytic amboceptor used. 
 As an excess of hemolytic amboceptor may produce hemolysis with a 
 decreased amount of complement, in a given test for free complement, as 
 in Wassermann's syphilis reaction, the patiei|t's serum may contain so 
 much natural antisheep amboceptor as to mate up for slight binding of 
 complement and give undue hemolysis or cveri. a false negative result. 
 
 Hemolysis of alien corpuscles by a normal'^erum is found to depend 
 upon the same mechanism of amboceptor ancf complement as in the ar- 
 tificial immune serums. The amboceptors are" easily removed by adding 
 the corresponding corpuscles to the cold serum' and centrif uging the mix- 
 ture after allowing it to stand at 0°-5° C. for'an hour or two. The su- 
 pernatant fluid will now be found to be free fr'om amboceptors, whereas 
 by adding a little normal complement serum to the corpuscles hemolysis 
 results, indicating that the amboceptors had l^een bound to these. If a 
 normal serum contains several different ambbuceptors for as many dif- 
 ferent bloods, all may be removed at the one time by adding the respec- 
 tive corpuscles to the serum and allowing su|ficient time to elapse for 
 the amboceptors to become linked to their corpuscles. 
 
 Normal serum, therefore, probably contains numerous antibodies of 
 the amboceptor type, adapted to dissolve various foreign substances 
 
NORMAL HEMOLYSIN^ 
 
 369 
 
 when they gain access to the blood. It is probable that, aside from 
 hemolysins, various normal and immune cytolysins play an important 
 part in the processes of immunity. 
 
 While the normal hemolysins that may be found in the serums of 
 various animals have not as yet been fully worked out, the following 
 table, compiled by Sachs ' is a resume of the work reported in the Utera- 
 ture on the subject: 
 
 TABLE 7.— NATURAL HEMOLYSINS 
 
 Hemoltze the 
 Ehythbocytes 
 
 OF THE — 
 
 The Seetjm op 
 
 Rabbit. . . 
 Guinea-pig 
 
 Dog 
 
 Man 
 
 Goat 
 
 Ox 
 
 Sheep. . . . 
 
 Hog 
 
 Horse. . . . 
 Goose. . . . 
 
 Duck 
 
 Pigeon . . . 
 Hen 
 
 
 
 + 
 (+) 
 
 + 
 + 
 
 + 
 
 + 
 
 + 
 + 
 
 + 
 
 + 
 + 
 
 + 
 
 
 
 + 
 + 
 
 n 
 
 ? 
 
 
 i 
 
 •^ 
 
 o 
 
 d 
 
 -d 
 
 K 
 
 3 
 
 o 
 
 
 
 en 
 
 K 
 
 + 
 
 + 
 
 =1= 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 + 
 
 ± 
 
 + 
 
 + 
 
 ^t 
 
 + 
 
 zt 
 
 + 
 
 — 
 
 + 
 
 + 
 
 + 
 
 (±) 
 
 + 
 
 — 
 
 ± 
 
 — 
 
 + 
 
 + 
 
 — 
 
 — 
 
 
 
 =fc 
 
 — 
 
 — 
 
 — 
 
 — 
 
 — 
 
 — 
 
 
 
 — 
 
 + 
 
 + 
 
 — 
 
 + 
 
 =t 
 
 + 
 
 
 
 
 
 — 
 
 z 
 
 — 
 
 — 
 
 — 
 
 — 
 
 ~ 
 
 
 
 
 
 
 
 
 
 + = Well-marked hemolysis. 
 
 (+) =Questionable or feeble hemolysis. 
 
 ± = Doubtful. 
 
 — = No hemolysis. 
 
 Kolmer and Casselman have titrated the natural hemolysins for the 
 corpuscles of various vertebrates in a large number of human serums. 
 Most interest centers about the occurrence of natural antishcep hemoly- 
 sin, because of the wide-spread use of an antisheep hemolytic system in 
 complement-fixation reactions, as, for example,, the Wassermaun syphilis 
 reaction. In over 80 per cent, of human serums there is present suf- 
 ficient natural amboceptor for sheep's cells to give well-marked or com- 
 plete hemolysis. Although this factor must be considered in using an 
 antisheep hemolytic system in complement-fixation reactions, yet with a 
 proper understanding of principles and the employment of a satisfac- 
 tory technic the danger of error is reduced to a minimum. In the fol- 
 lowing table the percentages of serums showifig 100, 75, 25, and per 
 
 'Sachs, H.: Hamdbuch der pathogenen Mikrobrganismen, Kolle and Wasser- 
 maun, 2. Auflage, 2, p. 799. 
 24 
 
370 
 
 HEMOLYSINS 
 
 cent, hemolysis, with the maximum dose of serum (0.2 c.c.) used in the 
 method of titration, are shown: 
 
 TABLE 8.— SUMMARY OF NATURAL HEMOLYSINS IN HUMAN SERUM 
 
 Blood 
 
 Number of 
 Serums 
 Tested 
 
 Per Cent. 
 
 Showing 
 
 100 Per Cent. 
 
 Hemolysis 
 
 Per Cent. 
 
 Showing 
 
 75 Pkr Cent. 
 
 Hemolysis 
 
 Per Cent. 
 
 Showing 
 
 25 Per Cent. 
 
 Hemolysis 
 
 Peb Cent. 
 Showing no 
 Hemolysis 
 
 Sheep 
 
 Dog 
 
 Ox 
 
 125 
 25 
 85 
 25 
 40 
 25 
 25 
 25 
 25 
 50 
 
 64 
 16 
 6 
 
 
 
 
 
 
 
 
 20 
 36 
 20 
 8 
 1 
 
 
 
 
 
 
 9 
 30 
 
 24 
 16 
 3 
 12 
 8 
 4 
 4 
 2 
 
 7.5 
 18 
 50 
 76 
 96 
 88 
 92 
 96 
 96 
 98 
 
 Goat 
 
 Hog 
 
 Rat 
 
 Chicken. . . . 
 
 Horse 
 
 Rabbit 
 
 Guinea-pig . 
 
 Kolmer and Williams ^ have likewise studied the hemolysins found 
 in normal rabbit serum, as preliminary to some; work concerning the site 
 of formation of immune hemolysins. The ffesults obtained with the 
 maximum dose of serum (0.2 c.c.) are given in the following table: 
 
 TABLE 9.— SUMMARY OF NATURAL HEMOLYSINS IN NORMAL 
 RABBIT SERUM 
 
 Number of 
 Blood Serums 
 Tested 
 
 s- 
 
 Per Cent. Per Cent.. 
 
 Showing Showing 
 
 100 Per Cent. | 75 Per CEN'f. 
 
 HEMOLYsie Hemolysis 
 
 Per Cent. 
 
 Showing 
 
 25 Per Cent. 
 
 Hemolysis 
 
 Per Cent. 
 Showing no 
 Hemolysis, 
 
 Goat 
 
 Sheep 
 
 Dog 
 
 Human .... 
 
 Hog 
 
 Ox 
 
 Chicken. . . . 
 Guinea-pig . 
 Rat, white . . 
 
 2.5 
 50 
 25 
 50 
 25 
 25 
 25 
 25 
 25 
 
 4 
 18 
 32 
 
 
 
 
 
 
 
 52 
 56 
 40 
 4 
 4 
 4 
 4 
 
 
 
 88 
 
 76 
 
 72 
 
 20 
 
 20 
 
 20 
 
 8 
 
 4 
 
 
 
 12 
 24 
 28 
 80 
 80 
 80 
 92 
 96 
 100 
 
 ISOHEMOLYSINS 
 In 1892 Maragliano^ directed attention to the fact that the blood- 
 serum of patients afflicted with various dise'ases exerted a hemolytic 
 influence on the blood-corpuscles of healthy persons. Later Ascoli'^ 
 
 ' Jour. Infect. Dis., 1913, xiii, No. 1, 96. 
 
 ' Deutsch. med. Wochen.schr., 1892, xviii, 411. 
 
 ' Munch, med. Wochenschr., 1901, xlviii, 1239. 
 
ISOHEMOLYSINS 371 
 
 found the serums of cancer, pneumonia, and Addison's disease to be 
 actively hemolytic for normal corpuscles. Weil, Crile, Blumgarten 
 and Whittemore, and others have found a large proportion of the serums 
 of lower animals and man suffering with malignant tumors to possess 
 hemolytic activity for normal erythrocytes, and that a reaction based 
 upon this property may have diagnostic value. While isohemolysins 
 are to be especially found in the serums of cancerous persons, and the 
 corpuscles of tuberculous persons are hypersensitive and readily hemo- 
 lyzed, reactions based upon these observations are not specific, although 
 they may have some diagnostic value in relation to other symptoms. 
 It is well to remember this property of cancer serum, especially in making 
 blood transfusions. 
 
 Production of Immune Hemolysins. — Hemolysins are readily pro- 
 duced by injecting suitable animals with several doses of red blood- 
 corpuscles. For this purpose rabbits are commonly employed. Some 
 hemolysins are more readily produced than others; for example, anti- 
 sheep hemolysin is easily prepared, whereas it is far more difficult to 
 secure a potent antihuman hemolysin. The various methods employed 
 in preparing hemolytic serums have been described in the chapter on 
 Active Immunization of Animals. Antisheep hemolysin for conducting 
 the Wassermann reaction is readily prepared by giving a rabbit three or 
 four intravenous injections of 5 c.c. of a 10 per cent, suspension of washed 
 sheep's cells in sterile normal salt solution at intervals of three days. 
 The blood-cells should always be washed three or four times with an 
 excess of salt solution to remove all traces of serum, in order that pre- 
 cipitins may not be produced and anaphylactic shock of the inoculated 
 animal avoided. 
 
 The portion of the erythrocyte that is responsible for the production 
 of hemolysins is a moot question. Bordet and von Dungern maintain 
 that the stroma is the exciting agent; Nolf and others believe that the 
 stromata produce hemagglutinins, and that t-he hemoglobin is chiefly 
 concerned in the production of the hemolysin. 
 
 General Properties of Hemolysins. — Hemolysins are highly resistant 
 antibodies, and are easily preserved. Sterile immune serum may be 
 inactivated by heating in a water-bath for half an hour at 56° C, and 
 may be preserved for many months if small amounts are placed in am- 
 pules and kept in a cold place. If an equal quantity of neutral glycerin 
 is added to the clear inactivated serum it will aid greatly in its preserva- 
 tion. The amboceptors resist drying to a well-marked degree, and filter- 
 paper saturated with the immune serum and dried, after the method of 
 
372 HEMOLYSINS 
 
 Noguchi, preserves the hemolytic activity in a remarkable degree. 
 Heating an immune serum at 56° C, for half an hour, as in the process 
 of inactivating complement, does not materially injure the hemolytic 
 activity of a potent serum. A temperature* of 70° C. or above may 
 cause deterioration and finally destroy the amboceptors. 
 
 As was previously mentioned, hemolj^tic amboceptors possess a 
 great affinity for the receptors of their homologous corpuscles, and will 
 readily unite with them at a low temperature. At incubator temperature 
 the union is quite rapid, so that corpuscles may be "sensitized" within 
 half an hour. 
 
 Source of Hemolysins. — As has been stated elsewhere, Metchnikoff 
 regards the leukocytes as the source of fixatcur or amboceptor forma- 
 tion. Bulloch found that the amount of hefnolytic amboceptor in a 
 serum runs parallel with the number of mononuclear leukocytes, and he 
 regards this as an indication of the activity of the lymphoid tissue in 
 general, which he considers as the main source of amboceptor formation. 
 While it is probable that endothelial cells and mononuclear leukocytes 
 are especially concerned in the process, our own investigations in this 
 field would indicate that the process is more general, being participated 
 in by cells of other tissues which possess suitable combining affinities 
 for the alien corpuscles. 
 
 Antihemolysins. — A further step in the study of hemolysins, but one 
 more of theoretic than of practical interest, was the discovery of anti- 
 hemolysins. By injecting guinea-pigs with normal rabbit serum con- 
 taining amboceptors for ox blood, Bordet secured a serum that inhibited 
 the action of anti-ox immune serum. EhrlicK and Sacks, by injecting a 
 goat with normal rabbit serum, likewise secured a serum that acted as 
 an anti-amboceptor against immune hemolytic amboceptors for ox 
 blood. Ehrlich argued that the anti-amboceptor acted against the com- 
 plementophile group of the amboceptor, which prevented union with a 
 complement from taking place. This view was advanced in support of 
 his theory concerning the two-armed character of the amboceptor and 
 that an anti-amboceptor may be produced against either the cj-tophile 
 or the complementophile group or both. In these particular serums, 
 however, the investigators may have been working with an anticomple- 
 ment instead of an anti-amboceptor. 
 
 A specific hemolysin — one, for example, specific for dog blood, de- 
 rived by treating a rabbit with dog cells — is highly toxic for dogs, being 
 capable of producing hemolysis in vitro and a- clinical condition known 
 as hemolytic jaundice. It is possible, however, gradually to immunize 
 
PEACTIGAL APPLICATIC^'S 373 
 
 a dog against this amboceptor for his own cells bj' starting -ndth very 
 small doses and gradually increasing these until it is found that the 
 animal tolerates amounts that would be fatal to non-immunized animals. 
 If a portion of this serum is now added to the specific hemolytic serum, 
 it will be found that the power of the latter is inhibited. Although this 
 action may likewise be due to anticomplement, it is probable that an 
 anti-amboceptor against the cytophile group of the amboceptor is also 
 formed, which prevents the amboceptor from upiting with the red blood- 
 eells, although conclusive experimental e\'idence of this has not been 
 adduced. 
 
 PRACTICAL APPLICATIONS 
 
 There is probably no other group of antibodies that possesses greater 
 diagnostic value than do the hemolysins, a fact that was demonstrated 
 in the practical apphcation of the Bordet-Gengou phenomenon of com- 
 plement fixation in the diagnosis of syphilis a^nd other infections. By 
 adding sensitized corpuscles — i. e., corpuscles with their homologous am- 
 boceptors — to a fluid, the presence or absence of complement may be 
 determined. If complement is present, hemdlj^sis will ocem- and be 
 complete or partial, depending upon the amotnt of complement avail- 
 able; if hemolysis does not occur", it may be concluded that free com- 
 plement is absent. This is the basis of the complement-fixation diag- 
 nosis of syphilis, gonorrhea, glanders, differentiation of proteins, etc. 
 T^Tien a proper amount of complement is added to a mixture of antigen 
 and its immune serum (containing amboceptors), it is bound to these 
 amboceptors, so that when corpuscles and h&piolj'tic amboceptor are 
 subsequently added, hemolysis does not occur, ^vace there is no available 
 complement, it having been "fixed" by the first amboceptors. If, how- 
 ever, amboceptors for the antigen in the first instance are not present, 
 as where a normal sermn is used, the complement remains free and 
 acts with the hemohi:ic amboceptor to produce hemolysis of the test 
 corpuscles. In this manner the hemolysins and their corresponding 
 corpuscles are employed as indicators or tests for the presence of free 
 complement, so that if an antigen is known, th'e antibody may be deter- 
 mined; or vice versa, by using a known antitfody, the antigen may be 
 determined, the criterion in each instance being whether complement is 
 or is not bound or "fixed," a fact that is dete^ined by the subsequent 
 addition of a hemolysin and its homologous corpuscles. 
 
 The practical applications and technic of these reactions are given in 
 subsequent chapters. 
 
374 HEMOLYSINS 
 
 The hemolysins have no therapeutic application or value. They 
 have their chief value in diagnostic reactions and in the study of cyto- 
 lytic phenomena in general. 
 
 Quantitative Relationship between Heniol3rtic Amboceptor and 
 Complement. — From a practical as well as a theoretic standpoint an 
 important property of amboceptor and of complement is the quanti- 
 tative relationship that each bears to the other. This is especially im- 
 portant in hemolytic reactions, where an excess of either may compen- 
 sate for a decrease of the other and yield fallacious results. If a certain 
 amount of guinea-pig's complement is necessary to lyse 1 c.c. of a 2.5 
 per cent, suspension of sheep's cells, along with hemoljd;ic amboceptor, 
 then double this amount of complement will'be required to lyse 2 c.c. 
 of the same blood, and so on. If a constant quantity of corpuscles and 
 hemolysin are added to a series of test-tubes, a"nd increasing amounts of 
 complement after incubating the mixtures for an hour the smallest 
 amount of complement that produces complete hemolysis is called a 
 unit, and in this manner the strength or activity of a serum complement 
 is measured or titrated. In a similar manner the hemolytic activity of a 
 serum or its measure of hemolysin may be determined by placing in a 
 scries of tubes, as previously directed, a definite and equal amount of 
 corpuscle suspension, and to each tube is then added an amount, also 
 definite and equal, of a normal serum as complement, which is known 
 to be incapable of causing hemolysis. There are next added decreasing 
 and graduated amounts of the immune serum whose native complement 
 has been destroyed by inactivation. After iqcubating the mixtures for 
 an hour, the smallest amount of inactivated immune serum that will just 
 produce complete hemolysis is known as the amboceptor unit of the serum. 
 In other words, there are three substances concerned in serum hemolysis: 
 the amboceptor, the corpuscles, and the complement. By taking two 
 of these as constants, e. g., the corpuscles and the complement, the unit 
 of amboceptor may be determined; or by taking the corpuscles and 
 amboceptor as constants the unit of complement may be determined. 
 Since the corpuscles and amboceptor are most stable, these may be 
 used as constants, and the unit of complement determined under these 
 conditions as preliminary to complement-fixation reactions. 
 
 It is important to bear in mind, in this connection, that the titer of 
 an immune hemolytic serum will vary with the complement used. For 
 example, an antisheep amboceptor is much more active when guinea-pig 
 serum is used as complement than it is when tested with the same quan- 
 tity of rabbit serum as complement. 
 
PEACTICAL APPLICATIONS 375 
 
 After determining the unit of amboceptor or complement — that is, 
 after adjusting the hemolytic system to exact> proportions — the results 
 that follow the varying quantities of complement and amboceptor re- 
 quire the most careful consideration. Less than one unit of amboceptor 
 with one unit of complement caiuiot yield complete hemolysis; Ukewise 
 if with one unit of amboceptor less than a unit of complement is combined 
 hemolysis is incomplete; with one unit of amboceptor and one unit of 
 complement and a double dose of corpuscles hemolysis will also be in- 
 complete. With less than a unit of complement and an excess of ambo- 
 ceptor, however, hemolysis may be complete. The complement may be 
 reduced to so small an amount that hemolysis is incomplete no matter 
 how much amboceptor is used, but the important fact to be borne in 
 mind is that a slight decrease in complement may be compensated for 
 by the presence of many units of amboceptor, so that complete hemolysis 
 results and a false reaction is secured. The converse of this is true to a 
 less marked extent — i. e., an excess of complement may compensate for 
 a decrease in amboceptor, but is less capable of doing so. 
 
 These facts are of the utmost importance in making hemolytic ex- 
 periments, as in complement-fixation reactions, where the entire test 
 depends upon demonstrating whether or not a portion or the whole of 
 the complement used has been fixed. Unless, in a series of hemolytic 
 reactions, the amount of amboceptor employed' is the same throughout, 
 the amount of complement acting in these cannot be determined by 
 comparing the degree of hemolysis. This is true especially in cases 
 where a small amount of complement is fixed, pe in the Wassermann re- 
 action, with a serum containing a small amount of syphiUtic antibody, 
 when the presence of an excess of hemolytic amboceptor may give 
 complete hemolysis and overshadow the fact that a small amount 
 of complement has been actually fixed by syphilitic antibody and an- 
 tigen. 
 
 Method of Titration of Immune Hemolysin. — Various methods have 
 been employed by different workers in this field, but all are based upon 
 the same principles as have been here outUned. 
 
 A small amount of immune serum is inactivated by heating in a water- 
 bath at 56° C. for half an hour. In testing the' serum of a rabbit during 
 the process of immunization 2 or 3 c.c. of blood are easily secured from 
 the ear, and the serum is separated. After it has been inactivated, the 
 serum is diluted to 1 : 100 (1 c.c. of scrum to &9 c.c. of salt solution, or 
 0.1 c.c. serum -1-9.9 c.c. of salt solution). 
 
 Fresh guinea-pig serum is secured for complement by bleeding a 
 
376 
 
 HEMOLYSINS 
 
 healthy pig under ether anesthesia into a Petri dish or centrifuge tube. 
 This serum is diluted 1 : 20, making a 5 per oent. solution, by adding 1 
 c.c. of serum to 19 c.c. of salt solution. Each cubic centimeter of this 
 dilution contains 0.05 c.c. of undiluted seriim, which experience has 
 shown is a satisfactory amount to use. 
 
 The corpuscle suspension is then prepared.. The blood used depends 
 upon the kind of amboceptor that is to be titrated. With sheep and ox 
 blood, a 2.5 per cent, suspension of washed cofpuscles may be employed. 
 With antihunian amboceptor, the corpuscles are usually used in 1 per 
 cent, suspension. (See Noguchi Modification of Wassermann Reac- 
 tion.) After the corpuscles have been washed three times, 1 c.c. is 
 placed in 39 c.c. of salt solution, or sufEcient salt solution is added to 
 2.5 c.c. of the corpuscles to make the total volume equal 100 c.c. 
 
 To a series of six sterile test-tubes increasing doses of the diluted 
 immune serum are now added, together with i c.c. of complement dilu- 
 tion, 1 c.c. of corpuscles suspension, and sufficient normal salt solution 
 to make the total volume in each tube about 3 or 4 c.c. The follow- 
 ing table shows the method of preliminary titration of a hemolytic 
 serum: 
 
 TABLE 10.— PRELIMINARY TITRATION OF A HEMOLYSIN 
 
 Ttjbe 
 
 Amoitnt of Inactivated 
 
 Immune Serum in C.c. 
 
 (1:100) 
 
 Dose op 
 Comple- 
 ment, C. 
 c. (1:20) 
 
 Dose of 
 Corpus- 
 cles, C.c. 
 (2.5 PEn 
 
 CENT.) 
 
 Normal 
 . Salt 
 Solution 
 
 RESuiyr OF Hemoltsib 
 
 after One Hour in the 
 
 Incubator (37° C.) 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 a. 
 
 0.1 (0.001 c.c. undiluted) 
 0.2 (0.002 c.c. undiluted) 
 0.4 (0.004 c.c. undiluted) 
 0.6 (0.006 c.c. undiluted) 
 0.8 (0.008 c.c. undiluted) 
 1.0 (0.01 c.c. undiluted) 
 
 
 
 q. s. 4 c.c. 
 q.„s. 4 c.c. 
 q. s. 4 c.c. 
 q. s. 4 c.c. 
 q. s. 4 c.c. 
 q. s. 4 CO. 
 
 No hemolysis 
 Partial hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 
 In this instance the unit of amboceptor is about 1 : 500, which is too 
 low for a satisfactory antisheep serum. The rabbit should, therefore, 
 receive another dose or two of corpuscles, and the serum be titrated again 
 in from four to seven days after the last injection has been given. In 
 this titration it will be well to use a higher dilution of the inactivated 
 immune serum, as 1 : 1000. This may be prepared by adding 1 c.c. of 
 a dilution of 1 : 100 with 9 c.c. of normal salt, solution and mixing well. 
 The titration is then proceeded with as follows : 
 
ft; 
 

 
 
 ^ 
 
 j 
 
 - j 
 
 0. 
 
 
 1 
 \ 
 
 
 Fig. 103. — Titration of Hemolytic Amboceptor. 
 
PRACTICAL APPLICATIONS 
 
 377 
 
 TABLE 11.— PRELIMINARY TITRATION OF HEMOLYSIN 
 
 
 
 
 Dose 
 
 
 
 
 
 Dose 
 
 OF 
 
 
 
 
 
 OF 
 
 Cor- 
 
 
 
 
 Amount of Inactivated 
 
 Com- 
 
 pus- 
 
 NORIVI4I4 
 
 Result of Hemoltsis 
 
 Tube 
 
 Immune Sebum in c.c. 
 
 ple- 
 
 cles, 
 
 S-ILT 
 
 after One Hour in the 
 
 
 (1:1000) 
 
 ment 
 IN C.c. 
 (1:20) 
 
 Co. 
 
 (2.5 
 
 FEH 
 
 CENT.) 
 
 Solution 
 
 Incubator at 37° C. 
 
 1 .. 
 
 0.05 (0.0001 c.c. undiluted) 
 
 
 
 q. S. 4=C.C. 
 
 No hemolysis 
 
 2 ,. 
 
 0.1 (0.0002 CO. undiluted) 
 
 
 
 q. s. 4 C.C. 
 
 No hemolysis 
 
 3 ,. 
 
 0.15 (0.0003 c.c. undiluted) 
 
 
 
 q. s. 4 C.C. 
 
 Beginning hemolysis 
 
 4 .. 
 
 0.2 (0.0004 c.c. undiluted) 
 
 
 
 q. s. 4 C.C. 
 
 Partial hemolysis 
 
 5 .. 
 
 0.25 (0.0005 c.c. undiluted) 
 
 
 
 q. s. 4 c.c. 
 
 Just complete hemolysis 
 
 6 .. 
 
 0.3 (0.0006 c.c. undiluted) 
 
 
 
 q. s. 4"c.c. 
 
 Complete hemolysis 
 
 7 .. 
 
 0.35 (0.0007 c.c. undiluted) 
 
 
 
 q. s. 4 c.c. 
 
 Complete hemolysis 
 
 8 .. 
 
 0.4 (0.0008 c.c. undiluted) 
 
 
 
 q. s. 4 c.c. 
 
 Complete hemolysis 
 
 9 ,. 
 
 0.45 (0.0009 c.c. undiluted) 
 
 
 
 q. s. 4 c.c. 
 
 Complete hemolysis 
 
 10 .. 
 
 0.5 (0.001 c.c. undiluted) 
 
 
 
 q. s. 4"c.c. 
 
 a 
 
 Complete hemolysis 
 
 The following controls should be set up at the same time: 
 
 1. 1 c.c. of corpuscles in 1 c.c. of amboceptor dilution. This tube 
 
 should show no hemolysis, as the serilm has been inactivated 
 and is too highly diluted for complement activity, even though 
 native complement were present. 
 
 2. 1 c.c. of corpuscles in 1 c.c. of complement dilution. This tube 
 
 may show a trace of hemolysis, due to the presence of a small 
 amount of natural amboceptor for the corpuscles used. As a 
 general rule, guinea-pig serum is free from natural antisheep 
 amboceptor, or the amount is so small under these conditions 
 that it is not necessary to remove it. 
 
 3. 1 c.c. of corpuscles in 3 c.c. of salt solution. This tube should show 
 
 no hemolysis, and serves to show that the diluent was isotonic. 
 
 In the foregoing titration it is found that 0.25 c.c. of 1 : 1000 dilution 
 of amboceptor is the unit, or the titer is 1 : 4000. This method is less 
 difficult, and probably more accurate, than preparing a series of dilu- 
 tions of amboceptor as a 1 : 100, 1 : 200, 1 : 500, 1 : 1000, 1 : 2000, etc., 
 using a cubic centimeter of each dilution. The method requires accu- 
 rate pipets and careful work, but yields uniform and satisfactory results 
 (Fig. 103). 
 
 The rabbit may now be bled mider anesthesia. The serum is sep 
 arated and inactivated and again titrated, as the final titration, for some 
 unknown reason, is likely to be a little lower than in the primary tests. 
 
 When suitably preserved, a hemol3rtic serum will maintain its activ- 
 ity for long periods of time; it should always, however, be titrated before 
 complement-fixation tests are undertaken. 
 
378 HEMOLYSINS 
 
 Methods for Removing Hemolysins from a Serum. — In general, 
 these aim to remove the natural hemolysins, such as natural antisheep 
 hemolysin, from human serums preliminary to making the Wassermana 
 test, or from a guinea-pig serum that is to foe used as complement. 
 
 The method of removal consists simply in adding corpuscles to the 
 serum, and allowing sufficient time for the, corresponding hemolytic 
 amboceptor to become attached and then removing both by centrifuging 
 the mixture. If the serum is fresh, it should be cooled to 0° to 3° C, 
 in order to inhibit complement activity, which would hemolyze a por- 
 tion of the corpuscles. 
 
 To remove natural antisheep hemolysin from a patient's serum place 
 a measured quantity of cold serum in a centrifuge tube and add four 
 volumes of a 2.5 per cent, suspension of sheep/s cells. This will make a 
 dilution of 1 : 5, so that 0.5 c.c. of the dilution is equivalent to 0.1 c.c. of 
 undiluted serum. After placing the tube in ice-water for from fifteen 
 to thirty minutes centrifuge thoroughly to remove the corpuscles. The 
 process may be carried out after the serum, has been inactivated, in 
 which case it is not necessary to work with cold serum. 
 
 In removing a natural hemolytic amboceptor from a guinea-pig 
 serum that is to be used as complement a measured amount of serum is 
 first removed to a separate tube and thoroughly chilled in a glass of 
 cracked ice. If a large amount of serum is to be used, for example, 5 c.c, 
 it is well to place about 0.1 to 0.2 c.c. of pure undiluted corpuscles, after 
 their last washing, in the bottom of a centrifuge tube. This quantity 
 of corpuscles does not materially affect the dilution of the serum. If a 
 smaller amount of serum is used, such as 1 c.c, it is well to add 8 c.c. of a 
 2.5 per cent, suspension of corpuscles, and after centrifuging the mixture 
 the final dilution of 1 : 20 is secured by adding 10 c.c. of salt solution to 
 the supernatant diluted serum. 
 
 Method of Determining Natural Hemolysins in Seriun. — To ascer- 
 tain whether or not a certain natural amboceptor is present in a serum it 
 is merely necessary to inactivate the serum, arid to a measured amount, 
 for example, 0.2 c.c, add 1 c.c. of complement serum (1 : 20) that is 
 known to be free of the particular amboceptor in question, and 1 e.c. 
 of a 2.5 per cent, suspension of the corresponding corpuscles. Sufficient 
 salt solution is added to bring the total volume to 3 or 4 c.c. The mix- 
 ture is then incubated at 37° C. for one or two hours, when the occur- 
 rence of hemolysis indicates the presence of the amboceptor for the 
 corpuscles employed. 
 
 To determine the amount of natural amboceptor by titration dilute 
 
SERUM DIAGNOSIS OF PAROXYSMAL HEMOGLOBINURIA 379 
 
 the serum with nine parts of normal salt solution, and to a series of test- 
 tubes add increasing amounts of 0.05 c.c, 0.1 c.c., 0.2 c.c., 0.4 c.c., 0.8 
 c.c, 1 c.c, and 2 c.c, corresponding respectively to 0.005, 0.01, 0.02, 
 0.04, 0.08, 0.1, and 0.2 c.c. of the undiluted serum. Add 1 c.c of a 5 
 per cent, dilution of fresh amboceptor-free guinea-pig serum as com- 
 plement, and 1 c.c. of a 2.5 per cent, suspension of the corpuscles; 
 sufficient salt solution is added to make the total volume about 4 c.c. 
 After shaking, the tubes are placed in the incubator at 37° C. for two 
 hours, removed, and the results read, or the tubes may be placed in a 
 refrigerator overnight and the results read in the morning. 
 
 SERUM DIAGNOSIS OF PAROXYSMAL HEMOGLOBINURIA 
 
 A hemolytic substance may be demonstrated in the blood-scrum of 
 most cases of paroxysmal hemoglobinuria at certain periods. Although 
 the exact nature of this substance is unknown, it has many of the prop- 
 erties of isohemolysins, being capable of sensitizing the red corpuscles 
 of the patient or those of a normal person at a low temperature, hemoly- 
 sis being effected in the presence of fresh serum, presumably with com- 
 plement, and best at body temperature. 
 
 According to Cook, about 90 per cent, of hemoglobinurics show a 
 positive Wassermann reaction. Landsteiner found that about 10 
 per cent, of paretics showed similar reactions, and other observers have 
 reported finding isohemolysins in epileptics and idiots. Malaria and 
 trypanosomiasis have also been regarded as causes of this condition, the 
 most evidence, however, indicating that the etiology has a luetic origin. 
 It is possible that the hemotoxin is similar to the hemolysin of cobra 
 venom, being in the nature of an amboceptor complemented by the 
 fatty acids or lecithin of red corpuscles (endocomplement) or by a serum 
 complement. 
 
 First Method. — According to Ehrlich, a small tourniquet should be 
 applied about the base of one of the patient's; fingers, and this is then 
 kept immersed in ice-cold water for half an houi'. Blood from the finger 
 thus constricted is then collected in a Wright capsule, and blood from a 
 finger of the other hand is used as a control. Both are allowed to clot 
 and are then centrifugalized. The serum from the finger held in iced 
 water is tinged red from dissolved hemoglobin, whereas the control 
 serum is not tinged or at least not tinged so deeply. 
 
 Second Method. — Donath and Landsteiner have applied Ehrlich's 
 method in vitro. Their method consists of collecting blood in a small 
 
380 HEMOLYSINS 
 
 test-tube, cooling to 0° C. for half an houK, heating subsequently to 
 37° C. for three hours. The presence or absen,ce of hemolysis is observed 
 and the results compared with those obtained from normal blood 
 treated in the same maimer and at the same t}me. 
 
 Third Method. — This technic is carried ottt in vitro in the following 
 manner: Pipet 2 c.c. of the patient's blood in a small test-tube and 
 separate the serum. At the same time place 1 c.c. of blood in a centri- 
 fuge tube containing 9 c.c. of a 1 per cent, scrlution of sodium citrate in 
 normal salt solution. Wash the corpuscles twice and suspend the sedi- 
 ment in 10 c.c. of normal salt solution. Then, secure a cubic centimeter 
 of a fresh serum from a normal person. Proceed to make the .following 
 mixtures: 
 
 Tube 1 : 0.2 c.c. patient's serum -|- 1 c.c. corpuscle suspension. 
 
 Tube 2: 0.1 c.c. patient's serum + 1 c.c. corpuscle suspension. 
 
 Tube 3: 0.2 c.c. normal serum -|- 1 c.c. corpuscle suspension. 
 
 Tube 4: 0.1 c.c. normal serum + 1 c.c. corpuscle suspension. 
 Tube 5: 1.0 c.c. corpuscle suspension. 
 
 Add sufficient normal salt solution to ea<?h tube to make the total 
 volume measure 2 c.c. Shake gently, and place in the refrigerator at a 
 low temperature (not higher than 4° C.) for an hour. Shake each tube 
 gently and place them in the incubator at 37° C. for two hours. The 
 tubes are then centrifuged and the presence or absence of hemolysis 
 is noted. Usually the patient's serum show^ hemolysis of greater or 
 less degree. 
 
 Similar mixtures may be made with the pEttient's serum and the cor- 
 puscles of a normal person. The hemoljrtic substance is capable of lysing 
 these to the same degree that it does the patient's own cells. 
 
 Method of Determining the Resistance of: Red Blood-Corpuscles. 
 Non-Specific Hemolysis 
 
 Various substances have been employed to test the resistance of the 
 red blood-corpuscles. Of these, the hypotonic solutions of sodium 
 chlorid, of varying strength, have yielded results of clinical importance, 
 especially in the study of paroxysmal hemoglobinuria, the primary ane- 
 mias, etc. 
 
 The following technic, slightly modified alter the methods used by 
 Smith and Brown, Gay, Moss, Karsner, and Pearce, is a ready means 
 for determining the resistance of human corpuscles to salt solutions of 
 different tonicities. Chemically pure sodium chlorid is dried for two 
 hours at 170° C, and immediately weighed in amounts necessary to 
 
SERUM DIAGNOSIS OF PAROXYSMAL HEMOGLOBINURIA 381 
 
 make 500 c.c. of salt solution, ranging from 0.1„to 0.6 per cent, in grada- 
 tions of 0.02 per cent. This means the preparation of twenty-six dif- 
 ferent solutions, which should be preserved in proper-sized bottles fitted 
 with tight rubber stoppers. 
 
 When the test is needed only occasionally, these solutions are readily 
 prepared by filling a 50 c.c. buret, graduated in one-tenths, with dis- 
 tilled water, and another with a 1 per cent, solution of pure dried sodium 
 chlorid. From these, the various solutions are readily prepared after 
 the following manner: 
 
 10 c.c. of 0.6 per cent, sodium chlorid = 6 c.c. of 1 per cent, salt 
 
 solution -t- 4 c.c. of 
 distilled water. 
 10 c.c. of 0.58 per cent, sodium chlorid = 5,i8 c.c. of 1 per cent, salt 
 
 solution -|- 4.2 c.c. of 
 distilled water. 
 10 c.c. of 0.56 per cent, sodium chlorid = 5.6 c.c. of 1 per cent, salt 
 
 solution -|- 4.4 c.c. of 
 distilled water. 
 10 c.c. of 0.54 per cent, sodium chlorid = 5.4 c.c. of 1 per cent, salt 
 
 solution -|- 4.6 c.c. of 
 distilled water. 
 10 c.c. of 0.52 per cent, sodium chlorid = 5.2 c.c. of 1 per cent, salt 
 
 solution -t- 4.8 c.c. of 
 distilled water. 
 10 c.c. of 0.5 per cent, sodium chlorid = 5 c.c. of 1 per cent, salt 
 
 solution -|- 6 c.c. of 
 distilled water. 
 Similar dilutions are made, until the final dilution is reached. In 
 many instances it may not be necessary to use so large a number of dilu- 
 tions, as from 0.5 to 0.2 per cent, may be sufficient range to indicate the 
 tonicity. 
 
 Five cubic centimeters of blood are aspir.ated, under aseptic pre- 
 cautions, from an arm vein of the patient, and immediately placed in 
 25 c.c. of sterile 1 per cent, sodium citrate in 0.85 per cent, sodium chlorid 
 to prevent coagulation. The flask or large centrifuge tube is well 
 shaken, and the mixture is centrif uged at sufficient speed to throw down 
 the corpuscles. The supernatant fluid is drawn off, and the corpuscles 
 are washed once or twice more with sterile normal salt solution. After 
 the last washing the supernatant fluid is removed, leaving the erythro- 
 cytes at the bottom of the tube. 
 
382 HEMOLYSINS 
 
 A series of small test-tubes (10 by 1 cm.) are marked appropriately 
 and placed in a rack. To each tube 3 c.c. of the various hypotonic salt 
 solutions and 0.05 c.c. of the red blood-corpuscles (about 1 drop) are 
 added. The salt solution and corpuscles in each tube are well mixed, 
 and the whole series is placed in the refrigerator for from eighteen to 
 twenty-four hours, after which the readings are made. 
 
 The tube of lowest dilution — even if it shows but a trace of hemoly- 
 sis — represents the point of minimal resistance. The strength of salt 
 solution in which all the corpuscles are hemolyzed represents the maxi- 
 mal resistance. 
 
 Normally, the minimal resistance is about 0.47, and the maximal 
 resistance about 0.3 (Morris). 
 
CHAPTER XXI 
 VENOM HEMOLYSIS 
 
 Nature of Venom Hemolysis. — In a previous chapter the statement 
 was made that certain snake poisons, and especially cobra venom, are 
 actively hemolytic. Flexner and Noguchi ^ first demonstrated that the 
 blood-corpuscles of certain species of animals undergo hemolysis when 
 a suitable serum is present, and believed that" the venom contained an 
 amboceptor that was active with serum complement. 
 
 Shortly afterward Kyes^ discovered that venom may hemolyze the 
 corpuscles of certain animals without the presence of serum, and be- 
 lieved that the complement^like activator was contained within the cor- 
 puscles, to which he accordingly applied the name endocomplement. 
 
 Later Kyes^ confirmed Calmette's observation that practically any 
 scrum, when heated to 65° C. and higher, showed an increased activity 
 in the process of venom hemolysis. Kyes and Sachs ' then concluded that 
 endocomplement was not of the nature of a thermolabile complement, 
 but was, rather, a combination of lecithin and the stromata of erythro- 
 C}rtes. 
 
 Kyes later succeeded in combining cobra venom and lecithin by shak- 
 ing a watery solution of venom with a solution of lecithin in ether, form- 
 ing cobra-lecithid, which was found to be actively hemolytic. 
 
 The erythrocytes of various animals differ'in their susceptibility to 
 venom hemolysis. For instance, those of the^ dog and guinea-pig are 
 most susceptible to the process ; those of the ox, goat and sheep are en- 
 tirely refractory, whereas those of the horse, rabbit, rat, pig, and man 
 occupy an intermediate position. Sacks suggested that the variation 
 in hemolytic resistance of red blood-cells from these species of animals 
 was dependent on the amount of lecithin contained in the cells. Kyes, 
 on the other hand, believes that since all erythrocytes contain sufficient 
 lecithin to activate cobra venom, the varying susceptibility depends 
 rather on the availability of the intracellular lecithin for the reaction, 
 i. e., whether the lecithin in the cell is available in a free state. 
 
 ' Jour. Exper. Med., 1902, vi, 277. 
 
 ^ Berl. klin. Wochenschr., 1902, xxxix, S86; ibid., 190.3, xlii, 21; ibid., 1903; xlii, 
 956; Biochem. Zeitschr., 1907, iv, 109; Jour. Infect. D,is., 1910, vii, 181. 
 ' Berl. kUn. Wochenschr., 1903, xUii, 21, 57, 82. 
 
 383 
 
384 VENOM HEMOLYST^ 
 
 According to this theory, therefore, any factor that modifies the 
 availability of the cell lecithin may modify the susceptibility of the cells 
 for hemolysis with cobra venom. 
 
 Noguchi^ has questioned the correctness of this view. He holds 
 that although lecithin exists in the stroma of all kinds of corpuscles, it is 
 not present in a form available for venom activation, and that the de- 
 gree of susceptibility to hemolysis depends chiefly upon the amount of 
 ether-soluble activators present in the cells, as, for example, fatty acids 
 particularly oleinic acid, and their soluble soaps. In his opinion heating 
 an inactive serum to 65° C. and higher renders it active with venom, 
 owing to the presence of a protein compound of lecithin. 
 
 A normal serum may, therefore, contain two activators, one being 
 thermolabile and resembling complement (inactivated by calcium chlo- 
 rid), and the other being thermostabile and a protein lecithin. By add- 
 ing oleinic acid or its soluble soap to a non-activating serum the latter is 
 rendered highly active so far as venom hemolysis is concerned. Hence 
 while Kyes regards lecithin as the chief component of endocellular com- 
 plement, Noguchi regards the fatty acids, neutral fats, and soluble 
 soaps as the active agents. 
 
 Other observers consider the fatty acids and soap as indirect activat- 
 ing agents in venom hemolysis, in that they possess the power of modi- 
 fying the cell and rendering the intracellular lecithin available for the 
 formation of complete hemolysin. 
 
 On the other hand, in susceptible cells the union of cobra venom and 
 lecithin occurs directly with the formation of the complete hemolysin, 
 Kyes' cobra-lecithid, due to the splitting of the fatty acid radical from 
 the lecithin. Ludecke, von Dungern, and Coca and Manwaring re- 
 gard this product as a venom-free lecithin derivative, and not as a leci- 
 thin. They prefer to call the active principle "cobralecithinase," and 
 the complete hemolysin" mono-fatty-acid-lecithin." 
 
 According to Kyes, the relative amounts of lecithin and venom am- 
 boceptor show quantitative relationship corpparable to serum ambo- 
 ceptors and complements, namely, that, withi^n certain limits, the larger 
 the amount of venom, the smaller the amoufit of lecithin necessary to 
 effect hemolysis; and, conversely, the larger the amount of lecithin, the 
 smaller the amount of venom required. 
 
 Further reference to the intimate relationship that exists between 
 lipoids and complements and hemolysis is alpo made in the discussion 
 on the nature of complements, on p. 329. 
 
 1 Jour. Exp. Med., 1907, ix, 436. 
 
VENOM HEMOLYSIS IN SYPHILIS 385 
 
 VENOM HEMOLYSIS IN SYPHILIS 
 The first application of venom hemolysis was made by Weil/ who 
 found, in testing the hemolytic powers of cobra, venom with cells derived 
 from persons suffering from different diseases, that the red cells of syphil- 
 itic individuals offered a characteristic resistance. Various explanations 
 have been offered for this phenomenon: 
 
 1. It was argued that the quantity of red-cell lecithin is actually 
 diminished in syphilis after the primary stagft because pallidum toxin 
 attacks the same lipoidal substances of tissue cells as does cobra venom, 
 in this way accounting for the diminished amount of lecithin that can 
 be extracted in syphilis, as compared with that obtained from normal 
 tissues. Accordingly, the increase in resistance is only apparent, and 
 is due rather to the fact that there is insufficient endocomplement for 
 the venom amboceptor. 
 
 2. Another explanation offered was that the increased resistance of 
 the red cells of syphilitic persons to venom hemolysis is due to the fact 
 that pallidum toxin attacks endocomplement, and that the cells become 
 specifically immunized to this deleterious influence in much the same 
 way that repeated injections of such a hemolytic agent as saponin leads 
 in rabbits to the production of red cells, which show a marked resistance 
 to saponin hemolysis but not to any other hemolytic agent. 
 
 3. Pallidum toxin was believed to so affect the lecithin content of red 
 cells as to render a smaller quantity of it available in a free state for 
 union with the venom amboceptor to form the hemolysin. 
 
 4. Another theory advanced was that pallidum toxin effects a dis- 
 sociation of red cells between lecithin and cbolesterin, the latter sub- 
 stance causing inhibition of hemolysis. 
 
 Whatever may be the true explanation, the fact has been quite well 
 attested that the red cells of a large percentage of persons in the tertiary 
 stage of syphilis exhibit a characteristic increased resistance to venom 
 hemolysis, and while the cobra hemolysis test in this disease is of second- 
 ary importance to the Wassermann reaction a'S a diagnostic procedure, 
 yet it represents one of the most interesting of biologic phenomena, and 
 may possibly be employed in other clinical methods. 
 
 Technic of the Cobra Venom= Tests 
 
 Preparation of Venom Solution. — A 1 : 1000 stock solution of dried 
 
 cobra venom is prepared by accurately weighiiig out 0.01 gram of dried 
 
 ' Proc. Soc. Exper. Biol, and Med., 1909, vi, 49; ibid., 1909, vii, 2; Jour. Infect. 
 Diseases, 1909, vi, 688. 
 25 
 
386 VENOM HEMOLYSIS 
 
 pulverized venom and dissolving this in 10 c.c. of normal saline solution 
 (1 : 1000). This stock dilution is best preserved in amounts of 1 c.c. in 
 sealed ampules, kept in the frozen state in the ice chest in a wide-mouthed 
 well-stoppered vacuum bottle containing salt and ice (Schwartz). 
 
 Each cubic centimeter is sufficient for makahg three tests, so that the 
 10 ampules will be enough for 30 tests, or 1 gram of venom for 3000 re- 
 actions. Or the dried venom may be weighed out in amounts of 
 0.0005 gram in test-tubes, and diluted, just before being used, with 1 c.c. 
 of normal salt solution (1 : 2000). Immediately before the tests are 
 conducted subdilutions are prepared of the stock dilution (1 : 1000), 
 using separate pipets for each, as follows: 
 
 Solution A: 1 : 10,000 = 1 c.c. stock solution -f 9 c.c. normal saline 
 
 solution. 
 Solution B: 1 : 15,000 = 2 c.c. solution A -}- 1 c.c. normal saline 
 
 solution. 
 Solution C: 1 : 20,000 = 1 c.c. solution A + 1 c.c. normal saline 
 
 solution. 
 Solution D: 1 : 30,000 = 1 c.c. solution B -|- 1 c.c. normal saline 
 
 solution. 
 Solution E: 1 : 40,000 = 1 c.c. solution C -f- 1 c.c. normal saline 
 solution. 
 These amounts are sufficient for making three tests; if more tests 
 are to be made, larger amounts of the various dilutions will keep fairly 
 well in a good refrigerator for several days, but it is always well to plan 
 the work so that the exact amount will be prepared and no waste occur. 
 Each lot of stock solution should be tested occasionally with the cells 
 of known normal and positive persons, to make certain that the venom 
 is active in these dilutions. These titrations are conducted in the 
 same manner as the test. 
 
 Preparation of Blood-cells. — With a sterile syringe blood is drawn 
 from a vein at the elbow and 2 c.c. placed in an accurately graduated 
 centrifuge tube containing 5 c.c. of a 2 per cent, solution of sodium 
 citrate in normal saline solution. The suspension should be shaken gently 
 to insure mixing and the prevention of coagulation, but defibrination 
 by means of whipping should never be practised. The cells may be pre- 
 pared at once or placed in the ice-chest overnight. Sufficient normal 
 saline solution is added to bring the total volume to 15 c.c. Mix gently 
 and centrifuge at low speed until the supernatant fluid is clear. Draw off 
 the fluid, add more normal saline solution, mrx up the cells, and centri- 
 fuge again until clear. Repeat this process once more so that all traces 
 
mm r "■" i 
 
 ■ ! ■ 
 
 1 
 
 ■ 
 j 
 
 ''^- S*r' '^ 
 
 i 
 
 i 
 
 J 
 
 1 
 
 i J j i:; '.i 
 
 ? 
 
 I II 
 
 Fig. 104. — Venom Hemolysis. 
 
VENOM HEMOLYSIS IN SYPHILIS 387 
 
 of serum will be removed. After completing the last eentrifugaliza- 
 tioii, which should be thorough (ten minutes), the cells are diluted with 
 25 volumes of normal saline solution, which makes a 4 per cent, sus- 
 pension — for instance, 0.8 c.c. of corpuscles would require 20 c.c. of 
 diluent. 
 
 Just what influence sodium citrate has upon the process is not 
 known, but it is certain that satisfactory results are seldom obtained 
 with blood defibrinated by whipping with rods of glass beads. Similarly, 
 if centrifuged too rapidly, cells are broken vip or rendered more sus- 
 ceptible to the venom ambocepter. 
 
 The Test. — Into a series of five test-tubes, (4 by J/^ inches) place 
 1 c.c. of each dilution of venom, and label each'tube correctly; add 1 c.c. 
 of the 4 per cent, suspension of cells to each tube, shake gently, and incu- 
 bate for one hour at 37° C. Except in cases where the venom dilutions 
 are being frequently used and are known to be reliable, controls should 
 be included. I usually add a normal control of cells from a healthy 
 person mth the 1 : 30,000 or 1 : 40,000 dilution and expect complete 
 hemolysis to occur. A preliminary reading is made at the end of an 
 hour; the tubes are shaken gently and placed in the refrigerator over- 
 night; the final reading is made next morning, and should tally quite 
 closely with the preliminary reading. 
 
 Reading the Results. — Unless the cells are derived from a very strongly 
 reacting case of syphilis, the 1 : 10,000 dilution, vnll be hemolyzed. The 
 normal control tube should show complete hemolysis. If the 1 : 10,000 
 tube is not hemolyzed and some of the higher dilutions show hemolysis, 
 an error in technic has occurred, and the test should be repeated with 
 fresh dilutions. 
 
 The reactions are read and recorded as follows (Fig. 104) : 
 
 A 
 
 B 
 
 C 
 
 D 
 
 E 
 
 H. 
 
 M. H. 
 
 — 
 
 — 
 
 — = strongly positive. 
 
 H. 
 
 H. 
 
 M. H. 
 
 _ 
 
 — = moderately positive. 
 
 H. 
 
 H. 
 
 H. 
 
 S. H. 
 
 — = weakly positive. 
 
 H. 
 
 H. 
 
 H. 
 
 H. 
 
 M. H. = neg;ative. 
 
 H. 
 
 H. 
 
 H. 
 
 H. 
 
 H. = certainly negative. 
 
 H. = complete hemolysis; M. H. = marked hemolysis; S. H.=sUght hemolysis; 
 the dash ( — ) = no hemolysis. 
 
 If complete hemolysis has occurred in all tubes after an hour's 
 incubation, the cells are regarded as being hypersensitive to cobra 
 venom. This occurs as a rule in primary syphilis and in active tuber- 
 culosis. 
 
388 venom hemolysin 
 
 Practical Value of the Venom Test in Syphilis 
 
 1. While the test is much simpler than the Wassermami reaction and 
 there is less possibility for errors in technic ts creep in, it possesses but 
 two other advantages, namely: (1) It may; react positively in latent 
 or tertiary syphilis when the Wassermann reaetion may be negative, and 
 (2) it maj' react positively in treated syphilitic cases when the Wasser- 
 mann reaction is negative, and thus point to a continuation of treatment. 
 Corson- White and Ludlum ^ found 94 per cent,, Schwartz^ 69.3 per cent., 
 and Stone and Schottstaedt' 90.9 per cent, of positive reactions in the 
 active stages of syphilis. 
 
 2. The test is positive in but about 20 per cent, of cases of tabes 
 dorsalis and general paralysis (White and Ludlum), a finding obviously 
 inferior to the Wassermann reaction. 
 
 3. During primary syphilis the cells are hiypersensitive and positive 
 reactions are but occasionally obtained. 
 
 4. Positive reactions may occur in cancer; but otherwise the test is 
 quite specific, and may, in selected cases, prove. a valuable adjunct to the 
 Wassermann reaction. However, with a more improved technic in 
 performing the Wassermann reaction, and especially if antigens re- 
 enforced with cholesterin are used, the venom test is inferior to the 
 Wassermann. In cases where syphilis or tuberculosis of the lungs is 
 to be differentiated, a negative venom test would indicate tuberculosis, 
 as in this disease the cells are hypersensitive. 
 
 THE PSYCHO-REACTION OF MUCH 
 Normal serum, when added to a lytic dose ®f cobra venom and human 
 red blood-cells, will not interfere with hemolysis. According to Much 
 and Holzman,* however, if the serum obtained from a patient suffering 
 from depressive mania or dementia prascox is .added to the mixture of 
 venom and human red blood-cells, the expected hemolysis does not take 
 place. 
 
 Technic. — A 1 : 5000 dilution of cobra venom is prepared by diluting 
 1 CO. of the stock dilution (p. 385) with 4 c.c. of normal saline solu- 
 tion. Enough of the patient's blood is collected from a vein at the elbow 
 to yield at least 1.5 c.c. of serum; heat the serum to 55° C. for an hour. 
 Prepare a 5 per cent, suspension of ivashed human blood-cells. An effort 
 
 'Jour. Nervous and Mental Diseases, 1910, xxxvii, 721. 
 
 " New York Medinal Journal, 1912, xev, 23. 
 
 ' Archiv. of Int. Wed., 1912, x, S. " Munch, med. Woohenschr., 1909, 20. 
 
THE PSYCHO-REACTION OF MUCH 389 
 
 should be made, if possible, to use as a control the cells of a person 
 which are known to be readily hemolyzed by 1 c.c. of the 1 ; 5000 dilution 
 of venom in half an hour. 
 
 Into a series of three small test-tubes place 0.4 c.c. of the patient's 
 serum and decreasing amounts of venom — 1, 0.8, and 0.5 c.c. respectively. 
 Next add to each tube 1 c.c. of the blood-corpuscle suspension. The 
 total volume in each tube is brought up to 2;5 c.c. by the addition of 
 normal saline solution. 
 
 A similar series of tubes should be set up as controls, the patient's 
 serum being omitted. The following table sho.ws the arrangement : 
 Tube 1: 0.4 c.c. serum -{- 1 c.c. venom ^- 1 c.c. blood. 
 Tube 2: 0.4 c.c. serum -|- 0.8 c.c. venom -|- 1 c.c. blood. 
 Tube 3: 0.4 c.c. serum -\- 0.5 c.c. venom -4- 1 c.c. blood. 
 Tube 4: 1 c.c. venom -(- 1 c.c. blood. 
 Tube 5: 0.8 c.c. venom -|- 1 c.c. blood. 
 Tube 6: 0.5 c.c. venom -|- 1 c.c. blood. 
 Tube 7:0 1 c.c. blood. 
 
 The tubes are shaken gently and incubated for an hour at 37° C, 
 when a preliminary reading is made. If the control tubes 4, 5 and 6 
 are hemolyzed, a positive reaction would be indicated by the inhibition 
 of hemolysis in the first three tubes, 1, 2 and 3. Control tube 4 at least 
 should be completely hemolyzed at the end of half an hour, and in a 
 positive reaction tube 1, containing the same amount of venom with the 
 patient's serum, will show inhibition of hemolysis. 
 
 Practical Value. — Corson-White and Ludlum^ have found the reac- 
 tion positive in 80 per cent, of cases of the catatonic form of dementia 
 prsecox and in 62 per cent, of the hebephrenic- type. Only 3 out of 37 
 cases of manic-depressive insanity reacted positively. Among controls 
 with serums of other diseases positive reactions were secured in one case 
 each of cerebrospinal syphilis, tertiary lues, fejcophthalmic goiter, and 
 confusional insanity, and in two cases each of general paralysis and 
 epilepsy. The afore-mentioned observers claim, however, that if the 
 venom is carefully standardized with blood-cells that are completely 
 hemolyzed in 1 : 5000 dilution of venom in thirty minutes, the reaction 
 possesses some diagnostic value, having been found, under these condi- 
 tions, to yield positive reactions in 87 per cent, of cases of dementia 
 prjecox and in 100 per cent, of catatonics. 
 
 According to Citron, the reaction is probably due to interference 
 with hemolysis by an increase in the cholesterin of the serum — a pos- 
 1 Jour. Nerv, and Mental Diseases, 1910, xxxvii, 721. 
 
390 VENOM HEMOLYSIS. 
 
 sibility more apt to occur in diseases of the central nervous system than 
 in any physiologic or other pathologic condition. 
 
 VENOM HEMOLYSIS IN TUBERCULOSIS 
 Calmette found that the blood of tuberculous patients may activate 
 cobra hemolysin in very small doses, and upon this observation he 
 devised a test that yielded about 65 per cent, of positive reactions in 
 tuberculosis. Positive reactions have, however, been found in other 
 diseases, and the practical value of the test has not been established. 
 
 VENOM HEMOLYSIS IN CANCER 
 
 Although the red blood-corpuscles of the horse may be hemolyzed by 
 venom without the aid of serum, Kraus, Graff and Ranzi '-•^ found that 
 about 70 per cent, of cancer serums considerably hastened and aided 
 the hemolytic process. 
 
 A 1 : 5000 dilution of venom is used. In; two series of four tubes 
 each place respectively 0.1, 0.2, 0.3, and 0.5 c.c. of the patient's serum 
 (heated) ; to the first series add 0.3 c.c. of the venom solution, and to the 
 second series 0.15 c.c. of the same. To each of the tubes in the series 
 add 5 drops of a 10 per cent, suspension of washed horse corpuscles; 
 shake thoroughly and incubate at 37° C. Inspect the tubes at the 
 end of fifteen and thirty minutes, and then after one, two, and three 
 hours. 
 
 Positive reactions have also been found in pregnancy after the fourth 
 month, in icterus, advanced tuberculosis, and other diseases. 
 
 » Wien. Idin. Wochenschr., 1911, No. 28. 
 
 2 Munch, med. Wochenschr., 1912, No. 59, 574. 
 
CHAPTER XXII 
 
 PRINCIPLES OF THE PHENOMENON OF COMPLEMENT 
 
 FIXATION 
 
 Historic. — With Bordet's discovery of the hemolysins in 1898, 
 and his demonstration of the role of the antibody or sensitizer and alexin 
 in the process, new light was thrown upon the bacteriolysins, and the 
 close analogy between hemolysis and bacteriolysis soon became apparent. 
 Bordet's discoveries were quickly verified by Ehrlich and Morgenroth 
 and the German school in general, although his views regarding the 
 mechanism of the processes were questioned: The controversy soon 
 centered upon the question of the unity or the multiplicity of alexins 
 or complements. Bordet at this time advanced^ his belief in the existence 
 of one alexin or complement that would act with any sensitizer or ambo- 
 ceptor, and he still maintains this view. One of the experiments con- 
 ducted by him, and later made in conjunction with his pupil, Gengou, 
 in support of his theory, is now known as the Bordet-Gengou phenomenon 
 of complement fixation. This has become widely known as the precursor 
 of all complement-fixation tests, and is the baSis of the well-known and 
 invaluable Wassermann reaction for the diagnosis of syphilis. 
 
 In devising the technic of this important method, Bordet's main 
 object was to show that the complement in a normal serum would unite 
 with either a bacteriolytic or a hemolytic amboceptor, and that, by 
 furnishing sufficient of either amboceptor, all the complement may be 
 "fixed." He argued that if two or more coOiplements existed in the 
 same serum, as was held by Ehrlich, they would demonstrate their 
 presence by exhibiting different affinities for these widely varying 
 amboceptors. 
 
 Prior to this Bordet had shown that the addition of a small amount of 
 normal serum to an immune hemolysin would result in lysis of the homol- 
 ogous corpuscles, and that the process could not take place without the 
 alexin. He then mixed an emulsion of pest bacilli with antipest serum and 
 added a small amount of normal, unheated guinea-pig serum to supply 
 the alexin or complement. After allowing the mixture to stand for four 
 hours at room temperature, it was sought to determine whether the 
 alexia had been fixed by pest antigen and pest amboceptor, or whether 
 
 391 
 
392 PHENOMENON OF COMPLEMENT FIXATION 
 
 it was free in the fluid. Bordet knew that th^ normal alexin serum used 
 in the experiment could produce hemolysis of corpuscles with a homol- 
 ogous amboceptor, so he tested for free alexin by subsequently adding 
 to the mixture anti-rabbit hemolysin and rabbit corpuscles. Hemolysis 
 did not occur, because the alexin or complement had been bound by the 
 pest antigen and amboceptor. When a normal serum was substituted 
 for the antipest serum, hemolysis occurred, because the normal serum 
 contained no sensitizers or amboceptors that could unite with the pest 
 bacilli and "fix" the alexin, which, therefore, remained free, and when 
 the hemolysin and red corpuscles were subsequently added, united with 
 them to lyse the red cells. In this way the corpuscles and hemolysin 
 served as indicators for free or unfixed alexiil or complement, just as 
 litmus or phenolphthalein may be used as a test for the presence of an 
 acid or an alkali. 
 
 By showing, in this manner, that the complement of a seruni could 
 be fixed by either bacteriolytic or hemolytic amboceptors, Bordet 
 endeavored to support his views on the unity of complement. Ehrlich 
 and Morgenroth later verified his findings, and in addition, by more 
 delicate and complicated experiments, showed* that many complements 
 may be present in a serum, a fact manifested by a different rate of 
 absorption, by the action of specific anticomplements, etc., as mentioned 
 in a previous chapter. 
 
 While Ehrlich's theory as to the multiplicity of complements has 
 been widely accepted, the subject really possesses greater academic 
 than practical interest, for experience has sho}vn that the results are the 
 same in complement-fixation tests at least,. regardless of whether we 
 believe in the unity or in the multiplicity of complement, as under 
 ordinary conditions the complement or complements in a given quantity 
 of serum are capable of being absorbed by bacteriolytic, hemolytic, 
 or other amboceptors. 
 
 Gengou showed later that not only cellular antigens, such as bacteria 
 and red blood-cells, are capable of stimulating the production of ambo- 
 ceptors, but that the proteins in solution, such as serum and milk, may 
 produce complement-binding amboceptors in addition to precipitins. 
 This subject was later studied more extensively by Moreschi, whose 
 interest became aroused as the result of his theoretic studies upon 
 anticomplements. This investigator observed that, upon mixing a 
 soluble protein with its antiserum precipitation occurred and the existing 
 complement disappeared, a coincidence that fed him to assert that the 
 complement disappeared because it was carried down mechanically in 
 
HISTORIC 393 
 
 the precipitate. This explanation naturally had the effect of leading 
 many to assume that the Bordet-Gengou phenomenon of complement 
 fixation may be the result of a similar precipitation process, and led to 
 many interesting and valuable investigations, especially those made by 
 Gay. It is now generally agreed, however, that protein amboceptors 
 are formed, and that actual complement fixation occurs independently 
 of precipitation. Later Neisser and Sachs elaborated on Gengou's 
 studies, and perfected a complement-fixation technic for the differen- 
 tiation of proteins that is much more delicate than the precipitin test, 
 and serves to demonstrate and differentiate traces of protein, as in 
 blood-stains, so minute in quantity as not to be appreciable by the 
 precipitin test. 
 
 Widal and Lesourd applied the Bordet-Gengou reaction to the 
 diagnosis of typhoid fever, using an emulsion of typhoid bacilli and the 
 serum of a typhoid-fever patient, and found that a positive reaction 
 occurred more frequently and earlier than the agglutination test. These 
 observations were made soon after Bordet and Gengou's discovery, and 
 were probably the first direct and practical application of a complement- 
 fixation technic in diagnosis. It was not until several . years later, 
 however, that the possibilities of the method were seriously considered. 
 
 Hitherto most experiments were conducted with known antigens and 
 their antibodies. It was shown, especially in the work of Neisser and 
 Sachs on protein differentiation, that when an antigen and its specific 
 antibody are present complement is absorbed, and the specific relation 
 existing between these bodies was again emphasized. Hence in a com- 
 plement-fixation test, if the antibody is known the antigen may be found, 
 or if the antigen is known the antibody may be found, the detection in either 
 instance depending upon whether or not complement is absorbed, this being 
 decided by adding corpuscles and their amboceptors to the mixture, the 
 absence or the occurrence of hemolysis determ^iing this point. In this 
 manner Neisser and Sachs were able to diagnose the nature of blood- 
 stains by using solutions of the suspected stain's as antigen, and adding, 
 in different experiments, known antiserums secured by injecting rabbits 
 with various bloods. When a positive complement-fixation reaction 
 occurred, they concluded that the antigen of the blood corresponded to 
 the known antibody, and they were thus able to identify the species of 
 animal from which the blood in the stain was derived. 
 
 Wassermaim and Sachs, encouraged by these results, endeavored, 
 by complement-fixation tests, to show the existence of antigens in dis- 
 eased organs, using tuberculous glands and lungs with an antituberculous 
 
394 PHENOMENON OF COMPLEMENT FIXATION 
 
 serum and the serums of tuberculous persons. Complement fixation 
 occurred under certain circumstances, and these are discussed more 
 fully in Chapter XXIV. These investigations finally led to the serum 
 diagnosis of syphilis, in which the antigen, was supplied by tissues 
 containing large numbers of the Spirocheta palhda. By furnishing the 
 antigen it was hoped that the luetic antibody could be detected in the 
 body-fluids through the absorption of complement, by the union of the 
 antigen and its antibody in the complement-fixation test. Although 
 the primary results were somewhat discouraging, the possibility was 
 shown to exist, and while the original theories regarding the specific 
 nature of the antigen-antibody reaction have been modified by subse- 
 quent discoveries, nevertheless this reaction of Wassermann, Neisser 
 and Bruck, and Detre has proved itself one of the most valuable diag- 
 nostic procedures known. 
 
 THE ORIGINAL COMPLEMENT-FIXATION METHOD OF BORDET 
 In a paper published in 1901 Bordet ^ gives the results attained with 
 three different antigens and their respective antiserums — pest, typhoid, 
 and Proteus vulgaris. The details of the technic employed with an 
 antigen of pest bacilli and an antipest horse serum are given. 
 
 (a) The antigen consisted of twenty-four-hour cultures of pest bacilli 
 in normal salt solution, making a somewhat concentrated emulsion. 
 
 (6) The antipest horse serum was heated for half an hour at 56° C, 
 to remove the alexin or complement. Normal horse serum (heated) 
 was also employed as a negative control. 
 
 (c) As alexin, the fresh serum of a normal guinea-pig was used. 
 
 (d) The substance sensibilisatrice or hemolysin consisted of the inac- 
 tivated serum of a guinea-pig injected with rabbit's red blood-cells. 
 
 (e) The corpuscles of the rabbit were washed, to free them of alexin, 
 and were used as the indicatory antigen. 
 
 To 0.4 c.c. of the pest emulsion 1.2 c.c. of inactivated antipest 
 serum and 0.2. c.c. of guinea-pig alexin were added. This mixture was 
 allowed to remain at the ordinary laboratory temperature (18°-20° C.) 
 for several hours. In order to ascertain whether or not the alexin had 
 been absorbed, hemolysin and erythrocytes were added to the mixture. 
 This was accomplished by sensitizing about 20 drops of washed rabbit s 
 cells with 2 c.c. of inactivated hemolysin for about fifteen minutes, and 
 adding two drops to each of the test-tubes. Hemolysis did not occur 
 1 Bordet: Ann. de I'lnst. Pasteur, 1901, xv, 19. 
 
ORIGINAL COMPLEMENT-FIXATION METHOD OP BORDET 395 
 
 because the alexin had been fixed by the pest antigen and antibody. A 
 similar test, conducted with normal serum, hemolyzed in a few minutes 
 because the complement or alexin remained free in the mixture. Even 
 at this early stage Bordet included the unportant controls on his antigen 
 and serums that are so necessary in all complement-fixation tests. 
 
 The following table gives the original details and results of the first 
 complement-fixation experiment with pest antigens and antipest serum: 
 
 TABLE 
 
 12.— THE 
 
 ORIGINAL BORDET-GEl^GOU COMPLEMENT- 
 FIXATION REACTION 
 
 Tube 
 
 Antigen, 
 C.c. 
 
 Serum 
 
 Alexin, 
 C.c. 
 
 Hemolysin and 
 Erythrocytes 
 
 Results 
 
 (a) 
 
 (b) 
 
 (c). 
 
 0.4 
 0.4 
 
 0.4 
 0.4 
 
 1.2 c.c. inactivated 
 
 antipest serum 
 1 2 c.c. inactivated 
 
 normal serum 
 1.2 c.c. inactivated 
 
 antipest serum 
 1.2 c.c. inactivated 
 
 normal serum 
 1.2 c.c. inactivated 
 
 antipest serum 
 1.2 c.c. inactivated 
 
 normal serum 
 
 0.2 
 
 0.2 
 0.2 
 0.2 
 
 2 drops sensitized 
 .rabbit's blood 
 2 drops sensitized 
 
 rabbit's blood 
 2 drops sensitized 
 
 rabbit's blood 
 2 drops sensitized 
 
 rabbit's blood 
 2' drops sensitized 
 
 rabbit's blood 
 2 drops sensitized 
 
 rabbit's blood 
 
 No hemol- 
 ysis 
 
 Complete 
 hemolysis 
 
 Complete 
 hemolysis 
 
 Complete 
 
 Id).. 
 
 (e) 
 
 hemolysis 
 No hemol- 
 
 (/)■■• 
 
 ysis 
 No hemol- 
 
 
 ysis 
 
 Mechanism^ of Complement Fixation. — The divergent views of 
 Bordet and Ehrlich on the mechanism of antigen-amboceptor action 
 have been given elsewhere. Bordet believes that the antibody unites 
 directly with the antigen, and serves to sensitize and prepare it for direct 
 union with the alexin or complement, in a manner similar to that of 
 using a mordant in aiding the penetration of a dye-stuff. In the absence 
 of the homologous and specific antibody (sensitizer), the antigen is 
 incapable of absorbing more than very small amounts of complement or 
 none at all. In the absence of antigen, the sensitizer and complement do 
 not unite, or unite to but a very slight degree. The important require- 
 ment for complement fixation is, therefore, an antfgen that has been 
 sensitized by the antibody, and in this manner has an increased combin- 
 ing affinity for complement. 
 
 According to Ehrlich and Morgenroth, however, the complement does 
 not unite directly with the antigen, but only indirectly through the 
 antibody, which acts as a connecting link or amboceptor between antigen 
 and complement. Antigen alone, or even amboceptor alone, binds the 
 complement only very slightly or not at all. The important require- 
 ment for complement fixation is an amboceptor attached to its homol- 
 
396 PHENOMENON OF COMPLEMENT FIXATION 
 
 ogous or suitable antigen, which increases the affinity and fixing power of 
 the amboceptor for the complement. (See Fig. 82.) 
 
 Complement-fixation tests also serve to demonstrate that absorption 
 of complement is not necessarily followed by lysis of the antigen. For 
 example, anthrax and pest bacilli, when mixed with their homologous 
 amboceptors and complement, show no bacteriolysis or but a very 
 slight reaction. The erroneous conclusion thus reached, that these 
 serums contained no amboceptors, was disproved by Bordet, who demon- 
 strated that they contained amboceptors and that complement was 
 absorbed or fixed although bacteriolysis had not taken place. Whether 
 the difference here depends upon variations in the nature of lytic and 
 non-lytic amboceptors or whether it is due to the relative amounts of an 
 amboceptor or the construction and constitution of the antigen is not 
 known. It would appear, however, that the last two possibilities are 
 largely concerned, although, so far as complement fixation is concerned, 
 it is inunaterial whether or not bacteriolysis occurs. 
 
 Complement-fixing antibodies, therefore, are probably all in the 
 nature of amboceptors, and these are to be found in varying amounts in 
 practically all immune serums, including antitoxic, agglutinating, and 
 precipitating serums. 
 
 NON-SPECIFIC COMPLEMENT FIXATION 
 
 The importance of having proper controls in practically all tests is 
 especially to be emphasized in complement-fixation work. While the 
 underlying principles are readily understood and the technic is compar- 
 atively simple, there aie, however, many sources of error that require a 
 thorough understanding in order that an intelligent and reliable com- 
 plement-fixation reaction may be secured. These refer mainly to non- 
 specific fixation of complement and to quantitative factors governing 
 complement-fixation technic. 
 
 Formed elements, such as bacteria and tissue-cells, as well as various 
 organic and inorganic material, may fix complement by themselves, i. e., 
 in a non-specific manner, and chemicals, such as acids and alkalis, may 
 destroy it. 
 
 1. An antigen alone in certain amounts may absorb complement. This 
 anticomplementary dose of an antigen, as it is called, must be determined 
 beforehand by a process of titration when increasing amounts of antigen 
 are mixed with a constant dose of complement; hemolysin, and corpuscles 
 and the anticomplementary action of the antigen noted by the results 
 
NON-SPECIFIC COMPLEMENT FIXATION 397 
 
 of hemolysis, i. e., if a small amount of complebaent is absorbed, hemol- 
 ysis \^t1I be correspondingly incomplete, if all complement is absorbed, 
 hemolysis does not occur at all. If, therefore,; in any complement-fixa- 
 tion test the antigen is used in an amount that will give this non-specific 
 absorption, even to a slight degree, a grave soul-ce of error is introduced. 
 
 As a general rule for all complement-fixation tests, the dose of antigen 
 employed should never be more than one-fpurth or one-half of its 
 anticomplementary dose (that amount which of itself is capable of 
 non-specific complement-fixation). 
 
 2. A serum may of itself absorb a smxill ampunt of complement, espe- 
 cially if it is old or infected with bacteria. This is loiown popularly as 
 the anticom'plementary action of a serum, and in every complement- 
 fixation test in order to detect this condition a proper control, consisting 
 of the dose of serum used plus complement, hemolysin, and corpuscles 
 is required, the non-specific absorption of complement being determined 
 by the results of hemolysis. 
 
 Moreover, perfectly fresh sermns may show this non-specific absorp- 
 tion of complement to a slight degree, especially in the presence of the 
 lipoidal extracts used as "antigens" in the Wassermann reaction. 
 
 Heatiug a serum to 56° C for half an hour largely removes this 
 anticomplementary effect of serums, unless tlley are quite old and in- 
 fected; accordingly, heated serums are used almost exclusively in com- 
 plement-fixation tests. This is usually called inactivation, or the removal 
 of native complement from a serum, but in the majority of instances 
 the complement of a serum generally deleiiorates rapidly, and the serum 
 is heated mainly for the purpose of removir^ its anticomplementary 
 action, i. e., its ability to effect non-specific absorption of complement. 
 
 By referring to the original Bordet experiMent, it will be observed 
 that this investigator controlled any non-specific absorption of comple- 
 ment by both the inunune and the normal serum in tubes C and D of 
 the series by using the full dose of these serums, with a similar amount of 
 complement, and noting that hemolysis was complete. His controls, 
 E and F, were to determine if the process of i^ijictivation or removal of 
 native complement from the two serums was cemplete, and the total ab- 
 sence of hemolysis showed that it was. His control on the anticomple- 
 mentary action of the antigen was also included in tube D, for if the 
 emulsion alone had absorbed complement to any degree, hemolysis 
 would have been incomplete. 
 
 Quantitative Factors in Complement-fixation Tests. — From what 
 has been said it will, therefore, readily be appreciated that complement- 
 
398 PHENOMENON OF COMPLEMENT FIXATION 
 
 fixation tests are largely quantitative. Equally fallacious results may 
 be obtained by using too large or too smafl amounts of the various 
 ingredients. 
 
 While it is possible to use too large quantities of antigen, so that 
 non-specific absorption of complement occurs, leading to false positive 
 reactions, it is also possible to use an amount- so small that any specific 
 absorption of complement by antigen and antibody cannot readily be 
 detected. 
 
 The same is true, but to a much less extent, of the immune serum, 
 for while too large amounts of serum may lead to non-specific fixation of 
 complement, surprisingly small amounts may give well-marked specific 
 fixation, this factor depending, of course, upon the quantity of anti- 
 bodies contained in the serum. 
 
 Of even greater importance are the quantity of complement em- 
 ployed and the proper adjustment of the hemolytic system, composed of 
 complement, hemolysin, and corpuscles. 
 
 Too large an amount of complement may furnish sufficient to sat- 
 isfy the amboceptors of an immune serum united with the antigen, 
 with enough free complement left over to produce partial or complete 
 hemolysis when corpuscles and hemolysin are subsequently added. In 
 this manner specific complement fixation would be overlooked and a 
 false negative reaction secured. 
 
 It is also possible to use too small an amount of complement, with 
 relatively large doses of serum and antigen, so that the complement 
 becomes unduly susceptible to non-specific fixation and consequently 
 false positive reactions may be secured. 
 
 It has previously been explained that an excess of hemolysin may 
 offset any slight deficiency in the amount of complement. For instance, 
 if a small amount of complement is specifically fixed by an antigen and 
 its amboceptor, the addition of too large an amount of hemolysin may 
 result in complete hemolysis of the corpuscle, and thus overshadow the 
 slight but specific fixation of complement. 
 
 On the other hand, hemolysis cannot be complete if the dose of 
 amboceptor is too small. With a given dose of corpuscles and comple- 
 ment a certain amount of hemolysin is necessary to produce hemolysis, 
 this dose being determined by a process of titration, as described in a 
 previous chapter. If less than this dose is y^ed, but the amounts of 
 corpuscles and complement remain the same,- hemolysis will be corres- 
 pondingly incomplete and lead to false positive reactions. 
 
 A very important feature of all complement-fixation tests will be 
 
PRACTICAL APPLICATIONS 399 
 
 seen to be a proper and accurate adjustment' of the hemolytic system. 
 Taking arbitrary amounts of corpuscles and hemolysin as constants, 
 the quantity of complement necessary to produce hemolysis may be 
 determined (titration of complement); or, taking corpuscles and com- 
 plement as constants, the amount of hemolysin necessary to effect 
 complete hemolysis may be determined. One or the other or both titra- 
 tions should be made before the main test is attempted, in order to 
 avoid using an excess or too little of either ingredient. If the exact unit 
 of complement and hemolysin are used, the results must be very care- 
 fully guarded, because in a general way all antigens and serums exert a 
 slight anticomplementary action that may yield results that will be 
 interpreted as weak positive reactions. For this reason the original 
 complement-fixation tests invariably called for a slight excess of com- 
 plement or hemolysin or both, to allow for possible non-specific com- 
 plement fixation, and this is a good general rule that makes the reaction 
 somewhat less delicate, but more reliable in the long run, especially for 
 inexperienced workers. 
 
 Complements of different species of animals=act differently in activat- 
 ing a hemolytic amboceptor and toward fixation by antigen-antibody com- 
 binations. For instance, a complement from one animal may readily 
 enough combine with a hemolytic amboceptor to produce hemolysis, 
 but will not lend itself for fixation, and is, therefore, unfit for comple- 
 ment-fixation tests. Noguchi and Bronfenbrenner have found guinea- 
 pig serum most suitable from all standpoints^ but it is important to 
 remember that the complementary activity of the serums from different 
 guinea-pigs varies, and, therefore, it is necessary to titrate each comple- 
 ment serum or hemolysin, i. e., adjust the henjolytic system, before the 
 main test is conducted. 
 
 These quantitative factors are of great importance, and complicate 
 any complement-fixation method, but efforts to circumvent or ignore 
 them are likely to lead to errors in technic. A proper understanding and 
 appreciation of these factors constitutes the basis for reliable work, 
 whereas less essential details may be altered to conform to the ideas and 
 convenience of the individual worker. 
 
 PRACTICAL APPLICATIONS 
 It will be understood, therefore, that complement-fixation reactions 
 may serve two primary purposes : 
 
 1. With a known antigen, the antibody may be found. This is the 
 
400 PHENOMENON OF COMPLEMENT FIXATION 
 
 usual order in diagnostic tests. For example, in the reaction for sjrphilis 
 the antigen is furnished and the antibody sought for in the body-fluids. 
 So specific has this test proved in the diagnosis of this disease that a 
 positive reaction secured with a proper techuic is regarded as strong 
 evidence of the existence of lues, even though the primary lesion had 
 occurred years before and the person is at the time in apparent good 
 health. In the gonococcus fixation test and other tests of a similar nature 
 the antigen is known and is furnished, and the antibody is tested for in 
 the serum. 
 
 2. With a known antibody the corresponding antigen may be found. 
 This order of events has less practical application, and is used principally 
 in the diagnosis of blood-stains and in the differentiation of proteins in 
 general. It is also used in making special b&cteriologic investigations, 
 when an organism may be identified by specific complement fixation 
 with its known antibody serum. In these instances the antibody serum 
 is" secured by immunizing rabbits with a knpwn antigen, the immune 
 serum then being used for selecting the antigen in unknown substances 
 and mixtures. 
 
 Complement-fixation methods have their greatest value, and are 
 probably best known, in the serum diagnosi& of syphilis — the biologic 
 syphilitic reaction of Wassermaim, Neisser and Bruck, and Detre. 
 Although originally believed to be a direct application of the specific 
 Bordet-Gengou phenomenon of complement fixation, subsequent inves- 
 tigations have shown that the antigen need not be specific, in the sense 
 of containing the Spirocheta pallida, but t6at lipoidal substances in 
 general may serve as "antigen," the peculiar and specific character of 
 the reaction depending upon the nature of tJhe antibody, which has a 
 strong affinity for lipoids, and in such a mixture is capable of absorbing 
 or fixing a considerable amount of complement. 
 
 In no other disease has the method been-so widely employed as in 
 syphilis, although it possesses value in the serum diagnosis of various 
 bacterial infections, such as gonorrhea, glanders, typhoid fever, echino- 
 coccus disease, etc., and in the diagnosis of blood-stains and in the 
 differentiation of proteins in general. 
 
 In the following chapter the Wassermann- syphilitic reaction will be 
 considered in some detail, as a thorough working knowledge of this test 
 is of great value, and serves as the foundation of complement-fixation 
 technic in general. 
 
CHAPTER XXIII 
 
 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 The Wassermann Reaction in Syphilis 
 
 Historic. — Following Bordet's important discovery of complement 
 fixation no practical applications wore made for several years until 
 Neisser and Sachs continued Gengou's studies on protein antigens 
 and amboceptors, and advocated complement fixation as a fine and 
 delicate method of control on the precipitin test in the detection and 
 differentiation of minute traces of proteinSj as in the recognition and 
 diagnosis of blood-stains. 
 
 Encouraged by these results, Wassermann used the method in an 
 attempt to discover in the blood-serum, during the course of an infection, 
 the bacterial proteins derived from a microorganism. Practical appli- 
 cation proved, however, that enough of these proteins did not exist free 
 in the blood to give definite complement fixation. 
 
 In 1905 Wassermann and Bruck found that bacterial extracts may 
 be substituted for emulsions of bacteria as antigen in performing the 
 Bordet-Gengou test, and that extracts of diseased organs containing 
 large numbers of bacteria or their products maty be employed. Accord- 
 ingly, these observers prepared aqueous extracts of tuberculous lungs 
 and glands and used them as antigens in the study of complement fixa- 
 tion in tuberculosis. Positive reactions were secured with an anti- 
 tuberculous serum and with the serums of persons who had received 
 injections of tuberculin. 
 
 At this time Schaudinn and Hofmann discovered the spirochete of 
 syphilis, a finding that served to focus the attention of the medical world 
 upon this disease. In cooperation with Neisser, who was conducting 
 extensive researches on experimental syphilis in monkeys, Wassermann 
 and Bruck apphed the complement-fixation method to the study of 
 these experimental infections, and published a report of their work on 
 May 10, 1906. 
 
 At first monkeys were immunized ^vith aqueous extracts of human 
 chancre, condylomata, syphilitic placenta, etc., and their serums, 
 mixed in vitro with these extracts, were found to give the complement- 
 26 401 
 
402 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 fixation reaction. Since these results may have been due to protein 
 amboceptors or precipitins produced simultaneously by the injection of 
 human serum contained in the extracts, the ejcperiment was carried out 
 with extracts of bone-marrow and other organs of syphilitic monkeys 
 used to obviate this error. It was found, however, that the inactivated 
 serums of syi^hilitic monkeys reacted positively with antigens of either 
 human or monkey lesions, and regardless of whether the monkeys had 
 been injected with human extracts, since, after ordinary cutaneous 
 infection, their serum would show complement fixation. These early 
 reports also showed a high specificity for complement fixation, as monkey 
 immune serum did not react with extracts ofr normal organs or normal 
 monkey serum with extracts of syphilitic organs. 
 
 Just fourteen days after Wassermann, Neisser, and Bruck published 
 their report, a second paper on the same subject appeared, showing the work 
 of Detre. Using aqueous extracts of luetic papules, liver, pancreas, and 
 tonsillar exudate as antigens, Detre performed the complement-fixation 
 method with the serums of six syphilitic and four normal persons, finding 
 positive reactions with two of the six luetic serums. 
 
 In 1906 Wassermarm and Plaut studied the cerebrospinal fluids of 
 41 cases of paresis, and found positive reactions in 32, 4 cases reacting 
 doubtfully and 5 negatively. In the following year Levaditi and Marie 
 and Schutze observed positive reactions with- the cerebrospinal fluid of 
 tabetics, whereas Morgenroth and Stertz confirmed the previous finding 
 in paresis. Since then numerous investigators have corroborated these 
 observations, and while all the evidence tended to strengthen the belief 
 in the luetic origin of general paralysis and tabes, decisive confirmation 
 was lacking until Noguchi and Moore, in 1913, demonstrated the pres- 
 ence of the Treponema pallidum in sections of the cerebral cortex. 
 
 In 1906 Wassermann, Neisser, Bruck, and Schucht applied the 
 complement-fixation test to a large number of cfases of syphilis in Neisser's 
 clinic. Aqueous extracts of luetic liver, placerita, glands, chancres, and 
 gimamata were used as antigens. Of 257 cases in all stages of the disease, 
 only 49 reacted positively. With but 19 per cent, positive reactions, 
 the method did not appear to have a promising future, although at the 
 present time, with a better understanding df the technic and of the 
 importance of quantitative factors that greatly influence the results, 
 the value of the test has been greatly enhanced. 
 
 As it appeared that, after all, no method of diagnosis was to be 
 secured as the result of the demonstration of the syphilitic antibody in 
 the body-fluids, Neisser and Bruck determined to return to earlier 
 
THE WASSERMANN REACTION IN SYPHILIS 403 
 
 methods and attempt to discover if luetic antigen could be demonstrated 
 in the serums of luetics through complementi fixation. Antigens pre- 
 pared of the red corpuscles of syphilitic persons gave positive reactions 
 with the serums of highly immxmized monkeys. Of 160 luetic patients, 
 in 70 per cent, either antigen or antibody was found. Later, however, 
 Citron showed that the extracts of corpuscles of normal persons yielded 
 similar results, which, in the light of subsequent discoveries, was due to 
 their content in lipoidal substances. 
 
 Up to this time the sjrphilitic reaction was considered as but a simple 
 and direct application of Bordet's phenomefibn, requiring a specific 
 syphilitic antigen before complement could be fixed with the sjTjhilis 
 antibody. In January, 1907, Weygandt reported that he had obtained a 
 positive reaction in tabes with an extract of normal spleen. Marie 
 and Levaditi, using an aqueous extract of normal fetal liver, secured 
 positive reactions with the cerebrospinal fluid of paretics, but observed 
 that it was necessary to use larger doses than when extracts of syphilitic 
 organs were used. Subsequently other investigators, as, for example, 
 Fleischmann, Michaelis, Landsteiner, and Plaut, found that watery 
 extracts of normal organs served to fix the complement with luetic 
 antibody. Finally, in December, 1907, a profound impression was cre- 
 ated by the discovery made by Landsteiner, Miiller, and Potzl, that an 
 alcohohc extract of guinea-pig heart yielded results equal to those 
 obtained with an aqueous extract of syphUijbic liver. These results 
 indicated that the antigenic principle was soluble in alcohol, and a pro- 
 longed series of investigations on the various lipoids and their relation 
 to the reaction was begun. These included the employment of leci- 
 thin by Forges and Meier; sodium taurocholate and glycocholate by 
 Levaditi and Yamonouchi; cholesterin and vaaelin by Fleischmann; 
 oleic acid by Sachs and Altman; acetone-insoluble fractions of alcoholic 
 extracts by Noguchi ; and many other combinsttions of various lipoidal 
 substances by different investigators. 
 
 These dealt a blow to the theory of the Wassermann reaction, which, 
 taken in conjunction with the wide-spread use of the test by inexperi- 
 enced and unskilful persons and the many sources of error, tended to 
 retard an earlier appreciation of the great value, of the test, and served to 
 swing the pendulum of medical opinion so far irt the wrong direction that 
 it is only now, with a better understanding of its possibilities and limita- 
 tions, that the method is being established in its proper sphere. Posi- 
 tive reactions were said to have occurred in frambesia, leprosy, malaria, 
 pellagra, pneumonia, scarlet fever, typhoid fever, malignant tumors, and 
 
404 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 practically every other disease liable tb afflict humanity. The careful 
 work of Citron was largely instrumental in preserving the importance 
 of the reaction until a better understanding of the technic resulted in 
 improved and more careful work, with a greater respect for the real value 
 of the Wassermaim reaction. 
 
 Although the true explanation of the mechanism of complement 
 fixation in syphilis is still lacking, sufficient work has been done to show 
 that the specific nature of the reaction is dependent upon a peculiar 
 luetic antibody, and that the older belief in the specificity of antigen, in 
 so far as it insisted upon the presence of the 'Treponema pallidum in the 
 tissues extracted for "antigen," is largely disjjroved. While the method 
 cannot be said to be absolutely diagnostic of syphilis, since positive 
 reactions were had in frambesia (yaws) and leprosy, yet it is practically 
 so, especially in those countries where these two infections are unknown 
 or are relatively infrequent. 
 
 Principles and Theories of the Syphilitic Reaction. — The discovery 
 that the antigen in the Wassermaim reaction is jiot necessarily biologically 
 specific, but may be furnished by a variety of different lipoids'^ from 
 normal or syphilitic tissues, opened up an entirely new phase of the 
 well-known theory of complement fixation, and separated the syphilitic 
 reaction for the classic Bordet-Gengou phenomenon, as based upon the 
 absorption of fixation of complement by a specific antigen and its anti- 
 body. 
 
 As is now well known, the lipoids have always been chiefly concerned 
 in the reaction, although Wassermann and Betre and their coworkers 
 naturally ascribed the complement-fixing powers of their extracts to the 
 presence of the Treponema pallidum. It is, indeed, fortunate that pure 
 cultures of the treponema were not available at the time the original 
 studies were made, for these would naturally have been employed as 
 antigen, and as subsequent work with pallidum antigens has shown 
 complement fixation to be quite irregular and less rehable than when 
 lipoidal extracts are used, this result, coupled with the imperfect under- 
 standing and faulty technic of the earlier investigations, would probably 
 have yielded results so discouraging as to constitute weighty drawbacks 
 to the full development of the reaction. 
 
 Notwithstanding the large amount of work that has been done in an 
 effort to ascertain the true nature of the syphilitic reaction, a correct 
 
 •The term "lipoid" ("fat-like") is applied to compounds that are soluble in 
 ether, alcohol, chloroform, and benzol, but every lipoid is not soluble in all these 
 reagents. 
 
THE WASSEKMANN REACTION IN SYPHILIS 405 
 
 explanation of its mechanism is still lacking,, as the large number of 
 theories advanced tend to show. 
 
 While lipcddal extracts, as well as normal and luetic serums, may sepa- 
 rately absorb or fix small amounts of complement, a mixture of a suitable 
 extract and syphilitic serum is capable of fixing large amounts of comple- 
 ment, and this constitutes the mam principle and all that is definitely 
 known of the syphilitic reaction. 
 
 The serum of a syphilitic is characterized, therefore, by the presence 
 of this hpodotropic, antibody-like substance, which has a great affinity 
 for lipoids and in mixture with them will cause the absorption or fixation 
 of complement to a well-marked degree. Instead of being an example 
 of complement fixation in a mixture of specific antigen with specific 
 antibody, as originally believed, it is technically a non-specific reaction, 
 but practically it is highly specific, since this peculiar antibody is found 
 in largest amount and most constantly in syphilis, and to a lesser extent 
 in practically only two other diseases, namely, leprosy and frambesia. 
 In countries and districts where these diseases are infrequent or unknown 
 with proper technic the reaction for syphilis is* highly specific. 
 
 As will be shown further on, the presence. of this lipodotropic sub- 
 stance is dependent upon the activities of the Treponema pallidum, 
 and when repeated tests continue to show its presence, there is evciy 
 reason to believe that a cure has not been effected, but that the patient 
 still harbors the living parasite. 
 
 Citron has advanced the hypothesis that the antibody-producing 
 antigen is a toxolipoid, which would explain the fact that while pure 
 lipoids, such as lecithin, cannot stimulate antibodies (Bruck), as the 
 toxolipoid does, they can, nevertheless, react with the lijjodotropic 
 antibodies in vitro, with fixation or absorption of complement. As 
 Sachs and Altman point out, an equally tenable theory would be that in 
 syphilis the tissues undergo such alterations tha't they can produce anti- 
 bodies to the lipoid substances as may be contained in the spirochetes 
 themselves. 
 
 While the production of this lipodotropic antibody is still unex- 
 plained, the fact remains, nevertheless, that it forms the basis of the 
 biologic syphihtic reaction, and in a mixture=with a suitable lipoid is 
 capable of absorbing or inactivating complement to a marked degree. 
 Whether or not it is a true antibody in the sense that it is inimical to the 
 spirochete is doubtful; by many it is regarded as a secondary product of 
 cellular activity, and has been called syphilis "reagin." 
 
 Since similar "reagins" arc to be found in other infections, notably 
 
40li THK TECHXir OF rO\iri.KMK\T-Kl\,\ PUIN lUlACTlONS 
 
 in fnunU'sia ;ind loprosy, invi\-^tisators in this liold anxiously awaited 
 the isolatiiui of tl>o Tivponmna pallidum in prtro cultuiv, iH-iiovinu; that 
 if this ivsult were socurod it would bo possible \o work with a spoeitii- anti- 
 gou, dctenniuo the nature of the true syphilis antibody, and possiiily 
 establish a oonipknnent-tixation lest speeitie for syphilis. 
 
 In UHlit Seheiesehewsky, usinu; an antiiie.n of an impure and non- 
 pathoiivnie oulture of a spiroehete rejiarded as the Trepontana pallidum, 
 reported positive eonipleinent-tixalion reaetilms with the majority of 
 serums tested. 
 
 In 1912 2Soiiuehi. having undoubtetily isolated the spiRH'hett> in 
 pure oulture, prepareil antigens and found tjiat, whereas certain loni;- 
 standing or treateil eas(>s of s\philis yielded positive reaetions with the 
 pallidum antigens, thi' reactions were uniforndy negative wiien the 
 lipoidal extracts were used. In priniar\- anfl secomlary syphilis the 
 reactions with pallidum antigens were unifonttl>- negafixe. w heicas with 
 the lijioidal exiiacts the\- were uniformly pitsilixe. .\s a result of his 
 experiments i\'oguchi eoneludetl that in sypliilis there is prixiuced a 
 ti'ue antibody that reads speeilically with paflidum antigen, in addition 
 to the lipodotropic "reagin," which reacts with lipoidal extracts, and 
 whereas {he la(t(M- indii'ates acti\it\- of the infeeting agent, the former 
 is a gage of the defensive acli\'ily of the infeeti'd host. 
 
 (.'raig and Nichols, using alcoholic extracts of pure cultures in ascites 
 kidney agar of Treponema pallidun\. Spirochete pertenuis, and Spiro- 
 chete microdentium, found similar positi\'e reacticnis ii\ ail stages of 
 syphilis with the three antigens, but the reactions were weaker .and less 
 constant as compared with those obtaineil wit If a slock of lipoidal extract. 
 
 Sinular studies conducted by Kolmer, Williams, and Laubaugh with 
 aqueous and alcoholic extracts of pallidum cultures siiowed positive 
 reactions in secondary, tertiary, and congenital syphilis. The aiiucons 
 extracts yielded better r(>acli()ns than the alcjoholie extracts; in pracli- 
 eally all inslani'cs, however, the reai'lions were weaker liiaii Ihosc ob- 
 tained with the ordinary li])oidal extracts, t'ontrol antigens of (\'plu)id 
 and cholera liacilli and sterile culture mediums demonstrated that all 
 eonta.ini'd lijioidal substances that may gix'c weak reactions with the 
 lipodophilic "reagin." This ma\' explain S|'her(>sehewsky's positive 
 reactions witi\ an antigen of a spirochete that in all ])rol>abilil'y was 
 Spirochete microdentium (Noguchi). 
 
 The true nature of the Wassei'mann-Detre t-eaction, therefore, caniiol 
 be said to have been determined. It is probable that, the ordinary 
 syphilis reaction is in itself not depeudeid ui)on,;i true antibody, and that 
 
GENERAL TECHNIC 407 
 
 the reaction is not an immunity reaction, but due rather to the presence 
 of peculiar tissue products (reagins) altered by the presence and activ- 
 ities of the spirochetes themselves, and that tile Wassermami reaction 
 is an expression of this acti^-e injury to tissue-cells. In addition to this 
 secondary product there is probably a true syp^iilis antibody that may 
 )ield specific complement fixation with pallidum antigens. 
 
 In so far as the Wassernaaim reaction is concerned, the true antibody 
 is entirely secondary in importance, and the whole question is intimately 
 concerned with the chemistry of lipoids. AViiile future researches in 
 immunochemistry may reveal the mechanism of the reaction, the prin- 
 ciples are at least well understood at present, so that the s>'iihilis reac- 
 tion is proving of great diagnostic and practical, value. 
 
 TECHNIC OF THE WASSERMANN REACTION 
 Glassware for Complement-fixation Reactions. — Tvst-tubes should be 
 of convenient sizes, — 12 by 1.5 cm., — perfectly clean, free from acids 
 and alkalis, and preferably sterile. They n^ed not be plugged with 
 cotton as it suffices to sterilize them in a wire basket with their mouth- 
 ends downward. Smaller test-tubes, as those used in the JS'oguchi 
 modification of the AYassermann reaction (S W 1 cm,), are wrapped in 
 newspaper in bundles of 25 and sterilized. 
 
 Pipds should be perfectly clean and preferably sterile. Three kinds 
 are required: The ordinary 1 c.c. pipet, gi-aduated to 0.01 c.c. and 
 calibrated to the tip ; a number of special pipets for the fourth method 
 and the gonococcus fixation test, of about the same length and external 
 diameter as an ordinary 1 c.c. pipet, but of much smaller caliber, so 
 that the pipet ^\dll hold 0.2 c.c; it should be giaduatod to 0.01 c.c. and 
 calibrated to the tip; 5 c.c. pipets divided into 0.1 c.c. Care should be 
 exercised in handling pipets to a\-oid breaking the tips. After use they 
 should be washed free from blood, serum, etc. 
 
 GENERAL TECHNIC 
 
 For testing for the Wassermann s^-philis pefaction five reagents are 
 used: 
 
 1. The fluid to be tested. 
 
 2. Complement. 
 
 3. Hemolytic amboceptor. 
 •I. Blood-corpuscles. 
 
 5. Antigen (organic extract). 
 
408 THE TECHNIC OF COMPLEMENT-Fi:j^TION REACTIONS 
 
 I. The Fluid to be Tested. — (a) Senim., — As a general rule, all 
 specimens of blood submitted for complement-fixation tests should be 
 collected aseptically in sterile containers. Tjhis is especially necessary 
 when there has been delay in transmitting the fluid to the laboratory, as 
 when sent through the mails from distant points. When the reactions are 
 to be conducted on the same or on the following day, the specimen of 
 blood may be collected in chemically clean but not necessarily sterile 
 containers. Bacterial contamination renders a fluid anticomplementary 
 and unfit for complement-fixation tests. Specimens should be kept on 
 ice until used, and the serum promptlj^ separlated from the clot. 
 
 Collecting Blood for the Wassermmm Readion. — In collecting blood 
 for the Wassermann reaction the following points should be remembered: 
 
 1. That during active antisyphilitic treatment the blood may react 
 negatively, whereas at a later period a true positive reaction is observed. 
 It is well, therefore, not to collect blood until all specific treatment has been 
 suspended for at least two weeks. 
 
 2. That blood collected during or immediately after an alcoholic 
 debauch may yield a false negative reaction (Craig arid Nichols). 
 
 3. That blood should not be collected just after anesthesia or while 
 the patient has a high temperature. 
 
 As a general rule, at least 1 c.c. of serum and 2 c.c. of cerebrospinal 
 fluid are required for making the sj-philitic reaction. From 2 to 3 c.c. 
 of blood are needed, these amounts being easily collected from adult 
 persons by pricking the finger deeply and filling a small test-tube or 
 vial, as shown on p. 32. This method is very convenient, especially 
 for physicians, hospitals, and dispensaries where direct access to a 
 laboratory can be had. When the treatment is to be guided by the 
 Wassermann reaction, a number of tests are required, and patients may 
 object to repeated venipuncture, whereas no objections will be raised 
 to simple puncture of the finger. 
 
 Larger amounts of blood are collected from a vein at the elbow under 
 aseptic precautions, as described on p. 33. As-a rule, it is well to collect 
 at least 5 c.c. of blood, especially if the specimen is shipped from a distant 
 point (Fig. 105). An excess of serum permits" the technician to repeat a 
 test when necessary, or to apply more than, one method, and thus at 
 times both the physician and the patient are saved the time and annoy- 
 ance incident to collecting another specimen (Fig. 106). 
 
 The Keidel tube, which is sterilized and ready for use, is quite a 
 convenience (p. 36). However, a test-tube or a centrifuge tube may be 
 used, or, when a specimen is to be mailed, a 5 or 10 c.c. vial of thick glass. 
 
GENERAL TECHNIC 
 
 409 
 
 stoppered with a cork or a rubber stopper is quite satisfactory (Fig. 105). 
 Vial, stopper, and needle are readily sterilized in boiling water, drained, 
 and cooled, the specimen collected, the vial tightly stoppered, and the 
 whole sent at once to the laboratory. Cotton stoppers are unsatisfac- 
 tory, as unless the tube or vial is maintained iii an upright position, the 
 fluid may be absorbed. When specimens of blood are to be mailed, it 
 is better to fill a small vial than to place the same amount in a large 
 container, for in the latter case agitation through handling may result 
 in so much mechanical hemolysis taking place 
 as to render the serum unsatisfactory for use* 
 Specimens so collected may be sent for long 
 distances, even in warm weather, and undergcf 
 no change. 
 
 In collecting blood from children, or where 
 the veins are small, a proportionately smaller, 
 needle may be used. In infants the cupping 
 apparatus of Blackfan is quite satisfactory (p. 
 36); frequently sufficient blood may be ob-. 
 tained from a great toe. 
 
 The specimen of blood should be kept in a 
 cold place, and the serum removed at the end 
 of twenty-four hours. Serum that is allowed 
 to remain with the clot for longer periods is 
 more Ukely to become anticomplementary, 
 especially if it becomes deeply tinged witlt 
 hemoglobin. In cases where the serum does 
 not separate the clot may be broken up gently 
 with a sterile glass rod and centrifugahzed.- 
 The serum should be clear and free from cor- 
 puscles. Opalescent and milky serums, ob- 
 tained during the period of digestion and from 
 nursing women, usually do not interfere with 
 
 the reaction; bile-stained serum may at times give marked non-specific 
 fixation of complement. 
 
 It is essential that all serums be heated at B5°~C. for half an hour imme- 
 diately before the test is made. This exposure to heat somewhat diminishes 
 the reacting power of a syphilitic serum, but, as shown by Seligman and 
 Pinkus, it is a necessaiy procedure, for a considerable proportion of nor- 
 mal serums or those from diseases other than syphilis will react positively 
 when unheated, whereas when heated, they will give a negative reaction. 
 
 Fig. 105.— a Vial to 
 Contain Blood for 
 
 THE WaSSERMANN Ke- 
 ACTION. 
 
 Tills is an ordinary 
 glass vial fitted with a 
 rubber stopper. It holds 
 5 c.c. to the mark, and 
 is readily packed for mail- 
 ing. Never stopper with 
 cotton. A good cork stop- 
 per may be used. 
 
410 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 Furthermore, serums that are sterile and- are kept for some time at 
 room temperature or even in an ice-chest acquire an anticomplementary 
 power that is destroyed by heating at 55° C. for half an hour. Therefore 
 when serums are preserved for a number of days they are heated not so 
 much for the purpose of removing native hemolytic complement, which 
 has probably already deteriorated, but to remove thermolabile anti- 
 complement. Serums that are old or contaminated with bacteria are likely 
 to be highly anticomplementary, and this property cannot be destroyed 
 
 Fig. 106. — Outfit for Collecting Blood for the Wassermann Reaction (Xew 
 York Board of Health). 
 The container is a centrifuge tube which holds about 15 to 18 c.c. of blood to the 
 mark. The needle is furnished in a separate tube, sterihzed and ready for use. This 
 outfit is not adapted for mailing. 
 
 by heating at 55° C. or filtration through a Berkefeld filter. In order to 
 conduct a reliable test it is usually necessary to secure fresh serum. 
 This anticomplementary action of serums is so important that in every 
 complement-fixation test there is a serum control tube containing all the 
 ingredients except antigen, the object being to discover any inhibitory action 
 of the serum itself upon the complement. 
 
 Wechselmann's method of converting syphilitic serums showing a 
 negative or weakly positive reaction to those exhibiting a marked positive 
 
GENERAL TECHNIC 411 
 
 reaction depends upon removing the excess of indifferent and inhibiting 
 serum components and upon the destruction or "diminution of the natural 
 antisheep amboceptor present in so large a percentage of human 
 serums. As Noguchi and Bronfenbrenner have pointed out, this method 
 may likewise remove the antibodies concerned in the reaction. To 1 c.c. 
 of heated serum add 3.5 c.c. of saline solution and 0.5 c.c. of a 7 per cent, 
 suspension of freshly precipitated barium sulphate; shake, and let it 
 stand for one hour at 37° C; centrifugalize, and pipet off the diluted 
 serum, which is now ready to be tested (1 c.c. = 0.2 c.c. of undiluted 
 serum). 
 
 Cadaver serums are likely to be highly discolored with hemoglobin 
 and quite anticomplementary. Such serums niay be tested in half the 
 usual dose, and while the results are quite specific, they are not so reliable 
 or constant as those obtained from the living. 
 
 The doses of serum used in testing for the syphilis reaction are given 
 with each method. In the original Wasscrmann test 0.2 c.c. was used. 
 As a rule, from 0.05 c.c. to 0.2 c.c. of serum are satisfactory; higher doses 
 may occasionally show a stronger positive reaction, but the serum niust 
 be perfectly fresh to avoid non-specific complement fixation, and the 
 natural antisheep amboceptor should first be removed. 
 
 (b) Cerebrospinal Fluid. — In certain nervous diseases the cerebro- 
 spinal fluid is examined for the syphilis reaction. Fluid is secured by 
 lumbar puncture, according to the method described on p. 37. If the 
 specimen contains blood, it should be centrifuged until it is clear. It 
 should not be heated before use, as it does not contain hemolytic comple- 
 ment, and fresh fluids from cases other than syphilitics do not react pos- 
 itively. Cerebrospinal fluids, as a rule, possess weaker deviating powers 
 than the corresponding blood-serum, and hence it is necessary to use 
 larger doses — at least 0.5 to 1 c.c, instead of 0.05 to 0.2 c.c, as in the 
 case of blood-serum. 
 
 (c) Other Fluids. — Positive syphilitic reactions have been described 
 as occurring with milk, pleural and peritoneal exudates, and albuminous 
 urine (Bauer and Hirsh) from luetic cases. The reactions with these 
 substances are conducted in the same manner as with cerebrospinal 
 fluid. The material should be perfectly fresh, as anticomplementary 
 action is likely to occur. 
 
 n. Complement. — While complement is to be found in the fresh 
 normal serum of practically all warm-blooded animals, not all are 
 suitable for complement-fixation tests. A suitable complement must 
 possess two important properties: (1) Complementary activities, or 
 
412 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 the power of activating a hemolytic amboceptor, and (2) fixability, or 
 the power of being "fixed" by antigen and. antibody. Noguchi and 
 Bronfcnbrenner have studied the complements of the dog, sheep, hog, 
 ox, rabbit, and other animals, and fountl that the complement of the 
 guinea-pig was best adapted, from all standpoints, for the complement- 
 fixation test. The complements of pigs and sheep are quite fixable, but 
 their weak hemolytic action and rapid deterio|-ation render them unsuit- 
 able for fixation purposes. Rabbit complement is quite active, but is 
 not easily fixable. Kolmer, Yui, and Tyau found rat complement 
 fairly well suited for making the syphilitic reaction with an 
 antihuman hemolytic system. 
 
 The hemolytic power of guinea-pig complement is not constant. 
 In unhealthy animals it is likely to be low, and even among normal 
 animals it may show some variation. For this reason the hemolytic 
 power of each serum is determined by a nlethod of titration before 
 complement-fixation reactions are conducted. Fixed doses of hemolysin 
 and corpuscles may be used, and the amount of complement necessary 
 for effecting complete hemolysis may be determined, or a fixed dose of 
 complement and corpuscles may be used with different amounts of 
 hemolysin, the chief object being to adjust all three factors of the hemo- 
 lytic system, namely, complement, corpuscles,- and hemolysin, to exact 
 and known proportions. 
 
 The complement in the serums of different guinea-pigs may show 
 considerable variation in fixability. The amount of complement in- 
 hibited by serum alone and organic extract alone, or by given constant 
 quantities of serum and extract, may vary rnore markedly than their 
 complementary activity. To reduce this error to a minimum it is advis- 
 able, whenever possible, to use the pooled serums of two or more pigs 
 for making complement-fixation tests. 
 
 Guinea-pig complement serum is collected by bleeding the animal, 
 under ether anesthesia, into a Petri dish or centrifuge tube. The large 
 vessels on both sides of the neck are quickly severed with a pair of 
 sharp-pointed scissors or a scalpel, care being, exercised not to sever the 
 trachea and esophagus. A funnel is used for collecting blood in centrifuge 
 tubes. It is well finally to sever the spinal cord, in order that the animal 
 may not recover from the anesthetic and thus Insure a painless operation 
 throughout. (See p. 46.) 
 
 It is best to keep the blood at room temperature for a few hours 
 until coagulation has occurred and the serum has separated; then place 
 the whole in the ice-chest until needed. Blood may be collected in 
 
Fig. 107. — Titration of Hemolytic Complement. 
 
GENERAL TECHNIC 413 
 
 centrifuge tubes and allowed to coagulate; it is then broken up with a 
 glass rod, centrifuged, and the serum secured at once; such complement, 
 however, is likely to be unduly hypersensitive to the anticomplement 
 action of organic extract and of mixtures of extract with normal se- 
 rums. As a general rule, therefore, it is good practice to bleed the animal 
 late in the afternoon preceding the day on which the experiment is to he 
 made, or at least some hours before the regular work of the day begins. The 
 serum should be clear and contain no corpuscles.. 
 
 Titration of Complement. — In the original Wassermarm reaction 
 fresh guinea-pig serum is used in a constant dose of 0.1 c.c. I have used 
 for several years just half the amount of corpuscles and complement 
 directed in the original technic, and find that the reactions are somewhat 
 sharper and clearer, besides being more econoraic. Using the corpuscles 
 and amboceptor as constants, the amount of complement necessary to 
 produce hemolysis may be determined after the manner described in 
 a previous chapter. To a series of test-tuVjes add increasing amounts of 
 complement serum diluted 1:20 as follows: 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 
 0:8, 0.9, 1, and 1.2 c.c. Add 1 c.c. of a 2.5 per Qent. suspension of sheep's 
 cells and an amount of hemolytic amboceptor" .equal to two units. To 
 this add sufficient salt solution to bring the total volume of each tube up 
 to about 3 c.c; shake gently and incubate for one hour at 37° C. That 
 tube showing just complete hemolysis contains one unit of complement 
 (Fig. 107). 
 
 And now we come to a very important question, namely, the amount 
 of complement that is to be used in conducting complement-fixation tests. 
 Many present-day observers use exactly one unit of complement and 
 one unit of amboceptor. This is permissible, providing the complement 
 is titrated in the presence of a constant dose of antigen and a constant 
 dose of serum, in order that due allowance for the anticomplementary 
 action of these may be made in the titration. Under these circum- 
 stances, however, it is necessary to titrate each patient's serum with the 
 complement, because one serum or even the pooled serums of different 
 persons should not be taken as a standard in the titration, for two impor- 
 tant reasons: (1) The patient's serum which we are about to test may 
 be more anticomplementary than the serum used in the complement 
 titration, and hence when used in the main test, with exactly one unit 
 of complement, mild degrees of inhibition of hemolysis will be secured 
 that may be interpreted as slightly positive reactions; or (2) the serum 
 used in the complement titration may contain more or less natural 
 hemolytic amboceptor than the patient's serum, and this factor exerts 
 
414 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 an influence on the titration, so that the miit varies with different serums. 
 For these reasons I have included here a fourth method for using a 
 single unit of complement under conditioils where the anticomple- 
 mentary action of each serum and the antigen are determined, in pref- 
 erence to titrating the complement with a ser^um and using this unit for 
 a mmiber of other serums that are sure to differ from each other. 
 
 In this titration, therefore, we determine the amount or unit of 
 complement necessary to produce hemolysis with fixed amounts of 
 amboceptor and corpuscles. It will be obseuved that I have used two 
 units of amboceptor, as determined by a previous titration. These two 
 units are equivalent to one dose, and the same would be true whether 
 three, four, five, or more units were used, because in this titration the 
 corpuscles and amboceptor are arbitrary and fixed constants, and are 
 used for determining the amount of complement necessary to bring 
 about complete hemolysis. 
 
 In conducting the main tests the dose of corpuscles and that of 
 amboceptor are the same as those used in the complement titration, but 
 instead of using exactly one unit of complement, it is necessary to use 
 one and one-half or two units, to allow for the anticomplementary 
 action of antigen and patient's serum. I have long used the former dose 
 when testing serums not more than three days old, as the extra half-unit 
 is all that need be allowed for these anticomplementary influences. 
 With older serums it is well to use two units, and this is true also when 
 using antigens rcenforced with cholesterin, as the latter is well known for 
 its antihemolytic properties. 
 
 As the result of a large number of comparative titrations and tests 
 I have found that 0.05 c.c. of complement serum (=1 c.c. of a 1:20 
 dilution) is a safe amount to use with 2.5 per cent, corpuscle suspension, 
 and equally good results are secured by adopting this amount as a fixed 
 dose in titrating the amboceptor before each day's work. If the pig 
 serum happens to be weaker or stronger in complement than it is ex- 
 pected to be, the differences are adjusted in the amboceptor titration. 
 Following the same rule, one and one-half or two units of amboceptor 
 are used in conducting the main test, the extra half, or one unit, over- 
 coming the anticomplementary action of antigen and patient's serum. 
 
 It is important to remember that, in conducting this titration, 
 amboceptor, complement, and corpuscles are to be mixed at once; if 
 amboceptor and corpuscles are mixed and allowed to stand for ten 
 minutes or more before receiving the complement the corpuscles become 
 "sensitized," and the amount of complement required for effecting 
 
GENEHAL TECHNIC 415 
 
 hemolysis will be less than if all three are niijfed one after another. If 
 this rule is not adhered to, an error in technic may result. On p. 447 
 another method of complement titration iss given, usiag corpuscles 
 previously sensitized for at least half an hoilr at room temperature; 
 this method is to be preferred, and is the one I usually employ when 
 titratiag complement. 
 
 in. Hemolytic Amboceptor. — Since the original work of Wasser- 
 mann appeared, the antisheep hemoh'tic system has been most widely 
 used in experimental investigations. 
 
 Antisheep amboceptor is readily prepared by immunizing rabbits 
 with washed sheep's corpuscles. A simple and efficient method is to 
 give intravenous injections of four doses of 5 c.c. each of a 10 per cent, 
 suspension every three or four days. Other methods and the details 
 of Jhe preparation and preservation of amboceptor are given in Chap- 
 ters rV and V. 
 
 The one objection to the use of the antisheep hemolytic system is 
 the presence, in a large proportion of human serums, of variable amounts 
 of natural amboceptor for sheep's cells. In about 70 per cent, of fresh 
 inactivated human serums sufBcient amboceptor is present partially 
 or completely to hemolyze the usual dose of sheep-cell emulsion -ndth 
 the customary amount of guinea-pig complement. In fact, the Bauer 
 and Hecht modifications of the W'assermann reaction utihze this natural 
 amboceptor, but, as vn\l be pointed out further on, this factor is too 
 variable to be employed in conducting the reaction, as non-specific or 
 false positive results are quite likely to occur. 
 
 As has been stated ui the preceding chapter, the delicacy and accuracy 
 of any complement-fixation test depend to a large extent upon proper 
 adjustment of the hemolytic system. It will readily be miderstood that 
 the presence of an unknown quantity of natural amboceptor in a serum 
 is a drawback to accurate quantitative estimations. The importance 
 of this hes in the fact that for some unknown reason an excess of ambo- 
 ceptor may completely hemolyze the corpuscles, even though a small 
 amount of the necessarj- complement has been specifically fixed by 
 antigen and syphilis antibody. In this mannei* negative reactions may 
 result with serums that would otherwise show a' slight positive reaction. 
 To remove this source of error Noguchi has ^dvocated the use of an 
 antihuman hemolytic system, which renders the reaction more dehcate. 
 However, comparative studies between antisheep and other hemohtic 
 systems demonstrate that, -nith proper technid", the influence of natural 
 amboceptors may be reduced to a minimum and rendered almost neg- 
 
416 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 ligible. At any rate it is very simple and but li'ttle trouble to remove the 
 antisheep amboceptor routinely from human serums previous to mak- 
 ing the tests (for tcchnic, see p. 378). ■ 
 
 A method for titrating immune amboceptor has been given on p. 375. 
 This important feature will be dealt with 4gain in giving a detailed 
 description of the various methods that follow. 
 
 IV. Red Blood Corpuscles. — Defibrinatetf sheep blood is washed 
 three times with an excess of sterile normal salt solution to remove all 
 traces of serum (p. 28). 
 
 In the original Wassermann reaction a 5 jjer cent, suspension in salt 
 solution is used in .doses of 1 c.c. This emialsion is quite heavy, and 
 sharper and clear results are secured by using Just half this amount and 
 at the same time sufficient cells are usetl to niake the readings easy and 
 distinct. Either 0.5 c.c. of a 5 per cent, or 1 c.c. of a 2.5 per cent, sus- 
 pension may be used. I have used the latter with entire satisfaction for 
 several years. 
 
 The emulsion of cells is prepared with sterile 0.85 per cent, sodium 
 chloric! solution. To 2.5 c.c. of corpuscles sufficient salt solution is 
 added to bring the total volume of the emulsion up to 100 c.c, or smaller 
 amounts may be prepared by suspending 1 c.c. of corpuscles in 39 c.c. 
 of salt solution. 
 
 Before each day's work the amount of corpuscle suspension needed 
 should be calculated, and sufficient for the day prepared at one time, for 
 if a fresh emulsion is prepared later, titration- with the complement and 
 amboceptor would be required. Attempts to count the corpuscles in 
 suspension can only be regarded as approximate and are unreliable. 
 By titrating each emulsion with the complement, and amboceptor to be used, 
 that particular emulsion is thereby adjusted, so that it is immaterial whether 
 a few more or a few less corpuscles are present. , 
 
 Sheep's blood is obtained either from an abattoir or by bleeding an 
 animal from the external jugular vein (p. 48.)'. The latter method is 
 preferable, but due care must be exercised not to bleed too frequently 
 or in excessive amounts, as if anemia occurs the corpuscles become 
 unduly fragile. 
 
 Sheep's cells are easily preserved in a satisfactory condition for 
 forty-eight hours by first washing them and then storing the sedimented 
 corpuscles in a good ice-chest. Suspensions > are less easily preserved. 
 It is best to use fresh corpuscles, and those that are several days old 
 and unduly fragile should never be used. Attempts to preserve cor- 
 puscles with the aid of mercury bichlorid, formalin, etc., have not yielded 
 satisfactory results. 
 
GENERAL TECHNIC 417 
 
 V. Antigen. — ^As was pre\'iously stated, the ordinary alcoholic 
 extracts of syphilitic livers used as " antigens " in conducting the Wasscr- 
 mann reaction are not biologically specific. It is generally accepted that 
 even in watery extracts of syphilitic hvers the main antigenic principles 
 are lipoidal substances, independent of the Treponema pallidum itself. 
 Next to pure cultures of paUida, these aqueous extracts of luetic livers 
 come closest to being a specific biologic antigen. Although alcoholic 
 extracts of luetic fiver may contain special fipoidal substances that 
 enhance their efficacy as antigens, yet, as shown by Noguchi, and as 
 confirmed later by us, alcohol does not serve well to extract pure cultures 
 of palUda, and therefore these extracts can hardly be regarded as specific, 
 in the sense that they contain antigenic principles of the spirochetes 
 themselves. The only specific biologic antigen is an aqueous extract 
 of a pure culture of pallida; this antigen is, however, much less service- 
 able than an ordinary organic extract, because the Wassermann reaction 
 depends upon the peculiar fipodophUic "reagin," which absorbs com- 
 plement with lipoids in a characteristic but. biologically non-specific 
 manner. 
 
 The term "antigen," as ordinarily used in the Wassermann reaction, 
 must therefore be regarded as a misnomer. It" is, however, so generally 
 used that it may be retained, with a distinct miderstancfing as to its 
 actual meaning. 
 
 With the discovery that alcoholic extracts of normal organs may serve 
 as antigen and that the chief antigenic principles reside in the lipoids, 
 it followed that extensive researches were undertaken in the hope of 
 discovering a lipoid, or a combination of lipoi(te, that would prove suffi- 
 ciently delicate to act specifically in the serum diagnosis of syphilis, and 
 not with normal serums or those of persons suffering from other dis- 
 eases. As a result, a large number of different extracts are in use. Each 
 has its own advocates, so that the general subject of antigens is the most 
 complicated one with which we have to deal in performing the Wasser- 
 mann reaction. 
 
 While various organic extracts may be used, practical experience has 
 shown that some are better than others. It is important to remember : 
 
 1. That all antigens are capable in theinselves of absorbing a certain 
 amount of complement. This is due to the presence of undesirable 
 extractives, some preparations containing more than others. In certain 
 doses, however, all antigens are capable of exerting this anticomple- 
 mentary action, and, other things being equal, lihat antigen is best which 
 shows this non-specification in the smallest degree. Before an antigen 
 27 
 
418 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 may be used in conducting any complement-fixation test it is necessary 
 to ascertain its anticomplementary dose, for. if this dose were used, a 
 portion of or all the complement would be fixed, in a non-specific manner, 
 so that hemolysis, being partial or absent, yields false positive reactions. 
 
 2. Most antigens, when in sufficiently large amounts, are hemolytic, 
 i. e., they may hemolyze corpuscles in a non-specific manner. This 
 hemolytic action is usually due to the presence of undesirable extractives, 
 and extracts of organs that have undergone advanced autolysis or fatty 
 degeneration are known to contain more of these hemolytic substances 
 than do extracts of normal organs. As a general rule, a highly anti- 
 complementary antigen is likely to be correspondingly highly hemolytic. 
 The hemolysis may be due to the presence of lipoidal substances or to 
 the alcohol used in preparing the extract. If an antigen were used in an 
 amount equal to its hemolytic dose, partial or complete hemolysis would 
 occur in all tubes, so that a false negative result would be secured. As 
 a rule, the hemolytic dose of an antigen as determined by titration in 
 the presence of serum is larger than the anticomplementary tlose, so that 
 if the latter is known, it is not always necessary to determine the former. 
 
 3. Practically every alcoholic organic extract will serve, in certain 
 amounts, to absorb complement in the presence' of the serum of a syphilitic 
 person. Some extracts, however, will do this better than others. The 
 Wasscrmann reaction depends upon the fact: that a larger amount of 
 complement is fixed by the syphilis antibodjj and extract than is fixed 
 by normal serum or the serum of a person with some disease other than 
 syphilis and this same extract. The only twcf notable exceptions to this 
 general rule are to be found with the serum o£ tuberous leprosy and that 
 of frambesia. The amount of antigen that is found, by a process of 
 titration, to fix a large amount of complement with a constant dose of 
 syphilitic serum is known as its antigenic dose. Not every lipoid serves 
 equally well as antigen, and therefore consiflerable research work has 
 been done in the hope of discovering an extract or a combination of 
 lipoidal substances that would show a constaiit reaction and would react 
 only with the syphilis antibody. Thus far this has not been accom- 
 plished ; unfortunately, pure pallida antigens are not entirely specific or 
 serviceable, and if the specific and irleal antigen is discovered in the future 
 it will probably be of the nature of a lipoidal substance, altered or pro- 
 duced in a specific manner by the Treponema pallidum itself. In the 
 meantime we have antigens sufiiciently dolicate and specific, when 
 properly used, to render the Wassermann reaction of great value in the 
 diagnosis of syphilis and to serve as a guide to its treatment. 
 
GENERAL TECHNIC 419 
 
 From a practical standpoint, therefore, tp be suitable for use as 
 antigen in the syphilitic reaction any extract or preparation must fulfill 
 the following requirements: 
 
 1. It should be largely free from anticomplementary action. 
 
 2. It should likewise be free from hemolytic action, in small doses at 
 least. 
 
 3. It should possess a high degree of sensitiveness for the syphilitic 
 antibody, i. e., be capable of absorbing relatively large amounts of com- 
 plement in the presence of syphilitic serum. A good antigen is one that, 
 in small amounts, is perfectly antigenic, and that does not become 
 anticomplementary or hemolytic until from' four to ten times this 
 amount is used. 
 
 4. It should be quite stable and not difficulfto prepare, and different 
 preparations should bear a certain relationship to one another in their 
 properties — that is they should keep well, and different extracts prepared 
 in the same manner should show fairly constant antigenic, anticomple- 
 mentary, and hemolytic doses. 
 
 Preparation of Antigens. — The following antigens have been most 
 widely used and recommended : 
 
 1. Aqueous extract of syphilitic livers. 
 
 2. Alcoholic extracts of syphilitic liver. 
 
 3. Alcoholic extracts of normal organs. 
 
 4. Alcohohc extracts of normal organs reenforced with cholesterin. 
 
 5. Acetone-insoluble lipoids. 
 
 6. Lecithin and cholesterin. 
 
 7. Aqueous extracts of pallidum culture. 
 
 1. Aqueous Extracts of Syphilitic Livers.^This is the original anti- 
 gen, as employed by Wassermann, Neisser, and Bruck; Wassermann still 
 uses these extracts in preference to others. They may contain spiro- 
 chetes or their direct derivatives, and, as sho?vn originally by Wasser- 
 mann and Neisser, may be true biologic antigens, for when injected into 
 monkeys, antibodies are formed. 
 
 No satisfactory analyses of these extracts have been made. Chem- 
 ically they differ in no essential respect from the liver of acute yellow 
 atrophy (Ehrmann and Stern; Seligman and Einkus). As antigen they 
 are more efficient than similar extracts of normal liver. The nature of 
 the specific factor has not yet been demonstrated with certainty. They 
 react with the serums of leprosy and yaws, and, as in the case of other 
 antigens, their main antigenic principle is apparently due to the presence 
 of lipoids. 
 
420 THE TECHNIC OP COMPLEMENT-FIX}i.TION REACTIONS 
 
 They are less stable than alcoholic extracts; and are likely to become 
 highly anticomplementary and lose their power of reacting with syphil- 
 itic serums. Citron is convinced that these changes are brought about 
 by careless handling of the extract, or its exposure to the light. He 
 reconmiends that the extract be kept constantly in the ice-chest, and 
 that it be kept out only long enough to remove sufficient for the day's 
 work. 
 
 Preparation. — The fresh liver taken from a syphilitic fetus, and showing the 
 presence of spirochetes by dark-ground illumination^ is weighed and cut into fine 
 pieces. Four times its weight of 0.5 per cent, phenol in= normal salt solution is added. 
 The mixture is placed in a brown bottle and shaken mechanically at room temperature 
 for twenty-four hours. It is then filtered through gauze, to remove the larger par- 
 ticles, and stored in a brown bottle in an ice-chest. After several days of sedimenta- 
 tion the fluid assumes a yellowish-brown copalescence and is ready for the preliminary 
 titration to determine its anticomplementary and hemolytic doses. The sediment 
 should not be disturbed, but the supernatant fluid should be carefully removed by 
 means of a pipet. According to Citron, extracts that must be used in quantities of 
 less than 0.1 c.c. are, as a general rule, unsatisfactory. Only such extracts should he 
 used as in doses of 0.4 c.c, will not interfere with hemolysis. The method of making 
 these titrations is given on page 428. 
 
 2. Alcoholic Extracts of Syphilitic Livers. — These antigens are 
 extensively used. They are not true biologic antigens, for they do not 
 give rise to antibodies (Schatilof and Isabohnsjsy; Seligman and Pinkus); 
 they are, however, usually better antigens than similar extracts of 
 normal liver, a fact that may be explained, in part at least, by chem- 
 ical changes, namely, fatty changes, autolysis, soaps (Beueker), excess 
 of cholesterin (Piglini), etc., which, while not specifically syphihtic in 
 nature, are often produced to a striking degree in congenital syphilis. 
 
 Preparation. — Fetal liver known to contain numerous spirochetes is used in 
 preparing this extract. Fresh organs may be examined at once by dark-field illu- 
 mination, or if this is impossible and the fetus shows signs of syphilis, the liver may be 
 cut into large pieces and preserved in 70 per cent, alcohol. After a few days a section 
 is removed and stained by the Levaditi method for spirochetes. If these microorgan- 
 isms are numerous, the liver is suitable for preparing the antigen; otherwise it should 
 be discarded. Very fatty livers are to be avoided, anS. those of still-bom fetuses are 
 to be preferred. 
 
 Ten grams of liver are minced, ground with quartz sand, and treated with 100 
 c.c. of absolute ethyl alcohol. The mixture is shaken mechanically with glass beads 
 for twenty-four hours, and extracted in the incubator for ten days. The containing 
 flask or bottle should be well stoppered to prevent undue evaporation, and should be 
 shaken up at least once a day. The extract is then filtered through fat-free paper or 
 paper washed with ether and alcohol to remove the hemolytic substances that may 
 be present. The filtrate is measured, and the loss by evaporation is made up by 
 the addition of more alcohol. If a shaking apparatus is not at hand, extractions may 
 be left in the incubator a few days longer. After standing a few days a sediment 
 forms, which should not be removed or disturbed. 
 
 3. Alcoholic Extracts of Normal Organs. — 'these are used extensively, 
 at present, and apparently yield results equal to those obtained with 
 extracts of luetic liver. It is certainly true that a good extract of a normal 
 organ is superior to a poor one prepared from luetic liver. Many, with 
 
GENERAL TECHNIC 421 
 
 the idea of specificity uppermost in their mind, adhere to the use of the 
 latter, whereas the results of research and of practical work shows that 
 lipoids from normal organs serve equally well as antigen in making the 
 syphilitic reaction, and, indeed, may prove superior if luetic liver is 
 used that has undergone advanced fatty changes or autolysis, when 
 undesirable hemolytic and anticomplementary derivatives are extracted 
 in excess. 
 
 Human, guinea-pig, and beef-heart muscle are usually employed. 
 The first is especially efficient and is to be preferred. 
 
 Preparation. — The organ is obtained fresh from the autopsy room. It is freed 
 from fat, and to each 10 grams of minced muscle 100 g.c. of absolute ethyl alcohol 
 are added. Extraction is conducted in exactly the same manner as was described in 
 the preparation of alcoholic extract of luetic liver. 
 
 If guinea-pig heart is employed, as much of the fat as possible should be removed, 
 otherwise the extract may be quite anticomplementary. 
 
 Boas prepared an extract of human heart by treating the ground muscle with 
 nine parts of absolute alcohol, shaking for an hour at room temperature, filtering, and 
 storing away in a stoppered bottle. He found that different extracts so prepared are 
 remarkably constant in their properties, although they deteriorate rapidly and should 
 be prepared freshly every few weeks, 
 
 4. Alcoholic Extracts of Normal Organs Reenforced with Choles- 
 terin. — Sachs advocated the addition of pure cholesterin to alcoholic 
 extracts of normal heart as a means of rendering these antigens more 
 delicate, without materially increasing their anticomplementary and 
 hemolytic properties. He found that such preparations possess proper- 
 ties equal to the best syphilitic extracts. This=work has been confirmed 
 by Hemlein, Altman, Mcintosh and Fieldes, DesmonUere, Walker, 
 and Swift. We have studied the subject with much interest, comparing 
 the results with those secured from other antigens, as alcoholic extracts 
 of syphilitic liver and acetone-insoluble lipoids. It is true that these 
 preparations are highly sensitive — so much so that I never employ them 
 alone in making diagnostic reactions, but always in conjunction with 
 other extracts as controls, in order to detect and avoid non-specific 
 reactions with non-luetic serums. We have found that they occasion- 
 ally give faint positive reactions with normal serums; on the other hand, 
 not infrequently, they react strongly positive in cases where, with other 
 extracts, the reactions are negative; in the majority of such cases 
 the serum is from a long-standing or a treated case of lues that needs 
 further treatment until the reaction with a chblesterin extract becomes 
 negative. These alcoholic extracts of normal organs have their greatest 
 value, therefore, with known syphilitic seruij.is when the reaction is 
 conducted as a guide to treatment. In diagnostic reactions, however, it 
 is my opinion that they should not be used alone, but together with less 
 
422 THE TECHNIC OP COMPLEMENT-FIXATION REACTIONS 
 
 sensitive antigens. In other words, one should use every -precaution and 
 exercise great care before making a diagnosis; when lues is known to be 
 present, however, the treatment should be thorough, and there would seem to 
 be no better criterion for judging the state of the infection than repeated 
 negative reactions with cholesterin extracts. 
 
 Preparation. — These extracts are prepared of human, ox, and guinea-pig heart. 
 Human heart usually yields the best extract. Care should be taken to use only 
 muscle and to avoid fat. To 10 grams of minced muscle add 100 c.c. of absolute 
 ethyl alcohol. Shake in a mechanical shaker for twenty-four hours, and continue 
 the extraction in the incubator for ten days or two weeks. Then filter through fat- 
 free filter-paper, and add absolute alcohol to make up for the loss through evapora- 
 tion. Add 0.4 gram of Kahlbaum's cholesterin (0.4 per cent.); shake well and stand 
 aside in the refrigerator for a few days. The cholesterin goes into solution slowly in 
 cold alcohol, and 0.4 per cent, of cholesterin usually- saturates the solution. After 
 a week the extract may be again filtered and stored in a tightly stoppered bottle. The 
 slight sediment that may form should not be disturbecf. 
 
 These extracts keep fairly well. Differejit preparations are quite 
 similar in their properties; they are usually found to be highly antigenic 
 and no more anticomplementary than crude, alcoholic extracts. They 
 constitute, therefore, inexpensive and very sensitive antigens. 
 
 5. Acetone-insoluble Lipoids. — As previously stated, crude alco- 
 holic extracts may contain an excess of undesirable constituents, such 
 as neutral fats, fatty acids, soaps, and certain, protein materials, which 
 are responsible for the untoward anticomplementary and hemolytic 
 effects. To eliminate these Noguchi advised the exclusive use of the 
 acetone-insoluble fraction instead of the entire unfractionated alcoholic 
 extract, especially if unheated human serums are used in conducting the 
 syphilis reaction. These extracts are compo^d essentially of lecithins, 
 which, when prepared from any one source^ consist of a mixture of 
 analogous bodies; lecithins from different sources vary in their compo- 
 sition. In speaking of lecithins, one is prone to regard them as chemicals, 
 and to overlook their biologic properties. Noguchi no longer employs the 
 term lecithin to designate the acetone-insolubte fraction of tissue lipoids. 
 
 These antigens are readily prepared of ox-heart or of human liver, 
 the former being preferable for use. Their main disadvantage is the 
 expense of preparation, for it may be necessary to prepare several 
 extracts before one that is satisfactory is secured. A good extract will, 
 however, keep well, and is a reliable and valuable antigen for the testing 
 for the syphilitic reaction. 
 
 Preparation. — A mashed paste of the muscle of ox-heart is extracted with 10 
 parts of absolute alcohol at 37° C. for four days. It" is then filtered through filter- 
 paper and the filtrate collected and brought to a state of dryness by evaporation. 
 The use of the electric fan is not necessary, for if poured into large flat dishes, the 
 filtrate will evaporate in from twelve to twenty-four hours. The residue is then 
 taken up with a sufficient quantity of ether, and the tujbid ethereal solution is allowed 
 
GENERAL TECHNIC 423 
 
 to stand for a few hours in a cool place until cleared. The clear ethereal portion is 
 then carefulb' decanted oil into another clean evaporating dish, and then allowed to 
 become concentrated by evaporating the ether off. The concentrated ethereal 
 solution is now mixed with about 10 volumes of pure acetone. A hght yellow precipi- 
 tate forms, which is allowed to settle, and the supernatant fluid is decanted off. Dis- 
 solve each 0.3 gm. of this substance in 1 c.c. of ether and add 9 c.c. of pure methyl 
 alcohol. As a rule, the greater part of the substance goes into solution. This alco- 
 holic solution remains unaltered for a long time, and is kept as a stock solution from 
 which the emulsion for immediate use may be prepared at any time by mixing 1 c.c. 
 with 19 c.c. of saline solution. This solution is then titrated for antigenic, anticom- 
 plementary, and hemolytic action. 
 
 According to Noguchi, if the extract is anticomplementary or hemo- 
 Ij-tic in doses of 0.4 c.c. of a 1 : 10 dilution, it is unsuitable. If it produces 
 complete inhibition of hemolysis -^-ith 0.1 c.c. of sj-philitic serum In doses 
 of 0.02 c.c. or less of the same dilution ( = 0.2 c.c. of a 1:100 dilution), it 
 is suitable. In making the fixation test, 0.1 c.c; of a 1 :10 emulsion is to 
 be used, thus emplojdng five times the minim-al antigenic dose which 
 does not cause non-specific fixation and is not unduly sensitive. 
 
 6. Lecithin and Cholesterin. — Browning, Cruickshank, andMcKenzie 
 advocate the use of a mixture of ox-liver lecithin and cholesterin as 
 antigen in testing for the syphilitic reaction. They find that the amount 
 of complement fixed by lecithin alone with gj'philitic serums is very 
 much increased by the addition of cholesterin;. that cholesterin does not 
 increase the anticomplementary action of the aptigen; that the binding 
 power or antigenic value of a mixture of lecithin and cholesterin is equal 
 in most cases to crude alcoholic extracts, but is less anticomplementary. 
 These observers use this mixture in their modified method, which con- 
 sists in the accurate estunation of the amount of complement fixed by 
 .extract and syphilitic serum. It would seem, that this extract should, 
 ifi the ordinary methods, be controlled with less sensitive antigens, and 
 I have found that ordinary cholesterinized alooholic extracts of himian 
 heart are equally efficient and less difficult to prepare in performing a 
 quantitative Wassermann reaction after the method just outlined. 
 
 Preparalum. — Minced ox-liver, obtained within three or four hours after death, 
 is digested with four parts of 95 per cent, alcohol for three or four days at room 
 temperature, during which time the mixture is stirred'yp at least once a day. The 
 filtrate is evaporated in an open flat porcelain dish on the water-bath at 60° C. for 
 four or five hours until a syrupy mass remains. Thistis rubbed up with washed and 
 dried quartz sand untU a firm mass results. The mixfure of dried extract and sand 
 ;(about 50 grams of sand to the residue of 1000 c.c. of extract) is placed in a spheric 
 flask, closed with a perforated rubber stopper through which runs a short piece of 
 quill tubing drawn out to a capillary point at the end.- This tube serves to prevent 
 the vapor in the flask from forcing out the stopper. Ethyl acetate i.^; placed in the 
 flask, wliich is then stoppered and immersed up to its neck in water at (iO° C. The 
 flask is shaken repeatedly, and after ten minutes the ethyl acetate is poured off into 
 a hot-water filter, the funnel jacket being kept at 60° C., and the solution filtered 
 through fat-free filter-paper. Another portion of ethyl-Scetate is added to the sand, 
 and the extraction repeated. After a third treatment practically all the soluble matter 
 will have been extracted. In all, about 170 c.c. of ethyl acetate should be used to 
 
424 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 extract the residue of 1000 c c. of crude extract. The gortion that is precipitated when 
 the ethyl acetate solution is placed in the ice-chest overnight is again dissolved in 
 ethyl acetate at 60° C. and the solution again placed in the ice-chest overnight. 
 Finally the portion insoluble in ethyl acetate in the cold is dissolved in water-free 
 ether (sp. gr., 0.717) at room temperature. To the ethereal solution in a glass cyl- 
 inder four volumes of acetone are added, causing a precipitate to be deposited. 
 Precipitation is aided by shaking the mixture for several minutes. Separation is 
 complete in ten minutes. The supernatant fluid is pqured off, and the crude precipi- 
 tate of lecithin is redissolved in ether and the precipitation with acetone repeated 
 twice. The precipitate is finally rubbed up with sand and the soluble portion taken 
 up by extraction with absolute ethyl alcohol for twenty-four hours at room temper- 
 ature. The last traces of lecithin may be removed by extracting the residue with an 
 additional small quantity of alcohol. The ordinary commercially "pure" reagents 
 are satisfactory for this method of preparation of lecithin. 
 
 The lecithin of 1000 c.c. of crude extract should be extracted with about 100 c.c. 
 of alcohol. The strength of the solution is estimated by evaporating 10 c.c. at 57° 
 C. and weighing. The alcoholic lecithin solution is kept in a stoppered bottle at room 
 temperature in the dark. After allowing it to stand for a week a 0.75 per cent, 
 solution in alcohol is diluted 1 : 7 with normal salt solution, to secure the maximum 
 turbidity, and titrated. Browning and McKenzie use 0.6 c.c. of this emulsion as the 
 ' antigenic dose. 
 
 7. Aqueous Extract of Pallidum Culttu-e. This antigen is prepared 
 by Noguchi, who uses pure cultures of Treponema pallidum in ascites 
 kidney agar. Preferably several strains should be used in the prepara- 
 tion, which corresponds quite closely to luetin. Cultures grown seven, 
 fourteen, twenty-one, twenty-eight, thirty-five, and forty-two days are 
 chosen and examined, and those that show the best growths in the agar 
 columns are selected. The oil is poured off, the tubes cut just above the 
 kidney, and the column of ascites agar between the piece of kidney and 
 the oil removed with particular care, so as not to include the kidney or 
 the oil. This substance is ground in a mill until the spirochetes show 
 disintegration. The thick emulsion is then diluted with normal salt 
 solution and heated to 60° C. for one-half hour; 0.4 per cent, phenol is 
 added as a preservative, and the emulsion titrated for its anticomple- 
 mentary dose. In conducting complement-fixation reactions with 
 pallidum antigen one-half of the anticomplementary dose is used, and the 
 serum must be inactivated. 
 
 Comparative Antigenic Values of Various Extracts 
 For several years past I have been particularly interested in studying, 
 from a practical standpoint, antigenic values^ of the extracts most com- 
 monly employed in the serum diagnosis of syphilis, and comparing them 
 with suitable alcoholic extracts of sj^philitic liver as a standard antigen. 
 For this purpose antigens were carefully chosen after titration, and 
 only those were employed that were safely free from anticomplementary 
 action and whose antigenic dose was known. A large number of serums 
 and cerebrospinal fluids from syphilitic and non-syphilitic persons were 
 
GENERAL TECHNIC* 
 
 425 
 
 tested with numerous diiferent extracts at the same tune, and under 
 similar conditions. A suitable alcohohc extract of syphilitic liver was 
 always included among the antigens in testing each serum or fluid, and 
 the other extracts compared with it in determining their antigenic value. 
 In the following table the results of such studies, covering a period 
 of two years, are given: 
 
 TABLE 12.— COMPARATIVE ANTIGENIC VALUES OF VARIOUS TISSUE 
 
 EXTRACTS 
 
 
 Antigenic Properties a8 Compared to Alcoholic Ex- 
 tracts OP Syphilitic Lives 
 
 Extra era 
 
 Equal 
 
 Stronger 
 
 Weaker 
 
 Negative 
 (Positive 
 with Alco- 
 holic Extract 
 of Syphihtic 
 Liver) 
 
 Positive 
 (Negative 
 with Alco- 
 hohc Extract 
 of Syphilitic 
 Liver) 
 
 Cholesterinized alcoholic ex- 
 tracts of human, pig, and 
 beef heart 
 
 .50,0 
 73.0 
 
 71.1 
 
 68.7 
 73.2 
 
 30.0 
 10.8 
 
 1.9 
 
 4.4 
 
 9.7 
 
 f3.4 
 
 18.9 
 2l5 
 
 3.2 
 5.7 
 6.6 
 3.5 
 
 20 
 
 Acetone-insoluble Lipoids . . . 
 Alcoholic extract of pig and 
 beef heart 
 
 1.8 
 
 7 6 
 
 Acetone extract of syphilitic 
 hver 
 
 Alcoholic extract of normal 
 liver 
 
 1.3 
 
 The results of these studies have shown : 
 
 1. That cholesterinized alcohohc extracts of human, beef, and guinea- 
 pig heart are far more sensitive than simple alcoholic extracts of syphil- 
 itic liver. Another pecuUar feature of these antigens is the fact that in 
 syphihs, if they react at all, they usually do so quite strongly. 
 
 2. That the addition of cholesterin to crude alcohohc extracts of 
 syphihtic Hver and of normal liver doubles their antigenic sensitiveness 
 without materially increasing their anticomplementary and hemolytic 
 action. 
 
 3. We have practically never found a serurn that reacted negatively 
 with a cholesterin extract and positively with an alcohohc extract of 
 syphihtic hver. On the other hand, ui about '20 per cent, of cases the 
 cholesterinized antigens wiU react positively, whereas with the plain 
 antigen of syphilitic hver the reactions are negative. In the majority 
 of such instances the person was known to b& luetic, but had received 
 treatment and was regarded clinically as cured", or the serum was that 
 of a long-standing and unrecognized case of lues. Unfortunately, slight 
 
426 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 reactions may be secured with about 5 per cent, of normal serums. For 
 this reason, when conducting tests for diagnostic purposes, I control the 
 cholesterinized extracts with less sensitive antigens, such as alcohohc 
 extract of syphilitic liver and acetone-insoluble lipoids. 
 
 4. It is highly important that these extracts be carefully standardized 
 and that any serum, even if but slightly anticomplementary, be dis- 
 carded. 
 
 5. In our experience, repeated negative reactions with satisfactory 
 cholesterinized antigens constitute the best evidences of the absence of 
 lues or testify to the recovery from a luetic infection. The treatment of 
 syphilis should be continued until the patient's serum reacts negatively 
 with alcoholic extract of syphilitic liver, and finally with cholesterinized 
 extracts. The disease cannot be regarded as cured until the reaction 
 has remained negative for a year or two at least, and treatment must not 
 be discontinued until this result is secured, or= it is shown that the se- 
 rum is " Wassermann fast" and that it is impossible to secure a nega- 
 tive reaction. 
 
 6. For the less experienced worker, or wheii but one antigen is being 
 used in conducting the reaction, a properly prepared alcoholic extract of 
 syphilitic liver is to be recommended. One drawback to the use of this 
 extract is the difficulty of obtaining suitable tissues for the preparation 
 of the antigen. It has been my practice for many years to preserve the 
 livers of as many still-born fetuses as I could obtain in 70 per cent, 
 alcohol, and to discard them later unless on section they showed the 
 presence of numerous spirochetes. These aaitigens are usually more 
 sensitive than similar extracts of normal livpr, but it is important to 
 remember that not every extract is satisfactory simply because it is 
 prepared of syphilitic tissues. 
 
 7. A suitable preparation of acetone-insoluble lipoids, prepared after 
 the method of Noguchi, constitutes a sensitive, reliable, and satisfactory 
 antigen. When properly titrated and standai?dized and used with inac- 
 tivated serums, this antigen may prove quite sensitive and safe. Nogu- 
 chi, in his efforts to simplify the technic of the syphilis reaction, impreg- 
 nated filter-paper with this antigen and allowe"d it to dry. This prepara- 
 tion is unstable and generally unsatisfactory.. The antigen is best pre- 
 served in a stock bottle or in ampules, and is, diluted with salt solution 
 just before being used in the test. Under these conditions we have found 
 these extracts to be quite stable. 
 
 4. Plain alcoholic extract of human, guinea-pig, and beef heart are 
 easily prepared, are quite inexpensive, and when properly standardized 
 
GENERAL TECHNIC 427 
 
 serve as satisfactory antigens. Boas uses alcoholic extracts of human 
 heart (Michaelis) exclusively, and has found, that they yield better 
 results than alcoholic extracts of syphilitic ifvei. Garbat and others 
 use and recommend similar extracts of guinea-pig heart. 
 
 8. Aqueous extracts of pure cultures of pallida have not thus far 
 yielded results equal or superior to ordinary non-specific antigens. As 
 compared with lipoidal extracts, they have generally yielded reactions 
 that are much weaker, and in primary and secondary syphilis may react 
 entirely negatively. Much is yet to be learned, however, of bacterial 
 antigens in general, and the subject must be regarded as still in the 
 experimental stage. 
 
 It may be said to he well proved that extract antigens in the syphilis 
 reaction are not biologically specific, and need not be extracts of syphilitic 
 tissues. An antigen cannot give reliable or satisfactory results unless it is 
 carefully titrated and its properties determined. Antigens may serve as a 
 frequent source of error when the complement-fixation reactions are con- 
 ducted by those possessing insufficient knowledge of their good and bad 
 properties. The test for the syphilitic reaction should not be undertaken 
 by any one not competent to titrate and judge of the qualities of the antigen 
 to he employed. 
 
 Method of Diluting Antigens. As a general rule, all organic extracts 
 must be diluted with normal salt solution before being used. 
 
 If extracts and diluent are mixed quickly, the emulsion is clear or 
 slightly opalescent. If the diluent is added slowly to the organic extract, 
 the resulting mixture becomes quite turbid and milky. As shown by 
 Sachs and Roudoni, the antigenic power of the extract is more marked 
 with the turbid than when the clear or opalescent emulsion is used. 
 For this reason, in testing for the syphilis reaction the emulsion of organic 
 extract should be made so as to secure the maximum amount of turbidity. 
 The required amount of antigen is placed in a test-tube, and the salt solution 
 is added slowly with a pipet; or the salt solution rnay be placed in a tube and 
 the extract floated on it and gradually mixed. 
 
 Although with each new extract it may be necessary to titrate with 
 various dilutions before one that is satisfactory is reached, experience 
 has shown in the majority of instances that the following dilutions are 
 usually correct: 
 
 1. Alcoholic extract of syphilitic liver: 1 part with 9 parts of salt 
 solution. Extracts of German manufacture are usually diluted six or 
 seven times. 
 
 2. Cholesterinized extracts and acetone-insoluble lipoids in methyl 
 
428 THE TECHNIC OF COMPLEMENT-FIXATION KEACnONS 
 
 alcohol require higher dikition, as 1 part of cxirait with 10 parts of salt 
 solution. 
 
 3. Plain alcoholic extracts of human, beef i\n(l fiuinca-piu; heart like- 
 wise require high dilution, as, for example; extract, 1 pai't and salt 
 solution, 19 parts. 
 
 4. Alcoholic (>xtract of human heart, prepared after the method of 
 Boas (quu'k methotl), is usetl in lower tlilution, as 1 part of extract witli 
 9 parts of salt solution. 
 
 5. The solution of lecithin and cholestt-rin used by Browning;, 
 Cruickshank, and Mackenzie is diluted with seven parts of salt solution. 
 
 6. Aqueous extracts of syphilitic liver and;extracts of pallida culture 
 are used undiluted, or may require dilution with four parts of salt solu- 
 tion. 
 
 Although these emulsions will keep Jor a few ddys if placed in the re- 
 frigerator, it is advisable to make up only the anionnt required for imme- 
 diate use, as freshly prepared emulsions are better than older ones. 
 
 Method of Titrating Antigens. Three values arc to be determined: 
 
 1. The anticomplementary dose, or that aniount of antigen that in 
 itself is capable of fixing or inactivating the complciiicnt. 
 
 2. The hemolytic dose, or that amount of antigen (hat in itself is 
 capable of lysing red blood-cells. This action is probably due to the 
 presence of certain lipoids and alcohol. 
 
 3. The antigenic dose, or that amount of antigen that serves tc 
 absorb or fix a certain and constant dose of complement with a definite 
 ariiount of syphilitic serum. 
 
 1. Anticomplementary Titration. The determination of the anti- 
 complementary dose is probal)ly the most iujportant, for if an extract 
 were used in an amount that was anticomplenu-ntary or cai)ablc of 
 fixing complement in a non-specific manner, all the tests would sliow 
 false positive reacitions, regardless of whether the serum was from a 
 normal or from a luetic person. 
 
 After determining this anticomplementary dose, in conducting the 
 main test the antigen may be used in one-fourth the amount. 
 
 All tests are conducted with chemi('ally ('lean and prefcTably sterile 
 test-tubes (12 cm. by 13 mm.) and graduilted pipets. Ac('ni!i,(:y in 
 measurements is very essential in performing all complement-fixation 
 work. 
 
 A preliminary titration of the hemolysin is made, with the coinpIein(mt 
 and corpuscle suspension to be us(m1 in titrating the antigen, in order to 
 determine its hemolytic dose, i. e., to adjust the hemolyti<' system. If, 
 
GENERAL TECHNIC 429 
 
 for instance, the hemolytic serum is known to have a titer of about 
 1 : 2000 (see p. 375), a stock solution is made by diluting 1 c.c. of the serum 
 with sterile salt solution to make a dilution of 1 : 200. In a series of six 
 test-tubes increasing amounts of this diluted- amboceptor are placed: 
 0.05 c.c, 0.1 CO., 0.15 c.c, 0.2 c.c, 0.3 c.c, and 0.4 c.c; to each tube add 
 1-c.c. of complement serum diluted 1 : 20 ( = (^05 c.c. undiluted serum) 
 and 1 c.c. of a 2.5 per cent, suspension of sheep's cells and sufficient salt 
 solution to bring the total contents up to 3 or 4 c.c. Each tube is shaken 
 gently and incubated at 37° C. for an hour. At the end of this time that 
 amount of amboceptor that just completely hemolyses the corpuscles is taken 
 as the hemolytic unit (Fig. 103). In conducting the antigen titrations 
 one and one-half or twice this amount is used as the dose. 
 
 The antigen extract is diluted 1 : 10 by placing 1 c.c. in a test-tube and 
 slowly adding 9 c.c. of normal salt solution. 
 
 Increasing amounts of this emulsion are placed in a series of test- 
 tubes: 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.5, and 2 c.c. To each tube are now 
 kdded 1 c.c. of the diluted complement serum ( = 0.05 c.c. undiluted se- 
 rum) and sufficient normal salt solution to bring the total volume up to 3 
 or 4 c.c. Shake each tube gently and incubate for one hour at 37° C. Then 
 add to each tube 1 c.c. of the corpuscle suspension and a dose of am- 
 boceptor equal to IJ^ units, as just determined by previous titration. 
 Shake gently and reincubate for another hour and a half, when a prelim- 
 inary reading of the results may be made. That amount of antigen that 
 shows beginning inhibition of hemolysis is regar&ed as the anticomplemen- 
 tary unit. The final readings are made after the tubes have stood over- 
 night in a refrigerator at low temperature (Fig. 108). 
 
 This titration may also be made in the presence of normal serum, 
 although this is not absolutely necessary. The serum must be perfectly 
 fresh, and must be that from a person known to be free from lues. It is 
 inactivated by heating to 55° G. for half an hour, and 0.1 c.c. is added 
 to each tube. Complement and salt solution are now added, and the 
 titration conducted in the manner just described. Normal serum may 
 iabsorb a small amount of complement in itself, and hence a titration 
 conducted with serum may show a slightly lower anticomplementary 
 dose. 
 
 The fo owing controls arc included: 
 
 1. A hemolytic system control, containing the complement, corpuscles, 
 and amboceptor in the same amounts as were used in conducting the 
 titration. This control should show complete l^emolysis. 
 
 2. A serum control, which is the same as the hemolytic system control 
 
430 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 plus 0.1 C.C. of the serum. This should show complete hemolysis, and 
 indicates that the serum was not anticomplementary. This control test 
 should never be omitted. 
 
 3. A corpuscle control, including 1 c.c. of the corpuscles in salt solu- 
 tion. This tube should show no hemolysis. 
 
 The following table gives the results of a titration with an alcoholic 
 extract of syphilitic liver diluted 1 : 10 (see Fig. 108) : 
 
 TABLE 13.— ANTICOMPLEMK.NTAHY TITRATION OF AN ORGANIC 
 
 EXTRACT 
 
 Tube 
 
 1 
 
 2 
 3 
 4 
 5 
 
 6: 
 
 7 
 
 8 
 
 9 
 
 10 
 
 Antigen 
 
 COMPLE- 
 
 Normal 
 
 (1:10) 
 C.c. 
 
 (1:20), 
 C.c. 
 
 Serum 
 C.c. 
 
 0.2 
 
 1 
 
 0.1 
 
 0.4 
 
 1 
 
 0.1 
 
 0.6 
 
 1 
 
 0.1 
 
 0.8 
 
 1 
 
 0.1 
 
 1.0 
 
 1 
 
 0.1 
 
 1.2 
 
 1 
 
 0.1 
 
 1.5 
 
 1 
 
 0.1 
 
 2.0 
 
 1 
 
 0.1 
 
 Control 
 
 1 
 
 — 
 
 Control 
 
 1 
 
 0.1 
 
 Anttshbrp 
 Hemolysin 
 
 Units 
 
 1}2 
 
 m 
 m 
 
 V:, 
 
 1}2 
 
 Sheep's 
 Corpus- 
 cles 
 
 2.5 PER 
 CENT., c.c. 
 
 Resttl/ts 
 
 Hemolysis 
 
 Hemolysis 
 
 Hemolysis 
 
 Hemolysis 
 
 Hemolysis 
 
 Slight inhibition of 
 hemolysis 
 
 ftlarked inhibition of 
 hemolysis 
 
 Complete inhibition 
 of hemolysis 
 
 Hemolytic control: 
 complete hemolysis 
 
 Serum control: com- 
 plete hemolysis 
 
 In this titration tube No. 6, containing 1.2 c.c. of the antigen emulsion 
 showed beginning inhibition of hemolysis and was recorded as the anti- 
 complementary dose. 
 
 2. Hemolytic Titration. As previously mentioned, organic extracts 
 are capable in themselves of hemolyzing red cells; this is due to the 
 hemotoxic action of lipoids and alcohol. Extracts of organs that have 
 undergone advanced autolysis and decomposition are very likely to be 
 hemolytic. 
 
 Serum exerts an inhibiting influence on thq lytic action of an organic 
 extract. Hence the hemolytic dose of an extract depends largely on 
 whether or not complement serum is used in the titration. 
 
 When an organic extract is titrated in the presence of complement, 
 the hemolytic dose is higher than the anticomplementary dose. In the 
 foregoing titration 3 c.c. of the extract emulsion showed beginning 
 hemolysis, and when 4 c.c. was used, hemolysis was complete. These 
 large amounts of emulsion give the tube contents quite a millfy appear- 
 ance, but close inspection shows that all the cells are broken up. 
 

 us 
 
 Fig. 108. — Tithation or Antigen for Axticumplejientary Unit. 
 
 ■vaae^- 
 
 J 
 
 
 f* 
 
 ?.// 
 
 
 
 i 
 
 
 -_: 
 
 
 " " 
 
 
 
 \ 
 
 Fig. 109. — Titration of Antigen for Antigenic Unit. 
 
GENERAL TECHNIC 431 
 
 As a general rule, the hemolytic titration is-not absolutely necessary. 
 It may be conducted with the anticomplementary titration by adding 
 another tube or two to the foregoing series, with higher doses of extract; 
 or this titration may be conducted separately, .and without complement 
 and hemolysin, by using the same doses of antigen with 1 c.c. of cor- 
 puscle suspension and sufficient salt solution to bring the total volume in 
 each tube up to 3 or 4 c.c. 
 
 3. Antigenic Titration. — As previously ststted, this titration is not 
 absolutely necessary, as one-fourth the anticomplementary dose of an 
 extract may be used in the main test. For instance, in the foregoing 
 titration 0.3 or 0.4 c.c. may safely be used in making the test for the 
 syphilitic reaction. Different extracts vary, however, in their anti- 
 genic value. Some may be highly anticonlplementary and have a 
 comparatively low antigenic value; purer extracts, such as acetone- 
 insoluble lipoids or cholesterinized alcoholic extracts of heart, are 
 largely free from anticomplementary action', and at the same time 
 possess a high antigenic value. It is advisable, therefore, to use an 
 antigen whose full antigenic as ivell as anticomplementary doses are 
 known, for, while it is necessary to use sufficient antigen, it is not advisable 
 to use a larger amount than is necessary. 
 
 For this titration all antigens except alcoholic extracts of syphilitic 
 hver, should be diluted 1 : 20 with normal salt solution. Usually the 
 antigenic unit is so much lower than the anticomplementary unit that 
 it is best determined with a more dilute antigen. 
 
 The titration is conducted in a manner similar to the anticomple- 
 mentary titration, except that 0.1 c.c. of fresh and inactivated serum 
 from a known and untreated syphilitic person is added to each tube. 
 Increasing doses of antigen, patient's serum, and complement are 
 mixed, shaken, and incubated for one hour. One and one-half doses 
 of hemolysin and corpuscles are then added, the tubes shaken and 
 incubated for another hour, after which the preliminary reading is 
 made. The final reading is taken after the. tubes have been placed 
 overnight in a refrigerator at low temperature. That amount of 
 antigen that shows just complete inhibition of hemolysis is taken as the 
 antigenic unit (Fig. 109). In conducting the syphilis reaction two to 
 four times this unit is used, providing that these amounts are at least four 
 or five times less than the anticomplementary do^e. This larger antigenic 
 dose is advisable, because the exact unit may not be sufficient with 
 serums containing but small amounts of syphilis antibody such as 
 those of treated or long-standing cases of lues. 
 
432 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 The following table illustrates this titration with the same alco- 
 holic extract of syphilitic liver (see Fig. 109) : 
 
 TABLE 14.— ANTIGENIC TITRATION OF AN ORGANIC EXTRACT 
 
 Tube 
 
 Anti- 
 gen 
 
 1: 10, 
 Co. 
 
 1 
 
 0.05 
 
 2 
 
 0.1 
 
 3 
 
 0.15 
 
 4 
 
 5 
 
 6 
 
 7 
 
 0.2 
 
 0.25 
 
 0.3 
 
 Control 
 
 8 
 
 Control 
 
 
 
 o 
 
 
 Sheep 
 
 >> 
 
 Syphi- 
 
 Com- 
 
 S3 
 
 Anti- 
 
 COK- 
 
 d 
 
 litic 
 
 ple- 
 
 sheep 
 
 PUSCLES 
 
 C 
 
 Sekum 
 
 ment 
 
 Hem- 
 
 2.5 
 
 
 (Inactive) 
 
 1:20, 
 
 > 0) 
 
 olysin, 
 
 .Peh 
 
 C.c. 
 
 C.c. 
 
 "B o 
 
 Doses 
 
 Cent., 
 C.c. 
 
 I 
 
 
 
 o-d 
 
 
 
 
 
 
 
 
 
 
 
 
 
 3 
 
 0.1 
 
 1 
 
 1,1-2 
 
 1 
 
 M 
 
 
 
 M a 
 
 
 
 V 
 
 0.1 
 
 1 
 
 
 i'A 
 
 1 
 
 a 
 
 
 
 
 
 1| 
 l1 
 
 0.1 
 
 1 
 
 
 IK 
 
 1 
 
 
 
 
 
 
 .3 
 
 
 
 3-^ 
 
 
 
 0.1 
 
 1 
 
 -g M 
 
 VA 
 
 1 
 
 -d 
 c 
 
 0.1 
 
 1 
 
 z^ 
 
 W2 
 
 1 
 
 0.1 
 0.1 
 
 1 
 1 
 
 'is 
 1 "^ 
 
 18 
 
 1 
 1 
 
 1 
 
 
 
 1 
 
 •s " 
 
 1}^ 
 
 1 
 
 1 
 
 
 
 3 o 
 
 
 
 
 
 CO *^ 
 
 
 
 H 
 
 Kesui/ts 
 
 Slight inhibition 
 
 of hemolysis 
 Marked inhibi- 
 tion of hemoly- 
 sis 
 Complete inhi- 
 bition of hemol- 
 ysis 
 No hemolysis 
 No hemolysis 
 No hemolysis 
 Serum control: 
 
 Hemolysis 
 Hemolytic con- 
 trol : Hemoly- 
 
 In this instance 0.15 c.c. of the emulsion represents the antigenic 
 unit. In performing the Wassermann reaction 0.3 or 0.4 c.c. was used, 
 and these amounts were about one-fourth the anticomplementary dose. 
 
 It is not unusual to find cholesteriniz'ed alcoholic extract and 
 acetone-insoluble lipoids perfectly antigenic in 0.05 c.c. of a 1:20 
 dilution, and not anticomplementary under 1 or 2 c.c. of a 1 :10 dilu- 
 tion. In these instances four times the antigenic dose, or 0.2 c.c. 
 can be used, and yet this amount is at least 10 times smaller than the 
 anticomplementary dose — a condition of affairs that constitutes a safe 
 and desirable antigen. 
 
 Each new antigen should be tested with a number of serums and 
 controlled by an older antigen of known value before being finally 
 accepted as satisfactory. 
 
 Antigen containers should be well stoppered and kept in the refrig- 
 erator. Deterioration may set in suddenly, and they should, therefore, 
 be re titrated every few weeks. 
 
 VARIOUS METHODS FOR CONDUCTING THE SYPHILIS REACTION 
 First Method. — The simplest technic, and the one best adapted for 
 inexperienced workers, is the original Wassermann reaction, performed 
 
METHODS FOR CONDUCTING THE SYPHILIS REACTION 433 
 
 with alcoholic instead of aqueous extracts of syphilitic liver as antigen. 
 It is true that this method is not an exact quantitative reaction, and 
 that it is probably less delicate than some of the modified methods, 
 but its advantages are that it is easy of manipulation, is readily 
 learned, and is especially recommended for persons who perform these 
 tests at irregular intervals, as false positive" reactions are less likely 
 to occur than when the more delicate methods are used. 
 
 Second Method. — In this method the technic is essentially the 
 same as in the first method, except that three different antigens are 
 used instead of one, namely, cholesterinized extract of normal heart, 
 alcoholic extract of syphilitic liver, and acetone-insoluble lipoids. 
 This method has three advantages: (1) It permits the use of a choles- 
 terinized extract under conditions where any tendency to non-specific 
 fixation is to be controlled; (2) an antigen may at any time suddenly 
 become anticomplementary and yield false results, whereas by this 
 method the source of error is detected and may be avoided, since it is 
 not dependent upon any one extract; (3) an = extensive study of the 
 comparative values of antigens has led to the- distinct impression that 
 the lipodophilic antibody in different syphilitic serums frequently 
 shows a special affinity for the lipoids in a certain plain antigen more 
 than it does for those in another antigen; in- fact, I have not infre- 
 quently found that, with weakly positive serums, if one antigen had 
 been employed, a false negative report would have been rendered, the 
 true reaction being given by the other two antigens. These results 
 could not be ascribed to faulty antigen, for with other weakly positive 
 serums the extract would be found to react satisfactorily. 
 
 As previously mentioned, cholesterinized alcoholic extracts are 
 very sensitive, so that from this standpoint additional antigens would 
 appear to be superfluous. This very property, however, in my 
 opinion, renders it advisable to control them with less sensitive ex- 
 tracts. In this way all the advantages of a. very sensitive antigen 
 may be secured, and the disadvantages avoided until more extended 
 use demonstrates whether or not it is entirely safe to use these extracts 
 alone. 
 
 With strongly reacting serums all antigeng possess equal antigenic 
 power. With the serums of long-standing or' treated cases of syphilis 
 the cholesterinized extracts may react strongly positive, whereas with 
 the aqueous and the alcoholic extracts the reactions are weakly 
 positive, or negative with one and positive with the other. In cases 
 of syphilis that have received considerable treatment the reaction 
 28 
 
434 THE TECHNIC OF COMPLEMENT-FIX^ATION REACTIONS 
 
 may be negative at first with the aqueous and the alcoholic extracts, 
 and as treatment is continued it may finaily be negative with the 
 cholesterinized extracts. In a certain percentage of cases, especially 
 those of old infections of the central nervous system, the reaction is 
 positive with the cholesterinized extract and negative with the other 
 extracts; strong reactions of this character psually indicate syphilitic 
 infection. Occasionally a weak (10 per cent, or less, inhibition of 
 hemolysis) reaction may be had with the serum of a person who denies 
 syphilis. 
 
 Third Method. — One disadvantage of the regular Wassermann 
 technic is that it may not readily show improvement of the patient 
 while the treatment is going on. For instance, if complete fixation 
 of complement occurs with 0.2 c.c. of serum, one does not know 
 whether this is the smallest fixing dose, or whether there might be a 
 fixation even with much smaller quantities, of serum. This is very 
 important in examining cases during the course of the treatment, as 
 otherwise improvement in the condition rng^y be overlooked. Just 
 as soon, however, as the reaction with 0.2 q.c. of serum is a degree 
 less than absolutely positive, then the various steps, down to com- 
 plete negative reactions, are readily observed and recorded by the 
 usual Wassermann technic. This disadvantage may be overcome 
 by using at least six different doses of serum: 0.01, 0.02, 0.04, 0.06, 
 0.08, 0.1 c.c. In this way a means is afforded for judging of the 
 strength of the reaction, and the effect of anti-syphilitic treatment is 
 readily observed. 
 
 Fourth Method. — It has previously been pointed out that the 
 syphilis reaction is dependent upon the fact that while hemolytic 
 complement may be rendered inactive or fixed by serum alone and 
 organic extract alone, it is characteristic of Syphilis that a mixture of 
 serum and extract will absorb or fix more complement than the sum of 
 the amounts absorbed by these two substances alone. In the foregoing 
 methods no attempt has been made to meateure the amount of com- 
 plement absorbed by serum and antigen alone, but sufficient comple- 
 ment has been furnished to allow for this non-specific fixation, and 
 we are content to show that the serum and antigen alone do not 
 absorb enough complement to interfere with hemolysis, so that any 
 inhibition of hemolysis may be interpreted as specific complement 
 fixation. 
 
 Browning and Mackenzie have devised a technic whereby it is 
 possible to estimate the actual amounts of complements absorbed. 
 
METHODS FOR CONDUCTING THE SYPHILIS REACTION 435 
 
 first, by the serum and antigen alone, and second by these two sub- 
 stances combined. The complement absorbed is measured in terms 
 of hemolytic doses. This method consumes a little more time and 
 more of the various reagents is required. It is, nevertheless, the 
 best quantitative method we have, and shows exactly the degree of 
 complement fixation in each case. 
 
 In conducting any complement-fixation test the following are 
 essential factors if success is to be achieved: (1) Reliable reagents, 
 particularly a good antigen must be had, for no matter how much 
 care is exercised, good results cannot be secured with indifferent 
 reagents; (2) the observer must possess a thorough working under- 
 standing of the underlying principles and particularly of the quanti- 
 tative relations of the various reagents; (3) tjiere must be an accurate 
 adjustment of the hemolytic system; (4) he must have a careful, 
 painstaking and accurate habit of pipeting small amounts. Accuracy 
 should never be sacrificed for speed, as the latter is properly acquired 
 only with experience. 
 
 Teceinic of the First Method 
 This is the original Wassermaim reaction, except that an alcoholic, 
 instead of an aqueous extract of sj'philitic liver, is used as antigen, 
 and the amounts of each reagent arc just one-half those originally 
 employed. This is the simplest of all technics and, when properly 
 performed, constitutes, in the final analysis,- a reliable test and one 
 especially adapted for those not constantly ejigaged in this work. 
 
 1. Complement. — Fresh clear serum (not over twenty-four hours 
 old) of a healthy guinea-pig. Dilute 1:20 by adding 19 c.c. of sterile 
 normal saline solution to each 1 c.c. of serum. Dose, 1 c.c. ( = 0.05 
 c.c. of undiluted serum). 
 
 2. Corpuscles. — Sheep's blood washed three times and diluted 
 to make a 2.5 per cent, suspension. For example, 1 c.c. of corpuscles 
 in 39 c.c. of salt solution makes up sufficient for a number of tests. 
 
 3. Hemolytic Amboceptor. — Serum of a rabbit immunized ■ndth 
 washed sheep's corpuscles. As stated elsewhere, this serum is heated 
 to 55° C. for half an hour, and an equal part of chemically pure 
 glycerine is added. ]\Iix well and preserve in sterile 1 c.c. ampules. 
 Each ampule will, therefore, contain 0.5 c.c, of serum. One stock 
 dilution is prepared in such manner that about 0.2 c.c. represents one 
 hemoh-tic dose. It is usually well to prepare a whole series of flasks 
 with various dilutions, and in making a titration to use 1 c.c. of each 
 
436 THE TECHNIC OP COMPLEMENT-FIXATION REACTIONS 
 
 dilution; I have found it much more accuratfe, simple, and economical, 
 however, to prepare one stock dilution, which is titrated with each 
 complement and corpuscle suspension before^ each day's work. For 
 example, if a serum is known to have a titer of 1 ; 2000, an ampule 
 (0.5 c.c. of serum) is diluted with 200-c.c. of salt solution; this gives a 
 dilution of serum approximately 1:400, of which 0.2 represents one 
 hemolytic dose. The titration must be repeated each time to make 
 sure of this, because the complement of different pigs may vary in 
 activity, and the chief object is to adjust tjie amboceptor and com- 
 plement to each other. 
 
 Titration of Amboceptor. — Into a series of six test-tubes place in- 
 creasing amounts of the amboceptor dilutiqn: 0.05 c.c, 0.1 c.c, 0.15 
 c.c, 0.2 c.c, 0.25 c.c, and 0.3 c.c Add 1 cc of complement (1 : 20) 
 and 1 c.c. of corpuscle suspension to each tube, and sufficient salt 
 solution to make the total volume in each tube about 4 c.c. Shake 
 gently and incubate for one hour at 37° C. At the end of this time 
 the tube showing just complete hemolysis contains one hemolytic 
 dose, or unit of amboceptor. In the tests double this amount, or two 
 units, is used. 
 
 The amboceptor titration is very important. Under no cir- 
 cumstances should the same dose be used day after day without 
 titration, because the complement of different guinea-pigs may vary 
 in its activity, and these variations would be detected and would be 
 adjusted in this titration. For example, with a weaker complement 
 the dose of amboceptor required to effect complete hemolysis becomes 
 higher; each new corpuscle suspension may also vary slightly in the 
 actual number of cells contained in 1 cc, but this makes no difference 
 when each suspension is titrated with the, complement and ambo- 
 ceptor to be used in the day's work. This titration is set up first, 
 and while it is in the incubator, the main tests are arranged. 
 
 4. Antigen. — Alcoholic extract of syphilitic liver or acetone- 
 insoluble lipoids of proved value may be used. It is well to estimate 
 just how much antigen will be required for the tests on hand, so that 
 no waste will occur, as fresh emulsions are better than old ones carried 
 over from day to day. The dose should be at least double the titrated 
 antigenic unit, or one-fourth of the anticomplementary dose. For 
 instance, if an alcoholic extract of syphilitic liver diluted 1 : 10 is 
 found on titration to be perfectly antigenic in doses of 0.2 c.c, and 
 not anticomplementary in amounts under 2, c.c, then 0.4 c.c. maybe 
 used in making the tests, as this amount is still about five times less 
 
METHODS FOR CONDUCTING THE SYPHILIS REACTION 437 
 
 than the anticomplementary dose, and well within the range of safety 
 against non-specific complement fixation. If 10 tests arc to be made, 
 then at least 4.4 c.c. of diluted antigen are required, including sufHcient 
 for the antigen control, or in round numbers, 0.5 c.c. of antigen plus 4.5 
 c.c. of salt solution in order to secure the maximum turbidity slowly added. 
 
 5. Serum.^Serum should be fresh and clear and heated in a water- 
 bath to 55° C. for half an hour before using. The temperature should 
 not go above 56° C. nor below 55° C. The complement in perfectly 
 fresh serum is usually inactivated at this temperature within fifteen 
 minutes, but it is better to adopt the period of one-half hour as a 
 routine, especially in removing heat-sensitive anticomplementary sub- 
 stances that develop in serums more than a day old. Dose, 0.1 c.c, 
 or if the serum is perfectly fresh, 0.2 c.c. may be used. 
 
 6. Cerebrospinal Fluid. — This should be fresh and free from blood. 
 It is used unheated, as spinal fluid contains little or no hemolytic 
 complement. The dose should be at least= four times that of the 
 serum, or from 0.4 to 1 c.c. 
 
 The Test. — A front and a rear tube for each serum are placed in a 
 rack. Each tube is marked plainly with the -patient's name or in- 
 itials, and in addition the front tube is marked with the number of 
 the antigen or with the letter "A," or the word "antigen" is written 
 on it, the rear tube being marked "control" (serum control). The 
 necessity for carefully marking each tube is nowhere more important 
 than in conducting Wassermann reactions with a number of serums, 
 as the slightest error or lapse of memory may result in confusion and 
 prove to be quite a serious matter. 
 
 In each series of reactions the serum from a known case of syphilis 
 that has given a positive reaction and the serum of a known non- 
 syphilitic person are included as positive and negative controls re- 
 spectively. 
 
 Into each front tube the proper dose of aijitigen is placed; to the 
 front and rear tubes 0.1 c.c. of the patient's serum is added. (If 0.2 
 c.c. is being used as the dose, this amount should be placed in both 
 tubes.) To all tubes 1 c.c. of the complement (1 : 20) and sufficient 
 normal salt solution are then added to bring the total volume in each 
 to about 3 c.c. 
 
 The rear tube of each set is the serum control; the positive and 
 negative serums are treated in just the same-manner as the patient's 
 scrum. In addition to these there are three other important controls 
 that should not be omitted : 
 
438 THE TBCHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 1. The antigen control: Dose of antigen plus 1 c.c. of complement 
 (1 ; 20) and a sufficient quantity of salt solution. 
 
 2. Hemolytic system control: 1 c.c. of complement (1 ; 20) and 2 
 c.c. of salt solution. 
 
 3. Corpuscle control: 1 c.c. of corpuscle" suspension plus 3 c.c. of 
 salt solution. 
 
 Each tube is gently shaken and incubal«d at 37° C. for an hour, 
 when two hemolytic doses of amboceptor and 1 c.c. of corpuscle 
 suspension (2.5 per cent.) are added to each tube except the corpuscle 
 control. Tubes are shaken and reincubated for an hour or an hour 
 and a half, depending upon the hemolysis of the serum controls, after 
 which a preliminary reading is made and recorded. With partially 
 positive reactions the tubes may be centrifuged in order to read the 
 relative amounts of hemolysis, and the final reading made at once, or 
 the tubes may be placed in the refrigerator (just above freezing- 
 point) and the final readings made the next morning. 
 
 TABLE l.-}.— SCHEME FOR CONDUCTLXG A ■VS^ASSERMANN REACTION 
 (FIRST METHOD) (SEE Fiji. 110) 
 
 Unknown Sertim, 
 Mil. B. 
 
 Unknown Cetie- 
 
 BROaPINAL FlUIiJ. 
 
 Mb. C. 
 
 Known Positive 
 Syphilitic 
 
 Serum 
 
 Kno^wn Negative 
 No*RMAL Sehum 
 
 Serum, 0.1 c.c. 
 
 + 1 
 
 Complement (1 j 
 
 c.c. of 1:20) +1 
 Salt solution 
 
 (q. s. 3 c.c.) 
 
 Antigen, 0.4 c.c. 
 
 + 
 Serum, 0.1 c.c.+ 
 Complement (1 
 
 c.c. of 1 : 20) + 
 Salt solution 
 
 fq. s. 3 c.c.) 
 
 Cerebrospinal 
 fluid, 0.8 c.c. + 
 
 Complement (1 
 c.c.of 1:20) + 
 
 Salt solution 
 (q. s. 3 c.c.) 
 
 Serum, 0.1 c.c. 
 
 + 
 Complement fl 
 
 c.c. of 1 : 20) + 
 Salt solution 
 
 (q. 8. 3 c.c.) 
 
 Serum, 0.1 c.c. 
 
 +• 
 Complement (I 
 
 c.c. of 1:20) + 
 Sak solution 
 
 (q. s. '■', c.c.) 
 
 0.4 
 
 0.4 
 
 .5. 
 Antigen, 
 
 c.c,+ 
 Serum, 0.1 c.c. 
 
 j Complement (1| 
 
 Antigen, 
 
 C.C.+ 
 
 Cerebrospinal 
 fluid, 0.8 c.c 
 
 + I ■ 
 
 Complement (1[ c.c.ofl:20j + 
 
 CO. of 1 : 20) +1 Salt solution 
 
 Salt solution i (q. s. 3 c.c.) 
 
 fq. s. 3 c.c.) 
 
 7. 
 Antigen 0.4 c.c. 
 
 + 
 Serum, 0.1 c.c.+ 
 C'omplemerit (i 
 
 c,c. of 1:20) + 
 Sa[t solution 
 
 (q. 8. 3 c.c.) 
 
 10. 
 Antigen control: 
 Antigen, 0.4 
 
 c.c. + 
 Complement (1 
 
 c.c, of 1:20) + 
 Salt solution 
 
 fq. s. 3 CO.) 
 
 9. 
 
 Hemolytic con- 
 trol: 
 
 Complement (^ 
 c.c. of 1:20) + 
 
 Salt solution 
 (q. 8. 3 c.c.) 
 
 Tubes are shaken gently and incubated at 37° C. for an hour, after which two 
 hemolytic doses of amboceptor and 1 c.c. of corpuscle, suspension are added to each. 
 They are then gently shaken and reincubated for an hour or an hour and a half, after 
 which a preliminary reading is made. 
 
 All the tubes in the rear row (upper row in table) (serum controls), the antigen 
 and hemolytic system controls, and the front tube with the negative normal scrum, 
 are completely hemolyzed. The front tubes with th^ unknown scrum and cerebro- 
 spinal fluid and the positive serum control show inhibition of hemolysis or positive 
 reactions. 
 
iV^ 
 
 '^ 
 
 3\\ ' S 
 
 Fig. 110- — ^^'ASSEE1IA^•^' Reaction (Fi-ust Method). 
 
 iV 
 
 'Ites*'' 
 
 ^/ 
 
 Fig. 111. — Reading the Wassehmann Reaction. 
 
METHODS FOR CONDUCTING THE SYPHILIS REACTION 439 
 
 This scheme illustrates the technic employed with an unknown 
 serum and cerebrospinal fluid. The proper dose of diluted antigen is 
 taken as 0.4 c.c, and two doses of hemolytic amboceptor determined 
 by titration as equivalent to 0.4 c.c. of the stock dilution. 
 
 Reading and Recording the 'Wasserjmann Reaction 
 
 1. The hemolytic system control is inspected first. It should show 
 complete hemolysis, indicating that the compilement and amboceptor 
 were active and have been used in sufiicient amounts. If a few cor- 
 puscles are found in the bottom of the tube, some error in pipeting 
 has probably occurred, too many corpuscles or too little complement 
 or amboceptor having been introduced. 
 
 2. The corpuscle control should show no hemolysis, indicating that 
 the solution is isotonic and that the corpuscles, are not unduly fragile. 
 
 3. The antigen control should show compjete hemolysis, indicat- 
 ing that the dose used was not anticomplementary. If this tube 
 shows incomplete hemolysis, due to the ailticomplementary action 
 of the antigen, all the front two tubes will also show some inhibition 
 of hemolysis, due to this non-specific complement fixation, and it is 
 necessary to repeat the tests with another extract. 
 
 4. The rear tubes of all serums should be completely hemolyzed, 
 indicating that the serums were practically free from anticomple- 
 mentary action as previously stated, most antigens and serums are 
 usually very slightly anticomplementary if small amounts of com- 
 plement are used with a close single unit of amboceptor, but in this 
 technic the complement and two units of amboceptor are sufficient, 
 under ordinary circumstances, to offset this influence. If, however, 
 a serum is more than normally anticomplementary, the rear tube 
 will show some inhibition of hemolysis, and, of course, in the front 
 tube a similar inhibition, and probably to a greater degree, will be 
 seen. If the serum is very slightly anticomplementary and the 
 front tube shows complete inhibition of hemolysis, the reaction is in 
 all probability positive. If the rear tube, however, shows marked 
 inhibition of hemolysis, indicating that it is highly anticomplementary, 
 the result cannot be determined, but a rete^ with fresh serum must 
 be made. This indicates the great importance of the "serum control" 
 and it may he stated that a test should never be made without it. 
 
 5. The front tube containing the known syphilitic serum should 
 show inhibition of hemolysis, indicating that the extract possesses 
 antigenic properties. 
 
440 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 As the complement is "fixed" by the syphilis antibody and ex- 
 tract, hemolysis could not occur when the corpuscles and amboceptor 
 were added. If a portion of the complement is fixed by antibody and 
 extract, then the unfixed portion will hemolyze some of the corpuscles, 
 the reaction being moderately positive, slightly positive, etc., depend- 
 ing upon the degree of hemolysis that takes place. This illustrates 
 the importance of observing exactness in pipeting, and the great 
 influence of quantitative factors in testing for the Wassermann reac- 
 tion, for if an excess of complement is used' there may be sufficient 
 for all the syphilis antibody, and enough unbound complement to 
 hemolyze all the corpuscles. In this manned a false negative reaction 
 will result. Corpuscles and sufficient hemolytic amboceptor are 
 added merely in order to test for any free complement. Under proper 
 conditions a total lack of hemolysis indicaies that there is no free 
 complement, but that it has been fixed by syphilis antibody and 
 extract, constituting a positive reaction (H — | — \- -f ). Complete 
 hemolysis indicates that complement was not bound and that syphilis 
 antibody was, therefore, absent from the fluid tested — a negative 
 reaction ( — ). Partial hemolysis indicates that a portion of the 
 complement has been fixed by smaller amounts of syphihs antibody 
 and of the extract, yielding partially positive reactions (+ + +; 
 
 + +; +; ^). 
 
 6. The front tube containing the known normal serum should show 
 complete hemolysis because, in the absence of syphilis antibody, the 
 complement remains free to hemolyze the cbrpuscles with the hemo- 
 lytic amboceptor. 
 
 7. Various methods have been proposed for recording the results 
 of hemolytic tests. The following scheme, after Citron, is widely 
 used (Fig. Ill): 
 
 + -|--1--|- = complete inhibition of hemolysis = strongly positive. 
 -|- -|- -|- = 75 per cent, inhibition of hemolysis = moderately positive. 
 -|- -|- = 50 per cent, inhibition of hemolysis = weakly positive. 
 -|- = 25 per cent, inhibition of hemolysis = very weakly positive. 
 =t = less than 25 per cent, inhibition of hemolysis = delayed 
 
 hemolysis or doubtful reaction^ 
 — = complete hemolysis = negative reaction. 
 
 Under the third method a scale is givefi. that is easily prepared 
 for making these readings. However, after some experience they are 
 readily made, and at first should be attem^jted only after the non- 
 hemolyzcd corpuscles have been centrifuged pr allowed to settle to the 
 
METHODS FOR CONDUCTING THE SYPHILIS REACTION 441 
 
 bottom of the tube. As stated elsewhere,„ this method is not an 
 accurate measure of the amount of syphilis antibody, but constitutes 
 a relative and convenient gage of value within certain limits. In 
 reporting reactions to the clinician, the plus signs should not be used, 
 or if used, should be interpreted by the terms "strongly positive," 
 "weakly positive, " etc. 
 
 Technic of the Second Method 
 
 Practically the same technic is used in this as in the first method, 
 except that three different antigens, instead of one, are used with each 
 serum, for the reasons previously stated. 
 
 This method is to be strongly recommended, as it is simple, ac- 
 curate, and reliable. Although a little more work is demanded and 
 a larger quantity of the various reagents is required, the results 
 warrant the expenditure of a little more labor, and the second ob- 
 jection is readily overcome by using half the quantities prescribed 
 in the original Wassermann technic, as given=in the first method. 
 
 1. I generally use the following three antigens: (1) A cholesterin- 
 ized alcoholic extract of human heart; (2) alcoholic extract of syphil- 
 itic liver; (3) acetone-insoluble lipoids. 
 
 As previously stated, these extracts are used in amounts equal to 
 from two to four times their titrated antigenic unit, providing these 
 doses are at least four times smaller than the anticomplementary 
 units. The amount of each antigen require'd for the work at hand 
 is calculated, placed in test-tubes, and slowly diluted with the 
 requisite amount of salt solution to secure maximum turbidity of the 
 emulsions. 
 
 2. The complement is diluted 1 : 20 and is used in doses of 1 c.c; 
 sheep's corpuscles are made up into a 2.5 per cent, suspension, and 
 used in doses of 1 c.c; antisheep amboceptor is titrated as in the first 
 method, and used in doses equal to 1^^ units; serums are heated to 
 55° C. for half an hour, and used in doses of 0.1 to 0.2 c.c; cerebro- 
 spinal fluid is used unheated in amounts of 0.8 c.c. 
 
 The Test. — For each serum four test-tubqs are arranged in a row 
 and marked with the patient's name or initials. The first tube is 
 marked "C. H.," and receives the cholesterifiized heart extract; the 
 second tube is marked "S" for the alcoholic extract of syphilitic 
 liver; the third is marked "A" for acetone-ipsoluble lipoids, and the 
 fourth is not marked at all or simply marked with the letters "S. C." 
 (serum control). 
 
442 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 To each of the four tubes 0.1 or 0.2 c.c. of the patient's serum is 
 added, or 0.8 c.c. of cerebrospinal fluid. 
 
 To each tube 1 c.c. of the diluted complement (1 : 20) and suffi- 
 cient salt solution to bring the total volume in each up to 3 c.c. are 
 now added. 
 
 Controls. — A known positive and negative serum should be included, 
 unless one is performing a large number of tpsts with reliable antigens 
 every week, in which case, among many serums, a few at least are 
 likely to be positive. Under these circumstances these controls may 
 be omitted ; as a general rule, however, they should be included. 
 
 To the hemolytic system control tube 1 c.c. of complement dilu- 
 tion and 2 c.c. of salt solution are now added. Three antigen control 
 tubes are set up for each antigen with the dose employed, plus 1 c.c. 
 of complement dilution and sufficient salt solution to make the total 
 volume about 3 c.c. The corpuscle control receives 1 c.c. of the sus- 
 pension plus 3 c.c. of salt solution. 
 
 All the tubes are shaken gently and placed in the incubator for an 
 hour at 37° C. At the end of this time the aihboceptor and corpuscles 
 are addeil to each tube except that containing the corpuscle control. 
 Each tube is shaken gently and reincubated for an hour or longer, 
 depending upon the hemolysis of the serum controls. A preliminary 
 reading is now made, and the tubes set aside in the refrigerator at low 
 temperature until the corpuscles have settled. As a general rule, the 
 two readings coincide quite closely. Occasionally, with serums of 
 vigorously treated cases of syphilis, at the preliminary reading the 
 antigen tubes will show very slight inhibition or delay in hemoly is, 
 whereas if allowed to settle overaight this may not be noticeable. 
 
 Reading the Results. — The readings are made in the same manner 
 as described in the first method, the controls always being inspected 
 first. The hemolytic, antigen, and serum controls and known nega- 
 tive serum tubes should all be hemolyzed. The antigen tubes con- 
 taining the positive syphilitic serum should* not be hemolyzed. Re- 
 sults with the unknown serums are depende'nt upon whether or not 
 the serums are luetic, and if they are, upon the quantity of syphihs 
 antibody present. 
 
 With strongly positive serums there is complete inhibition of 
 hemolysis with all three antigens. With serums of long-standing or 
 treated cases of syphilis containing smaller amounts of antibody the 
 reaction with the cholesterinized extract is usually strongly positive, 
 whereas with the other two antigens the degree of inhibition of hemol- 
 
METHODS FOB CONDUCTING THE SYPHILIS REACTION 443 
 
 ysis is less marked and variable (see Fig. 112). In from 15 to 20 
 per cent, of cases the cholesterinized extract shows a 50 per cent, or 
 more inhibition of hemolysis, whereas with the other two antigens the 
 reactions are negative. In our experience the majority of such 
 serums were taken from patients giving a frank history of syphilis of 
 many years' standing and from known cases undergoing treatment, 
 further therapy being indicated until the reaction finally becomes 
 negative when cholosternized extracts are used. In a small pro- 
 portion of cases a feebly positive reaction of 25 per cent, or less in- 
 hibition of hemolysis may be found with the cholesterinized extract 
 alone. Many of these reactions occur with serums of treated cases 
 of syphilis; on the other hand, a similar reaction may occur with 
 about 5 per cent, of normal serums, so that if the history and clinical 
 conditions are clearly negative, a slight degree of inhibition of hemoly- 
 sis (5 to 10 per cent.) with the cholesterinized extract and marked 
 hemolysis with the other two antigens may bejnterpreted as a negative 
 reaction. 
 
 After a new antigen has been prepared and titrated, it should be 
 tested out in this manner by placing it in the series along with at 
 least two other older antigens of proved value, and used in the ex- 
 amination of a large number of serums before it is finally accepted as 
 reliable. 
 
 Technic of the Third Method 
 
 As previously stated, it may be desirable to measure the quantity 
 of syphilis antibody in a patient's serum more accurately, especially 
 when observing the effect of treatment. This is readily accomplished 
 by using decreasing doses of serum with constant doses of antigen and 
 complement. In those tubes showing partial inhibition of hemolysis 
 the degree of hemolysis is determined by comparison with a hemo- 
 globin scale (Boas). 
 
 The technic is quite similar to that followed in the second method. 
 
 1. One antigen is employed, either an alcoholic extract of syphilitic 
 liver or acetone-insoluble lipoids. The same" general rule as to dosage 
 is employed, namely, from two to four times the titrated antigenic 
 unit, providing these amounts are not more than one-fourth the 
 anticomplementary dose. 
 
 2. 0.5 c.c. of each serum is heated to 55° C. for half an hour and 
 diluted 1 : 10 by adding 4.5 c.c. of salt solution. 
 
 3. Fresh guinea-pig serum complement is diluted 1 : 20 as usual, 
 and used in doses of 1 c.c. ; sheep corpuscles are made up in a 2.6 per 
 
444 THE TECHNIC OF COMPLEMENT-FIXATION EEACTIONS 
 
 cent, suspension and used in doses of 1 c.c; antisheep amboceptor is 
 adjusted to the complement and corpuscles by a method of titration, 
 as given with the first method, and used in a dose equal to 13^ hemo- 
 lytic units. 
 
 The Test. — Seven test-tubes are arranged in a row and marked 
 with the patient's name; in each of the first six tubes the dose of 
 antigen is placed. The following amounts of diluted serum (1 : 10) are 
 then added: 
 
 First tube: 0.1 c.c. serum diluted 1 : 10 = 0.01 c.c. serum. 
 
 Second tube: 0.2 c.c. serum diluted 1 : 10 = 0.02 c.c. serum. 
 
 Third tube: 0.4 c.c. serum diluted 1 : 10 = 0.04 c.c. serum. 
 
 Fourth tube: 0.6 c.c. serum diluted 1 : 10 = 0.06 c.c. serum. 
 
 Fifth tube: 0.8 c.c. serum diluted 1 : 10 = 0.08 c.c. serum. 
 
 Sixth tube: 1.0 c.c. serum diluted 1 : 10 = 0.1 c.c. serum. 
 
 Seventh tube: 1.0 c.c. serum diluted 1 : 10 = 0.1 c.c. serum (control). 
 
 The seventh tube contains no antigen, and is the serum control 
 with the maximum dose employed. 
 
 In testing cerebrospinal fluid the following doses may be used 
 (undiluted): 0.1 c.c, 0.2 c.c, 0.4 c.c, 0.6 c.c, 0.8 c.c, 1 c.c, and 
 1 c.c. for the seventh or control tube. 
 
 To each tube 1 c.c. of the diluted compleraent (1 : 20) is now added, 
 and sufficient salt solution to bring the total volume in each up to 3 c.c. 
 
 Controls. — Unless one is examining a large number of serums and 
 is sure that the antigen is satisfactory, known positive and negative 
 serum controls may be included in the se"ries. As a general rule, 
 however, they should not be omitted. 
 
 The hemolytic system control receives at this time 1 c.c. of the 
 complement dilution and 2 c.c. of salt solution. The antigen control 
 receives the dose employed plus 1 c.c of complement and sufficient 
 salt solution to bring the total volume up to 3 c.c. The corpuscle 
 control receives 1 c.c. of the suspension and 3 c.c. of salt solution, and 
 the tube is plugged with cotton to show that it is finished. 
 
 All tubes are shaken gently and incubated for one hour at 37° C, 
 at the end of which time IJ/^ units of ambo£eptor and 1 c.c. of the 
 corpuscle suspension are added to all except the corpuscle control. 
 Each tube is shaken and all are reincubated for an hour or an hour 
 and a half, the length of time depending upon the hemolysis of the 
 serum controls when all are placed in the refrigerator at a low tem- 
 perature overnight, readings being made the next morning. 
 
 Reading the Results. — As is usual, the controls are examined first. 
 
Fig. 112. — Wassekhann Reaction (Si-jcokd Method). 
 
Fig. 113. — Wasseemann Reaction (Thied Method). 
 
METHODS FOR CONDtJCTING THE SYPHILIS REACTION 445 
 
 The hemolytic, antigen, negative serum, and 51II serum controls should 
 be completely hemolj'-zed. The corpuscle control should show no 
 hemolysis, indicating that the salt solution was isotonic and the cor- 
 puscles free from undue fragility. 
 
 A 1 per cent, hemoglobin solution in distilled water is prepared by 
 mixing 1 c.c. of the washed corpuscles used in preparing the suspension 
 with 99 c.c. of distilled water. Into a series of tubes dilutions of 
 this solution are placed as follows : 
 
 Tube 1 
 
 10.0 c.c. 
 
 Tube 2 
 
 9.0 c.c. 
 
 Tube 3 
 
 8.0 c.c. 
 
 Tube 4 
 
 7.0 c.c. 
 
 Tube 5 
 
 6.0 c.c. 
 
 Tube 6 
 
 5.0 c.c. 
 
 Tube 7 
 
 4.0 c.c. 
 
 Tube 8 
 
 3.0 c.c. 
 
 Tube 9 
 
 2.0 c.c. 
 
 Tube 10 
 
 1.0 c.c. 
 
 Tube 11 
 
 0.4 c.c. 
 
 Tube 12 
 
 
 of hemoglobin solution 
 
 n. 
 
 
 
 = 100 
 
 1.0 C°.c. 
 
 distilled water 
 
 - 90 
 
 2.0 c.c. 
 
 
 
 = 80 
 
 3.0 c.c. 
 
 
 
 = 70 
 
 4.0 c.c. 
 
 
 
 = 60 
 
 6.0 clc. 
 
 
 
 = 50 
 
 6.0 c.c. 
 
 
 
 = 40 
 
 7.0 c.c. 
 
 
 
 = 30 
 
 8.0 c;c. 
 
 
 
 = 20 
 
 9.0 c.c. 
 
 
 
 = 10 
 
 9.6 c.c. 
 
 
 
 = 4 
 
 10.0 c.c. 
 
 
 
 = 
 
 This scale is not permanent, and must be; prepared anew for each 
 set of reactions. 
 
 A negative reaction is indicated in the sixth tube, which contains 
 the maximum dose of serum (0.1 c.c), by complete hemolysis or by 
 hemolysis ranging from 80 to 100, according to the scale. Inhibition 
 of hemolysis in this tube giving a hemoglobin scale ranging from 70 
 to 4 (inclusive) is regarded as a positive reaction. Absolute lack of 
 hemolysis in this tube is according to the scale, but usually the 
 patient's serum or complement serum is sufficiently tinged with hemo- 
 globin to give a slight color to the supernatant fluid, so that an ab- 
 solute positive reaction may range from to 4. For instance, the 
 serum of an untreated case of secondary syphilis gave the following 
 reading (see Fig. 113). 
 
 Tube 1: 0.01 c.c. serum 100 
 
 Tube 2: 0.02 c.c. serum 100 
 
 Tube 3: 0.04 c.c. serum 30 
 
 Tube 4: 0.06 c.c. serum 10 
 
 Tube 5: 0.08 c.c. serum •. 4 
 
 Tube 6: 0.1 c.c. serum 4 
 
 Tube 7: 0.1 c.c. serum (control) 2 
 
446 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 Although these figures suffice for recording purposes, they are not 
 adapted, without some qualifying phrase, for rendering reports to 
 clinicians. According to the inhibition of hemolysis or the degree of 
 hemolysis in the sixth tube containing the maximum quantity of 
 serum the following scheme may be used in reporting the results: 
 
 to 10 = strongly positive =(+ + + +) 
 10 to 30 = moderately positive = (+ + +) 
 30 to 50 = slightly positive = (++)- 
 50 to 80 = very weakly positive = (+) 
 80 to 90 = doubtful or delayed hempjysis = (±) 
 100 = negative = ( — ) 
 
 Technic of the Fourth K&thod 
 
 In this method the amount of syphilis, antibody in a serum is 
 measured according to the number of hemolytic doses of complement 
 absorbed or fixed with a constant amount of antigen. As previously 
 stated, any organic extract used as antigen may of itself fix a certain 
 amount of complement; a non-syphilitic serum may do the same, 
 and a mixture of the two may fix still more, though the amounts may 
 be relatively small. A peculiarity possessed by a syphilitic serum is 
 that it fixes a large amount of complement when mixed with antigen; 
 as a result, the test becomes a quantitative and not a qualitative reac- 
 tion. The only practical means we possess of estimating the amount 
 of complement in a fresh serum is to ascertain the hemolytic dose, 
 i. e., to find the smallest quantity of serUm that is just sufficient 
 completely to lyse the test amount of sensitized corpuscles. When 
 this has been done, the quantity of complement used in the reaction 
 may be expressed in terms of hemolytic doses fixed, and not in terms 
 of the amount of fresh serum. 
 
 When properly performed according to. this method, which has 
 been modified after the technic of Browning and Mackenzie, the 
 syphilis reaction becomes quite delicate and accurate, but is more 
 complicated than the other methods, and stould not be attempted 
 until one is accustomed to the .simpler test and thoroughly under- 
 stands the underlying principles of the syphilis reaction and knows 
 the many sources of fallacy. The greater amount of work that it en- 
 tails and the larger quantities of complement-serum and amboceptor 
 that are required may serve as factors against its adoption as a routine 
 method. 
 
 1. Hemolytic Amboceptor and Corpuscles. — A stock dilution of 
 antisheep amboceptor is titrated according tp the technic given in the 
 
METHODS FOR CONDUCTING THE SYPHILIS REACTION 447 
 
 first method. It is well to prepare this dilution in such a manner 
 that the hemolytic dose will not be more than 0.1 c.c. If the am- 
 boceptor is being constantly used, so that its titer is well known, it will 
 not be necessary to titrate, but 5 times this unit is Used in sensitizing the 
 corpuscles. Otherwise small amounts of complement and corpuscle 
 suspension must be prepared, and the amboceptor titrated as follows : 
 Sufficient complement is prepared by diluting 0.5 c.c. of fresh 
 guinea-pig serum with 9.5 c.c. of salt solution (1 : 20), each cubic 
 centimeter of which contains 0.05 c.c. undiluted serum. It is well 
 at this time to prepare only sufficient 2.5 per cent, suspension of 
 sheep's cells for the titration, which is then conducted after the 
 technic given in the first method. For instance, if 0.1 c.c. Of ambo- 
 ceptor dilution represents one hemolytic unit, 0.5 c.c. would be used 
 in sensitizing each dose of corpuscles. If this quantity of ambo- 
 ceptor dilution is added to a 2.5 per cent, suspension of cells, the 
 emulsion would be considerably reduced, which would not be advis- 
 able. It is well, therefore, to calculate the amount of amboceptor and 
 corpuscle suspension required for the work on" hand, measure out the 
 amboceptor dilution in a flask, and add the corpuscles and then 
 sufficient salt solution to bring the total volunft to the required amount 
 to make a 2.5 per cent, suspension of cells. To continue the fore- 
 going example: five hemolytic doses of amboceptor and sufficient 
 corpuscular suspension are required for 100 tubes; this requires 50 c.c. 
 of amboceptor dilution, 2J^ c.c. of washed corpuscles, and sufficient 
 sterile normal salt solution to bring the total volume up to 100 c.c. 
 Permit the mixture to remain for at least half an hour at room tempera- 
 ture for the process of sensitization to take place before the complement 
 titration is performed. 
 
 2. Complement. — Fresh clear guinea-pig serum is diluted with 
 four parts of normal salt solution and titrated to determine the hemo- 
 lytic unit. 
 
 Complement Titration. — Into a series of eight test-tubes place 
 increasing amounts of the diluted complement serum (using the 
 special 0.2 c.c. pipets graduated in 0.01 c.c), as follows: 0.01, 0.02, 
 0.03, 0.04, 0.05, 0.06, 0.08, 0.1 c.c; add 1 c.c. of sensitized corpuscles 
 and sufficient salt solution to make the total volume about 3 c.c. 
 After incubating for one hour at 37° C, the smallest amount of comple- 
 ment giving complete hemolysis represents one hemolytic unit. 
 
 3. Antigen. — Browning and Mackenzie use a 0.75 per cent, dilu- 
 tion of lecithin and cholesterin in alcohol, prepared according to their 
 
44S THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 method. This is diluted 1:7 with normal Salt solution to secure the 
 maximum turbidity, and is used in a constaift dose of 0,G c.c. 
 
 A cholesterinized ak-holic extract of human heart, acetone-insolu- 
 ble lipoids, or plain alcoholic extract of sypKHitic liver ma>- he used in 
 four tin\cs their titrated antigenic dose, with' satisfactory results. 
 
 4. Fluid to be Tested.— Serum is heated at 55° V. for half :ui 
 hour and used in dose of 0.1 c.c. Cerebrospinal fluid should be fresh, 
 and is used unhcated in dose of O.S or 1 c.c. 
 
 5. The Test. — Two series of tubes are required. 
 
 Series A : These tubes should contain the* dose of antigen employed 
 with one, two and three hemolytic doses of complement respectively, 
 and sufficient salt solution to bring the total volume in each up to 3 c.c. 
 These are the antigen controls to determinfe how much complement 
 may be fixed by antigen alone. Usually but one or at most a portion 
 of two doses are fixed. 
 
 Series B: For each serum 12 test-tubeS are arranged in a row. 
 They are labeled with the initial of the patieait's name and numbered. 
 Into each is placed 0.1 c.c. of the jiatient's serum. Into each of the 
 first nine tubes are placed the dose of antigen and 2, 4, 6, S, 10, 12, ITi, 
 18, 20 hemolytic doses of complement resi)fctively. The hist three 
 tubes are the serum controls, to determine the amount of compleniimt 
 fixed by serum alone, and receive 1, 2 and 2f hemolytic units of com- 
 plement respectively. Sufficient salt solution is atided to each tube 
 to bring the total volume up to 3 c.c, and all are incubated for an 
 hour at 37° C. 
 
 Controls. — Series "A" contains the antigen controls, and these 
 suffice for making any number of tests with the same complement and 
 sensitized corpuscles. 
 
 (2) Each serum is controlled in the last three tubes of series B. 
 
 (3) A known positive syphilitic Serum may be included and tested 
 in the usual manner. 
 
 (4) A known negative normal serum may be included, but it is 
 not necessary to use more than 2 to 4 and 6 hemolytic units of com- 
 plement instead of running out to 18 or 20 units, as is done with an 
 unknown serum or with cerebrospinal fluid. 
 
 (5) A hemolytic control is set up with one dose of complement and 
 stifficient salt solution to make 3 c.c. 
 
 (6) A corpuscle control may be included, containing 1 c.c. of 
 sensitized corpuscles and 3 c.c. of salt solution. This tube is plugged 
 with cotton to indicate that it is finished. It controls the tonicity of 
 the salt solution and should show no hemohCsis. 
 
/ 
 
 VS^Sji^^/ 
 
 iTm 
 
 
 ■ 
 
 . J 
 
 ' 
 
 8 
 
 ^Vi 
 
 Fig. 114. — Wasseemann Reaction (Fourth Method). 
 
MODIFICATIONS OF THE WASSBEMANN REACTION 449 
 
 At the end of an hour 1 c.c. of sensitized corpuscles is added to all 
 the tubes. These are then gently shaken and reincubated for an 
 hour and a half, after which time the results are read. A final 
 reading made after the tubes have been allqwed to stand overnight, 
 usually tallies very closely with this reading. 
 
 Reading the Results. — The controls are first inspected. The 
 corpuscle control should show no hemolysis, and the hemolytic control 
 be just hemolyzed. In series A of the antigen controls it is usually 
 found that in the first tube hemolysis is incomplete, whereas the 
 second and third tubes show complete hemolysis, indicating that the 
 antigen alone fixed a unit or one hemolytic dose of complement. The 
 last three tubes of series B are the serum control tubes, and the first 
 tube usually shows incomplete hemolysis, its degree depending upon 
 the age of the serum. Occasionally the second tube is also incom- 
 pletely hemolyzed, indicating that about two units of complement 
 have been absorbed. Unless the serum is quite anticomplementary, 
 the third tube is completely hemolyzed. 
 
 The first nine tubes of series B are now examined. Browning and 
 Mackenzie have made an arbitrary rule to. regard the reaction as 
 positive when lysis is incomplete with five hemolytic doses of com- 
 plement, in addition to the sum of the amounts inhibited by serum 
 and by antigen alone. When an alcoholic extract of syphilitic liver 
 is used as antigen, I have found this rule to be too lax, and I regard 
 the reaction as slightly positive when lysis is incomplete with two or 
 three doses of complement, in addition to the sum of the amounts inhib- 
 ited by serum and by antigen .alone. For example, if a given serum 
 and antigen each show fixation of but one dose of complement, and 
 hemolysis is slight or entirely inhibited in the second tube of the se- 
 ries B containing serum and antigen with four doses of complement, 
 the reaction is usually weakly positive. More strongly reacting 
 serums will absorb from six to ten doses of complement, and not in- 
 frequently the serum of an active case of syphilis will fix more than 
 20 hemolytic doses of complement (see Fig. 114). 
 
 MODinCATIONS OF THE WASSERMANN REACTION 
 Modification of Nogughi 
 Among the large number of modifications of the original syphilis 
 reaction that have been devised, that of Noguchi has proved of dis- 
 tinct value. In this method an antihuman hemolytic system is 
 29 
 
450 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 employed that eliminates one possible source of error, due to the 
 natural antisheep amboceptor in human serum. 
 
 Noguchi advocated the use of active serum for this test, with an 
 antigen of acetone-insoluble lipoids. Active serum yields a more 
 delicate reaction, but may give false or proteotropic complement 
 fixation, especially when crude alcoholic extracts are used as antigens. 
 Before I began using cholesterinized alcoholic extracts in making the 
 Wassermann reaction I not infrequently found that the Noguchi test 
 with active serum was more delicate than the Wassermann reaction, 
 but with cholesterinized extracts the results ran closely parallel. It 
 is a good practice to conduct both a Wassermann and a Noguchi test 
 with each serum, as a negative Noguchi test with active serum is a 
 better indication of the absence of syphilis than is a negative Wasser- 
 mann reaction. A positive Wassermann reaction, however, is better 
 evidence of the presence of syphilis than is a- positive Noguchi reaction, 
 because of the possibility of false complement fixation occurring in 
 the latter when active serums are used. I may state, however, that 
 ■when a good antigen of acetone-insoluble lipoids is used, the per- 
 centage of false reactions is relatively small, being less than 2 per 
 cent. The Noguchi test, on the other hand, may be conducted with 
 inactivated serums, when the danger of false reactions is removed, 
 but the delicacjr of the test is likewise ditninished, so that it more 
 closely approaches the Wassermann reaction. 
 
 Noguchi endeavored to simplify the technic of the syphilis reaction 
 by preparing complement, antigen, and afnboceptor dried on filter- 
 paper. These were titrated and so adjusted that a certain measure 
 of paper represented the required amount of each reagent. In this 
 manner it would be possible for a large central laboratory to prepare 
 and standardize these reagents, putting tHem up ready for use in the 
 simplest possible form, and thus making them available for the 
 practising physician. Complement, however, deteriorates so rapidly 
 that the paper is useless unless it is freshly prepared. The antigen 
 slips likewise deteriorate, but not so rapidly as the complement; the 
 amboceptor, however, is well preserved by this method, and forms 
 a simple method for titrating and handling this important reagent. 
 
 Technic of the Noguchi Modificatioii. — (1) Complement is fur- 
 nished by the fresh, clear serum of the guinea-pig, put up in 40 per 
 cent, solution, prepared by diluting 1 part of serum with l}i parts of 
 normal salt solution. Dose 0.1 c.c. (5 drops from a capillary pipet). 
 Whenever possible it is always best to use a mixture of the serums 
 from two or more guinea-pigs. 
 
MODIFICATIONS OF THE WASSEEMANN REACTION 451 
 
 2. Human Corpuscles. — These are washed three times with normal 
 salt solution, and used in dose of 1 c.c. of a 1 per cent, suspension. 
 To a graduated centrifuge tube containing 9 c.c. of sterile 2 per cent, 
 sodium citrate in normal salt solution add 1 c.c. of blood and shake 
 gently. This amount of blood is easily secured by pricking the finger 
 with a lancet and collecting the blood in the centrifuge tube up to the 
 mark 10. This is centrifuged thoroughly, and the supernatant fluid 
 drawn off. More saline solution is then added to the corpuscles stirred 
 up and the mixture centrifuged. The washing is repeated once more, 
 and the supernatant fluid discarded. The corpuscles are then sus- 
 pended in sufficient salt solution to make a total volume of 100 c.c. 
 
 3. Hemolytic Amboceptor. — Antihuman hemolysin is prepared by 
 immunizing rabbits with human cells, as described on p. 72. It is a 
 difficult matter to secure a potent amboceptor; human erythrocytes 
 are more toxic than sheep's cells for rabbits, and most animals produce 
 but small amounts of the amboceptor. Hemagglutinins are produced in 
 large amounts, and when using a low titer h^paolytic serum, the test- 
 corpuscles are quickly agglutinated in small dense masses that are 
 broken up with difficulty and that interfere greatly with hemolysis. 
 With serums having a titer of 1 : 1000 or over, the agglutinins are not 
 so much in evidence; a satisfactory reaction ia best observed, therefore, 
 with a potent amboceptor (1 : 1000) serum. 
 
 The hemolytic serum may be preserved in 1 c.c. ampules after 
 adding an equal part of glycerin, and a stock dilution prepared and 
 titrated in the usual manner. The serum is aiso well preserved dried 
 on filter-paper, as described on p. 79. A trial titration should always 
 be made to determine the potency of the serum before the paper slips 
 are prepared. 
 
 If paper amboceptor is used, the uniform rule of titrating it vnth the 
 complement and corpuscles on hand should be observed before the actual 
 tests are made. This is done chiefly because, where one guinea-pig 
 serum is used for complement, it may occasionallj' happen that the 
 serum is less active than usual, so that if fixed doses of complement and 
 amboceptor are used, the reactions may at times prove to be incomplete 
 and inaccurate. The process of titration is so simple that any one may 
 readily conduct it, and thus fulfil the most important requirement of any 
 complement-fixation test, namely, adjustment of the complement, 
 ainboceptor, and corpuscles to one another. 
 
 Titration of Serum Hemolysin. — Prepare a 1 : 100 dilution by mixing 
 0.1 c.c. of immune serum (inactivated) with 9.9 c.c. of saline solution. 
 
452 THE TECHNIC OF COMPLEMENT-FIX5i.TION REACTIONS 
 
 To a series of six small test-tubes add increasing amounts of this diluted 
 serum as follows: 
 
 Tube 1: 0.1 c.c. amboceptor serum (1 : 100) +0.1 c.c. complement 
 
 (40 per cent.) + l c.c. corpuscle suspension (1 per cent.). 
 Tube 2: 0.2 c.c. amboceptor serum (1 : 100) +0.1 c.c. complement 
 
 (40 per cent.) + l c.c. corpuscle suspension (1 per cent.). 
 Tube 3: 0.4 c.c. amboceptor serum (1 : 100) +0.1 c.c. complement 
 
 (40 per cent.) + l c.c. corpuscle suspension (1 per cent.). 
 Tube 4: 0.5 c.c. amboceptor serum (1 : 100) +0.1 c.c. complement 
 
 (40 per cent.) + l c.c. corpuscle suspension (1 per cent.). 
 Tube 5: 0.8 c.c. amboceptor serum (1 : 100) +0.1 c.c. complement 
 
 (40 per cent.) + l c.c. corpuscle suspension (1 per cent.). 
 Tube 6: 1 c.c. amboceptor serum (1 : 100) +0.1 c.c. complement 
 
 (40 per cent.) + l c.c. corpuscle suspension (1 per cent.). 
 
 Sufficient saline solution is added to the first tubes of the series to 
 bring the total volume up to 2 c.c. The tubes are then shaken gently 
 and placed in the incubator at 37° C. for two hours (or one hour in 
 water-bath at the same temperature), during which time they should be 
 inspected and shaken gently several times. At the end of the period of 
 incubation that tube which shows just complete hemolysis contains one 
 amboceptor unit, and double this amount is used in making the main 
 tests. If the serum has a titer of less than 1 : 500, it should not be used 
 either in preparing the amboceptor slips or in conducting the reaction. 
 
 Titration of Dried Amboceptor Paper. — After the paper (S. & S. No. 
 597) has been evenly saturated with immune serum and dried, the sheets 
 are cut into 5 mm. strips and standardized by placing increasing lengths 
 of paper into a series of tubes as follows: 
 
 Tube 1: 1 mm. paper+0.1 c.c. complement (40 per cent.) (5 drops) 
 
 + 1 c.c. corpuscle suspension (1 per pent.). 
 Tube 2: 2 mm. paper+0.1 c.c. complement (40 per cent.) (5 drops) 
 
 + 1 c.c. corpuscle suspension (1 per cent.). 
 Tube 3: 3 mm. paper+0.1 c.c. complement (40 per cent.) (5 drops) 
 
 + 1 c.c. corpuscle suspension (1 per cent.). 
 Tube 4: 4 mm. paper+0.1 c.c. complement (40 per cent.) (5 drops) 
 
 + 1 c.c. corpuscle suspension (1 per cent.). 
 Tube 5: 5 mm. paper+0.1 c.c. complement (40 per cent.) (5 drops) 
 
 + 1 c.c. corpuscle suspension (1 per cent.). 
 Tube 6: 6 mm. paper+0.1 c.c. complement (40 per cent.) (5 drops) 
 
 + 1 c.c. corpuscle suspension (1 per cent.). 
 
 One cubic centimeter of saline solution is^ added to each tube, and 
 the mixture shaken gently and incubated at 37° C. for two hours or 
 one hour in a water-bath. At the end of this time the tube just completely 
 
MODIFICATIONS OF THE WASSERMANN REACTION 453 
 
 hemolyzed contains one amboceptor unit, and in' performing the test double 
 this amount is used. (See Fig. 115.) 
 
 This titration should always be conducted before the actual tests 
 are set up, as is the rule in conducting the Wassermann reaction. When 
 the titer of the paper is known, it may not be iiecessary to set up all the 
 tubes of the foregoing series, as a few only are necessary to determine 
 if the same amount of paper as was used in the previous tests will suffice 
 with the new complement and corpuscle suspension at hand. 
 
 All titrations and the main tests may be conducted in a water-bath 
 (37° C). With the aid of a good thermometer a satisfactory bath is 
 easily improvised. In fact, I believe that better results are secured 
 in the water-bath than in the incubator. It is possible, therefore, to 
 conduct these reactions in a perfectly satisfactory manner without the 
 aid of an expensive incubator. 
 
 4. Antigen. — Acetone-insoluble lipoids (Noguchi) are to be used 
 exclusively if the tests are conducted with active scrums. When heated 
 serums are used, any extract may be employed, as in making the Wasser- 
 mann reaction, but the same antigen gives excellent results, and I use it 
 exclusively in conducting the Noguchi reaction with both active and 
 inactivated serums. 
 
 The antigen must be titrated as usual, and its anticomplemen- 
 tary hemolytic and antigenic properties determined. According 
 to Noguchi, an extract is satisfactory if it ig antigenic in 0.02 c.c. 
 of a 1 : 10 dilution, and not anticomplementary or hemolytic in 
 amounts under 0.4 c.c. (1 : 10). In conducting the tests five times 
 the antigenic unit, or 0.1 c.c, is employed; this do.se is at least four 
 times smaller than the anticomplementary unit, and is therefore safe 
 and satisfactory. 
 
 The antigen is best preserved in methyl alcohol, as described on 
 p. 422. Dried on paper and properly preserved in sealed tubes in a cold 
 place it will retain its activity for several months, but as a general rule 
 it is best to use fresh emulsions of the alcoholic; solution. 
 
 Titration of Antigen. — The anticomplementary, hemoljiiic, and 
 antigenic doses of an extract are determined in the same general manner 
 as was described under the Wassermann reaction. 
 
 1. Anticomplementary Titration. — A portion of the stock alcoholic 
 solution of acetone-insoluble lipoids is diluted with 9 parts of saline 
 solution. This is the emulsion that is employed in conducting the 
 Noguchi reaction, and contains 0.3 per cent, of the original lipoidal 
 substances. 
 
454 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 Sufficient emulsion for these titrations is» prepared by diluting 0.4 
 c.c. of the alcoholic solution with 3.6 c.c. of saline solution. 
 
 Into a series of seven small test-tubes plate increasing amounts of 
 this emulsion as follows: 0.1, 0.2, 0.3, 0.4, O.g, 0.8, 1.0 c.c, add 0.1 c.c. 
 (5 capillary drops) complement (40 per cent.)- to each; also 1 c.c. of a 
 1 per cent, suspension of corpuscles and sufficient saHne solution to 
 make the total volume in each tube about % c.c. Incubate at 37° C. 
 for one hour (one-half hour in water-bath), and add two units of ambo- 
 ceptor. Shake the tubes gently and reincubate for two hours (one hour 
 on water-bath). That tube showing beginning inhibition of hemolysis 
 contains the anticomplementary dose, which' should not be under 0.4 
 c.c. In the tubes containing the larger doses sUght hemolysis may be 
 noticed, which is evidence of the hemolytic action of the extract. 
 
 An eighth tube should be included, containing 0.1 c.c. diluted com- 
 plement, two units of amboceptor, and 1 c.c. oft the corpuscle suspension. 
 This is the hemolytic control and should show«complete hemolysis. 
 
 2. Antigenic Titration. — Since the extract, is likely to have a high 
 antigenic value, it is necessary to dilute the antigen still further by plac- 
 ing 0.5 c.c. of the foregoing emulsion in a test-tube and adding 4.5 c.c. 
 of sahne solution (1 : 100 dilution of the antig-en). Into a series of six 
 test-tubes place increasing amountsiof this cWiulsion as follows: 0.05, 
 0.1, 0.2, 0.3, 0.4, 0.6 c.c. To each tube add four drops (0.08 c.c.) of 
 inactivated or one drop (0.02 c.c.) of fresh active syphilitic serum; also 
 0.1 c.c. (five capillary drops) of complement (40 per cent.) and 1 c.c. of 
 1 per cent, coipuscle suspension. Then add sufficient salt solution to 
 bring the total up to 2 c.c. 
 
 Two controls should be included: (1) The= serum control, containing 
 the dose of serum, 0.1 c.c. of the complement, 1 c.c. of corpuscle sus- 
 pension, and saline solution; (2) the hemolytic control, containing at 
 this time 0.1 c.c. of complement, 1 c.c. of corpuscle suspension, and 
 sufficient saline solution. 
 
 All tubes are incubated for one hour at 37° C. (one-half hour in 
 water-bath), after which two units of amboceptor are added to each 
 tube. The tubes are then shaken gently and reincubated for two hours 
 (one hour in water-bath). At the end of this time the two controls 
 should be completely hemolyzed, and in the series proper that tube 
 showing just complete inhibition of hemolysis contains one antigenic 
 unit. Usually the first and second tubes show some inhibition of 
 hemolysis, and in the third and other tubeS hemolysis is completely 
 inhibited. In this case 0.2 c.c. of this emulsion would be one anti- 
 
Fig. 115. — Titkatiox of Axtihumax Hemol^'tic Amboceptor. 
 
 Fig. 116. — Xoguchi ^Modification of the Wassermaxn Reaction 
 
MODIFICATIONS OP THE WASSERM'ANN REACTION 455 
 
 genie unit ( = 0.02 c.c. undiluted antigen); five times this amount equals 
 0.1 c.c. of the first emulsion (1:10), which is the amount to be used in 
 making the main tests. 
 
 Unless the antigen shows signs of deterioration, these titrations need 
 be made only about once a month. 
 
 If paper antigen is employed, both titrations are conducted in 
 exactly the same manner by adding increasing lengths of a strip of 
 dried paper 5 mm. in width, impregnated with the antigen. 
 
 5. Fluid to be Tested. — If active serum is used, it should be fresh, 
 free from hemoglobin, and preferably not over twenty-four hours old. 
 The dose is 0.02 c.c, or one capillary drop; inactivated serums are used 
 in doses of 0.08 c.c, or four capillary drops. Cerebrospinal fluid is used 
 unheated in doses of 0.2 c.c, or 10 capillary drops. Sufficient blood 
 for this test may be collected in a Wright capsule. (See p. 32.) 
 
 6. The Test. — The complement, amboceptor, antigen, and serums 
 may be conveniently measured by drops froni a capillary pipet (Fig. 
 2). In placing a drop the pipet should be held uniformly at an angle 
 of 45 degrees, or else the size of the drop will differ, depending on whether 
 the pipet is held vertically or horizontally. 
 
 Arrange four pairs of small test-tubes (10 -by 1 cm.) in a rack con- 
 taining two rows of holes. Into each of the tubes on the front row 
 place five drops (0.1 c.c.) of antigen emulsion (alcoholic solution, 1 part, 
 with saline solution, 9 parts); then add five drops (0.1 c.c.) of com- 
 plement (40 per cent.) to all the tubes. Into each of the first pair of 
 tubes place one drop (0.02 c.c.) of active or = four drops (0.08 c.c.) of 
 inactivated patient's serum, and mark the front tube with the patient's 
 name. To each of the second pair of tubes add an equal amount of 
 syphilitic serum known to give a positive reaction (positive control), and 
 to each of the third pair add normal serum known to give a negative re- 
 action (negative control). Mark the tubes in the front row of each pair 
 respectively. The front tube of the fourth pair is the antigen control, 
 and the rear tube the hemolytic control, and each should be so labeled. 
 Into each tube place 1 c.c. of the 1 per cent, corpuscle suspension and 1 
 c.c. of saline solution, making the total volume^ in each tube about 2 c.c. 
 Shake each tube and incubate at 37° C. for one hour (half an hour in the 
 water-bath). At the end of this time add two units of amboceptor to 
 each tube, shake gently, and reincubate for two hours (one hour in the 
 water-bath). During this time the tubes should be shaken gently 
 once or twice to break up any masses of agglutinated corpuscles. 
 
 The following chart, after Noguchi, illustrates the various steps to 
 
456 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 be taken in making the test with one patient's serum. Of course, any 
 number of serums may be examined with the same controls (Fig. 116). 
 
 TABLE 16 
 
 — NOGUCHI MODIFICATION OP THE WASSERMANN 
 
 
 REACTION 
 
 
 Set fob 
 
 Positive 
 
 Negative Antigen and Hem- 
 
 
 
 Diagnosis 
 
 Control Set 
 
 Control Set ' oIytic Controls 
 
 
 
 
 2. 
 
 . . ^- 1 ^- 
 
 8. 
 
 S 
 
 j3 
 
 t! 
 
 Unknown se- 
 
 Positive serum: 
 
 Normal serum : 
 
 Hemolytic con- 
 
 o 
 
 a 
 
 a 
 
 rum : 1 drop ' 
 
 1 drop 
 
 1 drop 
 
 trol: ' ^ 
 
 o 
 
 t 
 
 Complement: 5 
 
 Complement: 5 
 
 Complement: 5 
 
 Complement: 5 2 
 
 
 a 
 
 drops 
 
 drops 
 
 drops 
 
 , drops j "l 
 
 S 
 
 2 
 
 1 per cent, cor- 
 
 1 per cent, cor- 
 
 1 per cent, cor- 
 
 1 per cent, cor- ^ 
 puscle sus- 1 y- 
 
 
 puscle sus- 
 
 puscle sus- 
 
 puscle sus- 
 
 O 
 
 J3 
 
 pension: 1 o.c. 
 
 pension; 1 c.c. 
 
 pension: 1 c.c. 
 
 pension: 1 c.c. 
 
 a 
 
 a 
 
 Salt solution 
 
 Salt solution 
 
 Salt solution 
 
 Salt solution i^ " 
 
 O 
 
 (q. s. 2 o.c.) 
 
 (q. s. 2 c.c.) 
 
 (q. s. 2 c.c.) 
 
 (q. 8. 2 c.c.) 
 
 
 c 
 
 . 
 
 1. 
 
 3. 
 
 5. 
 
 7. 
 
 ii'r^ 
 
 Antigen: 5 
 
 Antigen : 5 
 
 Antigen: 5 
 
 Antigen con- (, 'g 
 
 l^i-i 
 
 drops 
 
 drops 
 
 drops 
 
 irol: S. '■^ 
 
 ^ !^ 
 
 Unknown se- 
 
 Positive serum: 
 
 Normal serum : 
 
 Antigen: 5 i^.S 
 drops 
 
 of a 
 ours 
 
 rum: 1 drop 
 
 1 drop 
 
 1 drop 
 
 Complement: 5 
 
 Complement: 5 
 
 Complement: 5 
 
 Complement: .5 f;; 
 
 M -^ 
 
 drops 
 
 drops 
 
 drops 
 
 . drops _ 
 
 •a g 
 
 1 per cent, cor- 
 
 1 per cent, cor- 
 
 1 per cent, cor- 
 
 I per cent, cor- ^ 
 
 3 , ^ 
 
 puscle sus- 
 
 puscle sus- 
 
 puscle sus- 
 
 puscle sus- -2 
 
 O 1 ^ 
 
 pension: 1 c.c. 
 
 pension: 1 c.c. 
 
 pension: 1 c.c. 
 
 pension: 1 c,o. .5 
 
 -*^ \J^ 
 
 Salt solution 
 
 Salt solution 
 
 Salt solution 
 
 Salt solution S 
 
 T3 ' ? 
 
 (q. s. 2 c.c.) 
 
 (q. s. 2 c.c.) 
 
 (q. s. 2 c.c.) 
 
 (q. s. 2 c.c.) p^ 
 
 < 
 
 « 
 
 At the end of the second incubation, or after two hours more at room 
 temperature, the tubes are inspected. The antigen and hemolytic 
 system controls, as well as all the rear tubes- or serum controls, should 
 be completely hemolyzed. The first tube coritaining a known syphilitic 
 sejrum shows inhibition of hemolysis; the front tube containing normal 
 serum is completely hemolyzed; the front tube containing the patient's 
 serum shows complete inhibition of hemolysis (strong positive), varying 
 degrees of inhibition (moderately or weakly positive), or is completely 
 hemolyzed (negative). The results may be recorded and reported after 
 the same manner described on p. 440. 
 
 Modification of Bauer 
 
 Bauer does not use rabbit-sheep amboceptor, but takes advantage 
 
 of the antisheep amboceptor normally prese4t in variable amounts in a 
 
 large proportion of human serums. Although this test is quite delicate, 
 
 the quantity of natural amboceptor in human serums is too variable a 
 
 factor to be depended upon, and the modification is not, therefore, in 
 
 general use. 
 
 ' Four drops if serum is inactivated. 
 
MODIFICATIONS OF THE WASSEEMANN REACTION 457 
 
 Modification of Hecht-Weinberg 
 In conducting the syphilitic reaction Hecht ^ utilizes not only the 
 natural antisheep amboceptor in human senun, but also the native 
 hemolytic complement. The serum must be- perfectly fresh, and, of 
 course, is used unheated. This modification has been said to be more 
 delicate than the Wassermann reaction, because none of the syphilis an- 
 tibody is destroyed or complementoids produced, as, presumably, will oc- 
 cur during inactivation (heating) of a serum. In my experience, this test 
 has proved quite delicate, but is open to the same error that may occur 
 whenever an active serum is used, with a crude alcoholic organic extract 
 as antigen — i. e., the appearance of false positive or proteotropic reac- 
 tions. As with the Noguchi reaction, using active serum, a negative 
 Hecht-Weinberg test has considerable diagnostic value in excluding 
 syphilis; a positive reaction must be, however, carefully controlled. 
 In performing the test I always use an extract of acetone-insoluble 
 lipoids as antigen. 
 
 The Hecht test is performed as follows: An alcoholic extract of human heart is 
 used as antigen. Each serum ■ is tested in four small, tubes. The serum must be 
 fresh (not over twenty-four hours old) and unheated. 
 
 Tube 1: 0.02 c.c. serum (1 drop) and 0.08 c.c. (4 drops) of 1: 50 dilution of heart 
 
 extract. 
 Tube 2: 0.02 c.c. serum and 0.08 c.c. of 1: 100 dilution of heart extract. 
 Tube 3: 0.02 c.c. serum and 0.08 c.c. of 1 : 200 dilution of heart extract. 
 Tube 4: 0.02 c.c. senun and 0.08 c.c. of normal salt solution. This is the one 
 
 control of the test and should show complete hemolysis. 
 
 The tubes are placed in the incubator for half an hour, or into a water-bath at a 
 temperature of 37° C. for ten minutes. One cubic centimeter of a 1 per cent, suspen- 
 sion of sheep's cells is added to each tube, and the tube shaken gently and reincubated 
 for half an hour or ten minutes, as the case may be. 
 
 Tube 4 must show complete hemolysis ; if it does not do so, the test is worthless. 
 In the test of a strongly positive acting serum all the other tubes show no hemolysis, 
 whereas a weakly acting serum shows lysis in all tubes but Tube 1. 
 
 This test is exceedinglj' simple, but is somewhat crude and unre- 
 liable. If employed at all, the results should always be controlled by 
 the Wassermann reaction. 
 
 It is necessary to avoid using too large dosBs of sheep's cells. Since 
 in the test just described, there is no way of determining beforehand the 
 amount of corpuscles a serum may hemolyze, Gradwohl ^ has modified 
 the technic so that the hemolytic index of each serum is determined 
 before corpuscles are added to the main tubes. His method is as follows : 
 
 "Place in a rack 14 small test-tubes. The first .10 of these tubes arc used to 
 determine the hemolytic index of the suspected blood. By this we mean the ex- 
 act amount of hemolytic amboceptor present in the given blood-serum. The last 
 
 1 Wien. klin. Woohenschr., 1909, xxii, 265. 
 ' Jour. Amer. Med. Assoc, 1914, kiii, 240. 
 
458 THE TECHNIC OF COMPLEMENT-FIX!A.TION REACTIONS 
 
 four tubes are used in the actual test. Add 0.1 c;o. of fresh unheated patient's 
 blood-serum to each of the first 10 tubes. Then add decreasing amounts of 
 normal salt solution to these tubes, beginning with \ c.c., then 0.9, 0.8, 0.7, 0.6, 
 0.5, 0.4, 0.3, 0.2, 0.1 c.c. to the succeeding nine tubes. Next add increasing 
 amounts of fresh 5 per cent, suspension of sheep's blood, starting with 0.1 c.c. and 
 ending with 1 c.c. Place the rack in the water-bath for one-half hour. The tube 
 which last shows complete hemolysis constitutes our "hemolytic index"; if it is 
 Tube 4, our index is 4, because this tube had received 0.4 c.c. of sheep's corpuscles. 
 The index determines the amount of sheep's corpuscles to be added to the last four 
 tubes. The first three tubes (11, 12, and 13) constitute the tubes for the actual test, 
 wiile the last tube in the rack (Tube 14) serves as oflr serum control tube. Tubes 
 11, 12, and 13 receive, therefore, the patient's serumj the proper amount of sheep's 
 corpuscles (dependent on the hemolytic index), rising strengths of antigen, but no 
 complement and no amboceptor. Tube 14 receives only sheep's corpuscles but no 
 antigen. In our technic we use 0.1 c.c. of a diluted antigen, determined by titration 
 in Tube 11, 0.15 c.c. antigen in Tube 12, and 0.2 c.c. in Tube 13. In order to equalize 
 the volume of fluid in all these tubes we add 0.2 c.c. jiormal saline to Tube 11, 0.15 
 c.c. to Tube 12, and 0.1 c.c. to Tube 13, and 0.3 c.c. to Tube 14. The tubes are theu 
 agitated and placed in the water-bath for half an hour. These last four tubes are 
 filled at the time we make the additions to the first 10 and are left with them in the 
 water-bath for half an hour for fixation of complement; the rack is then taken out 
 and the hemolytic index computed. If the index is low, say from 1 to 4, we add 
 0.1 c.c. of sheep's blood to the last four tubes. If the index is between 5 and 7, we 
 use 0.15 c.c. sheep's blood, and if it is between 7 and 10, we use 0.2 c.c. In our ex- 
 perience in this country we have never foimd an index above 10, although in France 
 it is not uncommon to obtain an index of 15 or 17. 
 
 " If the patient's serum has an index below 3, we regard the reaction as of doubt- 
 ful value. If it is above 3, we regard it as absolute. The reaction is read off exactly 
 as is the Wassermann, that is, inhibition or non-iriliibition of hemolysis." 
 
 Modification of Steru 
 Margaretta Stern devised a modification of the Wassermann reac- 
 tion, using fresh active serum and the patient's complement, and over- 
 coming non-specific reactions by using f tp, -|- of the usual dose of 
 extract, and three or four times the amboceptor unit. This method is 
 open to defects inherent in the use of variable amounts of complement 
 and excessive amounts of hemolj^tic amboceptor, which makes it im- 
 possible to test a specimen a few days after it has been collected. 
 
 Modification of Tcherno.gubou 
 
 Tchernogubou proposed an antihuman hemolytic system mth active 
 serum. Blood is collected in sodium citrate, and therefore contains 
 erji-hrocytes, complement, and syphilis antibody if the patient is luetic. 
 Antigen is added, and after sufficient time has elapsed for fixation of 
 complement to take place, antihuman amboceptor is added to test for 
 free complement. There are many objections to this method, the chief 
 ones being the variable amount of complement present in human serum, 
 the large amount of hemolytic amboceptor required, the absence of a 
 suitable control on the antigen, and the fact, that old blood is entirely 
 unsuited for making the test. 
 
 Tchernogubou has also proposed a system in which the natural 
 amboceptor and complement of human se'rum are utilized against 
 
MODIFICATIONS OF THE WASSERMANN REACTION 459 
 
 guinea-pig corpuscles. These factors are so variable that this modified 
 test has been largely abandoned. 
 
 Modification of Detre and Brezovsky 
 In this modification an antihorse hemolytic system is used with 
 rabbit's complement. The chief objections are the variations in the 
 activity and fixability of rabbit complement, and the difficulty of ob- 
 taining horse blood. In addition to these, this method possesses no 
 advantages over Wassermann's antisheep system. 
 
 Modification of Browning and Mackenzie 
 These investigators use an antiox hemolytic system, and have 
 modified the original Wassermann technic so as to make the method an 
 accurate quantitative test. (See p. 446.) They claim that with their 
 modified technic the results secured are practically the same with the 
 antiox and antisheep systems, although the human serums contain 
 much less natural antiox amboceptor and, theoretically, therefore this 
 system is better. 
 
 Modification of von Dungern 
 More recently von Dungern has proposed a modification similar to 
 that of Noguchi. Like Tchernogubou, he uses active serum only, and 
 utilizes the patient's own blood-cells. The blood is defibrinated and 
 used in doses of 0.1 c.c. Complement in the human blood is disregarded, 
 and is furnished by guinea-pig serum in dried-paper form. Alcoholic 
 extract of syphilitic liver is used as antigen, and this opens up an avenue 
 for false positive or proteotropic reactions to creep in. Antihuman 
 amboceptor is prepared by immunizing goats, but, as Noguchi has shown, 
 this amboceptor gives a much weaker hemolytic reaction than that de- 
 rived from the rabbit. Von Dungern generally omits the important 
 control with known syphilitic serum; there is no direct control on the 
 antigen, and old bloods cannot be used. 
 
 The "Wassermann Reaction in the Various Stages of Syphilis 
 1. In Primary Syphilis. — As would be expected, a certain degree of 
 tissue change most occur before syphilis reagin appears in the blood. 
 Even with the best technic there is a limit to the sensitiveness of the 
 Wassermann reaction, so that while the reagin may be produced at the 
 very onset of an infection, time and further tissue changes are required 
 before sufficient reagin is produced to yield a complement-fixation 
 reaction. 
 
 Since, therefore, the results of the Wassermann reaction in primary 
 
460 THE TECHNIC OF COMPLEMENT-FIXii^TION REACTIONS 
 
 syphilis are dependent upon the virulence of the infection, the time at 
 which the reaction is made, and the delicacy of the technic, it is not 
 surprising that the results of different uivestigators vary in the propor- 
 tion of positive reactions obtained. While positive reactions have been 
 said to have been secured before the appearance of the initial lesion, 
 these are rare, and there is always the likelihood that an earlier infection 
 was overlooked. A careful review of our own work and the literature 
 upon this subject establishes the following: 
 
 (a) A positive reaction may be secured as early as from four to five 
 weeks after infection has occurred. In such cases, however, it is often- 
 times probable that the time of infection in reality antedates the time 
 given by the patient. Craig has reported a positive reaction occurring 
 five days after the appearance of the initial lesion. Levaditi, Laroche 
 and Yamanouchi, and others have recorded many positive reactions 
 occurring in ten days or more after the chancje made its appearance. 
 
 (6) As a general rule, the Wassermann reaction becomes positive 
 during about the seventh to the eighth week after infection, or just a 
 week or two before the onset of the secondary eruption. In other words, 
 the reaction is usually secured first late in the primary stage, and in a 
 large proportion of eases before the secondary s3anptoms appear. 
 
 (c) In general, in primary syphilis the Wa,ssermann reaction will be 
 positive in about 80 per cent, of cases; where cholesterinized extracts 
 are used as antigens, or with the Noguchi system, using active serum, 
 the reactions are secured earlier and in a larger percentage of cases. 
 
 (d) It is generally agreed that a diagnosis should be made as early as 
 possible, and vigorous treatment instituted. A Wassermann reaction 
 may be performed, and if it shows a positive ^result, this indicates the 
 presence of syphilis, even if the lesion under suspicion is not specific, the 
 reaction being due to a previous infection. A negative reaction, how- 
 ever, does not exclude syphilis, and if it is at all possible, a microscopic 
 examination, using the dark-ground illuminator, should be made for 
 the treponema. In primary syphilis a micrcfe'copic examination of the 
 secretions of the lesion by a competent person is usually more valuable 
 than the serum test; as a general rule, both examinations should be 
 made, especially with patients in whom the chancre is almost healed or 
 atypical. 
 
 (e) The cerebrospinal fluid of persons in the primary stage of syphilis 
 has always reacted negatively (Boas). 
 
 2. In Secondary Syphilis. — It is in untreated cases of secondary 
 syphilis that the remarkable specificity of the Wassermann reaction 
 
MODIFICATIONS OF THE "WASSERMANN REACTION 461 
 
 is SO well demonstrated. The initial lesion may have been inconspicuous 
 and hence have been overlooked, and the secondary lesions may be 
 quite mild and inconclusive; in either case the Wassermann reaction 
 will usually establish the diagnosis. 
 
 (a) In untreated secondary syphilis the reaction is positive in from 
 92 to 100 per cent, of cases. In the examination of 437 serums from 
 untreated cases Boas has never had a negative reaction, and my own 
 experience has been the same. 
 
 (6) With the serums of patients who have received some treatment, 
 the percentage of positive reactions will be slightly lower. Of 310 such 
 cases examined by Boas, 97.6 reacted positively. The influence of 
 treatment upon the reaction is to be remembered, and a single negative 
 reaction does not by any means exclude the possibility of syphilis. 
 
 (c) The intensity of the reaction does not" bear any direct relation 
 to the severity of the infection: a mild infection with indefinite signs 
 may react quite strongly and absorb a large number of units of comple- 
 ment, whereas a severe case may react quite mildly. 
 
 (d) In secondary syphilis without cerebral symptoms the cerebro- 
 spinal fluid is practically always negative (Plant, Boas and Lind); 
 conversely, cases showing cerebral involvement usually react positively. 
 More recent work has shown that the cerebrospinal system is involved 
 early and in a relatively large number of cases (Craig and Collins^). 
 Udo J. Wile has found that about 30 per cent, of secondary syphilitics 
 give a positive reaction with cerebrospinal fluid. 
 
 3. In Tertiary Syphilis. — It is probably in tertiary syphilis that the 
 Wassermann reaction has its greatest value. Lues is so diverse in 
 character, and may be resfHDnsible for so many diverse clinical conditions, 
 that the reaction has become well-nigh indispensable as a diagnostic 
 aid. There is no limit to the time following infection in which positive 
 reactions may not be found. 
 
 (a) In cases of untreated and active tertiary syphilis the reaction is 
 positive in about 96 per cent, of cases. 
 
 (6) In cases receiving more or less antispecific treatment the reac- 
 tions are positive in about 75 per cent. In geijeral, therefore, a positive 
 reaction in tertiary syphilis may be expected in from 80 to 85 per cent, 
 of cases. 
 
 (c) In a large percentage of cases of syphilitic aortitis, aortic aneu- 
 rysm, aortic insufficiency, gummas of various organs, etc., the reaction 
 IS positive and possesses great diagnostic value. 
 
 ' Jour. Amer. Med. Assoc, 1914, fxii, 1955. 
 
462 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 (d) The Wassermann reaction has been especially valuable in the 
 study of the so-called parasyphilitic diseases.. In general paralysis or 
 paralytic dementia the serum reacts positively in about 100 per cent, of 
 cases, and the cerebrospinal fluid reacts positively in about 92 per cent. 
 The final and conclusive proof of the syphilitic nature of this disease 
 has been furnished by Noguchi and Moore, who found the Treponema 
 pallidum in sections of the brain. In certairt cases of general paralysis 
 the blood-serum may react negatively, whereas with the cerebrospinal 
 fluid the reaction is positive. 
 
 The fact that the blood-serum of a patient with a nervous disease 
 reacts positively does not necessarily indicate that the nervous disease 
 is of syphilitic origin, as the reaction may be due to specific infection of 
 some other structure; if, however, the ceretrospinal fluid also reacts 
 positively, then it is almost certain that syphilitic infection of the 
 central nervous system is present. 
 
 In untreated and active cases of tabes dorsalis the blood-serum reacts 
 positively in from 96 to 100 per cent, of cases. In treated cases the 
 number of positive reactions drops to about 40 to 50 per cent; in general, 
 therefore, a positive reaction with the serums of tabetics may be ex- 
 pected in 73 per cent, of cases. With the cerebrospinal fluid the per- 
 centage of positive reactions is somewhat lower, being about 60 per 
 cent. The positive Wassermann reaction is less constant in locomotor 
 ataxia than in general paralysis, due probably 1;o the fact that the former 
 is more chronic and that intercurrent periods of arrest are more prone 
 to occur. 
 
 In cerebral syphilis the blood-serum, and" particularly the cerebro- 
 spinal fluid, will react positively less frequently than in general paralysis. 
 In some instances a positive reaction is found with the cerebrospinal 
 fluid and not with the serum, a matter diflicult to explain and believed 
 to be due to the confining of the reacting substances in the subarachnoid 
 space. On the other hand, the lesions are probably not brought in 
 direct contact -nath the spinal fluid. 
 
 There is much evidence to indicate that localization of syphilis in the 
 nervous system is dependent upon a particular strain of Treponema 
 pallidum; other strains appear to possess a special afiinity for the 
 visceral organs, bones, etc. 
 
 (e) In tertiary syphilis not accompanied by lesions of the central 
 nervous system the Wassermann reaction with cerebrospinal fluid may 
 be positive in a relatively large percentage of cases. 
 
 4. In Latent Syphilis. — In cases of latent syphilis the Wassermann 
 
MODIFICATIONS OF THE WASSERMANN REACTION 463 
 
 reaction may constitute the only evidence of the existence of the disease, 
 and prompt institution of treatment may prcixent the development of 
 tertiary lesions, which are so likely to follow.* When the spirochetes 
 are few in number and are dormant, there is Ijttle tissue destruction or 
 alteration, and, as a result, so little reagin is frequently present in the 
 body-fluids that the Wassermann reaction will fail to detect the disease. 
 
 (a) In 363 cases of early latent syphilis, or those included within a 
 period of three years after infection. Boas found positive reactions in 
 about 40 per cent. ; in latent cases of long standing, or in those following 
 manifest tertiary lesions, the same investigator found 22 per cent, of 
 positive reactions among those who had received proper treatment; of 
 those receiving indifferent treatment, 74 per cent, reacted positively, 
 giving a general average of about 48 per cent.» 
 
 (b) The reaction with cerebrospinal fluid depends upon whether or 
 not the central nervous system is involved in the syj^hilitic process. Of 
 104 latent cases of syphilis in whom the spinal fluid was examined by 
 Altman and Dreyfus, positive reactions were found in about 10 per cent. 
 
 5. Congenital Syphilis. — The Wassermann reaction has thrown 
 considerable light upon the suliject of congenital syphilis. Wliile, in 
 general, the majority of cases react positively, the results are largely 
 dependent upon the time when the examinations are made, a fact brought 
 out by the highly instructive and systematic investigations of Boas and 
 Thomsen. These investigators divided their cases into three groups: 
 (1) Newly born children and their mothers; (2) two-year-old children; 
 (3) older children with congenital syphilis. 
 
 (a) Of 88 children born of syphilitic mothers and examined at birth, 
 the reaction was positive in 31 and negative in 57 cases. Of the 31 
 positive cases, 4 showed no symptoms of syphilis for a period of observa- 
 tion covering from three to nine months, and it is possible that the 
 sjrphilis reagin, and not the spirochetes, from the blood of the mother, 
 passed into the circulation of the child ; on the.other hand, all four cases 
 may have been examples of retarded congenital"syphilis. The remaining 
 27 cases either developed symptoms of syphilis .or died later with syphil- 
 itic manifestations in various organs. 
 
 Of the 57 children reacting negatively at birth, 42 showed no symp- 
 toms of syphilis during a period of three months of observation; 2 died 
 with evidences of syphilis in the internal organs; 13 developed sjonptoms 
 a,fter birth and gave positive reactions. 
 
 It may therefore be stated that a Wassermarm reaction of the mother 
 and of the child at the time of birth in cases where syphilis of the mother 
 
464 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 is suspected has considerable prognostic value. A large majority of 
 children reacting positively develop symptoms of syphilis; on the other 
 hand, the majority reacting negatively remain healthy. While an 
 examination of the mother alone does not warrant an absolutely definite 
 prognosis for the child, in general it may be said that a positive reaction 
 does not constitute a favorable prognostic sign for the child. 
 
 (6) The Wassermann reaction has also shed new light upon the 
 interpretation of Colles' law. Since the "apparently healthy mother 
 of a syphilitic child could suckle the child without being infected, whereas 
 the child is capable of giving syphilis to others," the most logical con- 
 clusion to draw is that the mother was gradually immunized against 
 syphilis during pregnancy, whereas we now know that the majority of 
 mothers show positive serum reactions and are really latent syphilitics; 
 in not a few such instances tertiary lesions have developed at a later date. 
 
 It is possible, however, for a syphilitic mother showing a positive 
 Wassermann reaction to give birth to a healthy child. Of 46 mothers 
 whose children showed no evidences of syphilis over a period of ob- 
 servation of three months, 17 reacted positively. Of 81 mothers giving 
 birth to syphilitic children, 61 reacted positively, and many of these 
 would naturally, in former years, have been regarded as examples of 
 Colles' immunity and considered free of syphilis. In many instances 
 the apparently healthy child of a syphilitic mother that could not be 
 infected by the mother {Profeta's law) has been shown by the Wasser- 
 mann reaction to be in reality a case of retarded congenital syphilis, 
 and that such children are not immunized, during intra-uterine life, 
 either passively or by means of pallidum toxins, against syphilis, as has 
 been so generally believed in past years. In other words, there appears 
 to be no lasting passive immunity in sjrphilis; it is doubtful if the toxins 
 of pallidum can pass between mother and child and immunize one or the 
 other without actual infection with the spirochetes themselves taking 
 place; that most examples of so-called immunity in syphilis in both the 
 mother (Colles' law) and the child (Profeta's law) are due to the actual 
 presence of pallidum in the tissues and are really latent infections. 
 
 (c) In manifest untreated congenital syphilis of children one year or 
 over in age the Wassermann reaction is positive in from 97 to 100 per 
 cent, of cases. The clinical manifestations may be quite varied and 
 clinically ill defined, so that the serum reaction possesses considerable 
 diagnostic value. In most instances the reactions are quite strong, and 
 while active treatment may improve local lesions, it is very difficult, 
 indeed, to secure negative reactions. 
 
MODIFICATIONS OF THE WASSEEMSInN REACTION 465 
 
 (d) In congenital mental deficiency the Wassermann reaction shows 
 that syphilis plays a larger part in the etiology of this condition than is 
 generally supposed. A not inconsiderable proportion of cases are of 
 infectious origin, and that infection is syphiUs. In Little's disease, 
 which is regarded as due to meningeal hemorrhage incidental to injury 
 received during labor, the serum reactions have shown that not in- 
 frequently the hemorrhage has a syphilitic origin. 
 
 The SPECiFicrrY of the Wassermann Reaction 
 The highly specific nature of the syphilis reaction has been proved 
 by very extensive investigations with the serunis of normal persons and 
 of persons afflicted with diseases other than syphilis. Unfortunately, 
 the reaction is beset by so many technical errors that a review of the 
 literature, and especially of the early literature, shows results that are 
 quite confusing and contradictory. Following the original communica- 
 tions of Wassermann and Detre, and especially after it was demonstrated 
 that the antigen need not be biologically specific, the subject was ex- 
 tensively investigated by various observers, who reported securing 
 positive reactions in many different diseases, results that we now know 
 inust have been due largely to technical errors At present it is known 
 that positive Wassermann reactions may occur in a few diseases other 
 than syphUis, but not to the extent that earlier investigators would have 
 us believe. In most of the diseases yielding positive reactions the 
 clinical symptoms are so marked that they may readily be differentiated 
 from syphilis, and accordingly the Wassermann reaction is of unequaled 
 and incalculable diagnostic value. 
 
 Positive reactions have been reported in framhesia (yaws), in which 
 the causal microorganism, the Spirochaeta pertenuis, is morphologically 
 almost indistinguishable from Spirochoeta pallidum. In leprosy of the 
 tuberous type positive reactions are frequently found, but cases of 
 anesthetic leprosy usually react negatively. Positive reactions have 
 been reported in cases of malaria during the febrile stage, when para- 
 sites are present, although the majority of cases react negatively. In 
 my own scries of 11 cases all the reactions were negative. Positive 
 reactions have also been found in some case^ of relapsing fever. 
 
 In scarlet fever the Wassermann reacticta is uniformly negative. 
 Owing to the original communication of Much and Eichelberg, however, 
 m the minds of many this disease is promih^ntly associated with a 
 positive reaction. While it is true that a positive reaction is very rarely 
 
 found, it is almost impossible entirely to exclude a diagnosis of con- 
 30 
 
466 THE TBCHNIC OF COMPLEMENT-FIXATION KEACTIONS 
 
 genital lues, at least in some of these cases. In my own series of 250 
 cases examined by the Wassermann and Noguchi methods, with antigens 
 of alcoholic extract of syphilitic liver and acetone-insoluble lipoids, the 
 reactions were positive in 5 cases, or 2 per cent. Similar results have 
 been secured by Boas, Browning and Mackeiizie, and others, so that it 
 may be said that the reaction in scarlet fever is uniformly negative. It is 
 also to be remembered that occasionally, or in about 1 to 2 per cent, of 
 cases, a positive reaction may follow ether or chloroform anesthesia, but 
 that this will later disappear. In -pellagra Fox, and later Bass, have 
 found occasional positive reactions. 
 
 Normal cerebrospinal fluid or the fluid from persons with ordinary 
 non-syphilitic diseases reacts negatively. Positive reactions have been 
 reported in leprosy and in frambesia. 
 
 The Effect of Treatment upon the Wassermann REAcnoN 
 Citron originally observed that during the mercurial treatment of 
 syphilis the Wassermann reaction gradually became weaker, and finally 
 disappeared. He also found that treatment was best governed by the 
 serum reaction, and that it should be persisted in until a negative re- 
 action was secured. His observations have in;the main been abundantly 
 confirmed by various observers the world over, although the extensive 
 series of observations now on record have given us a fuller understanding 
 of its principles. 
 
 The Wassermann reaction is the most constant and delicate single 
 symptom of syphilis, and whenever a serum is found to react positively, 
 antisyphilitic treatment is indicated, and should be persisted in until 
 the reaction becomes negative and remains so for a sufficiently pro- 
 longed period of observation. It is now quite generally believed that 
 a persistently positive reaction indicates the presence of living spiro- 
 chetes, and that treatment should be continued until the blood reacts 
 negatively. The reports of observers from all parts of the world indi- 
 cate quite clearly and conclusively that the. schematic, symptomatic, 
 intermittent, and hard and fast rules of treatment of former days are 
 not suflicient. They would also tend to show that the Wassermann 
 reaction is the most delicate symptom and the last to disappear, and 
 that treatment should be continued until this reaction disappears 
 entirely and permanently. It has been abundantly proved, however, that 
 in syphilis a single negative reaction is not sufficient or definite evidence 
 that a cure has been effected, for the disease may recur after treatment is 
 discontinued, at least to the extent that the Wass,ermann reaction reappears, 
 
MODIFICATIONS OF THE TVASSERMANN REACTION 467 
 
 followed by clinical manifestations. It is necessary, therefore, that sitc- 
 cessive examinations be made during a period of at least two years, and off 
 and on during the remainder of life. Recent work indicates that certain 
 strains of SpirochsEta pallida have an apparent selective affinity for the 
 tissues of the central nervous system; the Wassermann reaction with 
 blood-serum may be negative, whereas with the cerebrospinal fluid it 
 may be positive. In cases, therefore, of tertiary syphilis, at least, it is 
 advisable to examine the spinal fluid and continue treatment in case it 
 shows a positive Wassermann reaction. 
 
 It should be the object of treatment, in every case, not only to dis- 
 sipate the external and obvious lesions of the disease, but to produce a 
 condition of the blood in which the Wassermann reaction is permanently 
 negative. It is quite generally agreed that the older methods of treat- 
 ment, consisting of the administration of mercury and the iodids over 
 fixed and arbitrary periods of time, or until all manifest symptoms have 
 disappeared, are insufficient, and that the criteria by which the effects of 
 treatment can best be judged are: (1) Continted absence of symptoms, 
 and (2) permanent negative Wassermann reactions. 
 
 It is to be remembered, therefore, that while a single negative 
 reaction is a satisfactory indication of the progress of treatment, it does 
 not signify that a permanent cure has been effected. The Wassermann 
 reaction cannot be regarded as sufficiently delicate to indicate that a 
 single negative reaction means that a patient is totally free from all 
 spirochetes, for in some instances the reaction and the clinical symptoms 
 may recur after the treatment has been suspended, but the reaction is 
 the first symptom to reappear and the earliest indication of an impending 
 lesion. For all practical purposes the occurrence of a negative reaction 
 after treatment indicates either complete destruction of all the spiro- 
 chetes, or at least that the parasites are being held in abeyance and 
 rendered potentially harmless. 
 
 It is, accordingly, reasonable to regard the" Wassermann reaction as 
 the most delicate indicator of generalized spii-ochetal infection or the 
 assumption of spirochetal activity. A positive reaction indicates that 
 serious effects and gross local lesions are likely to occur at any time, and 
 that treatment should be continued. For all practical purposes a con- 
 tinned absence of symptoms and a permanently negative reaction are 
 strong presumptive evidences that a cure has been effected. 
 
 The serum should be tested every six months during the treatment, 
 and at periods of at least six months to a year after treatment has been 
 discontinued for several years. Persistently positive reactions during 
 
468 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 treatment would indicate that more active measures or a change in 
 therapy are needed. The occurrence of a positive reaction after treat- 
 ment has been discontinued is an indication for its resumption. 
 
 For a control on treatment the Wassermann reaction should be made 
 as delicate as possible, for while more prolonged treatment may be some- 
 what irksome to the patient, it is clearly indicated as a preventive of 
 serious after-effects, especially of involvement of the central nervous 
 system. It is in this branch of the work I have found that the use of 
 sensitive cholesterinized extracts as antigens in making the Wassermann 
 reaction or the Noguchi modification with the use of active serum, of 
 great value as the most delicate indicators. 
 
 One fact is to he clearly emphasized, namely, that the earlier energetic 
 treatment is begun, the more likely it is that a permanent cure will be effected. 
 Energetic treatment with mercurials or salvarsan, or, better, with a 
 combination of both, begun early and continued long, will in the majority 
 of cases restore the serum to its normal condition. In general, the 
 greater the interval of time allowed to elapse between infection and in- 
 stitution of treatment, the more difficult it is to restore the serum to 
 normal. Tertiary cases are cured only as the result of most persistent 
 treatment, and not infrequently in congenital syphilis, locomotor ataxia, 
 and general paralysis all one can hope to accomplish is to check the 
 progress of the disease. The most favorable cases are those in which 
 early diagnosis is made possible by clinical manifestations, preferably 
 confirmed by a demonstration of pallidum, and in which treatment is 
 undertaken before the serum has begun to react positively, and in which the 
 reaction remains negative throughout. 
 
 Treatment will, however, at least influence the Wassermann j-eaction 
 in practically all stages of syphilis. In a serks of 435 cases of syphilis 
 in all stages reported by Boas, a negative Wassermann reaction was 
 secured in no less than 80 per cent., and all but one of the remaining cases 
 showed a weaker reaction. The figures of different observers are not 
 all so favorable as these, a factor dependent to some extent, at least, 
 upon differences in the technic of the reaction. In general, however, 
 Boas' observations have been confirmed by other competent workers. 
 
 The effect of any treatment is greatly influenced by the individuality 
 of the host, certain persons possessing tissues more amenable to the 
 effects of the therapeutic agent than those o£ others. The therapeutic 
 effect is also dependent upon the virulence of the parasite and the 
 apparent selective affinity of certain strains of pallidum for particular 
 organs, and upon the method of treatment selected. 
 
MODIFICATIONS OF THE WASSEEMANN REACTION 469 
 
 The influence of salvarsan and neosalvarsan as agents in the treat- 
 ment of syphilis is considered in more detail in the chapter on Chemo- 
 therapy. My experience has shown that the earlier belief in the com- 
 plete sterilization of the human patient by a single dose was generally 
 unfounded, and that repeated smaller doses of the drug, used in con- 
 junction with mercurials, are necessary. Potassium iodid alone may 
 favorably influence the clinical symptoms and weaken the Wassermann 
 reaction in a small percentage of cases, and the same result has been ob- 
 served with such arsenical preparations as Bowler's solution, atoxyl, 
 arsacetin, and arsenophenylglj^cin. 
 
 It is to be remembered that, during or iihmediately after active 
 treatment with salvarsan or mercury, the Wassermann reaction may be 
 negative, even though the patient is not cured. As a general rule, a 
 negative reaction under these conditions should not be considered of 
 value unless all treatment has been omitted" for at least two weeks; 
 even then the test, if negative, should be repeated a month or so later. 
 Craig has recently drawn attention to the fact that in frank untreated 
 cases the degree of the reaction may vary within wide limits, and this is 
 especially true if the patients are receiving active treatment. 
 
 Provocatory Stimulation. — Paradoxic as it would at first appear, 
 antisyphilitic treatment may convert a negatively reacting serum into 
 a positive one. In not a few cases of latent sjrphilis reacting negatively 
 the administration of a specific spirillicidal agent, such as mercury or 
 salvarsan, is followed by positive reactions, due probably to the libera- 
 tion of endotoxins from destroyed spirochetes or to a stimulation of the 
 spirochetes by a dose of drug that did not suffice to kill them. This 
 condition is analogous to the Herxheimer reaction, or the aggravation 
 of skin lesions sometimes observed to follow the administration of 
 mercury or salvarsan. The fact possesses practical value, for in cases 
 where lues is known to have been present or is strongly suspected, and 
 th,e Wassermann reaction is indefinite or negative, the administration of 
 salvarsan or mercury, either internally or by inunction for a period of 
 from ten days to two weeks, followed a week after the last dose by a 
 Wassermann reaction, may now show a positive reaction and thus in- 
 dicate a latent syphilis requiring further treatment. 
 
 PltACTICAL VALXJE OF THE WASSERMANN REACTION 
 
 As previously stated, the Wassermann reaction serves two important 
 purposes: (1) As an invaluable aid in the diag^nosis and (2) as a guide 
 in the treatment of syphilis. 
 
470 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 The reaction may be of great value in determining the diagnosis of 
 extragenital sores and of atypical lesions in all stages of syphilis. A 
 negative reaction, however, has less value than a positive one, and 
 whenever possible, a microscopic examination of the secretions with the 
 dark-field illuminator should be made in order to confirm the diagnosis. 
 In early latent syphilis, after the initial lesion has healed, and before 
 the secondary eruption appears, the Wassernianii reaction is frequently 
 the only means of making the diagnosis, especially if the chancre has 
 been small, atypical, and practically neglected. 
 
 Indefinite symptoms and clinical unrecognizable cases constitute a 
 considerable proportion of cases of syphilis, and, as is true in all other 
 infections, this class constitutes the greatest menace to pubHc health. 
 Many patients are sincere in denying knowledge of infection and early 
 symptoms may be overlooked, the Wassermanii reaction being the sole 
 means of diagnosis and serving in this connection as an invaluable aid, 
 
 Usually the symptoms of syphilis are so well marked in the secondary 
 stage that the reaction is in most instances but confirmatory evidence. 
 However, in doubtful cases a negative reaction excludes syphilis with 
 almost absolute certainty, especially if the reaction is repeatedly 
 negative. 
 
 In the late latent and tertiary stages of sj^jhilis the Wassermann 
 reaction may be the only available basis on which to establish a diagnosis. 
 When one remembers how varied are the clinical manifestations of 
 chronic syphilis, how wide-spread is the disease, and how frequently the 
 reaction establishes the true diagnosis, the reaction must be regarded as 
 being of great value and as an indispensable diagnostic aid. It must not 
 be forgotten that patients showing an early involvement of the central 
 nervous system, and even those showing no such symptoms, may react 
 negatively with blood-serum and positively with spinal fluid; in all 
 such cases the spinal fluid should be examined wheriever possible. 
 
 A positive reaction occurring in aborting women is an indication for 
 treatment and may protect the fetus. Similarly a positive reaction ia 
 either parent of a seemingly healthy infant is an indication for treatment 
 of the child especially if the mother reacts positively. 
 
 In this connection, however, one point is worthy of special emphasis, 
 namely, that although a positive reaction indicates that the patient is 
 luetic, it does not necessarily mean that a particular lesion is syphilitic. 
 For example, a person may be luetic and yet have a cancerous ulceration 
 of the larynx. The mere fact that the lesion^ does not improve under 
 antisyphilitic treatment does not detract f rom_ the value of the Wasser- 
 
MODIFICATIONS OF THE WASSEKMANN REACTION 471 
 
 mann reaction, but is a warning that more care" is required in making the 
 clinical examination. I have seen a number of such cases in which a 
 positive Wassermann reaction was held a priori-SLS evidence of the syphil- 
 itic nature of a lesion that later proved to be either malignant or tuber- 
 culous. A weak positive reaction, associated with an active ulcerating 
 lesion, very frequently indicates that the lesion is not syphilitic, for 
 active lesions usually yield, strongly positive reactions. 
 
 In this connection may also be mentioned the growing importance 
 the Wassermann reaction has assumed in life-insurance examinations. 
 Statistics show that from one-tenth to one-third of all persons infected 
 with syphilis die as the results of the disease, and the death-rate among 
 5000 syphilitics accepted for insurance was one-third over expectation 
 (Brockbank). 
 
 An important question, especially from the standpoint of thera- 
 peutics is: Does a positive reaction invariably indicate the presence of 
 living spirochetes? May the reaction remain positive for an indefinite 
 time after the patient has been cured, just as agglutinins and antitoxins 
 may persist in the blood for some time after recovery from typhoid fever 
 and diphtheria has taken place? The sum total of the experience of 
 investigators from all parts of the world would indicate that a persist- 
 ently positive reaction means the presence of living spirochetes some- 
 where in the body. The lesions may not be active; the patient, while 
 clinically healthy, may be infective, and is always subject to possible 
 recurrences of clinical sj^philis. 
 
 Although gummas are slightly infectious, it is now known that they 
 contain living spirochetes, and the former view,.which regarded them as 
 sequels, rather than as actual active lesions of syphilis, is no longer 
 tenable. 
 
 Just how long the reaction may remain positive after the patient is 
 actually cured and all spirochetes are dead is, of course, difficult to state, 
 but experimental studies on the lower animals has shown that the reagin 
 disappears somewhat quickly under these conditions. 
 
 Although a persistently negative reaction is of good prognostic 
 importance, it is not so conclusive in the information it yields as is a 
 positive reaction. In other words, an occasional active lues may react 
 negatively, and not infrequently active syphilitic lesions are found at 
 autopsy in persons whose blood reacted negatively during life. While 
 it is true that great harm may result from a false positive diagnosis due 
 to faulty technic, yet it must be admitted that the Wassermann reac- 
 tion is not too delicate, and that we are just as prone to err on the side 
 
472 THE TECHNIC OF COMPLEMENT-FIXJVTION REACTIONS 
 
 of securing too many negative reactions. Every effort should be made 
 to render the test as dehcate as is possible with specificity. 
 
 While the value and dependability of the*Wassermann reaction are 
 based upon skilful technic that will eventually limit the performance 
 of the test to specially trained persons in central laboratories, every 
 effort should be made to render accessible tq all persons this valuable 
 diagnostic test of a disease that has such g.reat social and economic 
 importance. At present many persons are unable to afford the expense 
 of a number of tests, or even of one test, as required in the modern 
 treatment of this disease. This deficiency should be corrected, and the 
 test made available in all free dispensaries, especially those under the 
 supervision of a Social Service Department. 
 
CHAPTER XXIV 
 
 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 (Continaed) 
 
 Specific Complement Fixation in Bacterial Diseases. — As has been 
 stated elsewliere, the first complement-fixation: tests were performed by 
 Bordet with bacterial antigens and antiserums (pest and typhoid). 
 Following the application of the principles of complement fixation in the 
 serum diagnosis of syphilis, it was but natural that the possibilities of 
 this method as a general means of diagnosis soon became appreciated, 
 and in a short time numerous infections were studied. 
 
 Probably in no disease has complement fixation proved so constant 
 or so valuable a diagnostic procedure as in syphilis. In this condition 
 the peculiar lipodophilic reagin is largely responsible for the marked 
 fixation of complement, and from our present knowledge on the subject 
 we learn that this phenomenon has practically no analogy in any other 
 disease except frambesia. 
 
 With few exceptions bacterial antigens are likely to yield weaker and 
 more inconstant reactions. This is due to the fact either that our 
 antigen lacks a more available and specific antigenic principle, or that 
 the amount of complement-fixing bodies is small and variable. For 
 these reasons it becomes apparent that the preparation of antigen and 
 delicacy of technic are highly important factors. 
 
 Preparation of Bacterial Antigens. — Either the endotoxins or whole 
 bacterial body may constitute the main portion of an antigen. Most 
 recent efforts have aimed thoroughly to disorganize the bacterial cell in 
 order to liberate the endotoxic substances that pass into solution and 
 constitute the antigen. Experience has frequently shown, however, 
 that the protein substances of the bacterial cell itself possess antigenic 
 properties, and accordingly I have generally fo'und that antigens com- 
 posed of cells and the products of cellular activity are usually more 
 satisfactory than those prepared of the endotoxic substances alone. 
 
 As a general rule, bacterial antigens should he 'polyvalent — i. e., made 
 up of a number of different strains of the same; microorganism. Recent 
 researches in bacteriology tend to show that different strains of the 
 same microorganism have particular and more" or less individual patho- 
 genic and sometimes biologic characteristics,, and it is reasonable to 
 
 473 
 
474 THE TECHNIC OF COMPLEMENT-FIXlATION REACTIONS 
 
 assume that the antibody will likewise show gidividual properties and a 
 special afiinity for its particular antigen. When, therefore, one antigen 
 is being used in complement-fixation work, the results are more likely 
 to be satisfactory if a large number of different strains are included in 
 the antigen, with the hope that at least one of them will show a par- 
 ticular affinity for the antibody in the patient's serum. 
 
 Bacterial antigens may be prepared in various ways. 
 
 First Method. — Cultures are grown in a suitable fluid medium, such 
 a« plain bouillon, for forty-eight hours, or upon a solid medium, and 
 washed off with a suitable quantity of normal saline solution. The 
 culture or emulsion is shaken for an hour or so to break up the clumps, 
 and then heated to 60° C. for an hour. It is preserved by the addition 
 of 1 per cent of glycerin and 0.5 per cent, of phenol. 
 
 This constitutes the simplest bacterial antigen. It is composed of 
 both bacterial cells and the products of bacterial activity, and frequently 
 yields uniform and satisfactory results. 
 
 Second Method. — Cultures are grown on a suitable solid medium for 
 from twenty-four to forty-eight hours. Growths are removed by adding 
 sufficient distilled water or normal saline solution to yield a milky sus- 
 pension. The emulsion is heated to 60° C. for two hours, and shaken 
 mechanically with glass beads for twenty-four hours, to facilitate disin- 
 tegration. It is then filtered through a sterile Berkefeld filter or thor- 
 oughly centrifugalized; the filtrate is preserved with 0.5 per cent, 
 phenol and used as antigen. 
 
 This antigen is composed essentially of endotoxic substances, and 
 is the one usually employed in the preparation of gonococcus antigen. 
 
 Third Method. — Cultures are grown on a •solid medium, washed off 
 with normal saline solution, and the emulsion centrifuged thoroughly. 
 The sediment is dried over sulphuric acid op calcium chlorid, and the 
 dried material thoroughly ground with crystals of sodium chlorid. Suf- 
 ficient distilled water is then added to render the solution isotonic, and 
 so that it will contain about 0.05 gram of dried material in each cubic 
 centimeter. This emulsion is then shaken for twenty-four hours, 
 filtered or centrifuged, the filtrate preserved with 0.5 per cent, of phenol, 
 and used as antigen. 
 
 Fourth Method. — Cultures are grown on a solid medium and washed 
 off with normal saline solution. Saline suspension is then precipitated 
 with an equal quantity of absolute alcohol and centrifugalized. The 
 sediment is dried in vacuo over sulphuric acid, weighed, and ground into 
 a fine powder with sufficient crystals of sodium chlorid to make a 2 per 
 
STANDARDIZING BACTERIAL ANTIGENS 475 
 
 cent suspension of dried material in isotonic saline solution. This 
 stock suspension is not filtered or centrifuged, but is further diluted 
 with saline solution, and constitutes the antigen (method of Besredka, 
 modified by Gay). The actual amounts of dry antigenic substance 
 contained in 1 c.c. of various dilutions are as follows: 
 
 1 c.c. of 1: 40 dilution = 0.5 mg. 
 
 1 c.c. of 1: 80 dilution = 0.2.5 mfel 
 
 1 c.c. of 1; 160 dilution = 0.125 mg. 
 
 1 c.c. of 1: 320 dilution = 0.062 mg. 
 
 1 c.c. of 1 : 640 dilution = 0.031 mg. 
 
 1 c.c. of 1; 1280 dilution = 1.0155 mg., etc. 
 
 Standardizing Bacterial Antigens. — After an antigen has been pre- 
 pared it is standardized by determining the anticomplementary dose — 
 i.e., the amount of antigen that just begins to show inhibition of hemol- 
 ysis due to non-specific complement fixation. This dose is easily 
 determined by adding increasing amounts of antigen to a series of test- 
 tubes with a constant dose of complement in each. As a general rule, 
 it is well to add to each tube a constant dose oi fresh normal inactivated 
 serum, e. g., as 0.1 to 0.2 c.c, when the anticomplementary action of 
 serum alone is allowed for. I would emphasize the necessity of doing 
 this in experimental work with rabbit, dog, or any other animal serum. 
 After incubating for one hour, one and a half units of hemolytic ambo- 
 ceptor and 1 c.c, of corpuscle suspension are added to each tube, and the 
 tubes are reincubated for an hour or two and the reading made. In 
 the main test, one-quarter to one-half the a?iticomplementary unit may be 
 used, as this amount is known to be free from any power of non-specific 
 complement fixation. The former dose is, of course, safer than the latter. 
 
 The standardization may be completed by determining the antigenic 
 dose of the antigen by titrating with a suitable and constant dose of 
 specific immune serum. This titration is conducted by placing in a 
 series of test-tubes increasing doses of antigen with a constant dose of 
 heated immune serum (usually 0.1 c.c.) and a constant dose of comple- 
 ment. After an hour the proper dose of hemolytic amboceptor and cor- 
 puscles is added. The readings may be madejan hour or two later, or 
 after the tubes have been allowed to settle in a refrigerator. That tube 
 showing just complete inhibition of hemolysis contains the antigenic dose. 
 For the main test, it is well to use double this amount, providing this 
 dose is not more, and preferably less, than half the anticomplementary 
 dose. 
 
 The antigenic titration is not always satisfactory, for when an arti- 
 ficial immune serum is used, the concentration of antibodies may yield 
 
476 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 a much stronger reaction than one would expect in testing human serums. 
 Further than this, the antibody content in antiserums varies considerably 
 so that the antigenic unit fluctuates according to the particular serum 
 used in making the titration. In general, therefore, it is sufficient to 
 determine the anticomplementary dose and Ip use half or quarter this 
 amount in performing the main test. 
 
 After antigens are prepared they may require further dilution with 
 saline solution. This can be determined only by experience and as the 
 result of a trial titration. 
 
 As watery extracts are prone to deteriorate, it should he made a rule 
 that the anticomplementary dose be determined each time before the main 
 test is conducted. 
 
 It has quite generally been proved that alcoholic extracts of bacteria 
 do not yield satisfactory antigens, despite the advantage to be gained 
 because of their stability. 
 
 Principles of Complement Fixation with Bacterial Antigens, — The 
 principles of complement fixation in general should be thoroughly under- 
 stood. 
 
 After making considerable comparative studies with various methods, 
 I am convinced that, in the final analysis, a simple technic is best. I 
 use a relatively small but safe dose of complement, — 1 c.c. of a 1 : 20 
 dilution ( = 0.05 c.c. undiluted serum), — and titrate the hemolytic 
 amboceptor with this constant dose or unit of complement and the 
 corpuscle suspension. This titration is made each time the reactions 
 are performed, and with each and every coriiplement serum and cor- 
 puscle suspension. In this way differences in the activity of different 
 guinea-pig serums are readily detected and adjusted in the amboceptor 
 titration. If exactly one unit of amboceptor is used in conducting the 
 main test, the controls are not likely to be completely hemolyzed unless 
 the serum contains natural antisheep amboceptor, for the serum alone 
 will probably be slightly anticomplementary. For this reason I am 
 accustomed to use 1}^ doses of amboceptor, and I secure reactions that 
 are very delicate and yet sharp and clear cut. When testing an old 
 serum, — one that is likely to contain considerable thermostabile anti- 
 complementary bodies, — I use two hemolytic doses of amboceptor. If 
 one wishes to use exactly one unit of complement and one unit of ambo- 
 ceptor, the serum and antigen alone should be controlled, as in the fourth 
 method of performing the Wassermann reaction (p. 446). I frequently 
 use this technic in the gonococcus-fixation test, but as a rule I have 
 found the simpler technic herein given equally sensitive and rehable. 
 
COMPLEMENT FIXATION IN GONOCOCCTJS INFECTIONS 477 
 
 In this chapter are considered the main bacterial infections in which 
 complement fixation has been shown to possess value as a means of 
 diagnosis. Complement fixation has also proved of value in the diag- 
 nosis of animal parasitic diseases, such as ecchinococcus infection, and 
 in the differentiation of the proteins. With each of these the special 
 methods for preparing the antigen, titrating the antigen, and conduct- 
 ing the test are given. 
 
 COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS 
 
 This was one of the first infections to be studied by means of the 
 complement-fixation technic, but the results secured were not generally 
 satisfactory until it was shown that the antigen must be polyvalent. 
 
 Historic — In 1906 Muller and Oppenheim^ applied the complement-fixation test 
 to the diagnosis of gonorrheal arthritis, using a culture of the gonococcus as antigen. 
 To these observers, therefore, belongs the credit of beicfg the first to record a cornple- 
 ment-fixation test in a gonococcus infection. A little later in the same year Carl 
 Bruch^ applied the reaction to three cases of gonorrhea, using the serum of immunized 
 rabbits, and reported favorable results. In 1907 Meakins' reported having secured 
 positive reactions in three cases of gonorrheal arthritis, which was the first report in 
 America published on this subject. Th. Vanned'' studied the specificity of the reac- 
 tion with the serums of rabbits inamunized with gonococcus protein and one of a 
 meningococcus, and reported that the meningococcus immune serum did not show 
 complement fixation with gonococcus antigen, and, vice versa, that gonococcus ambo- 
 ceptor was not bound by meningococcus antigen. Wollstein* (1907), in a study of 
 the biological relationship of the gonococcus and meningococcus, reported findings 
 differing from those of Vanned. The former observer'found that bacteriolytic am- 
 boceptors in the serums of rabbits immunized with these cultures were closely related 
 and yielded fixation of complement with either antigen. Teaque and Torrey,* in 
 1907, issued a very important communication showing that the differences in. results 
 of previous investigators were probably duo in part to the use of single strains of the 
 organisms m. the preparation of antigens and immune serums. They emphasized 
 the fact that the gonococcus belongs to a heterogeneous family, and that in attempt- 
 ing to formulate a diagnosis of gonorrheal infection by the complement-fixation 
 method, the extracts of several different strains should be used. Naz Vanned and 
 later Watabiki' found that the gonococcus and meningococcus antibodies were quite 
 specific for their homologous antigens in complement-fixation reactions. 
 
 Particular attention was drawn to the gonococcus complement-fixation test by 
 the work of Schwartz and McNeal.* These investigators emphasized the necessity 
 of using polyvalent antigens, and their encouraging reports have stimulated renewed 
 interest in this subject. They found that if the infection is confined to the anterior 
 urethra, a positive reaction is not obtained; that a strong reaction is not to be ex- 
 pected before the fourth week of the infection, and then; only in acute cases with com- 
 plications. They regard a positive reaction as indicating the presence or recent 
 activity in the body of a focus of living gonococci, although a negative reaction does 
 not exclude gonococcus infection. The test, therefore, has a more positive than a 
 negative value. With Flexner's antimeningococcus serum positive reactions re- 
 
 ^ Wien. klin. Wochenschr., 1906, 19, 894. 
 
 ^ Deutsch. med. Wochenschr., 1906, 70, 36. 
 
 'Johns Hopkins Medical Bull., 1907, 18, 255. 
 
 ^Zeitschr. f. Bakter., 1907, 44, 10. ' Jour. Exp. Med., 1907, 9, 588. 
 
 = Jour. Med. Research, 1907, 17, 223. ' Jour. Infect. Diseases, 1910, 7, 159. 
 
 *Amer. Jour. Med. Sci., May, 1911; ibid., September, 1912; ihid., December, 
 
478 THE TECHNIC OF COMPLEMENT-FIJ^ATION REACTIONS 
 
 suited with their gonococcus antigen; with serums fropj cases of cerebrospinal menin- 
 gitis (meningocoecic) the results were negative. 
 
 In the succeeding years numerous investigators, including Swinburne, Gradwoh!, 
 O'Neil, Gardner and Clowes, Thomas and Ivy, Kolrher and Brown, have reported 
 favorably upon the practical value of the gonococcus complement-fixation test, 
 particularly as an aid in determining whether or not a patient is cured of the 
 infection. 
 
 Technic. — Since, because of the comparatively slight cellular in- 
 volvement, the quantity of antibody produced in a localized gonococcus 
 infection is probably small, the complement-fixation reactions are 
 generally weak, and consequently require the closest technical attention, 
 especially as regards the preparation of antigen and accurate adjust- 
 ment of the hemolytic system. 
 
 Hemolytic System. — As a rule, the antisheep hemolytic system is 
 employed; the various ingredients may be used in one-half the quantity 
 employed in the original Wassermann reaction, as given in the preceding 
 chapter, with the technic of the syphilis reaction, or one-tenth the quan- 
 tity employed in the original Wassermann technic may be employed. 
 I prefer to employ the larger amounts because the readings are usually 
 easier to interpret. 
 
 Fresh guinea-pig complement serum is diluted 1 : 20 and used in dose 
 of 1 c.c. ( = 0.05 c.c. serum); sheep's corpuscles are made up in a 2}/^ per 
 cent, suspension and used in dose of 1 c.c.^ antisheep amboceptor is 
 titrated (see p. 377) and used in an amount equal to IH hemolytic 
 doses in conducting the antigen titration and in the test proper. 
 
 Kolmer and Brown have compared the practical value of the anti- 
 sheep and antihuman hemolytic systems in the examination of a number 
 of serums. When the latter were used, some of the reactions were 
 somewhat stronger and yielded slightly better results, showing the 
 influence, probably, of natural antisheep amboceptor present in a large 
 proportion of human serums. 
 
 Antigen. — This constitutes the most important ingredient of the 
 test. As Teague and Torrey and Schwartz and McNeal have em- 
 phasized, the antigen should be prepared of many different strains of 
 gonococci. The difficulty of isolating this organism and the constant 
 care required in subculturing and keeping a large number of strains alive 
 render it practically impossible for many persons to prepare a gonococcus 
 antigen. Therefore until simpler methods are devised, this antigen is 
 best prepared in large central laboratories, where the cultures are handled 
 and preserved by specially trained persons. 
 
 The gonococci are well grown on a salt-free veal agar, neutral in 
 
o.. 
 
 oj 
 
 I 
 
 1 
 
 Fig. 117. — Anticomplementary Titration oi^ a Gonococcus Antigen. 
 
COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS 479 
 
 reaction to phenolphthalein, and to which a few drops of sterile hydrocele 
 fluid may be added. After culturing for frc«n twenty-four to forty- 
 eight hours, the growths are washed off with distilled water, and the 
 emulsion is heated in a water-bath for two hours at 56° C. It is then 
 centrifugalized and passed through a Berkefeld filter. A small amount 
 of preservative, as, e. g., 0.1 c.c. of a 1 : 100 dilution of phenol to each 
 cubic centimeter of antigen, may be added. The antigen is then well 
 preserved in small amounts in ampules that are sealed and heated to 
 56° C. for half an hour on three successive days. Just before being 
 used the antigen is made isotonic by adding 1 part of a 10 per cent, salt 
 solution to 9 parts of antigen. I preserve the antigen in ampules con- 
 taining 1 c.c, and after removing the antigen from the ampule to a large 
 test-tube, add 1 c.c. of 10 per cent, salt solutipn, and dilute the whole 
 1: 10 with the addition of 8 c.c. of normal salt solution, after which the 
 anticomplementary titration is made. 
 
 In this method of preparing antigen the endotoxins constitute the 
 main antigenic principle. Kolmer and Brown, after an experimental 
 study of the various antigens, found that a simple suspension of gono- 
 cocci in salt solution yielded slightly better results. The various 
 strains are grown for from forty-eight to sev-enty-two hours, and are 
 then washed off with sterile saline solution, observing particular care 
 not to include portions of the culture-medium. The suspension is 
 then shaken to break up clumps, and heated to 56° C. for an hour. A 
 small amount of preservative is now added, and the antigen stored in 
 1 c.c. ampules. Before using it is diluted 1 : 10 or 1 : 20, and titrated 
 for the anticomplementary dose. 
 
 Alcoholic extracts of gonococci have very little practical value, as 
 alcohol is not satisfactory for extracting the antigenic principles of 
 bacteria. 
 
 The anticomplementary dose of the antigen should be determined, 
 and one-half or one-quarter this amount should be used in conducting 
 the main test. An antigenic titration may also be conducted fidth an 
 antigonococcus serum, to determine the antigenic value of the antigen, 
 but in practice it is sufficient to use one-half the anticomplementary 
 dose. This titration should be conducted and the antigen standardised 
 before the main tests are adjusted. 
 
 In the following table the results of an anticomplementary titration 
 of a gonococcus antigen are given, the approximate dose having been 
 ascertamed in previous titrations (Fig. 117). 
 
480 THE TECHNIC OP COMPLEMENT-FIXATION REACTIONS 
 
 TABLE 17.— ANTICOMPLEMENTARY TITRATION OF A GONOCOCCUS 
 
 ANTIGEN 
 
 Tube 
 
 Antigen, 
 1:10, C.c. 
 
 Com- 
 ple- 
 ment, 
 1:20, 
 C.c. 
 
 Saline solution in each tube to 2 c.c; 
 tubes are shaken and incubated for 
 1 hour at 37" C. 
 
 Anti- 
 sheep 
 Ambo- 
 
 CEPTOB, 
 
 Units 
 
 Sheep's 
 Cor- 
 pus- 
 cles 
 f2.5 
 Per 
 Cent), 
 C.c. 
 
 t 
 
 03 -1-= 
 
 Is 
 £ 
 
 S 
 
 E- 
 
 Results 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5.. 
 
 6 
 
 0.2 
 0.4 
 
 0.6 
 0.8 
 
 1.0 
 
 
 
 1 
 1 
 1 
 1 
 
 1 
 
 1 
 
 IK 
 Wz 
 
 1^2 
 U2 
 
 1 
 1 
 1 
 
 1 
 
 1 
 
 1 
 
 Complete hemolysis 
 Complete hemolysis 
 Complete hemolysis 
 Slight inhibition of 
 
 hemolysis 
 Marked inhibition of 
 
 hemolysis 
 Hemolytic control 
 Complete hemolysis 
 
 If the antigen is new and the anticompletnentary dose is entirely 
 unknown, it may be necessary, in making this titration, to use a different 
 dilution, with higher and lower doses. In conducting the main test the 
 foregoing antigen could be used in dose of 0.2 or 0.4 c.c. of this dilution. 
 
 The Test. — The serums should be fresh and clear, and heated to 56° C. 
 for one-half hour. For each serum use four test-tubes (12 by 1 cm.), 
 arranged in a row. Into each of the first three; place the dose of antigen 
 and increasing doses of serum — 0.05 c.c, 0.1 Ic.c, 0.2 c.c; the fourth 
 tube is the serum control, and into this is placed the maximum dose of 
 serum (0.2 cc.) but no antigen; 1 c.c. of complement diluted 1 : 20 is 
 added to each tube. The following controls are included : 
 
 1. A positive control with an antigonococcus,serum or with the serum 
 of a patient who reacted positively on a former occasion. 
 
 2. A negative control with the serum of a healthy person. 
 
 Both of these controls may be set up with but the maximum dose of 
 serum (0.2 c.c). 
 
 3. The serum control of each serum is conducted in the fourth tube of 
 each series. At the completion of the test this tube should show com- 
 plete hemolysis and thereby indicate that the serum was not anticom- 
 plementary. 
 
 4. The antigen control at this time includes the dose of antigen and 
 complement. 
 
 5. The hemolytic system control at this time receives the dose of 
 complement. 
 
 6. The corpuscle control receives 1 c.c. of the corpuscle suspension. 
 To each tube sufficient saline solution is added to bring the total 
 
Fic. 118. — GuNococuus Complement-fixation Reai-tion. 
 
COMPLEMENT FIXATION IN GONOCOCCUS INFECTIONS 481 
 
 volume up to about 2 c.c. The tubes are shaken and incubated for one 
 hour at 37° C, when IJ^ units of antisheep amboceptor and 1 c.c. of 
 sheep corpuscle suspension are added to each tube except the corpuscle 
 control. The tubes are gently shaken again and reincubated for an 
 hour or longer, depending upon the hemolysis of the controls, after 
 which the results are recorded. This secondary incubation may be 
 omitted and the tubes placed in a refrigerator overnight and the re- 
 sults read the next morning. Under these conditions hemolysis occurs 
 slowly, and according to some workers in this field the reaction becomes 
 more delicate. 
 
 The following table is an example of a gonoeoccus fixation test with 
 the serum of a case of gonorrheal arthritis (Fig. 118). 
 
 TABLE 18.— GONOCOCCUS COMPLEMENT-FIXATION TEST 
 
 
 
 
 I 
 
 
 Sheep 
 
 
 
 
 
 fl 
 
 
 Con- 
 
 
 
 
 Com- 
 
 
 Anti- 
 
 PDS- 
 
 
 Patient's Serum, 
 
 Antigen, 
 
 ple- 
 
 
 sheep 
 
 CLE8 
 
 Results Afteh One and a 
 
 C.c. 
 
 1: 10, C.c, 
 
 ment, 
 
 T3 
 
 Ambo- 
 
 (2.5 
 
 Half Hours Incubation 
 
 
 
 1:20, 
 
 
 
 CEPTOK, 
 
 Peh 
 
 
 
 
 C.c. 
 
 03 
 S 
 
 Units 
 
 Cent), 
 C.c. 
 
 
 
 
 
 
 
 0.05 
 
 0,3 
 
 
 
 Ihz 
 
 
 Slight inhibition of hem- 
 olysis 
 
 0,1 
 
 0.2 
 
 
 ■4-=' o 
 
 1'" 
 
 iVi 
 
 
 Marked inhibition of 
 hemolysis 
 
 0.2 
 
 0.2 
 
 
 1^ 
 
 1J2 
 
 
 Complete inhibition of 
 hemolysis 
 
 0.2 
 
 
 
 
 ij^ 
 
 
 Serum control: hemoly- 
 
 
 
 
 
 
 sis 
 
 Positive serum, 
 
 
 
 53 
 
 
 
 
 0.2 
 
 0,2 
 
 
 p 
 
 15^ 
 
 
 Complete inhibition of 
 hemolysis 
 
 0.2 
 
 
 
 
 C^l® 
 
 ^¥2 
 
 
 Serum control: hemoly- 
 
 Negative serum, 
 
 
 
 =5J 
 
 
 
 sis 
 
 0,2 
 
 0.2 
 
 
 
 IK 
 
 
 Hemolysis 
 
 0.2 
 
 
 
 
 O 
 
 I'i 
 
 
 Hemolysis 
 
 
 
 0.2 
 
 
 3 
 1 
 
 n2 
 
 
 Antigen control : hem- 
 olysis 
 
 
 
 
 
 1 
 
 
 Wi 
 
 
 Hemolytic control : 
 
 
 
 
 
 
 hemolysis 
 
 
 
 
 
 
 
 C3 
 
 
 
 
 Corpuscle control : no 
 hemolysis 
 
 In reading the results the controls are first examined and the test 
 reported as negative, weakly positive, moderately positive, or strongly 
 positive, as the case may be. 
 
 GONOCOCCUS Fixation Test, Using One-tenth the Usual Amounts 
 This technic is employed for purposes of economy, especially since 
 the antigen is likely to be expensive. Otherwise the method is less 
 31 
 
482 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 desirable than the preceding one, as the results are more difficult to 
 read. 
 
 Complement serum is diluted 1 : 10 and used in dose of 0.1 c.c; 
 corpuscles are made up in a 10 per cent, suspension and used in dose of 
 0.1 c.c, the amboceptor is titrated with these amounts of complement 
 and corpuscles, and used in dose equal to one and one-half units. Each 
 day, before the main tests are undertaken, the anticomplementary dose 
 of antigen is determined by placing increasing doses of diluted antigen 
 with complement and salt solution in a series of tubes, incubating for an 
 hour, adding one and one-half units of amboceptor and the corpuscles, 
 followed by incubation for another hour. One-half or one-quarter of 
 the anticomplementary dose is used in making the main test. The 
 serums are inactivated and used in three ascending doses, — 0.005, 0.01, 
 and 0.02 c.c, — equivalent respectively to 0.5, 1, and 2 c.c. of a 1 : 100 
 dilution (0.1 c.c. serum, 9.9 c.c. salt solution). The fourth tube of each 
 series contains the maximum dose of serum without antigen, and is the 
 serum control. The other controls, general technic, and readings of the 
 reaction are the same as those previously described. 
 
 Specificity of the Gonococcus Complement-fixation Test 
 Viewed from a practical standpoint, the reaction is highly specific. 
 While complement-fixation experiments with antigens of gonococci and 
 meningococci and their respective immune serums have demonstrated a 
 biologic relationship between these microorganisms, yet practically with 
 human serums an antigen of pure cultures of gonococci will fix com- 
 plement only with the gonococcus antibody (amboceptor). In this 
 technic a specific antigen is employed, and it=is, therefore, a true applica- 
 tion of the Bordet-Gengou reaction of complement fixation by specific 
 antigen and specific antibody (amboceptor). Obtained under proper 
 technical conditions, a positive reaction is invariably reliable, and indi- 
 cates the presence of a focus of living gonococci. 
 
 PRACTICAL Value of the Gonococcus Complement-fixation Test 
 From our present knowledge of this reaction, it may be stated: 
 
 1. That the difficulty of isolating and preserving a sufficient number 
 of cultures of true gonococci in order to prepare a satisfactory poly- 
 valent antigen constitutes a weighty drawback to the practical use of 
 the test. 
 
 2. Because the gonococcus antibody, unless complicated by wide- 
 spread gonococcal metastases, is produced in small amount in the 
 
COMPLEMENT FIXATION IN GONOCOCCTJS INFECTIONS 483 
 
 majority of cases, the degree of complement fixation is usually much less 
 than that which occurs in the syphilis reaction, and accordingly the 
 reactions are usually weaker and often indefinite. 
 
 3. The reaction is seldom positive during tlie first four to six weeks 
 of an acute anterior or posterior urethritis, in the absence of complica- 
 tions. In acute exacerbations of a chronic urethritis the reaction is 
 positive in about 80 per cent, of cases. In ordinary chronic urethritis 
 with mild infection of the prostate gland the reaction is positive in from 
 30 to 40 per cent, of cases. In chronic urethritis complicated by marked 
 involvement of the prostate gland and epididymitis the reactions are 
 frequently positive, occurring in from 50 to over 80 per cent, of cases. 
 
 The test possesses considerable value in determining the fitness of an 
 applicant for a marriage license, and will, no doubt, be employed for this 
 purpose quite extensively, as a positive reaction is now generally re- 
 garded as indicating the presence of a focus of active gonococcal 
 infection. 
 
 4. During the course of an acute or a subacute urethritis the occur- 
 rence of an acute complication, such as prostatitis, epididymitis, etc., 
 is likely to result in a positive fixation test. 
 
 5. A positive reaction may persist for several weeks after the patient 
 is clinically cured. Torrey ' has shown experimentally that the antibody 
 persists in the blood of rabbits artifically immunized for from ten days 
 to six or seven weeks. Usually, under proper treatment, the reaction in 
 ordinary cases of urethritis disappears in from two to three weeks; if, 
 however, a positive reaction persists, a focus, of infection is probably 
 present, and the patient should be kept under, further observation and 
 the treatment persisted in. 
 
 6. In women the reaction is seldom positive until the infection h.-is 
 reached the cervical canal. In the case of little children, however, v/e 
 have known positive reactions to occur in axaite and chronic vulvo- 
 vaginitis, indicating either that the disease is "more severe in children, 
 with more antibody formation, or that it may' reach the cervical canal. 
 
 The reaction is positive in about 60 per cent, of cases of pyosal- 
 pingitis, and the test may prove of value in making the differentiation of 
 inflammatory lesions from certain cystic and neoplastic conditions, and 
 in establishing the gonorrheal basis of many of these infections. 
 
 7. Cases of gonorrheal urethritis yield from 80 to 100 per cent, of 
 positive reactions, and the complement-fixation test has considerable 
 value in establishing the diagnosis of these infections. 
 
 ' Jour. Med. Research, 1910, i, 95. 
 
484 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 8. The administration of gonococcus baeterin and antigonococcus 
 serum is likely to be followed by positive reactions. Just how long the 
 antibodies may persist in the blood after a cliilical cure has been effected 
 it is difficult to state ; at least from six to twelve weeks' time should be 
 given for them to disappear. 
 
 9. In medico-legal cases the courts may not accept the usual evi- 
 dence offered by a bacteriologic diagnosis based upon stained smears of 
 a secretion, and cultures are frequently differentiated from other Gram- 
 negative diplococci, only with difficulty. Conducted with the proper 
 technic, the gonococcus fixation test is highly specific and much less 
 difficult to perform. 
 
 10. Finally, it must be emphasized that the reaction has a far more 
 positive than negative value. The reaction is highly specific, but there 
 is a limit to its delicacy, so that a negative reaction in urethritis does 
 not exclude the possibility of gonococcal infection. 
 
 COMPLEMENT-FIXATION TEST m GLANDERS 
 
 The complement-fixation test is used extensively by veterinarians 
 in making a laboratory diagnosis of glanders. The test has been found 
 very reliable, and is usually more delicate than the agglutination test 
 and the Strauss guinea-pig test. It has also" been used successfully in 
 the diagnosis of human glanders. 
 
 Preparation and Standardization of Antigen. — The antigen should be 
 polyvalent, and composed of at least several different strains. Cultures 
 of Bacillus mallei are grown on slants of glycerin agar (1 per cent, acid) 
 for from forty-eight to seventy-two hours. The growths are then 
 removed, and sufficient distilled water added to give a milky suspension. 
 This suspension is sterilized by heating the tubes to 60° C. for two hours. 
 They are then shaken mechanically with glass beads for a few hours on 
 two successive days. Enough sodium chlorid is added to make the 
 solution isotonic, and the whole is preserved, with 0.5 per cent, phenol 
 and stored in a dark, cold place, where it will keep for many months. 
 
 A simpler antigen is prepared by growing the bacillus in glycerin 
 bouillon for seventy-two hours, sterilizing by heating to 60° C. for two 
 hours, and preserving with the addition of 0.5 per cent, of phenol. 
 
 The anticomplementary dose is then deteripined by titration. The 
 antigen is diluted 1 : 20 by mixing 1 c.c. with 19 c.c. of normal saline 
 solution. To a series of seven test-tubes add increasing amounts of 
 diluted antigen as follows: 0.1, 0.2, 0.4, 0.6, 0,8, 1, and 2 c.c. Add 1 c.c. 
 
COMPLEMENT-FIXATION TEST IN GLANDEES 485 
 
 of complement serum (1 : 20) to each tube, and sufficient salt solution 
 to bring the total volume in each up to 3 c.c. Incubate for an hour at 
 37° C, and add IJ^ units of antisheep amboceptor titrated just previous 
 to making the test (see p. 377) and 1 c.c. of sheep corpuscle suspension. 
 Beincubate for an hour or an hour and a half. At the end of this time 
 that tube showing beginning inhibition of hemolysis contains the anticom- 
 plementary dose, and one-fourth to one-half this amount is used in making 
 the main test. 
 
 An antigenic titration may also be made, but this is not absolutely 
 necessary. To a series of tubes containing 0.05, 0.1, 0.15, 0.2, 0.25, and 
 0.3 c.c. of diluted antigen add 0.1 c.c. of fresh heated glanders serum 
 (known to yield a positive reaction) and 1 c.c. of complement serum 
 (1 : 20) and sufficient salt solution. Incubate for one hour and add 13^ 
 units of hemolytic amboceptor and corpuscles. After a second incuba- 
 tion of from one to two hours, that tube shovring just complete inhibition 
 of hemolysis contains the antigenic dose, and double this amount may be 
 used in performing the main test, providing that it is still one-half or, 
 better, but one-quarter the anticomplementary dose. 
 
 A hemolytic system control is included in both titrations, and in the 
 antigenic titration an additional control on the serum, to determine 
 whether it is free from anticomplementary action. 
 
 The dilution here advised may be too low; if this is the case, the 
 titrations must be repeated with the antigen diluted 1 : 50 or 1 : 100. 
 
 The Test. — The external jugular vein of the horse is punctured with 
 a sterile needle, and from 5 to 10 c.c. of blood is collected in a centrifuge 
 tube or other glass container, which preferably should be sterile. The 
 clear serum is heated to 55° C. for one-half hour just before the tests are 
 conducted. 
 
 Into a series of four small test-tubes place the following doses of 
 serum: 0.05, 0.1, 0.2, and 0.2 c.c. To the first three tubes add the 
 proper dose of antigen; to all tubes add 1 c.c. of complement (1 : 20) and 
 sufficient salt solution to bring the total volume up to 3 c.c. 
 
 The following controls should be included : 
 
 1. The serum control on each serum is conducted in the fourth tube 
 of each set. 
 
 2. A known positive serum should be tested in the same manner. 
 
 3. A known negative serum should be tested in the same manner. 
 
 4. The antigen control, which at this stage contains the dose of 
 antigen, complement, and saUne solution. 
 
 5. The hemolytic control, which at this time contains but 1 c.c. of 
 complement plus saline solution. 
 
486 THE TECIINIC OF COMPLEMENT-FIXATION REACTIONS 
 
 6. The corpuscle control, containing 1 c.c. of corpuscle suspension 
 and 3 c.c. of saline solution. This tube should be plugged with cotton. 
 
 All tubes are gently shaken and incubated for an hour, after which 
 \}4. units of hemolytic amboceptor and 1 c.c. of corpuscle suspension are 
 added to all except the corpuscle control. The tubes are gently shaken 
 and reincubated for an hour or two, depending upon the hemolysis of 
 the controls. 
 
 The controls are first inspected. They should all show complete 
 hemolysis, except the first three tubes of the positive serum series and the 
 corpuscle control. Inhibition of hemolysis in the first three tubes of 
 the series containing the unknown serum indicates a strong positive 
 reaction. Complete hemolysis in all tubes indicates a negative reaction. 
 Partial hemolysis in the first three tubes indicates a partially positive 
 reaction. If the serum control or antigen con4.rol tubes should show in- 
 hibition of hemolysis, these were probably ariticomplementary and the 
 test should be repeated. 
 
 COMPLEMENT-FIXATION TEST IN CONTAGIOUS ABORTION 
 
 It is now generally conceded among veterinarians that the Bacillus 
 abortus of Bang is the specific cause of contagious abortion of cows. 
 
 Evidence is gradually accumulating to show that an organism be- 
 longing to the paratyphoid group is frequently the cause of a similar 
 condition among mares (Kilbourne and Smith,^ Liguierer,^ Liguierer 
 andZabala; Good;' VanNeelsbergen;* deJong;^ Meyer and Boerner)^ 
 Meyer and Boerner, who have studied this bacillus with particular care, 
 classify it with the paratyphoid-enteritidis group (Bacillus aborti equi). 
 
 Veterinarians are generally agreed that in contagious abortion of 
 cows the complement-fixation test is highly specific, and is frequently of 
 considerable value in establishing a diagnosis (Meyer and Hardenburgh 
 and others). 
 
 Meyer and Boerner have found fixation Of complement to occur in 
 contagious abortion of mares with an antigen of Bacillus abortus equi, 
 and recommend the test as diagnostic aid in this infection. 
 
 1 Kilbourne and Smith : United States Department of Agriculture, Bulletin No. 
 3, 1893, 49 and 53. 
 
 "^ Liguierer; Rec. Med. veterinaire, Ixxxii, 1905. 
 
 3 Good: Kentucky Agriculture Exper. Station Bulletin No. 165, 1912 
 
 * Van Neelsbergen: Tijdschrift. v. Veeartsenijk., xxiv, 1912. 
 
 '' de Jong: Archiv. f. Wissenshaftl. u. prak. Tierheilkunde, xxv, 1900. 
 
 ° Meyer and Boemer: Jour. Med. Research, 191S, xxix. No. 2, 325. 
 
COMPLEMENT-FIXATION IN BOURINE 487 
 
 Preparation and Standardization of Antigens. — The antigen of 
 Bacillus abortus (Bang) for use in the complement-fixation test in 
 contagious abortion of cows is prepared by cultivating a number of 
 strains of the bacillus, which have been trained to grow aerobically, 
 upon slants of glycerin agar for seventy-two hours. The growths are 
 then washed off with sufficient normal saline solution containing 2 per 
 cent, phenol to yield a cloudy emulsion. Shake briskly in order to 
 break up the clumps of bacilli, and filter through paper. Place in a 
 refrigerator for several days to complete the sterilization, and titrate 
 the anticomplementary dose each time before the main test is conducted. 
 
 The antigen may also be prepared by cultivating a number of strains 
 in glycerin-serum bouillon for five or six weeks. Centrifuge thoroughly 
 and wash the bacilli once or twice with normal saline solution, to remove 
 all traces of serum. Dilute the washed bacifli with sufficient normal 
 saline solution to give an emulsion equal in density to a twenty-four- 
 hour bouillon culture of Bacillus coli, and add Q.4 per cent, of phenol as a 
 preservative. 
 
 The antigen of Bacillus abortus equi for making the complement- 
 fixation diagnosis of contagious abortion of mares is prepared of eigh- 
 teen- to twenty-hour-old glycerin bouillon cultures, with an addition 
 of 0.5 per cent, of phenol. These antigens are less anticomplementary 
 than shake extracts, and keep their titer unaltered for many weeks 
 (Meyer and Boerner). They may also be used for making the mac- 
 roscopic agglutination test. 
 
 The anticomplementary dose is determined' each time, and one-half 
 this amount is used in performing the main test. The technic is the 
 same as that employed in the titration of glanders antigen. 
 
 The tests and controls are conducted with descending doses of fresh 
 inactivated serum (0.05, 0.1, and 0.2 c.c), in exactly the same manner 
 as the glanders reaction is performed. 
 
 COMPLEMENT FIXATION IN DOURINE 
 Dourine, or horse syphilis, is a specific infectious disease of the horse 
 and ass, transmitted from animal to animal by the act of copulation, 
 and caused by the Trypanosoma equiperdum. It is characterized by an 
 irregular incubation period, the localization of the early symptoms to 
 the genital organs, and, finally, by complete paralysis of the posterior 
 extremities, a fatal termination ensuing in from^ix months to two years. 
 The disease is especially prevalent among horses in the northwestern 
 
488 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 states, and may occur in such various and atypical forms as to render 
 clinical diagnosis difficult. 
 
 Complement-fixation methods of diagnosis have been tried by Pav- 
 losvici, Winkler and Wyschelersky, MoUer, Watson, Brown, and in a 
 large series of cases with good results by Moiler, Eichhom, and Buck.' 
 These last-named investigators examined 8657 specimens of blood from 
 horses in Montana and North and South Dakota, and of these, 1076 
 yielded positive reactions. 
 
 In most of these experiments the results were corroborated by clinical 
 and pathologic findings, and the investigators conclude that the com- 
 plement-fixation test is of great value, especially in countries where only 
 one of these protozoan diseases exists. 
 
 Preparation and Standardization of Antigen. — This is the most 
 difficult part of the technic, because the trypanosome is not readily 
 grown on artificial culture-media. Watery, alcoholic, and acetone 
 extracts of various organs of horses dead of the disease do not yield 
 satisfactory antigens. Since the reaction is a group reaction, and 
 dourine is the only trypanosome infection in this country, Moiler, 
 Eichhorn, and Buck selected the surra organism for the preparation of 
 antigen. After infecting a dog and at the height of infection with- 
 drawing 200 c.c. of blood into potassium citrate and hemolyzing with 
 0.5 gram of saponin, the trypanosomes were secured after thorough 
 centrifugalization and washed three times. After the last washing the 
 trypanosomes were emulsified in 50 c.c. of salt solution and preserved 
 with phenol. This antigen yielded highly satisfactory results, but the 
 difficulty of preparing it, and the small quantity secured, made it 
 necessary that another method be used. 
 
 An extract of the spleen of a rat just dead of surra was found to yield 
 a satisfactory antigen. The extract does not keep well, and must be 
 prepared freshly every few days and carefully standardized. Gray or 
 white rats are infected with surra by injecting 0.2 c.c. of blood from a 
 rabbit with this disease. If a large number of tests are to be made, the 
 rats should be so infected that one or two are available each day for the 
 preparation of the antigen. 
 
 The spleen from a rat with a small amount of salt solution added is 
 ground in a mortar until a pulpy mass results. More of the salt solution 
 is added from time to time, and the suspension thus obtained is filtered 
 twice through a double layer of gauze and diluted with salt solution to 
 40 c.c. 
 
 1 Amer. Jour. Veter. Med., 1913, viii, ,581. 
 
COMPLEMENT-FIXATION TEST IN TYPHOID FEVER . 489 
 
 The anticomplementary and antigenic doses are then determined, 
 and the extract used in double the antigenic unit, providing that this 
 amount is not more than half the anticomplementary dose. If a posi- 
 tive serum from an infected horse is not available, the anticomplementary 
 dose may be determined and half this amount used in conducting the 
 main test. 
 
 Anticomplementary Titration. — Into a series of six test-tubes place 
 increasing amounts of antigen — 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 c.c; add 1 
 CO. of complement (1 : 20) and sufficient salt solution to bring the total 
 volume in each tube up to 2 c.c. Incubate for one hour. Add 1^ or 
 2 units of antisheep amboceptor (determined by preliminary titration) 
 and 1 c.c. of 2.5 per cent, sheep's corpuscles. Incubate for one hour, 
 after which the reading is made. 
 
 The Test. — The serum is inactivated and used in dose of 0.15 c.c, 
 since it has been found that fixation in this quantity is obtained only 
 with serums of horses affected with dourine. In some instances the 
 serum of horses has reacted in doses as small as 0.02 c.c, and the reac- 
 tion may be conducted with increasing doses of serum — 0.05, 0.1, and 
 0.15 C.c. — in exactly the same manner as when the glanders reaction is 
 performed. The same Controls are included. 
 
 COMPLEMENT-FIXATION TEST IN TYPHOID FEVER 
 This was one of the original diseases in which Bordet and Gengou 
 first demonstrated the occurrence of complement fixation. Widal and 
 Lesourd attempted to make practical application of this method in the 
 diagnosis of the disease, but their results were indifferent, and since then 
 numerous writers have expressed various opinions as to the value of the 
 test. Garbat has secured uniform and reliable reactions with a poly- 
 valent antigen, and emphasizes the importance of this factor. 
 
 The antigen is prepared of numerous strains of typhoid bacilli — the 
 more the better. Cultures arc grown on slants of agar for forty-eight 
 hours, washed off with small quantities of sterile distilled water, heated 
 to 60° C. for two hours, shaken mechanically fqr twenty-four hours, and 
 either filtered through a sterile Berkefeld filter or thoroughly centrif- 
 ugalized. The filtrate is preserved with 0.5 per cent, phenol and used 
 as antigen. 
 
 I have secured good results by removing the growths with small 
 amounts of normal salt solution, and placing them in a shaking flask, 
 and shaking for an hour to break up the clumps. After heating to 60° C. 
 
490 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 for an hour, 1 per cent, glycerin and 0.5 phenol are added as preservatives, 
 and the mixture stored away in ampules containing 1 c.c. each. The 
 emulsion should be slightly milky in appearance. 
 
 The antigen is diluted 1 : 10 or 1 : 20, arid the anticomplementary 
 dose determined by titration before the main tests are conducted. The 
 technic of the reaction is exactly similar to thp gonococcus fixation test. 
 
 The ease with which the Widal reaction is performed renders it the 
 method of choice. Nevertheless the complement-fixation test is quite 
 delicate, and will frequently aid, where the agglutination test is negative 
 or absent and in making the differential diagnosis from paratyphoid 
 fever. The strongest reactions are secured late in the disease. 
 
 COMPLEMENT-FIXATION TEST IN TUBERCULOSIS 
 
 It was the original studies in complement fixation in tuberculosis 
 made by Wassermann and Bruch that later mduced these workers, in 
 cooperation with Neisser, to apply the method to the diagnosis of syphilis. 
 
 Antigens were prepared of tuberculous glands and lungs, and com- 
 plement fixation was found to occur with an* artificial immune serum 
 (Hochst) and with the serums of persons who^had received injections of 
 tuberculin, but not the serums of other tuberculous persons who had 
 not received tuberculin. 
 
 It would appear, therefore, that tuberculin may stimulate the pro- 
 duction of tuberculin antibodies in the nature" of amboceptors (Citron). 
 These amboceptors will frequently fix complement in vitro with a suitable 
 tubercufin antigen. 
 
 According to Citron, a tuberculous focus may contain tuberculin, 
 and antituberculin is probably produced by healthy cells in or about the 
 focus, which are capable of reaction. The production of antituberculin, 
 however, is a transitory process, arising only when tuberculin has reached 
 the circulation, either spontaneously or artificially. During this stage 
 the serum of the patient may yield a positive complement-fixation test. 
 Following this stage of activity there comes a period of quiescence dur- 
 ing which no free antituberculin can be demonstrated in the blood-serum. 
 The cells, however, arc sensitized, and possess many sessile receptors 
 that possess a high affinity for tuberculin and produce antituberculin 
 much more readily than do normal cells. Hence when a small amount 
 of tuberculin is injected it is bound by the sensitized cells in the zone 
 surrounding the tuberculous focus, and thus explains the heightened 
 action at this point, with the production of antituberculin. 
 
COMPLEMENT-FIXATION TEST IN TUBERCULOSIS 491 
 
 Further discussion on Citron's tuberculin theory is reserved for the 
 chapter devoted to this subject. Evidence indicates, however, that 
 immunity in tuberculosis is not dependent sole-ly upon the development 
 and presence of antituberculin, but that other antibodies are likewise 
 concerned. 
 
 As a practical test, complement fixation has not been successful in 
 tuberculosis. As previously stated, this may; be due to the fact that 
 antituberculin production is transitory and variable, and hence if the 
 amboceptors are absent or present in but very minute amounts, com- 
 plement fixation in vitro cannot occur. 
 
 Considerable attention has also been given the preparation of the 
 antigen, in the belief that a special endotoxic substance or part of the 
 bacillus is required. Various antigens have been used, such as Bacillen 
 emulsion, old tuberculin, tuberculin filtrate, and a watery emulsion of 
 tubercle bacilli. A mixture of Koch's old and new tuberculins has also 
 been used. Antigen may also be prepared after the method of Besredka 
 (p. 474), in which the bacilli are dried, ground, and used as a fine suspen- 
 sion of the bacterial substance. 
 
 More recently Hammer^ has reported favorably upon an antigen 
 composed of an alcoholic extract of tuberculous tissue and old tuberculin. 
 The tissue is extracted for five days with four parts alcohol and filtered. 
 To each cubic centimeter of extract, 0.1 c.c. o£ old tuberculin is added; 
 the anticomplementary dose is determined, and one-half this amount is 
 used in conducting the main test. 
 
 At present, however, complement-fixation tests have yielded in- 
 different results, although a test sufficiently delicate and constant to 
 permit early infections to be detected would be of inestimable value. 
 If the failures of the past have been due rather to faulty antigen than 
 to absence of tuberculin amboceptors, researches in the future will 
 probably solve the problem. 
 
 THE COMPLEMENT-FIXATION TEST IN THE* STANDARDIZATION OF 
 IMMUNE SERUMS 
 
 The technic of complement fixation has also been employed as one 
 means in effecting the standardization of antimeningococci and anti- 
 gonococcic serums. Since, however, the amount of complement-fixing 
 amboceptors in a serum is no index to its therapeutic and prophylactic 
 value, a measure of this one factor is not a reliable standard, 
 
 » Munch, med. Wochenschr., 1912, 5b, 1750. 
 
492 THE TECHNIC OF COMPLEMENT-FIjdA.TION REACTIONS 
 
 The technic consists in preparing the antigen and in determining its 
 anticomplementary dose. Whatever this is", one-half to one-quarter 
 this amount is added to increasing quantities of heated immune serum, 
 ranging from 0.001 to 0.1 c.c. Complement and saline solution are 
 added, and after incubating one hour at 37° C, the amount of comple- 
 ment fixation is determined by adding hemolytic amboceptors and cor- 
 puscles. 
 
 While this titration is one measure of the reaction of the animal used 
 in the immunization, better evidence of the, therapeutic value of the 
 serum is obtained by determining the content in bacteriotropins, by 
 testing the serum with the antigen in susceptible animals, or by a com- 
 bination of all methods. 
 
 THE COMPLEMENT-FIXATION TEST IN ECHINOCOCCUS DISEASE 
 Complement-fixation tests have been advocated as aiding the diag- 
 nosis of echinococcus disease. In making the tests, hydatid cyst fluid 
 of the human or sheep is used as antigen (Ghedini). Reports upon the 
 specificity and usefulness of this reaction are" somewhat contradictory, 
 although, as a rule, they are generally favorable. Cases of recent in- 
 fection with fresh active lesions may react negatively, a result that is 
 dependent presumably upon non-absorption of antigen and slight 
 antibody formation (Gaetano,' Weinberg and Bordin,^ Kurt Meyer'). 
 Thomsen and Magnussen,^ in a recent study of 12 cases, found that 
 10 reacted positively. Of 55 control cases (32 of whom reacted posi- 
 tively to the Wassermann reaction), all were negative except one. Kurt 
 Meyer found the serums of echinococcus infected persons to react posi- 
 tively with antigens of other taenia (Taenia solium and Taenia saginata), 
 and, conversely, the serums of persons infected with Taenia saginata and 
 Taenia solium to react ^vith an echinococcus antigen. Thomsen and 
 Magnussen, however, could not support thesa findings, and report most 
 favorably upon the specificity of the reaction.* Of 10 persons infected 
 with Taenia saginata, 2 with Taenia solium, and 1 with Bothriocephalus 
 latus, all reacted negatively with echinococcus antigen. 
 
 The antigen is best prepai'ed of the fresh fluid of an echinococcus 
 cyst of man or sheep. It should be filtered, if' necessary, preserved with 
 
 1 Gaetano: Riforma Medica, 1910, Nos. 39 and 40. Reference in Deut. med. 
 Wochenschr., 1910, No. 44. 
 
 ^ Weinberg and Bordin: Compt. rend. soc. biol., 1909, Bd. 66. 
 
 = Meyer (K) : Berl. klin. Wochenschr., 1910, No. 28. 
 
 ^ Thomsen (O.), and Magnussen (G.): Berl. klin. Wochenschr., 1912, No. 25. 
 
COMPLEMENT-FIXATION TEST IN ECHINOCOCCTJS DISEASE 493 
 
 0.5 per cent, phenol, and kept constantly at a low temperature. It is 
 highly important not to use the antigen in an anticomplementary dose, 
 hence it should be titrated before each test is made. 
 
 Anticomplementary Titration. — Dilute the fluid 1 : 10 with normal 
 sahne solution, and into a series of eight small test-tubes place increasing 
 amounts as follows: 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, and 3 c.c. Add 1 c.c. of 
 complement (1 : 20) and sufficient saline solution to bring the total 
 volume in each tube up to 4 c.c. Shake gently, incubate for one hour, 
 and then add 1^ units of antisheep amboceptor (previously titrated) 
 and corpuscles. Reincubate for one to two hours; the tube showing 
 beginning inhibition of hemolysis contains the anticomplementary dose, 
 and in performing the main test, one-half to one-quarter this amount 
 may be used. 
 
 If titration with diluted fluid does not give the anticomplementary 
 dose, the titration should be repeated with undiluted fluid. 
 
 AlcohoUc extracts of daughter cysts, the hydatid wall, or the fluid 
 have been found to give positive reactions in- syphilis (Israel, Brauer, 
 Henins), and are, therefore, unsatisfactory. 
 
 Thomsen and Magnussen speak favorably of antigen paper infil- 
 trated with cyst fluid and titrated. 
 
 The Test. — The patient's serum should be fresh, and should be heated 
 to 55° C. for half an hour. Into a series of five test-tubes place 0.025, 
 0.05, 0.1, 0.2, and 0.2 c.c. of serum. To each of the first four tubes add 
 one-half the anticomplementary dose of antigen; to all the tubes add 
 1 c.c. of complement (1 : 20) and sufficient saline solution to bring the 
 total volume up to 3 c.c. 
 
 The fifth tube is the serum control; an antigen, hemolytic system, 
 and corpuscle control should be included, as is usual in complement- 
 fixation tests. A normal serum may be incljided, and, if possible, a 
 known positive serum. 
 
 After incubating for an hour at 37° C, add IJ^ units of hemolytic 
 amboceptor and 1 c.c. of the corpuscle suspension to each tube, Re- 
 incubate for an hour or longer, depending upon the hemolysis of the 
 controls. Marked or complete inhibition of hemolysis in the fourth 
 tube (0.2 c.c. patient's serum), with lesser degrees of inhibition in the 
 other tubes of the series, indicates a positive reaction. Larger doses of 
 patient's serum should not be used on account of the probability of non- 
 specific complement fixation. 
 
494 THE TECHNIC OF COMPLEMENT-FIXfATION REACTIONS 
 
 PROTEIN DIFFERENTIATION BY COMPLEMENT FIXATION 
 The Determination of an Antigen by Complement Fixation. — In the 
 
 tests hitherto considered the antigens were known, and the suspected 
 antibodies sought for in the blood-serum or other body-fluid. In making 
 the reactions it was necessary to bring the serum to be tested into con- 
 tact with the antigen specific for the suspected antibody, in the presence 
 of complement, and at a suitable temperature. At the end of an hour 
 the mixture was tested for free complement by adding hemolytic ambo- 
 ceptor and red blood-corpuscles. This order may be reversed, and with 
 a known antibody the suspected antigen may be detected. The anti- 
 gen to be detected, as in a solution of blood or bacterial extract, is 
 brought into contact with its specific antibody in the presence of com- 
 plement. At the end of an hour, at a suitable temperature, the mixture 
 is tested as previously for free complement, by adding corpuscles and 
 hemolytic amboceptors. Under proper conditions complement fixation 
 would indicate a specific reaction between the antibody and its antigen, 
 and thus serve to identify the latter. 
 
 This method has clinical applications similar to those in which the 
 precipitin reaction is used : 
 
 1. In the differentiation of blood-stains a solution of the stain con- 
 stitutes the unknown antigen. By furnishing a known antiserum the 
 antigen is detected, i. e., the animal from which the blood was derived 
 is ascertained. 
 
 2. In the recognition and differentiation of meats. 
 
 3. In the detection of bacterial antigens in the blood-serum of 
 patients, or with highly immune serums an unknown bacterial antigen 
 may be identified and the test employed as a means of differentiation 
 among bacterial species. 
 
 4. Similar applications of the test may be made in the differentiation 
 of milks, seminal stains, and other albuminous substances. 
 
 As compared with precipitin reactions, the complement-fixation test 
 is probably more delicate and reliable and easier of interpretation. The 
 technic of the latter method is, however, more compHcated, and the 
 liability to error is greater unless the principles of complement fixation 
 in general are thoroughly understood and the importance of quantitative 
 factors is appreciated. 
 
 1. Complement Fixation for the Identification of Blood-stains. — The 
 application of the technic of complement fixation to the determination 
 of specific protein antigen, such as human or animal blood, was demon- 
 
PROTEIN DIFFEKENTIATION BY COMPLEMENT FIXATION 495 
 
 strated by Gengou in 1902, The principles worked out by him were 
 extensively studied and practically applied by Neisser and Sachs in the 
 forensic differentiation of animal proteins. 
 
 Hemolytic System. — Complement is furnished by the fresh serum of 
 a guinea-pig diluted 1 : 20 and used in dose of 1 c.c. ( = 0.05 c.c. serum); 
 washed sheep's corpuscles are made up in a 2.5 per cent, suspension and 
 used in dose of 1 c.c; antisheep amboceptor should be highly potent, 
 and is titrated after the method previously given (p. 377). In the 
 following titrations and in conducting the main test the hemolytic 
 amboceptor is used in an amount equal to 13^ or 2 units. 
 
 Specific Antiserum.. — This is obtained from a rabbit immunized 
 with the protein for which the test is to be made, namely, human or 
 animal blood-serum. In forensic tests it may be necessary to prepare 
 a number of these antiserums with the serums of man and the ordinary 
 domestic animals. The technic of mmunization is the same as that 
 employed for the preparation of precipitins (p. 70). An antiserum for 
 forensic tests must be sufficiently potent to fix complement with 0.0001 
 c.c. of its antigen. This is determined by a process of titration. If, for 
 example, an antihuman serum is to be titrated, the method of procedure 
 is as follows : 
 
 Secure 0.1 c.c. of fresh human serum and dilute 1 : 1000 by adding 
 99.9 c.c. of normal saline solution. Of this dilution, 0.1 c.c. is equivalent 
 to the standard dose of 0.0001 c.c. of undiluted serum. The antiserum 
 is heated to 55° C. for half an hour and diluted 1 : 10 (1 c.c. immune 
 serum plus 9 c.c, of sahne solution). Decreasing doses of immune serum 
 are mixed with a constant dose of antigen and complement. At the 
 same time the anticomplementary titration of the inunune serum is 
 made by substituting salt solution for antigen. The doses to employ 
 and the results of an actual titration are shown in Table 19 : 
 
 Tube 16 is the hemolytic system control, and shows complete hemol- 
 ysis; tube 15 is the antigen control, and shows complete hemolysis, 
 as the quantity of serum is too small to exert an anticomplementary 
 influence; tubes 11 to 14 are the tests for anticomplementary action of 
 the antiserum. In the present instance the serum was several months 
 old and the maximum dose of 1 c.c. ( = 0.1 c.c. undiluted serum) was 
 very slightly anticomplementary. A fresh serum is practically never 
 anticomplementary in this dosage, but these tubes should, nevertheless, 
 be included in each titration. Tubes 1 to 10 include the antigenic titra- 
 tion, and show that the antiserum is perfectly antigenic in dose of 
 
496 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 0.3 c.c. of this dilution ( = 0.03 c.c. undiluted serum). In 'perf arming 
 the main test double this quantity, or 0.6 c.c, would be used. 
 
 
 TABLE 
 
 19.— TITRATION OF AN IM1 
 
 VIUNE 
 
 SERUM 
 
 
 
 
 
 9 
 
 
 Sheep's 
 
 
 
 
 
 
 
 CoR- 
 
 
 
 Anti- 
 
 Seeum 
 
 Com- 
 ple- 
 
 ^ 
 
 Anti- 
 sheep ' 
 
 PUB- 
 
 Results after Incu- 
 
 Tube 
 
 serum, 
 
 
 ment, 
 
 
 Ambo- 
 
 (2.5 
 
 bation FOR One and 
 
 
 1:10, C.c. 
 
 C.c. 
 
 1:20 
 
 o 
 
 ceptor? 
 
 Per 
 
 One-half Houhs 
 
 
 
 
 C.c. 
 
 CD 
 
 3 
 
 a 
 
 Units 
 
 Cent.), 
 C.c. 
 
 
 1. 
 
 1.0 
 
 0.1 
 
 
 1>2 
 
 
 Inhibition of hem- 
 
 
 
 
 
 
 
 olysis 
 
 2 
 
 0.9 
 
 0.1 
 
 
 IH 
 
 
 Inhibition of hem- 
 
 
 
 
 
 
 
 
 olysis 
 
 3 
 
 0.8 
 
 0.1 
 
 
 a 
 a 
 
 13-2 
 
 
 Inhibition of hem- 
 olysis 
 
 4 
 
 0.7 
 
 0.1 
 
 
 j5 . 
 
 13-2 
 
 
 Inhibition of hem- 
 olysis 
 
 5 
 
 0.6 
 
 0.1 
 
 
 
 IV2 
 
 
 Inhibition of hem- 
 olysis 
 
 6 
 
 0.5 
 
 0.1 
 
 
 
 Wi 
 
 
 Inhibition of hem- 
 olysis 
 
 7 
 
 0.4 
 
 0.1 
 
 
 Xi 
 ?. 
 
 yy2 
 
 
 Inhibition of hem- 
 olysis 
 
 8 
 
 0.3 
 
 0.1 
 
 
 o 
 
 W2 , 
 
 
 Inhibition of hem- 
 olysis 
 
 9 
 
 0.2 
 
 0.1 
 
 
 
 W2 
 
 
 Partial inhibition of 
 hemolysis 
 
 10 
 
 0.1 
 
 0.1 
 
 
 & 
 
 1V2 
 
 
 Slight inhibition of 
 
 hemolysis 
 Very slight inhibi- 
 
 11 
 
 1.0 
 
 
 
 
 a 
 
 i>i 
 
 
 
 
 
 
 
 
 
 tion of hemolysis 
 
 12 
 
 0.8 
 
 
 
 
 
 
 m 
 
 
 Complete hemolysis 
 
 13 
 
 0.4 
 
 
 
 
 S 
 
 1Y2 
 
 
 Complete hemolysis 
 
 14 
 
 0.2 
 
 
 
 
 OJ 
 
 IK 
 
 
 Complete hemolysis 
 
 15 
 
 
 
 0,1 
 
 
 
 iH 
 
 
 Complete hemolysis 
 
 16 
 
 
 
 
 
 
 o3 
 
 iM 
 
 
 Complete hemolysis 
 
 Each antiserum is tested in a similar maimer. In forensic blood 
 tests an antihuman serum is, of course, employed first; if this is negative 
 and it is desirable to determine the source of the blood, other antiserums, 
 so that the ox, horse, dog, etc., are prepared, titrated, and tested with a 
 solution of the blood-stain. 
 
 The Blood-stain. — It is first necessary to ascertain that the stain is a 
 blood-stain; this is done by performing the hemin crystal test (p. 303). 
 The stain is then extracted in normal saline solution, as described on 
 p. 304. A 1 : 1000 dilution is made approximately by so diluting the 
 extract that it just gives a slight opalescence when boiled with a few 
 drops of acetic acid, and a slight foam persists after shaking. Unless 
 it is perfectly clear, it should be filtered. 
 
PROTEIN DIFFERENTIATION BY COMPLEMENT FIXATION 497 
 
 The Test. — Into a series of six small test-tubes place increasing doses 
 of extract of the blood-stain (antigen), as follows: 0.1 c.c, 0.2 c.c, 0.4 
 CO., 0.6 c.c, 0.8 c.c, 1 c.c; add double the titrated dose of antiserum 
 and 1 c.c. of complement (1 : 20), with sufficient salt solution to bring 
 the total volume in each tube up to 3 c.c. 
 
 The following controls are included: 
 
 1. Antigen control; 1.0 c.c. of the blood extract plus 1 c.c. of diluted 
 complement and salt solution. 
 
 2. Antiserum control: double the titrated dose plus 1 c.c. of diluted 
 complement and salt solution. 
 
 3. Hemolytic control; at this time, 1 c.c. of diluted complement and 
 salt solution. 
 
 4. Corpuscle control: 1 c.c. of corpuscle suspension and salt solution. 
 The tube should be plugged with cotton. 
 
 Shake all the tubes gently and incubate for an hour at 37° C. Add 
 13^ units of hemolytic amboceptor and 1 c.c. of corpuscle suspension to 
 each tube except the corpuscle control. Shake gently and reincubate 
 for from one to two hours, depending upon the degree of hemolysis 
 present in the controls. 
 
 The readings are made at once, and again after the tubes have been 
 allowed to settle in the refrigerator overnight. Inhibition of hemolysis 
 with the smallest dose of blood extract — 0.1 c.c. ( = approximately 0.0001 
 c.c. of blood) — indicates that the blood extract is most certainly the 
 antigen for the antiserum employed. Even with the maximum dose of 
 extract — 1 c.c. ( = approximately 0.001 c.c. of blood) — inhibition of 
 hemolysis serves to show the nature of the blood. With an antihuman 
 serum, for instance, a similar specific reaction would be possible only 
 with the bloods of the higher apes. 
 
 In making blood tests for medicolegal purposes the antiserum 
 should not only be standardized with a definite .dilution of human serum, 
 but the whole test should first be conducted with a known dried human 
 blood-stain, and it must be borne in mind that extreme accuracy in all 
 manipulations is essential. 
 
 In Table 20 are shown the method and the" results of an actual test, 
 using a dried human blood-stain and the, same antiserum as previously 
 directed. 
 
 I prefer this complement-fixation test to the precipitin reaction in 
 the differentiation of proteins, as the readings are sharper and more 
 definite. This test is fully as reliable as the precipitin test, and there 
 is less danger of group reaction. 
 .32 
 
498 THE TECHNIC OF COMPLEMENT-FIXATION REACTIONS 
 
 TABLE 20.— FORENSIC BLOOD TEST 
 
 1 
 
 2 
 3 
 
 4 
 5 
 6 
 7 
 8 
 9 
 10 
 
 Extract 
 
 OF 
 
 Anti- 
 
 Bjlood- 
 
 STAIN, 
 
 1: 1000 
 Co. 
 
 serum 
 1:10, 
 C.c. 
 
 0.1 
 
 0.6 
 
 0.2 
 
 0.6 
 
 0.4 
 
 0.6 
 
 0.6 
 
 0.6 
 
 0.8 
 
 0,6 
 
 1.0 
 
 0.6 
 
 1.0 
 
 
 
 
 
 0.6 
 
 
 
 
 
 
 
 
 
 Com- 
 ple- 
 ment, 
 1:20, 
 Co. 
 
 J2 
 
 ~ 
 
 is 
 
 Anti- 
 
 COE- 
 
 PU8- 
 
 sheep e 
 Ambo- 
 ceptor,* 
 Units" 
 
 CLES 
 
 (2..5 
 
 Feb 
 
 Cent.), 
 
 Co. 
 
 1>2 
 
 
 iK 
 
 
 IH 
 
 
 i'A 
 
 
 132 
 
 
 IM 
 
 
 I'A 
 
 
 W2 
 
 
 lA 
 
 
 
 
 
 Results After One 
 
 AND One-Half Hours' 
 
 Incubation 
 
 Marked inhibition 
 
 of hemolysis 
 Marked inhibition 
 
 of hemolysis 
 Complete inliibition 
 
 of hemolysis 
 Complete inhibition 
 
 of hemolysis 
 Complete inhibition 
 
 of hemolysis 
 Complete inhibition 
 
 of hemolysis 
 Antigen control: 
 
 hemolysis 
 Antiserum control : 
 
 hemolysis 
 Hemolytic control: 
 
 hemolysis 
 Corpuscle control: 
 
 no hemolysis 
 
 2. Complement-Fixation Method for the Identification of Meats. — 
 
 The technic is essentially similar to that used in the foregoing test. 
 Antiserums are prepared by immunizing rabbits with the serums of 
 various animals, as the ox, horse, dog, cat, or any other animal the 
 presence of whose flesh is to be identified in sausages, bologna, etc. It 
 is not necessary to immunize with an extract of these meats themselves, 
 as the blood or blood-serums will suffice. The technic of immunization 
 is the same as that employed in the preparation of precipitin serums. 
 Each antiserum is titrated with its antigen^ as previously described, 
 and is used in double the titrated dose in conducting the main test. 
 
 An extract of the flesh to be examined is prepared as described on 
 p. 310. 
 
 The test is then conducted in exactly the same manner as previously 
 described. 
 
 3. Complement-Fixation Method for the Identification of Bacterial 
 Antigens. — As a means of diagnosis, this test has very limited practical 
 value. It aims to detect, by means of complement fixation with a 
 known antiserum, a soluble bacterial antigen in the blood-serum of a 
 patient. For example, in typhoid fever the 'patient's serum is mixed 
 with a potent antityphoid serum in the presence of complement. After 
 
COMPLEMENT-FIXATION TEST fN CANCER 499 
 
 incubating for an hour at 37° C, amboceptor and corpuscles are added 
 to test for free complement. An absence of hemolysis indicates that 
 complement has been fixed by the antiserum and soluble typhoid antigen 
 in the serum of the patient. As a rule, and for purposes of diagnosis, 
 this order of procedure is reversed: the antigen is furnished and then 
 sought for in the patient's serum (p. 473), as ifi the gonococcus fixation 
 test, syphilis reaction, etc. 
 
 An immune serum is prepared by immunizing rabbits with increasing 
 doses of an emulsion of the bacteria which we wish to test for in the 
 patient's serum. 
 
 The bacterial extract is prepared as follows: Make cultures of the 
 bacteria on slants of agar; wash off a sufficient number with normal 
 saline solution until 20 or 30 c.c. of a heavy emulsion are secured; add 
 0.1 per cent, of phenol, and shake mechanically with glass beads for 
 twenty-four hours; then heat to 60° C. for aa hour, and either centri- 
 fuge thoroughly or filter through a Berkefeld filter. The clear filtrate 
 should bo preserved in a tightly stoppered bottle in an ice-chest. It is 
 well to titrate this extract for its anticomplementary dose. As a rule, 
 these extracts are free from anticomplementary action until relatively 
 large doses are employed. 
 
 The antiserum is heated to 55° C. for half &n hour and titrated with 
 0.01 c.c. of the bacterial extract (0.1 c.c. of a 1: 10 dilution) in 10 doses, 
 ranging from 0.1 c.c. to 1.0 c.c. Double the dose giving complete fixa- 
 tion of complement is used in testing for the bacterial antigen in human 
 serum. 
 
 In conducting this test the patient's serum is heated to 55° C, for 
 half an hour, and decreasing doses, ranging from 0.5 c.c. to 0.01 c.c, 
 are placed in a series of test-tubes together with double the titrated dose 
 of antiserum. Complement and salt solution ske now added, and after 
 incubating for an hour at 37° C., amboceptor and corpuscles are added 
 and the tubes reincubated. The general tecFmic and controls are the 
 same as those previously described. 
 
 The test has some value in special research work, but for practical 
 use it has given way to the agglutination reactions and complement- 
 fixation tests for the detection of antibody with a known antigen. 
 
 COMPLEMENT-FIXATION TEST IN CANCER 
 None of the various complement-fixation methods that have been 
 advocated from time to time in the diagnosis of cancer have proved of 
 
500 THE TECHNIC OF COMPLEMENT-FIJ^TION REACTIONS 
 
 practical value. More recently von Dungern' has advocated a method 
 that yielded 90 per cent, of positive reactions in known cases of cancer. 
 Positive reactions have also occurred in tuberculosis and syphilis, and 
 the reports of various other investigators are somewhat contradictory. 
 Whitman, in a study of 30 cases, found the method highly satisfactory. 
 
 Preparation of Antigen. — Two different antigens may be employed. 
 Of these, von Dungen prefers the first. 
 
 First Method. — Place 10 c.c. of blood (preferably obtained from a 
 patient suffering from general paralysis) in a centrifuge tube containing 
 0.1 c.c. of a 20 per cent, solution of sodium oxalate. Wash the cells 
 three times with normal saline solution. Aftet the last washing, measure 
 the corpuscles and add 20 parts by volume of chemically pure acetone. 
 Let this stand for three days at room temperature, giving it an occasional 
 shaking. Filter and evaporate the filtrate to dryness in the incubator, 
 and take up the residue at once with enough 96 per cent, alcohol to form 
 a 1 per cent, solution. This usually requires about 10 times as much 
 alcohol as the amount of acetone used. Before using, the alcoholic 
 solution is mixed slowly with four parts of normal saline solution and 
 titrated for its anticomplementary dose. Half this amount is used in 
 making the main test. 
 
 Second Method. — The tumor tissue, freed as much as possible of fat 
 and degenerated portions, is minced and treated with 20 volumes of 
 acetone and extraction continued for one or two weeks at room tempera- 
 ture with an occasional shaking. The extract Is then filtered, evaporated 
 to dryness at 37° C, and the residue taken up with half the amount of 
 absolute alcohol. This extract is then diluted 1 : 10 or 1 : 20 with salt 
 solution, and titrated for its anticomplementary dose, one-half this 
 amount being used in making the main test. 
 
 The Test. — The serum should be fresh. Add two volumes of 55 
 caustic soda (jq soda diluted with four volumes of normal saline solu- 
 tion), and inactivate the mixture for one-half hour at 54° C. (not 56° C). 
 The soda solution must be prepared accurately from chemically pure 
 substances, must be free from carbonate, and must be protected from 
 the air. 
 
 Into a series of five test-tubes place 0.075, 0.15, 0.3, 0.6, and 0.6 c.c. 
 of the serum-soda mixture. To the first four tubes add the proper 
 amount of antigen. To all tubes add 1 c.c. of,complement diluted 1 : 20 
 and sufficient salt solution to bring the total volume up to 3 or 4 c.c. 
 
 1 Miinch. med. Wochenschr., 1912, .59, 65, 1098, 2854. 
 
COMPLEMENT-FIXATION TEST In CANCER 501 
 
 The following controls arc included : 
 
 1. The fifth tube is the serum control. 
 
 2. A known positive serum should be inclucfed and set up in the same 
 manner. 
 
 3. A known normal serum should be used. 
 
 4. The antigen control, containing at this time antigen, complement, 
 and salt solution. 
 
 5. A hemolytic control, containing at this time complement and salt 
 solution. 
 
 Each tube is gently shaken and incubated"for an hour, after which 
 13^ to 2 units of amboceptor and 1 c.c. of corpuscles are added to each 
 tube. After one to two hours' incubation, the results are read, inhibition 
 of hemolysis with satisfactory controls indicates a positive reaction. 
 With these antigens, it is not strange that positive reactions may occur 
 in syphilis. 
 
CHAPTER XXV 
 CYTOTOXINS 
 
 In Chapter XXIII the cytolysins in general were considered, 
 especially their theoretic structure and the mechanism of their action. 
 
 It will be remembered that the general name "cytolysin" is applied 
 to an amboceptor or antibody of the third order of receptors that is 
 capable of preparing its antigen for the disintegrative or lytic action of a 
 complement. The two best known and most important members of 
 this group of antibodies have been considered, namely, the hemolysins 
 and the bacteriolysins. 
 
 Following the discovery of the hemolysins and the bacteriolysins 
 and of the mechanism of their action, it was not long before similar 
 studies were undertaken with other cells, with the result that attempts 
 have been made to prepare immune cytolytic; serums for practically 
 every organ of the body. This outcome was but natural, in view of the 
 enormous theoretic importance of specific cytolysins, not only from the 
 additional light that may be thrown upon physiologic and pathologic 
 processes in general, but also from the standpoint of specific therapeutics. 
 
 Nomenclature. — While actual lysis or solution of erythrocytes and 
 bacteria may be brought about by antibodies of this order, yet actual 
 solution is not apparent with most other body-cells, although a distinct 
 toxic action may be observed. For instance, an antispermatozoa 
 serum will cause these cells to lose their motility, but does not actually 
 dissolve them. Hence the name cytotoxin has been applied to these 
 immune serums. This is probably a better term than cytolysin; but 
 it is to be remembered that, so far as is now known, both cytolysins and 
 cytotoxins are antibodies that possess the same nature and structure, 
 except that in the former group the process is complete and ends in 
 actual lysis of the cell. The term cytolysin is, therefore, more appro- 
 priately applied to the bacteriolysin and hemolysins; whereas the term 
 cytotoxin is reserved for those immune serums that injure their cells mthout 
 complete lysis (a toxic action), such as nephrotoxin, hepatotoxin, etc. 
 This chapter is mainly concerned with the latter group. 
 
 Nature and General Properties of exotoxins. — As previously 
 stated, cytotoxins are amboceptors or antibodies of the third order, and 
 
 502 
 
METHODS OF STUDYING CYT(StOXINS 503 
 
 possess the same general properties as the bacteriolysins and hemoly- 
 sins, except that, as will be pointed out later, they do not possess the 
 same specificity. Without the presence of a complement they are 
 inactive. They are thermostabile, possess the same general affinity for 
 their antigen, and may be removed from a serum by saturation with 
 the antigen in a manner similar to that used for the removal of a 
 hemolysin. 
 
 Preparation of Cytotoxins. — Cytotoxic serums arc prepared by im- 
 munizing an alien animal with fine suspensions =of cells of the particular 
 organ being studied. (See p. 73.) Every effort should be made to 
 remove all traces of blood, and to secure as pure an emulsion of the same 
 cells and to work as aseptically as possible. Injections are best given 
 intraperitoneally, rabbits being well adapted for the preparation of 
 these serums. 
 
 Beebe ' has made the statement that more specific serums are obtained 
 if the nucleoproteins are isolated and used in the process of immuniza- 
 tion, than if the cells themselves are used. Wells, ^ however, believes 
 that the nucleoproteins of cells are not specifiqiu character, and Pearce, 
 Karsner, and Eisenbrey ' found that nephrotoxic and hepatoxic serums 
 prepared by the injection of the nucleoproteins of these cells, were no 
 more toxic than serums prepared from the globulins and albumins of the 
 same organs. 
 
 Methods of Stud3ring Cytotoxins. — ^While the phenomena of hemolysis 
 and bacteriolysis may readily be observed in experiments in vitro, the 
 influence of other cytotoxins on the particular cells used as antigens is 
 more difficult to detennine. The methods of exposing cell emulsions 
 to the action of the serum have not been found satisfactory for testing 
 the specificity of cytotoxins. 
 
 The technic generally employed consists in making subcutaneous, 
 intraperitoneal, or intravenous injections of the immune serum into the 
 animal, or into the arteries leading to particular, organs. Functional dis- 
 turbances and delicate histologic changes in various organs have served as 
 criteria for determining the degree of specificity that exists. The loss of 
 some manifestation of vitality on the part of the cell, as a loss of motility 
 (spermatozoa) or an inability to proliferate, may aid in studying the 
 effect of these serums. 
 
 More recently several other methods have been used, especially the 
 
 1 Jour. Exper. Med., 1905, vii, 733. 
 
 ^ Chemical Pathology, 1914, Second Edition, W. B. Saunders Co. 
 
 ' Jour. Exper. Med., 1911, xiv, 44. 
 
504 CYTOTOXINS 
 
 complement-fixation and the epiphanin reactions and that of observing 
 the influence of cytotoxic serums upon cells grown in vitro (Lambert). 
 
 Specificity of C3rtotoxins. — As has been stated elsewhere, the hemoly- 
 sins and the bacteriolysins are highly specific, especially the former 
 group. With the cyiotoxins, however, this ^ecificity is not observed. 
 Most cjdiotoxic serums are also hemolytic, notwithstanding the fact that 
 careful precautions have been taken to remove, so far as possible, all 
 traces of blood from the inoculum during the process of immunization. 
 Metchnikoff found a spermatotoxic serum to be also hemolytic, but he 
 believed that this property could be removed by treating the immune 
 serum with the corresponding corpuscles, and in this manner dissolve 
 out the hemolysin. Numerous other investigators have found, however, 
 that cytotoxic serums may attack the cells of other organs, as well as 
 those that have been used as their antigens. 
 
 The subject has been very carefully investigated by Pearce.' The 
 injection of an antidog nephrotoxic serum prepared by immunizing 
 rabbits with washed dog kidney is followed by the development of a 
 tubular nephritis, with albuminuria and occasionally hemoglobinuria, 
 and accompanied by granular degeneration of the liver. These serums 
 are usually hemolytic in vitro. Similarly, in a study of hepatotoxic 
 serums, Pearce found that the most striking lesions were referable to 
 the hemagglutinating and hemolytic properfies of the serum, causing 
 thrombosis, embolism, and hemorrhages, whereas secondary necroses 
 may be caused by a direct toxic action of the serum on certain parenchy- 
 matous cells. 
 
 Pearce, Karsner, and Eisenbrey found that the serums of rabbits 
 injected repeatedly with the nucleoproteins, globulins, and albumins of 
 the liver and kidney of the dog, gave no evidence of organ specificity 
 in vitro or in vivo experiments. These investigators were not able to 
 support the view put forward that nucleoproteins play an important 
 part in the production of cytotoxic immune serums. 
 
 Lambert^ has recently studied the subject with cultures of rat sar- 
 coma and rat embryo skin and their immune serums, and found that 
 these cytotoxins were not specific for the tissue injected. 
 
 These results are not surprising when it is remembered that all the 
 body-cells have a common origin, and that, although the cells of various 
 organs may differ considerably in morphologic^and functional characters, 
 they have certain receptors in common, and, as Pearce originally main- 
 
 1 Jour. Exper. Med., 1914, xix, 277. 
 
 2 Univ. Penna. Med. Bull., 1903, xvi, 217; Jour. Wed. Research, 1904, xii, 1 
 
ATJTOCYTOTOXINS 505 
 
 tained, it is hardly conceivable that specific somatogenic cytotoxins 
 can be produced. 
 
 Autocytotoxins. — While these toxins possess some theoretic interest, 
 they are of very rare occurrence in experimental work. Certainly cells 
 of the kidney, liver, and other organs are constantly dying and being 
 replaced by new cells; the receptors of these cells are thereby set free, 
 and are capable of forming a union with the receptors of other cells, and 
 the possibility for the formation of autocytotoxins is established. Ac- 
 cording to Ehrlich, however, the side-arms anchoring the receptors of 
 the dead cells are sessile in nature, and are unlikely to cause overproduc- 
 tion, as in the case of antibodies for bacterial substances or for the cells 
 of other species. On the other hand, a simultaneous production of 
 antiautotoxins that counteract the autotoxins and preserve a delicate 
 physiologic equilibrium may occur, the whole, subject being, however, 
 still in the experimental stage. 
 
 Of most interest in this connection are the theoretic autonephrotoxins. 
 These may be produced when part of a kidnrey becomes disorganized 
 in the living body, as by means of a toxin. Theoretic autotoxins may 
 then be produced, which, acting upon other kidney cells, institute a 
 vicious cycle. Acting upon these assumptions, Ascoli and Figari ^ and 
 Lindeman ^ have proposed a new theory as to the pathogenesis of certain 
 of the nephritides. These observers would account for the cardiac 
 hypertrophy of nephritis by attributing it to the action of the nephro- 
 toxic serum in causing contraction of the peripheral vessels, with con- 
 sequent increase of blood-pressure; the nephritic nervous symptoms, 
 they believe, are due to the fact that the seriim contains a neurotoxic 
 constituent. 
 
 Lindeman has produced a toxic nephritis in dogs by giving them 
 injections of potassium bichromate, and found that the serum, although 
 free from the chromate, was toxic to other do^s, a finding he believed 
 due to the presence of autonephrotoxins produced in the first dog as a 
 result of the destruction of kidney cells. According to this view, the 
 original toxic cause of a degenerative nephritis would be less responsible 
 for the continuance of the process than would the formation of an 
 autonephrotoxin. While these conclusions are somewhat far-reaching, 
 they serve to indicate that the same processes operative in bacterial 
 infection and immunity may have an important relation to other patho- 
 logic conditions. 
 
 1 Berl. klin. Wochenschr., 1902, xjodx, 634. 
 ' Ann. de I'lnst. Pasteur, 1900, xiv, 49. 
 
506 
 
 CYTOTOXINS 
 
 It is not rarely observed that in large tlimors and similar lesions 
 certain groups of cells may undergo digestion, but in these the lysis is 
 commonly ascribed to the action of ferments liberated upon the death 
 of cells. 
 
 Isoc3rtotoxins have been produced experimentally, as, for example, 
 by Ehrlich, who produced isohemolysins by injecting goats with goat 
 blood, and by Metchnikoff, who prepared isuspermatoxic serums. 
 
 Anticytotoxic serums have likewise been- prepared by careful im- 
 munization with cytotoxic serums. 
 
 Varieties of Cytotoxins. — As previously stated, attempts have been 
 made to prepare cytotoxic serums for practically all the organs and tis- 
 sues. Since none of these has been found to be absolutely specific, 
 and hence since they possess little or no practical value, they will receive 
 but brief consideration here. 
 
 1. Spermatotoxin. — This serum was prepared simultaneously by 
 Metchnikoff^ and Landsteiner in 1899, and was one of the earliest 
 cytotoxins to be studied. It is a hemolytic serum, and causes sperma- 
 tozoa to lose their motility. It would seem to affect also the vitality 
 of the spermatozoa in vivo, inasmuch as De Lester, by the injection of 
 this serum, rendered male mice sterile for from sixteen to twenty days. 
 
 2. Epilheliotoxin. — A C3d;otoxic scrum for the ciliated epithelium of 
 the trachea was prepared by von Dungern.^ The cells became dis- 
 integrated in the peritoneal cavity of the immunized animal, but not in 
 that of the normal animal. This serum also proved to be hemolytic. 
 
 Similar serums have been prepared with cancer cells, in the hope 
 of establishing a specific serum therapy, but all efforts have thus far 
 proved futile. 
 
 3. Leukotoxins. — This serum was first prepared by Metchnikoff' 
 and Besredka* by injecting the spleen of rats into guinea-pigs. The 
 serums have also been prepared by effecting immunization with exudates 
 rich in Ieukocji;es or with the emulsion of lymphoid organs (Flexner 
 and Ricketts). They are usually hemolytic, and also attack endothelial 
 cells. Their action may be observed in vitro^ when the leukocytes lose 
 their ameboid motility and the protoplasm swells, clears, and may dis- 
 integrate, leaving the nucleus. 
 
 4. Nephrotoxin. — This serum is best adapted for experimental studies 
 
 1 Ann. de I'Inst. Pasteur, 1900, xiv, -369. 
 
 2 Munch, med. Wochenschr., 1899, xlvi, 1228. 
 2 Ami. de I'Inst. Pasteur, 1899, xiii, 737. 
 
 * Ann. de I'Inst. Pasteur, 1900, xiv, 402. 
 
VARIETIES OF CYTOTOXINS 507 
 
 of the cytotoxins. As shown by Pearce, with the aid of this serum 
 physiologic and anatomic alterations and lesions are readily studied. 
 The injection of a nephrotoxic serum produces: a tubular nephritis, with 
 albuminuria and possibly hemoglobinuria. The serums are usually 
 hemolytic, and frequently cause degenerative lesions in the liver, due in 
 part to hemolysis and hemagglutination of red corpuscles. 
 
 5. Hepatotoxin. — Delezenne ^ was the first tp work with the so-called 
 hepatotoxin, which, he claimed, possessed absolute organ specificity. 
 Subsequent investigations, however, have brought forth contradictory 
 findings. Pearce found that hyaline thrombi,= formed of agglutinated 
 red corpuscles, are primarily responsible for the areas of necrosis and 
 hemorrhage, with secondary effects, which maybe ascribed to a cji;otoxin 
 liberated chiefly by the cells in the thrombus,= and acting on the liver- 
 cells. These findings and views have recently received support from 
 the investigations of Karsner and Aub.^ 
 
 6. Gastrotoxin. — Gastrotoxic serums have been studied by Bolton,' 
 who immunized rabbits with emulsions of the mucosa of the stomach 
 of the guinea-pig. The injection of this serum into guinea-pigs was 
 followed by the development of areas of hemorrhage, necrosis, and ulcer 
 formation that resembled peptic ulcers. According to Bolton, if the 
 gastric secretions were neutralized with large quantities of alkali, the 
 ulcers did not develop, indicating that the peptic ferments may be oper- 
 ative in the digestion of the cells after their destruction by the immune 
 serum. Gastrotoxic serums were found to produce precipitates with 
 clear filtrates of gastric cells, and were also shown to be hemolytic. 
 
 7. Synocytotoxin. — This serum has been produced experimentally 
 by immunization with an emulsion of placental cells. According to 
 Liepmann,^ it produces a precipitate with a filtrate of placenta cells, and 
 at one time it was believed that it might constitute a diagnostic test for 
 pregnancy. 
 
 Syncytotoxins are interesting as considered in reference to eclampsia 
 and other toxemias of pregnancy. As is well known, placental cells 
 may become detached and, gaining entrance tp the circulation, become 
 lodged in remote organs (Schmorl). This has given rise to the theory 
 that a placentotoxin is developed that produces the nephritis of preg- 
 nancy and necrotic lesions in the liver. Weichardt asserts that, by 
 digesting placenta in vitro with an active placentotoxic serum and in- 
 
 ' Semaine Med., 1900, xx, 290. ' Jour. Med. Research, 1913, xxviii, 377. 
 
 ' Proc. Roy. See, Ixxvii, 426, and Ixxix, 533. 
 * Deutsch. med. Wochenschr., 1902, xxviii, 911. 
 
508 CYTOTOXINS 
 
 jecting the digestate, he produced symptoms resembhng eclampsia in the 
 lower animals. It was hoped that an anticjd;oJoxic serum might be pre- 
 pared to combat the effects of the placentotoxin, but this hope has not 
 been realized. Renewed interest in this, particular subject has 
 been manifested by the recent studies of Abderhalden in ferments as 
 applied to the diagnosis of pregnancy (p. 248). 
 
 8. Neurotoxin. — This toxin has been prepared and studied by Dele- 
 zenne, Centanni, Delille, and others, by immunization experiments with 
 emulsions of cerebrum, cerebellum, and spinal cord. When injected 
 into the brain direct, these serums may cause, profound intoxication of 
 the nerve-centers, with torpor or convulsions, subnormal temperature, 
 and death. When injected directly into the veins, they are usually 
 without effect. In addition to their neurotoxic action, they are generally 
 hemoljrtic, and frequently endotheliotoxic and leukotoxic. 
 
 9. Thyrotoxins. — This serum is prepared by immunizing animals 
 with emulsions of thyroid gland. Thyrotoxins were quite prominently 
 before the profession a few years ago, owing to the work of Beebe, who 
 advocated their use in the treatment of various goiters. They have not 
 fulfilled their expectations, however, since they may also produce de- 
 generative changes in the various organs, as the liver, spleen, and kid- 
 neys. 
 
 ROLE OF CYTOTOXINS IN IMMUNITY 
 It is apparent that, according to our present knowledge, the cyto- 
 toxins proper, although they possess great tjheoretic importance from 
 their possible relationship to the removal and disposal of enfeebled and 
 dead cells, occupy a subsidiary place in the processes of immunity. The 
 processes governing these changes are finally and delicately balanced, 
 and although obscure, they offer an intricat^' but fascinating field for 
 research. 
 
 If the ferments concerned in Abderhalden's studies are related in 
 a;ny way to the cytolysins, the subject becomes of great interest, and a 
 new field, with immense possibilities, is opened for further study. 
 
 Practical Applications. — (1) In therapeutics the cytotoxins have not 
 established a place for themselves and their use has been disappointing. 
 As previously stated, the use of thyrotoxic serums has not met with 
 considerable success; epitheliotoxic seriima have likewise not been 
 efficient in the treatment of cancer. They possess theoretic interest, 
 however, from the possibility of their so injuring glands that their func- 
 tions may be studied; theoretically, the use of minute and carefully 
 
CYTOTOXIC REACTIONS 509 
 
 graded doses of hemolytic serums may effect the production of anti- 
 hemolysins and aid in the treatment of certain anemias. 
 
 (2) In diagnosis cj^totoxic reactions have been employed by Freund 
 and Kaminer. These observers used the cytotoxins as a diagnostic 
 aid in cancer, but with indifferent success. Abderhalden's pregnancy 
 test possesses some practical value, and a similar technic has been suc- 
 cessfully employed in the diagnosis of cancer. The work of Abderhalden 
 has served to open up a comparatively new and intensely interesting 
 field, which, when fully developed, as the result of overcoming difficult 
 technical procedures, may offer additional mfeans in the diagnosis of 
 various diseases. This test has been described elsewhere under the head 
 of Ferments (p. 252). 
 
 CYTOTOXIC REACTIONS 
 
 Cytolytic Cancer Diagnosis of Freund and Kaminer.' — This reaction 
 is based upon the observation that while normal serum has the power 
 to dissolve cancer cells, the serum of cancerous persons lacks this prop- 
 erty, and has the power to inhibit the destruction of such cells by normal 
 serum. 
 
 The same authors have also observed that when cancer serum is 
 mixed with an extract of cancer cells a precipitate forms. (See p. 314.) 
 They claim to have secured 88 per cent, of positive reactions in 113 
 cases examined, and believe that the reaction occurs early enough and 
 is sufficiently specific to render it of practical value. These observa- 
 tions, however, have not been sufficiently confirmed. 
 
 All emulsion of cancer cells is prepared by grinding the undegenerated 
 portions of a tumor, freed as much as possible from fat and fibrous 
 tissue, in a mortar and adding about five volumes of 1 per cent, sodium 
 biphosphate. The suspension is filtered through several layers of gauze, 
 and after the cells have become precipitated, the supernatant fluid is 
 decanted. The residue of cells is washed with 0.6 per cent, sodium 
 chlorid and allowed to settle again, the supernatant fluid is decanted, 
 and the residue covered with 1 per cent, sodium fluorid. The last- 
 named fluid must first be neutralized against alizarin until only a trace 
 of the violet color remains. The emulsion will keep for several weeks 
 in an ice-chest. 
 
 Serum. — The patient's serum should be collected just a few hours 
 
 (not over twenty-four) before the test is to be made, and must be clear 
 
 and free from cellular elements. 
 
 ,„ ' Biochem. Zeitschr., 1910, 26, 312; Wien. klin. Wochenschr., 1910, 23, 378, and 
 1221; iUd., 1911, 24, 1759. 
 
510 CYTOTOXINS 
 
 The Test. — To 10 drops of the patient's serum add one drop of 0.5 
 per cent, solution of sodium fluorid. Then add one drop of the cancer- 
 cell emulsion so diluted that when one drop of the mixture is placed in a 
 blood-counting chamber, about 10 to 20 cancer cells will be found in a 
 field of four large squares. Close the counting chamber carefully, ring 
 with vasclin to prevent evaporation, and place in the incubator for 
 twenty-four hours. 
 
 A second slide is prepared from a mixture composed of one volume 
 each of normal serum, cancer serum, and 0.6 per cent, sodium chlorid 
 and sufSoient cell emulsion. 
 
 A third slide is prepared with a fresh normal serum in the same 
 manner as when the patient's serum is used. 
 
 All slides are incubated for twenty-four hours at 37° C. and the cells 
 counted. A material reduction in the number of cells with the normal 
 serum will be noted; if the patient has carcinoma, the first and second 
 slides will not show this reduction, whereas if the patient is free from 
 cancer, similar reductions will be found in all three slides. 
 
 The authors recommend that both the cytolytic and the precipitin 
 test be conducted when enough serum is available for both. 
 
CHAPTER XXVI 
 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 While at the present time Ehrlich's side-chain theory best explains 
 the specificity and mode of action of various antibodies, there is a grow- 
 ing tendency to explain many of these reactions on a physicochemical 
 and colloidal basis. 
 
 From the fact that, without exception, antigens are colloids, and that 
 antibodies also are colloid in their chemical characters, it is advisable 
 to review briefly some of the main facts and theories concerning these 
 bodies and their reactions. 
 
 Varieties of Colloids. — Colloids may be composed of two different 
 classes of substances: 
 
 1. Organic substances, as, e. g., all forms of proteins and also gums, 
 starch, glycogen, tannin, chondrin, the greater, number of organic dyes, 
 and probably the enzymes. 
 
 2. Inorganic substances, as, for example, the inorganic colloids, such 
 as silicic acid, ferric hydroxid, arsenic sulphidy and many other similar 
 compounds. 
 
 Since the living tissues and fluids are, without exception, colloids and 
 colloidal solutions, the -properties of the cells arb largely the properties of 
 colloids. 
 
 Nature and Properties of Colloids. — Since Graham, in 1861, studied 
 the differences between the substances that did or did not diffuse readily 
 through animal or parchment membranes, soluble substances have been 
 classified in two main groups : (a) Colloids, or those substances that were 
 dissolved to the extent of showing no visible particles in suspension, but 
 that did not pass through diffusion membranes at all, or did so very 
 slowly indeed, and (&) crystalloids, or solutions that diffuse through 
 membranes quite readily. 
 
 On the other hand, we may have substances that are quite insoluble 
 when aggregated in masses, but when derived in pure form by me- 
 chanical means, can be suspended and uniformly distributed through a 
 fluid without showing any marked tendency to precipitate. Such 
 ^spensions or emulsions contain particles that are visible under the 
 microscope; they usually appear turbid, do not transmit electricity, and 
 
 511 
 
512 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 are not diffusible. Colloids occupy a place between the true solutions of 
 crystalloids and the emulsions. Sharp boundaries cannot usually be 
 drawn between any of the members of the series. They differ quanti- 
 tatively in some manner from the true solutions and the emulsions, but 
 may approach them closely, and sometimes resemble them so strongly 
 as to be almost indistinguishable from them. For the most part, 
 however, they show decided characteristics that will differentiate them 
 from the crystalloids, on the one hand, and the suspensions, on the other. 
 
 Those colloids that closely resemble the^ true solution have been 
 designated "colloidal solutions," and those resembling more closely the 
 suspensions, "colloidal suspensions." Of the two types, the colloidal 
 solutions are far more important biologically, since the colloidal suspen- 
 sions are usually prepared artificially and seldom occur in nature. 
 
 Colloids, therefore, appear to be suspensions of masses of molecules, 
 or perhaps of very large single molecules. When these aggregations 
 are sufficiently large, we have an ordinary suspension. 
 
 1. Colloids are usually amorphous in character, and with few excep- 
 tions do not present a typical structure; they are not crystalline under 
 any visible condition. This, however, is not invariably the case, for 
 we may have a protein, like hemoglobin, which resembles a typical col- 
 loid in every respect, and may yet form crystals readily and abundantly. 
 
 2. Colloids do not form true solutions, but the solvent is probably an 
 important factor in determining whether or not a substance is colloidal 
 in nature; e. g., soaps form true solutions in alcohol and colloidal solu- 
 tions in water; rubber forms colloidal solutions in ether, but not in 
 water. The term colloidal solution does not,= therefore, refer to a true 
 solution in the sense of a crystalloid, but to a colloidal state of suspen- 
 sion (the so-called colloidal solution). 
 
 3. Colloids are non-diffusihle, or lack the power of passing through 
 animal and parchment membranes. Not all colloids possess the same 
 rate of diffusion, this property being relative, rather than absolute; 
 however, solutions of salts (crystalloids) pass through so readily that 
 they are easily separated from proteins (colloids) by dialyzation, a 
 process that is in constant practical use. 
 
 4. Colloids have an extremely small osomotic pressure. They may, 
 to a very slight degree, exert some influence upon osmotic pressure, the 
 freezing and boiling-points of fluids, but in all cellular processes in which 
 manifestations of osmotic pressure or diffusion are present the crystal- 
 loids may be considered as almost entirely responsible for these. 
 
 5. The colloids exhibit surface tension to a high degree — in other words, 
 
NATURE AND PROPERTIES OP CX)LLOIDS 513 
 
 colloid fluids possess the force that strives to reduce its free surface to a 
 minimum. As partial expressions of this force, the formation of emul- 
 sions when oil and water are mixed and the ameboid movements of the 
 ameba and leukocytes may be mentioned as examples. 
 
 6. Colloids do not separate freely into ions whten dissolved, and accord- 
 ingly do not conduct electricity to an appreciable extent. When an electric 
 current is passed through a colloidal fluid, most of the colloids move 
 toward the anode; this phenomenon, known as cataphoresis, is also 
 generally exhibited by suspensions, and in this particular the colloids 
 resemble suspensions. 
 
 7. Colloids are usually easily precipitahle and coagulable, and this is 
 readily understood when the slender margin that exists between many of 
 the colloids and the suspensions is borne in mind. Relatively slight 
 changes, such as exposure, gentle heat, the presence of large quantities 
 of crystalloids, the action of enzymes, etc., may throw an organic colloid 
 out of solution, and when once precipitated, it is often incapable of again 
 dissolving in the same solvent. Colloids are also precipitated by many 
 electrolytes, apparently through the formation of true ion compounds. 
 
 8. The physical structure of colloids. This subject has been studied 
 extensively by Hardy. ^ Cells contain but one type of colloids, the 
 proteins that form non-reversible coagula. Sp long as a colloid is in 
 solution it is structureless ; but such solutions may become solid as the 
 result of changes of temperature and other physical means and from 
 admixture with certain chemical fixing agents. The structure of the 
 coagula varies according to the concentration, of the colloidal solution 
 and the nature of the coagulant, but in general the figures obtained in 
 the solidification of protein solutions by such fixing agents as mercury 
 bichlorid and formalin bear a striking resemblance to the finer structure 
 of protoplasm as described by cytologists. These facts, no doubt, have 
 an important bearing upon the various "foam," "reticular," and 
 "pseudo-alveolar" structures of the protoplasm of cells described by 
 Biitschli, Fromann, Arnold, Reinke, and others, and may indicate the 
 effect of fixatives upon colloid solutions, explaining the usual time-worn 
 objections to theories of protoplasmic structure as based upon arti- 
 ficial conditions not present in the normal living cell, and variously- 
 interpreted according to the fixative employed. 
 
 9. Colloids may he precipitated by electrolytes, of opposite sign, as well 
 
 ' A good general outline of the subject of colloids may be found in Pauli's " Physi- 
 cal Chemistry in the Service of Medicine," 1907, translated by Fischer (Chapnaan 
 and Hall). 
 
 33 
 
514 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 as by colloids. In a colloidal solution surface tension constantly tends 
 to make the particles of colloid approach one another, so that the surface 
 may become as small as possible, and in this manner brings about pre- 
 cipitation or coagulation. 
 
 In a stable solution this action is counterbalanced by a force of 
 electric repulsion. Pure colloids do not carry an electric charge and 
 are not conveyed by an electric current; their apparent charge depends 
 upon the nature of electrolytes that may be present. Traces of acid 
 and of acid salts give it a positive charge, whereas alkalis and alkaline 
 salts do the opposite (Pauli). 
 
 The process of coagulation of proteins, therefore, must depend upon 
 the neutralization of their electric charge, anci, as previously stated, this 
 can be accomplished either by electrolytes or by colloids: 
 
 (a) Precipitation by electrolytes is best illustrated by the action 
 of a strong acid on an albuminous solution. The negatively charged 
 particles attract to themselves the positively charged hydrogen ions; 
 their charge is now neutralized, and the force of attraction due to their 
 surface tension is no longer counterbalanced' by an electric repulsion. 
 The particles are drawn together, form largei; and larger masses, which 
 finally come under the influence of gravit;^ and precipitation takes 
 place. 
 
 (b) Precipitation of colloids by colloids is illustrated by the precipi- 
 tation of albumin by acetic and ferrocyanic acjds. The colloid must be 
 of opposite sign. As a result of the acid the |)articles acquire a positive 
 charge, if they are not so charged already. This charge is then neutral- 
 ized by the colloidal ferrocyanic acid of negative sign; the surface ten- 
 sion is no longer neutralized by an electric repulsion, and particles come 
 together to form larger masses that are finally deposited as a precipitate. 
 
 Instead of precipitating the other, an excess of one colloid may act 
 in a reverse manner. For example, as Neisser and Friedmann have 
 shown, a suspension of particles of mastic in water (made by dropping 
 an alcoholic solution in water) takes on a ne,gative charge, and can be 
 precipitated by positive colloids or ions, such as ferric chlorid. If the 
 dose of ferric chlorid is increased gradually, the precipitate becomes 
 more and more abundant, until an excess of ferric chlorid is present, 
 when the reaction ceases and the precipitate may be redissolved. This 
 has been explained on the assumption that when two colloids of opposite 
 sign are mixed, they tend to fuse and form masses; the addition of an 
 excess of either colloid tends to electrify the masses, causing mutual 
 repulsion and possibly resolution of the masses. Hence the precipitate is 
 
ANALOGY BETWEEN KEACTIONS 515 
 
 soluble in an excess of both substances, just as a precipitate is soluble 
 in an excess either of precipitin or of its antigen. 
 
 10. Absorption is the taking up of dissolved or volatile substances 
 by finely divided or colloidal bodies. It is a combination between two 
 substances dependent on physical attraction rather than on chemical 
 affinity, and taking place in variable ratios, rather than in simple and 
 constant ones, as occur in a true chemical union-. It is believed by many 
 that the two substances entering into the phenomenon of absorption 
 exist as such side by side in the compound, which is to be regarded 
 as an intimate admixture of the two, rather than as a new compound. 
 
 The lack of definite ratios by which colloids are absorbed has been 
 shown by Bordet in the amount of hemolytic itnmune body that can be 
 taken up by a given volume of corpuscles-^', e., the amount varies 
 according to whether the corpuscles are added at once or in successive 
 small portions. Thus, in one example, 0.4 c.c. of a hemolytic serum 
 dissolved 0.5 c.c. of corpuscles if added at once; but if 0.2 c.c. of cor- 
 puscles was added first and successive amounts of 0.1 c.c, then put in, 
 no solution took place after the one that followed the addition of the first 
 portion. This was explained by Bordet according to the principles of 
 absorption, this observer comparing it with the absorption of a dye by 
 filter-paper. While other explanations are possible, yet exactly analo- 
 gous phenomena may be seen in the mutual absorjjtion of colloids of 
 opposite sign. Thus, as we have previously stated, the addition of 
 a solution of an electropositive colloid to a soluJtion of an electronegative 
 one tends to repel the particles, with the formation of masses for the 
 puipose of self-jjrotection, and in this manner the process of agglutina- 
 tion and precipitation is begun. But if a small, amount of a second col- 
 loid is added to the same volume of the others, hew aggregates of the two 
 arc formed that are less favorable to precipitation and require more of the 
 second colloid to bring about complete precipitation. 
 
 ANALOGY BETWEEN THE REACTIONS OF IMMUNITY AND COLLOIDAL 
 
 CHEMISTRY 
 
 With these few brief remarks on the properties and nature of colloids 
 
 and the close resemblance of cellular protoplasm and fluids to colloids, 
 
 We may consider briefly the apparent similarity that exists between the 
 
 colloidal reactions and some of the reactions of immunity. This is 
 
 especially pertinent for several reasons: it has. been shown that cellular 
 
 protoplasm is colloidal in nature; that antigens are certainly colloidal, 
 
 and that antibodies, while they may or may not be solutions of colloids. 
 
516 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 are, in the final analysis, products of cellulUr activity, and therefore 
 derived from colloidal solutions. 
 
 1. Antitoxins. — The side-chain theory of Ehrlich was first applied 
 in explanation of the principles of inununity as affording an explanation 
 of the action of toxins, the formation of antitoxin, and the interaction 
 between these. Ehrlich has placed the various phenomena of immunity 
 upon a chemical basis, bringing forward new theories to explain the 
 variovis discrepancies that were found. For ^ample, it was soon found 
 that both toxin and antitoxin were unstable, and that neutralization of 
 a toxin by the addition of antitoxin was not=a simple process, like the 
 neutralization of an acid by an alkali, but, on the contrary, was likely to 
 be exceedingly complicated. This was explained as being due to the 
 degeneration of toxin into various toxoids, which were able to neutralize 
 antitoxin without being in themselves toxic wjien in a free state. They 
 were likewise found to have a greater affinity for antitoxin than the toxin 
 itself, so that when a toxin was tested and its toxicity determined, it was 
 discovered that more antitoxin wa,s needed to neutralize the mixture than 
 was originally calculated, because the toxoids took no part in testing the 
 toxin, but were active in uniting with antitoxin, and in this manner 
 leaving true toxin unneutralized, and therefore. toxic, unless an excess of 
 antitoxin was used. 
 
 Analogous conditions may be observed among colloidal solutions. 
 Thus, Danysz has shown that more toxin is neutralized if antitoxin is 
 added at once than when it is added in successive doses. As stated 
 elsewhere, this is explained by Ehrlich upon the assumption that time 
 is allowed for the degeneration of toxin into toxoids to take place, the 
 latter having a greater affinity for the antitoxin. It has been shown, 
 however, that in some cases the addition of a small amount of a second 
 colloid of opposite sign to a colloidal solution may render the solution 
 more stable and protect it from precipitation by an excess of the second 
 substance. Similarly, the amount of colloid necessary to precipitate 
 a constant amount of another colloid is reduced to a minimum if the 
 addition is made at once, and is rendered much greater if the colloid 
 added is made slowly in small amounts, an interval being allowed to 
 elapse after each addition. This is closely analogous to the Danysz 
 reaction, and explains the latter as being du&, when antitoxin is added 
 slowly to toxin, to the formation of transitronal compounds of toxin 
 and antitoxin of diverse nature, requiring more antitoxin for complete 
 neutralization of the toxin than if the antitoxin were added at once and 
 in one dose. 
 
ANALOGY BETWEEN REACTIONS 517 
 
 The difference between the Lq and L+ dosa of a toxin also has an an- 
 alogy in the reaction of simple colloidal substances. Thus, Bilty has used 
 ferric hydroxid, which neutralizes arsenic trioxid (the antidote for acute 
 arsenical poisoning), and found that the addition of one lethal dose of 
 arsenic to a neutral mixture of the two did not render the mixture toxic, 
 but that several lethal doses were required, just' as it is necessary to add 
 several instead of one lethal dose of diphtheria toxin to the Lq dose. 
 
 Thus it would appear that the neutralization of a toxin by an anto- 
 toxin has analogies among the known and simple colloidal reactions. 
 One objection to placing the toxin-antitoxin reaction upon a colloidal 
 basis is that both have the same electric chargg, i. e., both move tov/ard 
 the cathode, and, as we have seen, for the neutralization and precipita- 
 tion of colloids the solution of colloids should be of opposite sign. It 
 must be remembered, however, that toxins and antitoxins react in very 
 complex fluids containing other substances consisting of both colloids 
 and electrolytes, and until the electric charge of these in pure form is 
 determined, the apparent similar electric charge of toxin and antitoxin 
 can hardly outweigh the otherwise remarkable analogy it bears to col- 
 loidal reactions. 
 
 2. Agglutinins and Precipitins. — Various theories in explanation of 
 the phenomenon of agglutination have been described in a previous 
 chapter. The theory of Bordet appears to be best, and is based upon 
 certain principles of colloidal chemistry. When bacteria are suspended 
 in a fluid free from salt, agglutination does not take place because the 
 bacteria carry a similar negative charge of electricity. When, however, 
 ions of positive charge are added, as, e. g., sodiujn chlorid, the bacteria or 
 other cells are repelled and coalesce to form masses, according to the 
 laws of surface tension, in an effort to protect themselves. Larger 
 masses may be formed that finally come withiri the influence of gravity 
 and are deposited at the bottom of the test-"tube. According to the 
 same laws, the addition of agglutinin removes the negative charge of 
 bacteria or other cells, with the consequent formation of clumps and 
 masses. Similar phenomena may be observed in the precipitation of 
 colloidal suspensions of clay in distilled water by the addition of a salt. 
 
 Solutions of inorganic colloids, as, for example, that of silicic acid, 
 may agglutinate red corpuscles; bacteria, such as suspensions of typhoid 
 and colon bacilli, may be agglutinated by solutions of the ferric salts. 
 
 Just as an excess of one colloid solution will charge masses of the 
 other, resulting in a repelling action and breakirtg up of the agglutinated 
 clumps, so the addition of an excess of agglutinin is found to prevent 
 
518 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 agglutination or to give but a slight reaction. This phenomenon has 
 been explained, according to Ehrlich's side-chain theory, as due to the 
 presence of agglutinoids that have a great affinity for the bacteria and 
 unite with them without being active in the free state, owing to a loss 
 of the agglutinophore portion of the molecule. Each cell united with 
 an agglutinoid is one cell less to undergo agglutination by agglutinin, 
 and accordingly in weak dilutions of serum agglutination is feeble or 
 absent whereas in higher dilutions the phenomenon may be clearly 
 observed. 
 
 Other explanations of the action of agglutinins and precipitins, 
 based upon colloidal reactions, have been advanced. Thus Neisser and 
 Friedmann have shown that suspensions of mastic may be "protected" 
 against the precipitating action of ferric hydroxid by the addition of a 
 small amoimt of organic colloid, such as serum, leech extract, or extract 
 of t3^hoid bacilli, regardless of whether this" colloid is charged posi- 
 tively or negatively or is neutral. The aforenamed observers believe 
 that normal bacteria may be surrounded by a similar protective envelop 
 that prevents the agglutinating action of substances of opposite sign. 
 The action of agglutinin, therefore, would be to remove this layer, so 
 that the ions of opposite electric charge can unite with the bacteria and 
 bring about their agglutination. This may be an explanation of the 
 role of salts in the phenomenon of agglutination, the agglutinins remov- 
 ing the protecting envelops and the salt furnishing the ions of opposite 
 charge that bring about agglutination. 
 
 Owing to the fact that a discrepancy arises here for the reason that 
 emulsions of red corpuscles are agglutinated by both positive and 
 negative colloids (ferric hydroxid and cuprum ferrocyanid), Girard, 
 Mangin and Henri have given the following explanation of agglutina- 
 tion: When a red corpuscle is suspended in a fluid, various salts, es- 
 pecially the sulphates of magnesium and calcium, are diffused, which 
 tends to facilitate the precipitation of negative and positive colloids, 
 so that each corpuscle comes to be surrounded by a layer of precipitated 
 colloid material. This zone of precipitated colloids of either negative 
 or positive charge determines agglutination in the presence of a colloid 
 solution of opposite charge, such as agglutinin or inorganic colloids 
 (silicic acid, etc.). 
 
 3. Hemolysins. — Reference has been made elsewhere to the original 
 observations of Bordet, showing that red corpuscles may absorb much 
 more hemolyliic antibody than is necessary to bring about their lysis, 
 and that this absorption is analogous to colMdal absorption. 
 
COMPLEMENT-FIXATION TEST AS A COLLOIDAL REACTION 519 
 
 Inorganic colloidal solutions, such as that of silicic acid, may produce 
 hemolysis of red blood-corpuscles, e. g., those 5f the rabbit. Its action 
 is manifested in extremely small doses. It is rendered inert by heat, 
 and gradually deteriorates at room temperature. Furthermore, this 
 inorganic colloid possesses some of the properties of a serum hemolysin; 
 thus mice red corpuscles that have been agglutinated by colloidal silicic 
 acid are dissolved by traces of lecithin or of fresEi serum, but not by serum 
 that has been heated to 60° C. (inactivated). An excess of silicic acid 
 tends to prevent hemolysis, which is another example of the action of an 
 excess of one colloidal solution upon another of opposite sign. 
 
 Probably saponin hemolysis and the influence of fatty substances, 
 such as lecithin and the fatty acids, upon the phenomenon of hemolysis, 
 are closely related to, or to be explained by, the action of organic colloidal 
 solutions. 
 
 THE COMPLEMENT-FIXATION TEST AS A COLLOIDAL REACTION 
 
 Many observations support the view that complement fixation by a 
 specific antigen and its antibody is really complement absorption by a 
 precipitate that forms when antigen and atitibody are mixed. As 
 previously stated, all antigens are protein in character. While there is 
 some evidence to show that lipoids, and even carbohydrates, may act as 
 antigens, there is no doubt but that the chief antigenic principles of any 
 antigen are of protein structure; hence when mixed with an immune 
 serum containing specific antibodies, it is believed that an invisible 
 precipitate is formed that absorbs the complement. With serum anti- 
 gens the quantity of protein is so large that a jM-ecipitate can readily be 
 seen (the precipitin test). A serum antigert and its antibody may, 
 however, be so highly diluted that, when mixed, a precipitate is not 
 visible, although complement may be fixed (complement-fixation test 
 for the differentiation of proteins). Moreschi and Gay have contended 
 for many years that complements may become entangled and absorbed 
 in such precipitates. Reasoning on the basis of the colloidal theory, 
 it is possible that transition compounds of very diverse nature are 
 formed when antigen, antibody, and a complement are mixed. This 
 view on the action of complements and anti-complements is supported 
 by numerous investigators who have examined the question from the 
 standpoint of colloidal reactions. Thus in a complement-fixation test a 
 mixture of antigen, antibody, and a complement in definite proportions 
 results in the formation of new compounds oft opposite electric charge, 
 
520 THE RELATTON OP COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 which tend to aggregate in masses (although- these may be so small as 
 to be invisible) and reduce their surface tension in just the same manner 
 as agglutination and precipitation are brought about after the colloidal 
 theories. When corpuscles and hemolytic antibody are subsequently 
 added, hemolysis does not occur because free complement is absent. 
 
 A process similar to complement absorption by a specific antigen 
 and its antibody is the Wassermann reaction. According to the col- 
 loidal theories, this reaction may be explained as due to the formation 
 of an invisible precipitate by interaction between some substance in 
 the serum of a luetic person (probably in the nature of an altered globu- 
 lin), complement, and lipoidal substances contained in an alcoholic or 
 watery extract of a normal or a diseased orgaH. This view is supported 
 by the fact that euglobulin is known to be generally increased in the 
 body fluids of sjT)hilitics, and by analogy with the various precipitin tests 
 that have been devised for the diagnosis of syphilis, as, for example, the 
 reaction of Porges and Meier, which is dependent upon the appearance 
 of a precipitate when luetic serum is mixed with an emulsion of lecithin 
 or sodium glycocholate, etc. The exact nature of the antibody in 
 sj^hilitic serums that forms these new conjipounds with lipoids and 
 complement, resulting probably in the absorption of complement, is 
 unknown. It is most likely in the nature of a globulin, its main char- 
 acteristic being the power it possesses of reacting with lipoids. Schmidt ' 
 ascribes the reaction to the physicochemical properties of the globulins 
 of the syphilitic serum, which he believes possess a greater aflfinity for 
 the colloids of the antigen than do normal globulins. This view is 
 supported by the common observation that the turbidity of the antigen 
 emulsion is closely related to its efficiency, since clear solutions are less 
 active. Since various lipoidal substances may be employed, the Was- 
 sermann reaction can not be regarded as spfecific in the immunologic 
 sense, although practically it is highly specific, as similar conditions are 
 to be found in only two other diseases with any degree of regularity, 
 namely, frambesia and tuberous leprosy. 
 
 THE RELATION OF LIPOIDS TO IMMUNITY 
 It is becoming more and more evident that lipoids bear an important 
 
 relation to various immunologic processes, especially to certain cytolytic 
 
 phenomena. 
 
 As stated in Chapter VIII, the results of some researches that go to 
 
 1 Zeit. f. Hygiene, 1911, 69, 513. 
 
THE RELATION OF LIPOIDS TO IMMUNITY 521 
 
 show certain lipoids and lipoidal substances may act as true antigens 
 and produce antibodies.^ This, however, has not been definitely proved, 
 and while it is of great importance, is not necessarily pertinent to the 
 subject in hand. As the relation of lipoids to various immunologic 
 processes has frequently been described in earlier chapters, as, e. g., 
 where the role of lipoids in venom hemolysis, in the Waesermann syphilis 
 reaction, and in the various precipitin reactions in syphilis were con- 
 sidered, a brief resume may be of service in directing attention to this 
 important and particular phase of immunity. 
 
 Relation of Lipoids to Hemolysis. — (a) From the standpoint of 
 immunity, venom hemolysis is of peculiar interest as indicating the pos- 
 sible important relation of lipoids to hemolytic complement. Granting 
 that venom contains a hemolytic amboceptoi* (Flexner and Noguchi), 
 the complementing substance nmst be derived from the corpuscles, and, 
 according to Kyes, this complementary agent is represented in lecithin. 
 Kyes was able to produce what he considers are compounds of the hemo- 
 lysin with lecithin, namely, "lecithids. " Wllether these "lecithids" 
 are true compounds of hemolysins and corpuscular lecithin or simply the 
 active hemolytic products of the cleavage of lecithin by ferments con- 
 tained in the venom, is at present unknown. Noguchi and Lieberman 
 have shown that not only lecithin, but soap as well, especially unsatu- 
 rated fatty acids, and probably protein compounds of soaps and lecithin, 
 may act as the hemolytic complement and activate the hemolysin of the 
 venom. Lipoids from bacteria and trypanosomes have been found to 
 possess similar properties. Hemolytic lipoids have been secured from 
 serum, and the complementary activity of a fresh normal serum may be 
 destroyed by fat solvents, e. g., ether. While other investigators have 
 not been able to confirm Noguchi's attempts to produce an artificial 
 complement of fatty substances of exactly the same properties as serum 
 complement, this work indicates most strongly the close relation of 
 serum complement to lipoids. 
 
 (6) The hemotoxic activity of various toxins is probably dependent 
 largely upon their action on the lipoids of red corpuscles. The saponin 
 substances,^ a group closely related to glucosids, and found in at least 
 46 different families of plants, are strongly hemolytic. Ransom^ has 
 
 ' Bibliography on Lipoids and Immunity given by Landsteiner, Kolle and 
 Wassermann's Handbuch, 1913, 2, 1240. Also review of literature by Landsteiner, 
 Jabresb. Immunitat, 19 10, 6, 209. 
 
 '^ Complete literature on saponin, see Kobert "Die Saponinsubstanzen," Stutt- 
 gart, 1904. 
 
 ' Deut. med. Wooheneohr., 1901, 27, 194. 
 
522 THE REIATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 found that an ethereal extract of red corpuscles contains a substance 
 that inhibits saponin hemolysis. This substance consists largely of 
 cholesterin, and it is the presence of cholesterin in normal serum that 
 inhibits saponin hemolysis. This may be demonstrated experimentally 
 by adding cholesterin to a solution of a saponin. Noguchi ^ has shown 
 that lecithin does not possess the same antihemolytic action on saponin. 
 It would appear, therefore, that saponin causes hemolysis by combining 
 with, altering, or dissolving the lipoids of the stroma of corpuscles. The 
 resistance of corpuscles to saponin hemolysis varies in certain diseases, 
 being especially low in jaundice (McNeil)^. 
 
 While saponins, solanins, phallin, and other vegetable poisons are of 
 relatively simple chemical composition and quite unlike proteins, 
 enzymes, or toxins, it is possible that bacterial and vegetable hemo- 
 toxins, such as tetanolysin, abrin, ricin, crotin, and robin, may produce 
 their effects by a similar action on the lipoids of the erythrocytes. 
 Noguchi has shown that cholesterin inhibits the action of tetanolysin. 
 Landsteiner and Bottori have found that protagon, a brain lipoid, 
 possesses the property of binding tetanus toxin, which indicates that 
 this toxin may produce its effects by some action upon the lipoids of 
 nerve-cells. 
 
 (c) The important relatioyi of lipoids to the Wassermann reaction and 
 certain precipitin or floccule-f arming reactions, (Klausner, Porges-Meier, 
 Hermann-Perutz) has been mentioned repeatedly. Just what role the 
 lipoids play in these phenomena is not known. While the globulins of 
 syphilitic serums are strongly suspected of being concerned in these 
 processes, their relation is not clear. Klausiier' now believes that the 
 precipitate that forms when distilled water is; added to syphilitic serum 
 is due to the high lipoid content. 
 
 The Epiphaotn Reaction 
 Principle. — This reaction is based upon the observation made by 
 Weichardt* in 1908; he found that diffusion is accelerated when dif- 
 ferently colored solutions of antigen and its specific antibody are brought 
 together. Changes in diffusion are associated with changes in the 
 surface tension, both of which depend on a change in the osmotic pres- 
 sure. This is the principle made use of by Ascoli in his miostagmin 
 reaction, which will be described further on. 
 
 1 Univ. of Penna. Med, Bull., 1902, 15, 327. 
 
 2 Jour. Path, and Bact., 1910, 15, 56. ' Biochem. Zeit., 1912, 47, 36. 
 ' Berl. klin. Wochensclir., 1908, No. 20; Centralbl. f. Bakteriol., xliii, 143; ibid., 
 
 .xlvii, 39; Zeitschr. f. Immunitiitsforsch., 1910, vi, 651j Deutsch. med. Wochensohr., 
 1911, No. 4, 154. 
 
THE KELATION OF LIPOIDS TO IMMUNITY 523 
 
 Later Weichardt made the reaction more accessible to practical use 
 by introducing into the solution of serums and aiitigen a system composed 
 of sulphuric acid and barium hydroxid, together with certain catalytic 
 agents. Using phenolphthalein as an indicator, he could show that 
 fresh serums in high dilutions alter the surface tension of the finely 
 divided barium sulphate particles by their colloidal action, so as to in- 
 crease the absorption of H-ions, thus rendering the solution more alkaUne. 
 
 This phenomenon has been utilized by Weichardt, under the name 
 of "epiphanin reaction," to determine the occurrence of such interac- 
 tion of antigen and antibody. The reaction probably depends upon 
 physicochemical principles of absorption, but the exact nature of the 
 change is not yet understood. The reaction is based upon the following 
 generalizations ; 
 
 1. Solutions containing colloids — i. e., antigen alone, antiserum alone, 
 ■ or antigen plus non-specific antiserum in certain dilutions — act in the 
 foregoing system by shifting the phenolphthalein end-point (the point of 
 neutralization when acid and alkali are brought together in the presence 
 of this indicator) in the sense of increased OH-ions (pink color) . 
 
 2. Specific antigens can inhibit the activity of their specific antise- 
 rums, the specific antigen-antibody combination then becoming evident 
 in vitro by a shift of the end-point in the sense of increased H-ion con- 
 centration (light color). 
 
 Specificity. — The specificity of the reaction has been confirmed by a 
 number of investigators who used the test for the identification of a 
 host of antigen-antibody combinations in vitro. The underlying prin- 
 ciples have been, confirmed by Kraus and Amiradzibi,^ Schroen,^ 
 Seifert,^ Mosbacher,* and others. The reaction has been applied to a 
 study of various antigens and their antibodies, such as diphtheria toxin, 
 -tetanus toxin, typhoid and tubercle bacilli, tumor extracts and placenta 
 extracts by Weichardt; extracts of syphilitic livers and serums of 
 syphilitic patients by Seifert, Keidel and Hurwitz.' 
 
 Technic. — The technic of this reaction haa been modified from time 
 to time. The method here given is essentially the latest given by 
 Weichardt,^ slightly modified by Keidel and Hurwitz. 
 
 ' Zeitschr. f. Immunitatsforach., 1910, vi, 16. 
 ' Miinch. med. Wochcnschr., 1910, 38, 1981. 
 ' Deutsch. med. Wochenschr., 1910, 50, 233,3. 
 ' Deutsch. med. Wochenschr., 1911, 22, 1021. 
 'Jour. Amer. Med. Assoc, 1912, lix, 12.57. 
 « Berl. klin. Wochenschr., 1911, 13, 1935. 
 
624 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 Five constituents enter into the test: 
 
 1. The Antigen. — This is an alcoholic extract of syphilitic liver, 
 prepared in exactly the same manner as for performing the Wassermann 
 reaction. High dilutions of the antigen, rangmg from 1 : 100 to 1 : 10,000, 
 are prepared with normal salt solution. As in the Wassermann re- 
 action, not every antigen is satisfactory, a point that can be determined 
 only by making preliminary tests. 
 
 2. The patient's serum should be fresh, unheated, and highly diluted, 
 the dilutions ranging from 1 : 100 to 1 : 10,000,000. Usually it is better to 
 use higher than lower dilutions. When too -concentrated solutions of 
 serums and of antigen are used, erroneous results are likely to be ob- 
 tained. 
 
 3. A normal solution of sulphuric acid. 
 
 4. A saturated solution of barium hydroxid made equivalent to the 
 normal solution of sulphuric acid. In the use of the barium hydroxid 
 it is imperative to prevent its exposure to the air. A solution that has 
 become cloudy, owing to the entrance of carbon dioxid, should not be 
 used. In carrying out the test it is best to pour out the amount of barium 
 hydroxid needed for the test into a rubber-stoppered bottle or test-tube, 
 so as not to contaminate the stock solution. 
 
 5. A 1 per cent, alcoholic solution of phenolphthalein containing 1 
 per cent, of a 10 per cent, solution of strontium chlorid. The strontium 
 chlorid has been found to catalyze the reaction. 
 
 The Test. — The test is conducted as follows: A number of clean 
 beakers of about 50 c.c. capacity are used. For each dilution of the 
 serum a separate beaker is required. One be5,ker is used for an antigen 
 control, and another to control the system, of barium hydroxid and 
 sulphuric acid. Five beakers may be used, Nos. 1, 2, and 3 constituting 
 the main test. No. 4 the antigen control, and No. 5 the system control. 
 
 The reagents are added by means of overflow pipets. To each of the 
 first four beakers is added 1 c.c. of the dilute antigen to be used in the 
 test (about 1 : 10,000). To beaker 5 is added 1 c.c. of the salt solution 
 used in making the dilutions of the antigen and the serums. Now 0.1 
 c.c. of the dilute serum to be tested is added^ to each of the first three 
 beakers, each beaker, however, containing the same serum in a different 
 dilution. To beaker 5 the same quantity of salt solution is added, but 
 to beaker 4 — the antigen control — no serum or salt solution is added. 
 
 To each of the five beakers the system of sulphuric acid and barium 
 hydroxid and phenolphthalein is now added carefully. First, 2 c.c. of 
 the normal suphuric acid solution are added to each; then 2 c.c. of the 
 
THE RELATION OF LIPOIDS TO IMMUNITY 525 
 
 barium hydroxid, and finally 0.1 c.c. of the phenolphthalein strontium 
 chlorid mixture. 
 
 It will be seen that beaker 4 — the antigen control — contains all the 
 constituents of the test-beakers 1 to 3 except serum. To make beaker 
 4 qualitatively as well as quantitatively equal to beakers 1 to 3, 0.1 c.c. 
 of the dilute serum (the average of the dilutions of serum which are used 
 in the test) is now added to beaker 4, the reaction having already taken 
 place. 
 
 The addition of the sulphuric acid and barium hydroxid requires 
 great care. Since the reaction depends on small differences in acidity 
 or alkalinity, it is obvious that slight errors will vitiate the results. For 
 the acid and the alkah separate pipets are used. After emptying the 
 pipet of its content of acid or alkali, the last traces adhering to the 
 inside of the pipet are removed by washing. These washings are later 
 added to the beakers to which they belong. In filling the pipets with 
 acid or alkali, the latter should first be drawn up into the pipet at least 
 once, and then emptied again before the pipet is finally filled for delivery 
 into the next test-beaker. Only by careful attention to these points 
 in the technic can reliable results be obtained. 
 
 Reading the Results. — If beakers 1 to 3 contained the antiserum to the 
 antigen used, a positive epiphanin reaction will- be obtained, and if the 
 barium hydroxid and sulphuric acid were previously carefully adjusted 
 to each other, it will be found that beakers 1 to- 3 will be lighter than the 
 antigen control, beaker 4. The presence of a specific antigen-antibody 
 combination has shifted the phenolphthalein end-point in the sense of 
 increased H-ion concentration. The exact differences in the alkalinity 
 between beakers 1 to 3 and beaker 4 can be quantitatively determined 
 by titration -with jqq suphuric acid, and the results expressed as a curve. 
 
 If the antigen and antibody were not specific, the epiphanin reac- 
 tion will be negative. Beakers 1 to 3 will be more alkaline than the 
 antigen control, beaker 4, because, as previously pointed out, serums 
 alone or antigen with non-specific serums shift the phenolphthalein 
 end-point in the sense of increased OH-ion concentration. 
 
 The results may be plotted as curves. The titration values in j^ 
 sulphuric acid are placed on the ordinates, and the serum dilutions on 
 the abscissae. The positive values are plotte4 above the line and the 
 negative values below the line. No reaction is regarded as positive 
 unless it gives a titration value of at least 0.05 c.c. ^ sulphuric acid. 
 Values below 0.05 c.c. are easily within the limits of error. 
 
526 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 The following method, employed by Seifert, is much simpler, but is 
 open to the error on account of using the antigen and serum in too con- 
 centrated a state. In a small test-tube place'0.1 c.c. of a 1 : 10 solution 
 of the serum in normal salt solution, and add 0.1 c.c. of an alcoholic 
 extract of syphilitic liver. To this slowly add 1 c.c. of decinormal 
 sulphuric acid and 1 c.c. of a solution of barium hydroxid of the exact 
 concentration needed to neutralize the sulphuric acid solution. On the 
 addition of the drop of the phenolphthalein solution the fluid turns red 
 when the serum is from a syphilitic, where^ no change in tint occurs 
 with non-syphilitic serum. 
 
 Practical Value. — The reaction appears to be of considerable value 
 in the diagnosis of syphilis. With serums atTd antigen in proper dilu- 
 tions, the results closely parallel those secured by the Wassermann reac- 
 tion. Keidel and Hurwitz report positive reactions with luetic serums 
 in about 75 per cent of their cases. The ifeaction was found highly 
 specific in that syphilitic extracts gave negative reactions with serums 
 of non-syphilitic persons and patients suffering from malignant disease. 
 Extracts of normal fetal liver and beef heart gave negative reactions 
 with serums of syphilitic persons. 
 
 Positive reactions have also been found in malignant disease, as with 
 the antigens of carcinoma and sarcoma. Keidel and Hurwitz obtained 
 16 positive reactions in a series of 24 serums of persons suffering with 
 definite or suspected malignant disease. Burmeister did not find the 
 reaction of value in cancer. 
 
 The epiphanin reaction has also been use'd in the diagnosis of preg- 
 nancy, but sufficient work has not been done. to render an expression as 
 to its merits of value at this time. 
 
 The Miostagmin Reaction 
 
 Among the very large number of immunity reactions employed in 
 attempts to secure a diagnostic test for cancer, the "miostagmin reac- 
 tion" of Ascoli and Izar' is the only one thus far devised that claims 
 the serious attention of the clinician. 
 
 Principles. — This reaction is founded on the fact, noted by Ascoli, 
 that by the mixing of an antigen and its corresponding antibody there 
 results a reduction of the surface tension of the liquid containing these, 
 which may be demonstrated by counting the number of drops of the 
 fluid in a given volume (usually 1 c.c), under constant conditions. 
 Normal serum diluted with salt solution is first tested, and the number of 
 
 1 Munch, med. Woohenschr., 1910, 57, 62, 182, and 403. 
 
THE RELATION OF LIPOIDS TO IMMUNITY 527 
 
 drops found in a cubic centimeter determined with a specially devised 
 instrument known as Traube's stalagmometer. The antigen is so diluted 
 that when mixed with this normal serum it does not increase the number 
 of drops more than one in a cubic centimeter. When properly diluted 
 patient's serum and antigen are mixed, it may be found that the number 
 of drops is increased from 2 to 8 in a cubic centimeter. This constitutes a 
 positive reaction. The reaction is apparently due to the lowering of sur- 
 face tension, so that more and smaller drops are found; hence the term 
 'Imiostagmin" has been applied to the test, the word being devised from 
 the Greek, meanmg "small drop." 
 
 The reaction is said to be sharply specific and very delicate, so that 
 antigens diluted up to 1 : 100,000,000 or higher may be detected. The 
 technic requires considerable practice and experience or erroneous 
 results are quite likely to occur. 
 
 The exact nature of the reaction is not known. The antigens are 
 soluble in alcohol, but their nature is obscure. The antibody involved 
 in the reaction is referred to as the miostagmin, but its relation to other 
 antibodies is also unknown. It is probably a physicochemical or col- 
 loidal reaction, and for this reason it has been placed in this chapter. 
 
 Technic. — The antigen is most difficult to prepare. A recent method 
 described by Ascoli is as follows: 
 
 1. Cut non-degenerated portions of malignant tumor (cancer or 
 sarcoma) into small pieces and dry in vacuo or spread out in a thin 
 layer on clean glass plates and keep at a temperature of 37° C. 
 
 2. Pulverize the dried substance and extract with pure methyl 
 alcohol (in the proportion of 5 gm. to 25 c.c.) for twenty-four hours at 
 50° C. in closed vessels, and shake occasionallJ^ 
 
 3. Filter while still hot, and allow the filtrate to cool, and then filter 
 again through Schleicher and Schull's filter-paper No. 590. 
 
 4. It is now necessary to titrate the antigen and to determine in what 
 dilution it should be employed. Various dilutions of the antigen are 
 made with distilled water, as, e. g., 1 : 10, 1 : 25, 1 : 50, 1 : 100, 1 : 150, 
 1 : 200, etc. A fresh normal serum is diluted 1 ; 20 with normal salt 
 solution, and 9 c.c. of this are mixed with 1 c.c. of the various antigen 
 dilution. Into another tube place 9 c.c. of the-diluted serum and 1 c.c. 
 of distilled water. All test-tubes, pipets, and other glassware used must 
 be perfectly dry. 
 
 The tubes are gently shaken and placed in an incubator at 37° C. 
 for two hours. The drop number for each fluid is then estimated by 
 Traube's stalagmometer. This instrument is merely a finely and 
 
528 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 elaborately graduated pipet with a central bulbous reservoir. The 
 dropping end of the instrument ends in a flattened ground base, thus 
 insuring uniformity in the size of the drops. The instrument is so 
 graduated that a fraction of a drop can be estimated. That antigen is 
 to be chosen that does not alter the drop number fur normal serum by more 
 than one drop in a cubic centimeter — the strongest dilution that fulfils this 
 condition being chosen. 
 
 The Test. — The patient's serum is diluted 1 : 20 with noi-mal salt 
 solution and its drop number determined. Then take two tubes, and 
 into one place 9 c.c. of diluted serum plus 1 c.c. of antigen dilution; 
 into the other place 9 c.c. of diluted serum plus 1 c.c. of distilled water. 
 A third tube may be prepared, which should" contam 9 c.c. of normal 
 serum (1 : 20) plus 1 c.c. of the same antigen = dilution. A fourth tube 
 contains 9 c.c. of a known positive serum (1 : 20) from a case of cancer 
 and 1 c.c. of the antigen dilution. 
 
 All tubes should be carefully labeled, their drop numbers determined, 
 and then placed in an incubator at 37° C. for two hours or in the water- 
 bath at 50° C. for one hour. At the end of this time they are removed, 
 allowed to cool, and the drop number of eachsis determined. 
 
 The controls are first examined to show that the antigen has not 
 undergone any change. Variations of the number above one and a half 
 or two drops {as compared with the control containing distilled water in- 
 stead of antigen) are regarded as positive reactions. The increase in drops 
 is seldom greater than eight. 
 
 TABLE 21.— MIOSTAGMIN REACTION IN CANCER 
 
 9 C.c. Serum (1:20) Plus 1 C.c. 
 Antigen (1:200) 
 
 Dhops Per C.c. 
 Aptbr Incuba- 
 tion AT 37° C. 
 
 FOR Two Hours 
 
 Drops Per C.c. 
 
 Controls 9 C.c. 
 
 Serum (1:20) Plus 
 
 l*C.c. Distilled 
 
 Water Afper 
 
 Incubation 
 
 Results 
 
 Normal serum 
 
 Known cancer serum 
 
 Unknown serum for diagnosis 
 
 Unknown serum for diagnosis 
 
 Unknown scrum for diagnosis 
 
 56.0 
 62.4 
 61.2 
 57.2 
 62.4 
 
 56.4 
 59.2 
 60.0 
 57.0 
 59.8 
 
 Negative 
 
 Positive 
 
 Positive 
 
 Negative 
 
 Positive 
 
 Other Methods for Preparing Antigens. — Various methods for pre- 
 paring antigen and conducting the test are to be found in the literature, 
 and it is extremely difficult to arrive at a correct conclusion as to which 
 is the best method for preparing antigen. Among these methods for 
 
THE RELATION OF LIPOIDS TO' IMMUNITY 529 
 
 the preparation of antigen other than those previously described are 
 the following: 
 
 1. After securing the alcoholic extract described elsewhere evaporate 
 it to dryness. Again extract in methyl alcohol and evaporate. Extract 
 with warm ether, renewed several times during the course of twenty- 
 four hours. Dry, and repeat the extraction several times until the 
 alcohol remains colorless. Evaporate the alcoholic and ethereal ex- 
 tracts at 50° and 37° C. respectively. A yellowish-red, sticky mass 
 results. Dissolve this in a large amount of water-free ether. Filter, 
 and evaporate at room temperature until a slight powdery precipitate 
 is deposited. This solution constitutes the stock antigen, which, how- 
 ever, may require still further concentration. 
 
 2. A synthetic cancer antigen may be prepared by grinding up 0.5 
 gram of lecithin (ovolecithin Merck, or lecithin Richter), and extract it 
 with 50 c.c. of acetone for twenty-four hours at 50° C. Filter through 
 Schcicher and Schull's filter-paper No. 590, until clear. Just before 
 it is to be used it should be diluted with water ia-such amount that 1 c.c. 
 will contain the largest amount that does not cause a marked reduction 
 of surface tension in normal serum. As a rule, this dilution is between 
 1 : 50 and 1 : 100 (Kohler and Luger)i. 
 
 3. A syphilis antigen may be prepared by extracting 0.5 gram of 
 dried and powdered syphilitic liver with 50 c.c. of absolute alcohol for 
 two hours at 37° C. with frequent shaking. Filter, and concentrate to 
 10 c.c. 
 
 4. A bacterial antigen, as e. g., one of typhoid bacilli, may be prepared 
 as follows: Wash off five forty-eight-hour agar cultures of typhoid bacilli 
 with 5 c.c. of normal salt solution for each tube. Cover the emulsion 
 with toluol, and shake vigorously for several hours. Place in an in- 
 cubator at 37° C. for forty-eight hours, and filter through a sterile 
 Berkefeld filter. This filtrate may be used as antigen, or it may be 
 used in preparing an alcoholic extract in the following manner ; To the 
 original aqueous filtrate add 50 c.c. of absolute alcohol. Allow the 
 mixture to stand for one-half hour, shake, cehtrifugate, and then mix 
 the sediment with 20 c.c. of absolute alcohol. Shake thoroughly once 
 more, and again centrifugate. Combine the two extracts, and con- 
 centrate on the water-bath to about 20 c.c. 
 
 Practical Value. — This test is quite delicate", and errors due to faulty 
 
 technic are quite likely to creep in. Unless all precautions are rigidly 
 
 observed, the results are worthless. Although an extensive literature 
 
 1 Wien. klin. Woohenschr., 1912, 25, 1114. 
 34 
 
530 THE RELATION OF COLLOIDS AND LIPOIDS TO IMMUNITY 
 
 has accumulated bearing evidence as to the value of the test as a diag- 
 nostic procedure, the method has not, however, come into general use. 
 
 Ascoli and Izar especially have advocated the test in the diagnosis of 
 cancer. In 100 cases of malignant tumors, they obtained 93 positive 
 reactions; in 103 cases of other diseases they obtained only one positive 
 reaction. Tedesko, Stabilini, Leitch, Kelling, and others have reported 
 favorably upon the practical value of the test in the diagnosis of cancer. 
 Burmeister^ has found that a negative reaction has some value in 
 excluding cancer, and is of more value in arriving at a diagnosis than a 
 positive reaction, i. e., it has a higher negative than a positive value. 
 
 The test has also been used in the diagnosis of typhoid fever, para- 
 typhoid fever, syphilis, tuberculosis (positive only in active cases) 
 echinococcus disease, etc. Obviously, other naethods of diagnosis, such 
 as the agglutination reaction and the Wassermann reaction, have super- 
 seded this test in practical diagnosis. The method possesses, however, 
 considerable theoretic interest and is worthy of further investigation. 
 
 1 Jour. Infect. Dis., 1913, 12, 459. 
 
CHAPTER XXVII 
 ANAPHYLAXIS 
 
 It is generally believed that when an animal previously injected with 
 an antigenic substance is subsequently reinjected with the same sub- 
 stance, the antibodies induced by the first injection are reenforced, and 
 that a continuation of the process of immunization will eventually lead to 
 a high degree of immunity. Under certain circumstances, however, this 
 is not the case, because severe and even fatal symptoms, as well as other 
 manifestations, may set in after the second ijijection, indicating that, 
 instead of being immune, the animal is indeed hj'persusceptible or 
 hypersensitive to the affects of the antigenic substance. 
 
 Similarly, it is a common observation that whereas certain infections, 
 such as smallpox, scarlet fever, and measles, confer a state of immunity, 
 others, as, for example, pneumonia, erysipelas,,: and influenza, not only 
 are not followed by immunity, but, indeed, that a decreased resistance 
 or predisposition to subsequent infection by the same microorganism 
 may be induced. 
 
 Before experimental investigation of this subject was undertaken, 
 not a few observations were made and described by the early workers 
 in the fields of bacteriology and immunity that- correspond exactly with 
 the phenomenon of hyper sensitiveness, as we understand it today, 
 although the true explanation of their unexpected results was not sus- 
 pected, and they were modestly ascribed to faulty technic, embolism, 
 toxicity of the inoculum, etc. From this it followed that the discovery 
 that the experimental injection of such ordin^iy innocuous substances 
 as normal serum and milk may produce violent symptoms and death 
 gave rise to much surprise and incredulity, since scientists had long been 
 accustomed to regard the reaction of an animal to an injection as a 
 process of immunization, or diminished sensitiveness, instead of one of 
 increased sensitiveness. Here, as Besredka remarked, the rules of 
 immunity are " standing on their heads." 
 
 To this state of hypersensitiveness Richet, one of the earliest in- 
 vestigators in this field, applied the term '^anaphylaxis," meaning 
 'without protection." While it appears to be the exact antithesis of 
 "immunity," which means "with resistance to infection," recent re- 
 
 531 
 
532 ANAPHYLAXIS 
 
 searches would tend to indicate that the two subjects are indeed closely- 
 related. An enormous amount of work has already been done on ana- 
 phylaxis, the subject being of the greatest importance, not only on ac- 
 count of its practical bearing on serum therapy, but because of its inti- 
 mate relationship to the subjects of infection and immunity, and the 
 new light that these studies may throw upon the nature and mechanism 
 of those processes. Although the phenomena of anaphylaxis are now 
 known to be due to the proteins, and while the symptoms and lesions 
 of the condition are fairly well understood, the" exact nature and mecha- 
 nism involved in the process are not established, and the entire subject 
 is fraught with so much interest as regards infection and immunity that 
 it affords a fruitful field for further research. 
 
 In this chapter will be presented the known facts regarding anaphy- 
 laxis and the theories that have been advanced in explanation of its 
 nature and mechanism, the consideration of anaphylaxis in its practical 
 application to medicine being left for the following chapter. 
 
 Historic. — The first observation of anaphylaxis as it occurs in an 
 infectious disease was probably made by Jenngr in 1798. This investi- 
 gator observed the sudden appearance of an "efflorescence of a palish 
 red color" about the parts where variolous mastter had been injected into 
 a woman who had had cowpox thirty-one years before. 
 
 In 1839 Magendi found that rabbits that had been injected with egg- 
 albumin died after a repetition of the injection, a phenomenon strikingly 
 similar to that observed sixty-five years later by Theobald Smith 
 following injections of horse serum. This phenomenon was subsequently 
 studied thoroughly by Rosenau and Anderson and Otto. 
 
 While the effects of diphtheria and tetanus antitoxins were being 
 studied, peculiar and apparently paradoxic results were occasionally 
 observed during immunization of animals with the bacterial toxins. 
 Thus in 1895 Brieger' reported the case of a goat that was highly im- 
 munized against tetanus and yet was subject to tetanus. In 1901 von 
 Behring and Kitashima^ reported similar findings with diphtheria in 
 horse immunized against that infection. At this time it was shown that 
 the results could not be due to the cumulative effect of the toxin, and 
 the explanation offered aimed to show that the process was purely his- 
 togenetic, and based upon the assumption that receptors attached to the 
 body-cells had a closer affinity for toxin than the free (antitoxin) re- 
 ceptors in the blood-stream. At the present- time toxin hypersuscep- 
 tibility is held by some to be a true anaphylactic reaction brought about 
 
 1 Zeitschr. f. Hyg., 1895, 101. ' Berl. klin. Wochenschr., 1901, xxxviii, 157. 
 
VON pirquet's early studies 533 
 
 by protein substances in the toxin filtrate; others regard von Behring's 
 explanation as satisfactory. Further reference to this subject will be 
 made later on in this chapter. 
 
 Richet's Studies. — The fundamental observations upon which our 
 present knowledge of anaphylaxis is based were made in 1898 by 
 Hericourt and Richet.^ These observers found that repeated injections 
 of eel serum into dogs gave rise to an increased susceptibility to this 
 substance, instead of immunizing the dogs against the serum. These 
 studies were continued by Richet^ and his assistants with extracts of the 
 tentacles of certain sea anemones. These studies showed that a second 
 injection of the poison into dogs, given after an interval of several days, 
 is followed by greater and more intense activity than marked the first 
 injection. If the animal survives, however, the disease is conquered 
 more readily after the second than after the" first injection. As pre- 
 viously stated, Richet coined the word "anaphylaxis," meaning "with- 
 out protection," and indicating that the first injection destroyed any 
 natural resistance that the animal might possess against the poison 
 (actionocongestin) . From these studies he concluded that two dif- 
 ferent substances are contained in eel serum and in the tentacles of 
 actiniens, one concerned in establishing an immunity, and the other in 
 calling forth a hypersensitiveness; thus far^ however, the separate 
 existence of these two hypothetic substances has not been proved. 
 
 Arthus Phenomenon. — In 1903 Arthus,^ atlhe instigation of Richet, 
 showed that similar results may be obtained with non-toxic substances, 
 like serum and milk. On injecting rabbits at definite intervals with 
 normal horse serum, he found that the first two or three doses were 
 absorbed, whereas subsequent injections, given subcutaneously, led to 
 increasingly severe local reactions (Arthus phenbmenon) . If the animals, 
 however, were first injected subcutaneously and later intravenously, 
 or intraperitoneally, serious symptoms of dyspnea, convulsions, and 
 diarrhea, and even death resulted. 
 
 Von Pirquet's Early Studies. — For a long time urticarial eruptions 
 were occasionally observed to follow transfusion of blood from lambs 
 and other lower animals to persons suffering from anemia and similar 
 conditions. Soon after diphtheria antitoxin was discovered the medical 
 
 ' Compt. rend. Soc. de Biol., 1898, 53. 
 
 " Compt. rend. Soc. de Biol., 1902, liv, 170; 1903, Iv, 246; 1904, Ivi, 302; 1905, 
 Ivui, 112; 1907, Ixii, 358, 643; 1909, Ixvi, 763; WOS", Ixvi, 810; 1909, Ixvi, 1005. 
 Am. de I'lnst. Pasteur, 1907, xxi, 497, 1908, xxii, 465. 
 
 ' Compt. rend. Soc. de Biol. 1903, Iv, 817; 1906, Ix, 1143. Archiv. intemat. de 
 Physiol., 1908-09, vii, 472. 
 
534 ANAPHYLAXIS 
 
 profession was shocked to learn of the sudden death of the healthy child 
 of an eminent German professor following a prophylactic injection of the 
 serum. In 1902 von Pirquet began the study of these clinical mani- 
 festations with a child in Escherich's clinic who, after receiving a second 
 dose of horse serum ten days after the first, on the same day developed 
 symptoms of fever and a rash. On the basis of this observation von 
 Pirquet ^ reached the conclusion that the prevailing views regarding the 
 length of the incubation period of an infectious disease could not be 
 correct. He therefore propounded the theory that the organism con- 
 cerned in the etiology of disease calls forth symptoms only when it has 
 been altered by antibodies, the period of incubation representing the 
 interval necessary for the formation of these antibodies. 
 
 In conjunction with Schick, von Pirquet endeavored to study all 
 infectious diseases from the same point of view, especially smallpox, 
 measles, recurrent fever, streptococcus infections, and the reactions to 
 eowpox virus, tuberculin, and mallein. Later these same observers^ 
 studied the symptoms following injection and reinjection of horse serum, 
 designating the train of symptoms "serum sickness." They empha- 
 sized that a single injection of serum may suffice to bring about the 
 symptoms, and that this immediate reactivity possesses diagnostic 
 value in so far as it enables us to decide whether a previous infection 
 has occurred. How near the astute Jenner came to reaching the same 
 conclusion is shown in the following abstract from his report in 1798: 
 
 " It is remarkable that variolous matter, when the system is disposed 
 to reject it, should excite inflammation on the part to which it is applied 
 more speedily than when it produces the smallpox. Indeed, it becomes 
 almost a criterion by which we can determine whether the infection imll be 
 received or not (italics ours). It seems as if a change, which endures 
 through life, had been produced in the action, or disposition to action, in 
 the vessels of the skin; and it is remarkable, too, that whether this 
 change has been effected by the smallpox or the eowpox, that the dispo- 
 sition to sudden cuticular inflammation is the same on the application 
 of variolous matter." 
 
 Von Pirquet at this time proposed the term "allergy," from ergeia, 
 reactivity, and alios, altered, meaning altered energy or a changed re- 
 activity, as a clinical conception expressing" a truth without binding 
 
 ' Zur Theorie der Infectionskrankheiten (Vorlaufige Mitteilung), Aprfl 2, 1903. 
 "Zur Theorie der Vaksination, " Verhandl. d. Gesellsch. f. Kinderh., Kassel, 1903. 
 
 2 Wien. klin. Wochenschr., 1903, xvi, 758, 1244; 1905, xviii, 531. Die Serum- 
 krankheit, Leipsic, Deuticke, 1905; Munch, med. Wochenschr., 1906, liii, 66. For 
 a full bibliography on this subject of allergy up to 1910 see von JPirquet, Aichiv. Int. 
 Med., 1911, vii, 259 and 383. 
 
DEFINITION 535 
 
 any one to a theory based upon bacteriologic^ pathologic, or biologic 
 findings. 
 
 Theobald Smith Phenomenon. — While von Pirquet was making 
 these studies, great impetus was given the experimental study of ana- 
 phylaxis by the observation of Theobald Smith, who found that guinea- 
 pigs that were used for standardizing the strength of diphtheria anti- 
 toxin after a second injection of serum frequently presented symptoms 
 of a serious character, such as great restlessness, dyspnea, itching of the 
 skin, and violent convulsive seizures. In fully 50 per cent of the animals 
 death occurred within half an hour. 
 
 Simultaneously Rosenau and Anderson^ in this country and Otto^ 
 in Germany undertook the study of this phenomenon. The first-named 
 investigators showed most conclusively, by a thorough series of experi- 
 ments, the action of horse serum and other saibstances in guinea-pigs, 
 and proved that serum sickness was due to some constituent of the 
 serum independent of the antitoxic antibodies, as normal horse serum 
 yielded exactly similar results. 
 
 Among the earlier studies of anaphylaxis of importance were those 
 of Weichardt.^ These were made with extracts of placental cells, and 
 later with the proteins of pollen, in relation to bay-fever. Wolff-Eisner ^ 
 wrote a treatise that had as its fundamental idea the belief that hyper- 
 sensibility was due to endotoxins liberated by a lysin formed as a result 
 of the first injection. Also among the earliest and most valuable studies 
 upon the nature of anaphylaxis, and showing the important relation of 
 proteins to the process, are those of Vaughan^ and his coworkers; indeed 
 the studies of Smith, Rosenau and Anderson^ Vaughan and Wheeler, 
 Gay and Southard, Auer and Lewis, and others have gained for America 
 a prominent part in the development of this important subject. 
 
 Definition. — By anaphylaxis, in the limited meaning of the term, as, 
 e. g., in that following the injection of horse serum in man or following 
 the experimental administration of practically any protein in the lower 
 animals, is understood the following train of phenomena. When a 
 foreign protein is introduced into the animal body, usually parenterally, 
 
 1 Bull. 29, Hyg. Lab., U. S. P. H. and M. H. S., 1906; Bull. 36, Hyg. Lab., April, 
 1907; Jour. Infect. Dis., 1907, iv, 552; Bull. 45, Hyg. Lab., June, 1908; Bull. .'')(», 
 Hyg. Lab., 1909; Jour. Amer. Med. Assoc, 1906, xlvii, 1007; Archiv. Int. Med., 
 1909, ill, 519. 
 
 "von Leuthold Gedenksfihrift, 1905, i; Mijnch. raed. Wocheuschr., 1907, liv, 
 1665; KoUe and Wa,ssermann, 190S, ii, 255. 
 
 'Berl. klin. Wochenschr., 1903, No. 1. 
 
 'Zeitschr. f. Bakterio!., 1904; Berl. klin. Woohenschr., 1904, xli, 1105, 1131, 
 1156, 1273; Milnch. med. Woohen.sohr., 1906, liii, 217. 
 
 ' Summarized in " Protein Split Products, " Lea and Febiger, 1913. 
 
536 ANAPHYLAXIS 
 
 after a time a specific hypersensitiveness of the animal for this pro- 
 tein will appear. After a definite interval, a second injection of the 
 same substance, harmless in itself, may produce an itching rash and 
 fever or violent symptoms of illness, and rapid death may even occur 
 in an animal so inoculated. In other words, the first injection of the 
 protein (serum, milk, egg-albumen, etc.) produces no symptoms, but 
 serves to alter the jjower of reaction on the part of the body cells by rendering 
 them unusually sensitive or susceptible to thei, same or to closely related 
 foreign protein. Therefore, as defined by Rosenau, anaphylaxis may be 
 considered as "a condition of unusual or exaggerated susceptibility of the 
 organism to foreign proteins." 
 
 Terminology. — As previously stated, the word "anaphylaxis "(ana, 
 against, and phylax, guard, or phylaxis, protection) was given to the 
 condition by Richet, because he considered it one "without protection," 
 or a state just the opposite to immunity, or prophylaxis. In the sense 
 in which the phenomenon is now regarded the word is a misnomer, for 
 we look upon the condition of hypersusceptibility as a step toward the 
 attainment of a state of immunity, and as a distinct benefit and ad- 
 vantage to the organism. The term "allergy," introduced by von 
 Pirquet, is more appropriate, as it expresses the condition of the body- 
 cells, i.' e., their hypersensitiveness or altered reactivity, regardless of 
 any theories we may entertain as to the manner in which this change is 
 brought about or manifested. The word anaphylaxis has, however, come 
 into general use, and with this explanation, we may continue to so use it. 
 
 The term a.naphylactogen is applied to thp protein, as serum, milk, 
 egg-albumen, etc., which sensitizes the body-cells; sentizer is also a good 
 word, and the process of rendering body-cells hypersensitive by ad- 
 ministering a foreign protein has been called sensitization. In von 
 Pirquet's nomenclature the protein would be called an allergen. 
 
 The term anaphylatoxin is applied to the toxic substance believed to 
 be formed at the time of reinjection of the protein, and is regarded as 
 responsible for the lesions and symptoms of ^-naphylaxis. In the belief 
 that anaphylaxis resembles an intoxication, the second inoculation of 
 protein is frequently spoken of as the intoxicating dose. 
 
 PHENOMENA OF ANAPHYLAXIS 
 
 The essential symptoms and lesions of anaphylaxis vary in the dif- 
 ferent animals, and, indeed, they have been found to vary among animals 
 of the same species under different experimental conditions. 
 
PHENOMENA OF ANAPHYLAXIS 537 
 
 Man. — When it is remembered that anaphylaxis and immunity are 
 closely interwoven, and that anaphylaxis may be but a step toward 
 securing prophylaxis and immunity, it will readily be understood that 
 under the varying conditions of different injections the phenomena may 
 be quite dissimilar. One of the best known examples of a general 
 anaphylactic phenomenon in man is that following the injection of a 
 foreign serum, as, e. g., horse serum (diphtheria antitoxin), which is 
 characterized by an itching urticarial eruption, fever, and joint pains, 
 and which is commonly known as "serum sickness." Fortunately, the 
 severer and fatal forms of anaphylaxis in man' are extremely rare, most 
 cases having occurred in persons known to be hypersensitive to horse 
 protein or in those suffering from the condition known as status lym- 
 phaticus. Famihar local anaphylactic reactions are the tuberculin, 
 inallein, and luetin reactions. With this brief statement we shall pass 
 to a consideration of anaphylaxis in the lower ^.nimals, experimentation 
 having given us some insight into the mechanism of the process. Ana- 
 phylaxis in man and the relation it bears to immunity and disease, will 
 be discussed again in Chapter XXVIII. 
 
 Guinea-pig. — This animal gives the most constant and the most 
 intense symptoms. According to Doerrr, guinea-pigs are four hundred 
 times as sensitive an anaphylactic reagent as the rabbit. 
 
 Horse serum, when injected into normal guinea-pigs, gives rise to 
 no symptoms. As much as 20 c.c. may be injected into the peritoneal 
 cavity, and small amounts may even be injected into the brain without 
 causing any untoward symptoms. 
 
 When a small dose of serum is injected intravenously, intraperito- 
 neally, or subcutaneously, and ten clays later a second injection is made, 
 the animal develops symptoms of acute anaphylactic asphyxia, which, 
 in the majority of instances, terminates fatally. " In five or ten minutes 
 after injection the pig becomes restless and then manifests indications 
 of respiratory embarrassment by scratching at the mouth, coughing, 
 and sometimes of spasmodic, rapid, or irregular breathing; the pig 
 becomes agitated, and there is a discharge of urine and feces. This 
 stage of exhilaration is soon followed by one of paresis or complete 
 paralysis, with arrest of breathing. The pig is unable to stand, or if it 
 attempts to move, falls upon its side; when taken up it is limp; spas- 
 modic, jerky and convulsive movements now supervene. This chain of 
 symptoms is very characteristic, although they do not always follow in 
 the order given. Pigs in the state of complete paralysis may fully 
 recover, but usually convulsions appear, and are almost invariably a 
 
538 ANAPHYLAXIS 
 
 forerunner of death. Symptoms appear about ten minutes after the 
 injection has been given; occasionally in pigs not very susceptible they 
 are delayed thirty to forty-five minutes. Animals developing late 
 symptoms are not very susceptible and da not die. Death usually 
 occurs within an hour, and frequently in less than thirty minutes. If 
 the second injection be made directly into the brain or circulation, the 
 symptoms are manifested with explosive violence, the animal frequently 
 dying within two or three minutes" (Rosenau). 
 
 H. Pfeiffer has shown that a depression o£ the temperature is a con- 
 stant finding in the severer forms of anaphylaxis in the guinea-pig. In 
 fatal cases this decrease may be as much as from 7° to 13° C. Some 
 relation exists between the extent and the duration of the fall of temper- 
 ature and the severity of the symptoms. During acute anaphylaxis the 
 blood shows a leukopenia, — a diminution in complement, — and, as shown 
 by Friedberger,! a delay in or a loss of coagulajbility. The most striking 
 change observed after death is permanent distention of the lungs, resem- 
 bling emphysema, described by Gay and Southard, and particularly by 
 Auer and Lewis. ^ The lungs do not collapse, but remain fully distended, 
 forming a cast of the pleural cavities. The alveoli are distended, and 
 in some instances the walls may be ruptured. The walls of the second- 
 ary and tertiary bronchi are contracted, with infoldings of the normally 
 thick mucosa, due to contraction of the smooth muscle by peripheral 
 action, death really resulting from inspiratory immobilization of the 
 lungs. The heart continues to beat long after respiration has ceased. 
 Rosenau* and Gay and Southard^ have also described minute hemor- 
 rhages in various organs and mucous membranes. 
 
 The amount of serum necessary to sensitize a guinea-pig is sur- 
 prisingly small. Rosenau and Anderson found one guinea-pig that was 
 sensitized by 0.000,001 c.c. As a rule they used less than 0.004 c.c. in 
 their experiments. Besredka places the minimum amount necessary 
 to secure uniform results at 0.001 c.c, whereas 0.0001 c.c. proved suf- 
 ficient in a considerable percentage of animals. The sensitizing dose of 
 horse serum ordinarily employed in experiments upon guinea-pigs is 
 0.01 c.c. ; amounts ranging from 0.001 c.c. to 1 c.c. are ordinarily followed 
 by an incubation period of from ten to sixteen days. Large doses also 
 sensitize, but a longer incubation period is required. In order to pro- 
 duce a fatal result, the second or intoxicating dose must be considerably 
 
 1 Zeitschr. f. Immunitatsf., 1 orig., 1909-1910, 8, 636. 
 
 2 Jour. Exp. Med., 1910, xii, 172. ' Bull. No. 32 of the Hyg. Lab., 1906. 
 ■■ Jour. Med. Research, 1908, xix, 1, 5, 17. 
 
PHENOMENA OF ANAPHYLAXIS 539 
 
 larger than the minimum sensitizing dose, the proportion between the 
 two having been placed by Doerr and Russ as 1 : 1000, i. e., if 0.001 c.c. 
 of serum is injected intraperitoneally in order to effect sensitization, 
 1 c.c. injected by the same route ten or twelve dsLja later would in all 
 probability kill a half -grown guinea-pig, whereas 0.1 c.c. subcutaneously 
 would be followed by serious symptoms. 
 
 Rabbit. — Reference has been made elsewhere to the pioneer work of 
 Arthus, who first described the local anaphylactic reaction about the 
 site of subcutaneous injection. He also described objectively the most 
 important symptoms of acute anaphylactic death in the rabbit, as well 
 as the more ordinary type, which ends in recovery. 
 
 In acute and fatal anaphylactic shock in the rabbit Auer ^ found slow 
 respiration, weak or absent heart action, with fall in blood-pressure, 
 general prostration, the sudden falling of the animal on its side, a short 
 clonic convulsion, increased peristalsis, and expulsion in feces and urine. 
 Death is ascribed to a vascular or cardiac shock or to a failure of the 
 heart action of peripheral origin, mostly affecting the right side, and due 
 to a form of chemical vigor. The muscle may be gray, stiff, very tough 
 to the finger-nail, and non-irritable. Further evidence of the importance 
 of heart failure in anaphylaxis in the rabbit is furnished by the electro- 
 cardiographic study of Auer and Robinson.^ Blood coagulability is 
 delayed. 
 
 Rabbits are by no means so easily sensitized nor to so high a degree 
 as guinea-pigs. Non-fatal anaphylaxis accoiripanied by fall in blood- 
 pressure, increased heart-rate, and active intetilinal peristalsis is readily 
 produced, but there is considerable uncertainty in inducing acute 
 anaphylactic death. Sensitization is usually effected by two or three 
 intravenous injections of 1 c.c. of serum at intervals of three days. In- 
 toxication generally follows an intravenous injection of 1 to 5 c.c. of 
 serum about four to six weeks later. Much smaller doses than these 
 may, however, be used, as was occasionally shown during irmnuniza- 
 tion of rabbits with erythrocytes for the production of hemolj^tic ambo- 
 ceptor, when minute traces of serum, escaping the washing process, 
 served to sensitize, and at a later injection produced acute anaphylactic 
 shock and death within a very few minutes. 
 
 Cats. — ^Anaphylaxis in the cat has been studied especially by 
 Schultz,^ who observed that cardiac disturbances followed. Horse 
 serum, however, was found markedly toxic in effect, even in the un- 
 
 ' Jour. Exp. Med., 1911, xiv, 476. ' Jouf. Exp. Med., 1913, x-viii, 450. 
 
 ' Jour. Phar. and Exper. Therap., 1911-12, 3, 302. 
 
640 ANAPHYLAXIS 
 
 sensitized animal, as little as 0.25 c.c. per kilo killing young, normal cats, 
 and 0.1 c.c. per kilo causing a fall in blood-pressure both in the normal 
 and in the sensitized animals. 
 
 Dogs. — In these animals the most constant symptom of anaphy- 
 laxis is an initial and transitory rise in blodd-pressure, followed by a 
 prompt fall of from 80 to 100 mm. of mercury. This was first described 
 by Biedl and Kraus,'^ and subsequently by Eisenbrey and Fcarcc,^ 
 Robinson and Auer.^ The general symptom's are not so violent as are 
 those that occur in the guinea-pig and death is infrequent. Following 
 intravenous injection of the intoxicating dose of serum there may be 
 great restlessness, marked prostration, and vcmiting, tenesmus, and in- 
 voluntary discharge of feces and urine. If dfcath does not occur, a con- 
 dition of hemorrhagic inflammation in both the large and the small 
 intestine may develop, called by Richet "chronic anaphylaxis," and by 
 Schittenhelm and Weichardt, "enteritis an*aphylactica. " Robinson 
 and Auer, by an electrocardiographic study* detected cardiac changes 
 consisting of disturbance of the heart impulses, abnormalities in ventric- 
 ular contractions, and other interferences with the mechanism of the 
 heart, due probably to the effect of horse serum on the peripheral car- 
 diac tissue, and independent of the drop in blood-pressure or any effect 
 upon the central nervous system. The heart changes do not appear to 
 exert a primary influence on the blood-pressure, which is due to an 
 effect upon the splanchnics, and is probably a secondary factor in ana- 
 phylaxis or vascular shock of the dog. In many instances there is 
 leukopenia, with loss of mononuclear cells. Coagulation of the blood is 
 delayed, a condition first described by Biedl and Kraus,^ who believed 
 it to be due, probably, to a decrease in tliromboplastin or an excess of 
 antithrombin. Pepper and Krumbharr^ have shown that, by adding 
 srnall amounts of thromboplastin to the non-.coagulating, post-anaphy- 
 lactic, oxalated plasma, the coagulability of the blood will be restored. 
 
 As stated elsewhere, it may be difficult to produce anaphylaxis in 
 a dog. Usually a subcutaneous injection of 10 c.c. of horse serum, 
 followed in from three to six weeks by 5 c.c. intravenously, will at least 
 cause a marked fall in blood-pressure or fatal anaphylaxis. 
 
 Other Animals. — White mice and rats, while they may not develop 
 acute anaphylactic asphjrxia, such as is observed in guinea-pigs, do react 
 
 1 Wien. klin. Wochenschr., 1909, xxii, .365. 
 
 2 .Jour. Pharmacol, and Expcr. Therap., 1912, iv, 27. 
 ' Jour. Exper. Med., 1913, xviii, .5.56. 
 
 « Wien. klin. Wochen.schr., 1909, ii, 36.3. ^ Jo\ir. Infect. Dis., 1914, xiv, 476. 
 
MECHANISM OF ANAPHYLAXIS 541 
 
 to horse serum, as was shown by Schultz and Jordan. This reaction is 
 evidenced by restlessness, marked irritability of the skin, involuntary 
 passage of urine and feces, and temperature and blood-pressure changes. 
 Anaphylactic reactions have also been observed to occur in numerous 
 other animals, e. g., in cows, sheep, horses, hens, pigeons, and in 
 certain cold-blooded animals, the symptoms varying according to the 
 species. These reactions have not as yet been carefully studied. 
 
 MECHAJMISM OF ANAPHYLAXIS 
 
 Whereas the lesions and symptoms of anaphylactic shock here 
 described in different species of animals are those commonly observed 
 with serum proteins, they vary in no essential when any protein agent 
 is used when the conditions of dosage and administration are the same. 
 It is evident, however, that no one symptom, or group of symptoms, 
 can be regarded as characteristic of anaphylaxis in all animals. The 
 various species present widely differing pictures with the same protein 
 substance, and these differences are best explained on the ground of 
 changes in the anatomic structure and physiologic reaction of different 
 animals. Thus, Schultz has shown that serum "anaphylaxis is essentially 
 a matter of hypersensitization of smooth muscle in general, and that, 
 during anaphjdactic shock, all smooth muscle contracts. In the guinea- 
 pig this effect is most evident in the bronchi, owing to the peculiar, 
 though normal, anatomic structure of the mucosa, which is relatively 
 thick as compared with the lumen, so that contraction of the smooth 
 inuscle throws it into folds that completely occlude the bronchi causing 
 death from inspiratory asphyxia. The bronchial mucosa of dogs, 
 rabbits, and rats, however, is relatively thin and poor in smooth muscle 
 tissue, which may account for an entire absence of transitory respiratory 
 difficulties during anaphylactic shock in these animals. In the dog the 
 most marked effect is apparent upon the smooth muscle of the gastra- 
 intestinal tract, contraction resulting in setting up vigorous intestinal 
 peristalsis, vomiting, and involuntary emptying of the urinary bladder. 
 The characteristic initial rise in blood-pressure may be due to con- 
 striction of the splanchnic, pulmonary, coronary, and systemic arteries, 
 followed by a condition of paresis and a fall in blood-pressure. The 
 cardiac muscle is also involved, particularly on the right side, as shown 
 by Robinson and Auer, and this favors a venous accumulation of blood. 
 In the rabbit a similar effect is noted upon the smooth muscle of the 
 blood-vessels, and particularly on the heart, as well as upon the gastro- 
 
642 ANAPHYLAXIS 
 
 intestinal tract. Our present knowledge would ascribe these effects, 
 therefore, to a local or peripheral action of the protein upon smooth 
 muscle, and not primarily on the central nervous tissues, as was originally 
 believed. 
 
 The fall in blood-pressure, therefore, appears to be a most constant 
 and primary factor. So far as I am aware, no blood-pressure studies 
 on the anaphylactic guinea-pig have been rnade. In this animal the 
 heart continues to beat after respiration cea'ses, but this phenomenon 
 may be due to mechanical and other factors dependent upon the extreme 
 pulmonary emphysema. Fall in blood-pressure and congestion of the 
 splanchnic area may produce cerebral anemia, and be responsible in some 
 measure for the respiratory disturbances, the retching, the involuntary 
 expulsion of urine and feces, the great depressicwi and muscular weakness, 
 and the speedy recovery when death does not result. 
 
 In man the marked urticarial and other fashes and the inspiratory 
 asthma of those peculiarly sensitive to a protein due to a narrowing of 
 the bronchi, the latter being analogous to the condition observed in 
 the guinea-pig, the diarrhea, and the secondary drop in blood-pres- 
 sure, all indicate a similar action on smooth muscle. This also pro- 
 vides an adequate pharmacologic explanation of the action of atropin, 
 sedatives, and anesthetics in alleviating or masking the symptoms of 
 acute anaphylaxis. (See Chapter XXVIII.) 
 
 Aside from the severe fall in blood-pressijre and temperature, other 
 effects of the anaphylactic poison are leukopenia, local and general 
 eosinophilia (Vaughan,^ Moschowitz,^ Schlecht and Schwenket'), and 
 reduced coagulability of the blood. Pf eiff er ^ found poisonous substances 
 in the urine during anaphylactic intoxication, and Hirschfeld^ detected a 
 pressor substance in the serum of intoxicated guinea-pigs. 
 
 A more critical study of the nature and varieties of anaphyladogens, 
 or substances capable of producing anaphylactic sensitization, will now 
 be made. This will include also a consideration of the nature of the 
 substances directly responsible for the anaphylactic intoxication, com- 
 monly known as anaphylactotoxins, and of the question as to whether 
 anaphylactic intoxication is the result of an interaction in the blood- 
 stream (humoral) or in the cells (histogenetic) or in both. 
 
 1 Zeitschr. f. Immunitatsf., 1911, 9, 458. 
 ^ New York Med. Jour., January 7, 1911. 
 3 Arch. exp. Path. u. Pharm., 1912, 68, 163. 
 * Zeitschr. f. Immunitatsf., 1911, 10, 550. 
 6 Zeitschr. f. Immunitatsf., 1912, 14, 466. 
 
ANAPHYL\CTOGENS, OR ALLERGENS 543 
 
 ANAPHYLACTOGENS, OR ALLERGENS 
 
 So far as is now known, only 'proteins may become anaphylactogens, 
 and with the exception of gelatin and a iew other proteins, practically 
 any soluble protein will produce sensitizationj and intoxication of sus- 
 ceptible animals. Bacterial substances, extfacts of plant tissues, 
 purified vegetable proteins, and proteins derived from invertebrates and 
 cold-blooded vertebrates have all been fouiid capable of acting as 
 anaphylactogens when introduced in a soluble and unaltered condition 
 into an animal. 
 
 The proteins concerned must be foreign to the circulating blood of 
 the injected animal, but they maj- be tissue proteins of the same animal — 
 e. g., sync^-tial cells — that are not normally present in the blood. Indeed, 
 Uhlenhuth and Haendel ^ claimed to have sensitized a guinea-pig with 
 the dissolved lens of one eye so that it reacted to a subsequent injection 
 of the lens of the other eye. Proteins in solution are more active than 
 those in suspension or in partial solution, and in general tissue proteins 
 are less active than proteuis in the blood, lyiuph, and secretions, but 
 even keratins may produce anaphjdaxis when dissolved (Krusins^), 
 Uhlenhuth ' has obtained positive results with, proteins from mummies. 
 As previously stated, the altered protein of an animal may be reinjected 
 again into the animal and induce an anaphylactic reaction. Recently 
 Richet* has directed attention to this phenomenon, which he calls 
 ''indirect anaphylaxis," through obser\'ing an intense leukocji;osis in a 
 dog which reached the maximum on the eighth day following a second 
 chloroformization. 
 
 Non-protein Anaphylactogens. — As with Qther inununologic reac- 
 tions, observations have been made that are interpreted as indicating 
 that non-protein substances are capable of* producing anaphylaxis; 
 thus Pick and Yamanouchi^ sought to demonstrate the antigenic 
 properties of alcohol-soluble constituents of horse and beef serum, but 
 conservatively concluded that their results may have been due to a com- 
 bined action of protein and fat combinations. Similar conclusions were 
 also drawn bj' Uhlenhuth and Haendel^ in their study of animal and 
 
 * Zeitschr. f. Immmiitatsf., 1910, -4, 761. 
 
 2 .\rch. f. Augenheilk., ^^uppl., UUO, 47,47. 
 5 Zeitschr. f. Immunitatsf., 1910, 4, 774? 
 
 * Quoted in Jour. Amer. Med. Assoc, 1914, bdi, 711. 
 
 * Zeitschr. f. Immunitatsf., 1909, .5, 676.. 
 
 * Zeitschr. f. Immunitatsf., 1910, iv, 761. 
 
644 ANAPHYLAXIS 
 
 vegetable oils and fats. Bogomolex^ is less conservative, and believes 
 that he has succeeded in producing lipoid a'naphylaxis; these claims, 
 however, could not be confirmed by Thiele and Embleton.^ Meyer' 
 was able to sensitize pigs with pure lipoids extracted from tape-worms, 
 but was unable to intoxicate them with the Same extracts, results that 
 may be understood, since White and Avery*- have shown that as little 
 as 0.0001 milligram of edestin will serve tq sensitize a pig, whereas 
 larger amounts of protein — more than is confained in Meyer's prepara- 
 tions — are necessary to produce intoxication. Finally, the studies of 
 Wilson^ and White' leave no doubt as to the fact that pure lipoids 
 cannot produce anaphylaxis. 
 
 It is possible, however, for non-protein substances to combine with 
 or alter the proteins of an animal and thus cause anaphylaxis. In this 
 way can be explained apparent anaphylactic reactions to iron, salvarsan, 
 iodin, arsenic compounds, and other medicinal agents. 
 
 Chemistry of Protein Anaphylactogens. — The purest known proteins 
 act as anaphylactogens or sensitizers; in fact, the purer the protein, 
 the more thoroughly it sensitizes the animal and the smaller is the dose 
 necessary to produce intoxication. The crystallized proteins of hemo- 
 globin, egg-albumen, and such pure vegetable proteins as edestin and 
 excelsin, are powerful sensitizers. According to Wells,^ nothing less 
 than an entire protein molecule will suffice to produce anaphylaxis, 
 although Zunz ^ claims to have observed typical reactions with the pro- 
 teoses of fibrin, and Abderhalden' obtained one with a synthetic poly- 
 peptid. It is not necessary, however, for a prptein, in order to be active, 
 to contain all the known amino-acids of proteins, for certain vegetable 
 proteins, e. g., hordein and gliadin, which lack one or more amino-acids, 
 such as glycocoll or tryptophane, may produce typical reactions. Pre- 
 sumably, the inability of pseudoproteins, such as gelatin, to act as 
 anaphylactogens depends upon their deficiency in aromatic radicals. 
 
 Wells has obtained negative results with purified nucleoproteins, as 
 well as with the isolated components of nucleins, such as histon and 
 nucleic acid. 
 
 1 Zeitschr. f. Immunitatsf., 1910, v, 121, ibid, 1910, vi, 332. 
 
 ' Zeitschr. f. Immunitatsf., 1913, xvi, 160. 
 
 3 Folia Scrologioa, 1911, vii, 771; Zeitschr. f. Immunitatsf., 1914, xxi, 654. 
 
 * Jour. Infect. Dis., 1913, xiii, 103. 
 
 ' Jour. Path, and Bact., 1913, xviii, 163. 
 
 « Jour. Med. Res., 1914, xxx, 383. ' Jour. Infect., Dis. 1913, xii, 341. 
 
 s Zeitschr. f. Immunitatsf., 1913, 60, 580. 
 
 9 Zeitschr. physiol. Chem., 1912, 81, 314. 
 
ANAPHYLACTOGENS, OR ALBERGENS 545 
 
 While, therefore, it is probable, although it has not been definitely 
 proved, that nothing less than the entire protein molecule is capable of 
 producing the typical reaction, the questions arise whether the whole 
 molecule, or only a certain group thereof, determines the specificity, 
 and whether the whole molecule, or only a portion, is concerned as the 
 sensitizing agent. It is now generally accepted that both the sensitizing 
 and the intoxicating agents are one and the same protein, and the older 
 view, which held that in a mixed protein substance, such as blood-serum, 
 corpuscles, egg-albumen, etc., one protein is present that sensitizes and 
 another that intoxicates, is probably erroneous. Besredka, for instance, 
 finds that when a protein used to produce intoxication is heated it is less 
 likely to prove fatal, and he concludes that proteins contain a thermosta- 
 bile sensitizing and a thermolabile intoxicating portion. Doerr and 
 Russ, however, have shown by carefully conflucted experiments that 
 heat affects both properties of proteins to the same degree. Since pure 
 proteins, as, e. g., highly purified edestin, which is believed to be a chem- 
 ical unit, act as exquisite sensitizers and intoxieants, it seems reasonable 
 to believe that the sensitizing and poisonous group are constituents of 
 the same protein substance. Whether or not both sensitizing and 
 intoxicating groups are contained in each single molecule of a pure pro- 
 tein is a question that cannot be answered until we can be certain that 
 absolutely pure proteins are secured to start with, and until our methods 
 of effecting its cleavage have been perfected. Vaughan and his cowork- 
 ers have long maintained that a sensitizing non-poisonous and a non- 
 sensitizing toxic portion are groups of the same molecule, which they are 
 able to obtain in vitro from animal, bacterial, and vegetable proteins by 
 a method of splitting with sodium hydroxid in absolute alcohol, as 
 described in the chapter on Infection. The toxic intramolecular group 
 is regarded as non-specific, and the same for alF proteins, which exjilains 
 the identity of the sjrmptoms of anaphylactic shock whatever the pro- 
 tein by which it is induced. The non-toxic sensitizing group, however, 
 is specific, although it may not itself be a protein, or at least a biuret 
 body. Whether or not all proteins contain a sensitizing group has not 
 been determined. In keeping with his theory of the role of the toxic 
 moiety of a split protein molecule in the production of disease, Vaughan 
 beheves that when proteins are introduced pflarenterally into animals, 
 the non-toxic portion stimulates the body-cells to elaborate specific fer- 
 ments, constituting the phase of sensitization, so that when this protein 
 IS subsequently introduced, digestion rapidly takes place with the 
 
 liberation of the toxic substance responsible for the characteristic symp- 
 35 
 
546 ANAPHYLAXIS 
 
 toms, which may terminate in death. This interesting and plausible 
 theory will be referred to again. It has received further experimental 
 support from the work of White and Avery ^ with split edestin and 
 split tubercle-cell substance;^ and from that of Zunz^ and Wells and 
 Osborne/ the last-named observers working* with vegetable proteins, 
 and concluding that although it is probable that the entire protein molecule 
 is involved in the anaphylactic reaction, only certain groups are specifically 
 concerned in the process. In other words, it would appear that anaphy- 
 laxis, — for example, serum anaphylaxis — is not due to one protein sub- 
 stance in the serum that sensitizes and another that intoxicates, both 
 properties residing in the same protein molecule. Whether they are the 
 same intramolecular substances existing side by side, — one the sensitizer 
 and the other the intoxicator, — as is believed by Vaughan, carmot be 
 definitely decided, although experimental work would tend to indicate 
 that the latter may be the true explanation. 
 
 Physical State of Anaphylactogens. — The results of experiments 
 all tend to support the theory that proteins in solution are most power- 
 ful in producing anaphylaxis, because they ard'able to come into intimate 
 contact with body-cells, and cell permeation^ is probably necessary for 
 the most complete sensitization. This explafns in part the conflicting 
 statements concerning the effect of heat on the sensitizing properties of 
 blood-serum. Rosenau and Anderson found that animals could not be 
 sensitized with serum that has been heated at 100° C, whereas Doerr 
 and Russ placed the point at 80° C. Besredka showed that the sensitizing 
 properties are in part at least, dependent upon the physical condition of 
 the protein, and that heating undiluted blood-serum coagulates the pro- 
 tein and leads to a decrease of its anaphylactogenic properties. Similarly, 
 Vaughan found that proteins that were insoluble in water, as, for ex- 
 ample, edestin, sensitize more readily when dissolved in salt solution. 
 The same factors are operative with the protein used for intoxication, 
 the physical state of the protein substance having a direct bearing 
 on the rapidity with which shock is produced. Temperatures high 
 enough to disrupt and destroy proteins are, however, equally destructive 
 to their sensitizing properties. 
 
 An interesting question in this connection is whether sensitization 
 and intoxication may occur with the parenteral introduction of protein 
 as with the food. In the great majority of instances the gastro-in- 
 testinal enzymes so completely disrupt the protein molecule that sen- 
 
 1 Jour. Inf. Dis., 1913, xiii, 103. « Jour. Med. Res., 1912, xxvi, 317. 
 
 ' Zeitschr. f . Immunitatst., 1913, xvi, 580. " Jour. Inf. Dis., 1913, xii, 341. 
 
ANAPHYLACTOGENS, OR ALCERGENS 547 
 
 sitization and intoxication do not occur. Guinea-pigs have, however, 
 been sensitized by feeding them meat or serum, and instances of buck- 
 wheat, fish, and egg idiosyncrasies would tend to indicate that intoxica- 
 tion may result from the ingestion of these substances in sensitive 
 persons. 
 
 Rosenau and Amos' have demonstrated that proteins in a volatile 
 state, as in the exhaled breath of men, when condensed and injected into 
 guinea-pigs will sensitize these animals to subsequent injections of human 
 serum. While it is doubtful if the complex molecule possesses the power 
 of passing into the air in a gaseous form, it may probably exist in col- 
 loidal solution. Rosenau was also able, by keeping guinea-pigs in 
 stables together with horses, to sensitize them to horse serum. These 
 experiments are of fundamental importance in explaining instances of 
 human anaphylactic phenomena among those sensitive to horse protein, 
 — as, e. g., persons seized with sneezing and asthma when they come 
 near horses, — and also tend to show how minute may be the quantity 
 of protein capable of sensitizing and intoxicating body-cells. 
 
 Bacterial Anaphylactogens. — All bacterial proteins are anaphy- 
 lactogens, although, on account of the physical state of the bacteria, 
 they yield reactions more irregular and weaker than those observed 
 with proteins in solution. The tuberculin, luptin, mallein, and similar 
 reactions are true anaphylactic phenomena. Rosenau and Anderson, 
 Vaughan and Wheeler, Kraus, and others have observed anaphylactic 
 reactions with various bacteria, such as Bacillus subtilis and colon, 
 typhoid, anthrax, and tubercle bacilli. Not infrequently reactions 
 occur during the therapeutic administration of tuberculin and bacterial 
 vaccines. 
 
 This brings up the interesting question as to whether toxins are 
 anaphylactogens, a subject previously irientioned in the historic review 
 of this subject. Instances of hypersensitivenpss to diphtheria and te- 
 tanus toxins were early observed in attempts to immunize horses in the 
 production of antitoxins. As it is extremely difficult, if not impossible, 
 to isolate a toxin free from other constituents of the medium into which 
 it was excreted by microorganisms, this question cannot be answered 
 m a definite manner. There is no direct proof, however, that toxins 
 sensitize, although the protein in the toxin filtrate may serve to do so. 
 In 1902 Vaughan and Gelston ^ showed that the poison contained in the 
 cellular substance of the diphtheria baciUus is, an entirely different one 
 
 ' Jour. Med. Research, 1911, xxv, 35. 
 
 2 Trans. Assoc. Amer. Phys., 1902, 17, 308. 
 
548 ANAPHYLAXIS 
 
 from the toxin elaborated by the same microorganism, results that were 
 confirmed in 1911 by Friedberger and Reiter,/ working with the dysen- 
 tery bacillus. Thus hyper sensitiveness to toxins is -probably not an 
 anaphylactic phenomejion, but is due to a greater affinity of the body- 
 cells for the toxin. This explains the so-called paradox of Kretz, who 
 found that while the injection of an accurately neutralized toxin-anti- 
 toxin mixture produces no bad results in a normal animal, in one that 
 has been previously actively immunized with toxin, the reverse occurs. 
 Apparentljr the sessile receptors have a stronger affinity for toxin than 
 have the free receptors, and accordingly the:toxin becomes dissociated 
 and combines with the cells. 
 
 This view is also substantiated by the observation that the symptoms 
 of intoxication caused by the toxin used for immunization are not those 
 of anaphylaxis, which, for a certain animal, are the same regardless of 
 the source of the protein. Toxin hypersensitiveness does not seem to 
 be transmissible to normal animals, whereas in anaphylaxis the condi- 
 tion may be transmitted (passive anaphylaxis). 
 
 Whether or not endotoxins act as anaphylactogens cannot be definitely 
 stated. If they do, their action and effects are intimately connected 
 with those ascribed to the protein contained in the bacterial cell. It is 
 unlikely, however, that they play any role in inducing hypersuscepti- 
 bility, as their toxicity is usually apparent soon after injection, and 
 before sensitization has occurred. 
 
 The necessary period of incubation between sensitization and in- 
 toxication, the symptoms, and the fact that in some cases an immunity 
 is induced, are results that strengthen the belief that the phenomenon of 
 hypersensitiveness has a practical significance in the prevention and cure 
 of certain infectious diseases. 
 
 ANAPHYLATOXIN (PROTEIN POISON) 
 
 The symptoms of anaphylaxis that follow injection of the protein 
 into an animal previously sensitized with the same protein are such as to 
 leave no doubt that a poison is the etiologic factor, although as yet this 
 poison has not been isolated in a pure state. 
 
 Vaughan and Wheeler were the first to demonstrate this poison in 
 
 vitro by splitting proteins with a solution of NaOH in absolute alcohol. 
 
 Friedmann was first to produce it in vitro through the action of ferments 
 
 contained in immune and normal rabbit serum upon ox corpuscles and 
 
 iZeitsohr. f. Immunitatsf., 1911, 11, 493. 
 
ANAPHYLATOXIN (PROTEIN POISOn) 549 
 
 seram precipitates. Weichardt also produced it by digesting placental 
 protein with the serum of rabbits immunized with placental cells. 
 Friedbergcr' studied the subject more extensively by observing the 
 action of normal guinea-pig serum upon seruin precipitates, and was 
 first to apply the term " anaphylatoxin " to the poison. At the time he 
 regarded it as a true toxin, similar to diphtheria and tetanus toxins, but 
 at present we know that this poison is not a true toxin, because it cannot 
 produce an antitoxin, is thermostabile in acid solution, and is not a 
 single specific substance, but a mixture of more or less closely related 
 substances in the nature of protein cleavage products, as first shown by 
 Vaughan and Wheeler, and since accepted by Friedberger himself and a 
 number of other investigators. In a strict sense, therefore, this term 
 is a misnomer, but it is in such general use that it need not be discarded 
 if we have a clear understanding that the smn total of independent 
 researches by numerous investigators shows that it is not a true toxin, 
 as tetanus toxin, for instance, but a protein poison. 
 
 As the symptoms of anaphylaxis are always the same in the same 
 animal, no matter what protein is used, it would appear that the protein 
 poison is either always the same or composed of a group of very closely 
 allied products, and, indeed, this seems to have been proved by an 
 extended series of researches with the most diverse proteins of animal, 
 bacterial, and vegetable origin. 
 
 It may be stated, therefore, that anaphylatoxins may be regarded as 
 protein poisons composed of protein cleavage products, and that these are 
 responsible for the lesions and symptoms of anaphylaxis. There is some 
 difference of opinion regarding the source of the protein matrix and the 
 mechanism of its cleavage with the production of the poison in anaphy- 
 laxis, and I shall consider this phase of the subject later. There is, 
 however, a striking uniformity of experimental evidence and opinion 
 regarding the role and primary importance of the protein cleavage 
 poisons in the anaphylactic process. Briefly summarized, the evidence 
 on this point is as follows: 
 
 1. As was just stated, the first to advance the theory regarding the 
 r61e of protein-split products in anaphylaxis, as well as in infection and 
 immunity in general, were Vaughan and Wheeler. In 1907 these 
 observers showed that proteins may be split by boiling with alcoholic 
 sodium hydroxid solution into two fractions — one non-toxic and alcohol 
 soluble and the other toxic and alcohol insoluble. The toxic fraction, 
 when injected into normal guinea-pigs in doses of from 8 to 100 mg., 
 1 Zeitschr. f. Immmiitatsf., 1910,* 4, 636. 
 
660 ANAPHYLAXIS 
 
 kills the animals, death being preceded by all the symptoms of acute 
 anaphylactic intoxication. This toxic moiety has been obtained by 
 this method from the most diverse proteins, and seems to be the same for 
 all, the specificity residing in the non-toxic a'ttached group. This and 
 other observations led Vaughan and his collaborators to formulate the 
 hypothesis that the non-toxic and specific intramolecular group of a 
 protein serves to stimulate the body-cells to produce specific proteolytic 
 enzymes, and that upon the injection of a second dose of the same 
 protein, these enzymes at once disintegrate it, setting free the toxic 
 group that produces the lesions and symptoms of acute anaphylactic 
 intoxication. This protein cleavage in vivo is entirely analogous to 
 the cleavage process occurring in vitro with the alcoholic sodium hydroxid 
 solution, and the poison in both instances is regarded as the same. 
 
 Many of the later studies in this field, especially those of Friedberger 
 and the investigations of Abderhalden on "protective ferments," have 
 given support to this hypothesis, so that in its fundamental principles 
 it constitutes the most plausible and generally accepted explanation of 
 the processes involved in anaphylaxis. 
 
 2. Mention has previously been made of the protein poison obtained 
 by Friedmann by digesting ox corpuscles With immune and normal 
 rabbit serum, and by Weichardt as the result of the digestion of placental 
 protein with immune rabbit serum. In 1910 Friedberger,^ by digesting 
 a serum precipitate with normal guinea-pig serum, obtained a similar 
 toxic substance; by means of ferments he secured a protein cleavage' 
 poison that, when injected into normal guinea-pigs, produced the 
 symptoms of acute anaphylaxis, the process being entirely analogous to 
 that obtained by Vaughan and Wheeler by protein splitting with alcoholic 
 sodium hydroxid solution. Subsequent studies by Friedberger and his 
 collaborators^ showed that similar protein poisons could be obtained by 
 digesting microorganisms, as, e. g., Bacillus prodigiosus, Bacillus typho- 
 sus, and Bacillus tuberculosis, with normal guinea-pig serum. Fried- 
 berger and Nathan^ obtained the poison by digesting normal horse serum 
 with fresh guinea-pig serum; Bordet,* by digesting agar with fresh 
 serum; Dold and Aoki,^ by digesting meningococci, streptococci, 
 pneumococci, gonococci, and various other microorganisms with fresh 
 normal gunea-pig serum. As was expected,^ cleavage could be facili- 
 
 1 Zeitschr. f. Immunitatsf., 1910, 4, 636. 
 
 2 Zeitschr. f. Immunitatsf., 1911, 9, 569. 
 
 3 Zeitschr. f. Immunitatsf., 1911, 9, 567. 
 
 * Compt. rend de Soc. de Biol., 191.3, Ixxiv, No. 5. 
 6 Zeitschr. f. Immunitatsf., 1912, 12j 200. 
 
ANAPHYLATOXIN (pROTEIN POISON) 551 
 
 tated and hastened by using immune serum specific for any pai-ticular 
 bacterial or other protein. 
 
 These results indicate that fresh normal guinea-pig serum contains a 
 normal thermolabile non-specific proteolytic ferment capable of split- 
 ting some, through probably not all, pioteins, a view advanced by 
 Vaughan many years previously. After sensitization, a specific proteo- 
 lytic ferment is produced for the particular protein injected, the presence 
 of the specific in addition to the normal ferments constituting the differ- 
 ence between normal and sensitized animals. 
 
 Since these studies show that, when incubated with normal serum, 
 bacteria and certain other proteins yield a soluble and active poison, 
 the question naturally arises why this reaction does not occur when 
 these proteins are first injected directly into the blood? ^'aughan has 
 answered this question by assuming that the ferment is in a more avail- 
 able form in the scrum than it is in the blood, since the ferment is prob- 
 ably largely a leukoprotease mainly derived from the disintegration of 
 leukocytes. Or it may be that the cleavage is carried on in the circulat- 
 ing blood beyond the point of the products constituting the protein 
 poison, or that the inclusion of the foreign protein by the phagocytes may 
 delay the disruption of the former. 
 
 While there is this consensus of opinion regai'ding the role of protein 
 poison in the production of anaphylaxis, there is some diversity of opin- 
 ion regarding the source of the protein matrix; i. e., the protein that is 
 broken down, especially in view of the fact that mixtures of kaolin and 
 normal guinea-pig serum produce the poison. It has been suggested — 
 (a) That the kaolin, agar, bacteria, etc., absorb the complement from 
 the serum, and that this renders the serum poisonous ; (&) that the poison 
 is preformed in the serum, but that its action is neutralized by some 
 other constituent of the serum that is absorbed ; (c) that the absorption 
 of some constituent of the serum leads to a breaking-up of serum pro- 
 teins, with liberation of the poison. The latter view, as shown by Job- 
 ling and Petersen, ' appears to be the most plausible. These writers 
 beUeve that the normal try[;)tic or proteolytic ferment of the blood is 
 held in check by an antif erment of the nature of unsaturated fatty acids, 
 and that laokin, bacteria, agar, etc., remove this antitryptic influence 
 by absorbing the lipoidal antif erment, settings the ferment free, which 
 then acts upon the serum protein, producing; the toxic protein poison 
 (serotoxin). While Vaughan, Friedberger, ^nd their collaborators 
 believe that the protein poison is derived from the injected protein, 
 1 Jour. Exper. Med., 1914, xLx, 459 and 480. 
 
552 ANAPHYLAXIS 
 
 Jobling and Petersen believe that the matrix is, indeed, the protein of 
 the animal's own serum. Doerr ^ likewise regards it as highly improbable 
 that the poison is generated by the bacteria or other foreign protein, 
 but believe that it is derived from the serum through the absorption of 
 inhibitory antibodies by the bacteria, precipitates, etc. There can be 
 no doubt, however, of the high specificity of the anaphylactic reaction, 
 which indicates that specific ferments are produced for the protein 
 injected, and while the discussion cannot be considered closed, it would 
 appear that, for the present, it would be best to accept the theory that 
 anaphylaxis is due to the cleavage of the injected anaphylactogen by 
 normal and specific proteolytic ferments. 
 
 3. Further evidence of the role played by protein cleavage products 
 in anaphylaxis is furnished by the action of /3-imidazolyethylamin 
 ("histamin"), an amin produced by splitting off carbon dioxid from 
 histidin by chemical or bacterial agencies. This substance, first pre- 
 pared synthetically by Windam and Vogt, ^ and studied by Ackermann, ' 
 Harger and Dale,^ and especially Dale anxl Laidlow,^ produces, in 
 doses of about 0.5 mg., effects resembling acute anaphylactic intoxica- 
 tion. Heyde* has described similar effects with methylguanidin, and 
 other amins may possibly be involved. 
 
 ANAPHYLACTIN (ALLERGIN) 
 
 There is a diversity of opinion concerning the nature of the substance 
 developed in the body under the influence of the anaphylactogen, which 
 is capable of cleaving the latter and producing the anaphylactic poison. 
 That such a body exists in the blood of the sensitized animal is shown by 
 the production of passive anaphylaxis in normal animals by injecting 
 into them a few cubic centimeters of blood or serum from a sensitized 
 animal. The animal so sensitized becomes at once or within a few hours 
 sensitive to the specific anaphylactogen, regardless of the species of 
 animal furnishing the immune serum. 
 
 Terminology. — Various names have been applied to this body. 
 Vaughan objects to the term "antibody," although this designation 
 would seem to be appropriate if we consider that it is a reactionary 
 product or antibody to the protein responsibic for its production, al- 
 
 ' Handb. d. path. Mikroorgan., Kolle and Wassermann, second edition, ii, 947. 
 2 Berichte, 1907, xl, 3691. ^ Zeitschr. f . physiol.Chem., 1910, xlv, 504. 
 
 " Proc. Chem. Soc, 1910, xxvi, 128. ^ jova. Physiol., 1911, Ixi, 318. 
 ^ Cent. f. Physiolog., 1911, 25, 441; 1912, 26, 401. 
 
ANAPHYLACTIN (aLLERGIN) 553 
 
 though, instead of being protective to the animal, its host, it is just the 
 reverse. Richet named it toxogen, because it is responsible for the pro- 
 duction of a poison. Otto speaks of it as "reaction body, " Nicolle as an 
 albuminolysin, Besredka as sensihilisin, and von Pirquet as allergin. 
 
 Nature of Anaphylactin. — A full discussion of the nature of this body, 
 which is believed to cleave the anaphylactogen and set free the anaphy- 
 latoxin or protein poison, would involve a review of the entire subject 
 of antibodies and immunity in general. Vaughan regards it as a ferment, 
 evidently meaning that an amboceptor and a complement act together 
 and produce lysis or disruption of the protein molecule. It is difficult, 
 and indeed impossible, at this time to discuss with any degree of definite- 
 ness the propriety of regarding an immune and cytolytic amboceptor 
 as a ferment, just as we regard trypsin as a ferment. If the ferments 
 are to be considered as identical with amboceptors, then we must regard 
 Abderhalden's protective ferments as cytolysins, but certainly, in the 
 light of our present knowledge, we are hardly justified in drawing hard 
 and fast lines. Therefore while I have confined the discussion of the 
 protective ferments to a separate chapter (Chapter XV), and have not 
 considered it under the general head of the cytolysins, it is to be 
 remembered that the ferments possess many of the properties of lytic 
 amboceptors, and they may be identical with them although apparently 
 different owing to the application of chemical methods, especially by 
 Abderhalden, in their study. 
 
 Most observers regard the anaphylactic antibody, toxogen or allergin, 
 as an amboceptor and a complement. The aetual antibody then must 
 be considered as an immune albuminolysin, for complement is present 
 in normal serum, and is not necessarily increased during the process of 
 sensitization. As in other lytic processes, however, complement or 
 alexin is of great importance, constituting, as it does, the actual lytic 
 agent, after the antigen, or, in this instance, the anaphylactogen of the 
 second injection, has been sensitized by the, amboceptor. Some ob- 
 servers believe that the complement is decreased during anaphylaxis, 
 presumably being used up in effecting lysis of the protein. Friedmann 
 claims that in allergy to red corpuscles there is a close parallelism between 
 the anaphylactic bodies and the hemolytic amboceptor. 
 
 With the more recent work of Abderhalden on protective ferments 
 and the development of a dialysis and optical method of detecting the 
 products of protein cleavage, it was hoped that the true and exact nature 
 of anaphylaxis would finally be established. It would appear possible 
 to determine the presence, in the blood-seruni of sensitized animals, of 
 specific proteolytic ferments capable of demonstrating their presence 
 
554 ANAPHYLAXIS 
 
 in vitro, and to show the products of protein cleavage just after anaphy- 
 lactic shock and a corresponding decrease or total absence of the fer- 
 ments as a result of their participation in the albuminolysis. Indeed, 
 Abderhalden^ has recently claimed that all these conditions have been 
 found to exist: (1) The serums from 12 guiflea-pigs sensitized to egg- 
 albumen, when mixed with antigen, showed' digestive power by both 
 optic and dialysis (biuret) methods; (2) similar serums, dialyzed alone, 
 showed digestive products in only one of six serums tested: (3) the serum 
 of six guinea-pigs taken at intervals of from fi'WQ minutes, to one and one- 
 half hours after the second injection (egg-alhumen) and dialyzed, gave 
 negative results after from five and fifteen minutes, whereas four taken 
 a,fter thirty, forty-five, sixty, and ninety minutes respectively were 
 positive. In each test the serum (10 c.c.) was dialyzed against distilled 
 water for sixteen hours at 37° C, and the presence of the products of 
 digestion determined by the biuret reaction. In this manner the final 
 evidence of the role played by the protein si^lit products in the produc- 
 tion of acute anaphylaxis has apparently been- furnished. These results, 
 however, cannot at the present time be regarded as final. Pearce and 
 Williams, ^ in a similar study with horse serum, employing the dialysis 
 technic and using ninhydrin as the indicator, were unable to demonstrate 
 the presence of the ferments after sensitization, although by using large 
 amounts of serum secured after anaphylactic shock had occurred they 
 observed positive reactions, which may have been due, in part at least, 
 to the presence of cleavage products. Zunz^ found that the protein- 
 splitting properties of blood-serum, as tested on the sensitizing protein, 
 increased in the anaphylactic state from the fifth to the twentieth and 
 sixtieth day, and were not recognizable in blood-serum taken during 
 or soon after anaphylactic shock occurred.. Pfeiffer and Mita and 
 Vaughan have observed the apparent disappearance of the ferment from 
 the blood, even though the animal is sensitized, and the last-named 
 observer explains this on the assumption thatf the ferment comes from 
 the fixed cells, which are stimulated to elaborate the ferment only when 
 the specific protein is brought into contact with them. 
 
 One of the older theories of anaphylaxis, regards the process as a 
 precipitin reaction. It is apparent that, in the serum of an animal 
 immunized with a soluble protein, such as a serum, a precipitin and com- 
 plement-fixing body, presumably an albuminolysin or so-called "fer- 
 ment," are produced, and exist together in the immune serum. While 
 
 1 Zeitsohr. f . physiol. Chem., 1912, S2, 109. = ,Toilr. Inteot. Dis., 1914, 14, 351. 
 3 Zeitsohr. f. Immunitatsf., 1913, 17, 241. 
 
ANAPHYLACTIN (aLLERGIN) 555 
 
 the r61e of precipitins themselves appear to have been excluded as directly 
 participating in the production of anaphylaotic shock, recent experi- 
 ments of Zinsser 1 anti others would tend to show that a precipitin 
 possesses the nature of a protein sensitizer of antibody that sensitizes 
 its antige,n, just as a hemolytic amboceptor sensitizes its corpuscles, 
 precipitation being a secondary phenomenon due to the colloidal nature 
 of the reacting bodies under conditions of quantitative proportions and 
 environment that favor precipitation. This would assign to the pre- 
 cipitins and agglutinins an active though secondary role in the processes 
 of anaphylaxis and immunity. 
 
 At the present time, therefore, we may tentatively assume that the 
 ahaphylactin or allergin is of the nature of a specific lytic amboceptor 
 or albuminolysin, which, in conjunction with ilon-specific complement, 
 constitutes what is called a "ferment," and is capable of splitting a 
 protein molecule with the liberation or formation of a toxic moiety re- 
 sponsible for the lesions and symptoms of anaphylaxis. Just as other 
 iiormal amboceptors, such as hemolysins, are present in normal serum, 
 so these protein amboceptors or albuminolysins may be present for 
 various proteins, explaining the production of the anaphylaxis poison 
 in vitro with a normal serum when the latter is fresh and active, i. e., 
 when complement is present. 
 
 Theoretically, at least, it should be possible to detect the anaphy- 
 lactic amboceptor in a serum by a method of complement fixation, 
 although practically this is not the case. THe whole subject of "fer- 
 ments" requires further study, and, as a result; our knowledge and views 
 of antibodies and the processes of immunity in general are likely to 
 undergo some change. 
 
 The Cellular Theory of Anaphylaxis. — Aft interesting question in 
 this connection is whether the anaphylactic reaction is humoral or occurs 
 in the blood-stream, i. e., between the free anaphylactin or antibody 
 and the antigen, or whether it is cellular and occurs between the attached 
 or sessile antibodies and the antigen. Friedberger and Girgolaff ^ now 
 support the cellular theory, basing their belief on the results of their 
 experiments, which consisted of thoroughly washing the organs of a 
 sensitized animal with salt solution, and then transplanting them to a 
 normal animal, when the latter became sensitized. The recent and 
 brilliant studjes of Dale, ' whose investigations were carried out by the 
 
 ' Jour. Kxp. Medicine, 1913, 18, 219. 
 
 ^ Zcitsehr. f. Immunitatsf., 1911, 9, .57.5. 
 
 ' .lour, of Pharm. and Kxper. Therap., 1913, 4, 167. 
 
556 ANAPHYLAXIS 
 
 graphic method, employing the excised uteri ol'sensitized pigs and apply- 
 ing the antigen direct, decided the question in favor of the cellular 
 theory. In a similar study Weil ^ reached the same conclusions, namely, 
 that the anaphylactic condition is entirely dependent upon the sensi- 
 tization of the cells of the body; that all conditions that in any way in- 
 fluence the degree of sensitiveness of the cells in the same degree alter 
 the anaphylactic state, or sensitiveness, of the animal. It does not seem 
 possible, however, entirely to exclude the agency of the antibodies in the 
 blood, as Weil apparently does, or we would be at a loss to explain the 
 mechanism of passive anaphylaxis. It would appear that both the 
 attached and the free antibodies, particularly the former, contribute 
 toward the production of the anaphylactic response. 
 
 THEORIES OF ANAPHYLAXIS 
 With the foregoing explanation of the most widely accepted theory 
 of anaphylaxis, I may briefly summarize all the more important theories 
 advanced from time to time in explanation of the process. These 
 include the following. 
 
 1. Richet held that the sensitizer, or anaphylactogen, contains a 
 substance which he called "congestion" (because he did his original 
 work with extracts of the tentacles of sea anemones, which are toxic 
 and produce congestion of the internal organs), and that this generates 
 in the animal another substance, known as the "toxogenin. " The 
 reaction between the latter and the homologous protein on reinjection 
 sets free a poison, " apotoxin, " which, because ^of its effect on the nervous 
 system, produces the symptoms of anaphylaxis. This theory is prac- 
 tically the same as that generally accepted to-^ay, except that the anti- 
 gen is not of necessity primarily toxic for the animal. 
 
 2. Hamburger and More suggest that the- first injection leads to the 
 formation of precipitins, and that on reinjection precipitates are formed; 
 these they contend, may, by the formation of capillary emboli, produce 
 acute anaphylaxis, or at least that precipitin formation runs parallel 
 with the antibody formation. The symptoms of anaphylaxis, however, 
 are not those of embolism, and there is no eyidence to show that pre- 
 cipitation occurs in vivo, although, as Zinsser j56ints out, precipitins may 
 play the role of sensitizers of the antigen, preparing them for final lysis 
 or cleavage by a complement. In other wordg, the precipitin would act 
 as an amboceptor, differing, however, from our general conception of 
 
 1 Jour. Med. Research, 1914, ,30, No. 2, 87. 
 
THEORIES OF ANAPHYLAXIS 557 
 
 tke nature of amboceptors by being active in the absence of complement, 
 unless precipitation is a secondary pliysical phenomenon in the nature 
 of a colloidal reaction. 
 
 3. Besredka taught .that the sensitizer contains two substances — 
 "sensibilisinogen" and " antisensibihsin. " When injected the first 
 time, the former develops in the body a substsmce called "scnsibihsin," 
 and on reinjection the sensibilisin and antisensibihsin continue to form 
 a poison that acts on the nervous system. 
 
 4. Gay and Southard believe that, as a result of the first injection 
 of serum, there remains in the circulation a protein substance called 
 "anaphylactin, " which is slowly absorbed and continues to stimulate 
 the cells, leading to an abnormal affinity for the homologous protein, 
 which, on reinjection, leads to anaphylactic shock. 
 
 5. Vaughan and Wheeler are of the opinion that, with the parenteral 
 introduction of a foreign protein, the body-cells are stimulated to produce 
 a specific zymogen or ferment that digests it. The protein of the first 
 injection is so slowly digested that the effects are not recognizable. After 
 the protein of the first injection has been disposed of, the new ferment 
 continues to be formed in the cells, and on the second injection, after the 
 proper interval has been allowed to elapse, this zymogen is activated and 
 spHts up the protein, which promptly and abundantly results in the pro- 
 duction of the symptoms of anaphylactic shock. Vaughan believes 
 that there is a non-specific poisonous group oir moiety in each protein 
 molecule which, when liberated by the ferment, is responsible for ana- 
 phylaxis. This poisonous group is held as being the same in all pro- 
 teins, and hence the similarity of lesions and symptoms of anaphylactic 
 intoxication in animals regardless of whether the protein is of animal, 
 vegetable, or bacterial origin. The nature of the ferment is not clear. 
 In 1907 they regarded it as a zymogen — a theoretic labile chemical body 
 resulting from intramolecular rearrangement in the protein molecules 
 of the cell. Little is known of the action of these ferments except that 
 in some manner they cause cleavage of the protein molecule and libera- 
 tion of the toxic moiety. Later, Vaughan speaks of the ferment as 
 consisting of an amboceptor and a complement, the ferment (pre- 
 sumably the complement portion) being inactivated by a temperature of 
 56° C. and reactivated on the addition of serum and organic extracts. 
 Although Vaughan's theory best explains the nature and source of the 
 anaphylactic poison, that of Friedberger explains the production of the 
 "ferment," or rather the protein sensitizer (ajnboceptor), which, with 
 
558 ANAPHYLAXIS 
 
 a complement, digests the protein and sets free or produces the protein 
 poison. 
 
 6. Friedberger has attempted to explain^ anaphylaxis on the basis 
 of Ehrlich's side-chain theory of the action of antigens and the produc- 
 tion of antibodies similar to toxin-antitoxin- immunity. This theory 
 assumes that, on the first injection, the protein finds but few groups of 
 cellular receptors with which it can combine, and for this reason it is 
 not poisonous. During the period of incubation the animal cells develop 
 receptors specific for the homologous protein; with a single small dose 
 of protein most of these receptors remain attached to the cell (sessile); 
 on repeated injections, the newly formed receptors are in large part cast 
 off into the blood, and constitute the precipitins. In this manner an 
 animal relatively insusceptible to a foreign protein is rendered highly 
 susceptible, and on the second injection the protein is anchored firmly 
 to the cell, just as the cells of an animal may anchor diphtheria toxin. 
 One of the essential features of this theory is that it assumes that, ordi- 
 narily, the receptors are not preformed in sufficient numbers to anchor 
 enough protein to injure the animal with the first injection, regardless 
 of the size of the dose. On the other hand, tetanus and diphtheria 
 toxins find large numbers of sessile or cellular receptors, and are highly 
 toxic on the first injection. As originally evolved, the theory did not 
 explain the nature of the toxic agent responsible for the lesions and symp- 
 toms of anaphylaxis, and made no mention of the protein poison. Never- 
 theless it affords the best explanation we have on the formation of the 
 "ferment" or protein sensitizer (amboceptor). At one time Fried- 
 berger believed that anaphylaxis could be explained on the basis of a 
 precipitin reaction. Anti-anaphylaxis was explained on the assumption 
 that the protein of the reinjection uses up the sessile receptors already 
 developed, and, accordingly, not enough are present at the end of the 
 period of incubation to produce anaphylactic shock. Passive anaphy- 
 laxis was explained on the ground that the free receptors in the blood 
 of a sensitized animal become, on injection into a fresh animal, anchored 
 to the cells, thus forming fixed or sessile receptors that anchor the pro- 
 tein on reinjection and lead to anaphylaxis. 
 
 Nolfi has proposed a theory of anaphylaxis that has come to be 
 known as the " physical theory. " It assumes tiiat the active constituent 
 of proteins is a thromboplastic substance that disturbs the colloidal 
 equihbrium of the blood and leads to the deposition, on the surface of 
 the leukocytes and the endothelial cells of capillaries, of a delicate film 
 1 Quoted by Vaughan, "Protein Split Products," 1913, 340. 
 
PASSIVE ANAPHYLAXIS 559 
 
 of fibrin. Thus stimulated, the cells pour out an unusual amount of 
 antithrombin. On account of the consumption' of a part of the fibrino- 
 gen and the increased formation of antithrombi» the blood fails to coagu- 
 late after anaphylactic shock or peptone poisoning. Owing to the 
 coagulation deposits on the endothelial cells, the viscidity is increased 
 and the leukocytes adhere to the vessel-walls, thus accounting for the 
 leukopenia observed after protein injection. The endothelial cells are 
 injured, and the walls of the capillaries become more readily permeable, 
 thus accounting for the local edema often seen in anaphylaxis. The 
 fine capillaries of a given area may be occluded bj^ thrombi, thus ex- 
 plaining the necrosis characteristic of the Arthus phenomenon. The 
 irritation of the endothelial cells extends to the smooth muscle, leading 
 to vasoparalysis and the characteristic fall in blood-pressure. The 
 affinity of the endothelial cells for the protein is stimulated by the first 
 injection, and acts in a fulminating waj^ on reiAjection, thus explaining 
 the suddenness of anaphylactic shock. 
 
 The theory, therefore, also assimies the formation of a ferment that 
 acts primarily upon the proteins of the blood, = leading to the formation 
 of fibrin, which, as it were, mechanically induces the lesions and symptoms 
 of anaphylaxis. "Utile it offers a plausible explanation, the theorj^ is 
 not well supported, and at best may be regarded as a modification of 
 Vaughan's theory, demonstrating one way in wtich the protein poison 
 may act, 
 
 PASSIVE ANAPHYLAXIS 
 
 Passive anaphylaxis is produced by the injection of normal animals 
 with the blood or serum of animals or persons already sensitized. It 
 is similar to ■passive immunization, and is specific for the anaphylactogen 
 mth which the donor is sensitized. 
 
 The second animal may be of the same or of another species. If 
 it is of the same species, the condition induced by the transference of 
 the serum is called homologous; if it is of a diff.erent species, it is known 
 as heterologous, or passive anaphylaxis. 
 
 Passive anaphylaxis was discovered almost simultaneously and in- 
 dependently by Gay and Southard,^ working with guinea-pigs, by 
 NicoUe,^ with rabbits, and by Otto,^ who, working with both guinea-pigs 
 and rabbits, showed that a rabbit immune serum would passivelj' sensi- 
 tize a guinea-pig. 
 
 1 Jour. Merl. Research, 1907, xvi., 143. ^ Aim. de I'lnst. Pasteur, 1907, xxi, 128. 
 ' Miinch. med. Wochenschr., 1907, Xo. 39. 
 
560 ANAPHYLAXIS 
 
 At this place it may be mentioned that the new-born of a sensitized 
 mother animal may be sensitive, and remain so for a longer or shorter 
 period of time. This is an illustration of passive homologous anaphy- 
 laxis, a process that has been especially studied "by Rosenau and Anderson, 
 Gay and Southard, and Otto, the last observer finding that young guinea- 
 pigs remained sensitive for as long as forty-five days after birth. This 
 function of transmitting the condition of sensitization is solely maternal: 
 the male takes no part whatever in the transmission of these acquired 
 properties. 
 
 The Production of Passive Anaphylaxis. — An important phase of 
 this subject over which there has been considerable difference of opinion 
 refers to the question whether some time must elapse between the in- 
 jection of immune serum and anaphylactogen before anaphylaxis is 
 produced, or whether intoxication may follow the simultaneous injection 
 of both antibody and antigen. Thus Gay and Southard, in their early 
 studies, found their recipients first sensitized on the fourteenth day 
 after injection of immune serum. Otto and Friedmann observed shock 
 twenty-four hours after injecting the anaphylactic serum subcutaneously 
 and antigen intraperitoneally. By injecting both serums intravenously 
 and simultaneously, Doerr and Russ finally succeeded in producing acute 
 anaphylaxis and almost immediate death. Weil, ^ however, believes that 
 the simultaneous injection of antigen and of antiserum into opposite 
 jugular veins in the guinea-pig never produces an anaphylactic reaction, 
 in spite of the use of wide quantitative variations in both substances. 
 On the other hand, if the antigen were injected a few hours after the 
 antiserum, in the same quantitative variations, the reaction occurred 
 regularly. From this it was concluded that. the body-cells anchor the 
 antibody during the latent period, and that anaphylaxis is the result 
 of an interaction between the cellular antibodies and the antigen. 
 
 It would appear that passive anaphylaxis is not wholly determined 
 by the amounts of antigen or antibody, but by the proportion that 
 exists between the two. For example, Friedmann,^ in his studies on 
 passive homologous anaphylaxis in rabbits, found that, by employing 
 2.5 c.c. of antiserum with from 2.5 to 0.25 c.c. of antigen, no results were 
 observed, whereas positive reactions were obtained when the amount of 
 antigen was reduced from 0.025 to 0.0025 c.c. 
 
 Ordinarily, normal guinea-pigs may be passively sensitized by 0.1 to 
 0.5 c.c. of serum injected intraperitoneally, and anaphylactized one or 
 
 I Jour. Med. Research, 1914, 30, No:. 2, 87. 
 
 " Jahr. u. d. Ergeb. d. Immunitatsf.f 1910, vi, 67. 
 
ANTI-ANAPHYLAXIS. 561 
 
 two days later by an intravenous injection (0.1 tp 0.5 c.c.) of the antigen. 
 The immune serum may be prepared by injecting rabbits with horse 
 serum, after the methods for the production o£ precipitins described in 
 Chapter 1\. 
 
 The duration of passive sensitization is quite variable. Weil* foimd 
 that guinea-pigs sensitized with a homologous serum, that is, ■«'ith the 
 serum of another guinea-pig sensitized with horse serum, remain typic- 
 ally anaphylactic as long as seventy days after the injection. With 
 heterologous sensitization, however, as "nith the serum of a sensitized 
 rabbit, hji^ersensitiveness is almost invariablj" lost by the tenth day. 
 One explanation of this would be that a heterologous serum is excreted 
 more rapidly than a homologous serum, a condition commonly observed 
 in serum therapy with any heterologous serum. 
 
 The Mechanism of Passive Anaphylaxis. — Presumably the mecha- 
 nism of passive anaphjiaxis is relatively simple, and consists in the 
 transfer of the specific protein sensitizer or amboceptor that unites the 
 anaphylactogen or protein antigen with a complement, bringing about 
 lysis or cleavage of the protein and liberation of the toxic moiety- re- 
 sponsible for the lesions and symptoms of anaphylaxis. In other words, 
 the mechanism of passive anaphjdaxis may bfe likened to passive anti- 
 bacterial imm unization, with, however, one important clinical difference, 
 namely, that whereas in the former the microorganisms are destroyed 
 mthout apparent injurj' to the host, in anaphylaxis the body-ceUs are 
 acutely poisoned. However, a similar phenomenon in the sermn 
 treatment of disease, -n-ith lysis of bacteria, may be overshadowed or 
 possibly prevented by a condition of anti-anajphylaxis. 
 
 ANTI-ANAPHYLAXIS 
 
 The term anti-anaphylaxis was first apphed by Besredka and Stein- 
 hardt to a condition of insensibility to further injection of the anaphylac- 
 togen that may follow recoveiy from anaphylaxis, or be induced arti- 
 ficially by a single or by repeated small injections of the anaphylactogen 
 during the incubation period follo'^ing the first injection, and before 
 sensitization is completed. The state is usually only temporary', the 
 animal gradually becomes sensitive again after three weeks. 
 
 Theobald Smith had observed that those guinea-pigs that had re- 
 ceived the largest dose of diphtheria toxinrantitoxin mixture more 
 frequently sundved the second dose than did tjiose that received smaller 
 
 I Jour. Med. Research, 1913, 2S, Zs'o. 2, 359. 
 36 
 
662 ANAPHY1.AXIS 
 
 doses. Rosenau and Anderson found that animals reinjected before 
 the end of the period of incubation did not become responsive until 
 some time later. Otto has also made this (3bservation, but the most 
 thorough study of the subject has come as the result of the researches of 
 Besredka and Steinhardt. 
 
 Experimental Production of Anti-anaphylaxis. — It has long been 
 known that the larger the first or sensitizing injection of antigen, the 
 greater must be the dose of the second or intoxicating injection, indicat- 
 ing that a large sensitizing injection introduces the factor tending to 
 produce the condition of anti-anaphylaxis.i Partial desensitization, 
 or anti-anaphylaxis, may be produced by tbe- injection of a sublethal 
 intoxicating dose of anaphylactogen during the period of incubation or 
 at its close. Quantitative relations between the size of the sensitizing, 
 intoxicating, and desensitizing doses of antigen and the period of in- 
 cubation have been worked out in a series of" studies by Weil,^ both in 
 the living guinea-pig and on the excised uterus, after the graphic method 
 of Dale.^ Weil found that a small sensitizing dose of horse serum (0.01 
 c.c. subcutaneously) is followed by a relatively prolonged period of 
 incubation (from fourteen to sixteen days) ; that the minimal anaphy- 
 lactic or lethal dose is small (0.02 to 0.05 c.c.) j, that the blood, as a rule, 
 does not contain more than one sensitizing unit; and that the minimal 
 desensitizing dose is small (0.01 c.c). Conversely, after repeated 
 large sensitizing doses (2 c.c. of serum subcutaneously on each of three 
 days in succession) the incubation period is shorter (about ten days 
 after the last injection) ; the minimal anaphylactic lethal dose is larger 
 (0.4 c.c), and the minimal desensitizing dose is at least more than 0.2 
 c.c. of serum given intravenously. 
 
 Besredka and Steinhardt observed that the refractory state could 
 easily be developed in sensitized guinea-pigs by one of the following 
 methods: (1) The intracerebral injection of 0.25 c.c. of horse serum 
 before the expiration of the period of incubation (twelve days). (2) The 
 intracerebral injection of less than the fatal dofee (from 0.002 to 0.025 c.c.) 
 after the period of incubation. (3) Rectal injections of from 5 to 10 
 c.c. of serum. (4) By slowly reinjecting small amounts while the 
 animal is deeply narcotized with ether or alcohol. As shown later by 
 Rosenau and Anderson, a narcotic may mask but does not prevent the 
 occurrence of severe or fatal symptoms. Of these methods, Besredka 
 
 1 Jour, Med. Research, 1913, 29, No. 2, 233;; 1914, 30, No. 3, 299. 
 
 2 Jour. Pharm. and Exper. Ther., 1913, iv, 1*67. 
 
SPECIFICITY OF ANAPHYLAXIS 563 
 
 prefers the rectal injection, or, better still, th'e subcutaneous injection 
 of less than a fatal dose. 
 
 The subject of anti-anaphylaxis is of great "itnportance from its rela- 
 tion to serum therapy. No satisfactory method for producing this 
 state in a sensitized person has as yet been devised, owing, probably, to 
 the important quantitative factors shown, by the studies of Weil, to exist. 
 This subject will be discussed again in the following chapter, under the 
 head of Serum Sickness. 
 
 The Mechanism of Anti-anaphylaxis. — A true explanation of this 
 phenomenon camiot as yet lie given. In the first place, the term anti- 
 anaphylaxis cannot be considered a proper one, as the animal is not 
 entirely and permanently anti-anaphylactic, but subsequently becomes 
 sensitive. The blood-serum of a refractory or anti-anaphylactic animal 
 does not confer a similar condition on a second sensitized animal. 
 
 As previously stated, Friedberger believes that the refractory state 
 is due to neutralization or absorption of the anaphylactic antibody by 
 the antigen, but this explanation does not fit. in with the facts, first, 
 because the serum of an anti-anaphylactic animal will still passively 
 sensitize a normal animal, and, secondly, as shown by Weil, passive 
 anaphylaxis of a guinea-pig, such as that induced by the injection of a 
 rabbit antihorse serum, may be prevented for at least eight days by a 
 previous injection of normal rabbit or sheep serum. In other words, 
 it would appear that normal rabbit and sheep serum maj'' protect the 
 body-cells of the guinea-pig against the anaphylatoxin produced by 
 horse protein and horse anaphylactin or antibody. In explanation of 
 this paradoxic and non-specific reaction Weil, J who believes in the cel- 
 lular theory of anaphylaxis, has tentatively advanced the hypothesis 
 that the indifferent serum persists in the body-cells and markedly lowers 
 the reactivity of the cellular antibodies. 
 
 SPECIFICITY OF ANAPHYLAXIS 
 The anaphylactin, or so-called anaphylaxis antibody, displays quite 
 the same characteristics of specificity as do the other immune anti- 
 bodies, in that proteins of closely related species tend to interact, whereas 
 proteins of very distinct biologic ov cliemical nature are easily distin- 
 guished. In other words, the ana-phylactic reaction is highly specific, and 
 of considerable value in the study and identification of different proteins. 
 For example, Dale has recommended the use of the graphic method in 
 
 ' Jour. Med. Research, 1914, 30, Ko. 3, 299. 
 
564 ANAPHYLAXIS 
 
 vitro with excised muscle (uterus) for the identification of the protein 
 substances, such as blood-stains. Guinea-pigs sensitized with human 
 serum will react best with human serum, and to a lesser extent with 
 that of the higher apes, but not at all with the serum of the dog, ox, or 
 fowl. Wells and Osborne,^ working with purified vegetable protein, 
 were able to demonstrate that a single isolated protein (hordein or 
 gliadin) may contain more than one antigenic radical, and that not the 
 whole protein molecule, but certain groups thereof, determine the 
 specificity. Wells ^ was able to distinguish in the hen's egg five dis- 
 tinctly different antigens, and these corresponded to five proteins that 
 were isolated by chemical methods, so that. it would appear that the 
 specificity of the anaphylaxis reaction is determined by the chemical struc- 
 ture of the reacting proteins, rather than by their biologic origin. Whether 
 the chemical differences that determine specificity are of Jifiiantitative 
 nature, or whether they are sometimes dependent upon the number and 
 relationship of the amino-radicals, as was suggested by Pick, remains 
 to be determined. 
 
 1 Jo»ir. Infect. Dis., 1913, 12, 341. ^ Jour= Infect. Dis., 1911, 9, 147. 
 
Part IV 
 
 CHAPTER XXVIII 
 
 ANAPHYLAXIS IN ITS RELATION TO INFECTION AND 
 IMMUNITY 
 
 In reviewing our knowledge of the nature and mechanism of anaphy- 
 laxis we found that ordinarily innocent substances, such as sterile normal 
 serum and egg-albumen, when injected into animals, may give rise to 
 severe and even fatal intoxication, not because these substances are 
 poisonous in themselves, but because the antibodies which they stimu- 
 late the body-cells to produce react upon the innocent protein, causing 
 its cleavage, with the liberation of a harmful poison. Here, indeed, we 
 have an example of antibodies apparently injuring our body-cells instead 
 of protecting them. And now the question very naturally arises, are 
 the lesions of the many diseases caused in a siinilar manner, the anti- 
 bodies splitting the bacterial cell and liberating the protein poison? In 
 view of the fact they may do actual harm, in sorae instances at least, are 
 antibodies really protective and beneficial, and if so, in what manner? 
 
 It is not my purpose to review here the general subjects of infection 
 and immunity, but to discuss briefly the intimalte and inseparable con- 
 nection of what we call anaphylaxis or allergy witli infection, and to show 
 that anaphylaxis is really the first step in th§ process of immunity; 
 indeed, that well-marked antibacterial immunitj- is an example of an 
 early and efficient "anaphylactic" reaction. In other words, while the 
 striking and severe symptoms of serum anaphylaxis in either man or 
 lower animals give us the impression that we are here dealing with some 
 new, distinct, and strange phenomenon, these are, in fact, but exag- 
 gerated and severe examples, largely dependent upon quantitative 
 factors of what occurs during each infection. More than this, these 
 symptoms are due in part to the action of a poison formed or released 
 through the destruction of the antigen by the antibody; the antibody 
 being, therefore, apparently imperfect in its action, as it does not neu- 
 tralize the protein poison. Taking, as an example, a substance, such as 
 sterile horse serum; while ordinarily harmless itself, bad effects may 
 follow its injection at once if antibodies are present in our body-fluids, 
 or later if some of the serum persists in the body until antibodies are 
 
 565 
 
566 ANAPHYLAXIS IN RELATION TO INFECI'ION AND IMMUNITY 
 
 produced. It would appear, therefore, that when the antibodies are 
 produced for a given infectious agent, then {he severity of the disease 
 becomes apparent; that the lesions and symptoms are due not only to 
 the infecting microorganism and its products,, but to the results of their 
 destruction. An invading army may do some' pillaging, but the greatest 
 injury is done when the defenders begin the attack, the resulting fire 
 and destruction doing more harm than the invaders themselves. 
 
 While Vaughan and his coworkers were studying the protein poison 
 171 vitro, and Friedberger was investigating it m vivo, the former and then 
 the latter aiming to show that the poison liberated from the protein 
 molecule through the action of specific ferments is responsible for the 
 lesions and symptoms of disease, von Pirquet Avas studying the question 
 from the clinical aspect, formulating a working hypothesis on the nature 
 of infection and immunity based upon the principles of anaphylaxis, a 
 theory that has been supported by experimental data and has thrown a 
 new light upon the nature and mechanism of these processes. 
 
 RELATION OF ANAPHYLAXIS TO INFECTIOUS DISEASES 
 
 Although we may not be in general accord regarding the mechanism 
 of anaphylaxis, there is, however, general agreement as regards the nature 
 of the anaphylatoxin responsible for the lesions and symptoms of ana- 
 phylactic intoxication, namely, that it is a protein cleavage product. 
 In other words, a foreign protein and, indeetS, the misplaced protein of 
 our own body-cells, may be disrupted or digested by an antibody, and 
 liberate or generate a poison that, being derived from protein, is known 
 as the protein poison. In the chapter on Infection it was stated that 
 Vaughan ami his collaborators regard this protein poison as the same 
 for all proteins, and as responsible for all infectious diseases, the par- 
 ticular lesions and symptoms of each disease being dependent upon the 
 site of the infection and, accordingly, upon the location of the protein 
 poison. Similarly, in the preceding chapter we asserted that anaphy- 
 laxis is ascribed to an exactly similar phenomenon, namely, the splitting 
 of the foreign protein by an antibody (ferment) and the liberation of a 
 protein poison. In studying serum sickness,, an anaphylactic phenom- 
 enon frequently observed in man following the administration of horse 
 serum, von Pirquet argued that the period of from eight to ten days 
 usually following the injection before the appearance of symptoms was 
 the time required for the production of the antibody, which then re- 
 acted upon the serum still remaining in the body-cells and fluids, and 
 
RELATION OF ANAPHYLAXIS TO INFECTIOUS DISEASES 567 
 
 that the products of this interaction caused the lesions and symptoms 
 of serum sickness. It was then but a short step to apply these prin- 
 ciples to other infectious diseases. This "period of incubation" was 
 formerly regarded as representing a stage during which the infecting 
 microorganisms multiply in the body of the infected individual, to that 
 point at which they could give rise to S3Tnptoms of disease through the 
 agency of their toxins or through interference with the metabolism of 
 the host in other ways. But, as von Pirqu"et has pointed out, this 
 theory does not hold in serum sickness, as the serum may be sterile and 
 no infecting microorganisms are at work. Instead, he and Vaughan 
 would have us believe that during this period antibody formation is 
 taking place, and that an antibody-antigen reaction will occur with the 
 development of pathologic changes and symptoms just as soon as these 
 changes have progressed to a certain point. The period of incubation 
 will vary not only in point of time of reaction, but also qualitatively 
 and quantitatively, and using this as a basis von Pirquet recognizes 
 three main groups, depending upon whether the antibody is present in 
 our body-fluids as the result of a previously acquired infection (acci- 
 dental or by vaccination), or whether it must first be developed. 
 
 Group I : Reaction appears after eight to twelve days, as in measles, 
 smallpox, whooping-cough, chickenpox, and other infectious diseases in 
 which the antibodies must be developed before the symptoms are pro- 
 duced. This interval corresponds quite clostly to that observed in 
 serum sickness. If at this time the antigen, — (', e,, either the albumins 
 of the horse serum, if we are dealing with serum injections, or the bac- 
 teria in case of an infection — has disappeared from the body, no symptom 
 will, of course, result; if, however, some of the material is still present, 
 a reaction occurs, during which the protein poison (anaphylatoxin) is 
 produced, and to which, in turn, the symptoms that then develop may 
 logically be attributed. 
 
 Group II: The reaction appears after three to seven days. If, on 
 the other hand, the secondary infection, as, e. g., pneumonia, erysipelas, 
 etc., is acquired after a lapse of months or several years, or if the second 
 injection of serum is given after this time, i. e., at a time when the anti- 
 bodies called forth by the primary infection or first injection have 
 disappeared, a certain interval of time will elapse before symptoms of 
 sickness develop, as in the case of the first group. This interval, how- 
 ever, instead of being from eight to twelve days, is now from three to 
 seven, a fact readily explained on the basis that a cell that has once been 
 stimulated to active antibody formation will subsequently respond to 
 
568 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 the same stimulus with increased activity. This has been called by 
 von Pirquet the accelerated reaction. 
 
 Group III: The reaction appears immediately. If the first injection 
 of horse serum or infection is followed by actual disease or vaccination, 
 the reinjection or reinfection is acquired at a time when the antibodies 
 are present in the circulation in considerable amount, a reaction will 
 occur either immediately or within the first twenty-four hours. This 
 reaction may be quite virulent in intensity^ although it is shorter in 
 duration than when it occurs in the first group, von Pirquet speaks 
 of this as the immediate reaction. It is to be observed in cases of serum 
 sickness where the symptoms develop almost immediately following an 
 injection of serum months and even years after a previous injection; 
 it also occurs in cowpox vaccination, where a local reaction takes place 
 very quickly and soon disappears after a previous attack of smallpox 
 or vaccination. 
 
 If the antibodies are present in lesser amounts, the reaction may occur 
 in from the second to the fourth day; this, is called the torpid early 
 reaction. 
 
 At the time of the second injection of serum or reinfection with bac- 
 teria a small amount of antibody may still be present; this will give an 
 immediate though mild reaction, and is not enough to neutralize the 
 total amount of foreign protein introduced. A portion of the latter, 
 therefore, will result in the production of an additional amount of anti- 
 body, which occurs in an accelerated manner, and coming in contact 
 with some of the free antigen, gives rise to the accelerated reaction. 
 Hence we may have an immediate, followed by an accelerated, reaction. 
 
 To illustrate these principles, von Pirqufett names vaccinia as an 
 example of an acute infection in which the processes may be observed 
 on the skin. As the result of vaccination a colony of microorganisms 
 is formed on the skin. For the first two days the local response is 
 evidently traumatic in character. After the third or fourth day the 
 specific reaction sets in, in the form of a small papular elevation sur- 
 rounded by a small areola due to the local action of toxins or protein 
 poison from disintegrated microorganisms. By the eighth day a 
 vesicle has formed, and from its contents new colonies can be grown on 
 thousands of other arms. But one or two days later the ferment-like 
 antibody appears. The colony is attacked, its contents are digested, 
 a toxic substance is formed that diffuses into the neighboring tissues, 
 and the intense local inflammation which we call the areola appears. 
 In addition the toxin enters the general circulation and fever sets in. 
 
RELATION or ANAPHYLAXIS TO INFECTIOUS DISEASES 569 
 
 Simultaneously the microorganisms are destroyed, and we may no 
 longer be able to vaccinate with the contents of the now yellow pustule. 
 After two or three days the real struggle is ended, although the local 
 lesion may be aggravated by secondary infection, and the body contains 
 the new antibody for a long time. 
 
 If we now revaccinate, the antibody present will at once attack and 
 digest the microorganisms introduced into the scarification, and, as 
 these do not have time to multiply, only an extremely small amount of 
 toxin is formed, which gives the "immediate or early reaction" in 
 vaccinia. If a number of years have elapsed between the first and the 
 second vaccination, antibodies may be absent' or present in only small 
 amount, but the body-cells have been "keyed up" by the first vac- 
 cination, and hence react more quickly to the second. The antibodies 
 are produced in from three to five days, and attack the microorganisms 
 before they have had time to multiply in sufficient numbers; the rel- 
 atively small amount of digestion product produces a comparatively 
 mild local inflammation and practically no general symptoms. This is 
 sometimes known as the "immunity reactiofi, " or vaccinoid, and is 
 illustrated in Fig. 131. 
 
 It would, of course, lead us too far were we to analyze all the different 
 infectious diseases along these lines; suffice it= to say that the anaphy- 
 lactic principle serves to explain many points in the clinical sympto- 
 matology of disease that were not explained heretofore, or were ascribed 
 to the effects of the bacteria and the bacterial products. I am not 
 among those who, with Vaughan, would ascribe all symptoms to the 
 protein poison alone; nor do I regard the part played by the microorgan- 
 ism as simply dependent upon whether or not it" can grow in the bodj^ and 
 produce the ferment antibody. In the acute toxemias, for instance, such 
 as tetanus and diphtheria, where the amount and toxicity of the toxin 
 are out of all proportion to the number of bacteria, and where the 
 symptoms develop after a very brief incubation period, tissue changes 
 and symptoms are in all probability not primarily due to any antigen- 
 antibody reaction, with liberation of the protein poison, but are rather 
 to be attributed to a direct action of the tqxin upon the body-cells, 
 especially upon those for which the toxin possesses a special affinity. 
 When the antibodies appear, we may rightfully assume that a ferment, 
 in the nature of a protein amboceptor or lysin, appears with the antitoxin, 
 and theoretically we may expect a clinical reaction due to the protein 
 poison or anaphylatoxin liberated from the bacilli. It may be that 
 diphtheria antitoxin is in the nature of this ferment, or antitoxin and 
 
570 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 ferment may exist together and in this maimer afford an explanation 
 for the local destruction of the bacilli and the, cleaning up of the mem- 
 brane, the effects of the anaphylatoxin being overshadowed by the true 
 toxin, or expressing itself in the paralyses and other symptoms commonly 
 ascribed to the toxones. von Pirquet conce'des that in scarlatina the 
 primary symptoms of the malady, i. e., the eruption and angina, are due 
 to a pure toxin effect, — to the true scarlatinal virus, — but that the sequelge, 
 and notably the nephritis, are the expression of the action of anaphyla- 
 toxius that are formed when the corresponding antibodies are produced. 
 Likewise in such infections as typhoid fever and pneumonia, I 
 cannot eliminate the action of true toxins arid endotoxins, and ascribe 
 all the symptoms to the protein poison or anaphylatoxin. It is not 
 always true that the incubation period is devoid of symptoms. Mild 
 and evanescent symptoms are frequently present, and it is but natural 
 to ascribe these to the effects of bacterial products themselves. After 
 a time, when the bacteria have multiplied and reached special tissues, 
 the symptoms become more intense and the typical lesions are produced. 
 These may be ascribed to the result of the combined action of toxins 
 themselves and the protein poison, rather than to the protein poison 
 itself to the entire exclusion of the metabolic products of the bacteria. 
 In the present state of our knowledge it is, of course, very difficult to 
 decide which symptoms in a given disease are due to bacterial toxins, 
 which to endotoxins, which to ptomains, which to a mechanical action 
 of the bacteria, and which to the protein poison or anaphylatoxin. 
 While it is perfectly true that we can now understand symptoms as due 
 to protein poison that cannot be asci'ibed entirely to toxins and endo- 
 toxins, it seems to me unwise to ascribe all lesions and symptoms to the 
 toxins, on one hand, or the protein poison, on the other. It is necessary 
 for us to know the manner in which the prbtein poison is produced, 
 how infection is so closely allied to what we know as anaphylaxis, and 
 that in any one disease, such as diphtheria, tetanus, and pneumonia, the 
 toxins and endotoxins are of primary and immediate importance, whereas 
 in others, such as smallpox, chickenpox, and wHooping-cough the protein 
 poison itself is of primary importance and that in all disease all factors 
 may be concerned to a more or less degree. 
 
 RELATION OF ANAPHYLAXIS TO NON-INFECTIOUS DISEASES 
 
 When we come to consider non-infectious diseases, — and by this I 
 refer particularly to the symptom-complex of serum disease and those 
 
SERTJM DISEASE 571 
 
 conditions commonly ascribed to idiosyncrasies toward certain sub- 
 stances, such as horse asthma, satinwood dermatitis, buclcwheat poison- 
 ing, urticarias due to the ingestion of strawberries, pork, and the like, — 
 the relation of anaphylaxis to the processes involved is more intimate. 
 Here, indeed, we may regard the symptoms as entirely anaphylactic in 
 origin and character, as the substances in themselves, such as serum, 
 egg-albumen, horse effluvia, etc., are not regarded as toxic and we have 
 not the coincident effects of toxins, endotoxins, and ptomains, as in 
 bacterial infections. 
 
 Here, indeed, we may accept Vaughan's and Friedberger's theory 
 as to the action of the protein poison in its entirety, regarding it the same 
 in all proteins, and set free from the protein molecule by the so-called 
 "ferment," which is in the nature of a protein sensitizer or amboceptor, 
 and which, with a complement, brings about' lysis or cleavage of the 
 molecule, just as a similar ferment or antibody, called bacteriolysin, 
 disrupts a bacterial cell or its protein constituents. In short, we may 
 say that the work of Vaughan and his collaborators has shown us the 
 presence of this poison in all protein material with a method of extracting 
 it in vitro. Friedberger and his collaborators have shown how this 
 poison is set free in the body through the agency of the "ferment." 
 von Pirquet has studied the question at the beflfeide, dispensing with the 
 microscope and test-tube and depending solely upon the vital processes 
 and reaction, skilfully combining laboratory findings with bedside ob- 
 servations — in fact, he built up his theoiy on the nature of infection and 
 immunity before the role of the protein poison was discovered, and 
 subsequent work has added support to his conceptions. Of clinical and 
 practical importance in this connection are serum disease and hyper- 
 sensitiveness or idiosyncrasies for other proteins and certain drugs. 
 
 SERUM DISEASE 
 This name was applied by von Pirquet and Schick^ to the various 
 clinical manifestations, such as eruptions, fever, edema, and pain in the 
 joints, following the injection of horse serum. These symptoms are due 
 to the horse serum itself, for, as was early shown by Johannessen,^ 
 Bokay,' and others, they may manifest themselves after the injection of 
 sterile normal horse serum. The serum, moreover, of certain horses 
 
 '"Die Serumkrankheit," Leipsic, Deuticke, 1905. 
 2 Deutsch. med. Wochenschr., 189,5, 21, 855 
 ' Jahresb. f. Kindesh., 1897, xliv, 133.- 
 
672 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 appears to be more likely than that of others to cause these symptoms, 
 thus accounting for the fact that one lot of antitoxin will cause a higher 
 percentage of serum sickness than will another. A concentrated serum 
 is not so likely to produce serum sickness as whole serum, owing partly 
 to the fact that smaller doses of it are given. According to RoUeston and 
 Ker, the frequency of serum sickness is, as a rule, in direct proportion to 
 the amount of serum given, and in inverse ratio to the severity of the 
 attack; in other words, we may expect to encounter it most often in 
 mild and moderately severe cases that have received very liberal dosages 
 of serum. 
 
 The Nattire of Serum Disease. — Serum sickness is a true anaphy- 
 lactic phenomenon. We are prone to call the severe, fatal, and rare 
 instances of death following serum injection" examples of anaphylaxis, 
 and to regard serum sickness as a different condition. Both are funda- 
 mentally the same, except that in the first instance the body-cells are, 
 for some unknown reason, unduly and highly susceptible to the protein 
 poison. Fortunately, this undue hypcrscnsitiveness is frequently fore- 
 shadowed by the asthmatic or hay-fever-like attacks which the suscep- 
 tible person may exhibit when he enters stables or is otherwise around 
 horses. It goes without saying that horse serum should never be given 
 to such persons. 
 
 While serum sickness is usually due to horse serum, for the reason 
 that the horse is so commonly employed in the preparation of various 
 curative serums, the serum of the ox, rabbit, tod other animals may in- 
 duce the same train of symptoms in addition, in some instances, to 
 producing a direct toxic effect. 
 
 The foreign serum introduced into the human circulation acts as an 
 antigen, and calls forth the production of an antibody, the so-called 
 "ferment," which reacts with the antigen (remaining serum), causing 
 its cleavage and liberating a protein poison that acts primarily upon 
 smooth muscle and is responsible for the legions and symptoms. An 
 immediate reaction rarely follows the first injection of serum unless the 
 patient is one of those unfortunate but rare persons who in some manner 
 have been rendered highly sensitive to horse protein. In the majority 
 of instances symptoms do not develop for from eight to twelve days, 
 during which time the antibody is being produced. When antibody 
 formation has reached a certain point, it reacts upon any of the horse 
 serum that may persist in the circulation, producing the anaphylactic 
 or protein poison. If the dose of serum has bcien small, antibody forma- 
 
SEEUM DISEASE 573 
 
 tion goes on as usual, but the serum may not persist in the circulation. 
 Hence symptoms do not develop in such a person, although if reinjected 
 subsequently symptoms will appear. This is one reason why a con- 
 centrated serum is not so likely to produce serum sickness, since a smaller 
 quantity of it is injected. 
 
 If, however, the patient has received an injection of serum some 
 months previously, a reinjection is likely to be followed by an immediate 
 reaction, that is, the symptoms appear within from twenty-four to forty- 
 eight hours. If the first injection had been given a year or more previ- 
 ously, no antibody may be present in the blood, so that an immediate 
 reaction does not occur. If, however, the cells once stimulated are 
 "keyed up" indefinitely, and, accordingly, antibody formation is quite 
 rapid, so that we find symptoms developing in from four to seven days 
 after injection — the accelerated reaction. Or a small amount of antibody 
 may be present which gives a mild reaction around the site of serum 
 injection, followed in from four to seven days by a general reaction, 
 this being an immediate followed by the accelerated reaction. 
 
 As was previously stated, anaphylaxis is specific — that is, a person 
 receiving ox serum in the first injection would not be affected subse- 
 quently by an injection of horse serum, but only by ox serum. For this 
 reason it has been recommended that diphtheria antitoxin to be used for 
 prophylactic purposes should be prepared by- immunizing cattle, re- 
 serving the horse serum for treatment if the disease should be contracted 
 subsequently. 
 
 Symptoms of Serum Disease. — The most typical of these symptoms 
 are rash, fever, and prostration, besides joint and muscle pains, edema, 
 and adenitis. 
 
 The most obvious and important of the symptoms are undoubtedly 
 the various forms of rash. In 1000 consecutive cases of diphtheria 
 treated with antitoxin in the Philadelphia Hospital for Contagious 
 Disease, a rash developed in 430, or 43 per cent. The time of appear- 
 ance of the eruption depends, as was just stated, upon whether or not 
 the patient has been injected on a previous occasion, and if so, the 
 length of the interval between the first and subsequent injection, and 
 to a lesser extent upon the amount of the first injection, these factors 
 influencing the quantity of antibody present at the time of reinjection. 
 Of the 430 cases just mentioned, the time of appearance of the rash, in 
 days, after subcutaneous injection of antitoxin^ was as follows: 
 
574 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 TABLE 22.— TIME OF APPEARANCE OF SEKUiM RASH IN 430 CASES OF 
 
 SERUM DISEASt; 
 
 Day upon which the 
 
 Rash AppEAREn after Injection 
 
 ToTAT, Number 
 
 
 
 OF Diphtheria Axtitoxin 
 
 SHfnviNG Rash 
 
 
 First . 
 
 4 
 6 
 
 0.9 
 
 fiecond - - - 
 
 
 
 1.4 
 
 Third 
 
 
 
 7 
 
 1.6 
 
 Fourth .... 
 
 
 
 30 
 
 6.9 
 
 Fifth 
 
 
 
 35 
 
 8.1 
 
 Sixth 
 
 
 
 75 
 
 17.6 
 
 Seventh. . . 
 
 
 
 65 
 
 15.1 
 
 Eighth .... 
 
 
 
 85 
 
 19.8 
 
 Winth .... 
 
 
 
 44 
 
 10.2 
 
 Tenth .... 
 
 
 
 39 
 
 9,0 
 
 Eleventh. . 
 
 
 
 22 
 
 5.1 
 
 Twelfth . . . 
 
 
 
 5 
 
 1.1 
 
 Thirteenth . 
 
 
 
 6 
 
 1.4 
 
 Fourteenth 
 
 
 
 5 
 1 
 1 
 
 1.1 
 
 Fifteenth. . 
 
 
 
 0.2 
 
 Sixteenth 
 
 0.2 
 
 In this table are included some cases of reinjection, as, e. g., scarlet- 
 fever patients who received a routine immunizing dose of antitoxin 
 upon admission, and another after having contracted diphtheria; also 
 cases of diphtheria that became reinfected within a few months after 
 their discharge from the hospital. As will be seen, about 63 per cent, 
 of cases develop a rash between the sixth arid the ninth day after the 
 injection of antitoxin. 
 
 Three main types of rashes are generally recognized : 
 
 1. Urticarial Rashes. — If we include in this group all eruptions that 
 present a resemblance to urticaria, these rashes are the most common, 
 constituting from 70 to 90 per cent, of all eruptions. They usually 
 appear after the seventh day, becoming manifest first about the site of 
 injection. Large, irregularly shaped, and scattered blotches appear, 
 frequently with true wheals in the center (Fig. 119), accompanied by 
 intense itching and irritation. Sometimes true wheals do not appear. 
 The rash is often very profuse, and fresh blotches may continue to ap- 
 pear for two or three days. Occasionally, the rash is quite sparse and 
 mild, and may disappear within twenty-four hours. 
 
 2. Multiform Rashes. — This type of rash is quite common. It is 
 often circinate in its arrangement, or occurs in large blotches mixed with 
 a scattered morbilliform or measly form of rash (Fig. 120). Different 
 parts of the body may present different appearances at the same time. 
 This rash may occasionally closely siiimlate true measles, especially 
 since it involves the face, and as the conjunctivae are likely to be con- 
 
; Fig. il9. — Urticarial Rash of Serum Sickness. 
 Case of laryngeal diphtheria; had ref:ci\ecl 40,000 ujiils of aiililoxin eight days 
 previously; urticarial rash first appeared about thirty hours l^efore this ilrawing 
 was made. 
 
I 
 
 A 
 
 , * '1 '^ 
 
 Fig. 120. — Multiform Rash of Serum Sickness. 
 Child with laryngeal diphtheria on the skth day after receiving 65,000 units of 
 
 antitoxin. 
 
SKRUM DISEASE 575 
 
 gested in almost any variety of scrum sickness. It is differentiated 
 from measles by the fact that Koplik's spots are absent, there is no pro- 
 dromal rise in temperature, the papules are not elevated above the skin 
 as much as in measles, and that the eruption frequently starts from the 
 site of injection, instead of on the face. 
 
 3. Smrlaliniforni Rashes. — This type of rash occasionally occurs, 
 and may bear so close a resemblance to true scarlet fever as to be indis- 
 tinguishable from it. The eruption usually appears early, that is, in 
 from the first to the sixth day after injection, and may vary from a 
 uniform erythema which first appears about the site of injection, to a true 
 punctate scarlatinal rash. The differentiation' of these rashes from the 
 true scarlet-fever eruption is one of the greatest sources of trouble in 
 hospital practice, especially when they develop in the diphtheria wards, 
 where occasional cases of true scarlet fever are: always likely to apijear 
 from time to time. The absence of the following symptoms, or their 
 occurrence only in mild degree, would favor a diagnosis of serum disease : 
 Fever, or, at least, the presence of but a mild pyrexia, the vomiting, the 
 typically fm'red tongue, the angina, or at least but a mild throat involve- 
 ment, and the leukocytic inclusion bodies — all forming the symptom- 
 complex of true scarlatina. In many instances, however, it is necessary 
 to isolate the patient, when speedy recovery, absence of complications, 
 and less definite desquamation, which does not involve the palms and 
 soles, indicate that the patient was suffering from serum disease and not 
 from scarlet fever. 
 
 Severe Serum Disease. — The severer forms of serum disease, 
 characterized by sudden onset, — often within a few minutes after the 
 injection of serum, — extreme dyspnea, prostration, and death are, 
 fortunately, rare, about 30 cases in all being now on record. While 
 physicians are justified in exercising care and caution in the administra- 
 tion of serum, there are no absolute contraindications to its use except 
 status Ijrmphaticus and those instances where the person is known to be 
 hjrpersensitive to horse serum. Occasionally sudden and severe dyspnea 
 and prostration accompany an immediate reaction when a reinjection 
 of serum is given within a few weeks after the first. Persons suffering 
 with asthma are also bad risks, especially since the effects of serum 
 disease upon the bronchial mucosa are likely to aggravate the already 
 existing condition. This subject will be discussed in greater detail 
 under the head of Contraindications to Passive Immunization in Chap- 
 ter XXX. 
 
576 ANAPHYLAXIS IN EBLATION TO INFECTION AND IMMUNITY 
 
 The Prevention of Senun Disease. — Anaphylactic phenomena can 
 usually be expected to occur if serum is given within a year or so follow- 
 ing a previous injection. Persons who experience discomfort when 
 about horses should always be closely questioned. Fig. 121 shows the 
 urticaria-like lesion that develops on the arm of one of my colleagues 
 following scarification and the thorough application of a drop of horse 
 serum. This man is also susceptible, to a lesser extent, to guinea-pig 
 and rabbit scrum, and is seized with sneezing and distressing dyspnea 
 after entering a house where these animals are kept. Thayer has re- 
 ported a case of buckwheat hypersensitiveneSs where vaccination with 
 the flour resulted in a local reaction. It would be well for physicians 
 to make this simple test whenever they suspect a patient of being hyper- 
 sensitive to horse serum. All that is necessary is to cleanse the arm with 
 alcohol, scarify, as when vaccinating with cowpox virus, and rub in a 
 drop of the diphtheria antitoxin with a tooth-pick or some suitable 
 instrument. The reaction usually appears within fifteen minutes, and 
 there are usually no, or but very slight, general symptoms. 
 
 Various means have been tried and advocated to bring about a state 
 of anti-anaphylaxis or desensitization of the patient: 
 
 1. A preliminary injection of 0.5 c.c. of serum as antitoxin may be 
 given, followed in three or four hours by the regular injection. While 
 it is difficult to produce anti-anaphylaxis (see Chapter XXIX), this 
 method is in common use. 
 
 2. According to Auer and Lewis, ^ Auer,^ Anderson and Schultz,' 
 Biedl and Kraus,^ and Karsner,^ atropin sulphate has a distinct protective 
 action against the asphyxia of acute anaphylaxis in the guinea-pig. It 
 may be well to administer from ttt to ttc grain of the drug hypoder- 
 mically before injecting the serum, and once or twice subsequently, 
 at intervals of twelve hours, in those cases in which hypersensitivencss is 
 suspected, especially when serum has been injected within a year's 
 time. 
 
 3. Rectal injections of scrum have been advised by Besredka, who 
 bases his recommendations upon the fact that by this route absorption 
 takes place slowly and desensitization is gradual. While in the chapter 
 on Serum Therapy I have frequently advised the intravenous injection 
 of serum, it is to be understood that this applies to first doses. It is 
 
 1 Jour. Amer. Med. Assoc, 1909, liii, 458; Jour. Exper. Med., 1910, xii, 151. 
 
 2 Amer. Jour. Physiol., 1910, xxvi, 439. 
 
 = Proc. Soc. Exper. Biol, and Med., 1910, vii, 32. 
 « Wien. klin. Wochenschr., 1910, xxiii, 385. 
 « Jour. Amer. Med. Assoc, 1911, Ivii, 1023. 
 
Fig. 121. — Local Sert:m Anaphylactic Reactions. 
 Dr. 'W. P., anaphylactic reactions fifteen minuteg after application of serum; 
 rabbit serum in the upper; horse serum in the lower, kjubject to asthmatic attacks 
 when in an animal house or stable where rabbits, horses, and guinea-pigs are kept. 
 
IDIOSYNCRASIES 577 
 
 far mare dangerous to give serum intravenously to those injected on a former 
 occasion; in these instances the -physician will do well to give his injections 
 intramuscularly or suhcutaneously , when, if symptoms develop, they will 
 not he explosive nor so dangerous. Since rectal injections are usually 
 refused, the serum may be administered very slowly suhcutaneously. 
 Friedberger and Mita have advocated the slow intravenous infusion of 
 serum — a drop at a time. 
 
 The Treatment of Serum Disease. — In the majority of instances no 
 treatment is necessary. Calcium chlorid and; lactate, in doses of from 
 3 to 5 grains, have been advocated as a prophylactic and as a cure, but 
 they are of doubtful utility. Soothing lotions, a brisk cathartic, and 
 sedatives are indicated, and occasionally an opiate is advisable. 
 
 In the rare acute attacks marked by extreme dyspnea or rapid and 
 shallow breathing with rapid and feeble pulse, atropin and caffein should 
 be administered hypodermically. 
 
 miOSYNCBASIES 
 
 Studies in anaphylaxis have also offered an explanation of many, if 
 not of all, of those peculiar instances in which the inhalation of some 
 animal effluvium or of the pollen of certain plants or the ingestion of 
 certain food-stuffs and drugs is followed by a train of symptoms, among 
 which asthma and an urticarial rash are usually quite prominent. 
 Hitherto these manifestations have not been understood, and were 
 simply classed as idiosyncrasies — a term that is correct if we can make it 
 mean hyper sensitiveness, for experimental investigation leaves little 
 doubt but that in these persons antibodies for the substances in question 
 are present which attack the particular protein when it gains access to 
 the body, liberating the poison responsible for the symptoms. One 
 remarkable feature of these instances of idiosyncrasy, however, is the 
 extreme hj^persensitiveness of the body-cells, especially in those cases 
 where the inhalation of such iufinitesimal quantities of protein as are 
 contained in the air will bring on a typical asthmatic attack in a person 
 hypersensitive to horse protein. 
 
 Examples of idiosyncrasy are relatively common. Susceptible per- 
 sons learn to know that the ingestion of this or that substance is sure to 
 be followed by various distressing symptoms. How and when these 
 persons became hjrpersensitive are usually not known. In some in- 
 stances the condition is found in one or both parents and in several 
 members of the same family, making it appear to be hereditary. It is 
 37 
 
578 ANAPHYLAXIS IN RELATION TO INFECTJON AND IMMUNITY 
 
 well known that animals may be sensitized by feeding them proteins 
 not usually present in their diet, as by giving guinea-pigs horse serum or 
 the flesh of other animals. 
 
 Among the commoner examples of this form of allergy or anaphylaxis 
 may be mentioned : 
 
 1. Horse asthma, observed among those persons who are seized with 
 sneezing, cough, dyspnea, coryza, and prostration when they come near 
 horses, as in a stable or when driving. 
 
 2. Hay-fever, first ascribed to the pollen of plants by Elliotson in 
 1831, and thoroughly studied by Dunbar. Wolff-Eisner was the first 
 to regard the reaction as a phenomenon of hj^ersensitivity or anaphy- 
 laxis. Individuals subject to hay-fever show a uniform series of symp- 
 toms at certain definite seasons, either in the early summer or in autumn. 
 These are a reddening, swelling, and watering of the eyes, sneezing, a 
 sore feeling in the throat and larynx, and asthmatic disorders. The 
 instillation into the eye of a 1 per cent, solutibn of pollen in physiologic 
 salt solution is usually sufficient to elicit a: typical attack. Certain 
 persons are susceptible to various weeds, and each usually knows the 
 particular weed to avoid. In hay-fever we have the most marked in- 
 stances of extreme hypersensitiveness; persons may be seized with an 
 attack when some distance from the particular weed in question. Similar 
 phenomena are occasionally observed among workers in satinwood 
 (satinwood dermatitis). 
 
 3. Certain foods, such as egg-albumen, buckwheat (phagopyrismus) , 
 pork, oysters, clams, lobster, cheese, and various fruits, such as straw- 
 berries, gooseberries, and even vegetables, may act as poisons when in- 
 gested by persons who are hypersensitive to them. The symptoms vary 
 from a feeling of "indigestion" and "heartburn" to severe diarrhea, 
 vomiting, asthma, and the development of aji itching urticarial rash. 
 In some instances it has been possible passively to sensitize guinea-pigs 
 against the particular protein by injecting a few cubic centimeters of the 
 patient's serum. Thus Bruck succeeded in doing this with the serum of a 
 person hj^ersensitive to pork. In many instances it is possible to 
 demonstrate the hypersensitive state by rubbing a small amount of the 
 substance into a superficial abrasion of the arm, as in the example of 
 horse serum anaphylaxis illustrated in Fig." 121, and, as shown by 
 Thayer, in a case of buckwheat hypersensitiveness. It may also be 
 demonstrated in hay-fever by making conjunctival instillations of the 
 particular pollen. 
 
 4. Certain drugs, such as iodoform, iron fitrate, and even atropm, 
 
IDIOSYNCRASIES 579 
 
 strychnin, morphin, etc., appear to develop a state of hypersensitive- 
 ness. Klausncr was able passively to sensitize guinea-pigs against 
 iodoform by injecting the serum of a person sensitive to this drug. 
 Examples of drug anaphylaxis or idiosyncrasy are more difficult to ex- 
 plain unless such drugs contain a protein substance. On the other hand, 
 a drug may alter a body protein, rendering it. really a foreign protein, 
 and this may sensitize body-cells in the same manner as in the "indirect 
 anaphylaxis" of Richet, referred to in the preceding chapter, following 
 the second chloroforming of a dog. 
 
 As was previously stated, anaphylaxis, or rather the anaphylactic 
 mechanism, may be considered one of the essential steps in affording 
 resistance to disease or the state of immunity. Broadly speaking, the 
 lesions and symptoms of infection may be ascribed to the effects of 
 soluble toxins, endotoxins, and a protein poison. According to our 
 present knowledge, the endotoxins are mainly liberated with Ij^sis or 
 disrupture of the bacterial cell. Similarly the protein poison is pro- 
 duced by cleavage of the bacterial protein Substance. Whether the 
 endotoxins and protein poison are identical it is impossible to state. 
 For the present it may be well to consider them as separate entities. 
 In certain infections, such as tetanus and diphtheria, the soluble toxins 
 are chiefly concerned, and these are neutralized by specific antibodies, 
 the antitoxins. The antitoxins are not similar to the "ferment" that 
 splits protein, because they are able to neutralize their toxins without 
 the aid of complement. In other infections, such as typhoid fever, 
 cholera, and pneumonia, the endotoxins and protein poison may be con- 
 sidered the main etiologic factors. The chief antibodies are a cytolysin 
 (bacteriolysin), which disrupts or kills the bacterial cells, and bacterio- 
 tropin, which brings about the same result by favoring phagocytosis. 
 Apparently the cytolysins and the so-called "ferments" responsible for 
 cleaving the protein substance are quite similar in their mechanism. 
 The former are amboceptors, thermostabile and inactive without the 
 presence of a complement. There is no doubt but that heating a serum 
 containing a bacteriolysin and a complement will render the serum in- 
 active through destruction of the complement. While the ferment con- 
 cerned in splitting protein is regarded by many as an amboceptor and 
 complement, there is no general agreement on this point. Some in- 
 vestigators, for example, believe that the ferment concerned in splitting 
 placental protein, as in Abderhalden's pregnancy reaction, is rendered 
 totally inactive by heating the serum. Others believe that heating 
 diminishes the activity of the ferment, but does not destroy it altogether; 
 
580 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 the point cannot be decided at present, mainly because of technical 
 difficulties, but, for the sake of simplicity at least, we may regard the 
 protein-splitting ferments as quite similar to the cytolysins; indeed, 
 they may be identical. 
 
 In anaphylaxis we recognize two primary factors: first, the anti- 
 body, which splits the protein substance, which may be either a harmless 
 sterile protein, such as horse serum or pathogenic bacteria, with the 
 liberation of a protein poison which intoxicdtes body-cells; second, a 
 state of hypersensitiveness of the body-cells to this poison. While it is 
 clearly apparent that the protein poison generated in the test-tube may 
 intoxicate normal animals with the first injection, yet to understand the 
 extreme sensitiveness of body-cells in persons susceptible to horse 
 protein, where, for example, a few inspirations of stable air are sufficient 
 to bring on an attack of asthma, we must recognize a peculiar hyper- 
 sensitiveness of these cells, due probably to the fact that protein ambo- 
 ceptors are attached to the cells and unite with the inhaled protein with 
 great avidity. 
 
 The relation of anaphylaxis to immunity' consists, therefore, in the 
 fact that the mechanism concerned in anaphylaxis is identical with that 
 concerned in antibacterial immunity. Vaughan believes that the same 
 mechanism is identical in all forms of immunity, but we cannot sub- 
 scribe to this view, because the mechanism concerned in anaphylaxis 
 does not explain antitoxin immunity, or at least the antibodies concerned 
 in neutralizing diphtheria toxin are different from those digesting or 
 splitting a bacterial protein, as, for example^ typhoid bacilli. In anti- 
 bacterial immunity, however, where the chief! action hes in digesting the 
 infecting cells, the mechanism may be regarded as identical with that 
 concerned in producing the anaphylatoxin or protein poison. The ef- 
 fects are, however, different. In infection we have the combined 
 action of toxins, endotoxins, and protein poison upon the body-cells; 
 in serum anaphylaxis we have the effects of the protein poison alone. 
 Lesions and symptoms of disease, therefore^ may be regarded as the 
 summation of the products of infection and anaphylaxis. 
 
 Wlien we inject a bacterial vaccine we inject so much bacterial pro- 
 tein. This protein sensitizes body-cells and causes them to produce an 
 amboceptor (sensitizer or the anaphylactic ferment); this antibody 
 serves to bring about death by lysis of any corresponding bacteria in the 
 body (therapeutic immunization), or of any that may subsequently 
 gain access (prophylactic immunization). The effects, if apparent. 
 
ANAPHYLACTIC OR ALLERGIC REjiiCTIONS 581 
 
 may be ascribed to the protein poison liberated, also to the hberated 
 endotoxins, if we consider these as separate from the protein poison. 
 
 In antibacterial immunity, therefore, we recognize lysis or digestion 
 of the infecting bacteria by an antibody as the chief means of defense. 
 This antibody is produced by previous injection of the bacterial protein 
 in the form of a vaccine, or as the result of a previous infection. This 
 is true also of serum anaphylaxis, or of egg, mjlk, pollen, or any other 
 form of anaphylaxis, and in this way anaphylaxis is brought into rela- 
 tion with immunity. 
 
 With the antibody in our body-fluids, the corresponding bacterium 
 is destroyed soon after it comes into contact with the antibody, but since 
 the amounts of protein poison and endotoxin released are small and 
 highly diluted, we experience none or but slight effects. If, however, 
 the infection or entrance of bacterial protein is strictly localized to a 
 small area, so that the liberated poisons are concentrated, a local reac- 
 tion is produced, such as is seen, for example, in the cutaneous tubercu- 
 lin, luetin, mallein, and similar reactions. 
 
 The question may now arise as to the manner in which the body 
 cells dispose of, or become accustomed to, or are protected from the 
 protein poison and endotoxin. There is no satisfactory answer to this 
 question. We have seen that repeated injections of the same protein 
 lead to a condition of decreased sensitiveness. The method by which 
 endotoxins and the protein poison are neutralized, and whether anti- 
 bodies for these are produced, are points that require further investiga- 
 tion. This subject has been discussed in the preceding chapter, under 
 the head of Anti-anaphylaxis. It is true that our body-fluids may con- 
 tain large amounts of the antibody; that protein, bacterial or other, 
 may be vigorously split, but still we do not suffer from the effects of the 
 protein poison or endotoxins. Whatever the mechanism, it in some 
 ,manner concerns a neutralization or depression of the susceptibility or 
 hypersensitiveness of the body-cells ; either the protein antigen is stored 
 m the cells and in some manner depresses cellular activity, as Weil 
 suggested, or else the protein is split beyond the toxic moiety by the 
 free amboceptor in the body-fluids, and in this manner prevents the 
 protein poison from reaching the sessile receptors (those amboceptors 
 still attached to the body-cells). This reasoning is based upon the 
 assumption that symptoms are produced only in case the poison be- 
 comes attached to the cells, according to the cellular theory of anaphy- 
 laxis (see the preceding chapter). 
 
582 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 Anaphylactic or Allergic Reactions 
 
 As was previously stated, if a protein, such as tubercle protein (tu- 
 berculin), syphilis protein (luetin), glanders protein (mallein), etc., is 
 concentrated and applied to the skin or mucous membrane in a local 
 area, and if the corresponding antibody or "ferment" is present in the 
 body-fluids, the protein will be digested or split and the liberated protein 
 poison and endotoxin will diffuse into the local tissues and produce a 
 local reaction, characterized chiefly by congestion and edema. This 
 local reaction, marked by paralysis of the vessel-walls with dilatation, 
 is due to the action of the protein poison on smooth muscle, and is 
 analogous to the urticarial or other eruptions accompanying general 
 serum anaphylaxis (serum disease). Since i&e antibody-protein reac- 
 tion is highly specific, these tests possess considerable diagnostic value. 
 The technic of application and the practical value of the more important 
 tests will now be considered. The same underlying principle governs all. 
 It would appear that these reactions should be obtained in all infections 
 where we can secure and cultivate the causative microparasite. Theo- 
 retically, this is true, although practically the problem is greatly compli- 
 cated, or indeed impossible, owing to technical difficulties and especially 
 to the fact that the protein' antibody for one strain of a particular micro- 
 parasite may not be identical for all strains. 
 
 TUBERCULIN REACTION 
 
 An account of Koch's discovery of tuberculin, in 1891, is given in 
 the chapter on Tuberculin Therapy. Suffice, it to say here that Koch 
 was most interested in the curative properties of tubercuUn, and while 
 he has accurately and clearly described the classic picture of the syste- 
 matic tuberculin reaction, he failed to appreciate the true significance 
 of the reaction at the site of injection, although its occurrence is carefully 
 noted. 
 
 The Tuberculin Reaction.^The reaction to tuberculin is character- 
 ized by three essential features: 
 
 1 . A constitutional reaction, consisting of fever and the accompany- 
 ing general symptoms of lassitude, anorexia, and rapid pulse, varying 
 in severity with the intensity of the reaction. 
 
 2. A local reaction at the site of administration, varying in intensity 
 from slight tenderness and redness to severe inflammation with adenitis. 
 
 3. A focal reaction about the tuberculous lesion. 
 
TUBERCULIN REACTION 583 
 
 These reactions do not by any means run parallel. An intense 
 local reaction may occur, with no or but slight constitutional disturbance. 
 Not infrequently, and particularly in slight pulmonary lesions, signs 
 indicating a focal reaction may not be elicited. 
 
 Nature of the Tuberculin Reaction. — Koch believed that the tu- 
 berculin reaction was due to a summation of the effects of the injected 
 toxin and the toxic bodies formed by the tubercle bacillus within the 
 infected host. Koehler and Westphal, in 1891, suggested that, by a 
 union of the tuberculin with the products of the tubercle bacUlus, a 
 third new body was formed in the tuberculous focus. Marmorek, in 
 1894, suggested that the tuberculin stimulated the tubercle bacilli to 
 secrete a fever-producing substance. 
 
 Finally, in 1903, von Pirquet and Shick Qxplained the reaction as 
 due to a "vital antibody reaction," and this explanation is the one most 
 generally accepted to-daj^ According to this conception, an antibody- 
 like substance produced by the bacilli and diffused through the tissues 
 enters into combination with the tuberculin, giving rise to the formation 
 of a toxic substance in the general circulation, as well as at the point of 
 inoculation of the tuberculin, von Pirquet's discover^' of the cutaneous 
 reaction, in 1907, was a result of this theory, and served to establish a 
 further analogy between cowpox vaccination, tuberculosis, and the 
 tuberculin reaction. According to the principles laid down in the earlier 
 portion of this chapter, the tubercle bacilli arei" considered as stimulat- 
 ing the body-cells to produce an antibody or a "ferment" in the nature 
 of an amboceptor, which splits the tubercle protein contained in tu- 
 berculin, liberating a protein poison, which produces a general, local, 
 and focal reaction. 
 
 The general reaction may be explained as due to a general effect of 
 the poison on body-cells. The local reaction is caused by a concentra- 
 tion of the poison at the site of administration of the tuberculin, and the 
 focal reaction is due to the fact that cells alxtut the lesions are more 
 sensitive to the effects of the poison than are other cells, probably because 
 they are most concerned in antibody production and are supplied with a 
 large number of sessile or attached receptors (amboceptors) for the 
 tuberculin. 
 
 Specificity of the Tuberculin Reaction. — The tuberculin reaction is 
 highly specific. This does not mean that every case of tuberculosis 
 will give a tuberculin reaction, and positive reactions are occasionally 
 found in apparently healthy persons and cattle. The conditions under 
 which a negative reaction may occur in the presence of tuberculosis 
 
584 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 are, however, fairly well understood, and physfcians should be thoroughly 
 acquainted with these. Likewise most instances in which a positive 
 reaction was observed in the apparent absence of tuberculosis have 
 usually narrowed down to the fact that the lesion was so small or so 
 situated as to escape detection, and, indeed, this has been shown so 
 conclusively by autopsies that, in .the presence of a tubereuliu reaction, 
 on the autopsy must rest the burden of proof and blame. When we 
 realize how small a lesion may produce hypersensitiveness, it will readily 
 be understood how easily the clinician and pathologist may fail to detect 
 the lesion. 
 
 A large part of our knowledge regarding, the specificity of the tu- 
 berculin reaction has been gained from veterirtary practice, as the results 
 of a test in an animal could immediately be controlled by the autopsy 
 findings. Thus Fraenkel ^ collected from the literature 8000 carefully 
 observed instances, and found only from 2 to 3 per cent, of differences 
 between the result of the tuberculin test and of the autopsy. Voges,^ 
 in 7327 instances, noted 2.7 per cent, of contradictions. Kuhnau,' 
 Bang,^ and von Behring ^ speak of similar experiences. 
 
 It has long been known that the prevalence of tuberculous findings 
 anatomically far exceeded the number of cases recognized clinically. 
 Among cattle, anatomic tuberculosis is found in from 12 to 25 per 
 cent., and about 80 per cent, or more react to tuberculin. In many of 
 the latter, however, the disease does not progress, but, on the contrary, 
 tends to recede. 
 
 Similar conditions exist in human pathology. That tuberculosis is 
 very frequent among adults is now well known. The figures of Nageli ^ 
 and Burkhardt^ showed that the increasing 'frequency of tuberculous 
 infection with advancing years reached over 90 per cent, among those 
 past the eighteenth year; these figures are now well corroborated. Ham- 
 burger,* in the published results of an analysis of 848 autopsies on chil- 
 dren, showed that tuberculosis was in evidence in 40 per cent., increasing 
 from 4 per cent, among infants under three months of age to 70 per cent. 
 among children from eleven to fourteen years. This explains, in part 
 
 1 Zeitsohr. f. Tuberk., 1900, i, 291. 
 
 2 "Tuberculin unci Organismus," Jena, 1905, 77. 
 
 3 Berl. tierarztl. Wochenschr., 1899, 78. 
 
 ' Sixth Intemation. Congress on Tuberculosis, 1908, 211. 
 
 6 Beit. z. exper. Therap., 1905, x, 1. 
 
 e Vircii. Arcii. f . patii. Anat., 1900, clx, 426. 
 
 ' Zeitsclir. f . Hyg. u. Infectionsk., 1906, liii, 139. 
 
 8 Wicn. klin. Woclienschr., 1907, xx, 1070. 
 
TUBERCULIN REACTION 585 
 
 at least, the relatively high resistance of children to tuberculin, the 
 difficulty there is said to be in eliciting reactions, and the necessity that 
 exists for using large doses. Usually, when children fail to react, it is 
 because they are not tuberculous or because the lesion is too small, 
 whereas in later years, until adult life is reached,^the reaction is observed 
 with increasing frequency and with srualler doses, because the incidence 
 of infection increases progressively from 5 per cent, during infancy to 
 90 per cent, and over in adult life. 
 
 The prevalence of tuberculosis, however, by no means indicates that 
 the infected individual suffers ill health or will succumb to the infection. 
 An individual may be enjoying excellent health, and still harbor a tu- 
 berculous lesion, and display a marked degree of hypersensitiveness to 
 tuberculin. Such a person is not usually regarded as tuberculous until 
 there are tangible symptoms referable to its existence. It is important 
 to remember that tuberculin is an index of tuberculous injection, and not of 
 disease in a clinical sense. Numbers of persons and cattle reacting to 
 tuberculin remain healthy and do not develop symptoms of disease, the 
 autopsy disclosing the presence of inactive or regressing lesions. 
 
 In former years it was considered possible to obtain false positive 
 reactions in convalescents and patients in an enfeebled condition who 
 were non-tuberculous, and also in other diseases, such as syphihs, 
 leprosy, and actinomycosis. More accurate anatomic statistics and 
 careful studies of the tuberculin test administered to a large number of 
 individuals, healthy, tuberculous, and sufferers from other diseases, 
 have gradually changed the attitude of the profession and served to 
 establish the high specificity of the tuberculin reaction. 
 
 Sources of Error in the Tuberculin Reaction. — From the foregoing 
 it will readily be understood that most errors in the tuberculin reaction 
 refer to false negative rather than to false positive reactions. 
 
 False positive reactions may be observed in leprosy, where the bacillus 
 bears such close morphologic and biologic resemblance to the tubercle 
 bacillus, and it is likewise true that massive doses of tuberculin injected 
 subcutaneously may produce a toxic fever in debilitated individuals, 
 but positive reactions in healthy individuals can usually be ascribed — 
 (a) to a small hidden tuberculous lesion or (6) .to a healed tuberculous 
 lesion. As just stated, tuberculin simply indicates hypersensitiveness to 
 the tubercle protein, and this may exist with a. very small unimportant 
 lesion, or persist after a lesion has been "healed" to the extent of en- 
 capsulation. 
 
 False negative reactions are much more likely to occur, and the various 
 
586 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 conditions that may be responsible for these should be well understood 
 and remembered. 
 
 1. In the final stage of tuberculosis, especially in miliary tuberculosis 
 and in tuberculous cachexia, as in the third stage of pulmonary tubercu- 
 losis, the tuberculin reaction may be negative, or be attained only after 
 the injection of very large doses. There is* a lessened cutaneous re- 
 activity (cachectic reaction), marked by the appearance of colorless or 
 pinkish spots, instead of an intense papillary eruption. Koch and Ehr- 
 lich have explained this by assuming that the tissues had become too 
 thoroughly saturated with tuberculin produced at the infected area to 
 respond to further artificial additions. This condition may be regarded 
 as analogous to a state of anti-anaphylaxis in which we may consider 
 the free and sessile receptors united with the tubercle protein or the 
 cells loaded with the antigen, with depression, of cellular activity. 
 
 2. In the first stage of infection. At this period the antibody has not 
 been formed in sufficient amounts, different authors obtaining such 
 findings in nurslings. 
 
 3. In small, completely healed lesions, especially in those showing 
 nothing but scar tissue, as in the apex of a lung. In these the antibody 
 and cellular hypersensitiveness have disappeared, and while the lesion 
 may have been tuberculous, one cannot tell anatomically, in a given 
 instance, whether or not hypersensitiveness should have been present. 
 
 4. During continued treatment with tuberculin, when the reaction 
 may be negative owing to a condition of anti-anaphylaxis. 
 
 5. During measles. As von Pirquet and Preisich have demonstrated, 
 the cutaneous reactivity disappears during the^ first days after the erup- 
 tion, reappearing after about a week. Greuncr showed that the "Siich- 
 reaktion" which occurs after large doses of tuberculin did not disappear 
 entirely, indicating that in measles there is a lessened activity, rather 
 than a total disappearance. 
 
 6. Finally, according to von Pirquet, there are a few cases in which 
 we have a minimal reactivity and to which none of the former explana- 
 tions can be applied. Some cases of active tuberculosis may show only 
 a slight hypersensitiveness, although they are not cachectic. 
 
 Of course, it can readily be understood that the use of weak or an 
 otherwise unsatisfactory solution of tuberculin and an improper dosage 
 and technic will greatly influence the results. Likewise errors in inter- 
 preting what constitutes a positive tuberculin reaction are to be guarded 
 against. This applies especially to veterinaiT: practice. For example, 
 cattle brought from the fields and confined in a stall for the purpose of 
 
TUBERCULIN REACTION 587 
 
 making a tuberculin test may exhibit a fever for several days without 
 apparent cause. On the other hand, dishonest dealers may force cattle 
 to drink cold water or have given cold water irrigations just before the 
 temperature is taken, preventing the registration of a febrile reaction. 
 
 Methods of Conducting the Tuberculin Test. — The object of the 
 tuberculin test is to introduce sufficient tubercle protein to react with 
 the tubercle antibody, with the formation of the protein poison, which 
 shows its presence and effects by a general, a local, or a focal reaction or 
 by a combination of these. Various methods have been proposed, of 
 which the following are best known : 
 
 1. The subcutaneous tuberculin test is the oldest test of its kind, having 
 been discovered by Koch^ in 1891. It consis'ts in the subcutaneous 
 injection of old tuberculin. A positive reaction manifests itself in a 
 constitutional disturbance, accompanied by fever, a local reaction at the 
 site of injection ("Stichreaction"), and frequently a focal reaction at the 
 site of tuberculous disease. 
 
 2. The cutaneous tuberculin test of von Pirquet,^ consisting in the 
 local application of old tuberculin to a superficial abrasion of the skin. 
 A positive reaction is indicated by redness, edema, and other inflamma- 
 tory phenomena. 
 
 3. The conjunctival tuberculin test of Wolff-Eisner^ and Calmette,^ 
 consisting in the local application to the conjunctiva of one eye of a drop 
 of 1 per cent, solution of old tuberculin or purified tuberculin. A positive 
 reaction is indicated by congestion and lacrimation. 
 
 4. The percutaneous tuberculin test of Moro and Doganoff,'^ consistmg 
 in the application of tuberculin ointment prepared by nrixing equal parts 
 of old tuberculin and anhydrous lanolin and applying it to the skin over 
 the upper portion of the abdomen or about the nipple. A positive reac- 
 tion is indicated by an efflorescence of papules upon the anointed skin. 
 
 5. The intracutaneous tuberculin test of Mendel^ and Mantoux,' 
 consisting in injecting into the superficial layers of the skin 0.05 c.c. of 
 diluted old tuberculin. A positive reaction is denoted by infiltration 
 and hyperemia about the site of injection, similar to the reaction to the 
 cutaneous test. 
 
 Comparative Delicacy and Relation of the Various Tuberculin Tests. 
 
 — In judging of the comparative delicacy of the various tuberculin 
 
 'Deut. med. Wochenschr., ISfll, xvii, 101. 
 
 2 Berl. klin. Wochenschr., 1907, xliv, 699. 
 
 'Discussion, Berl. klin. Wochenschr., 1907, xliv, 70. 
 
 *Presse mMcale, 1907, xv, 388. ' Wien. klin. Wochenschr.. xx, 933. 
 
 «Med. Klin., 1908, iv, 402. 'Mtinch. med: Wochenschr., 1908, No. -10. 
 
588 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 tests by a review of the literature, it must be remembered that results 
 will vary according to the portion of the body inoculated, as sensitive- 
 ness of the cells varies in different parts of the body, and there are indi- 
 vidual differences in various persons that are difficult or impossible to 
 explain. 
 
 By comparing the figures obtained as the result of different tests 
 upon the same person with the relative frequency of individual tests, 
 Ham man and Wolman^ have drawn the following conclusions: 
 
 "1. The intracutaneous and subcutaneous local tests are the most 
 delicate we possess. They reveal practically the full percentage of 
 tuberculosis-infected individuals. 
 
 "2. In the order of their sensitiveness, the tests arrange themselves 
 as follows: 
 
 Intracutaneous Test. 
 Subcutaneous Local Test. 
 Cutaneous Test. 
 Subcutaneous Test. 
 Percutaneous Test. 
 Conjunctival Test. 
 
 "3. There is a definite but not a constant relation between the vari- 
 ous tests. An individual reacting to the conjunctival test will, as a rule, 
 give all the others, but not always. The cutaneous or the subcutaneous 
 tests may be negative when the conjunctival is positive. The sub- 
 cutaneous positive when the cutaneous is negative, etc. Some of the 
 unusual variations may, no doubt, depend upon faulty technic in per- 
 forming the tests, but all can certainly not be thus explained. Local 
 changes in sensitiveness and variation in the facility of absorption are 
 probably factors, but the exact conditions are"not understood. 
 
 "4. We have been unsuccessful in an attempt to make the cutaneous 
 test with different strengths of tuberculin equivalent to the conjunctival 
 test." 
 
 Although the subcutaneous test may be dangerous on account of the 
 harm that may result from too severe focal reactions, yet, when carefully 
 conducted, it is frequently the method of choice, especially in the diag- 
 nosis of an obscure pulmonary lesion, and in bone, joint, skin, and other 
 local infections, where the focal reaction may be detected by direct 
 examination. It is to be emphasized, how,ever, that the absence of 
 focal changes during a constitutional reaction does not exclude the tu- 
 berculous nature of a suspected lesion. In children, as shown by Hamill, 
 1 Tuberculin in Diagnosis and Treatment, 1912, Appleton, 167. 
 
TUBERCULIN REACTION 589 
 
 Carpenter, and Cope/ the various tuberculin reactions are likely to 
 yield results that are quite similar. 
 
 The Value of Tuberculin in Diagnosis. — As previously stated, a reac- 
 tion to tuberculin means essentially that the individual reacting has a tu- 
 berculous infection, and in itself means nnthinq more. Since tubercuUn 
 tests disclose inactive and relatively benign tuberculous infections, it 
 has little value, in doubtful cases, in aiding usl to decide whether or not 
 the individual has active disease, which clinically is the tj^pe of infection 
 about which we are most concerned. Lack of critical discernment in 
 the interpretation of the reaction and its apparent indefiniteness have 
 contributed toward diminishing its diagnostic value. 
 
 A positive tuberculin reaction is to be regarded as a symptom, or as 
 another link in the chain of clinical evidence, but is not in itself indisputable 
 evidence that a certain lesion is tuberculous, for it can never decide with 
 certainty an otherwise doubtful diagnosis. A similar example is that of 
 a positive Wassermann reaction in a patient with a lesion in the throat; 
 such a reaction does not necessarily mean that the lesion is S3rphilitic, 
 for the lesion itself may be cancerous, although, coincidentally, a latent 
 syphilitic infection may be present. 
 
 If tuberculin could differentiate between aptive and inactive lesions 
 according to the degree of reaction, its value would be greatly increased. 
 While the studies of Krompecker and Romer upon animals indicate that 
 the more virulent the infection the greater the' degree of hypersensitive- 
 ness, no such fixed relation exists in man. AH that may be said is that, 
 in general, the severer the reaction, the more acute the infection. On 
 the other hand, acute miliary tuberculosis or chronic cachectic cases 
 may react negatively. 
 
 The conditions under which a negative reaction may be obtained in a 
 tuberculous person are to be carefully borne in mind, for if these can be 
 excluded, a negative tuberculin reaction precludes, in all probability, an 
 active or clinically important tuberculous lesion. Tuberculin has, there- 
 fore, a higher negative than a positive value in diagnosis. 
 
 While it is obviously beyond the scope of this volume to discuss the 
 diagnostic value of tuberculin in tuberculous infection of the different 
 organs, I may briefly refer to a few of the more important conclusions 
 reached by individual investigators of large experience in this particular 
 field: 
 
 1. In the diagnosis of pulmonary tuberculosis, while a positive consti- 
 tutional or local tuberculin reaction is never conclusive evidence that a 
 » Archiv. Int. Medicine, 1908, ii, 405. 
 
590 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 definite pulmonary lesion is tuberculous, a focal reaction, on the other 
 hand, tells definitely of the presence of the disease, and shows, in some 
 measure at least, its extent. In these questionable cases, therefore, the 
 subcutaneous injection of tuberculin finds its most important applica- 
 tion, since a definite focal is of more value thaii a local reaction. 
 
 Tuberculin may also be of service when the symptoms suggest the 
 presence of a pulmonary tuberculous lesion, but the physical signs are 
 indefinite. Here the focal reaction is likely to be slight and to escape 
 detection, so that one of the cutaneous tests are usually employed. 
 
 2. In the diagnosis of bone, joint, and glandular tuberculosis the sub- 
 cutaneous test is likely to be most valuable, on account of the focal reac- 
 tion of hyperemia, swelling, heat, and pain about the lesions. The 
 cutaneous test is also valuable, but since this may react on account of 
 the presence of a lesion situated elsewhere, the focal reaction is more 
 conclusive. According to Hamman and Wolman, (a) a focal reaction 
 confirms the diagnosis of tuberculous bone or joint disease; (6) an al> 
 sence of reaction to the subcutaneous test excludes, with the highest 
 probability, the presence of tuberculosis. 
 
 3. In the diagnosis of genito-urinary and pflvic tuberculosis a positive 
 tuberculin reaction is of little value unless the subcutaneous method is 
 employed and the physician is sure of his ability to detect a focal reac- 
 tion, a proceeding that may be very difficult or indeed impossible. A 
 positive cutaneous test indicates the presentje of a lesion somewhere 
 in the body, without disclosing the nature of the renal or pelvic lesion. 
 A negative reaction, however, is of more value, especially when the 
 physician bears in mind the conditions under which a falsely negative 
 result may occur. 
 
 4. In the diagnosis of tuberculosis of the eye, ear, and larynx, the tu- 
 berculin reaction usually has a limited value, because the nature of the 
 disease can be so readily detected by direct inspection. In tuberculosis 
 of the larynx a focal reaction may be dangerous on account of edema. 
 Similarly in advanced tuberculosis of the ear a focal reaction may lead 
 to extension of the process to the meninges. In these instances, there- 
 fore, a cutaneous test should first be made, and if it is found to be nega- 
 tive or inconclusive, the subcutaneous test should be alpplied with extra 
 caution. 
 
 5. In the diagnosis of tuberculosis of the skin tuberculin may occasion- 
 ally prove of value — especially the focal reaction following subcutaneous 
 injection or a direct local application upon the lesion with a weak dilu- 
 tion, such as a 1 per cent, solution of old tuberculin. 
 
TUBERCULIN REACTION 591 
 
 6. In the diagnosis of tuberculosis of a serous membrane tuberculin 
 usually possesses a limited value. In tuberculous meningitis the sub- 
 cutaneous test is contraindicated, as a focal reaction may do harm. 
 Owing to the acute infection the cutaneous test may be negative, and 
 even if positive, would not aid greatly in the diagnosis because the 
 meningeal condition is always secondary to a primary focus. Tubercu- 
 lous pleurises, dry or with effusion, and unaccompanied by evident pul- 
 monary disease, are frequently associated with a low-grade tuberculin 
 hypersensitiveness. According to Hamman and Wolman, in a large 
 proportion of cases of pleurisy with effusion the conjunctival test is 
 negative and the cutaneous test but mildly positive. Bandelier and 
 Roepke assert that in a dry pleurisy increased pain and more pro- 
 nounced and extensive friction may occur during a constitutional 
 reaction to the subcutaneous test and indicate a focal reaction. In 
 tuberculous peritonitis the tuberculin test is frequently negative. A 
 positive reaction has far more value, especiallj^ in virulent types of the 
 disease, which come on insidiously with little or no constitutional dis- 
 turbance. 
 
 The Value of Tuberculin in Prognosis. — As previously stated, tu- 
 berculin may not react in the very early and very late cases of tubercu- 
 losis. In patients with rapidly advancing lesions the power to react 
 tends to decrease and frequently is absent. But this condition is 
 apparent without the aid of tuberculin. Whila Wolff-Eisner and Stadel- 
 mann' laid some stress upon the conjunctival reaction in prognosis, 
 others have been unable to confirm the results, and, as stated by Ham- 
 man and Wolman,^ tuberculin fails to yield us information of prog- 
 nostic value that other methods of clinical observation do not bestow. 
 
 The Dangers of TubercuUn. — Practically, the only danger lies in 
 the subcutaneous and conjunctival tests. With the subcutaneous 
 test, the greatest danger in pulmonary tuberculosis is the possibility 
 of overdosage, with the production of an extensive focal reaction which 
 may bring on hemorrhage or lead to local extension of the lesion. Be- 
 cause of its very important bearing on tubereulin treatment, the sub- 
 ject will be discussed in the next chapter. The same danger of excessive 
 focal reaction holds for tuberculous meningitis (increased intracranial 
 pressure), in tuberculous laryngitis (edema), and in tuberculosis of the 
 ear and nasal accessory sinuses (extension to meninges) . 
 
 The cutaneous, intracutaneous, and perj3utaneous reactions are 
 
 'Deutsch. med. Wochenschr., 1908, xxxiv, 180. 
 
 2 "Tuberculin in Diagnosis and Treatmelit," 1912, 179. 
 
592 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 practically devoid of danger, providing that a careful technic is ob- 
 served. 
 
 Regarding the dangers of the conjunctival test, opinions differ. 
 There can be no doubt, however, but that distressing sequelae, such as 
 severe recurring conjunctivitis, phlyctenular eonjunctivitis, and comeal 
 ulcers with permanent opacities have resulted from the use of this test. 
 Most of the unfavorable results have followed instillation in already 
 diseased eyes, or of too strong solutions, but this is not true of all cases. 
 As will be pointed out further on, in the first instillation not over 
 1 per cent, of old tuberculin should be used, and a second instillation 
 should never be made in the same eye for at least several years. 
 
 Since old people are especially prone to conjunctival inflammation 
 and corneal ulceration, it is probably better to exclude them from the 
 test. Another drawback to this test is the possibility of a "flare up" 
 in the eye following subsequent subcutaneous administration of tuber- 
 culin, either for diagnostic or therapeutic purposes. Hamman and 
 Wolman, however, do not consider this dangerous, and have observed 
 but two cases in an extensive experience. 
 
 The Subcutaneous Tuberculin Reaction 
 
 As has been stated repeatedly, the subcutaneous injection of tu- 
 berculin is resorted to at the present time mainly for the purpose of 
 eliciting a focal reaction, and, as a rule, this is more easily appreciable 
 when the general reaction is well marked. If tuberculin is used simply 
 to establish whether or not a person is hypersensitive, this fact may he 
 demonstrated by employing much smaller doses, as by the cutaneous, intra- 
 cutaneous, or conjunctival tests, the patient being spared the discomfort 
 of the constitutional symptoms. 
 
 Variety of Tuberculin. — Koch's old tuberculin is now used almost 
 exclusively. The technic of its preparation is given in the chapter on 
 Tuberculin Therapy. 
 
 Manufacturing chemists usually market this tuberculin in a series 
 of dilutions, labeled and accompanied by explicit directions, so- that the 
 physician may administer practically any dose desired by injecting 
 so many minims of such or such dilution. Otherwise a series of 
 dilutions are readily prepared in the physician's office or dispensary, 
 according to the method followed by Hamman and Wolman: 
 
 (a) Seven wide-mouthed bottles of about from 12 to 15 c.c. ca- 
 pacity, and fitted with ground-glass or rubber stoppers, are sterilized, 
 labeled, and numbered from 2 to 8. 
 
TUBERCULIN REACTION 593 
 
 (6) The diluent is sterile 0.8 per cent, salt solution with 0.2.5 per 
 cent, pure phenol. This is readily prepared by adding 8 grams of 
 pure sodium chlorid and 2.5 c.c. of pure phenol tb' 1000 c.c. of distilled 
 water. Dissolve, and filter into one large Erlennieyer flask or, prefer- 
 ably, into ten smaller flasks. Sterilize in the Arnold sterilizer for one 
 hour, or in the autoclave for twenty minutes, or by gently boiling for 
 fifteen minutes on each of two' consecutive days. 
 
 (c) Into each bottle place 9 c.c. of the diluent with a graduated and 
 sterile pipet. Bottle 1 contains pure tuberculin. To bottle 2 add 1 
 c.c. of tuberculin and shake carefully; to bottle 3, 1 c.c. from bottle 2 
 and shake; to bottle 4, 1 c.c. from bottle 3 ar«^l shake; continue in 
 this manner to bottle 8, from which 1 c.c. is discarded. 
 
 (d) We now have the following dilutions : 
 
 No. 1 — pure tuberculin. 
 
 No, 2—0.1 
 
 c.c. 
 
 tuberculin 
 
 in each cubic centimeter. 
 
 No. 3—0.01 
 
 c.c. 
 
 
 
 No. 4—0.001 
 
 c.c. 
 
 
 
 No. 5—0.0001 
 
 c.c. 
 
 
 
 No, 6—0.00001 
 
 c.c. 
 
 
 
 No. 7—0.000001 
 
 c.c. 
 
 
 
 No. 8—0.0000001 c.c. 
 
 (e) These dilutions are usually prepared every two weeks. When 
 not in use, the bottles are kept in a cool, dark place. It may not be 
 necessary to prepare all dilutions. For example, dilutions No. 3 and 
 No. 4 are sufficient for diagnostic purposes, as 0.1 c.c. of No. 4 equals 
 0.1 mg. of tuberculin and 1 c.c. of No. 3 equals 10 mg., thus affording 
 an ample range of dosage. 
 
 Method of Conducting the Test. — 1. The patient's temperature and 
 pulse-rate should be taken every two hours for from four to seven days. 
 This is easily accomplished in a hospital; ambulatory patients can 
 usually be readily trained to take their own temperature. All observa- 
 tions should be recorded in writing, and preferably on a temperature 
 chart. The patient's temperature must be constantly below 99° F. 
 before beginning the test, and, if necessary, prolonged rest in bed should 
 be enforced to overcome any existing fever. The test may be given 
 in spite of a daily rise of not over 100° F., but tuberculin by subcutaneous 
 injection should be given only exceptionally to febrile patients. 
 
 2. A very careful physical examination should be made, and the 
 results recorded just before and just after the test in order to detect 
 a focal reaction. This is extremely important, for it is our main justifi- 
 cation for injecting the tuberculin. 
 38 
 
594 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 3. Injections are made subcutaneously in the region of the back, 
 below the angle of the scapula, or in the arm. The skin needs no prep- 
 aration other than to be rubbed with alcohol. The injections are 
 best given during the late evening hours or early in the morning, in 
 order that the temperature and pulse changes may be observed, espe- 
 cially twelve hours after the injection. Records of temperature and 
 pulse should be made every two hours during the day and night for 
 forty-eight hours following an injection. Hamman and Wolman 
 recommend the "Tuberculin Sub 2 Syringe^" made by the Randall, 
 Faichney Co. Each syringe should be sterilized by boiling prior to 
 use, and it is recommended that a separate syringe be provided for each 
 dilution. 
 
 4. Considerable controversy has arisen over the size of the doses 
 to be employed. Koch's directions called for one milligram at first, 
 then for five, then for ten, and if no reaction followed this dose, it was 
 repeated. There is a decided tendency, however, to use smaller doses. 
 If the object is to establish the presence or absence of tuberculin hyper- 
 sensitiveness, mild reactions will suffice, and for this purpose small 
 doses repeated or in gradually increasing amounts may be given. If 
 one aims to produce a focal reaction, larger doses and more rapid in- 
 crease are desirable. Hamman and Wolman advise the following plan 
 for adults: For the first dose, ^ mg. is given. If there is a slight febrile 
 reaction of about one-half a degree, and especially if this is accompanied 
 by a local reaction at the point of injection, the second dose, which 
 is the same as the first, is given at the end of forty-eight hours. The 
 reaction will now most likely be more conclusive. If there is no appre- 
 ciable reaction after the first dose, the second, consisting of one milli- 
 gram, is given at the end of forty-eight hours, and the third dose, if 
 one is necessary, consists of five milligrams'. Occasionally the third 
 dose must be repeated, or even ten milligrams given if the negative result 
 is at variance with the clinical impressions. 
 
 If the temperature shows any irregularities, the intervals between 
 injections should be prolonged to three or four days or more. 
 
 Roth-Schultz advise the following doses:' 0.5 mg.; 1.25 mg., and 
 2.5 mg. as the terminal dose. 
 
 For children under fifteen years of age smaller doses are indicated — 
 an initial dose of one-tenth milligram and a terminal dose of one milli- 
 gram, with one or two intervening doses. Baldwin has advised 0.05, 
 0.2, 0.5, and 1 mg. 
 
 The Reaction. — The constitutional reaction is quite variable. Fever 
 
TUBERCULIN REACTION 595 
 
 is the most delicate indicator. A rise of 1° F. or more above the previ- 
 ous maximum is considered positive, especially if it is accompanied by a 
 local and a focal reaction. A definite febrile reaction due to tuberculin 
 is rare without the presence of a local reaction to the same or the pre- 
 ceding doses. General symptoms of headache, muscle pains, anorexia, 
 nausea, etc., may accompany the reactions. The local reaction consists 
 of redness and pain at the site of injection, with tenderness of the neigh- 
 boring lymph-glands, and is absolutely specific of tuberculin hyper- 
 sensitiveness. The focal reaction consists of an inflammatory reaction 
 with the production of rales, change in breath-sounds, etc. 
 
 The Intracutaneous Tuberculin Reaction 
 Variety of Tuberculin. — Koch's old tuberculin is used either in one 
 dose of 0.005 mg., or preferably in three different doses injected simul- 
 taneously; these will be described further on. 
 
 Method of Conducting the Test. — The skin of the forearm is cleansed 
 with alcohol and then dried. A small glass syringe fitted with a fine 
 needle is used. A separate syringe is used for the control fluid, con- 
 sisting of sterile salt solution, and three others for each of the different 
 dilutions used. In performing this test Hamman and Wolman make 
 four simultaneous injections: 
 
 First: 0,05 c.c, of sterile normal salt solution (control). 
 
 Second: 0.05 c.c. of a 1 : 1,000,000 dilution of old tuberculin, 
 
 which equals 0.00005 mg. This equals a dose of 0.05 c.c. of 
 
 dilution No. 4, just described in the preceding test. 
 Third: 0.05 c.c. of a 1 : 100,000 dilution, or=0.05 c.c. of dilution No. 
 
 3, equivalent to 0.0005 mg. 
 Fourth: 0.05 c.c. of a 1 : 10,000 dilution, or 0.05 c.c. of dilution No. 
 
 2, equivalent to 0.0005 mg. 
 
 Mantoux uses the last or fourth dose only. The injections are given 
 with the skin held taut or pinched up in a fold -between the index-finger 
 and thumb. The needle is inserted superflcially, with the ajjerture 
 directed toward the outer surface of the skin. If the point of the needle 
 is in the skin, a white elevation occurs immediately upon the introduc- 
 tion of the solution; if it is in the subcutaneous tissue, no infiltration is 
 apparent. 
 
 The Reaction. — The reaction consists of infiltration and hyperemia 
 about the site of injection, similar to the reaction in the cutaneous test. 
 It appears in from six to eight hours, reaches its maximum intensity 
 in from twenty-four to forty-eight hours, and usually disappears in 
 
596 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 from six to ten days. The salt solution generally produces a trau- 
 matic reaction, similar to a mild tuberculin reaction, which subsides 
 in forty-eight hours. 
 
 The reactions are best recorded after twenty-four hours, and the 
 simplest method of recording the results is to- measure the width of the 
 area of infiltration of each reacting point. 
 
 The Cutaneous Tuberculin Reaction (von Pirquet) 
 Variety of Tuberculin. — Undiluted old tuberculin is now used almost 
 exclusively in conducting the test. 
 
 Method of Conducting the Test. — 1. The flexor surface of the fore- 
 arm is chiefly used for making the applications, but it should be re- 
 membered that tests performed on different portions of the body are 
 not strictly comparable. 
 
 2. The skin is cleansed lightly with alcohol and dried. Three abra- 
 sions are made, about IJ/^ or 2 inches apart, with a von Pirquet skin 
 borer (Fig. 122) or with a needle, small lancet, or blood sticker. The 
 object is to scarify the superficial layers of the skin, avoiding as much as 
 possible bleeding, although a few small poinds of blood should appear. 
 To the upper and lower abrasions add a drop of tubercuUn; after ten 
 minutes wipe away the excess with a bit of cotton. No shield or pro- 
 tective dressings are required. The middle abrasion is the control, 
 and shows the amount of traumatic reactioti following the scarifying 
 process. Due precautions should, of course, be observed that none of 
 the tuberculin flows down the arm and reaches this spot. 
 
 3. The tests are inspected at the end of twenty-four hours. 
 
 The Reaction. — The traumatic reaction as shown in the control 
 area may present an inflammatory areola with, at times, slight infiltra- 
 tion. Before a test may be considered positive its areola should be at 
 least five millimeters wider than the control area. The reactions are 
 usually designated as follows: 
 
 1. Negative Reaction. — No appreciable difference between the tu- 
 berculin areas and the control. 
 
 2. Slight Reaction. — Definite but slight redness with some infiltration. 
 
 3. -f Reaction. — ^A wider area of redness, with definitely raised 
 centers. 
 
 4. -(-+ Reaction. — ^Wider area of redness, with more marked in- 
 filtration than +. 
 
 5. -l--f + Reaction. — Unusual redness and a wide area of infiltra- 
 tion, all cases which go on to vesiculation. 
 
TUBERCULIN REACTION 
 
 597 
 
 The usual or normal reaction begins to appear in from four to six 
 hours, reaches its maximum intensity in from twenty-four to forty- ' 
 eight hours, and then fades rapidly, although the infiltration may persist 
 for some days. Special types of the reaction have been described as 
 follows: 
 
 Fig. 122. — Method of Performing a von Pirquet Tuberculin Test. 
 
 The abrasion is being made over the insertion ol the deltoid muscle. The 
 borer is held firmly and perpendicular to the arm. A quick rotatory motion serves 
 to remove a circular area of epidermis. 
 
 The borer is shown in the upper right-hand comer.. 
 
 1. The premature reaction, characterized by a rapid course and slight 
 intensity. This type is supposed to occur in patients with manifest 
 tuberculosis who are not doing well. 
 
 2. The persisting reaction, which reaches its maximum intensity 
 about the second day and persists for a week or longer. 
 
598 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 3. The late reaction, which appears after twenty-four hours and de- 
 velops and recedes slowly. These last two types are believed to occur 
 in patients having inactive lesions. 
 
 4. The cachectic reaction, which is characterized by infiltration with 
 little or no redness. This type is common in the late stages of tubercu- 
 losis. 
 
 5. The scrofulous reaction, which is characterized by numerous small 
 elevated nodules, which may also appear on the extremities and trunk. 
 This reaction is peculiar to children and rare in adults. 
 
 The Conjunctival Tuberculin Reaction (Calmette) 
 
 Variety of Tuberculin Used. — A 1 per cent, solution of Koch's old 
 tuberculin is now generally used, as it is quite reliable, least expensive, 
 and the results that follow its use are more regular than those ob- 
 tained with purified tuberculin (0.5 to 2 per cent, aqueous solution). 
 
 Method of Conducting the Test. — The conjunctivae of both eyes 
 are inspected to ascertain if there is any evidence of disease and if they 
 are strictly comparable in color. The lower lid of one eye is drawn for- 
 ward to form a little pouch, and the patient is directed to look upward; 
 one drop of a 1 per cent, dilution of old tubeiiculin is then applied from 
 an ordinary eye-dropper to the lid at the; inner canthus. Profuse 
 lacrimation impairs the test, and it is useless to attempt it with weeping 
 or resisting children. If no reaction is apparent and it is still desired 
 to further the test, a drop of a 5 per cent, dilution may be placed in the 
 opposite eye. 
 
 The Reaction. — In a positive reaction the conjunctiva begins to 
 redden in from six to eight hours, and reaches its maximum in from 
 twenty-four to forty-eight hours, and then rapidly subsides and dis- 
 appears in from four to six days. In mild reactions the inner canthus 
 is the seat of the most marked changes. Positive reactions have been 
 classified as follows : 
 
 -|- Reaction: Definite palpebral redness. 
 
 4- -f Reaction : More marked palpebral redness with secretion. 
 
 -|--|-+ Reaction: Palpebral and bulbar* redness with subjective 
 symptoms and well-marked secretion. (See Fig. 124.) 
 
 Precautions. — On account of severe reactions and the danger of 
 inflicting permanent injury on the cornea there is a growing tendency 
 to regard this reaction with disfavor. Hamman and Wolman, however, 
 believe that, with proper precautions, these risks may be minimized, 
 if not completely avoided; they beheve, moreover, that the risk in- 
 
/■ 
 
 ^^^ 
 
 %^-:% 
 
 Fig. IXi. — A Positive Citaneous Tuberculin Reaction (von Pirquet). 
 
 Child with incipient pulmonary tuberculosis; a + reactioii; The control scarification 
 
 is barely to be seen, and is midway Ijetween the tuberculin reactions. 
 
Fig. 124. — A Positive Conjunctival Tuberculin Reaction (Wolff-Eisneb- 
 
 Calmette). 
 
 Severe reaction (+ + +) in a tuberculous cow; appearimce of the eye about 
 
 fourteen hours after second instillation of tuberculin. 
 
». . 
 
 Fig. 125. — A Positive Percutaneous Tuberculin Reaction (Moro). 
 
 E. MoK., adult male with arrested early pulmonary, tuberculosis. Lesions about 
 
 sixty hours after application of tubcrejjhn ointment. 
 
TUBEECULIN REACTION 599 
 
 curred is not great enough to warrant the abandonment of a procedure 
 that has proved itself of such great value in diagnosis. Nevertheless, 
 they emphasize the necessity for observing proper precautions, and 
 give the following rules to be adopted: 
 
 1. The conjunctival test should never be repeated in the same e5^e. 
 
 2. A solution stronger than 1 per cent, original tuberculin should 
 never be used for making the first instillation. 
 
 3. Any existing inflammatory disease of the eye is an absolute con- 
 traindication to the test. 
 
 4. The test should not be given to manifestly scrofulous children. 
 
 5. Skin diseases in which the lesions are situated upon the face, near 
 the eye, especially when these are suspected of being tuberculous, pre- 
 clude the application of the test. 
 
 6. It is safest not to give the test to elderly persons, and particularly 
 to arteriosclerotics, as they are unduly prone to develop corneal ul- 
 ceration. J 
 
 The Percutaneous Tuberculin Reaction (Moro 
 Variety of Tuberculin Used. — This consists of 5 c.c. of old tuberculin 
 and 5 grams of anhydrous lanolin thoroughly mixed. The ointment 
 retains its potency for many months when preserved in a cold dark 
 place. Manufacturers market the preparation in small collapsible 
 tubes containing sufficient for at least one or two tests. 
 
 Method of Conducting the Test. — A piece of ointment about the 
 size of a pea is thoroughly rubbed for at least a minute into an area of 
 skin about two inches in diameter over the upper abdomen or near a 
 nipple. The patient may be instructed how to apply this ointment, or 
 the physician may do it himself, protecting the finger with a rubber cot. 
 The Reaction. — This maj^ appear within twenty-four hours, or be 
 delayed for as long as from four to six days. It consists of an eruption 
 of slightly elevated papules situated upon a hyperemic base, which 
 vary in size from a pinhead to large areas of infiltration (Fig. 125). It 
 subsides in from three to ten days. Moro described these grades of 
 reaction as follows : 
 
 1. Mild reactions: From 1 to 10 scattered papules. 
 
 2. Moderate reactions: About 50 or more papules, partly discrete, 
 partly confluent, and with a general reddening' of the skin. There may 
 be well-marked itching. 
 
 3. Severe reactions: Numerous large, extremely red papules or 
 vesicles up to 8 mm. in diameter, having an intensely reddened base. 
 
600 anaphylaxis in relation to infection and immunity 
 
 Tuberculin Reactions Among the Lower Animals 
 
 In veterinary practice tuberculin is used almost solely for diagnostic, 
 and only occasionally for therapeutic purposes. 
 
 Four reactions are in common use : 
 The Subcutaneous Test. 
 The Conjunctival Test. 
 The Cutaneous Test. 
 The Intracutaneous Test. 
 
 The technic for conducting these tests and the reactions secured are 
 quite similar to those just described, and I would refer the veteriaary 
 surgeon to these respective descriptions. Differences in technic are 
 confined principally to dosage. 
 
 The Subcutaneous Tuberculin Test. — This is conducted with Koch's 
 old tuberculin. The method of preparation is given in the succeeding 
 chapter, under Tuberculin Therapy. Concentrated tuberculin is pre- 
 pared by concentrating the bouillon filtrate to one-tenth its original 
 volume. Diluted tuberculin is the concentrated product diluted to its 
 original volume by mixing 1 part of the dilutibn with 9 parts of 0.5 to 1 
 per cent, phenol in sterile normal salt solution or distilled water. 
 
 The injections are always given subcutaneously in some convenient 
 area, preferably around the shoulder, which ha's been shaven and cleaned 
 beforehand with a solution of creolin. 
 
 The first dose for horses and cattle is usually 0.4 c.c. of concentrated 
 or 4 c.c. of diluted tuberculin; the second dosais usually 0.8 c.c. of con- 
 centrated or 9 c.c. of the diluted tuberculin. 
 
 The general local and focal reactions are similar to those previously 
 described. 
 
 The Conjunctival Tuberculin Test. — For this test Koch's concen- 
 trated tuberculin may be used. Preference is usually given to the puri- 
 fied product, prepared as follows: Mix one part of Koch's concentrated 
 tuberculin with 20 parts of absolute alcohol. The precipitate that forms 
 is filtered off and dried over sulphuric acid. This powder is then made 
 up into a 4 per cent, and 8 per cent, solution in. sterile distilled water. 
 
 Two or three drops of the 4 per cent, dilution are placed in the inner 
 canthus of one eye, to sensitize the tissues. After twenty-four hours, 
 unless a positive reaction is present, two or three drops of the 8 per 
 cent, solution are instilled in the same eye in the same manner. The 
 reaction is usually apparent in from six to twelve hours (Fig. 124). 
 
 For the types of reaction and precautioijs to be observed see the 
 previous description. 
 
LUETIN REACTION 601 
 
 One advantage of this test is that the animal will give a reaction in 
 cases where, prior to the test, dishonest dealers have injected tuberculin. 
 
 The Cutaneous Tuberculin Test. — In making this test some con- 
 venient area is shaved and scraped slightly until serum exudes. A small 
 amount of Koch's old tuberculin is applied to the prepared area. In a 
 positive case a well-marked area of congestion and hyperemia appear 
 at the end of twenty-four hours. This may also be accompanied by a 
 rise in temperature. 
 
 The Intracutaneous Tuberculin Test. — In performing this test 
 from 0.2 to 0.4 c.c. of Koch's concentrated tuberculin are injected into 
 the skin through a fine needle. A white swelling should appear while 
 the injection is being given; if it does not appear, the injection is sub- 
 cutaneous and unsatisfactory for this test. The appearance of hyper- 
 emia and redness with a rise in temperature indicates a positive reaction. 
 
 LUETIN REACTION 
 
 The clinical course of syphilis indicates that the infecting micro- 
 parasite, Treponema 'pallida, possesses all the qualities essential to the 
 development of an anaphylactic condition in sj^philitic patients. Thus 
 the primary lesion appears after an incubation period of two or three 
 weeks. The secondary stage is manifested by periodic waves of various 
 general symptoms; the primary, secondary, and tertiary lesions show 
 a qualitative difference. Stimulated by von Pirquet's discovery of a 
 specific cutaneous reaction for tuberculosis, a number of investigators 
 (Finger and Landsteiner, Wolff-Eisner, Nobe, Ciuffo, Nicolas, Favre, 
 and Gauthier) attempted to obtain a specific reaction for syphilis by 
 applying extracts of syphilitic tissues — prepared from sj^philitic fetal 
 liver or chancre — to the skin of syphilitic patients. In spite of some 
 encouraging effects their results were, on the whole, contradictory. 
 :Further, Neisser and Bruck found that a reaction similar to that pro- 
 duced with syphilitic extract can be obtained also with a concentrated 
 extract of normal liver. This peculiarity of the skin of syphilitics is 
 ascribed by Neisser to what he calls the state pf " Umstimmung" in the 
 later stages of syphilis. Both Neisser and von Pirquet expressed the 
 hope and belief that a reaction may be secured by employing an extract 
 of pallida free from tissue constituents; this' was first and finally ac- 
 complished by Noguchiji in 1911, first with syphilitic rabbits and then 
 with human patients. Noguchi gave the appropriate name of "luetin" 
 'Jour. Exper. Med., 1911, 14, 557; Jour. Amer. |kled. Assoc, Iviii, 1163. 
 
602 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 to the extract of pallida. Theoretically, one should not expect to 
 obtain an allergic reaction in syphilis so long as the activity of pallida 
 is maintained at its maximum, or in the very! early stages, before there 
 is sufficient time for antibody formation. One can reasonably expect 
 the appearance of the phenomenon when the activity of the micropara- 
 site begins to abate through a gradually acquired defensive power of 
 the host, or under an effective therapeusis, as in the later stages of the 
 disease and in hereditary syphilis. Practical results have borne out 
 these theoretic expectations. 
 
 Preparation of Luetin. — At least six different ^tjrains of pallida in pure cul- 
 ture are being used by Noguchi in the preparation of luetin. These are cultivated 
 in ascites-kidney agar for periods of six, twelve, twenty-four, and fifty days, at 37° 
 C, under anaerobic conditions. The tubes showing large numbers of spirochetes are 
 then selected, the oil is poured off, the tube is cut, the agar column is removed, and 
 the tissue then cut off. The ascites-agar cultures are then carefully ground in a 
 sterile mortar, the resulting thick paste being gradually diluted with a fluid culture 
 until a homogeneous liquid emulsion is secured. The preparation is next heated 
 for an hour in a water-bath at 60° C, and then tricresol or phenol added to make 0.5 
 per cent. Cultures are made from this suspension, and rabbits inoculated intra- 
 testicularly ; both, after suitable intervals, must show an absolutely sterile preparation. 
 
 The luetin should be kept in the refrigerator when not in use. So 
 far as I am aware, all "luetin" is prepared in the Rockefeller Institute, 
 under Noguchi's own supervision. The isolation of Treponema pallidum 
 in pure culture is a difficult procedure, and, obviously, a luetin must be 
 prepared of pure cultures and from as many different strains as possible. 
 While pallida quickly loses its pathogenicity in artificial culture-media 
 and is also highly susceptible to the influence of germicides, its preparar 
 tion, nevertheless, is an important matter requiring skilful supervision. 
 
 A control fluid prepared of sterile agar and bouillon in exactly the 
 same maimer as luetin was originally advised by Noguchi, but recently 
 he claims that its use is not necessary. 
 
 Method of Application. — Luetin is not applied to an abrasion of the 
 skin, but is injected intracutaneously with a v&y fine needle and a sterile 
 syringe. According to directions from the Rockefeller Institute, the 
 luetin is to be well shaken, diluted with an equal part of sterile salt 
 solution, and 0.07 c.c. injected (0.035 c.c. undiluted). A slightly 
 smafler dose, as, e. g., 0.05 c.c, may be used for children. The skin of 
 the upper arm is usually selected as the site for inoculation. If a 
 control fluid is used, the luetin may be injected into the skin of the left 
 and the control fluid into the skin of the right arm, or both injections 
 
LUETIN REACTION 603 
 
 may be given in the same arm, about two inches apart, the control 
 being above the luetin. 
 
 After cleansing the skin with alcohol it is drawn taut or pinched up 
 between forefinger and thumb and the needle introduced, with the 
 aperture directed toward the outer surface of the skin. If the point 
 of the needle is in the skin, a white elevation occurs immediately upon 
 injecting the solution; if it is in the subcutaneous tissue, no infiltration 
 is apparent. 
 
 A special tuberculin syringe may be used, and the luetin drawn 
 directly into the barrel from the stock bottle" and diluted with sterile 
 normal salt solution. To obviate waste and the likelihood of contamina- 
 tion, I dilute a portion of luetin (1 c.c.) with an equal amount of sterile 
 salt solution (1 c.c.) in a sterile vessel, and then add 6 c.c. more of sterile 
 salt solution, shaking thoroughly and placing 0.2 c.c. in each of 30 small 
 sterile ampules, which are then sealed. Before using, the ampule is 
 shaken thoroughly, opened, and the contents aspirated into the syringe ; 
 0.1 c.c. is allowed for waste in loading the syringe and displacing air; 
 0.1 c.c. is injected, and this is practically equivalent to 0.035 c.c. of the 
 undiluted luetin, the dose recommended. When so prepared, the dilu- 
 tion keeps well, is especially adapted for dispensary use, and the phenol 
 is so diluted as to exclude any traumatic reaction. 
 
 Reactions. — Normal or Negative Reactions. — In the majority of 
 normal persons the injection of luetin is followed by a very slight trau- 
 matic reaction, or a small erythematous area appears, after twenty-four 
 hours, at and around the point of injection. No pain or itching sensa- 
 tion is experienced; the reaction recedes in forty-eight hours and leaves 
 no induration. In certain individuals the reaction may reach a stage 
 of small papule formation after from twenty-four to forty-eight hours, 
 which subsides withifi seventy-two hours, leaving no induration. 
 
 Positive reactions have been classified by Noguchi into three main 
 varieties : 
 
 (a) Papular Form. — "A large, raised, reddish, indurated papule, 
 usually five to ten millimeters in diameter, makes its appearance in 
 twenty-four to forty-eight hours. The papule may be surrounded 
 by a diffuse zone of redness and show marked telangiectasis. The 
 dimensions and the degree of induration slowly increase during the fol- 
 lowing three or four days, after which the inflammatory processes begin 
 to recede. The color of the papule gradually becomes dark bluish red. 
 The induration disappears within one week, except in certain in,stances 
 m which a trace of the reaction may persist for a longer period. The 
 
604 ANAPHYLAXIS IN RELATION TO INFECT^ION AND IMMUNITY 
 
 latter effect is usually met with among cases of secondary syphilis under 
 regular mercurial treatment in which there are no manifest lesions at 
 the time of making the skin test. Cases of congenital syphilis also show 
 this reaction. 
 
 " (b) Pustular Form. — The beginning and course of this reaction 
 resemble the papular form until about the fou/th or fifth day, when the 
 inflammatory processes commence to progress. The surface of the 
 indurated round papule becomes mildly edematous, and multiple 
 miliary vesicles occasionally form. At the same time a beginning cen- 
 tral softening of the papule can be seen. Within the next twenty- 
 four hours the papule changes into a vesicle, filled at first with a semi- 
 opaque serum that later becomes definitely purulent. Soon after this 
 the pustule ruptures spontaneously or after slight friction or pressure. 
 The margin of the broken pustule remains indurated, while the defect 
 caused by the escape of the pustular content becomes quickly covered 
 by a crust that falls off within a few days. About this time the indura- 
 tion usually disappears, leaving almost no scar after healing. There 
 is a wide range of variation in the degree of intensity of the reaction 
 described in different cases, as some show rather small pustules, while 
 in others the pustule is much larger. This reaction was found almost 
 constantly in cases of tertiary syphilis, as well as in cases of secondary 
 or hereditary S3rphilis which had been treated with salvarsan. 
 
 "(c) Torpid Form. — In rare instances the injection sites fade away 
 almost to invisible points within three or four days, so that they may 
 be passed over as negative reactions. But sometimes these spots sud- 
 denly light up again after ten days or even longer and progress to small 
 pustular formation. The course of this pustule is similar to that de- 
 scribed for the preceding form. 
 
 "This form of reaction has been observed in a case of primary 
 sjrphilis, in one of hereditary syphilis, and in two cases of secondary 
 syphilis, all being under mercurial treatment.'' 
 
 Aside from these three types of reactions, there have since been 
 described several cases of the formation of a hemorrhagic exudate, the 
 lesion usually rupturing spontaneously and not running a more severe 
 or a longer course than the pustular. Two ^ch reactions have been 
 reported by Kilgore.^ 
 
 "Neither in syphilitics nor in parasyphiEtics did a marked consti- 
 tutional effect follow the intradermic inoculation of luetin. In most 
 positive cases a slight rise in temperature took»place, lasting for one day. 
 1 Jour. Amer. Med. Assoc, Ixif, 1236. 
 
Fig. 126.— a Positive Litetin Reaction. 
 
 E. C, adult male; tertiary syphilis with detachment of the retina; papular lesion 
 
 of moderate severity; about forty-eight hours after injection of 0.07 c.c. hietin. 
 
LXJETIN REACTION 605 
 
 In three tertiary cases and in one iiereditary case, however, general 
 malaise, loss of appetite, and diarrhea were noted." 
 
 As was previously stated, Noguchi now asserts that the control 
 fluid may be omitted. In syphilitic persons this fluid may give a 
 reaction that is less intense and that is not followed by induration, but 
 is due probably to a true allergic condition, whereas in normal persons 
 none or but a slight traumatic reaction occiifs. It may at times be 
 difficult, however, to distinguish a slight luetih reaction from a well- 
 marked traumatic reaction, and in these instances an opinion may be 
 withheld until controlled by a second injection in the other arm with 
 control fluid. In other words, while the contuol fluid need not be used 
 routinely, it is well to have it at hand to be used in these doubtful cases. 
 
 In view of the occasional instances in which the reactions are re- 
 tarded a patient should be observed for two weeks before a reaction is 
 regarded as negative. 
 
 Results. — 1. The reports of Noguchi and of a number of different 
 observers show that the luetin reaction is generally negative in the 
 primary and secondary (untreated) stages of sj^philis. 
 
 2. In latent and tertiary syphilis Noguchi has reported positive 
 reactions in from SO to 95 per cent, respectively, and the reports of 
 others have showed from 64 to 100 per cent, of positive reactions. 
 
 3. In cerebrospinal syphilis positive reactions have been reported 
 in from 42 to 80 per cent, of cases. 
 
 4. In congenital syphilis the results have varied within wide limits — 
 10 to 96 per cent, of positive reactions. In cases under one year of age 
 Noguchi has reported about 23 per cent., and among later cases 96 
 per cent., of positive reactions. 
 
 5. While a few observers have reported positive results in diseases 
 other than syphilis, it is frequently very difficult absolutely to exclude 
 syphilis, and the general consensus of opinion is unmistakably to the 
 effect that in this country, at least, the luetifi reaction is specific for 
 syphilis. Slight reactions may be obtained in frambesia or yaws and 
 leprosy. 
 
 6. Second injections of luetin apparently do not give positive reac- 
 tions in non-syphilitic cases. 
 
 Practical Value. — It may be stated in general that the chief value of 
 the luetin reaction is in the diagnosis of those occasional cases of latent, 
 tertiary, or congenital syphilis that fail to react positively with the 
 Wassermann reaction. I am quite convinced that in the majority of 
 cases a negative Wassermaim reaction, carefully and skilfully per- 
 
606 ANAPHYLAXIS IN RELATION TO INFECTJON AND IMMUNITY 
 
 formed, and especially with an antigen reenforced with cholesterin, is 
 stronger evidence of the absence of syphilis then is a luetin test. On the 
 other hand, a definitely positive luetin reaction may be regarded as 
 indicating that the patient is or has been syphilitic, even though the 
 Wassermann reaction is negative. 
 
 2. Results indicate that in syphilis the luetin reaction persists longer 
 than does the Wassermann reaction. This is to be expected when we 
 assume that the substance in the blood-serum of a syphilitic responsi- 
 ble for the Wassermann reaction, is a separate reactionary product of 
 the body-cells to pallida toxin, the allergic luetin reaction being due 
 to the true pallida antibody. The toxin is believed to disappear from 
 the body-fluids within a few weeks after all the spirochetes have been 
 killed, whereas the allergic antibody may persist for longer periods of 
 thne, as it does in other conditions and diseases. In a case of treated 
 syphilis, therefore, showing a persistently negative Wassermann reaction 
 with both serum and spinal fluid, a positive luetin reaction does not 
 necessarily indicate that further treatment is required. 
 
 3. Briefly stated, to determme if a frankly syphilitic patient requires 
 further treatment the Wassermann reaction with serum and cerebro- 
 spinal fluid is the better test. To determine- more definitely whether 
 a given person showing a negative Wassermann reaction has ever had 
 syphilis the luetin reaction is the better and" more conclusive test; or 
 if in such a person the Wassermann reaction is positive, the luetin test 
 may be used for control and as corroborative evidence. 
 
 MALLEIN REACTION = 
 
 Mallein is a glycerin extract containing the toxic principles of the 
 Bacillus mallei, the microorganism causing glanders. It is used entirely 
 as a diagnostic agent in veterinary practice, but may also be used for 
 the diagnosis of human glanders, the dosage being the same as that of old 
 tuberculin. 
 
 Two methods are commonly employed: (1) The subcutaneous in- 
 jection and (2) instillation into the eye (ophthalmic test). 
 
 Method of Preparing Mallein.'— A pure culture of Bacillus mallei is usually ob- 
 tained by injecting a male guinea-pig with infected material, and at the end of 
 twenty-four or forty-eight hours, isolating a pure culture from the testicle. 
 
 The microorganism is grown for from six to eight weeks in special bouillon 
 containing 5 per cent, glycerin, at 37° C, similar to tuberculin. Unlike the tubercle 
 
 ' Technic employed by the Penna. State Live Stock and Sanitary Board, Dr. 
 A. B. Hardenburgh, Director. 
 
ALLERGIC REACTIONS IN TYPHOID FEVER 607 
 
 bacillus, glanders bacillus grows evenly through the bouillon instead of upon the 
 surface. 
 
 At the end of six or eight weeks the flasks are removed from the incubator and 
 placed in a sterilizer at 100° C. for at least two hours. This process kills the bacilli 
 and extracts the toxic pi-inoiples. The entire solution is evaporated down to yV of 
 its volume, filtered in small bottles, and sterilized. This is called concentrated mal- 
 lein, and is kept in this form until ready for use. 
 
 Before using it is diluted to its original volume with 0.5 per cent, phenol solution, 
 and passed through a porcelain filter. This process removes all the bacilli, and ren- 
 ders the solution aseptic and ready for use. 
 
 Ophthalmic mallein is prepared by taking one part of concentrated 
 mallein and adding 20 parts of absolute alcohol. This forms a precipi- 
 tate that is filtered and dried in a desiccator over sulphuric acid. A 
 5 per cent, solution of the powder is made with sterile water. 
 
 Subcutaneous Mallein Reaction. — The doSe of mallein for a horse 
 k 0.4 c.c. of concentrated mallein, or 4 c.c. or 1 dram of the diluted 
 product. The dose for a retest is 0.8 c.c. of concentrated or 8 c.c. or 2 
 drams of the diluted solution. Mallein is injected subcutaneously in 
 some convenient area, such as around the shoulder, which has been 
 shaved and cleaned. 
 
 A positive reaction is based on the same principle as tuberculin, 
 that is, a rise of temperature within twenty-four hours following the 
 LQJection, with a local inflammatory reaction at- the site of injection. 
 
 Ophthalmic Mallein Reaction. — Two or three drops of the aqueous 
 solution are dropped into the inner canthus of one eye. In case of a 
 positive reaction this is followed by a marked "conjunctivitis, associated 
 with a purulent exudate extending from the inner canthus similar to the 
 reaction shown in Fig. 124. In most cases there is also a rise of tempera- 
 ture. Only one dose is to be applied in the ophthalmic mallein test. 
 It is not considered necessary to sensitize the eye, as in tuberculosis. 
 The ophthalmic mallein test has the same advantages as the ophthalmic 
 tuberculin test, that is, one can obtain a reaction when dishonest horse 
 dealers have injected mallein prior to a subcutaneous mallein test, 
 also in cases of far-advanced glanders, which at times give no reaction 
 to a subcutaneous injection of mallein. 
 
 ALLERGIC REACTIONS IN TYPHOID FEVER 
 In 1907 Chantemesse^ observed characteristic inflammatory symp- 
 toms follow the instillation of typhoid-bacilli extract into the eye of 
 1 Deut. med. Wochenschr., 1907, 33, 1572. 
 
608 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 patients suffering from typhoid fever. Krails' and his associates, re- 
 peating these experiments, could not convince.themselves of the specific- 
 ity of this reaction, stating that healthy individuals also give it to some 
 extent, and that other bacterial extracts cause similar symptoms in 
 typhoid-fever patients. In addition he tried a cutaneous reaction, 
 but without result. Zupnik,^ on the contrary, states that a cutaneous 
 reaction is useful, while the ophthalmic reaction is not useful. Deehan' 
 obtained a weak to moderate reaction in 12 cases of typhoid fever, 
 whereas eight control cases showed none. Flojd and Barker * obtained 
 positive results in 19 out of 30 cases and none' in 18 controls, including 
 two cases of paratyphoid fever. Chauffor4 and Trosier^ reported 
 unfavorably on the reaction. 
 
 Austrian^ has reported very favorably upon an ophthalmic reaction 
 in typhoid fever following the installation of " typho-protein " prepared 
 by cultivating a large number of different strains of typhoid bacilli, 
 precipitating the protein with alcohol, drying- the precipitate, and re- 
 dissolving in water so that from one-third to one-half milligram is 
 contained in each drop. In typhoid-fever patients reaction of the 
 palpebral conjunctiva of the lower lid and of the caruncle appears on 
 an average of two and a half hours later, reaching the maximum about 
 the sixth hour, and usually subsiding within forty-eight hours. In 75 
 cases of typhoid fever this test was found positive in 71 and negative 
 in four. In three cases the eye test antedated the Widal reaction, and 
 in only 23 per cent, was the Widal reaction positive at as early a date 
 as the eye test. A study of 190 persons normal or ill with diseases 
 other than typhoid has convinced Austrian of the specificity of the test, 
 and he recommends it as an aid to diagnosis on account of its simplicity 
 and the absence of any discomfort to the patient. 
 
 More recently. Gay and Force ^ have reported favorably upon a 
 cutaneous reaction indicative of immunity against typhoid fever. The 
 preparation which they used, "typhoidin," is prepared in the same man- 
 ner as Koch's old tuberculin: 250 c.c. of a 5 per cent, glycerin bouillon 
 is inoculated with Bacillus typhosus and cultivated at 37° C. for five 
 days. It is then reduced, without filtration, to one-tenth of its original 
 
 1 Wien. klin. Woohensohr., 1907, 20, 1335. 
 
 2 Mtlnch, med. Wochenschr., 1908, 45, 148. 
 ' Univ. Peim. Med. Bull., 1909, 22, 6. 
 
 « Amer. Jour. Med. Sci., 1909, 38; 188. 
 6 Compt. rend. Soc. de Biol., 1909, Ixvi, 519. 
 6 Bull. Johns Hopkins Hosp., 1912, 23, 1. 
 ' Archiv. Int. Med., 1914, 13, 471. 
 
ALLERGIC REACTIONS IN OTHER DISEASES 609 
 
 volume by evaporation over an acetone bath for about eight hours. 
 A control solution of sterile 5 per cent, glycerin bouillon is prepared in 
 the same manner. 
 
 The skin of the forearm is cleansed with alcohol, and two abrasions 
 are made with the von Pirquet borer, as describqicl under the cutaneous 
 tuberculin test. The "typhoidin" is applied to one cut and the control 
 fluid to the other. The reactions are observed six and twenty-four 
 hours later. Occasionally there is a traumatic reaction in the control, 
 but a positive reaction may be detected by a wider areola and increased 
 induration. 
 
 Positive reactions were secured in 95 per cent, of cases that had re- 
 covered from typhoid fever, two of the cases having had the disease 
 respectively forty-one and thirty-three years before. The reaction was 
 found negative in 85 per cent, of individuals not having tjrphoid fever. 
 Of 15 persons immunized by the army method, from four and three- 
 quarter years to eight months previously, nine gave a positive skin 
 reaction. Twenty-four individuals immunized by a sensitized vaccine 
 (Gay and Claypoole) for from one to eight months previously reacted 
 positively. The test is advocated as a means of determining whether 
 or not a person possesses immunity to typhoid fever, either acquired 
 by recovery from the disease or by artificial immunization. 
 
 ALLERGIC REACTIONS IN OTHER DISEASES 
 
 Gonococcus Infections. — In 1908 Irons ^ reported general and local 
 reactions in persons suffering from gonococcus infections following the 
 subcutaneous injection of gonococcal vaccines. This reaction has been 
 observed by Bruck^ in epididymitis, by Reiter^ in pelvic infections in 
 women, and also by other observers in other conditions. 
 
 Experiments with glycerin extracts of the gonococcus prepared from 
 several strains, singly or combined in one preparation, have yielded 
 Irons ^ well-defined cutaneous reactions. These= tests were conducted 
 after the method of von Pirquet's tuberculin test. 
 
 Diphtheria. — Shick has advocated the intracutaneous injection of a 
 minute dose of diphtheria toxin as a test for antitoxin in the serum of an 
 individual. If sufficient antitoxin is present, the toxin is neutralized 
 and no local disturbances are apparent; otherwise local inflammatory 
 
 ' Jour. Infect. Dig., 1908, v, 279. ^ Deut. med. Wochenschr., 1909, xxxv, 470. 
 ' Zeitschr. f. Geburtsh. u. Kinderh., 1911, Ixviii, 471. 
 ^ Jour. Amer. Med. Assoc, 1912, Iviii, 931. 
 39 
 
610 ANAPHYLAXIS IN RELATION TO INFECTION AND IMMUNITY 
 
 areas may be observed. This test is not regarded as an allergic reac- 
 tion; a further description of it will be found, in Chapter XXX, under 
 Diphtheria. 
 
 Allergic reactions have also been observed, mostly in experimental 
 animals, in leprosy, sporotrichosis, in diseases due to hyphomycetes, and 
 in pregnancy. Further investigations will, no doubt, disclose reactions 
 of practical value in other diseases, such as rabies, scarlatina, measles, 
 etc., following the successful isolation and cultivation of their respective 
 causative microparasites. 
 
 ALLERGIC REACTIONS AS A MEASURE OF IMMUNITY 
 Based upon the assumption that the antibodies (anaphylactins) 
 concerned in splitting their specific protein antigen at the local site of 
 application (as tubercuhn, mallein, luetin, and typhoi din), with the libera- 
 tion or formation of the toxic material (anaphylatoxin) responsible for 
 the local reaction of infiltration, edema, pain, and redness of the skin, 
 are the same as those which bring about the destruction of this protein 
 antigen in a living state as in the form of living microparasites, it is at 
 once apparent that these reactions are of value=not only in the diagnosis 
 of various infections, but also as a measure of the defensive power or im- 
 munity of a person to a certain disease after recovery from this disease or 
 after active immunization by means of a vaccine. 
 
 In other words, an allergic reaction may have a prognostic value. As 
 pointed out by Gay and Force, in typhoid immunization the continued 
 ability of a person to react positively to typhoidin may be accepted as an 
 indication of the presence of antibodies and of sufficient immunity 
 against typhoid fever; when, however, a persoh.fails to react, this may be 
 regarded as an indication for further immuni'zation. Likewise in cow- 
 pox vaccination an immediate or "immunity reaction," consisting in the 
 development of a small papule at the site of inoculation within twenty- 
 four to forty-eight hours after vaccination, indicates that the person 
 possesses the specific antibodies and does not "take" in the usual sense 
 of the term, because the virus or antigen has been acted upon at once by 
 the antibodies present. For this reason vaccinations should be inspected 
 within a few days after inoculation, as after five days this "immunity 
 reaction" disappears and the result may be interpreted as a failure to 
 vaccinate successfully, whereas, on the contrary, it may actually indicate 
 a state of immunity. 
 
CHAPTER XXIX 
 
 ACTIVE IMMUNIZATION.— VACCINES IN THE PROPHYLAXIS 
 AND TREATMENT OF DISEASE 
 
 While the importance of natural immunity: must not be under- 
 rated in the protection it gives us after bacterial" invasion has occurred, 
 this immunity is, however, usually relative and- seldom absolute, and 
 may afford insufficient protection if the invading'bacteria are numerous, 
 or particularly virulent, or if the natural resistance of the organism 
 is weakened by fatigue, disease, or injury. 
 
 Usually the best and most lasting immunity is that actively ac- 
 quired, in which our own body-cells are stimulated or trained, as it 
 were, to produce specific antibodies against the offensive forces of a 
 particular bacterium or other pathogenic agent. A well-marked and 
 lasting degree of this form of immunity usually follows recovery from 
 many of the acute infections, particularly the acute exanthemata, such 
 as smallpox, scarlatina, measles, typhoid fever, typhus fever, etc. In 
 other infections, such as erysipelas, gonorrhea, and pneumonia, the 
 immunity is less complete, of short duration, or entirely absent, and, 
 indeed, a state of hypersusceptibility may actually follow. 
 
 It is very important, in this connection, to remember that the degree 
 of immunity is not necessarily in proportion to the severity of the disease ; 
 thus a mild infection may be followed by the niuch-desired immunity, 
 and while in general there is not considerable 'protection without infection, 
 the latter does not necessarily imply that a virulent infection, or even 
 the actual disease itself, is present, for discoveries have shown that an 
 active immunity may be acquired by inoculation with the antigen so 
 modified or attenuated that it can stimulate the production of specific 
 antibodies without producing the disease or otherwise greatly disturbing 
 the health of the individual. 
 
 Historic. — The facts here detailed are well illustrated in the history 
 of vaccination in smallpox and the development of vaccine therapy 
 in general. Hundreds of years ago the people of the eastern countries 
 were accustomed to expose their children to a mUd case of smallpox in 
 order that a similar mild infection might be acquired and a lasting im- 
 
 611 
 
612 ACTIVE IMMUNIZATION 
 
 munity thus secured with the least danger to" life. This practice, how- 
 ever, was not without risk, as the mild disease not infrequently became 
 a virulent one. Later the dose of infectious' agent was decreased by 
 applying the virus to a small abrasion on t^ie skin, and the resulting 
 mild but genuine attack of smallpox usually cjonferred the much-desired 
 immunity. But here again the severity of the disease was not under 
 control, as the virus occasionally assumed increased virulence and in- 
 duced severer infections than were desirable. Finally, Edward Jenner 
 observed and showed experimentally (1796) that when cowpox virus is 
 inoculated into the human being, a trivial infection, since called "vac- 
 cinia," is induced, and that this is followed by an absolute or nearly 
 absolute immunity of manj' years' duration against smallpox. In other 
 words, the virus, in its passage through the cow, becomes so modified 
 that it can no longer produce smallpox, but; is still able to stimulate 
 the production of the specific antibodies against this disease. Jenner 
 worked so hard to establish the truth of this finding that he had little 
 time to devote to the mechanism involved in the process. 
 
 In other words, the work of Jenner was jargely empirical, and the 
 explanation was not forthcoming until many years later, when Pasteur 
 laid the basis of scientific immunization by discovering that light, high 
 and low temperature, and exposure could so reduce the virulence of a 
 microorganism that while its injection into an animal was practically 
 without danger or ill effect, it could still stimulate the protective mech- 
 anism of the host and induce a high degree of iiiimunity. 
 
 That this could be done was a fact discovered accidentally by Pasteur 
 in 1879 while working with the organism of chicken cholera. After 
 an absence from home he found, on examining his cultures on his return, 
 that they had become innocuous — that hens could bear without any ill 
 effect inoculation of what would formerly have been a lethal dose. The 
 prolonged cultivation of the microorganism bad caused its attenuation 
 and Pasteur immediately grasped the far-reaching importance of this 
 discovery. He conjectured that it might be possible to produce a mild 
 and modified form of chicken cholera with a vaccine of the attenuated 
 microorganism which would afford protection to the fowl against the 
 severe form of the disease. This proved to be the case, and established 
 the possibility of so modifying or attenuating -the virulence of a virus or 
 germ that, while its administration is not followed by the actual disease, 
 it is capable of so stimulating the body-cells that the specific antibodies 
 are produced. This discovery formed the basis of prophylactic im- 
 munization or bacterin therapy in general. 
 
HISTORIC 613 
 
 Following this discovery, much work was done, for it appeared that 
 the question of prevention of any bacterial disease simply depended 
 upon whether the bacterium could be cultivated and so modified or 
 attenuated that while its injection would not be followed by disease or 
 other harmful effects, it would be capable of causing the production of 
 specific antibodies. 
 
 Naturally, most of the earlier work was done with the infections of 
 the lower animals, and, consequently, most discoveries were directly 
 beneficial to them. Pasteur soon devised a method of attenuating 
 anthrax bacilli by exposing them to certain temperatures for varying 
 lengths of time, so that a vaccine could be prepared that has proved of 
 great value. Later the same observer discovered a method of attenuat- 
 ing the virus of hydrophobia by a process of drying, and devised a 
 practical method of prophylactic immunization against this disease. 
 In addition to these his vaccines against swine erysipelas, symptomatic 
 anthrax, aid rinderpest have become well known. 
 
 The knowledge gained from a study of the diseases of the lower ani- 
 mals and the aid given them has been applied to human medicine with 
 considerable benefit, not only in prophylactic immunization, but also 
 in therapeutics (bacterin therapy). The latter application is a more 
 recent discovery, for which we are mainly indebted to the researches 
 of Wright, Leishman, Douglas, and their colleagues. 
 
 Nomenclature. — The word vaccine is from the Latin vacca (a cow). 
 Cowpox was called "vaccinia," or the cow disease, and Jenner designated 
 protective inoculation against smallpox with cowpox virus as vaccination. 
 With true courtesy Pasteur adhered to Jenner's nomenclature and 
 applied the term vaccine to erimlsions of dead or attenuated bacteria. 
 This is unfortunate and tends to create confusion, as the term vaccine 
 is inseparably associated with cowpox virus or lymph. The term bac- 
 terial vaccine has become widely known, and is used to designate bacterial 
 suspensions prepared for purposes of immunization. There is no essen- 
 tial difference, however, between cowpox vaccine, which contains the 
 modified germ or virus of smallpox in a diluent of lymph, and a bacterial 
 vaccine containing the germ, modified by some physical or chemical 
 agency in a diluent of saline solution or bouillon. It is, however, well 
 to reserve the unqualified term "vaccine" foil cowpox virus, and to 
 retain the designation "bacterial vaccine" for suspensions of attenuated 
 or dead bacteria. More recently the term bacferim has been applied 
 to the latter, but this would imply an extract of bacteria, as, e. g., tu- 
 berculin, which is not always the case. 
 
614 ACTIVE IMMUNIZATION 
 
 Some confusion likewise exists as regards the terms serum and vaccine 
 therapy. Serum therapy is a process of passive immunization induced 
 for either protective or curative purposes by the injection of the blood- 
 serum of another animal that has been actively immunized by inocula- 
 tion with bacterial toxins or the bacteria themselves, as, for instance, 
 the injection of diphtheria or tetanus antitoxins. Vaccine or bacterin 
 therapy is a process of active immunization brought about by the injec- 
 tion of the bacteria or their products directly into a patient. Bacterial 
 vaccines that are simple emulsions of dead- or attenuated bacteria 
 are not, therefore, serums, and the indiscriminate use of the two terms 
 is much to be regretted. 
 
 Method of Preparing Vaccines. — It may be stated that, in general, 
 the specific microorganism or virus used in a vaccine should be modified 
 as little as possible, or just sufficient to rob it of its disease-producing 
 power. For example, typhoid bacterial vaccine is prepared by suspend- 
 ing the bacilli in salt solution and exposing them to just enough heat 
 to modify them so that they can no longer multiply. The less modifica- 
 tion, the better the vaccine. If the exposure is too prolonged or the 
 temperature too high, the vaccinogenic power of the bacilli is destroyed, 
 and the suspension in salt solution is no more" potent or of no greater 
 value than the salt solution itself. Therefore the nearer the vaccine 
 approaches the fully viable virus or microorganism, the more potent it 
 will be. The proper preparation of a vaccine, therefore, is the first 
 step to successful vaccine therapy. 
 
 Vaccination, using the term in its broadest sense, may be performed 
 for prophylactic purposes and curative immunization in the following 
 ways: 
 
 1. The living microorganism may be inoculated. This is the ideal 
 method, but for obvious reasons has not been generally used and is still 
 in the experimental stage. It is based upon experimental observations 
 made on the lower animals that an organism, may be so introduced as 
 to render it incapable of producing disease, but may, however, stimulate 
 the production of specific protective antibodies. Most work on typhoid 
 fever is at present being done in the Pasteur Institute at Paris. Evidence 
 thus far indicates quite conclusively that the typhoid bacillus is unable 
 to produce typhoid fever unless it is introduced into the gastro-intestinal 
 tract, and the subcutaneous injection of living bacilli, modified only to a 
 slight extent by artificial cultivation, is not followed by ill effects and 
 produces a high grade of immunity. The principle is a good one, i. e., 
 in a vaccine the microorganism should be modified as little as possible. 
 
HISTOEIC 615 
 
 For obvious reasons this method is being extensively tried out on the 
 lower animals, especially the chimpanzee, before it is applied to human 
 medicine. 
 
 2. By inoculation with a modified virus or with microorganisms at- 
 tenuated or modified according to various methods. 
 
 (a) By passing the virus through a lower animal, as the passage of 
 smallpox through the heifer when the virus is incapable of producing 
 smallpox, although vaccinia confers a specific immunity against small- 
 pox. A vaccine for swine erysipelas is prepared in the same manner 
 (Pasteur) by passing the bacillus through the rabbit several times, 
 which increases its virulence for the rabbit but'decreases it for swine, 
 
 (&) By exposing suspensions of microorganisms to heat. They are 
 usually grown on a suitable solid medium, suspended in salt solution, 
 and exposed to a temperature at or just above their thermal death-point 
 for just sufficient time to kill or attenuate them in so far that they can- 
 not multiply. The same result may be secured by longer exposure to a 
 lower temperature. To secure a potent vaccine the principle of minimum 
 exposure at the minimum temperature should he observed, the question of 
 viability being controlled by culturing the vaccine. Most bacterial 
 vaccines are prepared in this manner. Of course, the fact that a vaccine 
 is sterile could be confirmed by exposing it to a very high temperature, 
 but in this case the product may be of no more value than so much salt 
 solution. Usually an exposure of 53° to 60° C, for from one-half to one 
 hour is sufficient, and only exceptionally are these limits exceeded. 
 
 (c) By exposing the microorganism to air and light. The first 
 bacterial vaccine (chicken cholera) was accidentally prepared by Pasteur 
 in this manner. 
 
 (d) By desiccating or drying the virus. This is the method of 
 vaccination in rabies, as the virus contained in the spinal cord of rabbits 
 is dried for varying lengths of time, emulsified, and injected. The longer 
 the period of drying, the greater the attenuation, and in this maimer the 
 strength of the vaccine and the progress of immunization are under 
 control. 
 
 (e) By exposing the microorganism to a high temperature for vary- 
 ing lengths of time. Anthrax vaccine, for the immunization of lower 
 animals, is prepared in several strengths by exposing suspensions of the 
 bacilli to 42° C. %r varying periods of time. 
 
 (/) By exposing the microorganisms or their products to certam 
 chemical germicides, as in the preparation of aiithrax vaccine by Roux, 
 
616 ACTIVE IMMUNIZATION 
 
 diphtheria and tetanus toxins (Behring), and in some preparations of 
 tuberculin. 
 
 3. By inoculating with bacterial constituents, as the soluble toxins, 
 bacterial extracts, and products of bacterial autolysis, as in the preparation 
 of Koch's tuberculin T. R., Koch's old tuberculin, mallein, diphtheria 
 and tetanus toxins, etc. 
 
 In Chapter XIII a method is given for preparmg bacterial vaccines, 
 of which typhoid vaccine is a type. Special methods of preparing cer- 
 tain bacterial vaccines and other vaccines, such as cowpox virus and 
 rabies vaccine, are given in this chapter. 
 
 Mechanism of Active Immimization. — As was stated in the chapters 
 on Immunity, in the presence of an infection the host endeavors to pro- 
 tect itself and overcome the invaders by vaijous means, among which 
 are phagocytosis and the production of more or less specific antibodies 
 that may neutralize the poisons of the parasite (antitoxins), directly 
 kill or destroy them (bactericidans), or so lower their vitality or re- 
 sistance that they are more easily phagocytcd (opsonins or bacterio- 
 tropins) . 
 
 During an infection, one or more of these protective forces, or all 
 of them, are brought into action. After th^ infection has been over- 
 come, the antibodies do not always disappear at once, but remain for 
 some time in the body-fluids and gradually diminish, so that if the host 
 is reinfected with the same parasite, the antibodies are at hand im- 
 mediately to overcome it and protect the host absolutely, or at least 
 so to modify the pathogenicity of the parasite^ or neutralize its products 
 that the host will suffer but mildly while the parasite is being finally 
 destroyed. The concentration and duration, of the various defensive 
 forces or antibodies vaiy in different indiviijuals and in different in- 
 fections, so that the degree and duration of an,active acquired immunity 
 are variable factors. Nevertheless — and this is the basis of active im- 
 munization — an animal or a person may have specific antibodies for a 
 certain parasite, produced by its own cells, without actually or necessarily 
 suffering from the disease, due to the effect's of inoculation with the 
 germ or virus in a modified or attenuated fonn. The dose of vaccine 
 may be so controlled that general symptoms the result of stimulation 
 of the body-cells are slight or not at all apparent, and, by gradually 
 increasing the dose, more and more antibodies may be produced until 
 a high degree of immunity is secured. 
 
 Active immunization may be practised for tvk) purposes: 
 
 (a) For prophylaxis, or the prevention of a disease, which is accom- 
 
HISTORIC 617 
 
 plished by the production of antibodies so that they may be at hand to 
 overcome an infection if it should occur. For example, the antibodies 
 specific against the virus of smallpox may be produced by inoculation 
 with cowpox virus, so that for years the system will be protected against 
 smallpox. Even if vaccination has been delayed until smallpox has 
 actually been contracted, iuoculation with cowpox virus early in the 
 period of incubation so stimulates the body-cdls that sufficient anti- 
 bodies are produced to modify and lessen considerably the virulence 
 of the smallpox virus. 
 
 This is especially true in rabies, when the vaccine is given in such 
 doses and at such intervals that sufficient antibodies are produced to 
 neutralize the effects of rabic virus and actually to destroy it during the 
 period of incubation or during the interval that elapses between the 
 time of infection and the appearance of the symptoms. In this way 
 the great majority of infected persons escape the sufferings of rabies 
 by enduring the relatively slight discomfort consequent to a series of 
 subcutaneous injections. 
 
 (b) For the treatment of disease. This is the bacterin or vaccine 
 therapy, a method that owes its origin to the researches of Sir Almroth 
 Wright and his colleagues. It was originally employed in the treatment 
 of those infections that showed a tendency to chroiiioity in which true toxins 
 played little or no part. Since recovery from an infection is in general 
 dependent upon the mechanical removal of the infecting agent, aided 
 by antibodies that facilitate phagocytosis or directly destroy the invading 
 bacterium and neutralize its products, Wright believed that in chronic 
 infections autovaccination, or stimulation of the body-cells to the 
 production of antibodies, by reason of the fact that it is irregularly 
 timed, is generally insufficient or altogether absent. For these reasons 
 he believes that any stimulus that will arouse the body-cells to throwing 
 into the circulation substances from the invading bacterium or diseased 
 tissues, may result in increased antibody formation, followed eventually 
 by clinical improvement or cure. In certain cases this stimulation may 
 be secured by judicious massage or manipulation of the diseased part, 
 by passive hyperemia (Bier), or by similar procedures. 
 
 However, if the microorganism is obtained and cultivated artificially, 
 it is possible, in many instances, so to modify or attenuate the bacilli 
 (usually by heat) that they may be reinjected ii|to the patient in suffi- 
 cient numbers to furnish the stimulus necessary for arousing dormant 
 or uninvolved body-cells to produce the opsonins and other antibodies 
 necessary for overcoming the infection. In other words, with each in- 
 
618 ACTIVE IMMUNIZATION 
 
 fection the host endeavors to protect itself by producing antibodies. 
 When the protection is insufficient, the infection will spread; when the 
 antibodies are in excess, the infection is overcome; when the forces are 
 about equal, a stage of chronicity may result in which the host becomes 
 accustomed, as it were, to the invaders, and, while the infection does 
 not spread rapidly, it does not, on the other hand, recede. In cases of 
 the latter type an extra dose of bacterial stimulant (a bacterin) may 
 arouse dormant or inactive cells to furnish an extra quantity of anti- 
 bodies and thus turn the tide. In acute infec'tions indifferent groups of 
 cells may likewise be brought into action, although it is more likely that 
 they are already involved, so that the extra stimulation in the form of 
 bacterin must be cautiously and carefully applied, if applied at all. 
 
 In therapeutic inoculation, therefore, the fundamental 'principle is to 
 stimulate in the interest of the infected tissues the unexercised immunizing 
 capacities of the uninfected tissues. This is especially true in chronic 
 infections, when the use of a bacterial vaccine may be likened to the 
 application of the whip to a lazy horse that is capable of further effort 
 and work. In acute infections, however, while the cells are at work 
 they may be capable of greater effort, but vaccines should be given 
 cautiously, as they may, to use the same simile, act as a whip to a willing 
 and well-worked horse that is unable to respond or does respond, with 
 resulting disastrous overexertion. 
 
 It should be remembered, in this connection, that usual forms of treat- 
 ment should be given while bacterin therapy is being instituted. For in- 
 stance, it is useless to administer a vaccine to a patient with a sup- 
 purative fistula or sinus if an infected silk suture is directly responsible 
 for the suppuration. The suture should be removed, if possible, and 
 after this is done, a vaccine may be of considerable aid in overcoming 
 the coincident infection. 
 
 In what manner can dead bacteria cause the production of anti- 
 bodies? The mechanism is similar to that involved during infection 
 with the living microorganism, and involves the first principles of im- 
 munity. The antigenic powers of a vaccine are probably always more 
 or less inferior to the living antigen, as some principle may be lost during 
 heating, drying, passage through animals, the action of germicides, etc. 
 For this reason living vaccines are to be preferred, although, for obvious 
 reasons, they cannot generally be employed in human practice. 
 
 Just what portion of the bacterial cells is mainly antigenic it is 
 difficult to determine, for it probably varies with different species. 
 With a true toxin, as, for example, the diphtheria bacillus, the toxin 
 
HISTORIC 619 
 
 constitutes the main principle, and causes the production of an anti- 
 toxin as its main antibody; with other bacteria, such as the typhoid 
 bacillus, a soluble toxin and an endotoxin in combination with the protein 
 of the bacterial cell are probably the main antige'nic factors responsible 
 for the formation of a bacteriolysin, opsonin, antitoxin, agglutmin, etc. 
 
 In brief, the antigenic principles of a microorganism are mainly 
 thermostabile and fairly resistant substances, so that a bacillus may be 
 so attenuated or altered that it cannot multiply of produce disease, and 
 yet is capable, through the agency of substances that have escaped 
 destruction, of causing the production of specific antibodies. 
 
 As was previously stated, vaccines may cause the production of 
 different antibodies. As curative agents, however, it would appear that 
 they are most efficacious in those infections in which phagocytosis is 
 known to be chiefly concerned in the defense of the host, e. g., in staphy- 
 lococcus infections. As shown by Wright and Douglas, Neufeld and 
 Kimpau, a bacterial vaccine facilitates phagocytosis, not so much 
 qualitatively or quantitatively as through the production of specific 
 substances that act directly and primarily upon the bacteria and render ' 
 them more vulnerable to phagocytosis (opsonin or bacteriotropin). 
 Wright has advised a method of opsonic measurement, previously 
 described, for measuring the immunity response,, but, as will be under- 
 stood, while the opsonin may be the chief antibody, it is seldom if 
 ever the only one, so that the opsonic index is but one measure of de- 
 fensive power. 
 
 According to Vaughan, a microorganism is dilrectly responsible for 
 the production of a specific proteolytic ferment Capable of causing the 
 disintegration or destruction of the bacterial cell and its products. The 
 ferment is the antibody, and is produced during an infection or by a 
 vaccine in just the same manner as antibodies in general are produced. 
 In other words, Vaughan regards antibodies as of the nature of pro- 
 teolytic ferments; thus the protein of the microorganism composing 
 a vaccine produces a specific proteolytic ferment capable of overcoming 
 its substratum when it meets the latter in the. form of the invading 
 rnicroorganism of an infection. 
 
 For example, the tissues affected may be unable to produce a suffi- 
 cient quantity of the specific ferment necessary to overcome the infec- 
 tion. The injection of bacterial protein in anotiier and healthier part 
 of the body leads to the production, in this locality, of a specific ferment 
 that is conveyed to the diseased area by way of the circulatory system, 
 
620 ACTIVE IMMTJNIZATltftsr 
 
 and aids in destroying the protein of the infecting microorganism and its 
 tissues. 
 
 Living versus Dead Vaccines. — Barring accidents, the employment 
 of a living virus is the most certain way of calling forth a maximum 
 output of antibodies. There is at present no satisfactory explanation 
 for this except that heat-labile substances destroyed in the ordinary 
 preparation of bacterial vaccines have antigenic properties (Smith). 
 Living vaccines are also capable of penetrating into deeper tissues, 
 whereas dead vaccines may remain where they are deposited. Similarly 
 living viruses are capable of exerting a continuous action and of de- 
 livering an infinite number of blows, whereas the injection of a dead 
 virus produces an interrupted action and deals but a single blow. The 
 actual dangers of using a living vaccine, as the possibility of it being 
 too virulent and thus producing disease, or of regaining virulence or 
 producing chronic "carriers" preclude their general employment in 
 human practice. 
 
 Sensitized Vaccines. — Besredka and Metchnikoff^ have suggested 
 a plan of injecting a vaccine composed of living bacteria that have been 
 immersed in their specific immune serum or, in other words, have been 
 sensitized (serobacterin) . They believe that such vaccines produce 
 practically no negative phase, but only slight local and general reaction, 
 and that the general response with antibody formation is facilitated. 
 This principle is supported by the observations of Theobald Smith, 
 who found that the experimental injection of a toxin-antitoxin mixture 
 aids the dissemination of the toxin through the body quite generally, 
 whereas the pure toxin is chiefly held at or near the place of injection. 
 This diffusion tends to cause maximum antibody formation over an 
 entire portion of the body by a relatively small amount of free or easily 
 dissociated toxin in the toxin-antitoxin mixture. Smith inclines to 
 the belief that a similar phenomenon of diffusion may occur with sensi- 
 tized dead bacteria. 
 
 In addition, the specific immune serum may aid in the disintegration 
 of the bacterial cell, either through the attachment of a bacteriolytic 
 amboceptor that would tend to lyse the bacterium with a complement 
 of the tissues, or through a preliminary action of opsonin which prepared 
 the bacterium for ultimate destruction and liberation of antigenic 
 principles. 
 
 Autogenous versus Stock Bacterial Vaccines. — It may be stated in 
 general that autogenous vaccines, i. e., those prepared from the pa- 
 1 Ann. de I'Inst. Pasteur, 1913, xxvii, 597. 
 
HISTORIC 621 
 
 tient's own bacteria, should be used whenever possible, especially in 
 the vaccine treatment of disease. To be successful, vaccine therapy 
 demands that the bacteria be as little changed as possible. Before 
 they are killed, the bacteria should be endowed with as many of the 
 potencies as possible with which they maintain themselves in the body. 
 As these potencies do not remain unchanged during artificial life, as 
 the loss of capsules, loss of virulence, etc., it is advisable to secure the 
 organism causing the infection as quickly as possible and prepare a 
 vaccine without undue delay. 
 
 Variants may occur among cultures of the same species, and the 
 injection of one strain may not protect against J another, as shown by 
 Neufeld for the pneumococcus. In the use of an autogenous vaccine 
 this risk of using an alien species or a different. strain is reduced to a 
 minimum. 
 
 In some cases the difficulty of securing and of identifying the in- 
 fective agent may be so great that much time is lost in preparing auto- 
 genous vaccines, as, for instance, in gonorrheal and tuberculous in- 
 fections, and in such cases it may be necessary to use a stock vaccine. 
 
 In protective immunization stock vaccines are used, as, for instance, 
 in the preparation of typhoid vaccine. In certain instances, as in 
 gonococcus and tuberculous infections, stock vaccines possess but 
 slightly inferior therapeutic value as compared with autogenous vaccines, 
 not to mention the delay and difficulty in cultivating and preparing 
 autogenous vaccines. In many other infections, as with the Bacillus 
 coh and streptococci, stock vaccines possess little or no value. 
 
 The wholesale manufacture of various bacterial vaccines and their 
 indiscriminate use have brought disappointment to many. Rational 
 and scientific vaccine therapy does not consist in the administration of 
 ready-made, uncertain, and oftentimes hit or miss mixtures, recently 
 so widely exploited. Especially is this true in the use of vaccines for 
 therapeutic purposes. The bacteriotherapeutist must possess sufficient 
 skill to enable him to make bacteriologic diagnosis, prepare autogenous 
 vaccines, and skilfully guard their administraticm. In most instances 
 these requirements are fulfilled by cooperation between the clinician 
 and bacteriologist, or, better, by one who is especially trained in vaccine 
 therapy. 
 
 The Negative Phase. — As has been stated in a previous chapter, 
 certain local, constitutional, and focal disturbances may follow the 
 injection of a bacterial vaccine. The first or local symptoms are not in- 
 frequently due to an excess of preservative in the fluid or to the presence 
 
622 ACTIVE IMMUNIZATION 
 
 of contaminating microorganisms, but abscess formation is distinctly 
 rare. I, in common with others, prefer to find slight focal and con- 
 stitutional symptoms following the first one or two doses of vaccine. 
 In furunculosis, for instance, when the old lesions discharge a little more 
 and one or two others threaten to develop during a day or so following 
 the injection of vaccine, I feel assured that the ultimate result will be 
 good. In tuberculin therapy, however, the trend of opinion is very 
 much in favor of administering doses so small that no appreciable focal 
 or constitutional lesions will follow. Both the pathologic and the 
 immunologic process concerned in tuberculosis are somewhat different, 
 and in ordinary bacterin therapy a slight focpJ disturbance is desirable 
 and indicates that the vaccine in question possesses some potency. 
 Allen, who has an exceptionally rich experience in vaccine therapy, 
 frequently mentions the desirability of administering doses sufficiently 
 large to evoke slight reactions. 
 
 Following the administration of a vaccine it is believed that the 
 quantity of opsonin in the body-fiuids is tempf rarily decreased, and that 
 the inoculated person is, therefore, more suscfeptible to infection (nega- 
 tive phase) (Fig. 56). It can readily be understood how a vaccine 
 may temporarily depress the cells and defensive mechanism in general, 
 but just how it may bring about an actual decrease in opsonin it is 
 more difficult to understand, as the actual amount that may be used 
 in dealing with the vaccine itself must be 'Small.' Veterinarians are 
 careful not to expose cattle to infection imme'diately after they are im- 
 munized with anthrax vaccine, on account of this hypersusceptibility 
 to infection. In general, however, I have observed that most im- 
 munizators are prone to regard the question lightly, and to neglect 
 the importance of the negative phase, whereas some deny that it ever 
 exists. 
 
 Contraindications to Active Immunization.!— It should be emphasized 
 that a properly prepared vaccine is a potent substance capable, when 
 given in excessive dosage or when otherwise injudiciously administered, 
 of doing much harm. A vaccine stimulates body-cells, and unless the 
 cell can withstand the stimulation, the administration of vaccine may 
 do actual harm. This is the main reason why vaccines should be used 
 very cautiously, if at all, in the treatment of 'severe generalized infections. 
 In passive immunization the conditions are different, as the body-cells 
 are not taxed, but rather, through the neutralization of the toxic sub- 
 stances which they are combating, they are relieved, and an antibody- 
 laden serum may, therefore, be freely administered in severe infections. 
 
PROPHYLACTIC IMMUNIZATION OR. VACCINATION 623 
 
 In active immunization for therapeutic purposes the conditions 
 should be carefully weighed and the treatment=conducted by one who is 
 qualified to judge of the potencies of harm and good in a vaccine, and 
 who has had sufficient experieiice to guide him' in dosage and frequency 
 of inoculation, the main objects being to tide over and aid nature during 
 an acute infection, and to arouse and stimulate her during a chronic 
 infection. 
 
 In prophylactic immunization the physician should satisfy himself 
 that the patient has no latent or active infectibn that may be rendered 
 worse during the temporary depression that follows inoculation. It is 
 true that this depression is fleeting and temporary, and that the possi- 
 ble harm incurred may be far outweighed by the ultimate good, but vac- 
 cines should be given with proper discernment and not carelessly and 
 injudiciously. These remarks have no relation to cowpox vaccination, 
 where the good so far overbalances the possible harm that in general 
 all persons should be vaccinated, especially if ^n epidemic is impending. 
 
 (a) Tuberculosis. — There is at present some discussion relative to 
 the harm that may be caused in tuberculosis by tjrphoid unmunization. 
 Probably all will agree that a patient with an active and acute tubercu- 
 lous lesion should be refused inoculation, but when the lesion is quiescent 
 or healed, or in the early latent stage, it is indeed difficult to understand 
 how a prophylactic dose of typhoid vaccine will do more or as much 
 harm as an attack of tonsillitis, rhinitis, or some similar acute infection. 
 
 (b) In diabetes, carcinoma, and other debilitating conditions vaccines 
 should not be administered unless the indications or requirements are 
 unusually urgent. 
 
 (c) Advanced nephritis, especially parenchjmiatous nephritis, may 
 be regarded as contraindicating the administration of a vaccine. 
 
 PROPHYLACTIC IMMXJNIZATION OR VACCINATION 
 Smallpox 
 Historic. — Just when and where smallpox vaccination was first 
 practised is not known. The original method of inducing immuniza- 
 tion against the disease by introducing the virus from a smallpox pa- 
 tient into a healthy person through an abrasion of the skin and thus 
 greatly diminishing the virulence of the dise&se was practised by the 
 Turks during the eighteenth century, the chief object being to preserve 
 the beauty of the young Turkish and Circassian women. In 1878 Lady 
 Mary Montagu, the wife of the British Ambassador at the Ottoman court 
 
624 ACTIVE IMMUNIZATION 
 
 in Constantinople, observing tliis practice among the Turks, had her 
 own son and daughter inoculated and was largely instrumental in estab- 
 lishing the practice in Europe. 
 
 As regards the prophylactic value of this^ method of inoculation in 
 England and continental Europe, statistics are incomplete, but the 
 literature of contemporary writers shows that protection was usually 
 complete. The induced disease was not, however, always mild, and not 
 infrequently assumed an unexpected virulence that not only proved dis- 
 tressing and even fatal to inoculated individuals, but also constituted a 
 source of infection to a community. While, therefore, the underlying 
 principles were sound, and while these early, attempts at preventive 
 immunization mark an epoch in the history of medicine and of the world, 
 it was not until Edward Jenner made his investigations into a theory 
 held by farmers and by experimental evidence established it as true that 
 a satisfactory method of inmiunizing the bpdy against smallpox was 
 introduced. 
 
 The peasantry in various parts of Europe, and especially in England, 
 had generally observed that those who had had= sores on their hands con- 
 tracted from similar lesions on the teats of cows, usually escaped small- 
 pox infection when the disease was epidemic in a community. In fact, 
 it is said that several farmers dehberately inoculated members of their 
 family with cowpox lesions and that these escaped smallpox. 
 
 Edward Jenner was a physician practising in Berkeley, Gloucester- 
 shire, and frequently used the method of dirept inoculation from a mUd 
 case of smallpox among his patients. While a- student he was impressed 
 with the traditions of cowpox vaccination, and finding that they were 
 largely true, determined to make experimental tests. On May 14, 
 1796, he vaccinated a boy, James Phipps, with virus from a cowpox 
 lesion on the hand of a dairy maid, Sarah Nehnes, and on July 1st he 
 inoculated the same boy with pus from a smallpox patient without re- 
 sulting infection. In 1798 he furnished further proof that cowpox will 
 afford protection against smallpox by inoculating a child direct from a 
 vesicle on the teat of a cow, and continued the inoculation from arm to 
 arm through a series of five children, after which all were inoculated 
 with smallpox virus, without a single case developing. In the same year 
 he published "An Inquiry into the Causes and Effects of the Variolse 
 Vaccinae," illustrated by four plates, and within a year or two vaccina- 
 tion became general over Europe. 
 
 Vaccination was introduced into the United States in July, 1800, by 
 Dr, Benjamin Waterhouse, Professor of Physic at Harvard University, 
 
PROPHYLACTIC IMMUNIZATION OR Vi..CCINATION 625 
 
 who vaccinated his own children. At about the same time John Red- 
 man Coxe, of Philadelphia, vaccinated his oldest child and then tested 
 the experiment by exposing him to cases of smallpox. This bold repeti- 
 tion of Jenner's experiment considerably strengthened public confidence 
 in the method and the practice spread rapidly. Thomas Jefferson, writ- 
 ing in 1806 to Edward Jeimer, said: "Future generations will know by 
 history only that the loathsome smallpox existed and by you has been 
 extirpated." 
 
 But Jeimer and his earlier supporters met with much opposition, 
 often bitter and unrelenting, and this is readily understood when it is 
 realized that even at the present day, over a hundred years later, cow- 
 pox vaccination still has its opponents, in spite of the fact that the 
 value of the method has been established, and it has been found the 
 greatest of all boons to the human race, and notwithstanding that it 
 has been definitely proved that a thorough ancj continuous practice 
 of the operation would quickly eradicate smallpox from the face of the 
 earth. This opposition is especially pernicious and unjust, since the 
 practice of former years of vaccinating by direct transmission from arm 
 to arm has been entirely abandoned, and that animal lymph, prepared 
 and collected under strict aseptic precautions, is being used exclusively. 
 
 The Relationship of Variola and Vaccinia.^The relationship of 
 variola to vaccinia has been discussed since Jenner's time, but no ade- 
 quate explanation has been found. 
 
 According to the general belief the smallpox virus, whatever it may 
 be, is altered in its passage through a lower animal, and loses forever its 
 power of producing smallpox, but is still so closely related that the anti- 
 bodies it produces are sufficient to protect against smallpox. 
 
 The close interrelationship existing between vaccinia and variola is 
 shown by the presence in the virus of both, and in section of the skin of 
 both, of microscopic cell inclusions, first described by Guarinieri in 1892. 
 This finding has been confirmed by Pfeiffer in Germany and Councilman 
 and his associates in this country. These investigators have made 
 extensive studies of these bodies, and believe them to be protozoa in- 
 timately associated with the etiology of vaccinSi and variola. More 
 recently, Fornet has described certain small, diplococcus-like bodies 
 that were found in cowpox vaccine and in smallpox lesions. These are 
 regarded as having an etiologic relationship to gmallpox, and if these 
 findings are confirmed, would prove the identity ctf variola and vaccinia. 
 
 Recent investigators, particularly Copeman, of England, and Brink- 
 erhofi and Tyzzer, of America, have shown, by carefully conducted ex- 
 40 
 
626 ACTIVE IMMUNIZATION 
 
 periments, that vaccination will protect monkeys against subsequent 
 inoculation with smallpox virus, and this completely confirms the early 
 experiments of Jenner and others who proved the efficacy of vaccination 
 by the "variolous test." 
 
 The Preparation of Cowpox Vaccine. — Inuring the early days of 
 vaccination it was customary to inoculate human beings with material 
 obtained from the pustules of those previously vaccinated. The old- 
 time physician carefully removed choice scabs' and carried them about 
 in a special case ready for inoculation. While this method served its 
 purpose well, there were several drawbacks to its use, the chief of which 
 was the danger of transmitting syphilis. It has now for many years 
 bfien the custom to use virus obtained from animals, the production of 
 which can be carefully controlled and tested, any danger of transmitting 
 syphilis being thus obviated, because the heifer or cow used in the pre- 
 paration of the virus is not subject to this disease. The opponents to 
 vaccination, however, persist in using old and obsolete statistics regard- 
 ing the transmission of syphilis to support their claims, although these 
 have absolutely no bearing upon the modem methods of preparing the 
 virus. 
 
 Seed Virus. — This refers to the virus for vaccinating the calves or 
 other animals used, and is a most troublesome factor to those engaged 
 in this work. According to Park and Huddleston, a sufficient amount of 
 vaccine virus should be on hand to vaccinate from 40 to 50 persons. 
 Five children in good health and not previously vaccinated should then 
 receive an inoculation, each spot being of the size of a ten-cent piece. 
 On the fifth day after vaccination the upper layer of the resulting vesicle 
 should be removed, and sterilized bone slips be rubbed on the base thus 
 exposed. From 100 to 200 slips on each side of the sUp may be charged 
 from each child. The slips should be allowed to dry for a minute, and 
 should then be placed in a sterilized box and preserved in cold storage, 
 where they will remain active for at least two or three weeks. The 
 aforenamed observers now use rabbits alternately to obtain seed virus. 
 
 Subsequent animals are vaccinated with any one of three vaccines — 
 (1) Slips charged from typical vesicles of a calf; (2) slips charged with 
 the serum from a calf after removal of the vesicles; (.3) the glycerinated 
 virus may be used to vaccinate succeeding calves, but in this case it is 
 necessary to keep the glycerinated virus for two or three months, smce 
 the use of fresh virus on a succession of calves leads to prompt degenera- 
 tion of the vaccine and to the production of infected vesicles. 
 
 The New York Vaccine Laboratory produces a virus that is never 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 627 
 
 ^more than four successive transfers from a human case of vaccinia, 
 and is guaranteed to give 100 per cent, of "takes" in primary vaccina- 
 tion. 
 
 Animals. — Various animals have been used, but female calves from 
 two to four months of age are preferable. Older animals may be used, 
 and in several European institutes cows are usually employed. With 
 properly constructed operating-tables, they may be handled with 
 comparative ease. Rabbits have also been used, especially in pro- 
 
 ^ pagating the seed virus and to obtain pure and highly active viruses. 
 
 The calves are kept under supervision for at least a few days. In 
 some institutes they are tested with tuberculin, although with good 
 veterinary inspection this test is not necessary. Soon after admission 
 the animal is clipped and given a thorough cle&nsing, which includes the 
 feet and the tail. 
 
 On the day before vaccination is to be performed the belly-wall is 
 cleanly shaved from the cuneiform cartilage to the pubis, and well up 
 on the iimer sides of the thighs and the flanks. The skin is then 
 thoroughly washed. Just preceding the vaccination the animal is 
 fastened to the operating-table and the abdomen and inner surface of 
 the thighs prepared as for an aseptic abdominal section, i. e., a thorough 
 scrubbing with hot water, green soap, and soft brush, followed by alcohol 
 and sterihzed water, the parts being then dried with a sterile towel. 
 All other parts are covered with sterile sheets, and the calf is now vac- 
 cinated under aseptic precautions. 
 
 Vaccination. — ^About 100 small scarifications are now made in these 
 areas, preferably by cross-scratches or in roVs of lines about one to 
 two centimeters square and at least one to two centimeters apart. 
 The scarification is simple, but usually brings, a small amount of blood. 
 After they have been made, they are mopped with sterile gauze and 
 rubbed with the charged slips, using one or two slips for each small area, 
 depending on the amount of virus each slip oontains. The lesions are 
 allowed to dry, and are then covered with sterile gauze or a simple 
 protective paste, or arc left entirely uncoveredt 
 
 Precautions should be taken to keep the animals as clean as possible. 
 Inoculated animals are to be kept in stalls or. stables apart from those 
 under observation. The stable should be so constructed that the floors 
 can be flushed daily with a hose and hot water*. Excreta should be re- 
 
 ^ moved promptly. No bedding is permissible, and means should be 
 provided for fastening the legs and preventing the animal from kicking 
 the scarifications. 
 
628 
 
 ACTIVE IMMUNIZATION 
 
 Collection. — Ordinarily, within forty-eight hours of vaccination, 
 the scratches are pinkish, slightly raised, and papular, and within five 
 or six days, depending upon the rate of development of the vaccine 
 vesicles, the virus should be ready for collection (Fig. 127). The calf 
 is killed and placed upon the operating-table. The appointments of the 
 operating-room are usually equal to those in a well-equipped hospital 
 
 Fio. 127. — Production of Cowpox Vaccine. 
 Note the lines of cowpox lesions over the abdomen and flanks of the calf. The 
 surgeon is about to cleanse this area in a thorough and careful manner, after which 
 the cowpox material is removed with a curet and collected in a sterile vessel. All 
 precautions are taken to insure as thorough aseptic technic as possible. 
 
 operating-room, being supplied with all conveniences and means for 
 carrying out a careful, painstaking, and aseptic technic. 
 
 The exposed parts are covered with sterile sheets. The operator 
 and his assistant are clad in aseptic gowns. The vaccinated field is 
 thoroughly scrubbed wath soap, sterile watej, and gauze, and mopped 
 with sterile gauze. Crusts are carefully picked off, and the soft, pulpy 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 629 
 
 mass cureted off with a special, spoon-like curet and collected in a 
 sterile vessel. After the curetage, serum exudes from the excoriated 
 base of the vesicle, and ivory tips may be charged in this. The sticky 
 and pulpy exudate is then mixed with four times its weight of glycerin 
 and water (50 per cent, glycerin, 49 per cent, water, 1 per cent, phenol), 
 and this is done most effectively by passing the mixture between the 
 rollers of a Doring mill. The glycerinated pulp is allowed to stand for 
 three or four weeks in order to allow bacteria, which are invariably 
 present, to undergo dissolution. At the end of this time the glycerinated 
 pulp is thoroughly titrated in specially constructed triturating machines, 
 and put up in small capillary tubes, which are; scaled, or "vaccine 
 points" may be prepared. If properly preserved in sealed tubes in 
 a dark, cool place the virus should remain active for at least three months. 
 
 According to Park and Huddleson, 10 grams of pulp and 200 charged 
 slips would be an average yield from a calf, and when made up should 
 suffice to vaccinate at least 1500 persons. Calves" vary greatly in their 
 yield of virus. Of two calves vaccinated in exactly the same manner, 
 one may furnish material for 500 vaccinations and the other for 10,000 
 iaoculations. 
 
 Testing the Vaccine. — The virus may be tested for its efficacy by a 
 variety of methods. Calmette and Guerin ' inoculate rabbits upon the 
 mner surfaces of the ears and estimate the potency of the virus from the 
 speed of development and the size of the resulting lesions. Guerin^ 
 estimates the potency of virus quantitatively by inoculating rabbits 
 with serial dilutions ranging from 1 : 10 to 1 : 100. Fully potent virus 
 should cause closely approximated vesicles in a dilution of 1 : 500, and 
 numerous isolated vesicles in a dilution as high as 1 : 1000. 
 
 Quantitative estimation of the bacteria in the glycerinated virus 
 is made by the plating method, and the vaccine used only when the 
 numbers of bacteria have been greatly diminished or are entirely absent. 
 The vaccine is also tested for tetanus by injecting relatively large 
 quantities subcutaneously into guinea-pigs and mice. 
 
 Under the Federal Law of July 1, 1902, and the regulations framed 
 thereunder, all firms manufacturing vaccine virus are required by the 
 Secretary of the Treasury to obtain a license before they may sell their 
 products in interstate commerce. The vaccine laboratories are care- 
 fully inspected by an official of the Hygienic Laboratory of the United 
 States Public Health and Marine-Hospital Service; the inspector 
 carries away with him as many samples of virus as he wishes, and addi- 
 
 ' Ann. de I'lnst. Pasteur, 1902. ^ Ann. de I'lnst. Pasteur, 1905. 
 
630 ACTIVE IMMTJNIZATIOlSr 
 
 tional samples are purchased in the open market in different parts of the 
 country. All these are subjected to a vigorous bacteriologic examina- 
 tion, especially for tetanus bacilli, by a laborlitory worker who devotes 
 all his time to this work. The federal regulations require each vaccine 
 institute to perform a careful autopsy on each calf after the vaccine 
 virus has been removed, and if any commumcalile disease is found or 
 suspected in the animal, the virus must notibe placed on the market, 
 but must be destroyed. In accordance with this law, permanent records 
 of the bacteriologic examinations of the virus and of the autopsy shall 
 be kept in each institute. 
 
 Technic of Vaccination. — The essential part of the process of vac- 
 cination is that the virus should be introduced through the epidermis 
 so as to be absorbed by the lymphatics and blood-vessels of the corium. 
 
 The site usually chosen is the skin of the outer side of the upper arm, 
 over the insertion of the tendon of the deltoid muscle. Sometimes, in 
 females, the outer side of the thigh or well above the knee on the inner 
 aspect of the thigh is used. Vaccination on the leg, however, is never 
 advisable, as it would appear that such vaccinations are more prone 
 to take on an excessive inflanomatory action owing to the greater con- 
 gestion due to the dependent position of the lower extremities; there 
 is also more likelihood of secondaiy infection and mechanical violence 
 occurring. 
 
 The skin is washed with alcohol and finally with water and then 
 dried, care being exercised not to rub too vigorously; if the skin is 
 reddened, it is best to wait until the hypereoiia subsides. Grasp the 
 arm with the left hand, rendering the skin tense, and with a sterile 
 needle or scalpel carefully remove the epidermis over a square area 
 measuring about one-eighth of an inch (Fig. 128). Bleeding should be 
 avoided — an abraded surface that just oozes- serum is especially to be 
 desired. The virus is then expressed or placed upon this area (never 
 blown out), and thoroughly rubbed in with a sterilized wooden tooth-pick 
 or the vaccine point. After allowing the lymph to dry, a light sterile 
 gauze dressing should be applied. 
 
 Some operators abrade through the lymph; others make one or two 
 straight lines of scratches and rub in the virus. 
 
 Another excellent method^ consists in abrading the arm with a light 
 
 rotatory motion of a von Pirquet chisel (sterilized in alcohol and in a 
 
 flame), measuring about 2 mm. in width. (See Fig. 122.) The virus 
 
 is then applied, and thoroughly rubbed in with a sterile tooth-pick. 
 
 1 Force, J. N.: Jour. Amer. Med. Assoc. ,.1914, bdi, 1466. 
 
PROPHYLACTIC IMMUNIZATION OR ¥ACCINATION 
 
 631 
 
 Cross-scarification, which is forbidden in Germariy, favors the growth of 
 anaerobic bacteria under the crust that forms on the surface of the 
 abrasion where the resistance is lowered by the action of the virus. 
 The circular scarification gives more control over the dosage, and there 
 is no tendency to the development of excessively sore areas. 
 
 Fig. 128. — Method of Vaccination Against Smallpox. 
 
 The skin is stretched, and a series of superficial and parallel scratches made through 
 
 the epidermis with a sterilized needle. 
 
 Usually one inoculation of the virus is sufficient, but in times of 
 threatened epidemic two or more inoculations are "made at the same time, 
 not only to insure a successful result, but rapidly to immunize the 
 patient. It would appear that the degree of immunity bears some rela- 
 tion to the number or size of the vaccination lesions, and this can readily 
 be understood if the infection is local and the body-cells are stimulated 
 by a diffusible toxin. If, however, the vaccination lesion is but the 
 
632 ACTIVE IMMUNIZATION 
 
 point of entry of what becomes a general infection, then a small lesion 
 should suffice. This point has not been definitely settled, but statistics 
 tend to show that persons vaccinated in two^or more areas develop an 
 immunity more quickly and that this immunity is more lasting. 
 
 The subsequent care of the wound is of considerable importance. The 
 operation is usually regarded as a trivial one, and justly so, but the 
 lesion requires judicious after-treatment instead of being entirely 
 neglected, as it so often is. The severe infections are usually attribut- 
 
 ■a- 
 
 able to gross and careless contamination of the wound. The best pos- 
 sible protection to the vaccinial ulceration is afforded by the forma- 
 tion of a hard, solid crust, due to desiccation of the contents of the 
 vaccine vesicle and pustule. Unless undue inflammation and suppura- 
 tion set in, such a crust will form. Care must be taken that the 
 crust is not subjected to mechanical violence calculated to loosen or to 
 detach it. 
 
 Constricting shields are likely to be unsatisfactory. The adhesion 
 of the crust to the sleeve or to a piece of protective gauze wUl often 
 lead to forcible dccrustation when the sleeve or the gauze is removed. 
 Schamberg and Kolmer^ have found that daily applications of a 4 per 
 cent, alcoholic solution of picric acid upon the vaccinated area after 
 the first forty-eight hours does not interfere with the success of the 
 vaccination, and lessens the degree of local inflammatory reaction and 
 constitutional disturbances by hardening the epithelial covering of the 
 vaccine lesion, and thereby decreasing the liability of extraneous 
 bacterial infection. 
 
 A point to be emphasized is that severe lesions are unnecessary, and 
 are usually due to scratching of the vesicle or pustule and consequent 
 introduction of dirt. No doubt tetanus bacilli may be introduced in 
 this manner, the resulting scab affording the necessary anaerobic con- 
 ditions for their development. 
 
 The Phenomena of Vaccination; Vaccinia. — Immediately follow- 
 ing vaccination a slight redness appears, which usually subsides rapidly. 
 After a short period of incubation — on or about the third day — a slight 
 red elevation makes its appearance, and the lesion begins to burn and 
 itch. On the sixth or seventh day the abrasiop becomes a small, silvery 
 gray, umbilicated vesicle with a sharply raised edge, filled with a clear 
 serum, and surrounded by a narrow red areola (Fig. 129). By the 
 tenth day the characteristic features are more rnarked, and the lesion has, 
 usually reached its height, being accompanied by a burning sensation 
 1 Lancet, London, Nov. 18, 1911, 1397. 
 
Fig. 129. — Vaccinia (.Seven-day Le.^^ion). 
 
 Fig. 130. — V.\ccinia (Xine-day Lesion). 
 
 Fig. 131. — ^'A^CINOID. A Vac- 
 
 CTNATiDN Scar. 
 
 Fig. 129 shows a se:'en-day ^■ac(■inat^on vesicle;* Fig. 130 .shows a nine-day 
 vaccination vesicle jnst before ]nistulation occurred. Fig. 131 shows a recent vacci- 
 nation scar with pitting and radiation; also a three-dav " vaccinoid'' or "immunity 
 reaction" with a small vesicle. 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 633 
 
 and an almost uncontrollable desire to scratch (Fig. 130). The areola 
 is now quite angry in appearance, and numerous minute vesicles are 
 seen on its surface. By the twelfth day the areola is smaller, the con- 
 tents become turbid and commence to dry, so that a few days later a 
 scab has formed that drops off in another week or two. 
 
 About the fifth day the child becomes restless and irritable and shows 
 a slight elevation of temperature. These symptoms may become more 
 pronounced until the end of the second week, when they subside rapidly. 
 
 Precautions should be taken to prevent scratching and infection of 
 the vesicle. The old-time "beautiful arms," with well-marked cellulitis 
 and adenitis of neighboring glands, were largely due to secondary in- 
 fection, and arc not at all necessary in the process of vaccination. 
 Evidence would tend to indicate that the vesicle is the typical lesion 
 of both smallpox and vaccinia, and that the pustules are simply infected 
 vesicles. Ordinary surgical care will do much to rob vaccination of its 
 discomfort and to render the operation a most harmless one. 
 
 The results of a vaccination, therefore, can be inspected and verified 
 on or about the seventh to the ninth day. With persons who have been 
 vaccinated successfully on a previous occasion the vaccinated area may 
 show a slight areola at the end of twenty-four hours, with or without a 
 papule, which subsides in seventy-two hours. This is called a "reac- 
 tion of immunity," and is due to the presence of antibodies against the 
 virus. Or a small, itchy, burning papule may^ form, which develops 
 into a small vesicle maturing on the fifth or sixth day, and then rapidly 
 subsiding, constituting the reaction known as vaccinoid (Fig. 131). 
 OccasionaUy vaccination is followed by the appearance of various 
 eruptions. 
 
 The appearance of the scar varies according to its age and to the 
 degree of tissue destruction. The physician is not infrequently re- 
 quested to examine a person and determine if the scar is satisfactory 
 evidence of successful vaccination. The typical good scar is circular, 
 and about the size of a ten-cent piece, with smooth, white, and depressed 
 center and a raised border. The border shows numerous radiations, and 
 the entire scar may show little pits of former hair-follicles when the 
 lesion was sufficiently destructive to remove the upper portion of the 
 corium. (See Fig. 131.) A burn or an ordinary pyogenic infection may 
 leave scars quite similar to those of vaccination, and vaccination scars 
 may show wide variation, but the circumscribed character, the raised 
 border with radiations and depressions, and the appearance of having 
 been stamped on the skin by a sharply cut die are quite characteristic. 
 
634 ACTIVE IMMUNIZATION 
 
 Poor scars are those that were said to have been the result of vaccination, 
 but in very many instances they are so indistinct as to make it difficult 
 or impossible to recognize them as vaccinatior|-marks. 
 
 Revaccination. — One successful vaccination does not necessarily 
 confer an absolute immunity against smallpox, and failure to recognize 
 certain limitations in this respect has done harm by enabling anti- 
 vaccinationists to create a distrust in the minds of the ignorant by 
 pointing to individual instances of failure. That a person who has 
 once been vaccuiated may afterward suffer from smallpox is undoubted, 
 but usually the vaccination was performed many years previously, 
 and in any case the disease, when it does occur, is relatively mild (vario- 
 loid). 
 
 There can be no doubt but that the immunity gradually diminishes. 
 Perhaps seven years may be taken as the average period of fairly com- 
 plete protection. Children should be vaccuiated within the first year 
 after birth, revaccinated upon entering school, and again after leaving it. 
 If smallpox is prevalent, all persons should be vaccinated, regardless 
 of the fact that they have previously been vaccinated. Only those who 
 have had smallpox may be excused. If, as a naatter of fact, persons are 
 still immune, the vaccination will not "take" and no harm is done, 
 whereas if it succeeds, such persons will have the satisfaction of knowing 
 that their immunity has been increased. Hence it cannot be too 
 strongly emphasized that not only vaccination, but revaccination, is 
 indicated to protect the individual and society against smallpox. Dwyer 
 claims that a person should be revaccinated repeatedly in succession 
 until he fails to react; even a slight "take" would indicate incomplete 
 immunity. 
 
 Occasionally a non-immunized person refuses to "take," but vac- 
 cination should be repeated three or four times, as failure is not infre- 
 quently due to old and inactive virus, and the" actual number of persons 
 absolutely insusceptible is very small indeed. 
 
 Risks of Vaccination. — When vaccination is properly performed with 
 a good virus, the risk of permanent injury to life or limb is almost 
 negligible. 
 
 1 . Tetanus. — The most serious of the inj uries that have been attributed 
 to vaccination is tetanus. The tetanus bacillus and its spores are so 
 wide-spread in nature that opportunity presents itself for contamination 
 of the vaccinal wound and the virus itself. Every precaution should, 
 therefore, be taken in the preparation of virus, and it is especially im- 
 portant that physicians and laymen should" realize the necessity for 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 635 
 
 observing ordinary care, and at least ordinary cleanliness, in the treat- 
 ment of the vaccinal wound. 
 
 It is exceedingly difficult to determine the source of infection in each 
 case of vaccinal tetanus, but experimental inyestigations would tend 
 to indicate that the virus itself is seldom, if ever, the vehicle of infection. 
 John F. Anderson, Director of the Hygienic Laboratory, in testimony 
 given before the Pennsylvania State Vaccination Commission, stated 
 that in experiments carried out on monkeys and guinea-pigs with vaccine 
 lymph purposely contaminated in the laboratory with countless numbers 
 of tetanus spores, it was found impossible to communicate tetanus 
 in this manner, although the vaccinations were more severe than the 
 ordinary vaccinations performed on man, in that several places were 
 inoculated and the areas abraded were large. Anderson stated that 
 his "conclusion from these experiments is that it is almost impossible 
 to produce tetanus, even with vaccine virus that contains tetanus 
 germs in it, by the simple act of vaccination." 
 
 Since 1909 there were approximately 100,000 specimens of vaccine 
 virus examined in the Hygienic Laboratory, particularly with the pur- 
 pose of determining the presence of tetanus germs or their products. 
 The vaccine was purchased in the open market, and the examinations 
 were made as thoroughly as it was possible to make them. To use 
 Anderson's words: " We have never succeeded in finding any evidence 
 of the presence of the tetanus organism or its products in vaccine virus." 
 (Report of the Commission.) 
 
 It would appear, therefore, that virus prepared according to modern 
 methods and with all recognized precautions is safe. In view of the 
 incidence of tetanus following other inj uries, it is reasonable to conclude 
 that most cases of vaccinal tetanus are secondary woimd infections, 
 and therefore largely preventable. 
 
 2. Syphilis. — With the use, years ago, of humanized virus, and par- 
 ticularly in the days of arm-to-arm vaccination, extremely rare instances 
 of the transmission of syphilis have been known to occur. Since, how- 
 ever, the use of calf virus, which is the virus exclusively employed in this 
 country, such an accident is absolutely impossible, as calves are not 
 susceptible to luetic disease. 
 
 3. Cancer, foot and mouth disease, tuberculosis, and various chronic 
 skin eruptions have been attributed to vaccination by its opponents; 
 none of these claims has, however, been substantiated. 
 
 Protective Value of Vaccination. — Of the value of the protection 
 afforded by vaccination against smallpox there can be no doubt in the 
 
636 ACTIVE IMMUNIZATl'ON 
 
 minds of right-thinlcing and unbiased persons. The history of the 
 world before the days of universal vaccination shows the wide prevalence 
 of smallpox and its fearful mortality. It was regarded as a disease of 
 childhood, owing to the fact that all contracted it at the earhest op- 
 portunity, and, accordingly, smallpox was the cause of a fearful infant 
 mortality. 
 
 At the present day, owing to the general employment of vaccination, 
 smallpox is a rare disease, but its very rarity has fostered a certain de- 
 gree of false security and carelessness in carrying out the process. A 
 young and new generation of non-vaccinated persons in any community 
 is a source of danger, and accordingly sporadic cases are often blessings 
 in disguise, from the fact that, when they appear, compulsory vaccina- 
 tion is then instituted and large numbers seek revaccination. 
 
 In Germany, where vaccination is compulsory, smallpox is now a 
 comparatively rare disease. While the general death-rate from all 
 diseases is lower in England and Wales than in Germany, the smallpox 
 mortality is seven and one-half times the mortality of Germany, and, 
 proportionate to the population, over 13 timeq. 
 
 Austria, one of Germany's neighbors, had, for the twenty years 
 following 1874, almost 30 times as high a smallpox mortality as Germany. 
 During this period 239,800 persons perished in Austria from smallpox 
 alone. 
 
 Physicians should carefully impress upon those over whom they have 
 any influence the necessity of being vaccinated, for only a thoroughly 
 vaccinated population can solve the problem of exterminating smallpox 
 as an epidemic disease. 
 
 Rabies 
 There are but few diseases more dreaded" by the laity than rabies, 
 or hydrophobia. Tales of the sufferings of infected persons, especially 
 those with the furious variety of this infection, characterized by maniacal 
 symptoms and dread of water (hydrophobia), have been thoroughly 
 disseminated, so that the cry of "mad dog!" on the public streets is 
 sufficient to arouse a general state of hysteric excitement in which an 
 otherwise harmless creature may be compelled to bite or snap for self- 
 protection. Not all dogs under these conditions are mad or infected 
 with rabies, and the bite of an angry dog, otherwise normal, is not 
 necessarily dangerous from the standpoint of rabic infection. However, 
 almost every one, upon being bitten by a dog, will promptly consult 
 his physician, and this is proper and to be encouraged. Genuine rabies 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 637 
 
 is an acute infectious disease in which the diagnosis is quite readily 
 made, and whenever possible an effort should be made either to confirm 
 or to disprove the diagnosis by making an examination of the animal's 
 brain, and if the dog is found to have been free from rabies, this fact 
 should be carefully impressed upon the patient,^ as otherwise the dread 
 of infection may weigh heavily upon the patient and lead to distressing 
 nervous disturbances. 
 
 While the infectiousness of rabies has been known for a great many 
 years and was proved experimentally by Galateir^ and Pasteur,^ it was 
 not until Negri, in 1903, described certain bodies (Negri bodies), seen 
 by him in large nerve-cells in sections of the central nervous system, 
 that anything was found that seemed absolutely specific for rabies. 
 Negri regarded these bodies as specific for rabies and probably of a 
 protozoan nature. Later investigations fully established the diagnos- 
 tic value of these bodies, and their definite characteristic morphology, 
 evidences of cyclic development, and staining qualities indicate a 
 protozoan structure resembling members of ther Rhizopoda, designated 
 by Anna Williams in 1906^ as Neurorrhyctes hydrophobim. 
 
 Rembringer,'' Poor and Steinhardt,^ Bertarelli and Volpino^ have 
 demonstrated the filterability of the rabic virus, and Noguchi^ has cul- 
 tivated from both "street" and "fixed" virug, very minute granular 
 and somewhat coarser pleomorphic chromatoid bodies which, on sub- 
 sequent transplantation, reappeared in the new cultures through many 
 generations and reproduced typical symptoms of rabies in dogs, rabbits, 
 and guinea-pigs. 
 
 To Pasteur is due the credit for having discovered (1880) the fact 
 that the disease may be prevented by conferring gradual inununization 
 with increasing doses of the attenuated virusj This treatment, with 
 some modification, is now used with evident success in all parts of the 
 world. 
 
 Nature of Rabies, — The virus or parasite is contained in the saliva 
 of the rabid animal, and infection is possible when the skin is abraded 
 by bites and scratches. The virus travels by way of the nerve-paths 
 
 1 Compt. rend. Acad. d. so., 1879, Ixxxix, 444. 
 
 ^ Compt. rend. Acad. d. so., 1881, xcii, 159. 
 
 5 Proc. N. Y. Path. Soc, 1906, vi, 77. 
 
 * Ann. de I'Inst. Pasteur, 1903, xvii, 834; 1904, xviii, 150. 
 
 ' Jour. Infect. Dis., 1913, xii, 202. 
 
 ' Centralbl. f. Bakt., Orig., 1904, xxxvii, 51. Bertarelli, ibid. 
 
 'Jour. Exper. Med., 1913, xviii, 314. 
 
638 ACTIVE IMMTJNIZATIpN 
 
 to the central nervous tissue, and, as in tetanus, the symptoms of the 
 disease are due to involvement of these tissue^.. 
 
 The period of incubation, or the time elapsing between the time of 
 injury and the first symptoms, is quite variable, ranging from twenty 
 to sixty days, although it may be as short as. ten days. As in tetanus, 
 this period depends upon — (a) the location of the injuiy ; (b) the quantity 
 or dose of virus; (c) the kind of animal responsible for the injury. Bites 
 about the face and fingers, especially if they are deep and lacerating, 
 are especially dangerous; bites about the back and lower limbs, espe- 
 cially if superficial, are much less dangerous, and accompanied by a 
 longer period of incubation. It is to be remembered that bites may be 
 infectious as early as nine days before the dogi shows well-marked symp- 
 toms of the disease. Not infrequently an animal is observed to be 
 surly and snappy for several days before rabid symptoms develop, and 
 a bite during this time should be regarded as dangerous. 
 
 Only about 16 per cent, of human beings bitten by rabid animals and 
 untreated appear to contract rabies. Since the establishment of the 
 Easteur treatment of the disease, the percentage of developed cases 
 after bites is much lower — about 0.46 per cent.. 
 
 Diagnosis and Management of Rabies. — Even though an animal 
 is unmistakably rabic, every effort should be rdade to destroy the animal, 
 not only in order to prevent further damage, but to corroborate the 
 diagnosis by microscopic examination of the nervous tissues for Negri 
 bodies. 
 
 1. As a general rule, all animal bites should receive surgical attention. 
 Wounds produced by animals clinically rabip should be cauterized at 
 once with fuming nitric acid or pure phenol. This is done to offset 
 the delay in securing the Pasteur treatment, and because there is evi- 
 dence to show that thorough cauterization of the wound is in itself 
 highly beneficial. 
 
 2. The animal should be promptly destroyed, not only to prevent 
 further damage, but in order to make a microscopic diagnosis by ex- 
 amination of the brain for Negri bodies. This examination is highly 
 important and should never be omitted, for if it shows the absence of 
 bodies, this fact should be carefully impre^ed upon the patient, as 
 there is no doubt that a neurotic element, amounting in many instances 
 to actual hysteria, may cause considerable harm to the patient even 
 though he is definitely free from rabic infection. 
 
 3. The whole dog may be packed in ice and shipped at once to a 
 ceiitral laboratory, or the head alone may be removed and packed in 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 639 
 
 ice or glycerin and promptly shipped. The brain should not be dis- 
 turbed. When it reaches the laboratory, the diagnosis should be made 
 at once by "smear" preparations and sections of the brain demonstrat- 
 ing the characteristic Negri bodies in the large ganglion-cells, and con- 
 firmed by inoculating emulsions of the brain into guinea-pigs or rabbits. 
 The latter requires from ten to twenty days before the result may be 
 known. A negative animal inoculation test is better evidence than a 
 negative smear or section; obviously, these examinations are to be 
 made only by properly trained persons. 
 
 According to Park, the value of the smear -method of diagnosis may 
 be sununarized as follows: 
 
 1. Negri bodies demonstrated, diagnosis, rabies. 
 
 2. Negri bodies not demonstrated in fresh brains, very probably 
 
 not rabies. 
 
 3. Negri bodies not demonstrated in decomposing brains, uncer- 
 
 tain. 
 
 4. Suspicious bodies in fresh brains, probably rabies. 
 
 4. If the animal was clinically rabid, the Pasteur treatment should 
 be commenced as soon as possible, without waiting for the laboratory 
 report, if this will be delayed for several days. Wounds about the face 
 and hands, where there is no clothing to retain the infectious saliva, 
 should receive intensive treatment; otherwise the milder course of 
 immunization will suffice. 
 
 5. As a general rule, it is well to send the patient to a regular Pasteur 
 institute, where there are special facilities for the proper treatment of 
 these cases. Otherwise the physician may treat the patient at home. 
 Several large manufacturing firms are prepared, to ship by mail the fresh 
 daily treatments properly preserved and ready for administration. 
 
 6. The Pasteur treatment should be given to every patient bitten 
 by a rabid animal or by one suspected of being rabid. Not all persons 
 are necessarily infected, even by bites of rabi4 animals, but this should 
 not unduly influence the physician, for he will not have fulfilled his duty 
 unless he carefully explains the etiology of the disease and advises im- 
 tuediate immunization. Aside from the actual benefits of the treat- 
 ment, the mental effect upon the patient is deserving of consideration. 
 Even slight wounds by rabid animals, wherever their location, should 
 be regarded as dangerous, and the Pasteur treatment advised in addi- 
 tion to routine cauterization. 
 
 7. With severe bites of angry but not necessarily clinically rabid 
 the treatment depends upon various factors. In any case the 
 
640 ACTIVE IMMUNIZATION 
 
 wound should be thoroughly cauterized and the animal carefully 
 guarded (not killed) for two or three weeks. If rabid symptoms appear, 
 the animal should be destroyed, the cerebral tissues examined, and the 
 Pasteur treatment of the patient begun. 
 
 8. All animals bitten by a rabid animal should, of course, be promptly 
 destroyed; even those bitten by a dog suspected of being rabid should 
 be destroyed or closely guarded until a definite diagnosis can be reached. 
 In England, where strict laws are enforced relative to the muzzling and 
 control of dogs, rabies is relatively infrequent, and it is especially urged 
 that similar measures be adopted and enforced in our own communities, 
 particularly during the summer months. 
 
 Principle of the Pasteur Treatment (Active Immunization) of Rabies. 
 -—This method is based upon the principle of stimulating the production 
 of rabic antibodies by injecting attenuated or. modified virus during the 
 period of incubation, so that the virus intreduced into the wound is 
 destroyed, neutralized, or its effects neutralized, while the virus itself 
 is finally destroyed. Pasteur worked out this theory and established 
 its truth by experiments upon the lower animals before applying the 
 treatment to man. 
 
 By passing the virus through a series of rabbits, the period of incuba- 
 tion is shortened to about six to seven days,, and at the same time its 
 pathogenicity for man is actually diminished, {virus fixe). By drying 
 the tissues containing the fixed virus attenuation is secured, so that it 
 is easily possible so to modify the virus that it cannot produce rabies in 
 man, but yet is able to produce the specific antibodies. The Pasteur 
 treatment is, therefore, a process of active inamunization with emul- 
 sions of a tissue (spinal cords of infected rabbits) in which the virus 
 has been attenuated by a process of drying and desiccation. The early 
 doses consist of highly attenuated cords, and^ succeeding doses become 
 gradually more potent, as is usual in the techni'c of any method of active 
 immunization. 
 
 Preparation of the Rabies Vaccine. — As a f)reliminary, it is necessary 
 to prepare or obtain "virus fixe." This m^ generally be procured 
 from a laboratory, or may be prepared by passing street virus from the 
 medulla of a rabic cow or dog through a series of young rabbits. After 
 from 30 to 50 passages the incubation period is gradually reduced to six 
 or eight days ("virus fixe"). 
 
 1. From an animal succumbing the day or night before, a piece of 
 the floor of the fourth ventricle measuring about 2 cm. in length is 
 emulsified in 1 c.c. of sterile bouillon, and three or four drops of this 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 
 
 641 
 
 emulsion are injected beneath the dura of a normal rabbit. In large 
 institutes two or more rabbits are injected daily. The inoculation is 
 quickly and easily performed by trephining a small area in the median 
 line of the forehead and injecting the emulsion beneath the dura mater 
 with a syringe. The whole operation must be. carried out in an aseptic 
 and practically painless manner. 
 
 2. After inoculation the animals are placed in clean cages; in from 
 six to eight days paralytic symptoms of rabies appear, followed in three 
 to four days by death. The hair is then sprayed 
 with a solution of lysol and the skin removed. 
 The cord and brain are then extracted under 
 aseptic precautions. The cord is severed just 
 below the medulla, a portion is snipped off into 
 sterile bouillon for culture, and then divided into 
 two equal pieces which are suspended by sterilized 
 sUk threads in a sterile glass jar containing flakes 
 of caustic potash (Fig. 132). The medulla is 
 placed in a sterile dish, and is used to continue 
 the inoculations, as was previously described. A 
 postmortem examination is finally performed, and 
 any cord in which the animal is found diseased or 
 in which the culture of the cord shows bacterial 
 contamination is rejected. 
 
 The jars with suspended cords are kept in a 
 special room at a temperature of about 20° to 25° C. 
 
 3. After a suitable period of drying pieces of 
 cord are prepared for injection. This is performed 
 in various ways at different laboratories; no at- 
 tempt at exact dosage is made. In the New York 
 Board of Health laboratories 1 cm. of the cord is 
 thoroughly emulsified in 3 c.c. of sterile saline 
 solution, the process being conducted in an aseptic 
 manner. If the material is to be shipped, an 
 
 addition of 20 per cent, of glycerin and 0.5 per cent, of phenol is 
 made. 
 
 Administration of the Vaccine. — Injections are given with a sterile 
 syringe. The abdominal region of the patient is bared, a spot touched 
 with tincture of iodin, wiped with alcohol, and the injection given sub- 
 cutaneously. Keirlc does not vary the dose according to the age, both 
 the old and the young receiving the same doss.- At times the injection 
 
 Fig. 132.— Prepara- 
 tion OF Rabies 
 Vaccine. 
 Note the cords 
 suspended within the 
 jar by means of sterile 
 silk tlireads; sticks of 
 sodium hydroxid to 
 absorb moisture and 
 hasten desiccation. 
 
642 
 
 ACTIVE IMMUNIZATION 
 
 is followed by redness and induration in the 'subcutaneous tissues, but 
 abscesses are never formed. 
 
 Cases of severe injury, such as deep bites about the face and fingers, 
 should be rapidly immunized {intensive treatment); in other cases the 
 treatment may be mild (mild treatment). The uniform dose of cord 
 emulsion, prepared as just described, is 2.5 c.t. The series of inocula- 
 tions given in the Research Laboratory of Now York in treating human 
 cases after an average bite are as follows: 
 
 TABLE 23.— SCHEDULE OF INOCULATIONS IN IMMUNIZATION 
 AGAINST RABIES 
 
 First 
 
 Second 
 
 Third 
 
 Fourth 
 
 Fifth 
 
 Sixth 
 
 Seventh. . , . 
 
 Eighth 
 
 Ninth 
 
 Tenth 
 
 Eleventh . . . . 
 
 Twelfth 
 
 Thirteenth. . 
 Fourteenth . . 
 Fifteenth . . . 
 Sixteenth. . . 
 Seventeenth . 
 Eighteenth. . 
 Nineteenth . . 
 Twentieth . . 
 Twenty-first . 
 
 Mild Treatment 
 
 INTENSIVE Treatment 
 
 14 and 
 12 and 
 10 and 
 8 and 
 6 day 
 .5 day 
 4 day 
 .3 day 
 2 day 
 4 day 
 .3 day 
 2 day 
 
 4 day 
 
 5 day 
 
 2 day 
 4 day 
 
 3 day 
 
 2 day 
 
 4 day 
 
 3 day 
 2 day 
 
 13 day cord 
 
 11 day cord 
 9 day cord 
 7 day cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 cord 
 
 12 and 11 day cord. Repeated in p. m. 
 10 and 9 da,y cord; 8 and 7 day cord p. M. 
 6 day cord 
 
 day cord 
 4 day cord 
 
 3 day cord 
 
 2 day cord 
 
 4 day cord 
 4 day cord 
 
 1 day cord 
 4 day cord 
 
 3 day cord 
 
 2 day cord 
 
 4 day cord 
 
 1 day cord 
 4 day cord 
 
 3 day cord 
 
 2 day cord 
 
 4 day cord 
 
 3 day cord 
 2 day cord 
 
 Results. — According to reliable statistics, the mortality of rabies 
 without the Pasteur treatment is about 16 per cent.; with the treat- 
 ment the average mortality is about 0.46 per cent. The mortality of 
 those bitten about the face or head is about* 1.2.5 per cent.; of those 
 bitten on the hand, 0.7.5 per cent.; of those bjtten on other parts of the 
 body, a little over 0.25 to 1 per cent. In the {"asteur Institute of Paris 
 only such persons are treated as have been lacerated, so that the virus 
 has gained entry into the wounds. Takinjz; into consideration only 
 those cases in which the diagnosis of rabies has been confirmed in the 
 animal by a competent examiner, the mortality of the cases treated at 
 the Pasteur Institute in Paris is only 0.6 per cent., which, compared to 
 the average mortality of 16 per cent, without vaccine treatment, speaks 
 most favorably for the value of Pasteur's antirabic immunization. 
 
PROPHYLACTIC IMMTJNIZATION OR VACCINATION 643 
 
 The bites of wolves are more fatal than those of dogs, the mortality 
 being about 10 per cent, in spite of the intensive treatment, and about 
 40 to 60 per cent, without treatment. 
 
 When symptoms of rabies have appeared, the treatment is unavail- 
 ing. Antirabic serums have been prepared fey immunizing animals, 
 such as sheep and horses, and these should h& tried in human patients 
 presenting symptoms, but the results in general have not been uniformly 
 encouraging. 
 
 TYPHom Fever 
 
 Our knowledge of vaccination in typhoid fever begins with the work 
 of Pfeiffer and Kolle.^ These observers, in 189!), immunized two volun- 
 teers with heat-killed cultures, and by complete laboratory investiga- 
 tions demonstrated the identity of the immunity following an attack of 
 the disease with the artificial immunity produced by inoculation. At 
 about the same time Wright,^ of London, inoculated two men with 
 killed cultures, and a year later published the results of the successful 
 vaccination of 17 persons. In 1898 he continued the work in India, 
 where 4000 soldiers were inoculated, with encouraging results. Later, 
 during the Boer war, Wright and Leishman treated 100,000 men, and 
 the results, while good, were not encouraging, due, as pointed out later 
 by Leishman,^ to the fact that the vaccine was damaged during its 
 preparation by overheating. Since 1904 an improved vaccine has been 
 used among the British troops in India in ever-increasing quantities, 
 with uniformly good results. 
 
 Antityphoid vaccination was begun in the United States army in 
 1908, the vaccine being prepared by Major Frederick F. Russel. Its 
 value has been established so clearly that vaccination is now compulsory. 
 The results obtained in the army have had considerable influence in 
 establishing a wide-spread general confidence iii antityphoid inoculation. 
 
 Preparation of Typhoid Vaccine.— Based upon general principles, 
 the vaccine should be prepared of typhoid bacilli as little changed by 
 heat or chemicals as possible. Russel has prepared the army vaccine 
 with a single avirulent culture which proved by animal experiments 
 and laboratoT-y methods capable of producing large quantities of immune 
 agglutinins and bacteriolysins. As a general rule, however, the vaccine 
 should be poljrvalcnt unless one obtains a culture of known value. 
 
 ^ Deutsch. med. Wochenschr., 1896, xxii, 735. 
 
 ^Lancet, London, September 19, 1896, 907; Brit. Med. Jour., January 30, 
 1897, 16. 
 
 ' Jour. Roy. Inst. 
 
644 ACTIVE IMMUNIZATION 
 
 The preparation of the vaccuie is comparatively simple. The bacilli 
 are grown on agar for twenty-four hours, washed off with sterile normal 
 salt solution, standardized by counting the bacilli, and killed by heating 
 to 56° C. for one hour. As a matter of safety^ 0.25 per cent, of tricresol 
 is then added (Russel) . The details of the technic are given in Chapter 
 XIII. 
 
 Metchnikofi has never fully accepted the belief in the value of heat- 
 killed vaccines, and at present is actively concerned with vaccines pre- 
 pared of living bacilli sensitized with their immune serum (sensitized 
 vaccines). Injection of these vaccines into chimpanzees is not followed 
 by any untoward effects, and apparently the bacilli so administered are 
 destroyed at once, as they have not been fouijd in the blood, urine, and 
 feces. Metchnikoff and Besredka have immunized persons according 
 to these methods and report excellent results. Obviously, there is some 
 reluctance in using a vaccine of living bacilli until extended animal 
 experiments have proved that they are harmless and more efficient than 
 the vaccines of killed bacilli. 
 
 As a general rule, the vaccine is prepared in two strengths: 500,000,- 
 000 bacteria per cubic centimeter for the first dose, and 1,000,000,000 for 
 the second and third doses. 
 
 Method of Inoculation. — The vaccine is best administered at about 
 4 o'clock in the afternoon, so that the reaction appears during the night 
 and is least likely to be disturbing. It is well to administer a cathartic 
 the day before the inoculation is made. Inoculations should not be 
 given during the menstrual period, as the general reaction is likely to be 
 somewhat severer at this time. 
 
 The skin over the insertion of the deltoid muscle is touched with 
 tincture of iodin and the injection given siibcwfaneoMsZj/. Intramuscular 
 injections should be avoided, as the reactions are more unpleasant and 
 accompanied by unnecessary pain on movement. In making deep 
 injections there is also danger of striking a netve, a proceeding that may 
 be followed by disagreeable neuritis. 
 
 After the injection has been given the ic[din is wiped away with a 
 pledget of cotton and alcohol; no dressing is necessary. 
 
 The syringe and needle should be sterile. Commercial firms have 
 placed the prophylactic on the market in sj^ringes with sterilized needles, 
 accompanied with f\ill instructions as to the technic of administration. 
 The vaccine should he well shaken before the injection is given. 
 
 When a large number of injections are to be given at one time, a 
 single syringe may be used with a large number of sterile needles, a 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 
 
 645 
 
 separate needle being used for each person. The vaccine may be put 
 up in individual anipules or in a stock bottle, the former being preferable. 
 
 Dosage. — Three injections are given at intervals of one week. For 
 
 adults (150 pounds) the doses used in the army have been as follows: 
 
 First dose: 600,000,000 bacilli. 
 
 Second dose: 1,000,000,000 bacilU. 
 
 Third dose: 1,000,000,000 bacilh. 
 
 These amounts are contained in 1 c.c. (about 15 minims). Children 
 receive doses in proportion to their weight ; if the dose cannot be divided 
 evenly, it is better to give a little more rather than a little less, for chil- 
 dren tolerate the injections remarkably well. 
 
 Reactions. — Persons in poor physical condition are more likely than 
 the robust to experience disagreeable after-effects. 
 
 The local reaction consists of a small red and tender area lasting about 
 forty-eight hours. Occasionallj'^ the edema and pain may be more 
 marked, but abscess formation is practically unknown. 
 
 The general reaction, when present, gives rise to headache, malaise, 
 and sometimes to fever, chills, and occasionally to nausea, vomiting, or 
 diarrhea. The severe reactions are not alarming and disappear quickly. 
 
 The inoculated person should abstain from severe exercise for the 
 following twenty-four hours and rest; in the great majority of instances 
 bur soldiers have not been inconvenienced and were able to continue 
 with their routine duties. 
 
 The following tables, compiled by Major Russel,' show the propor- 
 tion of reactions in adults and children: 
 
 TABLE 24.— PERCENTAGE OF GENERAL REACTIONS IN ADULTS 
 (128,903 DOSES) 
 
 Dose 
 
 None 
 
 Mild 
 
 Moderate 
 
 Seveke 
 
 First 
 
 68.2 
 71.3 
 78.0 
 
 28.9 
 25.7 
 20.3 
 
 2.4 
 2.6 
 1.5 
 
 0.3 
 
 Second. . . 
 
 0.2 
 
 Third. . . 
 
 0.1 
 
 
 
 TABLE 25.— PERCENTAGE OF GENERAL REACTIONS IN 359 CHILDREN, 
 TWO TO SIXTEEN YEARS QF AGE 
 
 Dose 
 
 None 
 
 Mild 
 
 Moderate 
 
 Severe 
 
 First 
 
 Second . . , 
 
 Third 
 
 73.. 54 
 86.26 
 92.56 
 
 24.51 
 
 11.99 
 
 0..38 
 
 1.67 
 1.75 
 1.06 
 
 0.28 
 
 > Jour. Amer. Med. Assoc, 1913, Ix, 344. 
 
646 ACTIVE IMMUNIZATION 
 
 A comparison of these tables shows that the general reaction is much 
 more infrequent or milder in children than in'adults, even after the first 
 dose; after the second and third doses the difference is more marked. 
 
 In former years considerable stress was laid upon the possibility of 
 a negative phase following the inoculation, during which a person was 
 believed to be more susceptible to infection. This is now believed by 
 Leishman, Russel, and others of extended experience to be incorrect, 
 the more general belief being that inoculatiohs may be made and are 
 especially indicated during epidemics of the disease. 
 
 Duration and Degree of Typhoid Immunity. — It should be empha- 
 sized that immunity following typhoid immunization is not absolute, 
 and an immunized person cannot afford to neglect ordinary precautions 
 against infection. A lowered state of general body health or a large dose 
 of infectious material may at any time result ill infection. 
 
 Tlie prophylactic treatment should be used in conjunction with 
 well-known sanitary precautions in order to obtain the best results. 
 
 The immunity is apparently manifest soon after the first and second 
 doses have been given. The duration is not known definitely. From 
 the rich experience of the British army in Indik Colonel Firth "■ concludes 
 that immunity begins to decline in about two and one-half years after 
 inoculation. However, even after four and five years the typhoid rate 
 among the inoculated is, estimated roughly, one-fourth that of unpro- 
 tected troops. 
 
 Results. — The value of the typhoid prophylactic therapy is best 
 shown in the army, where conditions are bette:^- controlled than is possible 
 in civilian life. In 1911, of a division of Uriited States troops, about 
 20,000 men along the southern boundary, only two cases of typhoid 
 fever developed and both recovered. During- this same period of time 
 49 cases were reported in the city of San Antcj-iio, with 19 deaths. The 
 soldiers mixed freely in the city, ate of fruits- and vegetables, drank of 
 the same water, and in this manner were freely exposed, although the 
 sanitary conditions in the camp were excellent. 
 
 In 1898, during the Spanish War, there were assembled at Jackson- 
 ville, Florida, 10,759 troops, among whom there were certainly 1729 
 cases of typhoid, and including the suspected cases, this figure reached 
 2693 cases, with 248 deaths. This camp coiitinued about as long as 
 that in 1911, the climatic conditions and water supplies being practically 
 the same, but the sanitary conditions were bad. The remarkable dif- 
 ference in the typhoid rate cannot, however, be reasonably explained by 
 ' Jour. Roy. Army Med. Corps, 19J1, xvi, .589. 
 
PROPHYLACTIC IMMUNIZATION OK VACCINATION 647 
 
 perfect camp sanitation, and the results in 1911 leave no doubt as to the 
 value of antityphoid vaccination. 
 
 Excellent results have been reported by Spooner, Hachtel and Stoner, 
 and others as to the prophylactic value of typhoid immunization in 
 hospital-training schools for nurses, insane asylums, and other public 
 institutions. 
 
 Recommendations. — In view of the satisfactory results obtained in 
 the army, typhoid vaccination is now obligatory on all members of the 
 army and navy corps. Protection of the individual by immunization 
 is the only measure of protection independeht of surroundings and 
 effective under all conditions. 
 
 Typhoid inoculation in civilian practice cannot be as wide-spread 
 or as readily performed as vaccination against smallpox, as the prophy- 
 lactic must be administered subcutaneously and more than one dose is 
 necessary. 
 
 1. Our various State and city boards of health should endeavor to 
 educate the laity, and, if necessary, offer the prophylactic free of charge 
 in order to build up a vaccinated community as far as this is possible. 
 
 2. Persons coming in intimate contact with typhoid patients, such 
 as physicians, nurses, and attendants in hospitals, should be immunized. 
 Hospital authorities are justified in making typhoid vaccination ob- 
 ligatory on all applicants for admission to training schools. 
 
 3. All inmates of asylums, homes, and other public institutions 
 under forty-five years of age should be immunized and the State should 
 be ready, if necessary, to furnish the vaccine. 
 
 4. The physician and nurse should urge vaccination upon all the 
 members of a family when there is a typhoid patient among them. 
 
 5. The physician should especially advisp immunization of those 
 about to leave their homes for a vacation in some neighboring seashore 
 or mountain resort. 
 
 6. In times of epidemics of typhoid fever ihe physician should urge 
 vaccination of all over whom he has influence. Thorough vaccination 
 with proper sanitary conditions offers the best hope of eradicating this 
 dreaded disease. 
 
 Plague 
 
 In view of the frightful infectiousness and mortality of plague, 
 
 prophylactic measures are highly desirable. Extermination of rats and 
 
 ground squirrels, especially of the former, about the wharves of seaport 
 
 cities and towns is highly essential, as the disease is transmitted by the 
 
648 ACTIVE IMMUNIZATION 
 
 fleas of these rodents. Aside from sanitary measures, plague vaccine, 
 especially that of Haffkine, has now been used extensively, with encour- 
 aging results. 
 
 Preparation of Plague Vaccine. — The Haffkine prophylactic is pre- 
 pared by growing pure cultures of Bacillus pestis in flasks of neutral 
 bouillon to which a few drops of sterile olive oil or butter-fat have been 
 added, to serve as floats from which the surface growth of the bacilli 
 can take place. The flasks are cultivated at from 25° to 30° C. for five 
 to six weeks, and are shaken every two or three days, by which the hang- 
 ing, stalactite-like colonies are thrown down, so that a new crop of the 
 bacilli can develop in contact with the air. 
 
 After growing for six weeks the purity of the culture in each flask is 
 tested by subcultures on agar and by direct smears. The masses of 
 bacilli are broken up by shaking, and the material is sterilized by heating 
 at 65° C. for from one to three hours. Phenol is added to the point of 
 0.5 per cent., and the fluid is tested for sterility by culture. If it is 
 found sterile, it is finally poured into small vials of from 10 to 30 c.c. 
 capacity. 
 
 Kolle prepares a vaccine by cultivating the bacillus for two days in 
 flasks of agar measuring 10 by 9.5 cm. Each surface of agar equals 
 about 15 ordinary agar slant cultures, and an agar slant holds about 
 15 loopsful of culture (4 mm. loop). A loop of this size holds about 2 
 mg. of organisms, and, accordingly, a flask of agar contains about 225 
 loopsful of culture, or 225 doses of 2 mg. each. Kolle prepares the 
 vaccine in amounts of 0.5 c.c. per dose (2 mg. <}f bacilli), and the growths 
 in each flask are removed with 112.5 c.c. of sterile normal salt solution. 
 The emulsion is shaken to break up clumps, heated for one hour at 70° 
 C, and tested for sterility. It is then preserved with phenol or tricresol 
 and placed in ampules containing 0.5 c.c. each. 
 
 The German Plague Commission strongly recommended the use of 
 twenty-four- to forty-eight-hour-old agar cultures instead of the old 
 bouillon cultures employed in the preparation of Haffkine's vaccine. 
 
 Kolle and Strong have also employed living cultures of greatly re- 
 duced virulence for the immunization of man. 
 
 Lustig and Galeotti prepare a vaccine of the toxic precipitate pro- 
 duced by dissolving the bacilli in a 1 per cent, solution of caustic soda 
 and neutralizing with 1 per cent, of acetic acid. This precipitate is 
 dried in vacuo and redissolved in a weak solution of sodium bicarbonate, 
 the dose for adults being 0.0133 gm. of solid substance. 
 
 Terni and Bandi inoculate rabbits or gumea-pigs intraperitoneally 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 649 
 
 with the bacillus, and just preceding or directly after deatfci they collect 
 the peritoneal exudate, in which the organisms are allowed to continue 
 growing for twelve hours more. The bacilli are then killed at a low 
 temperature, and the fluid thus obtained, after a preservative has been 
 added, constitutes the vaccine. 
 
 Dosage. — The ordinary dose of Haffkine-s prophylactic for adult 
 males is from 3 to 3.5 c.c. ; for adult females, from 2 to 2.5 c.c. Haffkine 
 himself has injected larger quantities without resulting barm. He 
 recommends giving a second injection after from eight to ten days. 
 The injections are given subcutaneously , with a sterile syringe, into the 
 upper arm or elsewhere in areas where the skin is not tightly bound down. 
 
 The local and constitutional effects are similar to those in typhoid 
 except that they are slightly intensified. The inoculation is followed 
 by redness and swelling at the seat of inoculation, and general syxap- 
 toms in the form of rise of temperature and a feeling of illness. The 
 latter pass off in twenty-four hours, but the patient should rest during 
 the first day after inoculation. 
 
 Duration of Immunity. — The immunity is apparent a few days after 
 inoculation, but is of short duration. In India the protection is believed 
 to last at least three months and possibly longer. In times of epidemic 
 the inoculations should be repeated at least two or three times a year. 
 The brief duration of the immunity is probably one reason why better 
 results are not secured. Best results are observed during epidemics when 
 protection is afforded for a short time, or until the danger is past. In 
 countries or localities where the disease is entjemic, persons may refuse 
 repeated inoculation and thus become susceptible to infection. 
 
 Results. — In the pneumonic variety of plague the prophylactic does 
 little or no good, a finding that has also been shown experimentally by 
 Strong and Teague.' 
 
 In the bubonic variety Haffkine's vacciiie has in general jdelded 
 encouraging results. The protection is not absolute; the immunity 
 IS of relatively short duration, and therefore good results are not so 
 readily appreciated when the disease is endenaic. The mortalitj' among 
 the inoculated is much lower, i. e., 11 to 41 per cent., as compared -ndth 
 50 to 92 per cent, among the non-immunized. Haffkine summarized 
 his results a few years ago as follows: Among 186,797 inoculated 
 persons there were 3999 attacks, or 1.8 per cent.; among 639,630 un- 
 inoculated persons there were 49,433 attacks, or 7.7 per cent., with 29,733, 
 or 4.7 per cent, of deaths. 
 
 ' Philippine Jour. Sci., 1913, vii. 
 
650 ACTIVE IMMUNIZATION 
 
 The Indian Plague Commission a few years ago reported as follows: 
 
 1. Inoculation sensibly diminishes the incidence of attacks of plague. 
 It is, however, not an absolute protection against the disease. 
 
 2. The death-rate is markedly diminishe.d by its means, not only 
 the incidence of the disease, but also the fatalitj', being reduced. 
 
 3. The protection is not conferred on those inoculated for the first 
 few days after the injection. 
 
 4. The duration of the immunity is uncertain, but it seems to last 
 for a number of weeks, if not for months. 
 
 After the disease has once developed, v.accination is of no avail. 
 When there is eminent danger of infection, vaccine and antiserum should 
 be given together. 
 
 Cholera 
 
 Protective inoculation against cholera was first practised by Ferran, 
 a Spaniard, in 1884, although little definite knowledge as to the value 
 of the procedure resulted from his work. He is said to have used impure 
 cultures of bacilli isolated from the feces of cholera patients. Broth 
 cultures were prepared, and the living organisms injected subcutane- 
 ously, using eight drops for the first and 0.5 c.e. for the second and third 
 doses, the injections being given at intervals of six or eight days. While 
 his method and results have been questioned, he was, however, the 
 first to use a method employed later, with some modifications, by Haff- 
 kiiie ill India with good results. 
 
 Preparation of Cholera Vaccine. — Haffkine, following Pasteur's 
 method with anthrax, uses two vaccines, — a weaker and a stronger, — 
 living microorganisms being used in both and. injected subcutaneously. 
 Vaccine No. 1 is weaker, and is obtained by growing the bacihi on agar 
 at a temperature of 39° C. Vaccine No. 2 is composed of more virulent 
 organisms, ijreparetl by passing the vibrios tiirough a series of guinea- 
 pigs until a strain is obtained which is invariably fatal to these animals 
 within twelve, or at least twenty-four, hours. Cultures are grown on 
 agar, washed off with 8 c.c. of sterile bouillon or saline solution, and 
 administered in doses of 1 c.c, which is equivalent to about two loopsful 
 (4 mm.) or 4 mg. of living bacilh. 
 
 Kolle has shown, however, both by animal experimentation and in 
 the human being, that heat-killed cultures aje equally good, and that 
 living cultures are unnecessary and may be undesirable. 
 
 By this method the vaccine is prepared by cultivating a virulent 
 strain on flasks of agar for twenty-four hourg, as described in the pre- 
 
PROPHYLACTIC IMMUNIZATION OR VACCINATION 651 
 
 paration of plague vaccine, and removing the growths with sufficient 
 salt solution so that 1 c.c. shall contain one loopful (4 mm.) of organisms 
 (2 mg.). The emulsion is then shaken to break up clumps, heated to 
 60° C. for from one-half to one hour, cultured to determine sterility, and 
 preserved with 0.5 per cent, phenol. 
 
 Strong has proposed the use of the products obtained by "autolytic 
 digestion" of the organism, i. e., by incubating an emulsion of them in 
 sterile water, in which they break up spontaneously. Twenty-four-hour 
 agar cultures are removed with sterile water, placed in a sterile flask, 
 and kept at a temperature of 60° C. for twenty-four hours. The mixture 
 is then put aside in the incubator for from two to five days. The best 
 results are obtained apparently after five days' autolytic digestion. 
 After such digestion the emulsion is filtered through a Reichel filter. 
 The fluid thus obtained must, of course, be examined for sterility and 
 carefully standardized before being used as a human vaccine. 
 
 Dosage. — Kolle's vaccine is given subcutanequsly ui two injections 
 about a week apart — 1 c.c. the first time and 2 c.c. the second time. 
 
 Haffkine's vaccines are given in the same manner at an interval of 
 five days. 
 
 The local effects are usually marked by more or less pain and edema, 
 which subside in forty-eight hours. The constitutional effects are not 
 infrequently severe, being marked by malaise, fever (100°-101° F.), 
 nausea and vomiting, followed the next day in about 10 per cent, of 
 persons by transient diarrhea. Usually all symptoms have disappeared 
 within seventy-two hours. 
 
 Results. — Haffkine's prophylactic vaccine has yielded favorable 
 results in India. Powell reports 198 cases of cholera among 6549 non- 
 immunized persons, with a total mortality of 124. Of 5778 inoculated 
 persons, there were 27 cases, with 14 deaths. Much better results were 
 obtained with Kolle's vaccine, and it is now generally used in preference 
 to the Haffkine vaccines. 
 
 Murata, during an epidemic in Japan in 1902, vaccinated 77,907 
 persons. Of these, 47, or 0.06 per cent., developed cholera, and 20, 
 or 0.02 per cent., died. Of 825,287 uninoculated persons, 1152, or 0.13 
 per cent., died. During a recent epidemic in Russia Franschetti inocu- 
 lated 11,178 persons. Of these, 8 contracted cholera and 1 died. In 
 St. Petersburg, during 1907-08, 30,000 persons were inoculated. Of 
 these, 12 developed cholera and 4 died. Of 10,000 uninoculated per- 
 sons, 68 contracted the disease. 
 
 It appears justifiable, therefore, to conclude that inoculation confers 
 
652 ACTIVE IMMUNIZATION 
 
 some immunity. This protection may be apparent after the first dose, 
 but is more marked after the second. The immunity conferred is far 
 from being absolute, and it is noteworthy that while the prophylactic 
 diminishes the liability of the inoculated per.s*on to cholera, it has less 
 influence on the mortality when the disease occurs in those who have 
 been vaccinated. , 
 
 Used in conjunction with modern sanitary regulations, however, 
 KoUe's vaccine certainly proves of value and should be used in combating 
 epidemics. 
 
 Other Diseases 
 
 Dysentery. — Protective vaccination against bacillary dysentery has 
 been attempted, but has not as yet yielded satisfactory results. Shiga 
 practised mixed active and passive immunization (vaccine plus immune 
 serum) on 10,000 persons, and while this did not decrease the number 
 of infections, a lower mortality resulted. The various types of the 
 dysentery bacillus and the high toxicity of the vaccines are obstacles 
 to the more general use of protective inoculation in this condition. 
 
 Cerebrospinal Meningitis. — Sophian and" Black ' have shown ex- 
 perimentally that a polyvalent meningococcic vaccine, heated to 50° C, 
 standardized in the usual manner, and given in three injections, in 
 doses of 100,000,000, 500,000,000, and 1,000,000,000, at intervals of a 
 week, appears to afford a high degree of protection. In the blood- 
 serums of inoculated persons these observers were able to demonstrate 
 opsonins, agglutinins, and complement-fixing amboceptors. All evi- 
 dence points to the efficacy of prophylactic vaccination, as only a 
 moderate degree of immunity may give complete protection against the 
 disease. The method has not thus far received ext,ensive trial, but in 
 the presence of an epidemic its harmlessness and apparent value should 
 be borne in mind. 
 
 Scarlet Fever. — Several Russian physicians, particularly Gabrick- 
 evski,^ Longovi, Nitikin, Shamarin, and others, have secured good 
 results from a method of prophylactic vaccination against scarlet fever 
 with a polj^alent vaccine of scarlet-fever streptococci. Heat^killed 
 vaccines were given in three successive doses. In this country the 
 method has been tried by Kolmer,' who found that while inoculations 
 with a heat-killed streptococcic vaccine cannot prevent scarlet fever 
 
 1 Jour. Amer. Med. Assoc, 1912, lix, .527. Black, ibid., 1913, Ix, 1289. 
 
 2 Russk. Vratch, St. Petersburg, 1906, x, 469. 
 = Penna. Med. Jour., February, 1912. 
 
PROTECTIVE IMMUNIZATION AMONG TIIe LOWER ANIMALS 653 
 
 itself, such inoculations may, however, prevent a severe attack of the 
 disease by producing some immunity against secondary streptococcic 
 infections. 
 
 PROTECTIVE IMMUNIZATION AMONG THE LOWER ANIMALS 
 
 Since discoveries in bacteriology and imrpunity have usually been 
 intimately associated with animal experimentation, it is not strange 
 that the lower animals should have been the first to benefit from the 
 knowledge thus gained. As a consequence, vaccine therapy, both 
 prophylactic and therapeutic, is being extensively used in veterinary 
 practice with good results. 
 
 Anthrax 
 
 This was one of the first vaccines studied by Pasteur, and as a prophy- 
 lactic measure, it has proved of great value. It finds its greatest field 
 of usefulness in case of an outbreak of anthra?, when it is used to pro- 
 tect the uninfected members of a herd, as well as any animals pasturing 
 on infected areas. 
 
 In preparing the vaccine Pasteur was hampered by the fact that the 
 spores of anthrax bacilli retain the virulence of the original bacilli. As 
 the result of extended experiments, however, he discovered a means of 
 attenuating the virulence of cultures by growing the bacilli at a tem- 
 perature of 42° C. ; he also found that inoculation with these attenuated 
 baciUi would effectively vaccinate sheep and cattle, and so protect them 
 against an attack of the disease. 
 
 One of the most dramatic stories^ in the history of science is the 
 account of the method by which Pasteur demonstrated his discovery to 
 the pubhc. Certain harsh critics, having heard of Pasteur's ability to 
 prevent anthrax in laboratory experiments, -and anxious to humiliate 
 Mm, sent him a public challenge to demonstrate the experiment on a 
 practical scale at a farm in the country. A number of farmers offered 
 to place 60 sheep at his disposal. The challenge was immediately 
 accepted, and Pasteur mapped out a plan of action, in which he safe- 
 guarded himself by making no half-statemeaits, but boldly promised 
 complete success. Of the total number, 25 sheep were to be vaccinated 
 and 25 were to remain unvaccinated. A fortnight later all 50 were to 
 receive a lethal dose of the fully virulent anthrax. He declared that 
 the 25 non-vaccinated sheep would die, whereas the 25 vaccinated would 
 temain alive and well. The remaining 10 sheep were to serve as controls. 
 1 Narrated by Elizabeth Fraser. 
 
654 ACTIVE IMMUNIZATION 
 
 The challenge and its acceptance were widely advertised in the 
 journals, and Pasteur was made the subject for many witticisms. Ex- 
 citement ran high, and a large crowd, comprised of physicians, veterin- 
 ary surgeons, journalists, farmers, etc., accb'mpanied Pasteur to the 
 farm (Pouilly le Fort) where he was to make the final test by inoculating 
 the deadly anthrax. One vaccinated animal^ developed a temperature 
 overnight, a fact that caused Pasteur much knxiety. On going to'thc 
 farm the next day, however, again followed by the crowd, he found all 
 the vaccinated animals well! Of the unvaccinated, 22 were dead, and 
 the others died during the following night; Pasteur's triumph was 
 complete, and the possibility of preventive vaccination was demonstrated 
 to the world. 
 
 The vaccine is prepared of attenuated cultures of virulent anthrax 
 bacilli. 
 
 Vaccine No. 1 is weakest or lowest in virulence, and the first to be 
 injected. This vaccine is prepared by growi|ig virulent anthrax bacilli 
 at a temperature of 42° C. for from six to ten weeks, or until to" loopful 
 of the culture, when injected into rabbits, guinea-pigs, and mice, will 
 show virulence for mice only, but not for guiijea-pigs and rabbits. 
 
 Vaccine No. 2 is prepared by growing virulent anthrax bacilli at 
 42° C. for about twenty days, or until to loopful is virulent for mice, 
 partly so for guinea-pigs, and not at all for rabbits. 
 
 Vaccine No. 3 is not generally used except for immunizing sheep and 
 goats. When, however, it is required, it is made as follows: Virulent 
 anthrax bacilli are grown at 42° C. until tV loopful, when injected into 
 mice, guinea-pigs, and rabbits, will be virulent for all the mice, all the 
 guinea-pigs, and some of the rabbits. 
 
 The vaccines are prepared in ampules containing 1 c.c. of the emul- 
 sion, each representing one dose, to be injected subcutaneously. Vac- 
 cine No. 2 is injected twelve days after Vaccine No. 1, and No. 3 after 
 the same interval following Vaccine No. 2. The resulting immunity 
 usually lasts about six months. 
 
 In instances where it is desirable to immunize a herd before turning 
 them out to pasture on infected areas it is well to inoculate the animals 
 in the early spring, keeping them in the stable during the time required 
 for at least two vaccinations, for the reasofi that, immediately after 
 vaccination, the animals may become hypersusceptible to infection. 
 
 When conducted in a careful manner by a competent veterinarian, 
 anthrax vaccination has proved fairly successful. 
 
ACTIVE IMMUNIZATION FOR THERAPEUTIC PURPOSES 655 
 
 Blackleg 
 
 Blackleg vaccine is used entirely as a pro{)hylactic agent, for the 
 disease runs too acute a course for the vaccine^ to exert any therapeutic 
 influence. 
 
 The vaccine is prepared by the Bureau of Animal Industry in the 
 following manner: The muscle tissue from a fresh blackleg tumor is 
 ground in a mortar, extracted or macerated with a little water, and 
 the fluid squeezed through cheese-cloth. The expressed fluid is then 
 evaporated at a temperature of 35° C. The dry brown scale is run 
 through a grinding mill and heated for six or seven hours at a teinijerature 
 of from 94° to 96° C. This process of heating attenuates the virulence 
 of the bacilli present so that, when injected, they produce but a mild 
 attack of the disease. The Department of Agriculture places the ground 
 material in packages containing a certain number of doses. These 
 packages are, upon request, mailed to veterinarians, who dilute the 
 ground muscle with as many cubic centimeters of sterile water as there 
 are doses in the package. One cubic centinleter of the suspension is 
 injected subcutaneously in some convenient area, as, e. g., about the 
 shoulders. 
 
 Blackleg vaccine should be applied in the spring, before young cattle 
 are turned out to pasture on infected areas. 
 
 In case of a fresh outbreak, all the healthy animals in the herd are 
 to be vaccinated as soon as possible. 
 
 Blackleg vaccination has been fairly successful, and usually confers 
 an immunity lasting for a period of about six rtionths. 
 
 ACTIVE IMMUNIZATION FOR THERAPEUTIC PURPOSESt— BACTERIAL 
 VACCINE THERAPY 
 
 Principles. — Although bacterial vaccines- have been extensivelj^ 
 employed in the treatment of various diseases,, it is difficult to express 
 an opinion as to their real value, and it is altogether impossible to make 
 dogmatic statements as to the percentage of cases in which they will be 
 helpful or effect a cure, or as to what result may be expected in an in- 
 dividual case. Following Wright's original aniiouncements, this special 
 therapy was enthusiastically received by the profession, and in a short 
 space of time the method was employed experimentally in many and 
 diverse infections. It may be stated that in many infections the vac- 
 cines may be beneficial, but they should be used only under proper 
 conditions, as was indicated in the first portion of this chapter. 
 
656 ACTIVE IMMUNIZATION 
 
 It may be stated in general that: 
 
 1. Vaccine therapy has a special field of usefulness in the treatment 
 of chronic infections. 
 
 2. Autogenous vaccines are to be preferred to stock vaccines, and in 
 some infections the former must be used. 
 
 3. It is not advisable to continue the usfe of the same vaccine for 
 more than several doses if no reaction and no improvement are noted. 
 New cultures should be made to determrae'if the right organism is 
 being used, or if reinfection with another organism has occurred. 
 
 4. It is essential that the vaccine be propej-iy prepared and not over- 
 heated. 
 
 5. It is highly important that the usual forms of treatment be employed 
 in conjunction with the vaccine therapy. Thus abscesses should be 
 incised; proper drainage of a discharging wound afforded; discharging 
 ears cleansed, etc. 
 
 6. While the dose should not be too larg§, neither should it be too 
 small, nor too far apart. There is a proper dose for each patient, and 
 this may be determined by starting with a ^mall dose and gradually 
 increasing it until some reaction is secured? An efficient dose must 
 necessarily produce some reaction, and increased doses are contra- 
 indicated so long as any sign of general or focal reaction is produced and 
 so long as steady progress is maintained. 
 
 Diseases of the Skin 
 
 Furunculosis. — Furuncles are usually caused by some member of the 
 group of staphylococci, and frequently the most rational and successful 
 form of therapy is by means of bacterial vaccines. A stock vacciae of 
 Staphylococcus aureus may prove satisfactory, and should be used while 
 an autogenous vaccine is being prepared. For adults the initial dose 
 may be 100,000,000 cocci, succeeding doses gradually increasiag until 
 1,000,000,000 are given at one time. The injections may be given at 
 intervals of from five to seven days. Following the first few doses, a 
 slight focal and some constitutional reaction should be secured. After 
 all the lesions have disappeared, one or two full doses at intervals of 
 several months will continue to fortify the patient against a recurrence. 
 
 Carbuncles. — These are invariably caused by the Staphylococcus 
 aureus, and exceptionally by a streptococcus. The urine should be 
 examined for sugar, and even if the patient is diabetic, small doses of 
 vaccine may be of value when used in conjunetion with the customary 
 treatment. 
 
ACTIVE IMMUNIZATION FOR THERAp'eUTIC PURPOSES 657 
 
 Sycosis. — Sycosis vulgaris is usually causeji by the Staphylococcus 
 aureus and albus, and such patients are frequently very rebellious to the 
 ordinary treatment. An autogenous vaccine* is very helpful in some 
 cases. Due care should be exercised in making cultures to secure pus 
 from a well-developed lesion. Relatively large doses of vaccine are 
 necessary, and treatment is usually prolonged, at least 12 injections being 
 necessary before the conclusion is reached that the vaccines are of no 
 service. As a rule, the condition will improve under vaccine therapy, 
 but only in exceptional cases does a complete cure result. 
 
 Acne. — Acne is frequently caused by two microorganisms — a staphy- 
 lococcus and the acne bacillus. In cases showing pustulation a staphy- 
 lococcus is invariably present, and exceptionally the bacillus may be 
 found alone in comedones. Cultures should be made from several 
 lesions, being careful to secure pus that has not been contaminated by 
 the skin. Stock vaccines maj^ be used, although autogenous vaccines 
 are likely to yield better results. It is well to administer a mixed vaccine 
 of the staphylococcus and acne bacillus, especially if the lesions are pus- 
 tular. It is highly essential that other forms <3f treatment be instituted 
 while vaccines are being tried, as the treatment is usually prolonged. 
 Exceptionally, however, a brilliant result may be observed after a few 
 injections. I generally prepare a mixed vaccine of 10,000,000 aenc 
 bacilli to each 500,000,000 cocci per 0.5 c.c. of vaccine. The first few 
 doses consist of 0.5 c.c, and later this amount Jnay be increased to 1 c.c. 
 per dose, which usually contains enough bacilli* and cocci for a nymber 
 of injections. Doses are given everj- five to seven days, or just when 
 retrogression is observed to occur. It may be necessary to use large 
 doses, and in anj^ case the treatment is prolonged over many weeks 
 and months. IMost cases will show improvement, but few are abso- 
 lutely cured Ijy a single series of injections. 
 
 Erysipelas. — This infection is usually caused by the streptococcus 
 erysipelatis. In severe cases an autogenous vaccine of al)out 20,000,000 
 cocci per cubic centimeter may be administered every three or four days, 
 and frequently aids in reducing the severity of the inflammation and 
 overcoming mental unrest and physical discomfort-. A vaccine may be 
 of aid in the treatment of subacute and chronic types of tliis disease. 
 Stock vaccines are of little or no value. 
 
 Genito-urinary Diseases 
 Cystitis. — Acute or subacute cystitis following catheterization after 
 labor or surgical operations or occurring in children is usually caused by 
 42 
 
658 ACTIVE IMMUNIZATION 
 
 a member of the group of colon bacilli. It is highly essential, in order 
 to attain success with vaccine therapy, that urine be collected aseptically 
 and the causative microorganism secured and used in the preparation 
 of a vaccine, as stock vaccines are of little or no value. Exceptionally 
 the infection may be due to another microorganism, either alone or in 
 conjunction with Bacillus coli. Treatment with an autogenous vaccine 
 may be of distinct aid in lessening the symptoms and in reducing the 
 amount of pus. The initial adult dose of Bacillus coh vaccine should 
 be about from 50,000,000 to 100,000,000 baciUi. In subacute cystitis 
 of the male, due to stricture of the urethra or enlarged prostate, or in the 
 female, due to perineal injuries, not much benefit follows its use until 
 the underlying cause is corrected or removedt. 
 
 Urethritis. — There is a considerable difference of opinion as regards 
 the efficacy of vaccines in the treatment of acute and chronic ure- 
 thritis of gonorrheal origin. A polyvalent stock vaccine of gonococci of 
 proved immunizing powers may be even more efficient than an auto- 
 genous one, especially if the latter must be prepared from a strain that 
 has been repeatedly subcultured in order to obtain the vaccine in a pure 
 state, or from one that has lost its virulence from long residence in the 
 infected urethra. Owing, therefore, to the difficulty of isolating and 
 cultivating gonococci, stock vaccines have been generally employed. 
 In subacute urethritis the initial dose may be 25,000,000; if complica- 
 tions threaten, less than this, and if no local reaction has followed more 
 than this, is given, the object being to secure a slight increase of the 
 secretion, which should become more purulent, and a little constitu- 
 tional disturbance, followed by lessening of local pain and tenderness. 
 
 In chronic urethritis the primary infection is gonococcal, although 
 other organisms, such as the Micrococcus catarrhalis, staphylococci, and 
 diphtheria bacilli may have some relation to the process. A stock 
 gonococcal vaccine may be used in sufficient dosage to evoke a reaction; 
 not infrequently, however, a stock and an aijtogenous vaccine or vac- 
 cines combined yield better results. Cultures of the urethra should 
 be made only after thorough cleansing of the meatus and flushing of the 
 urethra with sterile salt solution and massage of the prostate, cultures 
 being made direct from the prostatic secretion or from the crypts through 
 the urethroscope. In any case expert local treatment should be given 
 while vaccines are being used. 
 
 Gonorrheal Arthritis. — Polyvalent stock vaccines of gonococci have 
 generally been found to be of distinct value in the treatment of this 
 troublesome and oftentimes chronic infection. Local treatment of the 
 
ACTIVE IMMUNIZATION FOR THERAfEUTIC PURPOSES 659 
 
 urethra and general therapeutic measures should be employed. It may- 
 be stated that vaccines should be used routinely in all cases, as under 
 any condition the infection is likely to be prolonged and tedious. In the 
 acute stages the doses should be relatively small, about 10,000,000 cocci 
 being given every three to five days. In the subacute stages from 
 50,000,000 to 100,000,000 may be given at initervals of from five to ten 
 days. 
 
 Vulvovaginitis of Children.— Stock gonococcal vaccines have been 
 used quite extensively in the treatment of this troublesome infection. 
 On the whole, good results have been reported, although in any case 
 final judgment must be reserved until thorough bacteriologic examina- 
 tion shows whether the tissues are really free from infection or whether 
 the infection has subsided and become chronic. Smears of the secretions 
 alone are insufficient to determine whether a cure has been effected. 
 Injecting a solution of 1 : 2000 bichlorid of niercury in normal saline 
 solution into the vagina, followed by immediate centrif ugalization of the 
 washings and smears of the sediment, will frequently demonstrate the 
 presence of gonococci that will not otherwise be found. If vaccines are 
 used, a dose of from 5,000,000 to 10,000,000 every five to seven days 
 may be employed. 
 
 Respiratory Diseases 
 
 Rhinitis. — ^The use of mixed stock vaccine's is being advocated for 
 prophylactic immunization against recurrent attacks of acute rhinitis. 
 With Weston, I have found autogenous vaccines of some value in lessen- 
 ing the severity and hastening the recovery from the acute rhinitis of 
 scarlet fever, so potent a factor in the dissemination of that disease. In 
 chronic rhinitis an autogenous vaccine, prepared by growing cultures 
 with the care previously described, may be of distinct value, but only 
 when an underlying factor, such as a malformation or adenoids, has been 
 removed, and only when used in conjunction with efficient local treat- 
 ment. 
 
 Bronchitis. — Allen speaks very highly of the value of autogenous 
 vaccines in the treatment of acute and chronic- bronchitis and broncho- 
 pneumonia of children. Various microorganisms are found in the 
 secretions, and the vaccines used are generally mixed. The usual thera- 
 peutic measures are employed simultaneously." It is claimed that vac- 
 cines lessen the discomfort and hasten recovery. 
 
 Pertussis. — A hemophilic bacillus closely resembUng the influenza 
 bacillus has been ascribed by Bordet and Wollstein as the cause of pertus- 
 
660 ACTIVE IMMUNIZATION 
 
 sis. Several investigators who have used a stock vaccine of the per- 
 tussis bacillus claim that, used in small arid appropriate doses, the 
 severity of the paroxysms of coughing is lessejied, and the whole course 
 of the infection shortened; besides this, they assert, it decreases the 
 danger of a complicating bronchopneumonia. When the disease is 
 unusually severe and the prognosis is bad, a vaccine may be admiaistered 
 in doses of 25,000,000 if the patient is over four years of age. The 
 pneumococcus and Bacillus influenzae are frequently associated, and a 
 mixed vaccine of these microorganisms may be of special aid ia the later 
 stages of the disease. The usual remedial measures should be employed 
 while vaccines are being tried. A stock vaccine has been advocated for ' 
 purposes of prophylactic immunization, especially in institutions, where 
 pertussis among children claims a high mortality. 
 
 Otitis Media. — Autogenous vaccines prepared from carefully made 
 cultures of the diseased tissues, secured with the aid of an ear speculum, 
 may prove of some value in the treatment of subacute and chronic 
 suppurative otitis media. Additional treatment may be carefully 
 given, but not infrequently more harm than good is done by careless 
 flushing and cleansing of the auditory canal, whereby deeper and healthy 
 tissues become infected. Free drainage should be afforded, and ia 
 chronic otitis media necrosed ossicles and granulations may require 
 surgical removal. Injections may be given every five to seven days. 
 A slight increase in discharge after the first orie or two doses is of good 
 import, and indicates a slight focal reaction. With Weston, I have 
 treated a large number of cases of suppurative otitis media following 
 scarlet fever, and while the results were seldom brilliant, in general the 
 duration and severity of the infections were fa,vorably influenced. 
 
 Acute General Infections 
 Vaccines have been advocated in the treatment of typhoid fever and 
 pneumonia. In the former an autogenous vaccine may be prepared 
 if a blood culture is made early in the infegtion. Otherwise a stock 
 vaccine prepared of a strain of known immunizing power may be used. 
 The doses should be small, and may be given at short intervals. The 
 object is to stimulate the body-cells to further effort in the production 
 of antibodies. On the whole, I am not in favor of giving vaccines in 
 typhoid fever, especially if the infection is severe or the general vitality 
 of the patient is low, because of the danger of doing actual harm at a 
 critical period. If vaccines are used, however, the initial dose should 
 be less than 160,000,000 bacilli, and their effect must be very carefully 
 observed. 
 
TUBERCULOSIS TtJBEECXTLIN THERAPY 661 
 
 Autogenous vaccines may be of value in the treatment of delayed 
 resolution in lobar pneumonia, cultures being secured by puncturing the 
 lungs. Usually several microorganisms are found, and a mixed vaccine 
 may be given. 
 
 Autogenous vaccines may also be of service in the treatment of 
 puerperal sepsis and idcerative endocarditis, especially after the more 
 acute symptoms have subsided. In the former condition a strepto- 
 coccus may be obtained by blood culture or by intra-uterine cultures; 
 in the latter, the infecting microorganism is, obtained only by blood 
 culture. Stock vaccines may be administered, but are not likely to 
 prove of value, as both infections are usually caused by streptococci, 
 pneumococci, or some similar microorganisms^ showing so much differ- 
 ence in individual properties as to make the use of autogenous vaccines 
 imperative. The initial doses should be small — not over 50,000,000 
 cocci; they may be repeated every three to five days, and are gradu- 
 ally increased as the conditions warrant. 
 
 TUBERCULOSIS.— TUBERCULIN THERAPY 
 
 Historic. — Within the last twenty years the subject of tuberculin 
 therapy has elicited considerable discussion in the diagnosis and treat- 
 ment of tuberculosis. The wide-spread prevalence of the disease, not 
 only in man, but in the lower animals as well, the distressing symptoms, 
 the gloomy prognosis, and the economic importance it possesses, are a 
 few of the factors that have stimulated investigators the world over to 
 zealous and persistent efforts directed toward discovering a means of 
 preventing and curing this great scourge. Owing to the nature of the 
 infection, which covers relatively long periods of time, and the fact that 
 EQuch time is required for the conduct of experimental studies, researches 
 are of necessity prolonged, tedious, and difficult. Within a period of 
 less than six years the cause of syphilis has beep/liscovered and isolated, 
 and valuable diagnostic reactions and a well-nigh specific therapy have 
 been devised. The discovery of an early and specific diagnostic and 
 therapeutic measure for tuberculosis will achieve still greater triumphs — 
 in fact, few could be greater or more beneficial. 
 
 Koch was the first to note the curative action of tuberculin, and it 
 may be well to refer here to the original description of his fundamental 
 experiments,^ which have been the basis as well as the starting-point 
 of the entire study of tuberculins. 
 
 ' Deutsch. med. Wochensehr., ISOlji xvii, 101. 
 
662 ACTIVE IMMUNIZATION 
 
 "When one vaccinates a healthy guinea-pig with a pure culture of 
 tubercle bacilli, the wound, as a rule, closes -and in the first few days 
 seems to heal. However, in from ten to fourteen days a hard nodule 
 appears which soon breaks down, leaving an" ulcer that persists to the 
 time of death of the animal. There is quite a different sequence of 
 events when a tuberculous guinea-pig is vacdinated. For this experi- 
 ment animals are best suited that have been- successfully infected four 
 to six weeks previously. In such an animal the inoculation wound 
 likewise promptly unites. However, no nodule forms, but on the nejct 
 or second day after a peculiar change occurs. The point of inoculation 
 and the tissues about, over an area of from 0.1 to 1 cm. ia diameter, 
 grow hard and take on a dark discoloration. Observation on subsequent 
 days makes it more and more apparent that "tJie altered skin is necrotic. 
 It is finally cast off, and a shallow ulceration remains, which usually 
 heals quickly and permanently without the neighboring lymph-glands 
 becoming infected. Inoculated tubercle bacilli act very differently 
 then upon the sldn of healthy and tuber'culous guinea-pigs. This 
 striking action is not restricted to living tubercle bacilli, but is equally 
 manifested by dead bacilli, whether they be ftilled by exposure to low 
 temperature for a long time or to the boiling temperature, or by the 
 action of various chemicals. 
 
 "After having discovered these remarkable facts, I followed them up 
 in all directions and was further able to show that killed pure cultures of 
 tubercle bacilli ground up and suspended in water can be injected in 
 large amounts under the skin of healthy guinea-pigs without producing 
 any effect other than local suppuration. Tuberculous guinea-pigs, on 
 the other hand, are killed in from six to forty-eight hours, according to 
 the dose given, by the injection of small quantities of such a suspension. 
 A dose which just falls short of the amount necessary to kill the animal 
 may produce extensive necrosis of the skin about the point of injection. 
 If the suspension be diluted until it is just visibly cloudy, the injected 
 animals remain alive, and if the administration is continued with one 
 to two day intervals, a rapid improvement in their condition takes 
 place; the ulcerating inoculation wound becomes smaller and is finally 
 replaced by a scar, a process that never occurs without such treatment; 
 the swollen Ijonph-glands become smaller;" the nutrition improves, 
 and the disease process, unless it is too far advanced and the animals 
 die of exhaustion, comes to a standstill. 
 
 "Thus was established the basis for a rational treatment of tuber- 
 culosis. However, such suspensions of killed tubercle bacilli are un- 
 
TUBERCULOSIS TUBERCULIN flTHERAPY 663 
 
 suitable for practical use, since they arc ncithea absorbed nor disposed of 
 ia other ways, but remain a long time unaltered at the point of inocula- 
 tion and occasion smaller or larger abscess. " 
 
 Koch showed further that while the injection of tuberculous guinea- 
 pigs with large doses of tubercle bacilli produced rapid death, frequently 
 repeated small doses exerted a favorable effect upon the site of infection 
 and the general condition of the animals. The same observer also 
 reahzed that the harmful effects of injections of dead tubercle bacilli 
 were due to the non-absorbable parts of the bacilh. He attempted to 
 extract the immunizing substances, and in tbis A\ay produ(.'ed his first 
 or Old Tuberculin. "When injected into tuberculous guinea-pigs, old 
 tuberculin produced a rapid general reaction A\ithout any local necrosis 
 or sloughing, whereas when injected into a he'althy guinea-pig, no re- 
 action, either local or general, was produced. The fact that the general 
 results produced by old tuberculin were analogous to those obtained by 
 his first vaccine, except that local necrosis did npt occur, induced Koch, 
 in 1891, to promulgate it as a specific cure for tuberculosis in human 
 beings. 
 
 It is hardly necessary to describe the hopeful anticipation with which 
 it was received, and the keen disappointment that followed its earlier 
 clinical use. Indiscriminate use, extravagant expectations, and ex- 
 cessive dosage combined to ;\-ield results so discouragmg as to swing the 
 pendulum of medical opinion so far the other way that even now the 
 very word "tuberculin" suggests to many minds failure, and something 
 to be avoided. 
 
 A few earher followers of Koch continued their studies in the endeavor 
 to discover the causes of failure in tuberculin therapy. Their researches 
 have led to new principles in treatment and to more exact knowledge of 
 its indications, as well as its contraindications. As now employed, its 
 use being restricted to suitable patients and administered in safe 
 graduated doses, and accepting as e^'idence only the statements of those 
 who have used tuberculin and not of those who believe it to be dangerous 
 and have never used it, one deduction is justififed: that while tuberculin 
 is not a specific "cure" for tuberculosis, — any more than hygiene, diet, 
 and climate are cures, — it helps to arrest the'ijisease and is in general 
 a useful factor in the treatment of certain tj^ieg of the disease. Clinical 
 studies have shoVn, however, that innnunizbtion of the tuberculous 
 patient is frequently a chfficult procedure, owing to the fact that such 
 patients are prone to develop a remarkable state of hypersusceptibility. 
 
664 ACTIVE I.MMrXIZATION 
 
 in consequence of which e^'eiy inoculation will produce a reaction that 
 may be injurious to the patient. 
 
 Preparation of Tuberculins. — The kno\Yleclge that tubercle bacilli 
 and their secretions as seen in vitro contain both desirable and undesir- 
 able substances has led Koch and others to adopt different methods of 
 preparing tuberculin in the endeavor to obtain the desirable immunizing 
 principles in as pure a state as possible. 
 
 As a consequence, a large number of preparations have been advo- 
 cated from time to time, all of which are said to possess some special 
 properties and ^'irtues. All tuberculins, whatever then" mode of pre- 
 paration and manufacture, are derived froni cultures of the tubercle 
 bacillus. So numerous have the tuberculins!" become, and so superior 
 are the advantages claimed for each new product over the older ones, 
 both for diagnostic and for therapeutic purposes, that only a few of 
 those possessing special interest and value can here be described. 
 
 1. Old Tuberculin (0. T.). — This is Koch's original tuberculin, and 
 is the variety regarded by many as the most useful both in diagnosis 
 and in treatment. Its manufacture was based upon the prmciple that 
 the toxins elaborated by the bacilli into the culture-medium or liberated 
 bj' disintegration .of the bodies were chieflj^* concerned in stimulating 
 body-cells to the formation of antibodies. Since the bacillaiy bodies 
 were regarded as mainly responsible for the production of abscesses at 
 the point of inoculation, they are eliminated by a process of filtra- 
 tion. 
 
 Old tuberculin is prepared as follows: Large shallow flasks contain- 
 mg 5 per cent, of glycerin alkaline broth are inoculated with a culture 
 of human tubercle bacilli and gro^ni at body tf mperatm-e for from six to 
 eight weeks, at the end of which time the bacilli have gro^^•n into a flat 
 sheet covering the surface of the fluid (Fig. 1*33). The entire contents 
 are then subjected to a current of stcnni ove^ a water-bath for the pur- 
 poses of sterilization and for concentration info one-tenth of the original 
 volume. The glycerin, which is not evaporated, thus constitutes 50 
 per cent, of the resulting mi.\ture. The bacilli are remo-s'ed by filtration 
 through a Berkefeld or Chamberland filter.- The filtrate is a clear, 
 brown fluid, of a characteristic odor, which- keeps mdefinitely and is 
 ready for use. 
 
 Koch considered the soluble toxins of the bacillus as the desirable 
 immunizing agents, and believeil that the enjotoxins were responsible 
 for the necrotic effects. Since, however, it was accepted that bacter- 
 iolytic substances would be formed only after the injection of intact or 
 
TUBERCULOSIS TUBERCULIN THERAPY 
 
 665 
 
 fragmented tubercle bacilli, — with their contfiined endotoxins, — Koch 
 added T. R. and later B. E. to his list, in order»to make the production 
 of antibacterial substances still more complete. Furthermore, in order to 
 obtain as varied a supply of antibodies as possible the use of se\'eral 
 tuberculins, such as old tuberculin and bacilhis emulsion, was recom- 
 mended for use in the same patient. 
 
 2. iV"e«' Tuberculin {Known as T. R. or TuberciiUn Residue). — This 
 was the next, tuberculin to be promulgated by Koch,i and is prepared as 
 follows: Virulent cultures of human tubercle bacilli are grown in flasks 
 of nutrient glycerin broth for from four to six ^^'eeks, the bacilli being 
 then filtered off and dried in a vacuum. One gram of the dried tubercle 
 
 Fig. 133. — Preparation of Tubercitlin. 
 A flask of bouillon culture of tubercle bacilli (three to four weeks) . 
 layer of bacUli with stalactite fonnations. 
 
 Note the surface 
 
 bacilli is ground in an agate mortar until all the bacilli have been broken 
 up. To the pulverized mass 100 c.c. of distilled water are added, 
 and the mixture is then centrifugalized. The clear supernatant fluid 
 is poured off, and is now known as Tuberculin Oberes (T. 0., not to be 
 confounded with 0. T.). It contains substances not precipitable by 
 glycerin. The sediment is again dried, powdered, taken up in a small 
 amount of water, rentrifviged, the supernatant" fluid poured off, and the 
 process repeated until no sediment is precipitated except that composed 
 of gross accidental particles. The fluids resulting from all the centrif- 
 ugalizations, except the very first, are poureH together and the total 
 should not measure more than 100 c.c. This opalescent fluid is preserved 
 with 20 per cent, of glycerin and is laiown as T. R. It should contain 
 ' Deutsch. med. Wochenschr., 1897' xxiii, 209. 
 
666 ACTIVE IMMUNIZATION 
 
 in CEbch cubic centimeter 2 milligrams of solids, representing 10 milli- 
 grams of dried tubercle bacilli. 
 
 3. Bacillen Enndsion {B. E.). — This was 'a still later form of tuber- 
 culin made by Koch/ and, as its name indicates, it is an emulsion of 
 tubercle bacilli. The culture is grown as for 0. T.; the bacilli are 
 filtered off, ground, but not washed, and on* part of the pulverized 
 material emulsified in 100 parts of distilled water; an equal part of 
 glycerin is then added, making a 50 per cent, glycerin emulsion, each 
 cubic centimeter of which contains the iimnuhizing substances of 5 mg. 
 of dried tubercle bacilli. Since B. E. was ncft washed, it was assumed 
 that it would retain all extractives and the entire contents of the bodies 
 of the tubercle bacillus. 
 
 While Koch was preparing these various tuberculins others were 
 being made, one of which, prepared by Denys, is used extensively at 
 present in the treatment of tuberculosis. 
 
 4. Bouillon Filtrate (B. F.). — This is practically Koch's old tuber- 
 culin unheated. It is prepared in the same- maimer as 0. T., except 
 that the bacillus-free filtrate — a clear fluid said to contain only the 
 soluble secretions of the bacilli, plus the meta'bolized culture-medium — 
 is not heated or concentrated and is ready for use without any further 
 modification. 
 
 5. Berancck's Tuberculin. — This is a preparation for which its 
 inventor claims only minimal toxicity and a high content of specific 
 substances. It is prepared by cultivating the feacilli on a non-peptonized 
 5 per cent, glycerin bouillon medium, which is not neutralized. The 
 filtrate from this culture is known as T. B*, or toxin bouillon. The 
 residue is shaken for a long time at from 60° to 70° C. with 1 per cent, 
 orthophosphoric acid. Equal volumes of the unheated toxin bouillon 
 and of the acid extract of the bacillary bodies: are combined to form the 
 whole tuberculin. 
 
 6. Von Ruck's Watery Extract.'^ — This tuberculin has been widely 
 used in the United States. It is prepared §,s follows: Concentrate a 
 culture in vacuo at 55° C. to one-tenth volurae (this requires about one 
 month). Filter through paper and then through porcelain. Pre- 
 cipitate with an acid solution of sorUc bismuth iodid. Filter and neu- 
 tralize the acid solution. Filter again. Ptecipitate with absolute 
 alcohol to make 90 per cent, alcohol and filter. Wash the precipi-_ 
 tate with absolute alcohol. Dry the precipitate and make a 1 per 
 
 ^- Deutsch. med. Wochenschr., 1901, xxvii, 839. 
 2 Zeitschr. f. Tuberk., 1907, xi, 493. 
 
TUBERCULOSIS TUBERCULIN THERAPY 667 
 
 cent, aqueous solution. Filter. The last filtrate is von Ruck's tuber- 
 culin. 
 
 7. Dixon's Tubercle-bacilli Extract.'- — Dixon has prepared a tuber- 
 culin by treating cultures of tubercle bacilli with ether- and extracting 
 in salt solution. This has yielded good results in the treatment of tuber- 
 culosis. This product is prepared from thci living organisms. The 
 tubercle bacilli are grown on 4 per cent, glyceriii veal broth for a period 
 of from six to eight weeks at a temperature of 37.5° C. They are re- 
 moved from the incubator and collected on hard filter-paper. The 
 filtrate of glycerin broth on which they are grown is discarded. An 
 equal quantity, by weight, of tubercle bacilli ©f the bo\'ine and human 
 type is used. The mass of organisms is partially freed from excess of 
 moisture by placing it between two sterile filter-papers, after which it is 
 placed in a dish in the incubator for from twenty-four to forty-eight 
 hours. The dried organisms are then washed in an excess of ether, 
 which is allowed to act until it has removed all the water and glycerin. 
 The organisms are then subjected to an excess of fresh ether and washed 
 in this for six hours, to soften the wax of the bacilli. This fat separates 
 so that it collects at the bottom of the vessel and is removed with a 
 Pasteur pipet. After the second addition of ether has been removed the 
 mass is allowed to dry until no ether odor is perceptible. After the mass 
 of tubercle bacilli has been thoroughly dried it is ground in a mortar and 
 suspended in physiologic salt solution in the proportion of 1 part of the 
 ground mass to 5 parts of an 8 per cent, salt solution. This suspension 
 is shaken in a shaking machine for from eight to ten hours, and is then 
 allowed to stand for several days at room tcimperature. It is finally 
 passed through impervious bacteria filters several times and the filtrate 
 examined microscopically, bacteriologically, and physiologically. Cul- 
 ture tests are made to determine its freedom ffom contaminating organ- 
 isms, and guinea-pigs are inoculated to ascertain that it contains no 
 living tubercle bacilli. One cubic centimeter of this extract represents 
 0.5 gm. of the organisms, and is knowm as the gjtock solution, from which 
 serial dilutions are made. This solution is sterile, but 0.5 per cent, of 
 phenol (carbolic acid) is added as a preservative to prevent subsequent 
 contamination. 
 
 While the tuberculins just described are those mainly used, many 
 
 others have been prepared and advocated in the diagnosis and treatment 
 
 of tuberculosis. The aim is always to obtain the specific substances, 
 
 with as little as possible of the toxic substaiwes^not only those con- 
 
 1 Penna. Health Bull., October, 1911. 
 
668 ACTIVE IMMUNIZATION 
 
 cerned in producing necrosis, but likewise" the protein constituents 
 responsible for specific sensitizing action and anaphylactic disturbances. 
 For example, tuberculocidin and tuberculol are examples of attempts at 
 isolating the pure immunizing principle; endotin, or Moeller's tuber- 
 culin, is an example of an endeavor to rid the culture fluid of protein 
 substances.-^ 
 
 Action of Tuberculin. — If a healthy or a tuberculous individual is 
 injected with old tuberculin, an immunity will be established only 
 against the substances contained in this preparation. That this does 
 not fulfil the requirements is proved by the fact that an animal im- 
 munized against this tuberculin will not be protected against a later 
 infection with \bnng tubercle bacilli. It was mainly for this reason 
 that tubercle emulsion and new tuberculin were devised and used, in 
 the effort to provide immunization not only against the products of the 
 bacilli, but against the bacilli themselves, and to bring about their actual 
 destruction. 
 
 Probably none of the various tuberculins: can be considered as re- 
 presenting the true toxins of the tubercle bacillus, although they simu- 
 late these substances with sufficient closeness to bring about partial 
 immunity against some of the poisonous products and to warrant their 
 use in tuberculosis. An individual may become immunized against old 
 tuberculin so that large doses will evoke no reaction, but this does not 
 necessarih' imply that a cure has resulted; in fact, the injection of another 
 preparation, such as new tuberculin or bacillus emulsion, may bring 
 about a reaction. 
 
 Partial immunization possesses, however", distinct advantages, in 
 that it lessens some of the symptoms of tuberculosis. In addition, 
 tuberculin immunization may give most important aid in walling off 
 tuberculous foci with fibrous tissue, and in this manner bring about a 
 condition of potential cure. Following the injection of tuberculin a 
 focal reaction occurs, characterized by hyperemia and exudation about 
 the diseased tissues. "While an excessive dose of tuberculin may produce 
 excessive hyperemia, exudation, and necrosis of tuberculous tissue and 
 lead to actual harm, these being some of th^ effects that followed the 
 early use of tuberculin and resulted in bringing it into high disfavor, 
 smaller and carefully graduated doses tend to produce a mild inflamma- 
 tory hyperemia leading to destruction of tuberculous tissue and the 
 
 1 For a full account of these and other preparation's I refer the reader to the book 
 of Hamman and W'olman, "Tuberculin in Diagnosis and Treatment," 1912, Appleton 
 &Co. 
 
TUBERCULOSIS — TUBERCULIN THERAPY 669 
 
 formation of connective tissue which encapsuhites the focus; with this 
 there is also associated the local production of antibodies. 
 
 The Use of Living Tubercle Bacilli. — Any vaccine that will give 
 complete immunization against the tubercle bacillus and all its products, 
 in addition to a healing focal reaction of the right degree, will prove 
 of the greatest value in active prophylactic and curative immunization. 
 
 As has been stated repeatedly, this is probably best obtained by using 
 living cultures in such form that they canijpt produce the disease. 
 Obviously, it is a difficult matter to find such a culture or to modify 
 one to meet the requirements at hand. When this problem is solved, 
 it may then be possible to provide universal prophylactic immunization 
 and to aid the actually diseased in overcoming their infection. At 
 present the tuberculins are not adapted for prophylaxis because they 
 possess only partial immunizing powers, althq,ugh these effects may be 
 of distinct aid to the tuberculous patient, in addition to producing a 
 focal reaction of value in waUing off a lesion and producing local anti- 
 bodies. 
 
 Probably the first work done with the living bacillus was that of 
 Dixon ^ with attenuated cultures. This worker found that animals 
 inoculated with an old culture containing club-shaped and branching 
 forms of tubercle bacilli would resist subsequent inoculation with viru- 
 lent organisms. Since then numerous investigators — Trudeau,^ Pear- 
 son,^ de Schweinitz,* McFadyean,' Levy,^ Pearson and Gilliland,' 
 Behring,8 Thomassen,^ Neufcld," Theobald Smith," Webb and Wil- 
 liams,^^ and others — have, either directly or indirectly, supported this 
 original work, thus indicating that the most effectual active immuniza- 
 tion is secured by using living cultures. The method employed by Webb 
 and Williams is worthy of special mention, inasmuch as the ascending 
 doses of bacilli are actually counted by an ingenious method devised by 
 
 1 Medical News, Philadelphia, October 19, 1889. 
 
 ^Amer. Jour. Med. Sci., August, 1906; June, U)07; New York Med. Jour., 
 July 23, 1893; Medical News, October 24, 1903. 
 
 ' Proc. First Internal. Vet. Congress, 1893. 
 
 ■• Medical News, December 8, 1894. 
 
 ' Jour, of Comparative Path, and Therap., June, 1901; March, 1902. 
 
 « Centralbl. f. Bakt., 1903. 
 
 ' Phila. Med. Jour., November 29, 1902; Univ. of Penna. Med. Bull., 190,5. 
 
 ' Beitrage z. cxper. Therapie, Reft. s. Marburg, 1902. 
 
 « Recuil de Med. Vet., January 1.5, 1903. 
 
 1" Deutsch. med. Woclienschr., September 1, 1902'S April 28, 1904. 
 
 " Jour. Med. Research, June, 1908. 
 
 " Trans, of the Sixth Internat. Congress on Tuberculosis, 1908; Jour. Med. 
 Research, 1911, xix, 1. 
 
670 ACTIVE IMMTJNIZATIOJSr 
 
 Barbour.^ These observers used this method quite extensively with 
 lower animals, and have also secured good results with persons willing 
 and anxious to take all possible risks for the possible good that may 
 result. In no case have harmful results followed the injections. 
 
 More recently the profession and laity have been agitated by the 
 extravagant claims of Friedmann for a vaccine of living, acid-fast 
 bacilli derived from the turtle. This culture is said to be avirulent for 
 human beings, and to be capable of stimulating specific antibodies and 
 thus bringing about a cure. The unfortunate methods by which these 
 claims have been exploited, and the more recent investigations, tending 
 to show that no good has followed its use, and that the vaccine is not 
 entirely harmless, preclude any statements at this time. The principle 
 involved, however, namely, the use of a living vaccine composed of 
 microorganisms so altered by passage through a lower animal that they 
 cannot produce tuberculosis in the human being, but yet resembling the 
 human bacillus closely enough to produce specific antibodies, is sound, 
 and should stimulate further experimental research in this direction. 
 
 Patients Suitable for Tuberculin Treatment. — In deciding which 
 tuberculous patients are best suited to receive tuberculin treatment it 
 must be borne in mind that tuberculin is not a;form of passive immunity, 
 depending upon antitoxins, bacteriotropins, and bactericidins, but that 
 it serves to stimulate the body-cells to produce these protective sub- 
 stances. The output of antibodies provoked by tuberculin is dependent 
 upon the condition of the body as a whole, and its administration can 
 be of no help if the resources of the body are exhausted and the cells are 
 incapable of beneficiary reaction. In other words, tubercuhn therapy 
 must be guided by the same considerations that influence the vaccine 
 treatment of acute infections in general. 
 
 1. Patients afflicted with incipient tubercillosis are proper subjects 
 for receiving tuberculin treatment, since it tends to protect them from 
 relapse, and insures, to a greater degree, their ability to continue work. 
 
 2. Advanced and moderately advanced cases may be given tuberculin 
 if the nutrition is fair, the febrile reaction mild, the pulse not very rapid, 
 and if the treatment is controlled by rest. Old fibroid cases with fair 
 nutrition are especially suitable, as such pa,tients become capable of 
 moderate activity and are much less likely to suffer from relapses. 
 
 3. Cases of tuberculosis of the lymphatic glands, skin, and special 
 organs may be benefited by prolonged and careful tuberculin therapy. 
 
 4. Cases of latent tuberculosis, especially the children of infected 
 
 1 The Kansas Univ. Sci. Bull., 1907, iv, No. 1. 
 
TUBERCULOSIS TUBERCULIN THERAPY 671 
 
 families who are below par physically and sh6w tuberculin hypersen- 
 sitiveness, with indefinite physical signs, ar& proper subjects for re- 
 ceiving tuberculin treatment. 
 
 5. The question arises as to whether tuberculin may be administered 
 to ambulant patients. Tuberculin should be regarded as but one factor 
 in the treatment of tuberculosis, and, as such, should be combined with 
 the best therapeutic measures available. Therefore the tuberculin 
 treatment is supplementary to rest, hygiene, and fresh air, and the 
 benefit of the sanatorium should not be denied to patients, especially 
 to the poorer ones. The treatment of a mildly progressive ambulant 
 case should be undertaken only when prolonged rest in bed has had no 
 visible effect, and when no measures can be devised for administering 
 the tuberculin while the patient is in bed, as, e. g., patients who have 
 been at the sanatorium and have returned to work, or those who cannot 
 be persuaded to enter a sanatorium or for whom no place can be found. 
 The tuberculin treatment of more chronic or lojcalized tuberculosis may 
 be successfully undertaken in the clinic or office. 
 
 ContrEiindications to Tuberculin Therapy. — Owing to the increased 
 focal hyperemia that follows the injection of tuberculin, hemoptysis has 
 been considered a contraindication to its use. In such cases it is well 
 to wait for some time at least and begin the injections with very small 
 doses, as the ultimate effect, namely, the production of fibrous tissue, 
 may be of great aid in prolonging life. 
 
 Various authorities have expressed different views regarding other 
 contraindications, such as marked general weakness, fever, cardiac 
 disease, nephritis, epilepsy, syphilis, hysteria^ etc. As was stated by 
 Hamman and Wolman, these are not contraindications, but unfortunate 
 complications that would embarrass any form of treatment. Tuberculin 
 may be given to any patient whose resisting powers have not been too 
 much depressed as the result of complications. For the beginner in 
 this form of therapy, however, it is advisable that he acquire experience 
 by undertaking the treatment of uncomplicated cases before assuming 
 the responsibility of treating the more difficult' ones. 
 
 The fact that the ophthalmic test has been made is no contraindica- 
 tion to treatment by tuberculin if the reaction has subsided, since a 
 flare-up rarely occurs except after large diagnostic doses (Hamman and 
 Wolman) . 
 
 Administration of Tuberculin 
 
 1. Subcutaneous Injection. — Of most importance in this connection 
 is the attitude of the therapeutist toward the question of reactions 
 
672 ACTIVE IMMUNIZATION 
 
 following the administration of tuberculin. He must know whether 
 be does or does not wish to obtain symptoms of a tuberculin reaction 
 during the treatment; the size of the initial and particularly of subse- 
 quent doses will depend upon his desire to obtain a reaction or upon his 
 anxiety to avoid it. 
 
 Reactions. — At the present time tubercujin is never used for the 
 purpose of obtaining strong reactions, such as Koch originaUy insisted 
 upon getting. Koch administered a dose large enough to elicit a strong 
 constitutional reaction, and repeated it at intervals of one or more days 
 until that dose no longer produced a reaction", after which a stiH larger 
 dose was given and the former procedure repeated. Many — too many — 
 were unable to pass through this therapeutic, furnace unsinged, and, in 
 fact, the results obtained led to the period known as the "tuberculin 
 delirium," ending, as Hamman and Wolman stated, "to the consequent 
 downfall of the arrogant therapy to an humble position, whence it is 
 but just emerging, chastened and refined, to .assert its modest but now 
 truthful claims to a therapy less spectacular but more healing, less 
 forceful but more gently persuasive : healing a few, helping many, and 
 hurting none. " 
 
 While there is this general unanimity of opinion regarding the harm- 
 ful effects of strong reactions, yet tuberculin therapeutists may be 
 divided into those who scrupulously avoid all reaction, those who are 
 a little bolder and do not object to a very=mild reaction, and to an 
 intermediate class. In the first group are Sahliand Trudeau; the former 
 claims that it is essential, first of all, to do' no harm, and that cases 
 treated cautiously attain a tolerance for high doses as soon as, and even 
 sooner than, those that are rushed. PetruscKky is an exponent of the 
 bolder method, believing that, by proceeding very cautiously, much 
 time is wasted and not enough focal reaction is produced to promote 
 healing. To the intermediate class, and approaching rather the timid 
 class, are Bandelier and Roepke, Hamman, and Wolman, and many 
 others. These observers adopt no scheme of fixed dosage, but study the 
 individual patient, remembering what is to be avoided, rather than the 
 high dose to be reached. 
 
 The conditutional stjmptom.s of a reaction are: temperature, loss of 
 weight, rapid pulse, and general symptoms, such as malaise, headache, 
 chilliness, arthritic pains, gastric or intestinal disturbances, nausea, loss 
 of appetite, insomnia, and skin eruptions. 
 
 Of these general signs, the most important are fever, loss of weight, 
 and symptoms of general depression. The temperature should be taken 
 
TUBERCULOSIS TUBERCULIN THERAPY 673 
 
 for several days preceding the initial dose, so that the patient's "normal" 
 limits are known before the tuberculin is given. When the usual maxi- 
 mum temperature is reached, it is especially necessary to watch closely 
 for additional signs of reaction. Without some constitutional dis- 
 turbance a slight pyrexia is of less significance, and it is to be remembered 
 that tuberculous patients may have a flare-up of fever when they are 
 not being treated with tuberculin. Denys refuses to consider any 
 temperature reaction as due to tuberculin that comes on more than 
 forty-eight hours after the injection has been given-. It is characteristic 
 of tuberculin reactions that the rise is abrupt, and not in step-like pro- 
 gression. Hamman and Wohnan would hesitate to ascribe an elevation 
 coming on suddenly in the midst of a perfectly smooth course of tuber- 
 ■culin treatment, and unaccompanied by a local reaetion, to the injections, 
 when the dose has not been unduly large. 
 
 Loss of weight is a delicate sign — more valuable as a sjTnptom of 
 overdosage late in the treatment than as a protection against the sud- 
 denly appearing reactions. 
 
 Bandelier and Roepke regard an increase in the pulse-rate as a sign 
 of great importance. Hamman and Wohnan and LawTason Brown 
 have not been able to observe thLs sign very frequently. 
 
 The local signs are: Pain, tenderness, and swelling at the site of 
 injection. These may consist of all gradations from simple thickening of 
 the skin to a wide, deep, hard, and painful node/, -nith or without in- 
 volvement of the neighboring Ijinphatics. 
 
 The local reaction has assumed great importance in recent years, 
 especialh' since Denys drew attention to it as servihg as a warning of the 
 approach of a general reaction. It is characterized b}- the development 
 of pain, tenderness, and redness about the site of a former injection. 
 It is more often solitary than any other sign, and, in the absence of a 
 temperature record, it is safe to proceed, the sole guidance being the 
 local reaction, both subjective and objective. Js large dose of tuber- 
 culia maj^ give a local reaction, due merely to its (bulk and concentra- 
 tion (500 to 600 mg.), simulating a true reaction;, this may be avoided 
 by dividing the dose into two injections, which ^re given at the same 
 time, but not into neighboring areas of skin. 
 
 The focal signs in pulmonary tuberculosis a^e: increased cough, 
 dyspnea, expectoration, thoracic pain, hemoptysis, and extension of 
 the physical signs. 
 
 Focal signs of a reaction are an indication that the treatment has 
 been conducted too rapidly. 
 43 
 
674 ACTIVE IMMUNIZATION 
 
 Unless one belongs to the ultra-conservative class of tuberculin 
 therapeutists, it is not a slight focal reaction that is to be avoided, but 
 those reactions that are large enough to manifest themselves by changes 
 in the physical signs or by decided symptoms. On the contrary, it is the 
 production of such slight hyperemia about the focus of disease that 
 constitutes the most valuable result of tuberculin therapy. It is well to 
 start with a minute dose, and push the dosage rapidly until a slight focal 
 reaction at the site of injection or mild pyrexia is observed. When a 
 mild focal reaction is not produced, the patient is not receiving his due 
 amount. 
 
 Dosage. — Since each patient is a law unto himself, the initial dose 
 should be so small that no harm can result froni its use. White and Van 
 Norman make the initial dose equal to tie quantity of tuberculin 
 which, when applied cutaneously, will elicit a minimal reaction after 
 seventy-two hours. 
 
 As regards the size of the initial dose, patients can be divided into 
 three classes: (1) Children; (2) patients who exhibit a slight pyrexia 
 or are not in good condition; (3) patients in good condition. The 
 following table of doses is that given by Hamman and Wolman, the 
 smaller initial dose being for classes 1 and 2, the larger for class 3. 
 
 TABLE 26.— INITIAL AXD MAXIMAL DOSES OF THE COMMOXLY USED 
 
 TUBERCULINS 
 
 Tuberculin 
 
 Initial Dose 
 
 ^Maximal Dose 
 
 O.T 
 
 T. R 
 
 B. E 
 
 B. F 
 
 Beraneok's 
 
 0.0000001 c.c. to 0.000001 c.c. 
 0.000001 CO. to 0.0001 " c.c. 
 0.000001 c.c. to 0.0001 c.c. 
 0.00000001 CO. to 0.0000001 cc 
 Of A/32, 0.0.5 CO. 
 
 1 c.c. 
 
 2 C.c. 
 2 CC. 
 1 C.C. 
 
 Of H, 1 c.c 
 
 Old tuherculin (0. T.) is put up in ampules holding 1 c.c. and 5 c.c. 
 From these, higher dilutions are prepared by adding sterile normal salt 
 solution, using sterile glassware, starting with a 1 : 10 (A) of the original 
 strength, then making a 1 : 10 from this (B), a 1 : 10 from that (C), a 
 1 : 10 from that (D), and so on to the desired dilution. As 1 c.c. of the 
 original product represents 1000 milligrams of the pure tuberculin, 1 c.c. 
 of dilution A will contain 100 milligrams; B, 10 milligrams; C, 1 milH- 
 gram; D, 0.1 milhgram; E, 0.01 milligram, and F, 0.001 milligram, 
 which is the higher of the initial doses just given. 
 
 New tuherculin (T. R.) is so prepared that 1 c.c. represents 5 milli- 
 grams of the dry powder. Dilutions may be prepared in a manner 
 
TUBERCULOSIS — TUBERCULIN THERAPY 675 
 
 similar to the foregoing by diluting the original product 1 : 10 (A) ; 
 1 c.c. of this is in turn diluted to 1 : 10 (B) ; of this, 1 : 10 (G) ; and of 
 this, 1 : 10 (D); 1 c.c. of A contains 0.5 milligram; 1 c.c. of B, 0.05; 
 1 c.c. of C, 0.005, and 1 c.c. of D, 0.0005 milligram. 
 
 Beraneck's tuberculin is marketed, ready diluted, in a series of syringes, 
 A/128, A/64, A/32, to A/4, A/2, A, B, C, to H. H is the pure tuber- 
 culin. Each solution is one-half the strength of the next stronger one. 
 The increase of dosage is usually by 0.1 c.c. until 0.5 c.c. is given. Then 
 0.1 c.c. of the next stronger dilution is given, and so on. 
 
 Dixon's tuberculin is supplied in syringes in the series of dilutions 
 given in the following table, so that the doses may be increased as is 
 found desirable : 
 
 TABLE 27.— ADMINISTRATION OF DIXON'S TUBERCULIN 
 
 
 Amount (in Ghams) 
 
 of 
 
 
 Amount (in Grams) of 
 
 ILUTION 
 
 Extract of Tubercle 
 
 Dilutions 
 
 Extract 
 
 OF Tubercle 
 
 No. 
 
 Bacilli Contained 
 
 IN 
 
 No. 
 
 Bacilli Contained in 
 
 1 
 
 0.001 
 
 
 11 
 
 
 .0.1 
 
 2 
 
 0.01 
 
 
 12 
 
 
 .0.11 
 
 3 
 
 0.02 
 
 
 13 
 
 
 .0.12 
 
 4 
 
 0.03 
 
 
 14 
 
 
 .0.13 
 
 .5 
 
 0.04 
 
 
 1.5 
 
 
 .0.14 
 
 6 
 
 0.05 
 
 
 16 
 
 
 .0.1.5 
 
 7 
 
 0.06 
 
 
 17 
 
 
 .0.16 
 
 8 
 
 0.07 
 
 
 18 
 
 
 .0.17 
 
 9 
 
 0.08 
 
 
 19 
 
 
 .0.18 
 
 10 
 
 0.09 
 
 
 20 
 
 
 ,0.19 
 
 It is suggested that in ordinary cases the injection of each dilution be 
 repeated at least five times before changing to the next number or next 
 stronger dilution. 
 
 As a general rule, the succeeding doses of a tuberculin may be in- 
 creased by 0.1 c.c, due care being exercised when the dilutions are 
 changed. If the patient is quite sensitive, it may be well to repeat the 
 first dose of each stronger solution once or more often as it is reached, 
 and, instead of proceeding to 0.2 c.c, give only 0.15 c.c, thus tiding over 
 the gap. If the patient is not so sensitive, a few leaps may be taken 
 ■with the weaker dilution, so as to test the patient's tolerance, and, if it 
 is good, the next higher dilution is given at once. 
 
 Site of Injection. — Injections are probably^ best given in the back, 
 at the lower angle of the scapula. Local reactions are more reliable 
 here than when the injections are given in the arm. All injections 
 should be given under aseptic precautions and with a sterilized syr- 
 inge, in exactly the same manner as any bacterial vaccine is given. 
 All injections should be subcutaneous; intramuscular injections are 
 
676 ACTIVE IMMUNIZATION 
 
 to be avoided, and no great advantage is to B6 gained from using intra- 
 venous injections. 
 
 Time of Injection. — There is some difference of opinion as to the best 
 time of the day for administering tuberculin therapeutically. If given 
 in the morning, a slight febrile reaction may occur during the evening 
 which would otherwise be overlooked. Brown is in favor of the after- 
 noon as the most suitable time, because it affords an opportunity for 
 omitting the dose in case there is an accidental rise of temperature on 
 that day. On the other hand, it is contended that the rest at night 
 would tend to prevent the occurrence of the reactions that might appear 
 if the patient were up and about. 
 
 While it is not essential that the patient rest for a few hours after a 
 dose has been administered, this is advisable", and where absolute rest 
 can be enforced, the dosage may be increased with greater rapidity than 
 in ambulant patients. 
 
 Interval Between Doses. — The interval betVeen injections is usually 
 from three to four days — i. e., two injections' a week. This interval is 
 merely tentative, and while it should not be shortened, it may be nec- 
 essary to prolong it. While most reactions set in within from twenty- 
 four to thirty-six hours, some may begin as late as from forty-eight to 
 sixty hours (L. Brown). By waiting at least three days we may be 
 assured that, if no reaction has occurred, none will take place. 
 
 The usual interval is maintained as long as the patient is doing well. 
 After a while the patient becomes intolerant and exhibits slight reac- 
 tions, depression, and loss of weight. In such instances the interval may 
 be increased to a week and the injections continued. Hamman and Wol- 
 man find this occurrence so frequent that they advise creasing the 
 dose interval to one week, when the dose of 100 mg. of 0. T. is reached, 
 200 mg. of T. R. and B. E., and 50 mg. of B. F. 
 
 2. Other Routes for the Administration of Tuberculin. — Oral 
 Route. — It has been shown that reactions may follow the oral 
 achninistration of tuberculin. But absorptien is so irregular that a 
 quantity of tuberculin may be absorbed suddenly and cause unexpected 
 reactions. Much depends, apparently, upon the state of digestion 
 and upon the condition of the alimentary tract. The oral route also 
 deprives the physician of the benefits to be obtained from using the local 
 reaction as a guide. Otherwise the method, is simple and the tuber- 
 culin may be administered in the form of tablets or in capsules. It 
 is important, however, to exercise supervision over the patient, o. 
 
TUBEECTJLOSIS — TUBERCULIN! THERAPY 677 
 
 Solis Cohen has used a modification of Latham's method, and reports 
 favorable results: "Tuberculin residue (T. R.) triturated with milk 
 sugar is given with skim milk, whey, or beef-juice. The initial dose is 
 0.000001 mg. Both subjective and objective symptoms of reaction are 
 watched for. The dose is repeated once or twice weekly, according to 
 results. It is gradually increased by increments of 0.000001 mg. to the 
 reaction point, and then dropped one point lower, and so continued for 
 some weeks. Later, a further increase is attempted, and if reaction is 
 not shown, is proceeded with in a similar gradual way. The arbitrary 
 increment of 0.000001 mg. is maintained during this remittent progres- 
 sion until 0.0001 mg. has been reached. After that the increment may 
 be raised to 0.00001 mg. Thus, by successive stages, a maximum dose 
 is attained at a point determined for each individual by all the factors 
 in the case, including the rapidity of increase, character and intensity of 
 reaction, and maintenance of tolerance, as well as the focal and general 
 signs of improvement. The treatment is continued with intermissions 
 for many months, and may be resumed, if necessary, from time to time 
 over a period of years. " 
 
 Tuberculin has also been administered intrabronchially and by the 
 rectum without good results. 
 
 Koch was the first to administer tuberculin intravenously, but this 
 route has not come into general favor, owing to the fact that even greater 
 control over dosage is necessary; there is, besides, no local reaction to 
 serve as a guide, and technically the administration is more difficult 
 than is subcutaneous injection. 
 
 The Intrafocal Route. — The use of tuberculin intrafocally, that is, a 
 method by which the tuberculin is brought into immediate contact with 
 the diseased area, has been advocated in the treatment of tuberculous 
 pleurisy, tuberculous peritonitis, and tuberculosis of other serous mem- 
 branes, such as those lining joints, the tunica vaginalis of the testicle 
 etc. Senger, Crocker, and Pernet advise the intrafocal use of tuber- 
 culin in the treatment of lupus; others have applied it directly to 
 broken-down glands and to sinuses. Williarft Egbert Robertson ^ has 
 reported very good results in two cases of tuberculous pleurisy as a 
 result of withdrawing a small amount of fluid and injecting 5 mg. of old 
 tuberculin through the needle, which is left in situ for that purpose. 
 After some hours there was a slight reaction; the remainder of the fluid 
 was rapidly absorbed, and convalescence was promptly established, 
 ' Personal communication. 
 
678 ACTIVE IMMUNIZATION 
 
 though, of course, a damaged lung remained. In a case of hydrocele the 
 injection of old tuberculin was followed by a sharp local and a moderate 
 general reaction, followed by absorption, and without the slightest 
 evidence of recurrence to date, now about a year since the injection was 
 made. 
 
 Results of Tuberculin Therapy. — The early and disastrous results 
 obtained with tuberculin in various types of tuberculosis cannot be used 
 at the present day as a measure for determining the therapeutic value of 
 tuberculin. 
 
 So much depends upon the individual case, the duration and activity 
 of the disease, the possibility of supplementing the active immunization 
 with sanatorium treatment, the skill and patience of the physician, etc., 
 that, from a prognostic standpoint, every case must be judged upon its 
 own merits. The results of the modern use* of tuberculin show quite 
 clearly that it is not a "cure" for tuberculosis, „but, rather, a rational and 
 useful therapeutic aid, the best results being secured when the treat- 
 ment is carried out in special institutions or by speciaUy trained physi- 
 cians who have a practical knowledge of the difficulties, dangers, and 
 possibilities of tuberculin therapy. 
 
 The value of this or of any therapy can be judged according to various 
 standards: (1) Working ability; (2) duration of life; (3) the presence 
 of tubercle bacilli in the sputum; (4) physical signs, and (5) the symp- 
 toms. 
 
 The first three are especially valuable; duration of life is, after all, 
 the most important criterion, as anything that prolongs life is, of course, 
 welcomed. 
 
 Kremser chose 110 patients expectorating tubercle bacilli and treated 
 55 unselected cases with tubercului; of these, 22, or 40 per cent., lost 
 the bacilli from their sputum; of those treated without tuberculin only 
 16, or 29 per cent., lost their bacilli. Phillippi found that 58 per cent, of 
 his second stage cases were rid of bacilli m the sputum under tuberculin 
 treatment, as against 19 per cent, without. Brown reports from Saranac 
 Lake that, in the incipient class, 67 per cent, of the tuberculin patients 
 were rid of bacilli; of the others, 64 per cent. In the moderately 
 advanced the figures are respectively 44 per cent, and 24 per cent. 
 Bandelier gives the reports of 500 cases, of whom 202 had tubercle ba- 
 cilli in the sputum. In the following table he compares the working 
 Capacity and sputum examinations of these patients under tuberculin 
 treatment : 
 
TUBERCULOSIS TUBERCULIN THERAPY 
 
 679 
 
 Complete earning capacity 
 
 on discharge . 
 
 Sputum changed from pos- 
 itive to negative 
 
 Total 
 
 Stagr I 
 
 Stage II 
 
 
 Per cent. 
 
 Per cent. 
 
 500 cases (69.8 
 per cent.) 
 
 So.4 
 
 80.7 
 
 202 cases (63.9 
 per cent.) 
 
 100.0 
 
 87.3 
 
 Per cent. 
 
 32.8 
 44.0 
 
 The parallelism between the bacillary content of the sputum and the 
 working capacity is close and shows the value, from a statistical point 
 of view, of sputum examinations. 
 
 Healing with and without tuberculin is qualitatively the same, 
 although, in the opinion of Ziegler, Petruschky and Rohmer, Pearson 
 and Gilliland, Jurgens, Neumann, and others, quantitatively the re- 
 sults are different, all authors agreeing on the presence of more fibrosis 
 about the lesions than is usual in the untreated cases. Autopsy findings 
 necessarily point with less favor to tuberculin" therapy than do clinical 
 facts bearing on the rapidity and permanency with which a lesion heals. 
 
 The influence of tuberculin cannot at present be judged from the 
 presence of antibodies in the serum, as data bearing upon the presence 
 or absence of antitoxins, opsonins, agglutinirfe, and bacteriolysins are 
 insufficient. 
 
 General symptoms and signs, such as fever, cough, loss of weight, 
 accelerated pulse, digestive disturbances, dysptiea, and pain, if they are 
 due to tuberculosis, as a rule improve under tuberculin treatment. 
 Under proper conditions the incidence of hemoptysis is not usually 
 increased, and the quantity of sputum and number of expectorated 
 bacilli gradually decrease. 
 
 In the treatment of tuberculosis of the eye tuberculin has yielded 
 exceptionally good results; in tuberculous adenitis considerable good 
 may be accomplished with those glands that have not as yet softened. 
 In hone and joint tuberculosis there is a growing opinion that surgery 
 may be obviated or supplemented by tuberculin. In tuberculosis of 
 the ear, tuberculin has yielded good results an.fl should always be used. 
 The results in tuberculosis of the skin have been generally disappointing, 
 and when tuberculin is administered, the treatment should be prolonged 
 and supplemented by the usual therapeutic measures. Tuberculosis of 
 the intestine and mesenteric glands may be benefited, and in subacute 
 tuberculous meningitis this method of treatment may also be tried, 
 combined with lumbar puncture to relieve intracranial pressure. In 
 
680 ACTIVE IMMUNIZATION 
 
 tuberculosis of the genito-urinary organs the prognosis is largely de- 
 pendent upon the accompanying pulmonary condition and upon the 
 extent of the local process. There is general recognition of the value of 
 tuberculin as an adjuvant to hygienic measures, although great difference 
 of opinion exists as to surgical intervention. If one kidney is involved, 
 most surgepns advise early operation, followed by tuberculin treatment; 
 if both organs are diseased, tuberculin may prove a valuable adjuvant 
 to the treatment. 
 
CHAPTER XXX 
 PASSIVE IMHtJNIZATION— SERUM THERAPY 
 
 Serum therapy may be said to have had its origin in 1890, when von 
 Behring discovered diphtheria antitoxin. He found that guinea-pigs 
 surviving a subcutaneous inoculation of living diphtheria bacilli may 
 harbor virulent bacilli at the site of injection without showing any 
 evidences of intoxication. Subsequent investigation showed that the 
 blood-serum of these animals contained the protective principles, for 
 when the serum was injected into other animals along with the diph- 
 theria toxin, symptoms of the disease did not "develop, and, indeed, as 
 was shown later, the immune serum was found capable of neutralizing 
 the toxin in the test-tube. Shortly afterward Kitasato made similar 
 discoveries in studying tetanus, and these antitoxins have since proved 
 of great importance, not only from the new light that has been thrown 
 upon the mechanism of immunity, — and they were used as important 
 arguments for the humoral as opposed to the phagocytic theory, and 
 form the very basis and starting-point of Ehrlich's researches,— but also 
 from the new and important field of therapy that was now opened, 
 which gave promise and hope for the discovery of a specific serum treat- 
 ment for each bacterial disease. 
 
 At the time it was thought possible to immunize animals with the 
 various microorganisms known to produce disease, and that the immune 
 serums so produced may be employed in the form of specific treatment. 
 This theory rested on the fact that they contained the antibodies that 
 would quickly overcome the infection. With a few of the genuinely 
 antitoxic serums these hopes have been realized; but many other 
 serums have not yielded the expected and wished-for results, although 
 at the present time the reasons for failure are being recognized and 
 gradually eliminated. 
 
 Definitions. — It will be remembered that in active immunization 
 our own body-cells are stimulated to produce antibodies, either by 
 reason of the presence of a disease or as the result of vaccination with the 
 antigen of the disease in a modified and attenuated form. In passive 
 immunization, however, our own body-cells do not produce the antibodies, 
 
 681 
 
682 PASSIVE IMMUNIZATION SERT5M THERAPY 
 
 but we receive them passively in the form of ah injection of an antibody- 
 laden serum. The antibodies are produced hy active immunization of 
 some other animal, usually a horse, and we recbive the antibodies or prod- 
 ucts of this immunization in a passive manner, i. e., our body-cells receive 
 protection against an infection and aid us in overcoming it through anti- 
 bodies produced in some other animal. For this reason the process is 
 called passive immunization; the particular kind of increased resistance 
 afforded against infection is known as passive immunity, and since blood- 
 serum contains the antibodies and is the usual vehicle by which they are 
 transferred, the method is called serum therapy. 
 
 PURPOSES OF PASSIVE IMMUNIZATION 
 Passive immunization may be employed for two main purposes: 
 
 1. To prevent disease {prophylactic immunization). 
 
 2. To cure disease [curative immunization). 
 
 In prophylactic immunization the antibodies are introduced into our 
 body-fluids before infection has actually occurred, or at least in the 
 earliest stage of infection, for the purpose of placing them on guard to 
 destroy the infecting microorganism or to neutralize its products before 
 it has had an opportunity to produce disease: In other words, we aim 
 to fortify our natural defenses by purchasing antibodies from another 
 animal. From the fact that these antibodies may be introduced in a 
 short space of time and that in this manner an immunity may be quickly 
 gained, passive immunization for prophylactic purposes is indicated 
 when the danger of infection is imminent, and when it is impossible, or 
 when there is not sufficient time, for us to stimulate our own body-cells 
 to produce our own antibodies by active immunization with a vaccine. 
 
 Since the antibodies are produced in another animal, the serum, when 
 introduced into our body-fluids, represents a foreign protein, and, ac- 
 cordingly, we find that the antibodies are retained for relatively short 
 periods of time and are quickly eliminated or destroyed. In active 
 immunization, however, the antibodies are in native surroundings, and 
 our body-cells continue to produce them for some time after active 
 stimulation has ceased, in this manner insuring a higher degree of 
 immunity and one of longer duration. For purposes of prophylaxis, 
 therefore, active immunization is always more desirable than passive im- 
 munization; not infrequently the two forms^ are used simultaneously, 
 as the antibody-laden serum will afford instant protection, while the 
 vaccine is stimulating our body-cells to produce antibodies that will 
 
VARIETIES OF PASSIVE IMMUNITY 683 
 
 increase and maintain the protection over a longer period of time. This 
 mixed form of immunization has recently received special study by von 
 Behring in immunization experiments against diphtheria, and will be 
 considered in detail in a later section. 
 
 In curative immunization the conditions are somewhat different. 
 During the course of an infectious disease oilr body-cells are actively 
 engaged in combating the infectious agent, se-that reenforcemcnts, in 
 the form of specific antibodies, are indicated and welcomed for the aid 
 they give in overcoming an infection and the relief they afford our hard- 
 pressed protective mechanism. 
 
 For these reasons it may be stated that the more acute the infection, the 
 greater is the indicatmi for introducing an antibody-laden serum. In 
 chronic infections and in some acute infections we may practise active 
 immunization by introducing a vaccine, with the purpose in mind of 
 stimulating dormant cells to produce antibodies; but, as a rule, it is 
 reasonable to assume that in a severe generalized infection our body- 
 cells are doing their utmost to overcome the infection, and extra stimula- 
 tion may be actually harmful. By introducing antibodies produced in 
 some other animal, however, practically no extra strain is thro'mi upon 
 the body-cells; on the contrary, they may be relieved when the new 
 antibodies overcome the products of infection, and in this manner afford 
 them an opportunity to recover. Unfortunately, this is actually the 
 case in but a few infections, such as diphtheria, tetanus, and cerebro- 
 spinal meningitis, and does not apply equally well to the larger number 
 of bacterial infections due to the pneumococcus, streptococcus, gonococ- 
 cus, and similar infections. The reasons for failure of passive immuniza- 
 tion in the cure of the last-mentioned infections are now being studied 
 and realized, and means are being discovered for overcoming the diffi- 
 culties, so that serum therapy, in a broad sense, is about to play a more 
 important role in the treatment of many bactgrial infections. 
 
 VARIETIES OF PASSIVE IMMUNITY 
 While, strictly speaking, all antibodies are probably inimical to their 
 antigens, from the practical standpoint of passive immunization three 
 are of primary importance, namely: (1) The antitoxins, (2) the bac- 
 teriolysins, and (3) the bacteriotropins (immune opsonins). The anti- 
 toxins neutralize their toxins; bacteriolysins cause the death of their 
 respective bacteria if suitable complements are present, and bacterio- 
 tropins accomplish the same end by lowering the resistance of the 
 
684 PASSIVE IMMUNIZATION — SERTJiVI THERAPY 
 
 bacteria, and in this manner facilitating phagcjcytosis. Other antibodies 
 may be operative and prove of assistance, as, e. g., agglutinins may aid 
 in bacteriolysis and anti-aggressins may aid in phagocytosis, but too 
 little is known at the present time to allow fine distinctions to be made, 
 although the indications are that not one hut several antibodies are present 
 in each immune serum, which, acting together, tend to overcome an in- 
 fection. 
 
 From the practical standpoint, therefore^ immune serums may be 
 used to produce two main types of passive immunization, namely: 
 
 1. Antitoxic immunization, due to antitoxins for the true or extra- 
 cellular toxins, as in diphtheria and tetanus (antitoxic immunity). 
 
 2. Antibacterial immunization, due mainly to bacteriolysins and bac- 
 teriotropins, as in meningococcus, pneumococcus, streptococcus, gono- 
 coccus, and similar infections (antibacterial immunity). 
 
 The mechanism of both of these varieties ot passive immunity will be 
 considered briefly under their respective headings. 
 
 INDICATIONS FOR PASSIVE IMMUNIZATION 
 
 For purposes of prophylaxis only two immune serums have proved 
 their efficiency, namely, the antitoxin of diphtheria and tetanus anti- 
 toxin. 
 
 As will be pointed out further on, diphtheria antitoxin, when ad- 
 ministered in sufficient amounts, affords protection for at least from four 
 to six weeks; mixed immunization, by means of the simultaneous in- 
 jection of a neutral mixture of the toxin and antitoxin, as worked out by 
 von Behring, has been found to yield equally" good and more prolonged 
 immunity, but because of certain technical difficulties has not as yet 
 been widely adopted. 
 
 Tetanus antitoxin has its greatest value as a prophylactic. When 
 symptoms of tetanus have once appeared, serum treatment may be of no 
 avail, whereas it has proved its efficiency beyond doubt in neutralizing 
 the toxin before it reaches or unites with the nervous tissue. In all 
 wounds likely to be infected with tetanus the physician should include 
 the administration of tetanus antitoxin as a matter of routine treatment. 
 
 Of the antibacterial serums; many have a prophylactic value in ex- 
 perimental animals, but none, with the exception of the antiplague 
 serum, is in general use as a prophylactic in human practice. The rea- 
 sons for this are apparent when it is remembered that pneumococcus, 
 streptococcus, and meningococcus infections are not sufficiently epidemic 
 
INDICATIONS FOR PASSIVE IMMUNIZATION 685 
 
 in character to demand passive immunization". Meningococcus men- 
 ingitis may, liowever, be an exception, but the method of active im- 
 munization advocated by Sophian is promising, easier to carry out, and 
 should be tried during times of epidemic meningitis. 
 
 In typhoid fever, cholera, and dysentery antibacterial serums have 
 not been generally used in prophylaxis, although it would appear that a 
 potent anticholera serum would prove of value in preventing epidemics 
 of this frightfully infectious disease. 
 
 With the exception, therefore, of the true intoxications, prophylaxis 
 is more readily secured by active than by passive immunization. This 
 is certainly true of typhoid fever, rabies, and smallpox. The antiserums 
 of other microorganisms, such as the pneumococcus, streptococcus, and 
 gonococcus, are being used exclusively for therapeusis, rather than for 
 prophylaxis, of their several infections. 
 
 In veterinary practice hog-cholera serum has proved of value as a 
 prophylactic means of combating and limiting epidemics of hog cholera. 
 It is not definitely known whether this serum, is antitoxic or antibac- 
 terial, but it is probably a combination of both. 
 
 In the treatment of disease immune serums have proved of value in 
 diphtheria, tetanus, cerebrospinal meningitis, and, to a lesser extent, 
 dysentery, pneumonia, streptococcus infections, and plague. 
 
 AVhile antipneumococcus, antistreptococcus, and antigonococcus 
 serums have proved of some value in the treatment of their particular 
 infections, the more recent work of Neufeld and Handel, Dochez and 
 Cole, and their coworkers in pneumonia indisates that there are wide 
 biologic differences among various strains of these microorganisms, and 
 that no curative properties can be expected from a given serum unless 
 this is homologous for the type causing the infection. Further than 
 this, it has been found impossible to secure serums as rich in antibodies 
 as are secured with diphtheria and tetanus antitoxins, and that the 
 serums must be given intravenously in relatively large doses. A method 
 for the quick recognition of types of pneumococci has been worked out 
 in the Rockefeller Hospital, and immune serums have been prepared for 
 the main types, and the results of the serum treatment of pneumonia 
 along these lines have been found to be most encouraging. While this 
 method is not adapted for general use, it hoMs out a promise for the 
 future of serum therapy, and opens up a wide jeld of investigation with 
 the group of streptococci, gonococci, and meningococci. 
 
686 PASSIVE IMMUNIZATION SERIJM THERAPY 
 
 CONTRAINDICATIONS TO PASSIVE- IMMUNIZATION 
 
 The chief contraindications to the therapeuHc use of a serum, if any 
 ever exist, are those dependent upon the serum itself, for, as will readily be 
 understood, the introduction of antibodies themselves does not mean an 
 extra strain upon our body-cells, but rather tlie reverse. The question 
 before us, then, is one regarding the possible contraindications to the in- 
 jection of a foreign serum, and the dangers dependent upon its use. It 
 rhay be stated at once that, in the great majority of cases, the adviinis- 
 tration of a carefully prepared and properly adn%inistered serum is free from 
 danger. Since the introduction of diphtheria antitoxin in the prophylaxis 
 and treatment of that disease nrany thousands of injections have been 
 given, in all parts of the world and under all sorts of conditions, and the 
 number of fatalities is so small as to be regarded as almost negligible. 
 Serum therapy should not, however, be abused to the extent of using 
 the serum indiscriminately. I am opposed to using the serum as a 
 prophylactic unless the indications for its employment are distinct; for 
 example, in diphtheria it suffices to immunize only those who have been 
 brought into immediate contact with the injection. When, however, 
 the indications are clear and the symptoms of infection are present, I 
 believe in using the serum early and generously. 
 
 1. Of all possible dangers consequent to the use of serum therapy, 
 that of anaphylaxis is uppermost in the minds of practitioners. ^A^hile 
 it is true that anaphylaxis has been the cause of some fatalities, the 
 likelihood of this accident taking place is so reipote, in the great majority 
 of cases, that it should not occupy a prominent place in the physician's 
 mind, nor interfere with the use of the serum, as, for instance, anti- 
 toxin in the treatment of diphtheria. It is true that serum sickness is 
 comparatively common, and while the symptoms are frequently dis- 
 tressing, they are not dangerous and do not constitute the dreaded and 
 fatal anaphylaxis. With a little discrimination and care on the part 
 of the physician the risk of anaphylaxis may be rendered still more re- 
 r&ote if attention is given to the following questions: 
 
 (a) Is the patient sensitive to horse protein? This is probably the 
 most important single question, as in several of the fatal cases of ana- 
 phylaxis on record it was learned afterward that the patient was usually 
 rendered uncomfortable, and that sneezing, asthma, or even an urti- 
 carial rash would develop when the patient came into close proximity to 
 horses, as in a stable, or when driving behind them, etc. Fortunately, 
 these cases are very few, but several of the fatal cases of anaphylaxis 
 on record occurred in just such persons, and at the present time a 
 
METHODS or INOCULATION 687 
 
 physician should generally be able to detect this susceptibility and 
 avoid the dangers of anaphylaxis. In Chapter XXVIII the subject is 
 discussed in greater detail, and a method of vaccination is described 
 by which it may be possible to detect this condition; this consists of 
 rubbing a little of the serum into an abrasion on the arm (Fig. 121) 
 
 (6) Has the patient been injected with a serum on any former oc- 
 casion? If an injection has been given, especially a few weeks earlier, 
 a reinjection of serum may cause well-marked serum sickness, but the 
 possibilities of alarming anaphylaxis are so remote that serum should 
 never be withheld if the clinical condition indicates that it should be 
 given. Not infrequently a child receives an immunizing dose of diph- 
 theria antitoxin, but develops the disease a month or two later, after 
 the immunity has disappeared. Under these circumstances antitoxin 
 should not be withheld. If time permits, the physician may inject 0.5 
 c.c. of the antitoxin for the purpose of producing anti-anaphylaxis, fol- 
 lowed in two or three hours by the remainder of the serum. // it were 
 'possible to obtain it, it would be good practice to immunize the patient with 
 an ox-serum antitoxin, and then, if it was found necessary later to use an 
 antitoxin, the usual horse serum antitoxin could he employed. This would 
 still further eliminate the possibility of the development of disagreeable 
 or dangerous complications. 
 
 2. If a patient suffers from idiopathic asthma and the condition 
 known as status lymphaticus develops, serum should be given cautiously 
 because of the increased respiratory difficulties that may follow. It 
 may be well to give a preliminary hypodermic ipjection of atropin and 
 caffein, and then, after a few minutes, give th^ serum, injected slowly 
 and subcutaneously. 
 
 3. Aside from these questions, the physician may be called upon to 
 decide if a patient is physically able to withstand the effects of an inocu- 
 lation, especially the intravenous injection of relatively large amounts of 
 serum, such as are given in the treatment of pneumonia. In diphtheria 
 in very young and weak children, when a large number of units or sev- 
 eral injections are to be given, concentrated antitoxin is to be preferred, 
 in order that injury to the subcutaneous tissues, pain, and shock may be 
 reduced to a minimum. 
 
 METHODS OF INOCULATION 
 
 Upon the nature and severity of the infection will depend the question 
 whether the serums are to be given subcutaneously, intramuscularly, 
 
688 PASSIVE IMMUNIZATION — SERTJM THERAPY 
 
 intravenously, or intraspinously {subdurally) . In diphtheria, the anti- 
 toxin may be given subcutaneously unless the infection is quite severe; 
 in the latter case it should be given intramuscularly or intravenously. 
 In tetanus the serum should be given subdurally and intravenously. 
 In epidemic cerebrospinal meningitis the serum is always given sub- 
 durally. In pneumococcus, streptococcus, and gonococcus infections, 
 while the serum may be given subcutaneously or intramuscularly, it is 
 best administered intravenously. It is important for the physician to 
 know and appreciate that the route and method of inoculation and the 
 amount of serum administered are important factors in determining the 
 success or failure of serum therapy. 
 
 i, Technic of Subcutaneous Iroculatior 
 Serum given subcutaneously is slowly absorbed, and a portion of the 
 antibodies may be destroyed before they reach the blood-stream. When 
 large quantities of serum are to be given, as in pneumonia and strepto- 
 coccus infections, this method may not be permissible on account of the 
 pain and injury to the subcutaneous tissues that may result, aside from 
 the more important question of slow absorption and anchorage or de- 
 struction of the antibodies in various tissues before they reach the blood- 
 stream or the focus of disease. 
 
 1. Injections should be given where the subcutaneous tissues are 
 loose, where movement is least marked, and preferably where pressure 
 upon the parts is least likely to occur, for some soreness, dependent upon 
 the bulk of the injection, is bound to follow. For these reasons injec- 
 tions may be given in the abdominal wall; some prefer the hack, in the 
 region of the lower angle of one of the scapulce, and the buttocks, but in a 
 bedfast patient pressure at these points cannot readily be eliminated. 
 
 2. The skin about the site for injection may be prepared by an ap- 
 plication of tincture of iodin; this is washed off with alcohol just before 
 the needle is inserted. After the injection has-been given the remaining 
 iodin should be removed with alcohol, to prevent the occurrence of a 
 dermatitis, and the puncture wound covered with cotton and collodion 
 or with sterile gauze fastened with adhesive straps. 
 
 3. The sjT-inge and needle should be sterile. Manufacturers of bio- 
 logic supplies furnish antitoxin in syringes ready for injection, and these 
 are usually convenient and satisfactory. The needle should be of 
 medium size, and larger than that used for ordinary hypodermic medica- 
 tion. All glass or glass and metal syringes that may be boiled are to be 
 preferred when a syringe is not furnished. Before boiling such a syringe 
 
METHODS OF INOCULATION 
 
 689 
 
 the piston should be removed from the barrel, as othervnse it may expand so 
 rapidly as to cause the latter to crack. 
 
 4. When all is in readiness, the syringe being loaded and the air ex- 
 pelled, the skin is pinched up between the fingers and the needle quickly 
 inserted into the subcutaneous tissues. The injection should be given 
 slowly, and during the operation, if the patient is a child, an assistant 
 should be on hand to prevent struggling. In the illustration (Pig. 134) 
 the needle is shown connected with the barrel of the syringe by means of a 
 
 Fig. 1.34. — Subcutaneous Injection of Serum. 
 The site of injection is painted with tincture of iedin and covered with sterile 
 gauze fa.stened with straps of adhesive plaster. Just before the injection is given 
 the iodin Ls wiped off with a pledget of cotton and alcohql. A fold of skin is pinched 
 up between the thumb and forefinger of the left hand, the needle inserted, and the 
 serum slowly injected. The needle is then quickly withdra-n-n, and the puncture 
 covered with the gauze and held in place by the adhesive plaster. 
 
 short piece of rubber tubing. This permits an injection to be given 
 without danger of the needle being broken off if the patient should 
 struggle. Most pain is experienced when the first few drops of fluid are 
 injected; after that the pain is not severe unless the tissues are suddenly 
 distended, as by a quick injection. 
 
 The amount of serum that may be injected in one area depends upon 
 the age of the patient. Due care should be exercised against injecting 
 too much serum in one area, because of slower absorption and possible 
 necrosis of the skin and subcutaneous tissues. 
 44 
 
690 passive immunization serum therapy 
 
 2. Technic of Intramuscular Inoculation 
 
 As shown experimentally by Meltzer and Auer, absorption occurs 
 much more quickly when inoculations are given into the muscles than 
 when they are given into the subcutaneous tissues. For this reason anti- 
 toxin should be given intramuscularly in severe cases of diphtheria, as 
 the technic is just as simple as that of a subcutaneous injection. When- 
 ever the physician desires more speedy absorption than that which fol- 
 lows a subcutaneous injection, and the intravenous route cannot, for 
 some reason, be adopted, the inoculation shoirld be given in the muscles, 
 preferably those of the buttocks. 
 
 The technic is the same as that employed for subcutaneous injections, 
 except that the needle is plunged deeply into the muscles. If the most 
 muscular portions are selected for injection,, there will be little or no 
 danger of injuring a nerve. If desired, the syringe may be detached 
 after the needle has been inserted to ascertain if a vein has been entered, 
 which would be shown by a flow of blood. If this occurs, the needle 
 should be withdrawn slightly and passed in another direction. 
 
 3. Technic of Intravenous Inoculation 
 The necessity of administering serum intravenously in order to 
 obtain the best results, or any result at all, is becoming more and 
 more apparent. In severe cases of diphtheria the best results are 
 obtained when the antitoxin is given intravenously; in the treatment 
 of tetanus the tetanus antitoxin should be administered intra- 
 venously as well as intradurally, and both antistreptococcus and anti- 
 pneumococcus serums should always be given by the intravenous route. 
 Recent reports indicate that the proper serum treatment of these in- 
 fections requires large doses given intravenously. Physicians should, 
 therefore, be prepared to give intravenous injections. Since the use of 
 salvarsan in the treatment of syphilis has become so popular many 
 workers have perfected themselves in the technic of intravenous adminis- 
 tration, but there is still great hesitancy about giving intravenous in- 
 jections, although the methods are relatively simple and easily mastered. 
 Syringe Method. — When small amounts of fluid are to be injected, 
 as from 5 to 20 c.c, a syringe is employed. 
 
 1. It is best to use an all-glass syringe, or at least one with a glass 
 barrel, for the physician can then assure himself that all air has been ex- 
 pelled and that the fluid is free from solid particles. Further than this, a 
 flow of blood into the syringe will indicate that the needle has entered 
 the vein. The syringes furnished by manufacturing firms are not 
 
METHODS OF INOCUl^ATION 691 
 
 well adapted for making these injections, as the rubber plunger fre- 
 quently adheres to the glass barrel, so that the injection will be jerky 
 and difficult, and, besides, it may be difficult to determine when the vein 
 has been entered. It is better to empty the contents of these syringes 
 into a large, sterilized, glass-barreled syringe, such as the Record, Luer, 
 and Burroughs-Wellcome syringes, which have a close-fitting but easily 
 working piston, and are attached to the needle by a flange and not by a 
 screw thread. (See Fig. 8.) The needle should be sufficiently large 
 and have a sharp but short beveled edge. A long point may pierce the 
 vein through and through, and permit perivascular bleeding or result 
 in a subcutaneous injection. 
 
 2. In young children with fat arms and a weak circulation it is usu- 
 ally necessary to expose a vein at the elbow by making a small incision. 
 In older children and adults a vein may stand out prominently enough 
 to permit the needle to be inserted directly through the skin without 
 making an incision. A firm rubber tourniquet is applied above the 
 elbow; a very simple one is constructed by a single turn around the arm 
 with a piece of ordinary soft-rubber tubing held in place by a hemostat. 
 After the vein has been entered the tourniquet should be quickly re- 
 moved and this is quickly and deftly accomplished by releasing the 
 hemostat. 
 
 3. The skin about the site of injection is cleansed with soap, water, 
 and alcohol, or merely painted with iodin, which is removed with alcohol 
 just before the injection is to be given, in order that the vein may become 
 visible. 
 
 4. An assistant steadies the patient's arm and should be ready to 
 release the tourniquet. 
 
 5. The operator then steadies the skin over a vein — usually the 
 median basilic or median cephalic — with the left thumb and forefinger, 
 and introduces the needle into the vein. A flow of hlood into the syringe 
 indicates that the vein has been entered. The tourniquet is then released 
 and the injection slowly given. Or the needle may be detached from 
 the syringe and passed into the vein ; when blood appears, the syringe 
 is quickly attached and the injection made. The puncture wound is 
 then sealed with a wisp of sterile cotton and collodion or with gauze and 
 a bandage. The syringe shown in Fig. 135 is well adapted for the in- 
 travenous injection of serum, and was devised for the administration of 
 concentrated solutions of salvarsan and neosalvarsan, but any reHable 
 and large glass-barreled syringe may be used. 
 
 Gravity Method. — Larger quantities of sejnm or other fluid are 
 
692 PASSIVE IMMUNIZATION — SERiyM THERAPY 
 
 better injected by the gravity method, the simple apparatus shown in 
 Fig. 136 being quite satisfactory for the purpose. This consists merely 
 of a graduated cylinder, which serves as a measuring funnel, rubber tub- 
 ing with a pinch cock, and is furnished with a metal tip that fits the 
 needle. The needle should be of proper size, and have a sharp but some- 
 what short beveled edge. It may be curved, as shown in the illustration, 
 or may be straight. The apparatus shown in Fig. 142 is adapted 
 
 Fig. 135. — Method of Making Intravenous Injec-tion et Means of a Stringb. 
 This syringe was devised for the intravenous administration of a concentrated 
 solution of salvarsan. It is provided with a three-way coclc, which permits drawing 
 fluid into the syringe and then injecting it into a vein. This injection may also be 
 given by any glass syringe; the particular advantage of this one is that the operas 
 tor may inject more than one syringeful of fluid withtout removing the needle. The 
 same syringe may be used for the intravenous injection of any serum, as diphtheria 
 and tetanus antitoxins. (Apparatus made by B. B. Cassel, Frankfort, Germany.) 
 
 for the administration of salvarsan, and has the decided advantage of 
 permitting the operator to give an intravenous injection to an adult 
 person without assistance. 
 
 1. The cylinder, tubing, and needle should be sterilized by boiling 
 prior to use. 
 
 2. The serum or other fluid may be warmed by placing the container 
 in water of a temperature not higher than 42° C. (just comfortably hot 
 to hold the hand in). 
 
METHODS OF INOCULATION 
 
 693 
 
 3. The injections are best given in a vein at the elbow. The arm 
 about this region should be scrubbed with hot Water and soap, followed 
 by alcohol and 1 : 1000 bichlorid of mercury solution, or liberally painted 
 with tincture of iodin. A firm tourniquet is then applied above the 
 elbow; a single firm turn of rubber tubing held by a hemostat is quite sat- 
 isfactory, as when the vein has been entered the tourniquet should be 
 quickly released with the least movement and disturbance possible, and 
 
 Fig. 136. — Method of Makino Intravekovs Injection by Gravity. 
 
 This method is suitable for the intravenous administration of salvarsan or anti- 
 streptococcus serum, etc. The needle has been entered into a prominent vein 
 (indicated by a flow of blood); the tubing has been attached by means of a metal 
 tip which fits the needle easily and snugly; the tourniquet has been loosened and the 
 injection is being given. 
 
 this arrangement answers all requirements. Sterile towels should be 
 placed about the arm and shoulder. 
 
 4. About 20 c.c. or more of sterile distilled water or normal salt solu- 
 tion are then poured into the cylinder, and th^ cock opened until all air 
 has been expelled from the tubing. The fluid, serum, or salvarsan is 
 then poured into the cylinder. It is a good practice to fitter the fluid 
 through several layers of sterile gauze, especially when salvarsan is being 
 injected, in order to remove any hits of glass or other foreign bodies that may 
 be present. 
 
694 PASSIVE IMMUNIZATION SER¥M THERAPY 
 
 5. An assistant holds the loaded cylinder and tubing; the operator 
 steadies the skin over a prominent vein and quickly inserts the needle. 
 A flow of blood indicates that the vein has bfeen penetrated. The tub- 
 ing is then quickly and carefully attached, the tourniquet released by 
 unfastening the hemostat, and the injection giyen. As a rule, an eleva- 
 tion of the cylinder of two or three feet is sufficient. If swelling occurs 
 about the site of puncture and the patient complains of pain, the in- 
 jection is entering the subcutaneous tissue; when this occurs, the pinch 
 cock should be closed and the needle removed. It is then necessary to 
 make the injection uito another vein or into the same vein at another 
 site. 
 
 Technic of Subdural Inocolation 
 
 In the treatment of epidemic cerebrospinal meningitis, influenzal 
 meningitis, and tetanus the specific serums afe administered subdurally 
 by means of a needle introduced in the lumbar region. Recentl}^ sub- 
 dural injections of salvarsanized serum and vteak solutions of salvarsan 
 itself have been advocated in the treatment of cerebrospinal syphilis, 
 tabes dorsalis, and paresis. Everj' practitioner should be prepared to 
 perform lumbar puncture for the purpose of securing cerebrospinal fluid 
 for making the Wassermann reaction and the bacteriologic, cytologic, 
 and chemical examinations, and the administration of serum is a rela- 
 tively simple matter when the puncture has been successfully made. 
 
 The technic of lumbar puncture for the purpose of securing fluid for 
 diagnosis is described on p. 37. But when administering serum, and 
 especially in the treatment of meningitis, the clinical condition of the 
 patient and the danger of sudden collapse render it advisable and neces- 
 sary that the inoculation be given with the patient lying on his side. 
 
 Methods. — Two methods are now being emjJloyed. The older method 
 consists in injecting the serum by means of a syringe, and the later one 
 is a method whereby the serum is allowed to flow in by gravity. 
 
 Not infrequently a patient will develop synaptoms of collapse during 
 a subdural injection, and these have been asoribed to undue pressure, 
 the injurious action of trikresol or other preservative upon the respira- 
 tory centers, too rapid injection, and the introduction of too large a 
 quantity of serum. It is now apparent that in the past too little atten- 
 tion has been paid to the patient while the injection was being made, and 
 serum has usually been administered according to more or less fixed and 
 arbitrary rules, instead of being guided by the clinical condition of the 
 patient. 
 
METHODS OF INOCULATION 695 
 
 If symptoms of collapse appear during a suBdural injection, they may 
 be relieved by allowing the fluid within the can'al to flow out again, and 
 this is best accomplished when the inoculation is given by the gravity 
 method. The latter method has been largely worked out and is highly 
 recommended by Sophian, these recommendiitions being based upon 
 his extensive experience in the recent Texas fepidemic of cerebrospinal 
 meningitis. It is also recommended by Flexner and the Hygienic 
 Laboratory, and is undoubtedly the method of'choice. 
 
 Blood-pressure as a Guide in Administering Serum Subdurally. 
 According to the older and customary method of injecting serum sub- 
 durally, fluid is permitted to flow from the needle until from 15 to 20 
 c.c. have been removed, and an equal quantity'of serum is then injected. 
 In severe cases, with thick plastic exudates, only a few cubic centimeters 
 of fluid may be withdrawn, and, indeed, no fluid at all may be secured. 
 To inject arbitrarily a fixed amount of serum under such conditions msiy 
 be highly dangerouss to the patient, on accoifnt of increased pressure. 
 On the other hand, when the flow is free, it may be dangerous to permit 
 the canal to drain until intraspinal pressure is reduced to the normal, a 
 fact indicated by the flow of a drop of fluid every three to five seconds. 
 
 With these considerations in mind, Sophian^ has studied the value 
 of cerebrospinal fluitl pressure and blood-pressure as controls on the 
 amount of fluid that may be safely withdrawn and on the amount of 
 serum that may be injected. Durins the study and treatment of 500 
 cases of epidemic cerebrospinal meningitis this last-named observer 
 found that the blood-pressure was a valuable guifle. Cerebrospinal 
 fluid pressure was found to be misleading, owirfg probably to a local dis- 
 tention of the sulmrachnoid space at the site of injection, which resulted 
 in readings that did not represent the true intfacranial pressure. 
 
 1. Usuallj^, upon the withdrawal of cerebrcjspinal fluid, a fall of 
 blood-pressure occurs. With the ordinary blood-pressure in an adult 
 patient — about 110 mm. of mercury — Sophian recommends stopping 
 the flow when there has been a drop in pressure of about 10 mm. of 
 mercury; in children, about 5 mm. In a few cases there is no change 
 in blood-pressure or even a slight rise; in these instances fluid may be 
 removed until the flow has diminished to the r^te of a drop ever}^ three 
 to five seconds. 
 
 2. With the injection of serum the blood-pressure drops still further. 
 Generally, the decrease in blood-pressure is proportional to the rapidity 
 with which the serum is injected and the amount injected. By the 
 
 'Epidemic; Cerebrospinal Meningitis, 1913, Mosby Co., St. Louis. 
 
696 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 gravity method, under ordinary conditions, at least ten minutes should 
 be consumed in administering 15 c.c. of serum. A total drop of 20 mm. 
 of mercury indicates that sufficient serum has been injected. If it is de- 
 sired to inject more, as in a severe case of meningitis; close watch should 
 be kept for other symptoms of collapse. 
 
 3. Usually, under these conditions, less serum is administered than 
 has been advocated heretofore. It is apparent that the more potent 
 the serum, the less bulk is required — and the bulk alone is an important 
 factor, for a large injection may so injure the patient as to counteract 
 any good that the serum may do. Unfortunately, there is no accurate 
 measure of the curative value of antimeningococcic serum. It is highly 
 desirable that a serum be as potent as possible, and the physician must 
 rely upon the reputation of the firm producing the serum. Efforts are 
 being made to concentrate these serums, much as antitoxin is concen- 
 trated, and this is an end very much to be desired. 
 
 4. Blood-pressure changes are not constant in the same patient upon 
 different occasions. The pressure should be taken, after each puncture 
 and inoculation, for the administration cannot be guided by observa- 
 tions made on a previous occasion. 
 
 Collapse during Subdural Inoculation. — Carter has shown, by ex- 
 periments on dogs, that the first mechanical effects of increased intra^ 
 spinal pressure were respiratory depression and marked cardiac inhibi- 
 tion. Sophian has found that similar effects may be produced during 
 subdural injections of scrum in the treatment of meningitis. 
 
 The syviptoms of collapse, such as stupor, superficial or deep, ir- 
 regular and slow respiration, and dilatation of the pupils, are fore- 
 shadowed by a marked drop in blood-pressure. The pulse may con- 
 tinue good or become slow and irregular. Incontinence of urine and 
 feces may occur. 
 
 The treatment consists primarily in discontinuing the injection. By 
 lowering the fuimel, fluid is allowed to flow from the spinal canal and 
 mix with the serum. If a syringe is being used, it should be detached 
 from the needle or gentle suction made. After a few minutes the symp- 
 toms may disappear and the inoculation may be cautiously resumed 
 until the desired amount of serum has been injected; otherwise the 
 needle should be withdrawn. 
 
 In addition to this procedure atropin and caffein may be administered 
 hypodermically in large doses, and artificial respiration resorted to if 
 necessary. It is well to have these drugs ready for injection before the 
 inoculation is begun, so that no time will be lost when they are needed. 
 
METHODS OF INOCULATION 697 
 
 Anesthesia for Subdural Inoculation. — There is no doubt but that 
 lumbar puncture and the subdural injection of fluid are painful, the 
 amount of pain depending to some extent upon the degree of meningitis, 
 the method of injection, and the skill of the operator. Severe cases of 
 meningitis that are stuporous or moribund may not evince any evidences 
 of added discomfort; less toxic and robust or nervous patients may, how- 
 ever, suffer considerably and prove difficult subjects for injection. 
 
 A local anesthetic is practically useless except for the mental effect 
 it. has upon the patient who may demand it. General anesthesia for 
 lumbar puncture in meningitis adds a considerable element of danger, 
 but if it is absolutely necessary, a few whiffs of ether or chloroform may 
 be given while the needle is being inserted. In giving subdural in- 
 jections in tetanus a general anesthetic is necessary. 
 
 Sophian has found that if water is given through a straw while per- 
 forming lumbar puncture patients will frequently drink large quantities 
 of it and keep very quiet. 
 
 Gravity Method. — The apparatus required is very simple, and con- 
 sists essentially of a proper needle and from 12 to 16 inches of soft- 
 rubber tubing attached to a container or funnel for serum and furnished 
 with a metal tip by which it is quickly and readily attached to the 
 needle. 
 
 Several manufacturers of biologic supplies are marketing antimenin- 
 gococcic serum in a special container, fashioned after that devised by 
 Sophian and Alexander, with the needle and tubing adapted for the 
 administration of the serum by the gravity method. Such an apparatus 
 is shown in Fig. 137. 
 
 The physician may, however, prepare an equally efficient apparatus, 
 similar to that shown in Fig. 137, which consists of the glass barrel of a 
 20 c.c. syringe attached to 16 inches of soft-rubber tubing fitted with a 
 metal tip that holds the needle firmly and snugly. The whole is steril- 
 ised by boiling, and any quantity of serum may be administered with it. 
 The apparatus is adapted for the administration of antimeningococcic 
 serum, tetanus antitoxin, influenza serum, salvarsanized serum, or any 
 other fluid, and has given uniform satisfaction,. 
 
 The needle should be from 10 to 11 cm. in length, with a wide, rather 
 than a narrow, lumen — about 1,5 to 2 mm. This is important in ad- 
 ministering serum to a case of meningitis, in which a needle with a nar- 
 row lumen may become plugged with exudate. The needle should be 
 fitted with a trocar. The tip should have a short bevel with a sharp 
 edge. 
 
698 
 
 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 Technic. — 1. The serum should be warmed to body temperature by 
 wrapping the sealed container of serum in towels wrung out of water 
 comfortably hot for the hands (about 42° C.)- Cold serum possibly 
 increases the pain caused by the injection, although the pressure upon 
 
 Fig. 137. — Outfit for Inth.^spinal Injection of Antimeningitis Serum by 
 
 Gravity (Sophian). 
 
 the sensitive nerve-roots is the chief source of pain and discomfort. Due 
 care must be exercised that the serum is not coagulated by too high 
 temperature. 
 
 2. The funnel or syringe barrel, tubing, and needle should be steril- 
 
METHODS OF INOCULATION 
 
 699 
 
 ized by boiling. The outfits furnished by the manufacturers are steril- 
 ized and ready for use. Two sterile graduated centrifuge tubes should 
 be on hand for collecting and measuring the spinal fluid. The apparatus 
 should be assembled and ready for injection, so that at the appointed 
 time the tubing may be attached to the needle and the inoculation given. 
 3. The patient should be placed on the left side, on the edge of a bed 
 or table. An assistant places the patient in such a manner as to arch 
 
 !"Wy«''72' fJfif^ 
 
 /:, 
 
 ..^«f J 
 
 Fig. 1.3S. — Intraspinal Injection by Ghavitt. 
 The line marks the crest of the iUum, and indicates the third lumbar interspace. 
 The needle has been inserted and cerebrospinal flnid withdrawn. Antimeningococcus 
 serum is being administered. The barrel of a 20 c.c. Rvcmd syringe is serving as a 
 funnel, and is attached to the needle by means of IS inches of soft-rubber tubing 
 furnished with a metal tip. The needle, as shown, is reduced to about four times its 
 actual size. This method may be used for making the subdural injection of tetanus 
 antitoxin and salvarsanized serum. 
 
 the back as much as possible. A second assistant takes blood-pressure 
 readings during the operation. 
 
 4. Towels wet with bichlorid are arranged about the site of inocula- 
 tion. It is well for the operator to locate the site of injection by pal- 
 pating the spinous processes and selecting the- widest interspace, which 
 is usually on a level with the crests of the ilia if the back is well arched. 
 The skin is then cleansed with soap, water, and alcohol, and bichlorid 
 solution or a coat of iodin applied. 
 
700 PASSIVE IMMUNIZATION SERTJM THERAPY 
 
 5. The operator must then choose between the median or lateral 
 route of puncture. The median is the easier, and should always be 
 adopted by the inexperienced operator. 
 
 Wash off the iodin with a pledget of cotton soaked in alcohol. Lo- 
 cate the chosen interspinous space, pressing well between the spines 
 with the left thumb or index-finger, and holdiiig the finger in place pass 
 the needle perpendicularly in the median line between the spines, or, 
 better still, at an angle of 45 degrees upward and inward. If an ob- 
 struction is felt, withdraw the needle slightly and pass it in a different 
 direction until it imparts a sense of "giving way," which indicates that 
 the subarachnoid space has been reached. Quincke has estimated the 
 depth of lumbar puncture in adults to be usually from 4 to 6 cm. ; in 
 large muscular men it is from 7 to 8 cm., and in fat persons, about 10 
 cm. 
 
 The needle should be inserted slowly and deliberately, rather than 
 quickly, as puncture of a bone is likely to be followed by a dull, aching 
 pain, and, indeed, the point of the needle may be bent or broken. 
 
 The fluid may fail to flow or flow very slowly. This may be due to 
 the presence of a thick exudate, impalement of a nerve filament, or ad- 
 hesions arising from a previous pimcture. The needle may be turned 
 gently or the trocar inserted to remove an obstruction, after which the 
 flow usually starts; if it does not do so, the needle may be withdrawn 
 slightly or cautiously inserted a little further. 
 
 6. Fluid is collected in the centrifuge tubes while blood-pressure 
 readings are being made. When the pressure drops 10 mm., or if the 
 flow is about a drop every thtee or five seconds, the tubing is coimected 
 and the serum injected verj' slowly. 
 
 As a general rule, as much fluid should be withdrawn as can be done 
 with safety, and the maximum dose of serum= given. When the flow is 
 scanty, a larger dose of serum may be given than counterbalances the 
 fluid removed, the injection being guided by the blood-pressure. When 
 the total drop reaches 20 mm. of mercury, the injection should be dis- 
 continued, or if continued, the patient should be watched closely for 
 other symptoms of collapse. 
 
 7. After the injection has been completed the needle is quickly with- 
 drawn and the wound covered with sterile gauze held in place by ad- 
 hesive straps. All iodin should be washed off with alcohol to avoid irri- 
 tation or an actual dermatitis. 
 
 Syringe Method. — 1. The technic is practically the same as that 
 just described, except that the injection is given with a syringe. 
 
METHODS OF INOCULATION 
 
 701 
 
 Manufacturing concerns market their products in syringes all ready 
 for injection. When injecting tetanus antitoxin, it is necessary to 
 empty the syringe into another sterile syringe, as shown in the accompany- 
 ing illustration (Fig. 139), which will fit an appropriate needle. Since 
 the plunger of the purchased syringe oftentimes adheres to the barrel 
 and renders the injection jerky and difficult, I frequently transfer the 
 serum to a sterile, all-glass syringe which I kndw will work smoothly and 
 satisfactorily. 
 
 FiQ. 139. — Intraspinal Injection bt Means or a Syringe. 
 The line indieates the crest of the ilium, and usually passes between the third 
 _and fourth lumbar vertebrae, which is the proper point for inserting the needle. The 
 site of injection has been painted with tincture of iodin after cleansing with soap, 
 hot water, and alcohol. An assistant holds the patient to prevent sudden jerking 
 and possible accident. 
 
 2. Lumbar puncture is performed as for the gravity method while 
 blood-pressure observations are being made. When sufficient fluid has 
 been removed, the loaded syringe is attached to the needle and the in- 
 jection slowly given. The physician is frequently tempted to inject 
 the serum and complete the operation quickly, but it is better to inject 
 It in amounts of 0.5 to 1 c.c. every half to one minute, being guided by 
 the blood-pressure readings and general condition of the patient. If 
 the pressure falls below 20 mm. of mercury or other symptoms of col- 
 
702 PASSIVE IMMUNIZATION SERTJM THERAPY 
 
 lapse appear, the fluid may be drained from the canal by gentle suction 
 with the piston. This is usually impossible when the manufacturers' 
 syringe is used. Otherwise the syringe is detached and the fluid col- 
 lected in tubes until the patient's condition itnproves and the injection 
 is resumed or the needle removed. 
 
 At times the patient is so restless that this slow method is not feas- 
 ible. In such instances the physician shouid make the injection as 
 slowly as possible, endeavoring to put into tlje canal as much serum as 
 fluid was removed or at least a reasonable amotmt. 
 
 Antitoxic Immunization 
 serum treatment of diphtheria 
 
 The discovery of diphtheria antitoxin and its use in the treatment 
 of this infection constitute one of the triumphs of modern medicine. 
 
 Twenty years ago diphtheria was one of the most dreaded of diseases, 
 accompanied ordinarily by a mortality of at least 30 per cent., while the 
 loss of life from the laryngeal form of the disease, particularly after 
 tracheotomy, was simply appalling. 
 
 Shortly after Roux and Yersin (1888) had demonstrated that the 
 symptoms of diphtheria were due largely to a soluble poison or toxin se- 
 creted by the bacilli, Ferran, and later Fraenkel and Brieger (1890), 
 undertook experiments in active immunization against diphtheria. 
 About the same time von Behring discovered the antitoxin, and in a 
 series of extensive researches with Wernicke he established experiment- 
 ally its prophylactic and therapeutic value in diphtheria. The first 
 attempt to apply this discovery to the cure of this infection of the human 
 being was made in von Bergmann's clinic (1891). The results, while 
 encouraging, were not altogether satisfactory, owing largely to the fact 
 that the serums were weak and the doses given too small. The dis- 
 covery, however, resulted in creating an extraordinary stimulus to re- 
 searches in immunity, and during the following two years more powerful 
 serums were prepared, so that in 1896 a marked drop in the mortality of 
 diphtheria was apparent in those places where the antitoxin was being used. 
 
 Since then diphtheria antitoxin has been the means of saving count- 
 less thousands of lives, and the treatment of diphtheria, instead of being 
 a reproach to medicine, has become the model of what the scientific 
 treatment of an infectious disease ought to Ije. Statistics and the in- 
 dividual experiences of those especially engaged in the treatment of 
 diphtheria show that when the antitoxin is used on the first day of the 
 
sekthm treatment of diphtheria 703 
 
 disease, practically no mortality occurs. Parents and guardians should 
 be taught this fact, and cautioned to seek medical advice promptly when- 
 ever a child complains of sore throat. While the use of diphtheria anti- 
 toxin is still decried and opposed by a few members of the medical pro- 
 fession, — and this is not to be wondered at when it is remembered that 
 cowpox vaccination still has its opponents,^i| is at least to be hoped 
 that no physician will deprive a patient suffering from diphtheria of the 
 benefits to be derived from the antitoxin treatment. Jn the absence of 
 special contraindications the refusal or neglect to use antitoxin in the treat- 
 ment of diphtheria would, in the opinion of mosi physicians, constitute an 
 act of criminal negligence and malpractice. 
 
 Preparation of Diphtheria Antitoxin. — The methods of preparing and stand- 
 ardizing diphtheria antitoxin are given in Chapter XIV. Briefly, these con- 
 sist in the preparation of a strong toxin by cultivating a virulent strain of the bacillus 
 in a suitable broth for ten days or two weeks; the culture is then passed through a 
 porcelain filter, which retains the bacilli, the filtrate beirig a strong solution of toxin. 
 This is standardized by the physiologic test of determining its action upon guinea- 
 pigs, the minimal lethal dose (M. L. D.) or toxin unit being the amount of toxin that 
 will cause the death of a 250-gram guinea-pig in four days. 
 
 Strong and healthy horses that have been proved by the tuberculin and mallein 
 tests to be free from tubercle or glanders, are then continuously immunized by a 
 series of injections of the toxin. The first doses are guarded by the simultaneous 
 injection of antitoxin, but after from four to six months the animals are able to tolerate 
 enormous quantities of pure toxin. The horses are then bled aseptically from a jugu- 
 lar vein, and the separated blood-serum, preserved With a small percentage of an 
 antiseptic, becomes the antitoxui of commerce. 
 
 Many manufacturing concerns market a concentrated diphtheria antitoxin 
 prepared by precipitating the globulin fraction of the raw serum with ammonium 
 sulphate, and redissolving it in a minimal quantity o{ salt solution. The globulins 
 carry with them most of the antitoxin, and in tliis inamier a serum may be concen- 
 trated so that a large number of units are contained in ^ small bulk of fluid, obviously 
 a most desirable feature in the treatment of diphtheria. Further than this, it has been 
 observed that these concentrated antitoxins are much less likely to produce serum 
 sickness, a train of symptoms due to substances present alike in normal and in 
 antitoxic horse serum, and independent of the antitoxin. 
 
 The antitoxic unit is the smallest amount of serum that will just neutralize 100 
 times the minimal lethal dose of toxin for a 2.50-gram guinea-pig. The adoption of 
 such a standard enables us to attain some accuracy in dosage. Before its introduction 
 antitoxin was given in so many cubic centimeters, jvst as antimeningococcus and 
 antistreptococcus serums are given today, but since some horses produce more 
 potent serums than others, the results were quite irregular. .\t the present time an 
 antitoxic serum is marketed according to the number of units it contains, irrespective 
 of the actual amount of serum, the constant endeavor being to produce as potent a 
 product as possible. 
 
 Since antitoxin gradually deteriorates with time,, physicians should carefully 
 observe the date printed upon each package of antitoxin, which is the time limit 
 calculated by the manufacturers beyond which they d6 not guarantee that the full 
 antitoxic strength is maintained. 
 
704 PASSIVE IMMUNIZATION SERIJM THERAPY 
 
 Nature of Diphtheria.^In the great majority of cases diphtheria is 
 a local infection of some portion of the upper respiratory tract. The 
 bacilli are usually inhaled, find lodgment upon a mucous membrane, 
 and secrete a toxin that produces necrosis of the cells of the mucosa and 
 effectually resists phagocytosis of the bacilli. From this area of infec- 
 tion, which now becomes a prolific source of toxin production, the toxin 
 or poison is absorbed by the body-fluids, and the resulting toxemia is 
 chiefly responsible for most of the symptoms of the disease. 
 
 Other microorganisms, such as staphylococci, pneumococci, and 
 streptococci, which may be unable to infect a healthy mucous membrane, 
 readily multiply in the necrotic tissue and add to the severity of the 
 local lesion, the lymphadenitis, and the toxemia. 
 
 Rarely the diphtheria bacilli gain access to the blood-stream. The 
 severity and danger of diphtheria are dependent primarily upon the 
 strength and amount of toxin produced by the bacilli, and secondarily 
 upon the size and location of the primary lesion and the amount of anti- 
 toxin present in the patient's blood. A lesioij in the larynx is far more 
 dangerous than one of equal size on a tonsil, because in the former the 
 edema and necrotic exudate obstruct the trachea and may produce 
 death by suffocation. On the other hand, the size of the local lesion alone 
 is not an indication of the severity of the infection, because virulent 
 bacilli in a small patch may produce more toxin than less virulent ones 
 in a larger area, and the degree of local tissue necrosis is not an absolute 
 indication of the toxicity of the soluble poison. Other things being 
 equal, the patient who has most antitoxin, either naturally or acquired 
 as the result of a previous injection with antitoxin or of an attack of 
 diphtheria, will present least evidences of toxemia, although the bacilli 
 causing the infection may be most virulent. For example, the highly 
 virulent strain of diphtheria bacillus used extensively in the past eighteen 
 years in the production of antitoxin was isolated by Park and Williams 
 from the throat of a patient presenting no clinical symptoms other than 
 redness of the fauces and slight toxemia. 
 
 The primary lesion of diphtheria is usually located in the throat 
 (tonsils, uvula, larynx), and frequently in the nose; more rarely the 
 ears, conjunctiva, vulva, prepuce, and wounds are the seats of primary 
 infection. 
 
 Treatment of Diphtheria. — If we were always certain of seeing our 
 patients on the first day of their illness, and if the disease could always 
 be diagnosed in this stage, the treatment of diphtheria would resolve 
 itself into an immediate dose of antitoxin and rest in bed for two or three 
 
SEKUM TREATMENT OF DIPHTHERIA 705 
 
 weeks. But patients freauently come under treatment comparatively 
 late in the disease, or the true nature of the condition may not be fully 
 diagnosed at first and treatment thus be delayed. Diphtheria is, there- 
 fore, still to be regarded as a dangerous infection, and while the proper 
 use of antitoxin constitutes the most important part of the treatment, 
 local applications, general constitutional measures, and the management 
 of the various conditions that may complicate the disease are all to be 
 considered. Here we will discuss only the serum treatment of the dis- 
 ease. 
 
 Bearing in mind the pathology of diphtheria,- serum treatment aims 
 to fulfil the following primary indications : 
 
 1. To neutralize all free toxin circulating in the body fluids, and 
 also to neutralize, as much as possible, the toxin that has already united 
 with the tissue-cells. 
 
 2. To cause the destruction or removal of the bacilli producing the 
 toxin as quickly as possible. 
 
 3. To furnish the patient with sufficient excess of antitoxin to neu- 
 tralize the toxin as rapidly as it is produced until the virulent bacilli 
 disappear. 
 
 Action of Diphtheria Antitoxin. — Diphtheria antitoxin best fulfils 
 these requirements. In fact, there are no substitutes. In former days 
 the powerful and irritant caustics and germicides that were freely ap- 
 plied to the throat, instead of limiting the disease and destroying the 
 bacilli, probably actually encouraged its extension by excoriating and 
 depressing the resistance of the surrounding mucous membranes. 
 
 1. The chief action of antitoxin is just what its name implies, namely, 
 a substance that neutralizes the toxin. This is regarded as a chemical 
 reaction analogous to the neutralization of an acicl by an alkali. When 
 the antitoxin molecule has united with the toxin molecule, it is believed 
 that the toxin is neutralized and that both are rendered inert. As the 
 result of experimental studies, however, we know that this union is not 
 always a firm one, and it is possible for the toxin to become dissociated 
 and attack body-cells or other molecules of antitoxin, thus explaining in 
 part the necessity for giving quite large doses of antitoxin — doses that 
 are out of all proportion to what we would expect to be necessary, 
 when considered weight for weight between g\iinea-pig and man, to 
 effect complete neutralization of the toxin. 
 
 It is reasonable to presume, and may be accepted as true, that a 
 stronger affinity exists between diphtheria toxin and antitoxin than be- 
 tween body-cells and toxin. Just as the union between this toxin and 
 45 
 
7D6 PASSIVE IMMUNIZATION — SERUM THEEAPY 
 
 its antitoxin is somewliat unstable, so, in like manner, it is probable that 
 the union of toxin and body-cells is not so firm but that it may, during 
 an early stage, be dissociated to some extent by the more attractive 
 antitoxin. This factor is to be borne in mind in the treatment of diph- 
 theria, and is an additional argument for the administration of large 
 doses of the serum. 
 
 2. Antitoxin pure and simple does not, however, constitute the 
 only factor of value in antidiphtheric serum. While the pure antitoxin 
 neutralizes the toxin, it has no injurious action on the bacilli themselves, 
 and, indeed, it is said that the bacilli may live and multiply in the pres- 
 ence of large amounts of antitoxin. How, tjicn, are we to explain the 
 gradual disappearance of the membranous exddate and the bacilli at the 
 local site of infection? It has been shown experimentally that virulent 
 diphtheria bacilli resist phagocytosis. This condition is probably due 
 to an actual leukotoxic action of the diphtheria toxin, aided by an ag- 
 gressin-like action of the toxin which neutralizes opsonin, and in this 
 manner prevents phagocytic activity. I iu: common with other ob- 
 servers, have shown that the antiserum as ordinarily produced neutral- 
 izes the antiphagocjrtic action of diphtheria bacilli, and enables the 
 polynuclear leukocytes to ingest them readily.^ AVhether this is brought 
 about through neutralization of the toxin by antitoxin, or is due to the 
 presence of an immune opsonin (bacteriotropin) that lowers the re- 
 sistance of the bacilli, or to the presence of an anti-aggressin that neu- 
 tralizes the intrinsic defensive mechanism of the bacilli and thus favors 
 phagocytosis, I am unable to state, but probably all three factors are 
 operative. 
 
 3. As ordinarily prepared, diphtheria antitoxin possesses little or no 
 bacteriolytic activity. I have found, however, that antitoxin will fix 
 complement with an antigen of diphtheria bacilli,^ indicating, therefore, 
 the presence of bacteriolytic amboceptors. Serums produced by im- 
 munizing horses with un filtered cultures of diphtheria bacilli instead of 
 with the filtered toxin alone have been advocated for the general and 
 the local treatment, in the hope of securing lysis of the infecting bacilli. 
 Certainly it would appear wise to raise the bacteriotropic and bacterio- 
 lytic content of an immune serum by injecting the horses occasionally 
 with unfiltered cultures, for it is highly probable that the action of the 
 antiserum depends not only upon an antitoxin, but also, to some extent 
 at least, upon a bacteriotropin and possibly a bacteriolysin. 
 
 Administration of Diphtheria Antitoxin. — Antitoxin is usually ad- 
 1 Jour. Med. Research, 1912, xxvi, 373. ' Joiif. Infect. Dis., 1912, xi, 44. 
 
SEEUM TKEATMENT OF DIPHTHEEIA 707 
 
 ininistered by subcutaneous injection into the> tissues of the back, ab- 
 Homen, or buttocks. Experimental studies have tended to show that 
 complete absorption does not occur until forty-eight hours after, al- 
 though it is common clinical experience to observe improvement take 
 place during the first twenty-four hours after injection. 
 
 As will be emphasized later, it is highly desirable arid necessary to get 
 antitoxin into the circulation as soon as possible after infection has occurred. 
 Usually this is best accomplished by giving antitoxin to every patient 
 even suspected of being diphtheric, and making the diagnosis afterward; 
 the next best method is to administer the antitoxin in such manner as to 
 favor quick absorption. For this reason intramuscular and intravenous 
 injection should be resorted to in all severe cases. As pointed out in 
 Chapter XXXI, the Schick reaction in diphtheria has indicated that 
 diphtheria toxin may be dissociated from tissue-cells by large doses of 
 antitoxin. Park and his associates have shown experimentally by this 
 reaction that 20,000 units of antitoxin given subcutaneously were neces- 
 sary to yield an effect equal to 1000 units given intravenously. 
 
 Intramuscular injections into the muscles of the buttocks are just as 
 readily given as subcutaneous injections, and are probably no more pain- 
 ful to the average patient. It insures quicker absorption, and may, 
 indeed, be adopted as a routine practice. 
 
 Intravenous injections are far more difficult, especially in children 
 with fat arms and feeble circulations. An ant3sthetic or ten minutes' 
 struggling may do the patient harm, and unless the injection can be 
 given with little disturbance and danger, the serum should be given by 
 intramuscular injection. Not infrequently, however, an intravenous 
 injection yields splendid results in severe and apparently hopeless cases, 
 and in older children and adults this route of administration should be 
 considered. 
 
 The syringe method is well adapted for the intravenous injection of 
 antitoxin, as the bulk method of serum is usually small, especially if a 
 concentrated antitoxin is being used. 
 
 The technic of these injections has previously been described. 
 
 Antitoxin has also been given by the mouth and even by rectal in- 
 jection. The presence of a preservative, usually phenol, renders the 
 oral administration objectionable. While a tiierapeutic effect may be 
 secured after large doses have been swallowed, there are veiy few oc- 
 casions when this should be the method of chcjice. 
 
 Importance of Early Treatment. — The mo.st important point to be ob- 
 served in the treatment of diphtheria is to give antitoxin at once. It may 
 
708 PASSIVE IMMUNIZATION SERTJM THERAPY 
 
 he fatal to wait for the result of a culture, except perhaps in the case of the 
 mildest of infections. In fact, the necessity for early administration of 
 antitoxin cannot he overestimated. When once suspicion is aroused, anti- 
 toxin should be given at once and the diagnosis may follow. A few hours 
 may make an enormous difference in tiie prognosis of any case, and wiiile 
 a dose of 2000 units of antitoxin may prove of the utmost value in 
 checking diphtheria, it will do no harm whatever in case the disease is 
 not present. 
 
 It is true that a physician will naturally hesitate to administer anti- 
 toxin unnecessarily; nevertheless, diphtheria is frequently a difficult 
 disease to diagnose clinically, and is quite likely to be mistaken for tonsil- 
 litis. For this reason many physicians prefer to wait for the result of a 
 culture, and this is proper, provided that the patient, especially in the 
 case of a child, is given the benefit of the doubt by receiving 2000 units 
 of antitoxin. It is to be emphasized that a single negative culture does not 
 exclude diphtheria. As ordinarily made, about 20 per cent, of primary 
 cultures from genuine cases of diphtheria fail to show the presence of 
 bacilli, whereas subsequent cultures will show them to be present in 
 large numbers. A primary negative culture is most likely to be obtained 
 from a patient having a heavy exudate, as the physician may rub lightly 
 over the membrane, culturing the microorganfsms of secondary infection 
 and overlooking the diphtheria bacilli in the dejjths of the membrane 
 adjacent to the diseased mucous membrane. To wait another twenty- 
 four hours for a second culture still further reduces the patient's chances 
 for recovery. In the vast majority of instance^, therefore, antitoxin should 
 he given at once and repeated as often as is necessary until the correct diag- 
 nosis is established, and not one hut at least two successive negative cultures 
 should be obtained before diphtheria is to be excluded with any reasonable 
 degree of safety. 
 
 The following table, compiled from the annual reports of the Phila- 
 delphia Hospital for Contagious Diseases, shows the decided influence 
 of early treatment upon the mortality of diphtheria. This table com- 
 prises cases of diphtheria alone, and does not include cases complicated 
 by other diseases, such as scarlet fever and : measles. It is worthy of 
 special notice that of 74^ cases treated with antitoxin on the first day of the 
 disease, not one died. Ker,i in an exceptionally rich experience, has 
 nevei- seen a fatal result occur in a case that developed in a hospital and 
 in which antitoxin was administered on the first day of the disease. 
 'Infectious Diseases, 1909, Oxford. Med. Press. 
 
SERUM TREATMENT OP DIPiItHERIA 
 
 709 
 
 TABLE 28.— MORTALITY OF DIPHTHERIA ACCORDING TO THE DAY 
 
 OF ADMISSION (WHICH ALSO INCLUDES THE TIME OF GIVING 
 
 THE ANTITOXIN) IN THE PHILADELPHIA HOSPITAL 
 
 FOR CONTAGIOUS DISEA*SES 
 
 
 Total 
 Number 
 OF Cases 
 
 MoTtTALiTT According to the Dat on which Antitoxin 
 WAS FiBST Injected 
 
 Average 
 
 Yeah 
 
 First 
 Day 
 
 Second 
 Day 
 
 Third 
 Day 
 
 Fourth 
 
 Day 
 
 Fifth 
 Day 
 
 Sixth 
 Day 
 
 After 
 Sixth 
 Day 
 
 in Diph- 
 
 THEHIA 
 
 Alone 
 
 1904 
 
 1905 
 
 1906-07 . . . 
 
 1908 
 
 1909 
 
 IfllO 
 
 1911 
 
 1912 
 
 1913 
 
 712 
 862 
 1239 
 1426 
 2153 
 1870 
 1895 
 1676 
 1273 
 
 
 
 
 
 
 
 
 
 3.92 
 
 4,09 
 4.43 
 3.45 
 6.62 
 4.61 
 5.50 
 6.91 
 2.92 
 6.51 
 
 13.72 
 
 9.22 
 12.90 
 4.7 
 7.13 
 5.13 
 9.41 
 6.33 
 6.52 
 
 17.54 
 
 16.66 
 
 10.85 
 
 10.60 
 
 10,72 
 
 8.41 
 
 7.12 
 
 8.0 
 
 6.87 
 
 14.75 
 
 13.04 
 
 13.08 
 
 11.71 
 
 9.35 
 
 13.74 
 
 11.04 
 
 9,53 
 
 4..76 
 
 19.44 
 
 9.52 
 
 29.41 
 
 21.43 
 
 7.25 
 
 6.04 
 
 7.22 
 
 14.97 
 
 12.5 
 
 14.2 
 
 12.94 
 
 22.89 
 
 6.09 
 
 23.96 
 
 13.33 
 
 8,33 
 
 2.66 
 
 9.63 
 
 18.8 
 
 10.81 
 10.09 
 8.70 
 8.55 
 6.60 
 6.42 
 6.86 
 6.02 
 7.63 
 
 Average. . . 
 
 13106 
 
 0.4 
 
 5.0 
 
 8.3 
 
 10,7 
 
 1L2 
 
 13,1 
 
 7,96 
 
 It is generally believed that the paralyses of diphtheria are due to a 
 toxone, and not to the true toxin. Ehrlich believes toxone to represent 
 a late secretory product of the diphtheria bacillus, whereas others regard 
 it as a modified toxin. It is certainly apparent, however, that the bacilli 
 should be gotten rid of as soon as possible, so as to eliminate the possi- 
 bility of toxone production. This is best accomplished by the early use 
 of large doses of antitoxin, aided possibly by judicious local treatment. 
 
 Dosage of Diphtheria Antitoxin. — While it? is now generally agreed 
 that the doses of from 100 to 200 units, such as were commonly given 
 during the early years of antitoxin therapy, were far too small, there is 
 still some difference of opinion regarding the proper doses to employ. 
 Since the severity of the disease varies so markedly, no hard and fast 
 rules can be given. The physician who has a clear idea of the nature 
 of diphtheria and of the action of antitoxin, and knows what to expect 
 of the latter in the treatment of the disease, should have no difficulty 
 in properly treating a case of diphtheria. 
 
 It is to be emphasized, however, that while^ antitoxin constitutes the 
 most important part of the treatment of diphtheria, it is not usually the 
 whole treatment. Absolute rest in bed, a genfjrous diet, combined with 
 the use of tonics and local applications, are all part of the treatment. 
 Special treatment of the laryngeal form of the disease and the treatment 
 of complications are matters of considerable importance that influence 
 the prognosis in a given case. I may mention, in passing, the value of 
 
710 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 continuous enteroclysis of normal salt solution during the early periods 
 of severe infections; this appears to dilute the toxins and aid in their 
 destruction and excretion. 
 
 In administering antitoxin the physician must be guided by the 
 clinical condition of the patient, as we have as yet no practical labora- 
 tory method for estimating the degree of the toxemia. Treatment may 
 be regarded as satisfactory when — 
 
 1. The local patch of exudate has ceased to spread and shows indica- 
 tions of disappearing. 
 
 2. The general condition of the patient is improved, i. e., the toxemia 
 is decreased, the pulse grows stronger and more regular, and the patient 
 feels more comfortable. 
 
 The temperature is not a reliable guide, for not infrequently in 
 severe infections it may be normal or subnorm"al throughout. 
 
 So long as the exudate shows no signs of loosening and disappearing, 
 but tends to spread, and so long as the general condition remains unim- 
 proved or grows worse, large amounts of antitoxin should be given. 
 No case should be regarded as hopeless until death supervenes. 
 
 Every case of diphtheria is to be treated individually, rather than by 
 any set rule. The amount of antitoxin given in the initial dose, and in 
 subsequent doses as well, is dependent on the following factors: 
 
 1. The situation and extent of the lesion. 
 
 2. The general condition of the patient. 
 
 3. The day of the disease. 
 
 4. The age of the patient. 
 
 1. The Situation and Extent of the Lesion. — In ordinary tonsillar 
 diphtheria in which there is a small patch on one tonsil and which is first 
 seen on the second day of the disease, the initikl dose should be at least 
 5000 units. When the exudate involves the; pillars of the fauces, the 
 uvula, the posterior pharyngeal wall, or is well forward on the palate, 
 this dose should be doubled, and 10,000 units be given. 
 
 Cases that present laryngeal symptoms in addition to faucial lesions 
 should never receive less than 12,000 units. In well-marked laryngeal 
 diphtheria with dyspnea and partial suffocation at least 20,000 units 
 should be given, preferably by intramuscular or intravenous injection. 
 
 In 7iasal diphtheria a distinction must be made between those cases 
 that exhibit merely a dirty, chronic discharge containing bacilli, in which 
 2000 units may suffice, and those that present an actual membrane ac- 
 companied by well-marked toxic symptoms, , when a large amount of 
 serum — at least 10,000 units — should be given. Owing to the ready 
 
SERtJM TREATMENT OP DIPHTHERIA 711 
 
 absorption of toxin by the nasal mucosa a small patch in the nose may be 
 accompanied by severe general toxemia and frequently requires ener- 
 getic treatment. 
 
 In diphtheria of the eye, vulva, or of wounds relatively large doses 
 should be given — at least from 10,000 to 20,000 units. 
 
 All these doses must be regarded merely as suggestions for the initial 
 dose in cases seen on about the second day of the disease. Physicians 
 with extensive hospital experience, for example. Woody and McCullom, 
 generally favor large doses, and while in private practice the question 
 of expense and economy may be a factor, the physician will be wise to 
 err on the side of safety and give a little too much serum rather than too 
 little, especially in the first dose, when so much depends upon how soon 
 antitoxin is introduced into the body-fluids. 
 
 2. The general condition of the patient, or the effect which the tox- 
 emia will have on the patient, is highly important in estimating the 
 dosage of antitoxin. A patient who is pale, drowsy, prostrated, and 
 has a weak and irregular pulse; who has large masses of glands around 
 the neck, or who has marked albuminuria, will require a much larger 
 dose than one who presents none of these signs. Two persons of about 
 the same age and suffering from the same lesions may show very differ- 
 ent degrees of toxemia. In the severe cases we must administer the 
 maximum dose and repeat it at suitable intervals until an effect is pro- 
 duced. 
 
 3. The day of illness on which the patient comes under observation 
 is important in deciding the initial dose of antitoxin. For corresponding 
 tonsillar lesions a dose of 2000 units on the first day may do more good 
 than 5000 units given on the fourth day. Ker gives second-day cases 
 of purely tonsillar diphtheria 3000 units, and adds an additional 1000 on 
 the following day. 
 
 4. If the age of the patient exerts any influence at all on dosage, it in- 
 dicates that more antitoxin should be given to children than to adults 
 with corresponding lesions, as the disease is more fatal in children. So 
 far as infants are concerned, Ker seldom gives =more than 4000 units at a 
 single dose, which should be an adequate amount when we consider the 
 small size of the patient. Children over one year of age may be given 
 from 5000 to 10,000 or more units, depending upon the location of the 
 lesion and the degree of toxemia. 
 
 Repeating the Dose. — Whether or not subsequent doses of antitoxin 
 will be required is dependent upon the circumstances of the individual 
 case. In ordinary cases if on the day after treatment is commenced 
 
712 PASSIVE IMMUNIZATION — SERIIM THERAPY 
 
 there is no diminution in the amount of membrane visible and the gen- 
 eral symptoms have not improved, the dose shoukl be repeated. If the 
 membrane has spread and the toxemia is woi'se, the second dose should 
 be larger than the first. In septic cases the second dose may be given 
 in from six to ten hours after the first. If the symptoms are less urgent, 
 the interval may be extended to twelve, but should never exceed twenty- 
 four hours. 
 
 As to the total amount of serum to he administered, continued injec- 
 tions at relatively short intervals are required until improvement has 
 taken place. So long as membrane is present and the patient is toxic 
 it is probably worth while to push the treatment unless these show a ten- 
 dency to clear away. Time must be allowed for absorption to take place, 
 and the serum should not be pushed so far as to be wasted, and, quite 
 possibly, excreted unchanged. The remedy is expensive, especially in 
 private practice, and it is obviously desirable to have due regard for 
 economy. While, as previously mentioned, physicians of such wide ex- 
 perience as McCuUom, of Boston, and Woody, of Philadelphia, fre- 
 quently give 200,000 or more units in severe cases of diphtheria, others, 
 e. g., Ker, of Edinburgh, have never given mbre than 64,000 units to a 
 single patient, and, indeed, several of my colleagues of wide experience 
 claim that they have secured excellent results in severe infections with 
 doses that seldom exceeded 10,000 units. 
 
 Treatment of Relapses. — Occasionally, after a patient has recovered 
 from an attack of diphtheria the infection rpcurs after several weeks. 
 It is in such cases that the physician hesitates to administer antitoxin, 
 on account of the discomforts occasioned by serum sickness. It is true 
 that serum siclmess is more likely to follow in these than in primary 
 cases, and the very profuse and itchy eruption, joint pains, and fever 
 do indeed render the patient quite uncomfortable. Since a relapse is 
 usually, although not always, comparatively mild, the serum may be 
 dispensed with if there is no involvement of the larynx and if there is 
 not much evidence of toxemia; otherwise full doses of antitoxin should 
 be given without hesitation. The subcutaneous injection of 0.5 c.c. 
 of the serum two or three hours before the ma^in dose is given may pos- 
 sibly produce a condition of anti-anaphylaxis and ward off the more 
 dangerous sjTnptoras. If respiratory difficulties should follow, a re- 
 injection of serum, atropin, and caffein should be administered hypo- 
 dermically. 
 
 Antitoxin Sequeloe. — A certain percentage of cases will present a 
 group of symptoms that constitute the condition known as serum sick- 
 
SERUM TREATMENT OF DIPHTHERIA 713 
 
 ness, occurring at vaiying times following the administration of anti- 
 toxin. This condition has been shown to be due to certain constituents 
 of horse serum other than the antitoxic antibodies. It is noteworthy 
 that the serum of one horse may cause more serum sickness than that of 
 another; in general, concentrated antitoxins produce fewer cases than 
 do raw serum. 
 
 This condition is characterized by the development of a rash (urti- 
 carial, multiform, or scarlatiniform), mild fever, joint pains, and possi- 
 bly adenitis. The scarlatiniform rash may be extremely difficult to 
 differentiate from that of true scarlatina, especially in the wards of a 
 diphtheria hospital, where outbreaks of scarlet fever are not imcommon. 
 
 This subject has been considered in greater detail in Chapter 
 XX^TII on Anaphylaxis. It may be stated here that while the patient 
 is decidedly uncomfortable, and even quite sick, for several daj's, serum 
 sickness is not a dangerous condition; the treatment is largely s}-mp- 
 tomatic and palliative. 
 
 Value of Diphtheria Antitoxin. — At the present day it seems hardly 
 necessary to introduce elaborate statistics to prove the value of anti- 
 toxin in the prophylaxis and treatment of diphtheria. 
 
 1. It is generally admitted that most of thfe reduction in the mortality 
 of diphtheria cannot be attributed solely to the use of antitoxin, for 
 unquestionably bacteriologic diagnosis has permitted the inclusion, in 
 our statistics, of a certain number of cases that, twenty years ago, would 
 not have been classed as diphtheria. Generally speaking, however, the 
 mortality of diphtheria, considering aU types of infection coming under 
 observation at varying intervals after the disease has developed, is at 
 least five times less under antitoxin treatment tKan when it is treated without 
 antitoxin. This proportion is true, wheth^ we compare the general 
 mortaHty before 1896 with the present rate, or whether we take a large 
 city and compare the mortalitj- imder both forms of treatment for a 
 single or for several years. For example, in Philadelphia the mortality 
 rates per 100,000 of population for the five years preceding the use of 
 antitoxin were as follows: 
 
 1891 127.4 
 
 1S02 156.3 
 
 1S9.3 10.3.9 
 
 1S;94 122.5 
 
 1S95 115.9 
 
 In five years following the general use t)f antitoxin the mortality 
 rates per 100,000 population were as follows: 
 
714 PASfSlAE IMMUNIZATION SERWJW THERAPY 
 
 190G 37.78 
 
 1907 _ 34.60 
 
 1908 33.35 
 
 1909 33.6 
 
 1910 31.7 
 
 In Philadelphia, during the years 1909-10 and 1911, the average mor- 
 tality of diphtheria treated with antitoxin in the Philadelphia Hospital 
 for Contagious Diseases was 9.9 per cent., and in the private practice 
 of physicians, 13.07 per cent. In contrast to these figures is the mor- 
 tality of 40.34 per cent, in the private practice of those physicians 
 (fortunately few) who refused to give antitoxin or in those families op- 
 posed to its use. 
 
 From Table 28 it will be seen that the average mortality in 13,106 
 cases of pure diphtheria treated with antitoxin in the Philadelphia Hos- 
 pital for Contagious Diseases during the pasfe ten years was only 7.96 
 per cent. As stated elsewhere, when this is compared with the average 
 mortality of about 41 per cent, when no antitoxin was used, it is not diffi- 
 cult to appreciate the therapeutic value of the remedy. 
 
 2. As was previously stated, it is also worthy of note that there is 
 practically no mortality in diphtheria cases receiving antitoxin on the 
 first day of illness. During nine consecutive years {1904--1913), covering 
 the treatment of 741 such cases in the Philadelphia Hospital for Contagious 
 Diseases, not a single death occurred. During 1913 two of the 51 first- 
 day cases died. 
 
 It wiU also be noted from Table 28 that the patient's chance for re- 
 covery grows steadily less with each day that the administration of 
 serum is delaj-cd, and this should be evidence enough to convince any 
 right-minded person that we possess in antitpxin a remarkable remedy 
 for the treatment of diphtheria. 
 
 3. The influence of antitoxin is also noted- in the mortality of laryn- 
 geal diphtheria. While the mortality in this„condition is still high, ow- 
 ing to the frequency and dangers of bronchopneumonia and the necessity 
 for operative measures, it has been reduced at least one-half since anti- 
 toxin came into general use. Prior to 1896 the mortality was at least 
 70 per cent,; since then it has been reduced Ijo 35 per cent, or less. As 
 shown in the following table, of 1207 cases treated in the Philadelphia 
 Hospital for Contagious Diseases, the average mortality was 35.6 per 
 cent. 
 
SBRXJM TREATMENT OF DIPHTBTEEIA 
 
 715 
 
 TABLE 29— MORTALITY OF LARYNGEAL DIPHTHERL^ (INTUBA- 
 TION CASES) IN THE PHILADELPHIA HOSPITAL FOR 
 CONTAGIOUS DISEASES 
 
 (This table excludes those patients dying in the ambulance and moribund 
 cases dying within twenty-four hours.) 
 
 
 Total 
 
 NtJMBER 
 OF 
 
 Mohtalitt According to the Day upon 
 TiON WAS Performed 
 
 WHICH 
 
 NTUBA- 
 
 Average 
 Mortality 
 
 Yeah 
 
 
 
 
 
 
 
 After 
 Sixth 
 Day 
 
 
 Cases 
 
 First 
 Day 
 
 Second 
 Day 
 
 Third 
 Day 
 
 Fourth 
 Day 
 
 Fifth 
 Day 
 
 Sixth 
 Day 
 
 
 1903 
 
 67 
 
 66.6 
 
 25.3 
 
 21.8 
 
 30.9 
 
 22.5 
 
 
 45.0 
 
 26.60 
 
 1904 
 
 125 
 
 
 39.20 
 
 29.03 
 
 73.68 
 
 33.33 
 
 70.0 
 
 36.84 
 
 39.20 
 
 1905 
 
 136 
 
 
 30.76 
 
 39.39 
 
 50.0 
 
 21.42 
 
 16.66 
 
 42.30 
 
 36.76 
 
 1906-07... 
 
 72 
 
 
 50.0 
 
 55.0 
 
 40.74 
 
 45.45 
 
 71.92 
 
 28..57 
 
 49.29 
 
 1908 
 
 162 
 
 
 29.26 
 
 50.0 
 
 33.33 
 
 30.76 
 
 9.09 
 
 38.88 
 
 33.95 
 
 1909 
 
 183 
 
 
 50.0 
 
 44.18 
 
 38.88 
 
 23.81 
 
 53.84 
 
 40.0 
 
 42.62 
 
 1910 
 
 89 
 
 100.0 
 
 55.0 
 
 33.33 
 
 28.58 
 
 50.0 
 
 
 36.46 
 
 39.34 
 
 1911 
 
 104 
 
 50.0 
 
 76.66 
 
 55.55 
 
 62.63 
 
 36.36 
 
 
 27.27 
 
 54.80 
 
 1912 
 
 133 
 
 30.0 
 
 58.33 
 
 41.93 
 
 48.27 
 
 50.0 
 
 63.67 
 
 50.0 
 
 48.87 
 
 1913 
 
 136 
 
 100.0 
 
 65.91 
 
 63.64 
 
 35.29 
 
 25.0 
 
 47.82 
 
 68.0 
 
 58.82 
 
 Average. . . 
 
 12C7 
 
 
 
 
 
 
 
 
 35.6 
 
 Formerly when a child contracted diphtheria the parents were 
 warned of the likelihood and danger of the infection involving the larynx 
 and trachea; nowadays this possibility is quite remote. 
 
 4. Finally the claim of the opponents of the serum therapy of diph- 
 theria that antitoxin increases the percentage of paralyses is without 
 foundation. While it is true that this percentage is somewhat higher 
 than was noted in former years, this increase is tq be explained by the 
 fact that antitoxin saves a larger number of sevfere cases long enough 
 for them to manifest paralyses, and, second, by the greater attention 
 that has recently been directed to its milder forms. Since diphtheric 
 paralysis is regarded as caused by toxone or a later secondary toxic 
 product of the bacilli, the indications are to rid the patient of the bacilli 
 as quickly as possible, and this is best and most siirely accomplished by 
 the proper administration of antitoxin. 
 
 Prophylactic Immunization Against Diphtheria 
 
 The subcutaneous administration of relatively small doses of anti- 
 toxin will usually confer a passive immunity against diphtheria lasting 
 from two to four weeks. 
 
 The object is to introduce antibodies (antitoxin and opsonin) into the 
 body-fluids in order that they may neutralize the toxin as rapidly as it 
 IS produced, aid in the destruction of the bacilli, and thus protect the 
 
716 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 individual in case virulent bacilli should be inspired or otherwise gain 
 access to the tissues. 
 
 The doses advised are relatively small, and the injection does not 
 usually produce any discomfort other than soreness about the site of in- 
 oculation. For infants under one year of age 500 units suffice; for 
 older children and adults from 1000 to 1500 uiiits should be given. 
 
 The duration of this passive immunity is relatively short, owing to the 
 fact that the antitoxin is eliminated rapidly, as it is part and parcel of a 
 foreign serum that tends to be excreted or destroyed soon after its intro- 
 duction into the body. It will endure, however, for at least two weeks, 
 and frequently longer. Since the incubatio'h period of diphtheria is 
 only a matter of a few days, this suffices, in the majority of instances, to 
 protect the individual. 
 
 The indications are to immunize all persons who have come in inti- 
 mate contact with a case of diphtheria. It is especially valuable in 
 families and small communities, such as go to make up hospital wards 
 and asylums. The physician who is attending a case of diphtheria in 
 a private home should urge immunization upon all members of the house- 
 hold. 
 
 The immediate results pxe usually good. The main disadvantage is 
 the short duration of the immunity, so that no matter how faithfully it 
 is carried out, persons do not remain immune for long periods of time, 
 and accordingly the total morbidity of the disease is not influenced to 
 any extent. In homes from which the case of diphtheria is promptly 
 removed to a special contagious hospital and in which the remaining 
 members are promptly immunized the percentage of secondary cases 
 is practically nil. Of 6772 patients who were removed to the Phila- 
 delphia Hospital for Contagious Diseases, the remaining members of 
 the family not being immunized, secondary cases developed in 164 per- 
 sons, or in 2.4 per cent. Of 4063 cases of diphtheria treated at home with 
 antitoxin, the other members of the family not being immimized, sec- 
 ondary cases developed in 219 persons, or in 5.3 per cent. Of 639 diph- 
 theric patients treated at home who did not rdceive antitoxin and where 
 immunization was not practised, secondary cases developed in 151 
 persons, or 23.6 per cent. These figures, compiled by Dr. A. A. Cairns, 
 chief medical inspector of Philadelphia, and taken from the annual re- 
 ports of the Philadelphia Bm'eau of Health for the years of 1909, 1910, 
 and 1911, show that the best results are obtained when the diphtheric 
 patient is promptly removed to a special hospital and the remaining 
 members of the household are immunized. iWen when the patient is 
 
SERUM TREATMENT OF DIPHTHERIA 717 
 
 removed promptly there is some danger of other persons having been 
 infected, and immunization should, therefore, always be promptly prac- 
 tised. TMien the patient is treated at home, other members of the 
 household, even if immunized, are liable to develop the infection, prob- 
 ably owing to the fact that the patient harbors virulent bacilli for varA'- 
 ing periods of time after the passive immunity in other persons has passed 
 away and the quarantine is broken. Certainly in those homes where 
 antitoxin is not used either for therapeutic or for prophylactic purposes, 
 the percentage of secondary infections is so high as to leave no doubt 
 as to the value of antitoxin. 
 
 In this connection I may mention the desirabihty of using an anti- 
 toxin prepared by immunization of cattle for* the general purpose of 
 prophylaxis, and especiallj^ for the treatment of those persons who are 
 hj^persensitive to horse serum. In these cases horse antitoxin could be 
 used later if a person contracted diphtheria without danger of anaphy- 
 laxis. 
 
 Behring's Method of Immvmization against Diphtheria. — Owing to 
 the fact that the antibodies produced through the activities of our o-mi 
 body-cells (active immunization) persist for longer periods of time than 
 those that are introduced passively (passive immunization), Behring and 
 his assistants have been working upon a method of active immuniza- 
 tion in diphtheria whereby our own bodj'-cells are to be stimulated to 
 produce our own antitoxin in sufficient amounts to protect us against the 
 disease. It has long been kno'mi that more or less balanced mixtures 
 of this kind produce inmiunity in animals, and in 1907 Theobald Smith'^ 
 suggested that it might be possible to employ this method for the pur- 
 pose of producing immunity in man. Subsequentlj- Smith^ studied the 
 effects of injections of neutral mixtures in guinea-pigs and horses, and 
 again pointed out the applicability of the method to human beings 
 
 Active immunization in diphtheria could probabl}^ be accomplished 
 by the administration of minute and increasing doses of toxin, but there 
 would be some danger of producing an acute toxemia or paralysis, and 
 the process maj' require so much time as to be useless in the presence of 
 epidemics. 
 
 Behring's method, according to his report read before the German 
 Convention on International Medicine in 1913, is based upon the prin- 
 ciple that the union of toxin and antitoxin is not stable, and when a 
 neutral mixture of the Uxo is injected into animals, sufficient toxin be- 
 
 ' Jour. Med. Research, 1907, xvi, 359. 
 
 -Jour. Exper. Med., 1909, xi, 241; Jour. Med. Re'search, 1910, xxiii, 433. 
 
718 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 comes dissociated to unite with body-cells and' stimulate the production 
 of antitoxin. 
 
 The mixture of toxin, and antitoxin is knbVn as T.-A., and several 
 preparations are being used; of these, T.-A. 8 possesses the lowest 
 toxicity, and has a value equivalent to that of the standard preparation, 
 M. M. 1, while T.-A. 7 is ten times and T.-A. 6 twenty times as strong 
 in its toxic and immunizing power for man. 
 
 Before using it in human practice, Behring very carefully tested his 
 mixtures on lower animals, and every new lot ai T.-A. is carefully tested 
 by the same means to make sure that it does not contain a trace of the 
 paralyzing element of the toxin, that its toxic power is exactly determined 
 by testing it on guinea-pigs, and finally that the mixture be known, by 
 trial on horses, to be capable of producing antitoxin. Further, Behring 
 requires the testing of every new lot by his standard preparation M. M.l, 
 which has retained its toxic and immunizing ^properties for over a year 
 unchanged. 
 
 The method has now been used for immanizing a large number of 
 persons, chiefly under the supervision of von Behring and his assistants.' 
 The natural antitoxin content of the blood is determined, usually by 
 the intracutaneous method on the guinea-pig, before and after immuni- 
 zation, and has shown uniformly a considerable increase, which persists 
 over many months. 
 
 Local and general reactions have been observed, especially in older 
 children and adults with doses intended for the new-born; this fact is 
 explained on the assumption of a specific sensitization in consequence 
 of the previous introduction of diphtheria bacilli (carriers), which latter 
 render the individuals hypersensitive to the T.-A. Reactions that are 
 regarded as non-specific have been observed in tuberculous and scrofu- 
 lous persons, and for the present von Behring prefers that the use of the 
 prophylactic in such persons, as well as in atrophic infants and infants 
 less than nine months old, be regarded as contraindicated. 
 
 The fear expressed by some that the prophylactic is contraindicated 
 in those persons who harbor diphtheria bacilli for fear of producing the 
 disease during temporary depression of the defensive mechanism has 
 been finally dissipated as the result of practical experience. Not one 
 of the numerous bacillus-carriers that have been injected with T.-A. 
 have sickened with diphtheria. Whether or.not the active immuniza- 
 
 iSee Semaine Medicale, 1913, xxjdii, No. 18; Berl. klin. Woohenschr., 1914, 
 li, 917; Therap. Monatssch., 1913, xxvii, No. 11. 
 
SERUM TREATMENT OP TETANUS 719 
 
 tion with T.-A. will help them to get rid of the bacilli is still an open 
 question. 
 
 The subcutaneous injection is recommended as the best method of 
 administration. There can be no doubt that, in many cases, a single 
 injection produces sufficient protection. Such persons are, as a rule, 
 those who have already been sensitized by diphtheria bacilli. For the 
 ordinary run of cases at least two injections should be given. The first 
 injection then plays the part of a sensitizer. Experience shows that 
 sensitization occurs after from ten to fourteen days, which makes it 
 necessary that the second injection should not be given until after an 
 interval of not less than ten days. 
 
 In this country the subject has been studied by William H. Park and 
 his associates, who found that this form of active immunization gave 
 rise to decided antitoxin production in 22 per cent, of susceptible per- 
 sons. The interval between vaccination and the development of im- 
 munity was generally long — as a rule, not less than two weeks. Under 
 conditions of exposure about 20 per cent, of those who failed to respond 
 were found to develop clinical diphtheria. 
 
 The remedy has not been used sufficiently often to enable us to ex- 
 press an opinion as to its value. Behring beheves that its proper use 
 may thoroughly eradicate diphtheria. Obviously, its preparation must 
 be very carefully controlled, and for the present it should be used only 
 in institutions where thorough studies of the blood of patients may be 
 made before and after immunization. 
 
 The Schick Test. — ThLs oonaists of the iMrar.Mnn.eous injection of a quantity 
 of diphtheria toxin equal to one-fiftieth of the minimal lethal dose. I dilute this in 
 such a manner that this amount is contained in 0.0.5 c.c. According to Schick, one- 
 thirtieth of a unit or more antitoxin per cubic centimeter of blood is sufficient to 
 neutralize this amount of toxin. If the patient has less than this quantity of anti- 
 toxin, the toxin is not neutrahzed and acts as a local irritant, producing an area of 
 superficial inflammation. Therefore, tin's test is a measure of iiiunwdln in diphtheria 
 and is advocated for testing the members of families and institutions, where it prob- 
 ably suffices to immunize with antitoxin those who react positively. (See p. 609.) 
 
 SERUM TREATMENT OF TETANUS 
 Tetanus antitoxin was discovered by Behring and Kitasato in 1892. 
 Since then it has proved of great value in the prevention of lockjaw. 
 While, in general, authoritative opinions regarding its curative value 
 vary, the statistics and the individual experience of many investigators 
 of more recent years show quite conclusively that tetanus antitoxin does 
 possess curative value, and is of distinct aid in the treatment of tetanus, 
 
720 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 especially when the nature of the disease is understood and the serum is 
 administered accordingly. 
 
 Preparation and Standardization of Tetanus Antitoxin. — This technio has 
 been described in Chapter XIV. Briefly, it consists in immunizing the horse 
 with gradually increasing doses of tetanus toxin over a period of several months, 
 until the blood of the animal contains the antitoxin in sufficient quantities for thera- 
 peutic use. The animal is then bled under aseptic precautions, and the serum, with 
 the addition of a small amount of preservative, constitutes the antitoxin of commerce. 
 Several manufacturers concentrate the antitoxin in the same manner as diphtheria 
 antitoxin is concentrated. In view of the very large doses required in the treatment 
 of tetanus this is quite desirable. 
 
 The American unit is defined as the amount of antitoxin required just to neu- 
 tralize 1000 fatal doses of tetanus toxin for a 350-gram guinea-pig. The United 
 States Government has adopted this unit, and supplies the different producers with 
 standardized to.xin for testing the antitoxin. 
 
 Action of Tetanus Toxin. — It may be well to recall briefly the main 
 features concerning the pathogenesis of tetanus, as successful treatment 
 depends upon a thorough understanding of these principles. 
 
 1. Tetanus is a local infection accompanied and characterized by a 
 general toxemia. The bacilli and spores nevw gain access to the blood, 
 and are never distributed through the tissues and internal organs, but 
 reside at the local site of infection, where they produce a powerful toxin, 
 which, when absorbed, is responsible for the rnain lesions and symptoms 
 of the infection. Therefore while the blood, of the tetanus patient is 
 sterile, it usually contains the toxin. Neisser has produced tetanus in 
 mice by giving them subcutaneous injections' of the blood of a tetanus 
 patient. 
 
 2. Tetanus toxin has a strong affinity for nerve tissue, and this con- 
 stitutes the most important feature in the pathogenesis of the disease. 
 The toxin is rapidly absorbed from the locaf site of infection into the 
 blood and lymph-streams, where it is distributed to other muscles and 
 reaches the central nervous system indirectly through absorption by 
 the end-plates of motor nerves. As expected, absorption is most hkely 
 to occur along the motor nerves supplying the parts injured, and for this 
 reason the muscles and nerves should be infiltrated with antitoxin as 
 soon as possible after an injury has been received. 
 
 According to Meyer and Ransom, Marie and Morax, absorption 
 occurs along the axis-cylinders of motor nerves, the intramuscular end- 
 ings of which the toxin penetrates. The experiments of Field, Cerno- 
 vodeanu, and Henni indicate, however, that the toxins are ahswhed by 
 way of the lymphatics of the nerves, and not byway of the axis-cylinders; 
 the latter view is now most generally accepted. 
 
SEETJM TREATMENT OF TETANUS 721 
 
 Even though the toxin gains entrance to the blood, it cannot injure 
 the motor nerve tissue directly, as, for example, by means of the blood- 
 vessels supplying the central nervous system. As was previously stated, 
 the toxin in the blood and lymph channels may reach the central nervous 
 system only in an indirect manner, through the end-plates or Ijnoaphatics 
 of other motor nerves. 
 
 Ascending centripetally along the motor plates and lymphatics, the 
 poison reaches the motor spinal ganglia on the side inoculated; it then 
 affects the ganglia on the opposite side, making them hypersensitive. 
 The visible result of this hypersensitiveness is the highly increased muscle 
 tonus — i. e., rigidity. If the supply continues; the toxin next affects 
 the nearest sensory apparatus: there is an increase in the reflexes, but 
 only when the affected portion is irritated. In the further course of the 
 poisoning the toxin as it ascends continues to affect more and more 
 motor centers and also the neighboring sensory apparatus. This leads 
 to spasm of all the striated muscles and general reflex tetanus (Park). 
 
 3. Regardless of the severity of the infection, there is always an in- 
 cubation period in tetanus during which the bacilli multiply and produce 
 toxin which is traveling toward the tissues of the central nervous system. 
 Antitoxin in sufficient amount will neutralize the toxin as quickly as it is 
 prodiuxd, and thus protect the infected individual until the leukocytes and 
 other body-cells have destroyed the bacilli and spores. 
 • In general terms, the severer the wound and the heavier the infec- 
 tion, the shorter will be the incubation period and the higher the mortal- 
 ity. In acute tetanus the incubation period is less than ten days; in 
 chronic tetanus this period is much longer and more variable. 
 
 The toxin is produced, and may he absorbed during or at least soon after 
 the first twenty-four hours following infection; this explains the necessity 
 for administering antitoxin as soon as possible after the injury has been 
 received. 
 
 Action of Tetanus Antitoxin. — 1. Tetanus antitoxin will neutralize 
 free toxin in a chemical manner similar in some respects to that by which 
 neutralization of an acid by an alkali is effected. It is generally believed 
 that as soon as the molecule of antitoxin has become united with a mol- 
 ecule of toxin, the latter is rendered inert. It may be possible, however, 
 for the toxin molecule to become dissociated and attack nerve-cells or 
 other molecules of antitoxin; this is one reason for the necessity of giv- 
 ing large doses of antitoxin in order than an excess may be at hand. 
 
 2. When tetanus toxin has once united with nerve-ceUs, it is difficult 
 or impossible for the antitoxin to effect its neutralization. Hence the 
 46 
 
722 PASSIVE IMMUNIZATION — SEEUM THERAPY 
 
 greatest value of the antitoxin lies in prophylaxis; when properly ad- 
 ministered, however, it is possible for the antitoxin to aid in the cure of 
 tetanus, and its use should never be omitted in the treatment of any 
 case. 
 
 The value of antitoxin in the treatment of tetanus is probably de- 
 pendent upon the following two factors: (1)= Neutralization of all free 
 toxin as quickly as it is secreted and before it is absorbed by the nervous 
 tissue; (2) actual dissociation or neutralization of some of the toxin 
 ".loosely united" with nerve-cells or suspended in the lymph after it 
 has left the capillaries and before it is taken up by the nerve-cells. 
 
 3. Aside from its chief antitoxic action, an.ti-tetanus serum probably 
 contains anti-aggressins or bacteriotropins that aid phagocytosis by 
 overcoming their repelling or negatively chemotactic influence. This 
 may, however, be accomplished by simple neutralization of the toxins, 
 which impairs their leukotoxic action sufficiently to permit living leuko- 
 cytes to engulf and destroy the bacilli. 
 
 Methods of Administering Tetanus Antitoxin. — Recent investiga- 
 tions and case reports show quite conclusively that in the treatment of 
 tetanus as much depends upon the method of administering antitoxin 
 as upon the quantity administered. 
 
 1. Absorption by the subcutaneous route is so slow that it should not be 
 depended upon in the treatment of tetanus. Wh'iie it is true that the mor- 
 tality of tetanus has been reduced about 20 pe? cent, by the administra- 
 tion of large amounts of serum by this route' it should be emphasized 
 that a smaller amount, given subdurally or intravenously, will yield even 
 better results. Knorr has shown experimentally that after subcutaneous 
 injection the maximum quantity of antitoxin is not found in the blood 
 until twenty-four hours have elapsed. Since every hour counts heavily 
 in the chances for recovery when symptoms of tetanus have appeared, 
 it may be laid down as a general rule that the first doses of antitoxin 
 should be given subdurally or intravenously*. The subcutaneous route 
 may be chosen when serum is given for prophylactic purposes at the time 
 of injury, but should not be relied upon in the treatment of tetanus. 
 
 2. Intramuscidar injections may be given to keep up the good effect 
 of antitoxin after the first doses have been given subdurally and intra- 
 venously, and are to be preferred to the subcutaneous route whenever 
 the physician is unable to inject the serum subdurally and intravenously. 
 
 3. For the rapid neutralization of toxin free in the body-fluids serum 
 should be given intravenously. In the treatment of tetanus from 10,000 
 to 20,000 units may be given by this route as early as possible. While 
 
SERUM TREATMENT OF TETANUS 723 
 
 it is conceded that when the toxin has become firmly bound to the tissues 
 of the central nervous system dissociation by the use of antitoxin is not 
 possible, yet one can never know, in a given case. Low firm this union 
 has become, and clinical results, supported by animal experimentation, 
 show that life may be preserved by large doses of antitoxin injected into 
 the blood. 
 
 4. The experimental work of Pennin,i Park and XicoU,^ supported 
 by^ the clinical results reported by various observers, shows quite con- 
 clusively that the subdural route is a very efficacious and valuable avenue 
 by which to administer antitoxin in the treatment of tetanus. The serum 
 should be given by the gravity method, in exactly the same manner as 
 in giving antimeningitic serum. To insure its thorough dissemination 
 throughout the spinal meninges the antitoxin should be diluted, if 
 necessary, with normal salt solution. As a rule,- the amount injected 
 should be slightly less than the amount of fluid withdrawn. In the 
 case of a "dry tap, " if the operator is reasonably sure of having entered 
 the canal, from 3 to 5 c.c. of serum may be injected. It is generally 
 necessary to repeat this injection within twenty-four hours. 
 
 The reason for administering antitoxin subdurall>' is apparent when 
 it is remembered that neither the central nervous system nor the peri- 
 pheral nerves take up antitoxin direct from the blood (Park). Only 
 after very large intravenous doses are traces of antitoxin found in the 
 cerebrospinal fluid, and animals passively and actively immunized may 
 be rendered tetanic if the toxin is injected directly into the central 
 nervous system or into the nerve. While antitoxin injected subdurally 
 finaOy passes over into the blood, it will neutralize any free or dissoci- 
 ated toxin before the latter has developed any harmful tendency. 
 
 5. To neutralize any toxin that may have been absorbed by a nerve 
 it may be advisable to inject antitoxin directly into the nerve, and these 
 intraneural injections under anesthesia are advised by Ashhurst, John, 
 and others as part of the rational treatment of tetanus. 
 
 The technic of these injections has been described elsewhere 
 Prophylaxis of Tetanus. — The most succcssfui preventive treatment, 
 and practically the only successful curative one after the disease has de- 
 veloped, is by means of tetanus antitoxin. As a prophylactic remedy 
 this antitoxin exceeds in value even diphtheria antitoxin; therapeutic- 
 ally, however, it is far inferior to the latter, for the reason that part of 
 
 1 Mitt. a. d. Grenzgebiet d. Med. und Chir., 1913. xxvii, 1. 
 
 2 Jour. Amer. Med. Assoc, 1914, Ixiii, 235. 
 
724 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 the toxin produced by the tetanus bacilli sobn unites with the nerve- 
 cells of the spinal cord. 
 
 One of the greatest dangers from this terrible infection lies in the 
 fact that while the local lesion may show no signs of disturbance, the 
 central nervous system may suddenly manifest symptoms of poisoning. 
 The wounds that are likely to contain the tetanus bacillus are the following: 
 All wounds that may contain dirt contaminated by manure, such as 
 that from the streets, stables, barns, and even fields; wounds made by 
 firecrackers or toy pistols; gunshot wounds, especially those made by 
 blank cartridges; crushing injuries, made by raachinery or in other ways. 
 The feet and hands are especially prone to be infected with tetanus germs. 
 Street injuries that are not deep or perforating, but grinding and lacerat- 
 ing, are very likely to develop tetanus infection. It has also been stated 
 that tetanus bacilli may be harbored in an old injury, and yet cause no 
 symptoms until some additional injury or general disturbance of the 
 body causes the normal protection against infection to be broken down, 
 when toxins from the bacilli may be al>sorbed and tetanic symptoms 
 develop. This theory would seem to be responsible for an otherwise 
 apparently unaccountable development of tetanus. 
 
 From what has been said it will l>e seen that any injuries received on 
 the street, or those inflicted on workers about horses or cattle and in 
 stables, are more liltely to develop tetanus than are injuries received 
 in other ways. New-born babies may be infected through the stump of 
 the umbilical cord. Likewise a suppurating wound, or even a fresh 
 wound, which may be innocent at first, may become infected with the 
 tetanus bacillus if the wound or suppurating" focus is improperly cared 
 for. Many cases of vaccinal tetanus can thus be ac:counted for, i. e., 
 due to negligence in the care and treatment of the wound. It is now 
 generally agreed that proper treatment of the original wound, combined with 
 the administration of tetanus antitoxin, will surely prevent the development 
 of lockjaw. 
 
 In former years Fourth of July wounds claimed a heavy toll of fatal- 
 ities due to tetanus. Owing to the efforts of the American Medical 
 Association municipalities have been urged to adopt legislative measures 
 for enforcing a saner form of celebration, and Efforts have been made to 
 educate physicians in the proper care of thege wounds and to impress 
 upon them the great prophylactic value of tetanus antitoxin. These 
 efforts have been crowned with success, as sfetistics collected from all 
 parts of the country will show. In 1903 there were in the United States 
 40b deaths from tetanus; in 1904, 91; in 190.5,.87; in 1906, 75; in 1907, 
 
SERTJM TREATMENT OF TETANUS 725 
 
 73; in 1908, 76; in 1911, 18 cases and 10 deaths; in 1912, 7 cases with 
 6 deaths, and in 1913, 4 cases with 3 deaths. 
 
 While it is not within the province of this chapter to deal with sur- 
 gical technic, the proper cleansing and care of a wound constitute so 
 important a part of the prophylaxis of tetanus that I shall refer to this 
 subject, quoting largely from the technic recently described by Ashhurst 
 and John.i 
 
 Surgical Treatment. — 1. The surrounding skin should be painted 
 with a 3 per cent, alcoholic solution of iodin. 
 
 2. All foreign material should be removed f'rpm the wound, ana to do 
 this properly all parts of the wound should be made accessible by wide 
 incision, under ether, if necessary. This is especially true of a puncture 
 wound. It should then be freed from all tags and loose shreds of tissue 
 by means of the scissors, and the whole wound swabbed with the 3 per 
 cent, iodin solution. The wound should next be dressed with gauze 
 soaked in the same solution. The use of strong caustics is inadvisable, 
 as they cause sloughing and tend to produce a good focus for the growth 
 of tetanus bacilli. 
 
 3. The wound should be dressed daily at first, being exposed and 
 thoroughly irrigated with hydrogen dioxid solution, and then dressed 
 v/ith the gauze saturated with the iodin solution. As soon as healthy 
 granulations have formed, balsam of Peri{ applications should be 
 made. 
 
 4. Antitetanic powders have been prepared, made up with anti- 
 septics, and although experimentally their use has seemed to be suc- 
 cessful in preventing the development or abs"orption of tetanus toxin, 
 still it has not as yet shown that these results were not merely due to the 
 strong antiseptic that was combined with the antitoxin powder. It 
 might, however, be well to apply tetanus antitoxin and antitetanic 
 powder to the open wound, but these remedies are not to he relied upon nor 
 accepted as substitutes for the injection of antitoxin. 
 
 Use of Antitoxin. — The prophylactic use of tetanus antitoxin has 
 not infrequently been unsuccessful, due probably to the fact that it was 
 used incorrectly. 
 
 1. Antitoxin should be given as soon as possible after the wound has 
 been inflicted, and best at the time the primary treatment is given. 
 The antitoxin should be injected "as near the wound as possible, so as 
 to flood the tissues in the immediate vicinity, " and, if possible, it should 
 be given intramuscularly, so that the motor neiwes may absorb it rapidly. 
 1 Amer. Jour. Med. Sci., 1913, ckLvI, No. 1. 
 
726 PASSIVE IMMUNIZATION SEKJJM THERAPY 
 
 If anj' nerves are exposed, antitoxin should tJ& injected into them. The 
 injection of 1500 units of antitoxin is generally advised as the first prophy- 
 lactic dose. 
 
 2. From the fact that tetanus antitoxin is one of the albuminous 
 constituents of horse serum that are foreign tp-the human system, in the 
 human being the antitoxin is rapidly eliminated in from eight to ten 
 days after the injection is administered. Knbrr has found, as the result 
 of animal experiments, that by the sixth day only one-third, and by the 
 twelfth day only one-fiftieth, of the optimum quantity remained in the 
 blood. Hence it is important, if antitoxin is to prove useful, that it should 
 be present in the system for two or three weeks after receipt of the injury, 
 especially as it cannot be determined when the tetanus bacillus first gained 
 access to the wound. There should be a second intramuscular injection 
 of 1000 units of antitoxin about the end of the first week, and perhaps a 
 similar dose at the end of the second week. WhUe certain individuals may 
 develop serum sickness, no dangerous symptoms have been observed 
 to result from the use of tetanus antitoxin. 
 
 3. If the surgeon is first consulted several days after the injury has 
 been inflicted, the wound should be opened and dressed as previously 
 described, and, in addition to the intramuscular injection of 1500 units 
 of antitoxin in the neighborhood of the wound, it will be good practice 
 to inject an additional 5000 or 10,000 units intravenously. It requires 
 at least twenty-four hours for the antitoxin to be absorbed from the 
 subcutaneous tissues, and immediate neutralization of any toxin present 
 in the blood may mean a great deal from the standpoint of prognosis 
 if tetanus should develop. 
 
 Treatment of Tetanus. — While the great value of antitetanic serum 
 as a preventive is unquestioned, as a specific cure it has fallen short of 
 the eai'liest expectations. It has been shown, experimentally, however, 
 that tetanus antitoxin may save the lives of animals already manifestinp; 
 the symptoms of an otherwise fatal intoxication. In order to accomplish 
 this result the serum must be given in doses several hundred times the 
 size required merely for protective purposes^ and it must be injected 
 within a short time — from twenty-four to thirty-six hours — after the 
 onset of the tetanus. Furthermore, statistics favor the use of the serum 
 as the mortality has been reduced from 80 to 85 per cent, to 60 or 65 per 
 cent, in cases receiving serum treatment. 
 
 The recognition of the natural limitations of the serum treatment of 
 tetanus will serve to emphasize the importance of its proper adminis- 
 tration. A large number of units must be given, and must be injected 
 
SERUM TREATMENT OF TETANUS 727 
 
 in places best suited to secure the maximum neutralizing action of the 
 serum. 
 
 Surgical Treatment. — 1. The site of the wound should be located, 
 and if possible incised under ether or chloroform anesthesia and thor- 
 oughly cleansed of foreign material and necrotic tissue. It should then 
 be swabbed with the 3 per cent, alcoholic solution of iodin, washed with 
 hydrogen dioxid solution, and packed loosely with gauze soaked in the 
 iodin solution. 
 
 2. Cauterization with pure phenol, followed by alcohol, may be em- 
 ployed, but, as a rule, the weaker germicide is*preferable in order not to 
 produce necrosis of the tissues, which furnishes pabulum for bacterial 
 growth. The wound should be dressed daily. 
 
 Serum Treatment. — A niaximum amount of antitoxin should be 
 given the patient as soon as possible, and the greater the delay in giving 
 the antitoxin, the greater is the amount required. Since absorption 
 after subcutaneous injection is very slow, valuable time may be lost, 
 and since enormous amounts must be given, at great expense, this route 
 possesses much less value than the intravenous and intraspinal methods. 
 
 1. Administer intravenously from 10,000 ta 20,000 units of antitoxin 
 at once, and repeat the dose if no effect is apparent or if the good effect 
 A\'ears off in about from eighteen to twenty-four hours (the technic is 
 tiescribed on p. 690). After one or two intravenous injections the good 
 effect may be maintained by direct intramuscular injections of from 5000 
 to 10,000 units for one or two doses. 
 
 2. From 3000 to 5000 units should be given intra.'S'pinally by means 
 of lumbar puncture. This dose should be repeated every twenty-four 
 hours unless the symptoms have markedly ameliorated. The technic 
 of this injection is described on p. 694. A quantity of cerebrospinal 
 fluid should be removed before the serum is injected. After the first 
 injection the fluid may be found to have become cloudy, with a large 
 increase of cells, especially of the polynuclear variety, although bac- 
 teriologically it may be sterile. This outpouring of leukocytes is prob- 
 ably a reaction to the irritant effect of the serum, and especially of the 
 preservative it contains. 
 
 3. If a surgeon is at hand, from 500 to 1000 units of antitoxin should 
 be injected slowly intravenously into the sheaths of the nerve-trunks 
 leading from the infected region. These inje33tions are directed to be 
 made as near the trunk as possible, and to distend the nerve so as partly 
 to neutralize and partly mechanically to interrupt the passage of toxin 
 to the cord or brain. 
 
728 PASSIVE IMMUNIZATION SERTJM THERAPY 
 
 Both the intraspinal and the intraneural injections are given under 
 light chloroform anesthesia. 
 
 General Treatment. — A large number of substances have been ad- 
 vocated in the treatment of tetanus; of these, the most common are 
 injections of phenol and intraspinal injections of magnesium sulphate. 
 While phenol may be well tolerated bj' tetaniis patients, Ashhurst and 
 John believe that all these treatments are of little value, and that spinal 
 injections of magnesium sulphate are dangerous. 
 
 1. Chloral hydrate and potassium bromid should be given by mouth 
 or by rectum, in sufficient quantities to produce sleep and quiet. Drugs, 
 such as those of antagonistic physiologic activity, are more or less suc- 
 cessful and frequently of aid when given in conjunction with the anti- 
 toxin. 
 
 2. While combating the disease, the general care of the patient should 
 not be forgotten. A purgative should be administered early. Simple, 
 nourishing, non-stimulating food should be given by the mouth, if 
 possible, or by the nasal tube, if necessary. Absolute quiet should be 
 maintained. Distention of the bladder from retention of urine should 
 be guarded against. If water is not well absorbed, and especially if 
 there is peritoneal or pelvic inflammation, saline injections into the colon 
 should be given. 
 
 Results of the Antitoxin Treatment of Tetanus. — As was previously 
 stated, the prophylactic value of tetanus antitoxin has been proved beyond 
 any reasonable doubt. This does not imply, however, that the simple intro- 
 duction of 1000 units of antitoxin beneath the skin will surely protect 
 the patient, as the percentage of cases developing tetanus even after the 
 serum has been given is altogether too high. As was pointed out under 
 Prophylactic Treatment, the wound must receive thorough surgical at- 
 tention, and the antitoxin must be injected in such places where it will 
 have the greatest opportunity to neutralize the toxin. Even if tetanus 
 should develop under these conditions, it is likely to be mild and the 
 prognosis would be much more favorable. 
 
 Tetanus antitoxin has likewise been very successful in veterinary 
 practice, especially after castration and other operations, in injuries, and 
 among horses used for the purpose of producing diphtheria antitoxin 
 and other immune serums. 
 
 While the curative value of tetanus antitoxin has not come up to expecta- 
 tions, more recent carefully prepared statistics indicate that, with serum 
 treatment, the mortality is reduced at least 20 percent. This includes cases 
 treated by the subcutaneous injection of antitoxin, and it must be em- 
 
SERUM TREATMENT OF TETANUS 729 
 
 phasized that, in order to secure the best results, tetanus must be treated 
 in a rational manner according to its pathology. Under these circum- 
 stances we can confidently expect a greater reduction in mortality. But 
 at any rate no physician should withhold antitoxin in the treatment if 
 there are any possible means of obtaining it. If only 3000 units may be 
 had, it is far better to inject this amount intraspinally than subcu- 
 taneously. 
 
 Pennin,' in a recent and thorough review, reaches the general con- 
 clusion that anti-tetanus serum has reduced the mortality of tetanus 
 approximately 20 per cent. He gives figures from Denmark that are 
 especially valuable, because they were gathered from a small area, and 
 hence represent fairly uniform conditions: Of 199 cases not receiving 
 serum, only 21 per cent, recovered; whereas of 189 cases treated with 
 serum, 42.3 per cent, recovered. Of 92 acute cases with an incubation 
 period of less than ten days 24.2 per cent, recovered when serum was 
 used, whereas of 94 cases treated without serum only 5.3 per cent, re- 
 covered. It is significant that these Danish figures correspond closely 
 to the American statistics and those of other countries. Irons^ has 
 recently tabulated the results of 225 cases of tetanus treated with anti- 
 toxin collected from hospitals and private records for the years 1907 to 
 1913. The mortality in all cases receiving serum was 61.77 per cent.; 
 in 21 cases without serum the mortality was 85.7 per cent. The latter 
 figures correspond quite closely with the general mortality of about 85 
 per cent, of tetanus treated without serum. Irons' figures also show the 
 influence of large doses of antitoxin; of 57 cases receiving a small dose 
 of antitoxin (3000 units or less subcutaneouslj[), the mortality was 73.7 
 per cent.; of 143 cases receiving large doses (over 3000 units subcu- 
 taneously, or 3000 or less intraspinally or intravenously), the mortality 
 was 57.3 per cent. Magnesium sulphate was given intraspinally in 18 
 cases which also received serum; in 4 cases^^2 acute and 2 chronic — 
 the patients recovered, giving a mortality for the group of 77 per cent. 
 In 2 cases death recurred shortly after injection with symptoms of re- 
 spiratory failure. 
 
 In view of this evidence in favor of antitoxin in the treatment of 
 tetanus it is apparent that the physician is- compelled to give every 
 patient with tetanus the opportunity to obtain this 20 per cent, or more 
 benefit by administering the serum promptly and correctly. 
 
 ' Mitt. a. d. Grenzgeb. d. Med. u. Chir., 1913, xxvii. 
 2 Jour. Amer. Med. Assoc, 1914, Ixii, 20, 25. 
 
730 PASSIVE IMMUNIZATION SERTJM THERAPY 
 
 SERUM TREATMENT OF DYSENTERY 
 
 Soon after the discovery of a bacillus of dysentery by Shiga in 1892 
 the treatment of bacillary dysentery by the use of immune serums was 
 undertaken. Following the discovery of the etiologic importance of 
 Shiga's bacillus in the dysenteries of Asiatic countries, similar investiga- 
 tions were made in various parts of the world and various bacilli were 
 isolated. At first these microorganisms were*all regarded as being iden- 
 tical, but further investigation has shown that marked differences are 
 apparent, and two main types are now recognized: one type (Shiga) 
 does not ferment mannite and produces a soluble or extracellular toxin, 
 and a second type (Flexner-Harris, Hissy, etc.) ferments mannite and 
 does not produce an extracellular toxin. A further discussion of these 
 baciUi will be found in Chapter VII. 
 
 Dysentery caused by the bacilli of the KrUse-Shiga type may be re- 
 garded as a form of intoxication analogous to the intoxication of diph- 
 theria. The intestine, where the bacilli lodge, corresponds to the throat, 
 which is the site of infection in diphtheria; here the bacilli develop and 
 produce their toxins, and these toxins, when absorbed into the circula- 
 tion, in turn produce the symptoms of the disease. 
 
 Antitoxin has been prepared for the bacilli of the Kruse-Shiga type, 
 and these have yielded fairly satisfactory results in the prophylaxis and 
 cure of this variety of bacillary dysentery. Antiserums for the mannite- 
 fermenting group of bacilli (Flexner, Harris, Hess, Duval, etc.) have not 
 proved of much value in the treatment of these infections. Bacilli 
 of the latter group are largely responsible for the dysenteries in this 
 country, and also for a percentage of cases of ileocolitis of infancy. 
 Since the antiserum of the Shiga bacillus is of practically no value in the 
 treatment of infections caused by bacilli of other groups, the serum 
 treatment of dysentery is employed mainly in European and Asiatic 
 countries, where infections with this group are common. After fairly 
 extensive trials in this country the serum treatment of infantile diarrheas 
 and true dysenteries has proved disappointing. 
 
 The preparation and standardization of dysentery antitoxin is de- 
 scribed in Chapter XIV. 
 
 Administration and Uses. — Dysentery anititoxin has been used both 
 in the prophylaxis and in the cure of this infection. The doses of serum 
 advised by various observers varj^ considerably, owing to the marked 
 differences that exist in the potency of these serums. Since the various 
 manufacturers do not employ the same standards, the physician should 
 
SJflKUM TREATMENT OF DYSENTERY 731 
 
 use the serum in accordance with the printed directions that accompany 
 each package. 
 
 For prophylactic purposes, usually from 10 td 20 c.c. are recommended, 
 and it is further advised to repeat the injection after two or three weeks, 
 as the protection lasts only a short time. 
 
 For curative purposes, Shiga has advised 10 c.c. for mild cases and 
 from 20 to 60 c.c. for severer cases. It may be necessary to repeat the 
 injections several times. Vaillard and Doyle have given as much as 
 from 80 to 100 c.c, and have repeated this dose on the following days. 
 When the serum is being used during an epidemic, it is advisable to as- 
 certain beforehand the nature of the infection, as the antiserum for the Shiga 
 bacillus is highly specific and is not likely to prove of value in the treatment 
 of infections caused by the Flexner type of bacillus. 
 
 The injections have usually been given subcutaneously. Better 
 results would, no doubt, be obtained in the treatment of severe infec- 
 tions by administering large doses of serum intravenously. 
 
 Results. — Adequate statistics regarding the value of the serum in 
 the prophylaxis and treatment of dysentery are- not yet available. From 
 the prophylactic standpoint, encouraging results have been reported by 
 Kruse, Shiga, Vaillard and Dopter, Kosculet, and others, and it would 
 appear that passive immunization is of valu'e in combating localized 
 outbreaks, such as occur in institutions and armies. 
 
 From the curative standpoint, most observers agree that the use of a 
 potent serum will reduce the mortality of acute cases at least from 30 
 to 50 per cent. Shiga reports a drop in the mortality in Japan of from 
 22 to 26 per cent, to 9 to 12 per cent.; Kruse obtained a reduction in 
 mortality of about 10 per cent. Vaillard and Dopter' treated 96 cases, 
 with but 1 death; Rosenthal^ treated 157 cases with 7 deaths, — a mor- 
 tality of 4.5 per cent, as compared with that of 10 to 11 per cent, oc- 
 curring in other German hospitals. Coyne and Auche' treated 11 cases 
 due to the Flexner type of bacillus and report good results. In a 
 thorough investigation made in the United States in 1903 by the Rocke- 
 feller Institute, under the direction of Flexner, it was found that the 
 results of the serum treatment of ileocolitis, among children at least, 
 were quite disappointing. This is largely due to the fact that several 
 different strains of bacilli may be the cause of an infection, and unless a 
 corresponding antiserum is employed for the particular type causing the 
 
 1 Ann. Inst. Past., 1906, xx, 321; 1907, xxi, 241. 
 
 2 Deut. med. Wochenschr., 1904, xxXj No. 19. 
 
 3 Compt. rend. Soc. Biol., 1906, Ix, No. 26. 
 
732 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 infection in a given case, good results cannqt be expected. Probably 
 if some means were discovered for making a prompt bacteriologic diag- 
 nosis, and if several immune serums were on hand for the treatment of 
 infections caused by the main types, after the methods worked out by 
 Neufeld, Dochez, and Cole in the treatment of pneumonia, good results 
 may be obtained. 
 
 The curative effect of dysentery antitoxin is shown by a reduction in 
 the number of stools, by the fact that blood and pus disappear from the 
 discharges, pain and tenesmus are relieved, the temperature becomes 
 normal, and the patient gains in weight. Individual observers are fre- 
 quently enthusiastic over the results obtainefl in individual cases, and 
 no doubt these are striking in those instances where the antiserum ap- 
 pears to be specific for the particular form of infection. 
 
 THE SERUM TREATMENT OF HOG CHOLERA 
 While the cause of hog cholera has not as yet been discovered, it is a 
 well-known fact that the virus is present in the blood of infected animals, 
 and it is possible, by immunizing healthy hogs with gradually increasing 
 doses of infected blood, to prepare a potent immune serum that will 
 prove of value in the prophylaxis and cure of hog cholera. The nature 
 of this serum is unknown. It possesses many of the features of an anti- 
 toxin, and for the present may be classed with these. 
 
 Production and Standardization of Hog Cholera Serum. — Healthy hogs weigh- 
 ing about 100 pounds are selected, and injected subcutaneously with 40 c.o. of 
 hog cholera serum per hundred pounds of weight. Two or three days following 
 this protecting dose they are injected intravenously with 3 or 4 e.c. of sterile, defibrin- 
 ated blood, obtained from an animal suffering from the disease; or the animals 
 may be exposed in pens known to be infected. If the animals live for one month 
 without showing evidences of toxemia, they receive another injection of 5 c.o. of 
 infected blood (virus). Two or three weeks later they receive another injection of 
 from 15 to 20 c.c. of infected blood; in from fifteen:to twenty-one days after this 
 inoculation they receive a final injection of from 4 to 5 c.c. of virus per pound of 
 body weight. Animals tolerating the last injection are said to be hyperimmune, and 
 are bled in ten days. In hyperimmunizing the animal some prefer to inject the virus 
 intraperitoneally instead of mtravenously. If this method is adopted, about double 
 the dose of infected blood (virus) is required. About 5 per cent, of animals 
 succumb during the period of immunization. 
 
 The immunized hogs are bled aaeptically from the tail by snipping off the tip, 
 and 5 c.c. of blood per pound of weight is collected in sterile vessels. Each animal is 
 bled once a week imtil three or four bleedings have been made. 
 
 In about one week after the last bleeding they are hyperimmunized again by 
 giving them 4 or 5 c.c. of infected blood per pornid of body weight, and additional 
 bleedings are made so long as the tail lasts. 
 
SERUM TREATMENT OF SNAKE-BITES 733 
 
 The serums secured in the several bleedings are mixed together. The third or 
 fourth bleeding is said to be most potent or to contain the greatest number of anti- 
 bodies. 
 
 In iesiing and standardizing the immune serum, six hogs, weighing about 100 
 pounds each, are placed in an infected pen. No. 1 receives no serum and is a control; 
 No. 2 receives 15 c.c. of serum; No. 3 receives 20 c.c; No. 4 receives 30 CO.; No. 5 
 receives 35 c.c; and No. 6 receives 40 c.c. of immune serum. The animals are 
 allowed to remain in the pens until the control succumbs and the protecting dose of 
 serum has been determined. In this maimer we can determine just about the amount 
 of serum required to protect 100 pounds of hog. The Pennsylvania Live Stock 
 Sanitary Board has found that it does not require quite 40 c.c. of serum, but this 
 amount is recommended for safety, and because the weight may not be judged 
 accurately. 
 
 Hog-cholera serum is used in both the prophylaxis and the thera- 
 peutic management of this disease. For prophylactic purposes for each 
 100 pounds of hog 40 c.c. of serum are injected. These injections are 
 usually given subcutaneously and occasionally intramuscularly. In 
 some instances active and passive immunization is practised by the 
 simultaneous injection of 2 c.c. of virus, together with 4Q c.c. of immune 
 serum per 100 pounds of weight. Owing to the danger of spreading the 
 disease, this method is not generally employed. 
 
 The results of prophylactic immunization are excellent, and the 
 method is valuable in checking epidemics. Immunity is said to last for 
 six months after vaccination. 
 
 For curative purposes the serum has yielded good results, providing 
 it is administered not later than the fourth day after the animal shows 
 evidences of the disease. Several injections may be required, and the 
 intramuscular route should be chosen, because quicker absorption is 
 thus insured. 
 
 Calf cholera serum has been prepared by immunizing horses 
 with strains of colon, paracolon, and other bacilli belonging to these 
 groups, isolated from calves dying of calf cholera. The immune serum 
 should agglutinate the microorganisms used in its production in dilu- 
 tions of 1:2000 to 1:500. 
 
 This serum has proved of value in the prevention of calf cholera, 
 and may be of benefit in the treatment, providing that it is prepared 
 with the same strain or strains of microorganisms responsible for the 
 infection. 
 
 SERUM TREATMENT OF SNAKE-BITES 
 The nature of snake venom is discussed in Chapter VII, and the 
 method of preparing antivenomous serum is described in Chapter XIV. 
 
734 PASSIVE IMMUNIZATION SER¥M THERAPY 
 
 Calmette's aritirenene for cobra venom is useful in the treatment of 
 cobra envenomation, but is not serviceable for the treatment of other 
 snake-bites, as shown by Martin for Australian serpents and by McFar- 
 land for American snakes. In the venoms of dur snakes, as, for example, 
 the rattlesnake, copper-head, and moccasin, the poison is essentially 
 locally destructive, the respiratory poison being of secondary impor- 
 tance. McFarland failed to immunize horses against this locally de- 
 structive poison. Later Noguchi and Madsden succeeded in producing 
 an antiserum, prepared by immunizing horses with venom after the 
 toxophorous groups of the molecules had been destroyed, capable of 
 neutralizing the hemorrhagin of the Crotalus venom. 
 
 The serums of Calmette, Noguchi, and others are useful in the treat- 
 ment of their respective envenomations, but aside from India and a few 
 other reptile-infested countries, as well as in zoological gardens and 
 laboratories where snakes are kept, the serums have a very limited sphere 
 of usefulness. 
 
 SERUM TREATMENT OF HAY-FEVER 
 
 The nature of pollen toxin has been discussed in Chapter VII, and the 
 preparation of antitoxin in Chapter XIV. 
 
 Dunbar has prepared an antitoxin for certain pollen; this may be 
 obtained commercially. The method of administration consists in 
 dropping the serum into the eyes and sniffing it .into the nose at the onset 
 of an attack. It is necessary for the patient to carry the scrum and 
 dropper about, as the effects produced are of short duration, and the 
 patient is subject to repeated reinfections. Subcutaneous injections 
 are not advisable, as considerable local edeina is produced, and the 
 amount of protection afforded is uncertain. 
 
 Many observers, as, for example, Semon,^ McBride,^ Knight,* 
 Throst,'' Weichardt,^ and others, report that the serum affords a tem- 
 porary relief which is grateful to the patient, but which cannot be said 
 to be curative of the disease. In some instances it fails altogether, and 
 in these it is reasonable to assume that the antitoxin has not been pre- 
 pared from the particular pollen that infected the patient. An intoler- 
 ance to the serum may be excited. 
 
 More recently the possibilities of effecting active immunization 
 
 1 Brit. Med. Jour., 1903, ii, 123, 220. ^ Edin. Med. Jour., 1903, ii, 7. 
 
 ' Med. Record, March 10, 1906. 
 
 * Miinch. med. Wocheuschr., June 9, 1903. 
 
 ' Centralbl. f. Bakt., Abst., 1906, xxxviii, 493. 
 
SERUM TREATMENT OF HAYrFEVER 735 
 
 against hay-fever have been shown by Claves ' with the pollen of rag- 
 weed. The method is still in the experimental stage, but it is reasonable 
 to assume that vaccines may be prepared for the pollen of various plants 
 usually responsible for the hay-fever and autumnal catarrhs of this 
 country. 
 
 Antibacterial Immunization 
 
 General Considerations. — It may be stated that antibacterial serums 
 have not been found of equal value to the antitoxins, cither in the 
 prophylaxis or in the treatment of their respective infections. It is 
 true, however, that antimeningococcus serimi Raa reduced the mortality 
 of epidemic cerebrospinal meningitis from 75 to 90 per cent, to 30 per 
 cent, and less, and has thereby firmly established its value in the treat- 
 ment of this dreaded infection. Recent work in pneumonia has de- 
 veloped a method of serum therapy that has proved of value in the treat- 
 ment of this disease, and it is likewise true that antistreptococcus and 
 antigonococcus serums yield at times and in individual cases most 
 prompt and happy results. But the expectations for serum therapy 
 that were aroused in 1894 with the discovery of diphtheria antitoxin 
 have not been fully realized, although at the present time the reasons 
 for failure are being studied, understood, and gradually overcome. 
 
 Granting that serum therapy could be redu-ced to the simple prop- 
 osition of bringing specific antibodies into relation with the micro- 
 organisms producing a given infection, the process remains quite intri- 
 cate, largely owing to the fact that although different strains of the same 
 microorganism may possess identical morphologic and biologic charac- 
 teristics, yet they vary not only in pathogenicity, but also in the speci- 
 ficity of the antibodies that they stimulate the body-cells to produce. 
 In other words, serum therapy is more specific than it is generally con- 
 sidered to be. For instance, the antibodies of one strain of pneumococcus 
 may have little or no action upon another strain, and the same is prob- 
 ably true of the various pathogenic bacilli and groups of streptococci, 
 gonococci, and to a lesser extent also of meningococci. This fact has 
 long been known, and an effort has been made to overcome the difficulty 
 by immunizing horses with a large number of different strains of the 
 same microorganism in the hope that the polyvalent serum so produced 
 would contain sufficient antibodies for all ojr most infections of the 
 various strains of the particular microorganisms in question. With 
 diphtheria and tetanus bacilli, the soluble toxin is apparently quite 
 1 Proc. Soc. Exper. Biol, and Med., 1913, x, 69. 
 
736 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 similar for all strains, so that immunization with the toxin of one yields 
 an antitoxin capable of neutralizing the toxins of all. With dysentery- 
 bacilli, snake venoms, and pollens, however, the toxins are more specific, 
 and produce more specific antitoxins. 
 
 Recent work in pneumonia by Cole and Dochez and their coworkers 
 in the Rockefeller Institute, and Neufeld in Germany, indicates a more 
 promising future for antibacterial immunization. These investigators 
 have been able to divide pneumococci inta four main groups, have 
 worked out a relatively quick method of determining the group to which 
 a particular pneumococcus belongs, and have prepared immune serums 
 for these main groups. By injecting the serum corresponding to the 
 strain producing a given infection, encouraging results have been ob- 
 tained in the treatment of pneumonia, whereas the polyvalent serums 
 have been found, after quite extensive use, to yield indifferent results, 
 due in part to a relatively low content in the paHicular antibodies for that 
 certain strain causing a given infection. 
 
 These investigations in pneumonia are of great importance because 
 they reveal an immense field of interesting and similar researches in 
 streptococcus, gonococcus, meningococcus, typhoid, and other infections. 
 While it is obviously impossible to prepare an immune serum for each 
 and every strain of microorganism, it may be possible to subdivide 
 strains into a few main groups and then discover a method for quickly 
 determining to which group a particular culture belongs, so that the 
 corresponding immune serum may be administered. In this manner 
 we may be able to reduce the 30 per cent, mortality still remaining in 
 epidemic meningitis and otherwise place the treatment of specific in- 
 fections upon a more strictly scientific basis, as in the serum treatment 
 of diphtheria. 
 
 As previously stated, the method and route of injecting an immune 
 serum are of considerable importance in serum therapy. Large doses of 
 serum should be given until the desired effect is secured, or until it 
 becomes evident that more can be produced. In the mean time manu- 
 facturers should make every effort to produce potent serums and to 
 concentrate them, if possible, just as diphtheria antitoxin is concentrated. 
 
 THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 
 During the pandemic of meningococcal cerebrospinal meningitis in 
 1904-05 several laboratories sought to produce an immune serum for the 
 purpose of treating human cases of this infection. 
 
THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 737 
 
 After pursuing experimental studies on the subject on the lower 
 animals, Jochmann/ in 1905, immunized a horse and used the serum in 
 the treatment of 38 cases of epidemic meningitis. At first he employed 
 the subcutaneous method of injection, and later he used the intraspinal 
 method. The results were quite encouraging, and during the foUo-ning 
 year 30 more cases were treated, with a resulting mortality of 27 per 
 cent., as against a mortality of 53 per cent, in untreated cases. 
 
 At about the same time Kolle and A\'assermann^ reported that they 
 had prepared an antimeningococcus serum, which had not, however, 
 up to that time been used in the treatment of human infections. A year 
 later serum was administered subcutaneously and then intraspinally, 
 mth encouraging results. 
 
 In this country Park had, in 1905, prepared an antimeningococcus 
 serum, which was used in the treatment of 20 cases in Hartford, Conn., 
 by subcutaneous injection, but without beneficial results. Jochmann 
 had, in the mean time, shown the superiority of intraspinal injections, 
 and this method soon supplanted the subcutaneous method. 
 
 In 1905 Flexner began a series of studies regarding experimental 
 meningococcus infections in the lower animals, and the therapeutic 
 value of antimeningococcus serum. These valuable experiments at- 
 tracted the attention of the world, and placed this method of treatment 
 upon a firm basis. In 1906 Flexner^ proved that a specific immune 
 antimeningococcus serum could be producecj that, if injected intra- 
 spinally, would save the lives of monkeys. Later horses were immunized 
 and tlie serum used in the treatment of human infections during an 
 epidemic in Akron, Ohio, in ^l-dy, 1907. In a short time the serum was 
 used extensively in other epidemics, and a report of these earlj^ cases was 
 made by Flexner* in 1907. A later report by Flexner and Jobling," 
 covering the treatment of 400 cases, showed that the mortality had been 
 reduced from 75 per cent, to below 30 per cent. In 1909 they reported' 
 upon 712 cases treated with the serum, with a mortality of 31.4 per cent. 
 In a more recent report Flexner' reviewed all the cases — 1300 iu number 
 ^gathered from all parts of the world, treated, with serum prepared in 
 the Rockefeller Institute. The general mortafity rate is given as 30.9 
 per cent., as against 75 to 80 per cent, among cases not receiving serum 
 
 1 Deut. med. Wochcnschr., 1906, xxii, Xo. 20, 7SS" 
 - Deut. med. Wochenst-hr., 1906, xxxii, Xo. 16. 
 ' Jour. Amer. ^led. Assoc, 1906, xlvii, 560. 
 
 'Jour. Exper. Med., 1907, ix, 16S. ^ Jour. Exper. Med., 1908, x, 141. 
 
 « Jour. .\mer. Med. Assoc, 1909, Uii, 1443. 
 'Jour. Exper. Med,, 1913, xvii, 553. 
 47 
 
738 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 treatment. Of 1394 cases treated with serum during the Texas epi- 
 demic' (1912), the mortality was 37 per cent., as compared with a mor- 
 tahty of 77 per cent, among 562 cases receiving no serum. 
 
 Good results have been reported by many observers with Joch- 
 mann's, KoUe and Wassermann's, Ruppel's, Paltauf's, and Dopter's 
 serums, and the serums have been prepared by several commercial 
 biologic laboratories, so that the curative value of antimeningococcus 
 serum is definitely established. 
 
 Nature of Meningococcus Meningitis. — JBacteriologic and patho- 
 logic evidence indicates that the first stage of "meningococcus meningitis 
 consists of a meningococcus bacteremia, the virulent meningococci 
 gaining access to the blood-stream through the upper air-passages. 
 Later the infection becomes localized in the spinal and cerebral meninges. 
 It is probable that the microorganism causes a primary nasopharyn- 
 gitis, and in some instances the meninges may be infected b^' direct 
 extension through the sphenoid and ethmoid sinuses. The main symp- 
 toms and lesions of the disease, and several of the complications, for 
 example, the paralyses, eye complications, deafness, hydrocephalus, 
 and mental disturbances, are probably direotly due to suppuration of 
 the meninges, with involvement of accessory and motor nerve-roots, 
 meningeal irritation, and pressure from the accumulation of exudate 
 in the ventricles and subarachnoid space. Complications, such as 
 arthritis, pyelitis, endocarditis, adenitis, etc., are due to the hacteremia, 
 which may become chronic and be accompanied by deposits of menin- 
 gococci in the various tissues and organs. In addition to these com- 
 plications there is probably a varying degree olF general toxemia, due to a 
 soluble toxin or endotoxin liberated through lysis of the cocci. 
 
 Treatment of Epidemic Meningitis. — ^Although cerebrospinal menin- 
 gitis may be considered primarily as a general infection, in the majority 
 of instances local suppuration of the meninges constitutes the main 
 lesion. For anatomic and physiologic reasons, however, it is impossible 
 to treat the disease according to the ordinary principles governing the 
 treatment of localized suppuration, as, for example, bj^ continuous 
 drainage and by cleansing the affected parts with germicidal solutions. 
 Unaided, the leukocytes and body-fluids are generally unable to destroy 
 the cocci and terminate the infection before sCrious harm to important 
 nerves and nerve-centers has resulted, so that epidemic meningitis, with 
 a mortality of from 75 to 90 per cent., and followed by more or less 
 
 1 Sophian: Epidemic Cerebrospinal Meningitis, SU Louis, 1913. Report of Dr. 
 Steiner, President of the Texas Ptate Board of Health. 
 
THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 739 
 
 serious sequelae, which few survive, is one of the most dreaded of in- 
 fections. 
 
 In administering antimeningitic serum we aim to assist the patient's 
 leukocytes and body-fluids to overcome the infection. Repeated spinal 
 punctures remove portions of the infective material mechanically, but 
 the greatest dependence in bringing about quick destruction of the 
 cocci and effecting recovery of the patient is to be placed upon the serum. 
 
 Preparation of the Antimeningococcus Serum. — Method of Flexner and Jobling. — 
 1. Many strains of meningococcus are used in order that a polyvalent serum may 
 be prepared. Fresh strains from new epidemics and sporadic cases are constantly 
 added. "Fast " strains, or those isolated from cases in which the serum has produced no 
 beneficial effect, are especially desirable. 
 
 2. Immunization is performed first with an autolysate of the meningococci, and 
 later with living cultures. 
 
 3. Stock cultures are kept alive by transplanting them every four days in slants 
 of ascitic glucose agar, neutral to phenolphtlialeiii. 
 
 4. In preparing the autolysate, the cultures are subcuitured first on glucose-agar 
 slants without serum. After twenty-four hours' growth about 3 c.c. of salt solution 
 are added to each slant, and the culture emulsified. One cubic centimeter is then 
 poured over the surface of glucose-agar slants in large 500 c.c. Blake bottles. After 
 twenty-four hours' incubation heavy, uniform, and diffuse growths are secured. 
 
 Add 10 c.c. of normal salt solution to each bottle and wash off the culture. If 
 necessary, a long heavy platinum loop may be used. Each bottle is tested for con- 
 tamination by staining a smear according to the method of Gram. Each bottle is 
 emptied into a common vessel; 2 per cent, toluol is addecl} mixed well, and incubated 
 for from eighteen to twenty-four hours. The toluol is then allowed to e\'aporate, or 
 it may be immediately filtered off through sterile gauze saturated with salt solution. 
 The preparation is kept in a refrigerator and should be prepared fresh every month. 
 
 5. The injections are given subcutaneously about the neck and abdomen. Young 
 and healthy horses are selected for the purpose. The first dose consists of 2 c.c. of 
 the autolysate, and this is gradually increased, depending on the manner m which the 
 animal reacts, miUl 10 c.c. are given in a single dose. Then inject 2 c.c. of living cul- 
 ture diluted with two parts of salt solution, and increase the doses, the same as with 
 the autolysate, until 10 c.c. of culture are given at one injection. Next inject living 
 cultures and autolysate alternately, until a maximum of from 30 to 35 c.c. are given 
 in one dose; this last in then used as the constant dose until the immunization has 
 been completed. 
 
 Injections are given every five to se^en days until the large doses are reached, 
 when they are given every ten days or two weeks. 
 
 Horses usually show quite marked reactions, such as fever, depres.'sion, and 
 induration about the site of injection, but if due care is exercised, few animals are 
 lost during the immunization. 
 
 6. Bleedings are usually begun about the fourth month after immunization has 
 been instituted. The horses are bled aseptically from a jugular vein about every 
 two weeks, from six to eight liters of blood being removed at each sitting. The serum 
 is separated and preserved with trikresol. Recent investigations indicate that trikresol 
 Tnay he partly responsible for paralysis of the respiratory ccinters, and at present every 
 effort should he made to collect and market the serum, in a strix.tly aseptic manner, so that 
 
740 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 none m hut -very little preservative is required.'- Efforts are being made at present to 
 discover an efficient volatile antiseptic that may be driven off by warming the serum 
 at body temperature. 
 
 Method of Kolle. — Kolle prepares antimeningococcus serum by immunizing 
 horses with heated and then with living cultures or with bacterial extracts. All 
 injections are given intravenously. 
 
 (a) With Cultures. — The first dose equals half a culture removed with normal 
 salt solution from a test-tube of slanted agar after twenty-four hours' incubation, 
 and heated at 60° C. for half an hour. A week later the dose equals that of a whole 
 slant. The dose is increased each week by one slant until 10 are given. The eleventh 
 dose equals one agar slant of limng culture, and ea'ch week the dose is increased 
 until the growths from 10 slants are given at one time. Fourteen days after the last 
 injection the horses are bled and the serum is tested. 
 
 (b) With Bacterial Extracts. — Horses are first injfected with two doses of heated 
 cultures, as just described. The third dose consists of 0.1 c.c. of bacterial extract. 
 Every two weeks the dose is increased by 1 c.c. of extract until 5 c.c. are given at one 
 time. 
 
 Standardization of Antimeningococcus Serum. — An accurate method of stand- 
 ardizing antimeningococcus serum has not as yet been devised. In the selection of a 
 serum physicians must, therefore, be guided by the reputation of the manufacturers. 
 
 An antimeningococcic serum of high antibody content has antitoxic, bacterio- 
 tropic, and bactericidal properties. Kraus and Dorr consider that the chief function 
 of the serum is antitoxic ; Flexner and Jobling, Neuf eld, Jochmann, and Wassermann 
 believe that its bacteriotropic properties are its most important qualities. 
 
 The following methods for testing an immune serum are in use or have been 
 advocated; none of them is, however, sufficiently reliable to serve as a definite 
 measure of antibody content or of curative value; two of them, the hacteriotro-pic and 
 the complement-fixation test, are most widely u^ed in laboratories for the purpose of estimat- 
 ing the antibody co7itent of a serum,. 
 
 1. Bacteriotropic Titration. — While the antimeningitic serum was being prepared 
 at the Rockefeller Institute, Jobling^ used the opsonic test in standardization as the 
 method of choice, on account of the part taken by specific opsonins in promoting 
 recovery from meningococcus infections. As a definite and suitable standard of 
 strength Jobling has suggested that a serum be accepted as satisfactory when it shows 
 unmistakable phagocytic activity in dilutions up to 1:-5000. The method of Neuf eld 
 is used, as described on page 200. 
 
 2. Complement-fixation Tests. — The advantages of these tests are that the same 
 polyvalent antigen may be used as is employed for purposes of immunization; the 
 technic is simple, and the reactions are usually sharp and definite. According to 
 Sophian, in a series of comparisons with opsonic and. complement-fixation tests the 
 results corresponded in every instance, a high opsonic content being accompanied by 
 a high complement-fixation reading. The latter indicates, at least, that the horse has 
 responded to immunization and that curative antibodies probably are present. Labor- 
 atories usually adopt their own standards in preparing antimeningococcic serum. In 
 the complement-fixation test Kolle requires complete inhibition of hemolysis with 
 0.1 c.c. of serum. 
 
 The technic of these tests is given on page 491. 
 
 3. Agglutination Tests. — These tests are readily conducted with the polyvalent 
 antigen used in immunization, a macroscopic technic, as that described on page 284, 
 
 1 Hall, W.: Bull. No. 91, Hyg. Lab., TJ. 6. P, H. S., 1914. 
 
 2 Jour. Exper. Med., 1909, xi, 6, 4. 
 
THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 741 
 
 being employed. KoUe regards a serum as satisfactory" when it causes agglutination 
 of meningococci in dilutions up to 1 ; 5000. 
 
 ■i. Animal Inoculation Teste. — These tests have been found quite irregular and 
 impracticable for general use. As stated by Jobling, not only does the pathogenicity 
 of the meningococcus vary considerably from day to day, but the resistance of animals 
 to this microorganism is also quite variable. By preparing a large quantity of bac- 
 terial emulsions and using sufficient controls to determine the fatal dose, and by 
 employing a standard lethal dose of emulsions mixed with varying quantities of 
 serum injected intraperitoneally into 250-gram guinea-pigs, some conception of the 
 protective value of a serum may be obtained. 
 
 Action of Antimeningococcus Serum. — As previously stated, ex- 
 periments in vitro show tliat a potent antimeningitic serum possesses 
 three chief antibodies upon which its curative powers probably depend, 
 namely: (1) Bacteriotropins (immune opsonins), which lower the re- 
 sistance of the meningococci and facilitate their phagocytosis; (2) 
 badericidins, which kill the cocci extracellularly, either with or without 
 final lysis; and (3) antitoxins, which neutralize the true extracellular 
 toxin, which some strains of meningococci apparently produce in varjdng 
 degree. Other than these are the agglutinins, which probably aid in 
 bacteriolysis, and anti-aggressi7is, which may assist in the process of 
 phagocytosis. 
 
 Microscopic examination of a direct stained smear of the sediment of 
 cerebrospinal fluid obtained from fresh cases will show large numbers of 
 polynuclear leukocytes and cocci, the majority of the latter being ex- 
 tracellular. As the case improves, whether under serum treatment or 
 spontaneously, the microorganisms diminish in number and become 
 intracellular, frequently appearing clumped and failing to grow in 
 culture. It would appear, therefore, that a cure is brought about 
 partly by means of phagocytosis aided by bacteriotropins; by bacte- 
 riolysis through the agency of specific bacteriolytic amboceptors in the 
 immune serum and complements in the spinal fluid and blood-serum, and 
 to some extent by neutraHzation of a toxin with antitoxin. 
 
 A potent antimeningococcus serum furnishes these main antibodies, 
 and since the first two must act locally upon the cocci infecting the 
 meninges, the serum must be appUed locally and directly by intra- 
 spinous and subdural injection, since only traces of immune serum could 
 eventually find their way into the cerebrospinal fluid if the serum were 
 injected subcutaneously or intravenously. On the other hand, in the 
 treatment of meningococcus bacteremia and tqxemia the serum should 
 be injected intravenously and subcutaneously. 
 
 Unfortunately, an immune serum may not contain the antibodies 
 
742 PASSIVE rMMUNIZATION — SER^M THERAPY 
 
 for the cocci producing a given infection, and hence the scrum, even 
 though it is skilfully administered in large do.ses, will have no influence 
 upon the disease. Apparently the cocci of these resistant or "fast" 
 strains are uninjured by the antibodies in th(;-serurn. To overcome this 
 difficulty, a large number of different strains of meningococci are used 
 in immunizing horses. If, however, the sejum of one laboratory is 
 found to exert no beneficial effect, the physician should use the scrum 
 of another, for different laboi'alories probably immunize their lnjrses 
 with cultui'cs not in common use. It is highly desirable to secure cultures 
 of these "fast" strains. These should he sent at once to laboratories engayed 
 in the produciion of anlimeningitic serum, for the larger the number of these 
 strains used in immunization, the more ^potent cmd valuable will the scrum 
 be. 
 
 Administration and Dosage of Antimeningitic Serum. — In. Acute 
 Meningilis. — As a rule, sernin. should be injected, into the spinal canal as 
 early in. the disease as possible, and in such maximum am,ount us is com- 
 patible with safety. Intraspinal injection is absolutely necessary, for 
 the serum must be brought into contact witlj the infected membranes, 
 and only a trace would reach the spinal fluid if the serum were injected 
 subcutaneously (ir intravenously. The advarjtaKes of early administra- 
 tion are obvious, and if the symptoms are* indefiiute, the physician 
 should not hesitate to perform lumbar punctu're and to secure fluid for 
 microscopic examination, just as he would take a throat or nose culture 
 to aid in the diagnosis of diphtheria. The maximum, or at least an 
 adequate, amount of serum should be inject<;d, care being observed to 
 avoid undue pressure as a result of injecting too quickly or too large an 
 amount. This administration of antimeningi-tSc serum is, therefore, an 
 important and delicate, though relatively sirrfple, procedure. 
 
 1. The technic of intraspinal injection haw been descril^cd on p. 694. 
 Whenever possible, the serum should be injected by the gravity method, 
 and the amounts of fluid withdrawn and serUm injected controlled by 
 blood-]3ressure readings. 
 
 2. Lumbar puncture is performed, and tffie fluid collected in gradu- 
 ated tubes. In the ordinary case, fluid may'be allowed to drain until 
 the blood-pr<'ssure drops about 10 mm. of mercury, or if the pressure is 
 unchanged or rises, until the fluid flows abwpt one drop evt'ry three 
 seconds, provided there are no other evidences of collapse, such as faint- 
 ness, headache, and great restlessness. 
 
 3. As a rule, the amount of serum injectof] should be shghtly less 
 than the amount of fluid withdrawn. When the injection is controlled 
 
THE SERUM TREATMENT OF MENINGodoCCTTS MENINGITIS 743 
 
 by the blood-pressure readings, — the amount -wanes considerably, — usu- 
 ally the injection should stop when the pi'essure falls another 10 or 15 
 mm. For adults, the dose of serum should be about 30 c.c.; for an 
 infant, about 15 c.c. 
 
 The serum should be allowed to flow in slowly, an ordinary injec- 
 tion consuming at least ten or fifteen minutes. If symptoms of col- 
 lapse should appear before an adequate amount of serum has been 
 injected, the funnel may be lowered and the spinal fluids allowed to 
 flow out. When the symptoms have disappeared, the injections may be 
 continued and satisfactorily completed, 
 
 3. A\Tien the physician cannot administer the serum by the gravity 
 method or under blood-pressure control, the ifijection may be given by 
 means of a syringe (see p. 700). It should be given slowly, and the 
 patient observed closely in order to detect the general symptoms of 
 collapse. The amount of serum injected should not be larger than the 
 amount of cerebrospinal fluid withdrawn. A"ccording to Sophian, the 
 average doses are as follows : 
 
 Do^-t OF ANTrMt:.M.NGiTic Amuunt of Fluid 
 Seru.\]5 Withdrawn 
 
 1 to 5 years 3 to 12 g* 12 c.c. 
 
 5 to 10 years 5 to 1 5 C.c. 1.5 c.c 
 
 10 to 15 years 10 to 20 c.c. 20 c.c. 
 
 1.5 to 20 years 1 5 to 25 c.c - 30 c.c. 
 
 20 years and over 20 to 30 c.c. 40 c.c. 
 
 The injection of too large a dose of serum may be followed by head- 
 ache, pain in the back and legs, and restlessness. W hen the amount of 
 scrum injected exceeds the amount of spinal fluid withdrawn the symp- 
 toms just named must be regarded as the signal to stop; otherwise they 
 may be disregarded. 
 
 Intravenous Injection. — During epidemics of meningitis it may be 
 possible to detect cases in the bacteremic stage when meningococci are 
 present in the blood and clear fluid is collecti,ng in the ventricles. In 
 these and in all severe fulminant infections it is good practice to inject 
 from 30 to 100 c.c. of serum intramuscularly, or intravenously. It is 
 advisable to secure a culture of blood in ascites dextrose broth in all 
 cases in adults and older children. If sufficient serum may be obtained 
 and the expense is a secondary consideration,, an intravenous or intra- 
 muscular injection, given at the outset and once or twice during the acute 
 stage, may benefit the patient by neutralizing toxins and possibly pre- 
 vent complications due to the entrance of meningococci into the blood- 
 stream. 
 
744 PASSIVE IMMUNIZATION SERtJM THERAPY 
 
 Repeating Doses of Serum. — It is the general rule to give an intra- 
 spinal injection of serum every day for four days, and then on alternate 
 days until the acute symptoms have subsided, and to resume the treat- 
 ment if an exacerbation or a relapse occurs. In severe fulminant in- 
 fections, and especially if the exudate is thick and only small amounts of 
 serum can be introduced under pressure, it is well to repeat the injection 
 every twelve hours until several doses have been given. Some cases 
 require daily consecutive injections for six or more days; the average 
 case will require from four to six injections if the treatment is begun 
 during the acute stage; in the subacute and chronic cases many more 
 treatments are required. There are two main indications and guides; 
 
 1. The condition of the cerebrospinal fluid. 
 
 2. The clinical condition of the patient. 
 
 1. In most instances the cerebrospinal fluid tends to clear up macro- 
 scopically as the disease improves. This is, however, occasionally 
 misleading, as the fluid may become more turbid as the result of an 
 aseptic meningitis or excitation of a polynuclear leukocytosis due to the 
 serum, while in reality the numbers of meningococci are diminishing 
 and the patient is improving. 
 
 More accurate information is obtained by the microscopic examina- 
 tion of a stained smear of the sediment of the cerebrospinal fluid with- 
 drawn. In fresh acute cases the cocci are numerous and mostly ex- 
 tracellular; improvement is indicated by a diminution in their number, 
 and by the fact that they are mostly intracellular. I generally determine 
 the phagocytic index or the relative proportion of leukocytes that have 
 enguKed cocci and the opsonic index or the relative number of cocci per 
 leukocyte as determined by counting a large number. When many cocci 
 are present, or if they are few in number but mostly extracellular, the 
 indications are to puncture next day, even if the clinical condition of the 
 patient is good and the temperature is lower. The number and position 
 of cocci are, therefore, of more importance as a guide to subsequent 
 injections than is the total number of pus-ceUs. 
 
 2. As an indication for repeating the doses of serum the clinical con- 
 dition is of most value when combined with the examination of the 
 cerebrospinal fluid. Occasionally the patient,'$ condition may seem to 
 improve, although the fluid may show numerous cocci, which wiU sub- 
 sequently aggravate the clinical condition unless the serum is adminis- 
 tered. In favorable cases there is usually a lower temperature the day 
 following an injection, and frequently delirium^ becomes less marked and 
 there is some return to consciousness. The complexion, which is often 
 
THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 745 
 
 cyanosed at first, regains a healthy color; the pain in the head, neck, 
 and limbs becomes less severe, although the neck and spine may remain 
 stiff for several days. Finally the mind becomes clear and the patient 
 is cheerful, and no longer irritable, apathetiic, and hypersensitive. He 
 feels better and his appetite returns. When this favorable outcome 
 supervenes, the serum injections may be discontinued, to be resumed, 
 however, upon the first evidence of a relapse. The physician should be 
 on his guard for the appearance of acute hydrocephalus, which condition 
 is relieved by repeated lumbar puncture. 
 
 The Serum Treatment of Cases with Thick Elastic Exudate. — In very 
 severe cases the exudate may be so thick that it will not flow from the 
 needle. In these cases the serum should be injected in small doses under 
 pressure, and the injections repeated every eight to twelve hours. As 
 they are hkely in any case to terminate fatally, the physician is justified 
 iQ taking the risk of increasing intracranial jjressure. It may l^e well 
 carefully to inject a small amount of warm sterile salt solution, which 
 will dilute the exudate and possibly start a flow; or a second needle may 
 be inserted higher up, when a thinner exudate is found, or washing may 
 be possible by injecting salt solution in the upper needle and draining 
 ^through the lower. 
 
 The Serum Treatment of Cases with a Dry Canal and Cases of Posterior 
 Basal Meningitis. — Occasionally a patient improves clinically and the 
 amount of cerebrospinal fluid becomes very scanty, the spinal tap being 
 dry, although it is certain that the needle has entered the subarachnoid 
 space. In such instances a small amount of serum may be injected, or the 
 injection may be dispensed with if the clinical condition continues to 
 improve. If, however, cases with dry canals present evidences of 
 toxemia and general aggravation of symptoms, small doses of serum 
 should be injected under pressure and the injections repeated as often as 
 necessary. The physician must be very cautious, however, for if there 
 are clinical evidences of severe intracranial pressure, it is probable that 
 there is an encapsulation of fluid within the ventricles, and shutting off 
 of the communication between the ventricles and the subarachnoid 
 space. In this posterior basic meningitis intraspinal injections are 
 dangerous and aggravate the process. In infants it is necessary to punc- 
 ture the ventricles through the anterior fontanel and in older children 
 and adults by trephining at Kocher's point,- removing the fluid, and 
 if it is found to be cloudy or purulent, injecting serum. It may be 
 necessary to tap both ventricles alternately at intervals of several days, 
 depending upon the reaccumulation of fluid and pressure symptoms. 
 
746 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 Permanent drainage may be instituted in severe cases by means of small 
 catheters. While the operation is not usually dangerous, the ultimate 
 prognosis is very unfavorable. 
 
 The Serum Treatment of Subacute and Chronic Meningitis. — If there 
 is no evidence of sepsis; if the mind is clear and the neck limber; if the 
 general conditions are good and the cerebrospinal fluid is practically 
 cleared up, the affection is most likely hydrocephalitic, and may be 
 relieved by repeated spinal punctures, with rimoval of as much fluid as 
 is safe, using blood-pressure as an index. If meningococci are present 
 in cultures of the fluid, small amounts of serum may be injected. The 
 prognosis in these cases, however, is generally" bad, as the process is pro- 
 longed and the patient finally succumbs. 
 
 In the second form of chronic meningitis, when the meningeal 
 symptoms are active, intensified, and persistent, serum should be ad- 
 ministered every few days in the same manner and in the same dosage 
 as in the acute cases. Improvement is, however, usually temporary, 
 and the ultimate prognosis is very grave. 
 
 Serum Sickness. — Intraspinal injections of serum result in the sen- 
 sitization of the patient in just the same manner as if serum were in- 
 jected by other routes. The percentage of cases developing serum sickness 
 is likely to be high, since antimeningitic serum is not refined (Sophian 
 reports 60 per cent, of his cases as developing the condition), and while 
 the symptoms are distressing, they are seldom alarming, and fatal 
 anaphylaxis is extremely rare. Occasionally the onset of serum sick- 
 ness may be confusing, but if the meningeal condition has been respond- 
 ing as well as could be expected, it is wise to let the patient alone, rather 
 than to make additional punctures and cause further depression. Local 
 sedatives, laxatives, atropin, sedatives, and, at times, morphin, are 
 indicated. 
 
 Results of the Serum Treatment on Meningococcus Meningitis.— 
 (a) Upon the Course of the Disease. — In the majority of cases the sub- 
 dural injection of a potent antimeningitic serum is followed by some 
 immediate improvemejit in the local suppurative meningitis and general 
 sepsis, for the temperature usually drops, the mental condition improves, 
 and delirium is diminished, although the Kerriig sign may persist, partly 
 as the result of meningeal irritation and partly on account of fear. 
 Hydrocephalus is generally relieved, as indicated by lessening of the 
 pressure s>aiiptoms, as, for example, severe headache, vertigo, and 
 vomiting; breathing becomes more regular, and the pulse also becomes 
 slower and more regular. The duration of the illness is usually shortened. 
 
THE SERUM TREATMENT OF MENINGOCOCCUS MENINGITIS 747 
 
 According to Holt, in the New York epidemic of 1904-05, antedating the 
 use of serum, among 350 cases that recovered tlie duration in 3 per cent, 
 was one week or less, and in 50 per cent, five weeks or longer. Of 288 
 cases reported by Flexner and Jobling, the average duration of active 
 symptoms in those receiving serum was eleven days. Sophian, in an 
 experience of several hundred cases, reports that many acute cases were 
 relieved in five or six days and discharged as cured in two weeks. 
 
 (b) Upon Complications. — Next to its influence upon mortality, the 
 good effects of antimeningitic serum are apparent in that it lessens the 
 incidence and severity of the terrible complications of this disease. The 
 most severe and permanent sequels are those resulting from affections 
 of the internal ear and the essential structures of vision. A conservative 
 estimate of the incidence of the former among cases not receiving serum 
 treatment is 12 per cent. (Goffert), whereas Fkxner's^ analysis of 1300 
 cases treated with serum shows that deafness occurred in but 3.5 per 
 cent, of cases. At least from 12 to 24 per cent, of cases not receiving 
 serum treatment will develop more or less serious eye complications, 
 whereas Flexner's report shows that impairment of vision among serum- 
 treated cases occurred in about 0.9 per cent, of cases. The latter report 
 shows the occurrence of arthritis in but 0.9 per cent, of cases, whereas 
 among cases untreated with serum this is a frequent complication. 
 Whereas chronic meningitis is relatively common among cases treated 
 without serum, it is uncommon among those treated with serum. 
 
 (c) Upon Mortality. — The gross mortality among cases treated 
 'without serum varies from 70 to 90 per cent.; among serum-treated 
 cases the mortality is about 30 per cent. For example, of 1294 cases 
 treated with serum prepared in the Rockefeller Institute, the general 
 mortality was 30.9 per cent. 
 
 The importance of early diagnosis and prompt institution of serum 
 treatment is shown in the following table : 
 
 TABLE 30.— MORTALITY ACCOKDING TO THE PERIOD OF INJECTION 
 
 OF SERUM 
 
 
 
 MOETALITT 
 
 Per Ce-n-t. 
 
 
 Time of Injection 
 
 Flexner, 
 1294 Cases 
 
 Dopter, 
 402 Cases 
 
 Netter and 
 Debre, 99 Cases 
 
 Sophian, 
 IBl Cases 
 
 First to third day 
 
 -Fourth to seventh day .... 
 Later than seventh day . . . 
 Average mortality 
 
 18.1 
 27.2 
 36.5 
 30.S 
 
 8.2 
 14.4 
 24.1 
 16.44 
 
 20,9 
 33.3 
 26.0 
 2,'<.0 
 
 9.0 
 14.9 
 22.6 
 l.-)..5 
 
 1 Jour. ExiDer. Med., 1913, xvii, 553. 
 
748 
 
 PASSIVE IMMUNIZATION — SERtjM THERAPY 
 
 The influence of age upon mortality was early pointed out by Flexner. 
 The very high mortality in infants and in old persons is due to their 
 lowered vitality and enfeebled resistance. An additional factor in 
 young children is their greater tendency to develop extreme hydro- 
 cephalus and convulsions. 
 
 TABLE 31.- 
 
 MORTALITY ACCORDING TO AGE 
 
 
 
 ReMbted bt 
 
 
 Flexner 
 
 Dopter 
 
 Netter 
 
 Sophian 
 
 Under one year 
 
 One to two years 
 
 Two to five years 
 
 49.6 
 31.0 
 28.4 
 15.1 
 29.4 
 
 38.2 
 
 48.6 
 
 20.1 
 
 9.3 
 
 8.5 
 
 10.2 
 
 14.1 
 
 50.0 
 
 0.0 
 
 16.6 
 
 12.5 
 
 0.0 
 
 0.0 
 
 50.0 
 
 21.2 
 
 17.5 
 
 9 
 
 Ten to twenty years 
 
 Over twenty years or age 
 not given 
 
 18.0 
 32.0 
 
 The statistics show indubitably that the mortality of epidemic 
 meningitis can be greatly reduced by the administration of serum treat- 
 ment. While the ordinary type of epidemic meningitis responds best 
 to the specific treatment, the fulminant cases may also receive some 
 of the beneficial influence of the serum. To quote from what Flexner 
 wrote in 1909, and repeated in 1913: "In view of the various considera- 
 tions presented, the conclusions may be drawn that the antimeningitis 
 serum, when used by the subdural method of injection, in suitable doses 
 and at proper intervals, is capable of reducing the period of illness; of 
 preventing in large measure the chronic lesions and types of the infection, 
 of bringing about complete restoration of health, thus lessening the 
 serious, deforming, and permanent consequences of meningitis; and of 
 greatly diminishing the fatalities due to the disease." 
 
 Prophylactic Immunization in Meningococcus Meningitis. — In Chap- 
 ter XXIX mention has been made of the probable value of active im- 
 munization against epidemic meningitis by the subcutaneous injections 
 of three doses of a polyvalent meningococcus vaccine at intervals of a 
 week. Sophian and others have shown experimentally that opsonins, 
 bacteriolysins, agglutinins, and other antibodies are produced, and 
 while meningococcus meningitis is ordinarily but mildly infectious (about 
 5 per cent, of secondary cases in homes), the method is practically 
 devoid of danger and worthy of trial, especially for physicians, nurses, 
 and members of a household who are exposed to the infection over a 
 period of many weeks. 
 
THE SBKUM TREATMENT OF INFLUENZAL MENINGITIS 749 
 
 Passive immunization by means of the subcutaneous injection of 
 antimeningitic serum was advised by Jochmann in 1906, but has not 
 come into general use. The immunity resulting from the injection of 
 from 10 to 20 c.c. of serum is only temporary, and probably lasts about 
 a month. Another drawback is the resulting serum sensitization, which 
 renders subsequent injections of serum more likely to be followed by 
 serum sickness. In the presence of an activ"e epidemic, such as that 
 which occurred in Texas during 1912, immediate passive immunization 
 of physicians, nurses, and attendants by the subcutaneous injection of 
 15 c.c. of serum, followed by active immunization with three doses of 
 vaccine (500, 1000, and 1000 millions) at intervals of a week, may be 
 advisable. While there are no available statistics to prove the value of 
 this procedure, it is, at least, a rational one, and since there is danger of 
 contracting the disease, especially after prolonged contact, physicians 
 should practise immunization during epidemics of this dreaded disease. 
 In mixed passive and active immunization the serum probably affords 
 immediate protection, and tides over any temporary negative phase or 
 period of lowered resistance following the injections of vaccine. 
 
 THE SERUM TREATMENT OF INFLUENZAL MENINGITIS 
 
 Since lumbar puncture as an aid to the diagnosis of meningitis is 
 ^ coming into more general use, the important fact has been revealed that 
 the influenza bacillus is not an infrequent cause of severe, and usually 
 fatal, seropurulent cerebrospinal meningitis. In 1911 Wollstein' col- 
 lected 58 cases of this infection, all but 6 ending fatally, and as the bac- 
 terial diagnosis of meningitis is becoming more widely known and more 
 commonly employed, the number of reported cases is increasing rapidly. 
 The mortality of 90 per cent., which is exceeded only by the tuberculous 
 -^ and pneumococcus infections of the meninges, and the encouraging re- 
 sults following the use of a specific anti-influenzal scrum in experimental 
 infections in monkeys, render this subject one of great importance from 
 the standpoint of serum therapy. 
 
 Influenzal Meningitis. — Like the acute meningeal infections in 
 general, influenzal meningitis is more prevalent among children than 
 among adults. 
 
 Infection of the meninges is probably always secondary to infection 
 of the respiratory tract with virulent influenza bacilli, the route of in- 
 fection being chiefly through the blood-current. Direct infection from 
 1 Jour. Exper. Med., 1911, xiv, 73; Amer. Jour. Dis. Child., 1911, i, 42. 
 
750 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 the nose cannot be excluded, and should be considered a possibility. 
 However, all or nearly all cases of spontaneous influenzal meningitis in 
 human beings are the result of influenzal batiteremia, since the bacilli 
 have been cultivated in large numbers from the heart's blood before and 
 after death. The same is true of experimental influenzal meningitis in 
 the monkey. 
 
 According to Flexner,i the cerebrospinal fluid removed by lumbar 
 puncture from human patients is always turbid, and deposits a yellowish 
 or whitish sediment on standing. "As the disease advances, the fluid 
 becomes more heavily charged with pus-cells, until toward the end, and 
 as late as the seventh day of illness, the puncture may yield merely a 
 viscid mass of purulent matter. The number ef influenza bacilli pre- 
 sent in the fluid is usually large, and the bacilli lie chiefly extracellular, 
 among the pus-cells, although a variable but small number is usually 
 found ingested by the leukocytes. In morphology the bacilli vary 
 somewhat, and in this respect the observer may readily be deceived as 
 to the nature of the bacteria present. While some of the fluids contain 
 the typical, minute rods, others show quite ixregular and knobbed or 
 even filamentous bacteria that have little resemblance to the influenza 
 bacillus as seen in recent cultivations. These bizarre or involution 
 forms, however, are met in old and exhausted cultures; and when they 
 are recultivated on a suitable hemoglobin medium, they yield the typical 
 minute rods." 
 
 The cerebrospinal fluid removed from monkeys inoculated by sub- 
 dural injection with virulent cultures of the influenzal bacillus resembles 
 in all essential particulars the fluid removed from patients with spon- 
 taneous infections. 
 
 The baderiologic diagnosis can usually be made by microscopic 
 examination of stained smears of the fluid, but whenever possible, the 
 diagnosis should be confirmed by 'cultural methods. 
 
 Anti-infiuenza Serum. — After having satisfactorily demonstrated 
 experimental influenza meningitis in the monkey, Flexner and WoUstein 
 prepared an immune serum and showed that the experimental infection 
 could be controlled and cured by injecting the serum directly into the 
 seat of disease by intraspinal inoculation. The immune serum was 
 prepared by the ordinary methods, first a goat and then a horse being 
 injected with non-virulent and finally with virulent bacilli, covering a 
 period of many months, until their serums showed the presence of 
 agglutinins and bacteriotropins. The serum lacked bacteriolytic prop- 
 1 Jour. Amer. Med. Assoc, 1913, Ixi, 1872. 
 
THE SERUM TREATMENT OF PNEUMOCOCCUS MENINGITIS 751 
 
 erties, and did not give rise to complement fixation in dilutions greater 
 than 1 : 100. 
 
 Following the administration of serum, the cerebrospinal fluid tends 
 to clear up ; the bacilli become fewer in number and are mostly ingested 
 by the phagocytes; the pus-cells become less numerous, and while the 
 bacUli may persist in the fluid for a longer period, they ultimately dis- 
 appear. 
 
 Administration of Anti-infiuenza Serum. — The Rockefeller Institute 
 has distributed serum throughout different parts of the country, and is 
 prepared to furnish it to physicians upon request. Physicians, and 
 especially pediatrists, should resort to lumbar puncture early in all sus- 
 pected cases of meningitis, for only in this manner may influenzal 7nenin- 
 gitis be detected early enough to derive any possible benefit from, serum treat- 
 ment. The serum should be injected directly into the s-pinal canal by the 
 gravity or syringe method in exactly the same manner and uyith the same 
 precautions as are observed- in administering serum in the treatment of 
 epidemic meningitis. Since the disease is usually accompanied by a 
 bacteremia, it is well to inject serum intravenously , although serum in- 
 jected intraspinally soon finds its way into the blood-stream. All data, 
 including the clinical history and the records of bacteriologic examina- 
 tions of cerebrospinal fluid and blood, the amounts of serum injected, 
 and the results obtained, should be sent to the Director of the Rocke- 
 feller Institute. 
 
 THE SERUM TREATMENT OF PNEUMOCOCCUS MENINGITIS 
 ^Meningitis is caused more frequently by the pneumococcus than by 
 the influenza bacillus. Its mortality is certainly no less than in influ- 
 enzal meningitis. 
 
 The few instances in which antipneumoqoccic serum has been em- 
 ployed have not j'ielded results that inspire confidence in its employ- 
 ment alone. As will be emphasized later,, in considering the serum 
 treatment of pneumonia, an antipneumococcuif^serum is at best active only 
 against the homologous organism or organisms, the types of which have been 
 employed in its preparation. Even when the homologous serum is used 
 in treating experimental pneumococcus meningitis in monkeys, the 
 fatal termination may be delayed, but is not prevented. For this 
 reason the outlook for its successful emplo^mient alone in human in- 
 fections is not encouraging. Recent investigations of Lamar ^ have 
 1 Jour. Exper. Med., 1911, i; i'M., .380; xiy, L'.')6; 1912, xvi, .iSl. 
 
752 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 shown, however, that mixtures of homologous" antipneumococcus serum, 
 sodium oleate, and boric acid exert a marlied=and decided curative in- 
 fluence upon a virulent experimental meningitis, and while this method 
 has not thus far been generally applied in the treatment of the disease 
 in humans, it is deserving of trial and offers considerable encouragement 
 for an otherwise highly fatal infection. 
 
 Pneumococcus Meningitis.^ — This infection is usually secondary, 
 and follows on pneumonia or on inflanmiations of serous membranes by 
 indirect transmission by the blood or by direct infection from the naso- 
 pharynx, mastoid cells, frontal, sphenoid and ethmoid sinuses, and 
 internal ear. 
 
 The diagnosis is usually made as the result of microscopic examination 
 of stained smears of cerebrospinal fluid removed by lumbar puncture. 
 Large numbers of poljmuclear leukocytes with intracellular and extra- 
 cellular Gram-positive diplococci, occurring in pairs or in short chains, 
 usually indicate a pneumococcus infection. Whenever possible, the 
 diagnosis should be confirmed by making cultures of the fluid on dextrose 
 blood agar, and by injecting portions intraperitoneally and subcutane- 
 ously in mice. 
 
 Pneumococcus infections of the cerebral meninges have been found 
 experimentally to be more refractory to treatment than infections of the 
 spinal meninges, hence human infections following injuries to the head, or 
 occurring as the result of direct extension from neighboring sinuses, are 
 likely to be more refractory than indirect infections by way of the blood. 
 
 Serum Treatment. — Numerous investigations by Conradi,i Korschun 
 and Morgenroth,^ Levaditi,^ and Noguchi* have shown that substances 
 may be obtained directly from tissue-cells and4eukocji;es or after auto- 
 lysis which are bactericidal and hemolytic, and, as shown by Noguchi, 
 are largely in the nature of higher saturated fatty acids or their alkaline 
 soaps. (For an account of their similarity to= complements see Chapter 
 XVIII.) As shown by Klotz,'^ soaps occur in inflammatory foci, and the 
 origin of the fatty acids and soaps is readily accounted for since Achaline^ 
 has shown the presence of lipase in such foci. With the death of leuko- 
 cytes in an inflammatory focus, brought about by a bacterial poison, 
 leukocidins, or lack of nutriment, disintegration occurs, and by auto- 
 
 1 Beit. z. chem. Phys. u. Path., 1902, i, 193. 
 
 2 Berl. klin. Wochenschr., 1902, xx-xix, 870. 
 
 3 Ann. de I'Inst; Pasteur, 1903, xvii, 187. 
 
 ^ Biochem. Zeitsohr., 1907, vi, 327. = Jour. Exper. Med., 1905, vii, 633. 
 
 « Compt. rend. Soc. de biol., 1899, li, 568. 
 
THE SERUM TREATMENT OF PNEUMOGOCCUS MENINGITIS 753 
 
 lysis and lipolysis fatty acids and soaps are produced that in themselves 
 seem to exert a destructive action upon the infecting bacteria. 
 
 With these considerations in mind, Lamar investigated the influence 
 of soaps upon pneumococci. Solutions of 0.5- to 1 per cent, of sodium 
 oleate were found rapidly to kill pneumococci; much weaker solutions, 
 as, e. g., 0.1 per cent., or even 1 part of soap in 10,000 parts of water, 
 were found to lessen their virulence, and, what is more important and 
 significant, to render the organisms peculiarly susceptible to lysis by a 
 homologous antipneumococcus serum. 
 
 A serious drawback to the application of these discoveries was that 
 protein constituents of serum and exudates were found to inhibit the 
 bacteriolytic and hemolytic action of unsaturated fatty acid soaps, as 
 was shown by Noguchi ^ and then by von Liebermann.^ The latter and 
 von Fenyvessy' later found that this inhibition can be prevented in the 
 test-tube and also in the animal body by adding a minute quantity of 
 horic add, which prevents the union of soap and protein matter when the 
 latter is not too greatly in excess. 
 
 The experiments of Lamar with mixtures of homologous antipneu- 
 mococcus serum, sodium oleate, and boric acid in the treatment of pneu- 
 mococcus meningitis in the monkey have yielded excellent results, 
 especially when used early in the infection. These experiments have 
 proved that sodium oleate lowers the virulence of pneumococci and 
 renders them peculiarly and highly susceptible to solution by bacter- 
 iolysins present in the serum, and that boric a'cid largely prevents the 
 inhibitory action of protein constituents upon this sensitizing action of 
 sodium oleate. 
 
 Practical Applications. — One drawback to the use of this method in 
 the treatment of human infections is the necessity of using an anti- 
 pneumococcus serum corresponding to the organism causing the infec- 
 tion. In the Rockefeller Hospital a method has been worked out where- 
 by the type of infection is quickly determined by agglutination reactions. 
 Fortunately, the number of types of pneumococci are relatively few, 
 and it is to be hoped that an efficient polyvalent serum will soon be made 
 available by the Rockefeller Institute. Until this desideratum is 
 attained, the commercial serums at present on the market may be used, 
 with the addition of sodium oleate and boric acid. 
 
 It is highly desirable that the treatment be administered as early as 
 possible, when the exudate is largely serous or at most seropurulent. 
 
 Loc. cit. ' Biochem. Zeitschr., 1907, iv, 25 
 
 ' Zeitschr. f. Immunitatsforsch., orig., 1909, ii, 436. 
 48 
 
754 PASSIVE IMMUNIZATION — SERUM THERAPY 
 
 The mixture should be injected into the spinal canal after the with- 
 drawal of the fluid by the gravity or syringe method and under blood- 
 pressure control, as previously described. The amount injected and the 
 number of injections depend upon the clinical condition of the patient, 
 and in general may be administered in the same way as is antimenin- 
 gitic serum. 
 
 The initial dose may be 20 c.c, and is prepared as follows: 
 
 Antipneumococcus serum (sterile) , 4 c.c. 
 
 5 per cent, aqueous solution of boric acid (sterile) 15 c.c. 
 
 2 per cent, aqueous solution of sodium oleate (Kahlbaum's 
 or Merck's) (sterile) » 1 c.c. 
 
 THE SERUM TREATMENT OF OTHER LOCALIZED PNEUMOCOCCUS 
 
 INFECTIONS 
 
 Lamar has also suggested that mixtures of 9,ntipneumoeoccus serum, 
 
 sodium oleate, and boric acid may be used in the treatment of pncumo- 
 
 coccus pleuritis, peritonitis, or other localized infections, such as arthritis 
 
 and sinusitis, where the exudate may be removed and the serum be 
 
 brought into contact with the infected tissues. 
 
 The Serum Treatment of Pneumonia 
 Acute lobar pneumonia, with its clear-cut clinical course, unsatis- 
 factory and difficult treatment, uncertain prognosis, and high mortality, 
 was one of the diseases in which the earliest effprts were directed towiud 
 discovering a specific serum therapy. Sinc^ the pioneer work of the 
 Klemperers in 1891, numerous investigators have prepared serums that 
 have yielded either indifferent results or prove(jl benefirial in but a limited 
 number of cases, so that there has been no well-established form of 
 specific therapy. 
 
 Recent investigations by Neufeld and Handel ^ in Germany, and by 
 Dochez,^ Cole,3 and Gillespie^ in the Rockefeller Institute, have dis- 
 closed several , reasons for the failure of serum therapy in pneumonia, 
 and have emphasized the importance of the fpllowing factors: 
 
 1. The serum should correspond to the type of pneumococcus causing 
 the infection. 
 
 1 Zeitschr. f. Immunitatsforsch. 1909, iii, 159; Arb. a. d. k. Gesundheitsamte, 
 1910, xxxiv, 169; ibid., 1910, xxxiv, 293; Berl. klin. Wochenschr., 1912, xlix, 6S0. 
 
 2 Jour. Exper. Med., 1912, xvi, 665, 680, and 693; Jour. Amer. Med. Assoc, 
 1913, Ixi, 727. 
 
 3 Jour. Exper. Med., 1912, xvi, 644; Arch. Int. Med., 1914, xiv, 56. 
 
 " Jour. Amer. Med. Assoc, 1913, Ixi, 727; Jour. Exper. Med., 1914, xix, 28. 
 
SERUM TREATMENT OF LOCALIZED PNEUMOCOCCUS INFECTIONS 755 
 
 2. The serum must be administered in large doses, and preferably 
 intravenously. 
 
 3. To be most effective the treatment should be given as early as 
 possible. 
 
 The investigators in the Rockefeller Institute have divided the 
 pneumococci causing lobar pneumonia into four main groups, and have 
 worked out a method for the rapid identification and classification of the 
 particular pneumococcus that is the etiologic factor in a given infection, 
 so that with the proper administration of the corresponding immune 
 serum very encouraging results have been obtained in the serum treat- 
 ment of pneumonia. These researches are of importance not only in 
 this connection, but also from the fact that thSy may have disclosed the 
 reasons for failure in the treatment of streptococcus and other infec- 
 tions, and that similar studies in these conditions may insure for serum 
 therapy a definite and valuable role in the treatment of disease. 
 
 The Nature of Lobar Pneumonia. — The frequency with which the 
 Diplococcus pneumonicE is found in the local lesion and in severe cases in 
 the blood-stream of pneumonia patients, and the more recent experi- 
 mental studies of Wadsworth,^ Meltzer,^ Wollstein and Meltzer,- Win- 
 ternitz, Kline and Hirschfelder,^ leave little doubt regarding the etio- 
 logic relationship of this microorganism to lobar pneumonia. Much still 
 remains to be learned, however, regarding the methofl of infection and 
 the nature of the resulting disease. While pneumococci are to be found 
 living in the upper air-passages as harmless parasites, it is probable that 
 those causing infection differ inherently as regards adaptation or viru- 
 lence for man. In addition, it is likely that general resistance is low- 
 ered in some more or less peculiar manner, and experimental studies 
 in animals, as well as the course of the disease in man, suggest most 
 strongly that local changes in the respiratory tract may precede the 
 infection, so that a combination of factors, such as the virulence of the 
 organisms and the diminished general and local resistance, plays a part 
 in the production of lobar pneumonia. 
 
 In whatever manner produced, the disease is finally to be regarded 
 as a general infection, with localization of the process in the lung. While 
 pneumococci may be found in the blood of the most severe cases, the 
 general symptoms are apparently due to intokication with a poison or 
 toxin derived primarily from the pneumococci, and secondarily from the 
 
 ' Amer. Jour. Med. Sri., 1904, exxvii, S51 . ' Jour. Exper. Med., 1912, xv, 133. 
 
 'Jour. Exper. Med., 1913, xvii, :!.53, RWR; ibid., 1Q13, xviii, 54H. 
 'Jour. Exper. Med., 1912, x-vii, 6.57; ibid., 1913, xviii, 50. 
 
756 PASSIVE IMMUNIZATION SERTJM THERAPY 
 
 exudate in the local lesions. The studies of Rowntree/ Medigreceanu,^ 
 and Peabody,' showing chloiin retention; ol Peabody,'' showing pro- 
 gressive loss in the oxygen-combining power of the hemoglobin, due to 
 the formation of methemoglobin ; of Medigrecaanu,^ showing a deficiency 
 of oxydase or lessened power of the tissues to "carry on proper oxidation, 
 and of Neufeld and Dold,^ Rosenow,' Cole,^ Jobling and Strouse,' 
 indicating the presence of endotoxins within the pneumococci — all these 
 support the view that in pneumonia there is 'well-marked intoxication, 
 and this, in addition to the effects of the local pulmonary consolidation 
 on the heart, respiration, and nervous system, constitute the main fea- 
 tures of the infection. 
 
 Regarding the mechanism of recovery from pneumonia, there is 
 little definite information. The recent studies of Neufeld, Dochez, and 
 Clough indicate that antibodies are produced at or about the time of the 
 crisis, and that these are probably responsible' for the destruction of the 
 bacteria in the circulating blood, and, to a greater extent, in ,the local 
 lesion. In the resolution of the local lesion it is probable that ferments 
 play an important part. That resolution does not occur earlier may be 
 due to the overbalancing of the leukocytic ferments by the antiferments 
 of the serum, and the lytic ferments become active only when they reach 
 a point of excess over the antiferments, causing a solution of the fibrin, 
 relieving tension, and affording an outlet for" the exudate. According 
 to Vaughan, the pneumococci may be considered as furnishing a ferment 
 that brings about the production of a specific antiferment, capable of 
 reacting upon its substratum, the ferment and the new bacterial tissue, 
 and causing its destruction by a process of solution. Pneumococci in 
 the resolving lesion are probably destroyed by leukocidins released 
 through disintegration of leukocjrtes, by fatty, acids, and probably by 
 antibacterial substances in the blood. 
 
 While it is true that immunity does not usually follow an attack of 
 pneumonia, and, indeed, the patient is apparently hypersusceptiblc, it 
 has been found experimentally that the antibodies are highly specific 
 for the particular organism causing an infection. Reinfection is, there- 
 fore, possible with an organism belonging to another group, and lia- 
 
 1 Bull. Johns Hopkins Hosp., 1908, xix, 367. ^ jg^r. Exper. Med., 1911, xiv, 289. 
 
 3 Jour, Exper. Med., 1913, xvii, 71. " Jour. Exper. Med., 1913, xviii, 7. 
 
 6 Jour. Exper. Med., 1914, xix, 309. 
 
 «Berl. klin.Wochensohr., 1911, xlviii, 1069, 
 
 ' Jour. Infec. Dis., 1911, ix, 190. s jour. Exper. Med., 1912, xvi, 644. 
 
 ' Jour. Exper. Med., 1913, xviii, ,597. 
 
SERUM TREATMENT OF LOCALIZED PNEUMOCOCCUS INFECTIONS 757 
 
 bility to reinfection may be increased because of lowered local and gen- 
 eral resistance due to the previous attack. 
 
 Antipneumococcus Serum. — The indications of specific serum 
 therapy, are, therefore, mainly twofold: first, to destroy any pneumo- 
 cocci present in the blood and in the local lesion; or if the latter is im- 
 possible because of mechanical obstacles that interfere with the circula- 
 tion and prevent access of the antibodies to the-cocci, to at least prevent 
 extension of the lesion by preventing the multiplication of organisms at 
 its margin; second, to neutralize the toxins produced during the course 
 of the disease. 
 
 It would, of course, be highly desirable to have at our command a 
 serum that would cause solution of the local exudate and bring about a 
 crisis and a cure. It is hardly reasonable to expect, however, that a 
 serum can be produced that will contain digestants for fibrin and leuko- 
 cytes. The local lesion is most likely to be harmful because of the toxic 
 substances that emanate from it, and not because so large an area of 
 lung is temporarily incapacitated and the heart? embarrassed. A serum 
 that will prevent general bacteremia, limit the extension of the local 
 lesion, and neutralize the toxins while nature is preparing to react upon 
 the exudate with a ferment, is probably fulfilling all that may be ex- 
 pected of a specific serum therapy. 
 
 Groups of Pneumococci. — Neufeld and Handel have shown that an immune 
 serum produced by the injeotioii of a given variety of pneumococci into an animal 
 was not effective against all forms of pneumococci. In the Rockefeller Hospital a 
 serum, known as Serum 1, prepared by immunising a horse with a culture obtained 
 from Neufeld, was found to protect against only about one-half the types of pneu- 
 mococci (Group 1) studied. By immunizing rabbits tcr each of the types that were 
 not acted upon by Serum 1, and testing the immune serum against all strains by 
 cross-agglutination and by cross-protection experiments^ it was found that a number 
 of the serums possessed the same properties, thus indicating that their respective 
 cultures belonged to the same general group (Group 2). By immunizing a horse with 
 one of these, Senun 2 was produced. In Group 3 are placed all the organisms of the 
 so-called Pneumococais mucosus type. In Group 4 are included all the varieties of 
 pneumococci against which Serums 1 and 2 arc not effective, and which, from their 
 other properties, do not belong in Group 3. Animals may readily be immunized to 
 any member of this Group 4, and the serum of the immunized animal is protective 
 against the strain used for immunization, but in no instance has this serum been 
 found effective against any other member of this group or against the organisms of 
 the other groups. While no cultural or morphologic differences between the members 
 of Group 1, 2, and 4 exist, it has been found possible to group them by the aggluti- 
 nation reaction in exactly the same maimer as by protection experiments. Of 
 24 strains studied in the Rockefeller Hospital, 47 per cent, belonged to Group 1, 
 18 per cent, to Group 2, 13 per cent, to Group 3, and 22 per cent, to Group 4. 
 
 Determining the Type of Pneumococcus. — For this purpose, Cole has given the 
 following method: "When a patient with pneumonia i^ admitted to the hospital, a 
 
758 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 culture is immediately made from the blood and also one from a portion of sputum 
 coughed up from the lung, or, when this ia not obtainable, a culture is made directly 
 from the lung by the insertion of a needle. This procedure seems to be without 
 . danger. When there are large numbers of organisms in the sputum, a culture may 
 be obtained most rapidly by injecting the washed sputum into the abdominal cavity of 
 a mouse. After four or five hours the peritoneal cavity may be washed out with salt 
 solution and the cells thrown down in the centrifuge; a suspension of the organisms 
 is thus obtained. In whatever way the culture is obtained, the agglutination test 
 is at once applied. If the organism fails to agglutinate with either Serum 1 or Serum 
 2, it is, of course, useless to undertake serum treatijient. If, however, one of the 
 serums agglutinates the organism, treatment may be commenced at once with the 
 appropriate one." 
 
 Preparation of Antipneumococcus Serum. — Horses are immunized with dead 
 and then with virulent living cultures. In \iew of the fact that the serum should 
 possess some antitoxic value, it is desirable that the animals be immunized also with 
 autolysates. The whole process may be conducted after the method described for 
 the production of antimeningococcus serum. 
 
 In the Rockefeller Institute diiTerent horses are immunized with strains belong- 
 ing to Groups I and 2. It would appear possible to produce a potent polyvalent 
 serum, and this is very much to be desired, especially if further studies continue to 
 show that li.j per cent, of infections are caused by organisms belonging to these two 
 groups. 
 
 Kolle, by immunizing horses with cultures secuTed from pneumonia patients, 
 produces an antipneumococcic serum. These cultures are grown in broth for forty- 
 eight hours, heated to 60° C, and 5 c.c. injected intrp,venously into a horse. The 
 dose is increased each week until 120 c.c. are given at one time. Then 5 c.c. of living 
 culture is injected, and the doses increased in a similar manner until 120 c.c. are given 
 at one time. About six months are consumed in the process of immunization, and 
 two weeks after the last injection has been given the sgrum is tested. 
 
 Standardization of Antipneumococcus Serum. — As previously mentioned, there 
 is at present no accurate method for standardizing an antibacterial serum. It 
 is possible, however, to obtain some measure of its protective and curative power by 
 employing various tests. 
 
 1. Protective Value. — The lethal dose of a living pneumococcus culture for mice 
 is determined, and from 10 to 100 times this amount of culture is mixed with decreas- 
 ing doses of immune serum and the mixtures injected subcutaneously or intraperitone- 
 ally into a series of mice in order to determine the dose of serum that will protect. 
 Dochez has found that when these mixtures are injected at once and in the same place, 
 the seiTim will obey the law of multiple proportions up to a certain limit. 
 
 Merck's antipneumococcus serum is so standardized that 0.01 c.c. injected 
 subcvitaneously protects a mouse inoculated intraperitoneally twenty-four hours 
 later with from 10 to 100 times the lethal dose of a living virulent culture. This is 
 known as a normal serum, 1 c.c. containing an iirununity unit (I. U.). The serum is 
 marketed in vials containing from 20 to 40 c.c. Kolle regards as satisfactory a serum 
 that, in doses of 0.001 c.c. and less, will protect mice. 
 
 2. Bacivriolropic Value. — Neufeld lays considerable stress upon this point. The 
 technio of this titration has been described elsewhere. 
 
 3. Com.ple7nent-fixation and Agglutination Testa. — While these tests are fre- 
 quently sharply cut, and while they serve as a measure for record in the laboratory, 
 they do not necessarily indicate the therapeutic value- of the serum. 
 
SERUM TREATMENT OF LOCALIZED PNEUMOCOCCXJS INFECTIONS 759 
 
 Action of Antipneumococcus Serum. — The curative and protective 
 value of this serum depend mainly upon bacteriolysins, bacteriotropins, 
 and antitoxins. The first are readily demonsti-ated ill protection ex- 
 periments and also in pneumonic patients when pneumococci in the 
 blood-stream are destroyed. Bacteriotropins may likewise be demon- 
 strated experimentally and in the blood of patients if the corresponding 
 organism is used in the tests (Neufeld, Strouse). The antitoxic prop- 
 erties are shown clinically and also in ritro by neutralization of the hemo- 
 toxic poison obtained by dissolving pneumococci in bile. 
 
 Administration of Antipneumococcus Seriun. — To obtain the best 
 results, the serum should he given as early as possible and intravenously. 
 In young children, when the giving of an intravenous injection is quite 
 difficult or impossible, the muscles of the buttocks should be substituted. 
 Certainly small doses given subcutaneously are almost devoid of effect. 
 The procedure in use in the Rockefeller Institute consists in injecting 
 0.5 c.c. of serum subcutaneously to discover if hypersensitiveness exists 
 and to produce anti-anaphylaxis. As soon as the type of organism has 
 been determined, from 50 to 100 c.c. of the serum, diluted one-half with 
 salt solution, are injected intravenously. The condition of the patient 
 serves as a guide in the later treatment. Usually the serum is not ad- 
 ministered oftener than once every twelve hourg. 
 
 It has been shown experimentally that, in the presence of a maximum 
 degree of infection, no amount of serum, however large, is effective. 
 This suggests that the body must furnish a second substance to act with 
 the antibodies in the serum, and indicates the early administration of 
 serum before the infection has reached too extreme a grade. I would 
 also suggest that the body may be deficient in bacteriolytic complements, 
 and that the effect of a serum may be enhanced by adding fresh sterile 
 guinea-pig serum — say 5 c.c. to each 100 c.c. of immune serum — just 
 prior to administration. 
 
 Results in the Serum Treatment of Pnemnonia. — It is hardly neces- 
 saiy to review the numerous reports that have been made in past years, 
 because in most instances the serum was administered subcutaneously 
 and in too small doses to be of value, even granting that it contained 
 antibodies for the particular infection. Of 23 patients, all seriously ill, 
 treated in the Rockefeller Institute during the past year, the results 
 were as follows: Of 15 cases due to pneumococcus 1, all recovered but 
 one — a mortality of 6.6. per cent., as compared with the mortality of 
 24 per cent, among 34 patients not receiving serum treatment; of 8 
 cases due to Type 2, all recovered but two, one of these refusing to con- 
 
760 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 tinue the treatment — a mortality of 25 per cent, as compared to 61 per 
 cent, among 13 patients not receiving serum. 
 
 Aside from this decided influence upon mortahty, the general effects 
 of the serum were good. In 10 cases pneumococci were isolated from 
 the blood before the treatment was begun. In all these patients the 
 blood had become sterile after the first treatment. Following the in- 
 jection of serum all the patients seemed to feel better, and in a number 
 of them there was an apparent lessening in the degree of intoxication. 
 While in no case was one injection sufficient to bring about a crisis, in 
 all except the fatal cases the serum had apparently an ultimate favorable 
 effect, lowering the temperature and shortening the course of the disease. 
 
 THE SERUM TREATMENT OF STREPTOCOCCUS INFECTIONS 
 
 The acute character of streptococcus infections and their relative 
 frequency and severity have made them the subject of numerous efforts 
 on the part of various investigators toward developing an efficient serum 
 therapy. To Marmorek belongs the credit of first attempting, in 1895, 
 to prepare a curative serum on a large scale. Since then Aronson, 
 Tavel, Krumbein, Moser, Meyer-Ruppcl, Menzer, and others have pre- 
 pared immune serums with various cultures and according to various 
 methods. While many antistreptococcus serums will show undoubted 
 protective value, especially against their horaologous cultures as tested 
 in experimental animals, the general opinion regarding their curative 
 value in streptococcus infections of man have been conflicting and as a 
 rule unfavorable. Occasionally the rapid improvement of a patient 
 following an injection of the serum would indicate that it has proved 
 beneficial, and the same is occasionally true of a particular group of 
 infections treated with a specially prepared serum. The tendency of 
 acute streptococcus infections to end spontaneously by crisis must, 
 however, be borne in mind, and the good result observed in individual 
 cases may be coincident with, rather than the result of, the administra- 
 tion of the serum. 
 
 Several causes for the failure of antistreptococcus serum therapy 
 are now understood, and if these can be eliriiinated, the value of this 
 form of therapy will be greatly augmented. 
 
 1 . The serum should be given in large doses, and hy intramuscular and 
 intravenous injection. In a true streptococcic infection the cocci are 
 likely at some time to be found in the blood-stream, and an attempt to 
 destroy these organisms or to limit a local infection by injecting 10 c.c. 
 
THE SERUM TREATMENT OF STREPTOliOCCTJS INFECTIONS 761 
 
 , of seram in the subcutaneous tissues is almost sure to result in failure. 
 As in the serum treatment of pneumonia, at least 100 c.c. of serum 
 should be given intravenously and the dose repeated if necessary. 
 
 2. The scrum should be used as early in the disease as possible, 
 instead of waiting until the patient has become moribund. In puerperal 
 sepsis and scarlet fever, for instance, the question of serum therapy 
 should be considered early, for when properly administered, the serum 
 will at least do no harm and may prove efficacious. In order to determine 
 the value of the serum a bacteriologic diagnosis should always be at- 
 tempted, especially by means of blood cultures obtained by placing from 
 2 to 5 c.c. of blood in a flask containing at least 100 c.c. of dextrose broth 
 just prior to injecting the serum. 
 
 3. The serum should be -polyvalent. Mariiorek maintains that all 
 streptococci are alike, and he has, accordingly, prepared his serum from 
 a single highly virulent strain. Other investigators question this asser- 
 tion, and at present the consensus of opinion is agreed that streptococci 
 from different infections, such as puerperal sepsis, scarlet fever, ulcer- 
 ative endocarditis, and erysipelas, exhibit certain immunologic differ- 
 ences, even though their biologic and morphologic characters are quite 
 similar. Thus far no adequate methods for differentiating between 
 these organisms have been discovered, but it is likely that future re- 
 searches will show that streptococci from different infections, and even 
 from cases of scarlet fever, possess different immunologic characters 
 similar to the variations observed among the pneumococci causing lobar 
 pneumonia. If this is found to be true, under these conditions, a sim- 
 ilar serum treatment, while complicated, is likely to prove valuable in 
 the treatment of streptococcus infection. For the treatment of strep- 
 tococcus infection in scarlet fever the serum should be prepared of nu- 
 merous strains isolated from patients having this disease. What has just 
 been said is also true of the other three infections so frequently strepto- 
 xoccal, namely, puerperal sepsis, phlegmonous cellulitis, and ulcerative 
 endocarditis. If not these four, at least two antistreptococcus serums 
 should be available: one for scarlet fever and the other for other infec- 
 tions; both, and especially the latter, should be prepared by immuniz- 
 ing horses with a large number of various strains. 
 
 Mode of Action of Antistreptococcus Serum. — Virulent strepto- 
 cocci exert a powerful negative chemotactic influence upon leukocj^es, 
 repelling them and effectively resisting phagocytosis for varying periods 
 of time. The early researches of Bordet showed that antistreptococcus 
 serum neutralizes this influence and promotes phagocytosis. Since 
 
762 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 then numerous investigators have supported Bordet's findings, so that 
 it may be accepted as true that one of the chief antibodies in antistrep- 
 tococcic serum is of the nature of a bacteriotropin or immune opsonin. 
 A potent serum also contains an antitoxin, as may be shown experi- 
 mentally by neutralization of the hemotoxic poison of streptococci, 
 and also clinically, when the rapid subsidence' of fever and general im- 
 provement of the patient are probably due, in-part, to neutralization of 
 streptococcal toxins. Thus far the presence of bacteriolysins has not 
 been definitely proved, although they may be present and operative 
 in vivo. I have found that antistreptococcus serum contains an anti- 
 body capable of fixing complement with streptococcus antigens. -"^ It 
 may be stated, therefore, that the action of antistreptococcus serum is 
 dependent primarily upon bacteriotropins, and secondarily upon anti- 
 toxins and, possibly, bacteriolysins. 
 
 Preparation of Antistreptococcus Serum. — Some differences of opinion have 
 been expressed regarding the advisability of passing cultures that are being used 
 for purposes of immunization through a lower animal in order to increase their \ iru- 
 lence. For example, the unsatisfactory results that have followed the use of Rlar- 
 morek's serum have been ascribed not only to the fact that it is monovalent, but also 
 to possible alteration of the strain in its biologic characteristics by animal passage, so 
 that its vinilence for the human being was diminished or lost, and, accordingly, 
 while the antiserum is protective for the animals through which the passage has been 
 conducted, it is inactive for the human being. Tavel,. I-iJumbein, and Paltauf have 
 prepared polyvalent serums with different strains from human infections without 
 animal passage. JMenzer has prepared a serum with strains of cocci derived from acute 
 rheumatic fever, and Moser with strahis obtained froni scarlet fever, wliich have also 
 not been passed through animals. Aronson has attempted a combined procedure, 
 making use of passed and unpassed cultures conjointly, and this appears to be the 
 method of choice. In other words, those who prepare £*itistreptococcus scrums should 
 use as many fresh strains as possible, and in several of the older cultures the virulence 
 should be increased from time to time by passage through animals. 
 
 In preparing the serum young and healthy horses should be used. The injections 
 should first commence of dead cultures given subcutaneously, then of autolysates, 
 and finally of living cultures administered intravenously. Occasionallj' severe local 
 and general reactions are observed, and the whole procedure should be conducted 
 under careful supervision. 
 
 First Method, — Cultures are grown on a solid medium, and an emulsion and 
 autolysate prepared as described for immunizing with meningococci. Begin by 
 injecting subcutaneously 5 c.c. of emulsion heated to 60° C. for an hour, and in- 
 creasing the dose each week by 5 c.c. until 100 c.c. are given at one time. If the 
 reactions are mild (general and local), the doses may be increased more rapidly. Then 
 begin with 2 c.c. of living culture and increase the dose each week, '\\lien a dose of 
 10 c.c. is reached, inject with autolysate and living cultures alternately, gradually 
 increasing the dose until a dose of 50 c.c. is reached. Living cultures are then given 
 intravenously and the dose rapidly increased until 100 c.c. and more arc given at 
 
 1 Arch, of Int. Med., 1912, ix, 220. 
 
THE SERUM TREATMENT OF STREPTOGOCCtJS INFECTIONS 763 
 
 one time. Intravenous injections arc not infrequently tolerated better than sub- 
 cutaneous injections. The horses may be bled severa-1 times during the course of 
 immunization and their serums tested. AA'hen the serum is to be used therapeutically, 
 the animals should not be bled in less than from ten to fourteen days after the last 
 injection was given. In view of the large doses required, a concentrated serum is 
 advisable (Heinemann and Gatewood'). Since trikresol has an inhibiting influence 
 on phagocytosis ("Weaver and TuimiclifP), the minufial quantity (0.2 per cent, or 
 less) should be used, or preferably no preservati^•e at 3.11. 
 
 Second Method. — KoUe prepares a polyvalent serum with cultures derived from 
 cases of erysipelas, puerperal sepsis, scarlet fever, etc. Their viruJence is increased 
 from time to time by passage through rabbits. 
 
 Horses are used for immunization purposes and all injections are given intra- 
 venously at intervals of a week. Cultures are grown on test-tubes containing a solid 
 medium, and immunization is started with half a culture, heated. The dose i.s 
 increased each week with an additional culture until it equals 16 cultures. Then 
 li\ing and killed cultures are mixed, giving in one week 2 living and 14 killed cultures 
 and so on until 10 living and 6 killed cultures are given; at a single dose. The horse.s 
 are bled two weeks after the last dose is administered. 
 
 Standardization of Antistreptococcus Serum. — There is at present no single 
 satisfactory method for standardizing these serums, although a satisfactory method 
 is a desideratum for testing serums placed on the market. In order to obtain an 
 approximate idea as to the value of a serum, the following tests may be employed: 
 
 1. Protective Value. — At the Serum Institute in Vienna a passed ciilture (one 
 used in the process of immunization) is selected, and th'kt dose which will kill a mouse 
 at the expiration of or just preceding the end of four days is regarded as a single lethal 
 dose. In testing an antiserum 10 times this quantity of culture is used with decreasing 
 doses of immune serum injected twenty-four hours previously or simultaneously. 
 A normal serum is one of which 0.01 c.c. will afford protection, and 1 c.c. of such a 
 serum is said to be one immunity unit, i. e., it affords protection against 1000 lethal 
 doses of culture. 
 
 2. Bacteriotropic Value. — The technic of Wright or Neufeld may be employed 
 with a virulent culture and human leukocytes. Weaver and Turmicliff have obser\^ed 
 better results when using one part of immune serum reactivated with nine parts of 
 fresh guinea-pig serum. 
 
 3. Complement-fixation Tests. — These tests may be employed with the bacterial 
 emulsion or autolysate used in immunization as the antigen. 
 
 The serum should be kept in a cool, dark place. After a few months it loses 
 some of its protective value, and much of it on the market is worthless. 
 
 Administration of Antistreptococcus Serum. — It must be emphasised 
 here that, in order to obtain the best results, antistreptococcus serum must 
 he given intravenously. In an adult patient with a severe general infec- 
 tion from 30 to 100 c.c. of serum, diluted with an equal amount of sterile 
 normal salt solution, may be given in one dose. If improvement follows, 
 subsequent doses should be given subcutaneously or intramuscularlj^ in 
 order to prolong the action of the serum. If no improvement follows in 
 from twelve to twenty-four hours, or if an acute exacerbation sets in, a 
 second dose should be given intravenously. Since the various manu- 
 ' Jour. Infect. Diseases, 1912, x. No. 3. ^ Jour. Infect. Diseases, 1911, ix, 130. 
 
764 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 facturers use different cultures in the preparation of these serums, it 
 would be well to use a different brand of serum if the first does not exert 
 a beneficial effect. In patients with severe infections the activity of the 
 serum may be enhanced by adding, just before injection, 5 c.c. of fresh 
 sterile guinea-pig serum to each 50 c.c. of the immune serum. 
 
 In the treatment of localized streptococcus infections, such as men- 
 ingitis and sinusitis, it may be well to mix antistreptococcus serum with 
 sodium oleate and boric acid, as previously described. 
 
 Value of Antistreptococcus Serum. — Although the serum has been 
 in use for almost twenty years, the true exact value of the remedy has 
 not as yet been estimated. It may be stated that a carefully prepared 
 and properly administered serum will do no harm and may do good, and 
 that its use should form a part of the treatment of severe streptococcal 
 infections. 
 
 In some cases of wound infections with severe cellulitis and septicemia 
 the serum may at times exert a most pronouiHjed and happy effect. In 
 other cases, and especially in those in whom the cocci are found in the 
 blood, repeated injections may be of no value. 
 
 In severe anginose or malignant scarlet fever large doses of serum 
 from horses especially immunized with strains of streptococci from 
 scarlet-fever patients have, on the whole, yielded favorable results. 
 Not all cases of severe scarlet fever, however, are due to secondary 
 streptococcal infections : those patients who are overwhelmed and pros- 
 trated at the very outset arc probably intoxicated with the true scar- 
 latinal virus, whatever that may be, and such cases are not likely to be 
 benefited by serum treatment. The patients most likely to improve 
 under serum therapy are those who become severely ill after the onset 
 of the disease and the appearance of the eruption. 
 
 In puerperal sepsis and endocarditis of streptococcal origin the results 
 of serum treatment have not been uniform, but are generally unfavorable. 
 If serum is administered at all, it should be given early, in large doses, 
 and intravenously. Not all cases of puerperal sepsis are streptococcic, 
 and while the physician may not be justified in withholding serum until 
 a bacteriologic diagnosis has been made, this factor must be considered 
 when estimating the value of a serum. 
 
 In erysipelas the results have been very indifferent, and the same may 
 be said of bronchopneumonia, laryngeal diphtheria, smallpox, and tuber- 
 culosis. 
 
THE SERUM TREATMENT OF GONOCOCCAL INFECTIONS 765 
 
 THE SERUM TREATMENT OF GONOODCCAL INFECTIONS 
 In 1906 Torrcy and Rogers' described the preparation of an anti- 
 gonococcus serum and advocated its use in the treatment of gonococcal 
 infections, and especially of its various complications and metastases. 
 In the following year these observers reported^ favorably upon the re- 
 sults of serum treatment in gonorrheal arthritis, and to a lesser extent in 
 infections of the genito-urinary organs. Uhle and Maokinney^ used 
 the serum in the treatment of 23 cases of gonococcal infection, and found 
 it beneficial in three cases of gonorrheal arthritis and in one of myositis, 
 whereas in epididymitis and urethritis no appreciable effects were ob- 
 served. Herbst and Belfield,'* Schmidt,^ and Swinburne ^ agree as to the 
 yalue of the serum in gonorrheal arthritis, whereas in other complica- 
 tions they secured somewhat conflicting results. In all instances the 
 serum was used in relatively small doses, namely, 2 c.c, injected subcuta- 
 neously each day or every other day, the number of injections depend- 
 ing on the clinical condition of the patient. 
 
 Recently Corbus ^ has reported more favorable results in the treat- 
 ment of 24 cases of gonococcus infection by using larger doses of serum — 
 from 36 to 45 c.c. — injected intramuscularly. This observer advocates 
 the use of the complement-fixation test as a reliable guide to the admin- 
 istration of the serum : the more intense the reaction, the more efficient 
 will the serum prove; if the reaction is negative, the serum should not 
 be used. 
 
 Antigonococcus serum is advocated in the treatment of the following 
 conditions: 
 
 1. In gonococcus bacteremia. 
 
 2. In those infections arising by extension through the lymphatics 
 or the circulatory system, as, for example, arthritis, iritis, endocarditis, 
 and pleuritis. 
 
 3. In those acute infections arising by direct extension from the 
 urethra, such as acute prostatitis and epididymitis; acute orchitis and 
 probably cystitis in the male; and acute salpingitis in the female. 
 
 The serum has not proved of value in the treatment of gonorrheal 
 
 conjunctivitis, although it would appear advisable to employ it in large 
 
 doses administered intravenously or intramuscularly. 
 
 ' Jour. Amcr. Med. Assoc, 1906, xlvi, 261, 273. 
 
 2 Jour. Amer. Med. Assoc, 1907, xlix, 918. 
 
 ' Jour. Amer. Med. Assoc, 1908, li, 105. ■" Illinois Med. Jour., 1908, xiii, 6S9. 
 
 ' Therap. Gaz., 1909, xxvi, 609. » Trans. Amer. Urol. Assoc, 1909, iii, 170. 
 
 ' Jour. Amer. Med. Assoc, 1914, Ixii, 1462. 
 
766 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 Preparation of Antigonococcus Serum. — Torrey's serum is prepared by 
 immunizing rams with gradually increasing intraperitoneal doses of dead, and lator 
 of living, cultures of gonococci. Larger amounts of serum may be secured by immu- 
 nizing horses according to the methods described for the preparation of meningococcus 
 and streptococcus mimune serums, and in view of the larger doses now advocated, 
 this is advisable. This serum has been successfully concentrated in the same manner 
 as is diphtheria antitoxin. 
 
 Mode of Action of Antigonococcus Serum. — According to Torrey, 
 this serum is largely bacteriolytic in nature. The presence of antitoxins 
 has not been demonstrated; small amounts of bacteriotropins are pres- 
 ent, so that a potent serum probably destroys' the cocci by extracellular 
 lysis and phagocytosis. 
 
 Administration of Antigonococcus Serimi. — The amount of serum 
 used and the method of inoculation are very important factors in the 
 success or failure of the treatment. If the serum is used at all, it should 
 be given in large doses. The original method of giving 2 c.c. subcu- 
 taneously has been found inadequate in most instances. 
 
 In acute gonococcal metastases in the joints or in the endocardium 
 or other serous membrane, from 30 to 50 c.c of serum should be injected 
 intravenously, or at least intramuscularly. When gonococci are found 
 in the blood, or when the patient is profoundly septic, from 50 to 100 c.c. 
 of serum should be given intravenously. In epididymitis, orchitis, and 
 other local complications from 30 to 50 c.c. of serum should be given 
 intranmscularly or intravenously. Other forms of treatment should he 
 instituted simultaneously. If necessary, the serum injections should be 
 repeated in twenty-four hours, and if a good primary effect follow.s an 
 intravenous injection, it may be prolonged l^y one or more subcutaneous 
 or intramuscular injections on subsequent days 
 
 THE SERUM TREATMENT OF STAPHYLOCOCCUS INFECTIONS 
 
 While several attempts have been made to treat staphylococcus 
 infections with an immune serum, the investigations have been too few 
 and too brief to warrant a statement in regard to the value of serum 
 therapy in these infections. Thomas ^ prepared a serum by immunizing 
 a ram with 18 different strains of StaphylocoGcus pyogenes aureus, and 
 reported good results in the treatment of 28 cases of furunculosis and 
 carbuncles. 
 
 It is probable that, with more extensive use of antistaphylococcus 
 serum, its value will be proved, especially in? the treatment of severe 
 1 Jour. Amer. Med. Assoc, 1913, Ix, 1070. 
 
THE SERUM TREATMENT OF ANTHRAX 
 
 767 
 
 furunculosis of infants as well as of adults, when the low general vitality 
 of the patient contraindicates the use of a bacterial vaccine. With 
 extended use it may also be found to be of benefit in staphylococcus 
 bacteremia, osteomyelitis, arthritis, carbuncle, and other severe in- 
 fections. Following recovery or relief from an acute infection it would 
 seem to be wise actively to immunize the patient with a vaccine, or 
 serum and vaccine may be used conjointly. 
 
 The activity of the serum is probably largely dependent upon the 
 presence of bacteriotropins and antitoxins, the former promoting phag- 
 ocytosis and the latter neutralizing the staphylolysins or hemotoxic 
 poisons produced by staphylococci. 
 
 THE SERUM TREATMENT OF ANTHRAX 
 
 Sclavo,! Mendez,'-^ and Deutsche have prepared anti-anthrax serums 
 by immunizing sheep, goats, asses, and horses with virulent cultures of 
 anthrax bacilli. In the treatment of human infections, only Sclavo's 
 serum has been used, the others being used in the treatment of anthrax 
 among the lower animals. An anthrax serum prepared by American 
 manufacturers is also on the market. 
 
 Sclavo's serum has been shown to possess protective and therapeutic 
 properties in experimental infections, and favorable results have been 
 recorded in cases of human anthrax. According to Sclavo's statistics, 
 the serum has reduced the average mortality of anthrax from 24 per 
 cent, to 5.3 per cent. Cigognani, Legge, Lockwood and Andrewos, 
 Stretton and Mitchell, and others have reported favorably as to the 
 value of the serum. 
 
 It is somewhat difficult to 'prepare a potent serum, and horses should 
 be immunized with a large number of strains from human infections over 
 a long period of time. The serum is probably largely bacteriotropic and 
 bacteriolytic in nature. 
 
 Administration of Anti-anthrax Serum. — In the Philadelphia Hos- 
 pital for Contagious Diseases a number of anthrax eases are treated 
 every year. Whenever blood-cultures have revealed the presence of the 
 bacilli in the circulation, the patient has usually succumbed to the disease 
 in spite of serum treatment; the doses employed — 10 to 20 c.c. — may, 
 however, have been too small. I would recommend the following 
 course of treatment in these cases: 
 
 1 Berl. klin. Wochensohr., 1901, 18 and 19, 481, .520. 
 
 2 Centralbl. f. Bakt., 1899, xxvi, Nos. 21 and 22. 
 5 Inipfstoffe u. Sera, Leipzig, 1903. 
 
768 PASSIVE IMMUNIZATION— SERUM THERAPY 
 
 1. If the lesion is relatively small and accompanied by but slight 
 glandular involvement, with little or no evidences of toxemia, a blood 
 culture should first be made by placing from 2 to 5 c.c. of blood in a flask 
 of neutral bouillon, followed by an intravenous or intramuscular injection 
 of from 20 to 50 c.c. of serum. The object sought is to introduce serum 
 before the lesion is handled, and the blood culture is the best indication 
 for subsequent injections of serum and serves as a guide to prognosis 
 
 2. The lesion should then be excised, leaving a wide margin so as to 
 include infected lymphatic channels. It should be handled as little as 
 possible. The wound is then dusted lightly with powdered calomel 
 and next heavily powdered with ipecac. Edema soon subsides, and the 
 wound usually heals rapidly, with surprisingly little scar-tissue forma- 
 tion. If the edema does not subside and the infection is spreading at the 
 margins of the wound, more tissue should be> excised or multiple local 
 injections with phenol and anti-anthrax serum should be made. 
 
 3. The blood culture may be examined within twenty-four hours. 
 If anthrax bacilli are present, from 100 to 200 c.c. of serum should be 
 given intravenously, the injection being repeated in twenty-four hours. 
 Daily blood cultures should be made, and the serum injections con- 
 tinued until the blood becomes sterile. Salvarsan may also be injected 
 intravenously. In our experience, cases with sterile blood cultures have 
 invariably recovered. 
 
 4. In internal anthrax all the physician can do is to administer large 
 doses of the serum intravenously. Salvarsan may also be tried. (See 
 Chapter on Chemotherapy.) 
 
 THE SERUM TREATMENT OF TYPHOID FEVER 
 
 The serum of Chantemesse is the only serum that has been used on a 
 large scale in the treatment of typhoid fever in man. 
 
 The serum is derived from horses that have been immunized for 
 several years with bouillon filtrates containing typhoid toxin, chiefly 
 endotoxin, and with typhoid bacilli. Kraus and von Stenitzer, Meyer, 
 Bergell, and Aronson use bouillon filtrates and aqueous bacterial ex- 
 tracts; Besredka injects dead and then livilig cultures; MacFadyen 
 uses an endotoxin secured by breaking up cultures frozen at very low 
 temperature. It would appear that a serum should be bacteriolytic and 
 endotoxic, and this probably is best secured by prolonged intravenous 
 immunization of horses with a large number of dead cultures and then 
 with autolysates and living cultures conjointly. 
 
THE SERUM TREATMENT OF tLAGXJE 769 
 
 According to Chanteiriesse, the subcutaneous injection of a few drops 
 of his serum produces leuliocytosis and raises the opsonic index of the 
 patient's serum. He emphasizes the fact that the serum should be given 
 early, — before the seventh day, — and reports that, by its use, the mor- 
 tality has been reduced from 17 per cent, to 4.3 per cent. These results 
 have not been generally confirmed, and the subject is still sub judice. 
 
 THE SERUM TREATMENT OF PLAGUE 
 Of the various antipest serums that have been prepared, that of 
 Yersin is probably best known. This serum, it would appear, possesses 
 some prophylactic and therapeutic value. For purposes of prophylaxis 
 from 10 to 20 c.c. of serum may be injected subcutaneously or intra- 
 venously; the period of protection is short, averaging from ten to four- 
 teen days. Combined active and passive immunization, effected by means 
 of injections of a pest vaccine and an antipest serum, will probably exert a 
 •protective action of several months^ duration, and should be used by physi- 
 cians, nurses, and others during epidemics of plague. When used for 
 therapeutic purposes, the results have been quite variable. If serum is 
 used in the treatment of plague, it should be giyen as early as possible, 
 in the form of intravenous or intramuscular injections of from 50 to 150 
 c.c, if this amount is available. Injections should be continued at 
 twelve- to twenty-four-hour intervals for two or more days until sup- 
 puration has been controlled and the disease shows signs of abating. 
 The Plague Commission of India has not issued very favorable reports 
 upon the use of either this serum or that of Lustig. 
 
 (a) In addition to Yersin's serum, which is prepared at the Pasteur Institute of 
 Paris by immunizing horses with dead and then with living cultures of pest bacilli, 
 other serums have been prepared. For example : 
 
 (h) Kolle immunizes horses with intravenou.s inject-ions of heat-killed cultures, 
 beginning with J^ agar slant culture and doubling the dose each week until 15 cul- 
 tures are given at one time. The horses are bled fourteen days after the last dose 
 is given. 
 
 (c) Lustig immunizes horses with pest-nucleoproteins, obtained by breaking 
 up the bacilli with 1 per cent, of potassium hydroxid and precipitating the proteins 
 with acetic acid. These are then suspended in sterile normal salt solution, as in the 
 preparation of Lustig's vaccine. 
 
 (d) Temi-Bandi immunizes donkeys and -sheep with aggressins obtained by 
 intraperitoneal injection of guinea-pigs with pest bacilli. 
 
 (e) Markl immunizes horses with filtrates of old pest bouillon cultures. He 
 believes that the value of pest serum is largely dependent upon antitoxins. 
 
 The serums are usually tested by injecting mice with lethal doses of pest culture 
 and decreasing doses of antiserum. The agglutinin content may also be measured. 
 49 
 
770 PASSIVE IMMUNIZATION SERUM THERAPY 
 
 Whenever cultures are used in immunization, the serum should always be cultured 
 carefully and tested by animal inoculation to guard against the possibility of living 
 bacilli being present. 
 
 THE SERUM TREATMENT OF- CHOLERA 
 In some respects cholera would seem to be due mainly to a toxin 
 elaborated by the bacilli in the intestinal tract of infected persons, 
 similar to the action of the toxin of the Kruse-Shiga type of dysentery 
 bacillus. Various attempts have been made to prepare an efficient 
 anticholera serum, but the only one that has yielded encouraging results 
 in experimental infections as well as in cholera, of human beings is that 
 prepared by Kraus. This serum is prepared by immunizing horses 
 with a true toxin derived from a cholera-like vibrio isolated by Gottsch- 
 lich from the intestinal contents of pilgrims dying at El Tor from a dys- 
 entery or cholera-like infection. According to Kraus, this antiserum 
 is largely antitoxic, and serves to neutralize the toxin of true cholera 
 more effectively than does the antiserum resulting from immunization 
 with cholera cultures. A serum that is antitoxic and is obtained by pro- 
 longed immunization of horses by intravenous.iAJections with dead cul- 
 tures of cholera, and later with living cultures, bacterial extracts, and 
 filtrates of old bouillon cultures conjointly, would seem to be a desider- 
 atum. 
 
 Reports from Russia, where Kraus' and Kolle's serums have been 
 employed, indicate that a reduction of about 10 to 20 per cent, in the 
 rnortality has been accomplished. Jcgunoff's method of administering 
 the serum seems quite rational, and consists in making intravenous in- 
 jections of 140 c.c. of serum with 500 to 700 c.c. of sterile salt solution, 
 followed by a similar or slightly lower dosage in from six to twenty-four 
 hours after the first dose. The intravenous administration of salt solu- 
 tion alone has proved of value in the treatment of cholera, and it is 
 reasonable to assume that a potent serum may be of service by neutraliz- 
 ing toxins, destroying the bacilli, and at least furnishing additional 
 fluids for the depleted tissues and circulation. 
 
 There are no available statistics as regards the prophylactic value 
 of anticholera serum, but combined active and passive immunization 
 by means of a subcutaneous injection of from 10 to 20 c.c. of serum, 
 followed by three doses of vaccine, would appear to be a rational pro- 
 cedure in the presence of an epidemic or of a threatened epidemic of 
 cholera. 
 
THE SERUM TREATMENT OF TUBERCULOSIS 771 
 
 THE SERUM TREATMENT OF TUBERCULOSIS 
 
 While numerous efforts have been made to prepare an efficient anti- 
 tuberculosis serum, only two — those of Maragliano and Marmorek — 
 have been studied and are familiar. 
 
 Maragliano's serum is prepared by immunizing horses for from four 
 to six months with a mixture of a toxin prepared by the filtration of cul- 
 tures only a few days old and concentrated in vacuo at a temperature of 
 30° C, mixed with that obtained by aqueous extraction of killed virulent 
 cultures and concentrated by heating on a water-bath at 100° C. for 
 three or four days. Maragliano assumes that the antiserum possesses 
 antitoxic, bactericidal, and agglutinating properties. One cubic centi- 
 meter of this serum is injected every other day for one and a half months. 
 The favorable action of the serum is reported on, especially by Mircoli 
 and other Italian physicians, but in Germany and France proof of its 
 value could not be established. 
 
 Marmorek's serum is now prepared by immunization of horses with 
 young tubercle bacilli, whose acid-fast character is still very slight or en- 
 tirely absent. When the horses have attained a high degree of immun- 
 ity, they receive injections of various strains of pure cultures of strep- 
 tococci obtained from the sputum of tuberculous patients. The serum 
 of these animals is, therefore, antituberculous and also antistreptococcic, 
 and is serviceable against a mixed infection. 
 
 The serum is administered daily, either by subcutaneous injection, 
 in doses of from 5 to 10 c.c, or by the rectum in doses of from 10 to 20 
 c.c. The latter form of administration is quite objectionable to most 
 patients, but is the one least likely to produce serum sickness. 
 
 While this serum has been used quite extensively, the evidence at 
 present is too conflicting to permit definite conclusions to be drawn as to 
 its value in treatment. It would, however, seem to be worthy of further 
 trial in cases of localized bone and joint tuberculosis and in the incipient 
 stage of pulmonary tuberculosis. Citron recommends its use in patients 
 who evince persistent rise of temperature, and in the very severe but 
 not hopeless cases where tuberculin therapy carmot be undertaken. In 
 some of these cases he has obtained very encouraging results. Citron 
 occasionally begins with the serum treatment, an(| later combines tuber- 
 culin administration with it, finally omitting the serum altogether. 
 
CHAPTER XXXr 
 SERUM THERAPY (Continacd) 
 
 NORMAL SERUM THERAPY 
 
 The field of serum therapy has been extended in recent years by the 
 successful use of normal serum in the treatment of various pathologic 
 conditions, particularly the hemorrhagic diseases, some toxicoses of preg- 
 nancy, and certain skin affections. 
 
 While human blood has also been administered, usually by direct 
 transfusion, in the treatment of pernicious anemia, the leukenaias, and 
 pseudoleukemia, permanent good results have not been secured, al- 
 though the immediate effects, owing probably to the introduction of 
 large numbers of normal erythrocytes, may be satisfactory. 
 
 Normal Serum in the Treatment of Hemorrhage 
 
 Numerous reports by Welch, ^ John,^ Franz,' Nohlia,* Reichard,* 
 Perkins,* Claybrook,'' and others have shown that injections of normal 
 human, horse, or rabbit serum are of considerable value in the treat- 
 ment of melena neonatorum, hemophilia, purpura hmnorrhagica, hem- 
 orrhagic retinitis, intestinal bleeding in typhoid fever and in connection 
 with cirrhosis of the liver, pulmonary tuberculosis, in some cases of uterine 
 hemorrhage, and in surgical operations upon icteric persons. Barringer' 
 has reported the successful treatment, by injections of fresh normal 
 human serum, of unilateral kidney hemorrhage in a hemophiliac, and 
 advises that this simple treatment should be tried in similar cases of 
 varicose veins of the renal papilla. Levison^ has reported the successful 
 checking of hemorrhage from the urinary bladder following a simple op- 
 
 ^Amer. Jour. Obst. and Dis. of Women, etc., 1912, Ixv, No. 412. 
 2 Miinch. mod. \\'oohenschr., 1912, lix, No. 4. 
 = Miinch. med. Wochenschr., 1913, lix, 2905t 
 « La Presse MMicale, 1913, xxi, No. 20. 
 
 5 Jour. Amer. Med. Assoc, 1912, lix, 1.539. 
 
 6 Jour. Amer. Med. Assoc, 1912, lix, 1.539. 
 ' Jour. Amer. Med. Assoc, 1912, lix, 1.540. 
 8 Jour. Amer. Med. Assoc, 1912, lix, 1.538. 
 « Jour. Amer. Med. Assoc, 1913, Ix, 721. 
 
 772 
 
NORMAL SERUM THERAPY 773 
 
 eration by performing cystostomy, removing clots, and filling the bladder 
 with sterile horse serum. 
 
 This form of treatment is so simple and has liqen so successful in check- 
 ing hemorrhage in melena neonatorum and in other hemophilias following 
 injuries or operations that it should never he omiJted. 
 
 Defibrinated human blood, human serum, or the serum of the horse 
 and rabbit may be employed. The doses advised have been from 10 to 
 20 c.c. for infants and children and from 20 to 50 c.c. for adults. 
 
 The technic is very simple. Sterile normal horse serum ready for 
 injection may be purchased in the open market. Human serum may be 
 secured by -uithdraw-ing blood into large centrifuge tubes (see p. 33) 
 and allowing the serum to separate, or the clot may be broken up after 
 an hour and the serum secured by rapid centrifugalization. For in- 
 travenous medication the serum should be free from particles of fibrin. 
 Indeed, the whole operation may be conducted at the bedside by with- 
 dra^ning blood from the donor into a flask containing sterile glass beads, 
 and after a few minutes of vigorous shaking the defibrinated blood is 
 injected subcutaneousbr or intramuscularly. "\Mienever human serum or 
 blood is used and time permits, a AVassermann. reaction should be per- 
 formed beforehand, and it should be determined, by hemolj'tic and 
 agglutination tests, that the donor's serum does not hemolyze or agglu- 
 tinate the recipient's erj-throcj-tes. (See p. 290.) Of course, all pro- 
 cedures should be conducted in an aseptic manner. 
 
 Normal Serum in the Treatment of the Toxicoses of Pregnancy 
 Feiux, Freund, Rongji, and others have found injections of fresh 
 normal human serum from pregnant women-, scrum from placental 
 blood, and even horse serum useful in the treatment of the vomiting of 
 ■pregnancy. Freund has likewise observed that injections of Ringer's 
 and Locke's solutions are sometimes efficacious,' he has found injections 
 of serum of some value in eclampsia, and tentative^ advises its use in 
 this condition. The same observer has employed injections of normal 
 serum for the relief of the itching of pregnancy, and reports success. 
 Similar observations have been made bj' A'eieP and ATolf,^ especially 
 after injections of serum secured from other heilthy pregnant or recently 
 delivered women. 
 
 To obtain placental serum, the following technic may be employed: 
 After the delivery of the child the cord is sponged with bichlorid solu- 
 
 ' Miinch. med. Wochenschr., 1912, lix, Xo. 35. 
 . 2 Berl. klin. Wochenschr., 1913, 1, NO. 36. 
 
774 SERUM THERAPY 
 
 tion, cut, and the maternal end bled into a sterile, wide-mouthed flask. 
 This is placed in the refrigerator until there is complete separation of 
 serum. In pipeting off the serum due care must be exercised not to 
 include corpuscles. If the serum is not pentectly clear, it should be 
 centrifuged with aseptic precautions. It is well to have each serum 
 tested by the Wassermann reaction and 1 c,c. cultured in 100 c.c. of 
 glucose bouillon to test its sterility. The serum is then preserved in 
 bottles or vials, with the addition of two drops of 5 per cent, phenol to 
 each 10 c.c. of serum. 
 
 Normal Serum in the Treatment of Skin Diseases 
 In addition to the good results obtained in the treatment of general 
 pruritus of pregnancy with normal serum, Linser,^Hench,^and Prsetorius^ 
 have had favorable results from injections of normal human serum or 
 horse serum in the treatment of urticarial and chronic obstinate itching 
 affections, especially senile pruritus, and also: in malignant pemphigus. 
 Even better results have been observed in thg treatment of pemphigus, 
 psoriasis, and other skin diseases by injections of the patient's own serum. 
 This subject will be referred to further on. 
 
 Mention may also be made of the results observed in the treatment 
 of acute and chronic nephritis by injections of blood-serum from the 
 renal vein of the goat, dog, or sheep. Teissier* was probably the first 
 to apply this form of therapy, and reports favorable results in the treat- 
 ment of seven cases. Spillman,^ Bisso,^ and Dominquez^ have also 
 published favorable reports. The treatment is based upon the assump- 
 tion that the blood in the renal vein contains some of the internal secre- 
 tion of the kidney, which acts favorably upon the liver and emunctories 
 in general. This method of treatment has been advocated by the pre- 
 viously mentioned observers in the acute exacerbations of chronic 
 nephritis, in acute or chronic nephritis with threatening uremia, and in 
 arterial hypertension presumably of renal origin. Amounts of serum 
 ranging from 10 to 50 c.c. have been injected subcutaneously, and re- 
 peated on several days or every other day until several doses have been 
 given. 
 
 1 Dermat. Wocherischr., March 30, 1912. 
 
 2 Miinch. med. Woohenschr., 1913, lix, No. 48. 
 = Miinch. med. Wochenschr., 1913, Ix, No. 16. 
 ^ Bull, de I'Acad. de M6d., 1908, Ixxii, No. 31. 
 5 La Presse Mddicale, 1909, xvii. No. 86. 
 
 " Semana Med., Buenos Aires, 1912,:xix, No. 7. 
 ' Rev. de Med. y Cir., Havana, 1913, xvii, No. 24. 
 
AXJTOSERUM THERAPY* 775 
 
 AUTOSERUM THERAPY 
 
 A number of observers have reported favorable results following the 
 administration of the patient's own serum in the treatment of various 
 diseases. In most instances serum is secured by withdrawing blood into 
 a sterile container, and defibrinating it with glass beads; this is followed 
 by thorough centrifugalization, or the serum is secured after the blood 
 has been allowed to coagulate spontaneously. In other instances the 
 serum has been obtained from blisters purposely produced by the appli- 
 cation of cantharides; in still others, and especially in tuberculosis of 
 serous membranes, good results have been observed to follow subcu- 
 taneous injections of small amounts of the patient's pleural or peritoneal 
 fluid. 
 
 AUTOSERUM IN THE TREATMENT OF SkIN DISEASES 
 
 Sprethoff,'^ Strumpke,^ Gottheil and Latenstein,'' and other ob- 
 servers have secured favorable results from this form of serum therapy 
 in the treatment of obstinate and chronic dermatoses, due to general, 
 rather than to local, causes, in which the usual therapeutic measures 
 are ineffectual or only partially successful, as, for example, psoriasis, 
 dermatitis herpetiformis, pemphigus, lichen ruber,- lichen planus, urticaria, 
 squamous eczema, etc. Blood is withdrawn from the patient in amounts 
 of from 50 to 100 c.c. While still fresh, the serum is separated and in- 
 jected intravenously in doses of from 30 to 40 c.c. These treatments are 
 repeated from two to six times at intervals of from three to five days. 
 
 AUTOSERtnvi IN THE TREATMENT OF ACUTE INFECTIOUS DISEASES 
 
 Favorable results have also been reported in the treatment of acute 
 infections, as, for example, scarlet fever with the serum of convalescent 
 scarlet-fever patients. Thus Reiss and Jungmann,^ and later Reiss,^ 
 have observed a marked amelioration in the gertqral symptoms of severe 
 cases of scarlet fever following the intravenous injection of from 50 to 
 100 c.c. of serum removed from two or more third to fourth week con- 
 valescents. These observers use this form of therapy in severe cases on 
 or before the fourth day of the disease. After blood is withdrawn from 
 convalescents the serums are separated, tested by the Wassermann re- 
 action, mixed, cultured to determine their sterility, put up in amounts of 
 
 ' Med. Klin., 1913, ix. No. 24. = j)put. med. Wochenschr., 1913, xxxix, No. 30. 
 ^ Jour. Amer. Med. Assoc, 1914, Ixiii, 1190. 
 
 * Deut. Archiv f. klin. Med., 1912, cvi, Nos. 1, 2. 
 
 * Therap. Monatsschr., 1913, xxvii. No. 6. 
 
776 SERUM THERAPY 
 
 50 c.c. in ampules with the addition of 5 drops of 5 per cent, phenol, and 
 kept on ice. 
 
 Teissier' has had favorable results from Ihe subcutaneous and in- 
 travenous administration of serum from smajipox convalescents in the 
 treatment of severe and hemorrhagic smallpox. The injections must be 
 given early in the disease. Improvement in the general symptoms, 
 diminished suppuration, and consequent lessened scar formation are 
 some of the good effects ascribed to this treatinent. 
 
 Favorable results have also been observed in the treatment of lep- 
 rosy with injections of serum secured by raising a blister with cantharides. 
 Similar reports have been made by Jez^ in the treatment of erysipelas, 
 and by Mordinos^ in that of typhoid fever, influenza, and Malta fever. 
 Other observers have also reported good results following the injection 
 of from 5 to 10 c.c. of the patient's serum in the treatment of gonorrheal 
 arthritis, typhoid fever, pneumonia, and other infections. Robertson* 
 states that he has never observed the slightest influence of autoserum 
 injections in the treatment of typhoid fever and pneumonia. 
 
 Autoserum (Salvarsanized) in the Treatment of Syphilis of the Brain 
 
 AND Spinal Cord 
 
 The treatment of syphilis of the central nervous system has always 
 been unsatisfactory. With the discovery of salvarsan and neosalvarsan, 
 the hope was fostered that these remedies would prove of therapeutic 
 value in the treatment of tabes dorsalis, paresis, and cerebrospinal 
 syphilis. Experience has shown, however, tllat while the progress of 
 tabes dorsalis may be arrested in the early stages by vigorous treatment, 
 in paresis the prognosis is much less hopeful. As these diseases are now 
 known to be truly syphilitic, the presence of Treponema pallidum having 
 been actually demonstrated in the cerebral, cortex, spinal cord, and 
 cerebrospinal fluid by Noguchi and Moore,, Nichols, Graves, Marie, 
 Levaditi, and others, the cause for failure in the treatment of these in- 
 fections must be ascribed largely to the fact, that the choroid plexus 
 filters out salvarsan and mercury, as well as antibodies, and prevents 
 these remedies from reaching the cerebrospinal fluid, just as it prevents 
 the entrance of serum, albumin, sugar, urea, ammonia, etc. Although 
 there can be no doubt as to the spirocheticidal properties of salvarsan, 
 and as to its ability to kill the treponema in the tissues, this much-desired 
 action does not seem to occur mainly because the drug cannot gain ac- 
 
 1 Quoted in Jour. Amer, Med. Assoc, 1913, Ix, 380. 
 
 2 Wicn. klin. Woohenschr., August 31, 1901. 
 
 = La Presse MiJdicale, 1911, xix, No. 96, 1009. * Personal communication. 
 
ATJTOSERUM THERAPY 777 
 
 cess to the parasites. For similar reasons antimeningococcic serum 
 must be injected intraspinally, because when given intravenously, the 
 plexus appears to withhold the antibodies. The same is true of tetanus 
 antitoxin. 
 
 Shortly after the discovery of salvarsan and neosalvarsan hope was 
 entertained that these remedies, when injected intraspinally, would 
 bring the drug into direct contact with the infected tissues. Investi- 
 gations by Wechselmann,! Marinesco,^ and Ellis and Swift ^ have shown, 
 however, that even minute quantities of either drug may be irritating 
 and may prove dangerous. 
 
 Plaut'' showed early that the serum of patients who have received 
 salvarsan exerts a definite antisyphilitic effect, whereas normal serum 
 displays no such activity. JMeirowsky and Hartman* and Gibbs and 
 Calthrop^ observed good results in the treatment of syphilis from sub- 
 cutaneous injections of serum from other patients who had received 
 salvarsan. Swift and Ellis' then showed that sermn taken from a pa- 
 tient within six hours after the salvarsan was injected inhibited the 
 gro's\i;h of Treponema pallidum, but that if taken before the salvarsan was 
 administered, or within from six to twenty-four hours after treatment, 
 there was no inhibition. These last-named observers also reported bene- 
 ficial efl'ects following the intraspinal injection of salvarsanized serum, 
 with but slight irritative phenomena. Further experimental studies on 
 the spirocheticidal activity of salvarsanized serum were made by Gonder,^ 
 Castelli,' and especially by Swift and Ellis,**' the last-named investiga- 
 tors also noting that heating the serum at 56° C. for half an hour mark- 
 edly increased its activitj', which was due in part to the destruction of 
 some inhibiting substance. 
 
 Shortly after this Swift and EUisi"^ published a report of the treat- 
 ment of a mmiber of cases of tabes dorsalis" and other syphilitic infec- 
 tions of the central nervous system wath intraspinal injections of sal- 
 varsanized serum. In practically all these cases clinical improvement 
 was obsei'ved, with total or partial disappearance of the positive sero- 
 
 1 "Ehrlich-Hata 606," Berlin, 1911, 11. Deut. med. Wochenschr., 1912, xxx\-iii, 
 1446. 
 
 2 Diiitet. u. physik. Therap., 1913, xvii, 194. 
 = Jour. Exper. Med., 1913, x^'iii, 428. 
 
 * Deut. med. Woehcnschr., 1910, xxx\-i, 2237. 
 
 5 Med. Klin., 1910, vi, l.i72. %Brit. Med. Jour., 1911, 1, 360. 
 
 ' Xew York iled. Jour. 1912, xevi, 53. 
 8 Zeitschr. f . Tmmmiitatsf ., Orig., 1912. xt, 57. 
 ' Zeitschr. f. Chemothcrap., Orig., 1912, 1, 122 and 167. 
 '" Jour. Exp. Med., 1913, .\^-iii, 435. " .\Tch. Int. "Sled., 1913, xii, 331. 
 
778 
 
 SERUM THERAPY 
 
 biologic findings in the cerebrospinal fluids. Subsequent reports by 
 Hough,! McCaskey,^ Riggs,^ Cutting and Mack,'' Eskucken,^ Boggs 
 and Snowden,^ and Mapothes and Beaton^ of a number of cases of 
 tabes dorsalis, paresis, and other syphilitic infections of the central 
 nervous system showed that this method of treatment possesses dis- 
 tinct value. It was advocated by Swift and" Ellis in the treatment of 
 these diseases as an adjuvant to intravenous injections of salvarsan, as 
 a part of an intensive medication aiming to bring salvarsan into intimate 
 contact with the parasites in the most direct and safest manner. While 
 this form of treatment has, in a large percentage of cases, effected a 
 marked improvement in the subjective symptoms and has modified the 
 underlying pathologic tissue changes, as evidenced by the disappearance 
 or improvement of objective signs and sergbiologic findings in the 
 cerebrospinal fluid, it must still be considered in the experimental stage, 
 for sufficient time has not yet elapsed to perrnit an estimate of its ulti- 
 mate effect to be made. It is especially indicated in early and inci- 
 pient cases of syphilitic infections of the nervous system. It is self- 
 evident that it cannot be expected to cure cases in which marked tissue 
 destruction has occurred, but if it serves to cure early and incipient cases 
 of tabes and paresis, or at least tends to arrest their progress and possibly 
 the further progress of more chronic cases, and gives symptoniatic re- 
 lief, then this mode of therapy is a valuable one. At present it is ap- 
 parent that, in the hands of careful and competent persons, and with the 
 strict observance of an aseptic technic, the method is relatively devoid 
 of danger and constitutes a new, rational, and valuable addition to the 
 treatment of diseases that may otherwise prove intractable to the ordin- 
 ary antisyphilitic measures. 
 
 Technic. — From 0.6 to 0.9 gm. of salvarsan or neosalvarsan is in- 
 jected intravenously. One hour later 40 c.c. of blood are withdrawn 
 directly into centrifuge tubes and allowed to coagulate, after which it may 
 be centrifugalized. The following day 12 c.c. of 'serum are pipeted off and 
 diluted with 18 c.c. of sterile normal salt solution. This 40 per cent, 
 serum is then heated at 56° C. for one-half hour; After lumbar puncture 
 the cerebrospinal fluid is withdrawn until the pressure is reduced to 
 30 mm. cerebrospinal fluid pressure. The barrel of a 20 c.c. Luer syr- 
 
 1 Jour. Amer. Med. Assoc, 1914, Ixii, 183. 
 
 2 Jour. Amer. Med. Assoc, 1914, Ixii, 187. 
 'JouT. Amer. Med. Assoc, 1914, Ixii, 1888. 
 * Jour. Amer. Med. Assoc, 1914, Ixvii, 903. 
 
 ' Milnch. med. Woohenschr., 1914, li, No. 14. 
 
 « Arch. Int. Med., 1914, xiii, 970. ' Lancet, London, April, 1914, 18, 1. 
 
AUTOSERUM THERAPY 779 
 
 inge (which has a capacity of about 30 c.c.) is attached to the needle by 
 means of a rubber tube about 40 cm. long. The tubing is allowed to fill 
 with cerebrospinal fluid, so that no air will be injected. The serum is 
 then poured into the syringe, and permitted to flow slowly by means of 
 gravity into the subarachnoid space. At times it is necessary to insert 
 the plunger of the syringe to inject the last 5 c.c. of fluid. It is im- 
 portant that the larger part of the serum should be injected by gravity, 
 and if the rubber tubing is not more than 40 cm-, long, the pressure cannot 
 be higher than 400 mm. Usually the serum flows in easily even under 
 a lower pressure. By the gravity method the danger of suddenly in- 
 creasing the intraspinous pressure to the danger-point, such as might 
 occur with rapid injection with a syringe, is avoided (Swift and Ellis). 
 
 The method of injection by gravity is described on p. 694. In the 
 absence of a suitable manometer for estimating' cerebrospinal fluid pres- 
 sure the blood-pressure may be taken as a guide ; in any event the scrum 
 should be injected slowly. 
 
 McCaskey consumes about six or seven minutes in administering 
 the salvarsan intravenously, and advises withdrawing the blood twenty 
 minutes thereafter, instead of waiting an hour in order to secure a 
 larger quantity of the drug in the serum. He injects 15 c.c. of serum in 
 50 per cent, dilution intraspinally, and has not observed any mcreased 
 irritative effects. 
 
 Swift and Ellis inject the serum in strengths of 50 to 60 per cent, or 
 even higher in patients who do not exhibit reactions following the injec- 
 tion of 40 per cent, serum. Boggs and Snowden withdraw from 75 to 
 100 c.c. of blood one hour after the salvarsan injection, secure the serum, 
 heat it at 56° C. for one-half hour, and inject the undiluted serum in a 
 dose equal to the amount of fluid withdrawn, whether this is only a few 
 or as much as 30 c.c. 
 
 The method employed by Marinesco and Minea,* whereby the 
 patient's scrum is salvarsanized in vitro, is not to be regarded as the 
 same as the method of Swift and Ellis. The first-named investigators 
 sought to administer larger doses of salvarsan by this method, but in a 
 preliminary report, covering the treatment of 20 cases receiving injec- 
 tions every seven to eight days, the results are said to have been disap- 
 pointing. The recent unfortunate outcome of this form of therapy in 
 the County Hospital of Los Angeles is ascribed to the oxidation and 
 consequent toxicity of the neosalvarsan as a result of allowing the sal- 
 varsanized serum to stand for twenty hours before injecting it. 
 1 Bull, de I'Acad. de M4d., 1914, Ixxvii, No. 7. 
 
780 SERUM THERAPY 
 
 After-treatment. — The patient should be kept in bed for twenty- 
 four hours and the foot of the bed should be elevated for part of this 
 time. 
 
 Usually the temperature reaction is mild. There is frequently some 
 pain in the legs, which appears a few hours after the injection is given. 
 The pain is more often noticed in tabetics than in patients with cerebro- 
 spinal syphilis. It can usually be controlled" by means of phenacetin 
 and codein, but occasionally inorphin is required. 
 
 In a few instances violent maniacal symptoms have developed, due 
 possibly to the direct irritant action of the drug on the tissues, aided by 
 the sudden liberation of endotoxins from myriads of killed spirochetes. 
 
 Serobiologic Findings in the Cerebrospinal Fluid. — In all cases before 
 the treatment is undertaken a Wassermann reaction should be performed 
 with the blood and cerebrospinal fluid of the patient. A total cell count 
 should also be made with a specimen of fluid, the percentage of lympho- 
 cytes ascertained, and a Noguchi butyric-acid-globulin test performed. 
 Normally, the cells number about 8 per cubic millimeter of fluid (counted 
 with a Fuchs-Rosenthal counting chamber) ; the presence of more than 
 15 cells in this quantity of fluid may be regarded as bordering on the 
 pathologic. In tabes and paresis the cells may vary in number from 
 50 to more than 100, and are mostly small lymphocytes. A normal cere- 
 brospinal fluid remains clear or shows a faint opalescence when tested 
 by the Noguchi butyric-acid test. In tabes,: paresis, etc., varying de- 
 grees of cloudiness and the presence of precipitates are observed. 
 
 Repeating the Dose. — Usually a number of treatments are required, 
 and these may be given at from one to three weeks' intervals, depending 
 upon the condition of the patient. The results are estimated from the 
 subjective and objective symptoms and from an examination of the 
 cerebrospinal fluid. A decrease in the degr.ee of positiveness of the 
 ^Vassermann reaction and diminution in cells and in globulins are fav- 
 orable signs. Theoretically, treatment should be continued until the 
 Wassermann reaction becomes negative, the cells reach normal propor- 
 tions, and the globuhns show no increase. Practically, it may be im- 
 possible to secure these results; in many instances the Wassermann re- 
 action is the first to disappear. A total cell count of the fluid is not to 
 be depended upon without a differential coun:t with stained smears, for 
 the injections may produce a form of aseptic meningitis, accompanied by 
 an outpouring of polynuclear leukocytes. This subject has previously 
 been referred to in a consideration of the serum treatment of epidemic 
 meningitis. 
 
AUTOSERUM THERAPY. 781 
 
 AUTOSERUM IN THE TREATMENT OF TUBERCULOSIS OF SEROUS MEMBRANES 
 
 Numerous investigators, as, for example, Gilbert,^ Marcon,^ 
 Schnutgen,' Fishberg," Pfender,'' Robertson,^ and others, have reported 
 favorable results in the treatment of tuberculous pleurisy with effusion, 
 cases that arise either insidiously or abruptly with pain in one already 
 tuberculous, or in one in whom tuberculosis is suspected, following 
 withdrawal of a portion of the fluid and immediate injection of from 2 
 to 5 c.c. into the subcutaneous tissues. In these cases there is usually 
 a sharp reaction, consisting of a rise in temperature, occasionally ac- 
 companied by chill, lassitude, and, in the majoiity of cases, diuresis or, 
 more rarely, diarrhea, followed by gradual absorption of the fluid within 
 the following few days up to two or three weeks. Fishberg mentions the 
 disappearance of pain, dyspnea, and prostration within two or three 
 days in favorable cases. 
 
 It is difficult to state whether the improvement is due to autotherapy 
 or simply to the puncture and removal of so much fluid. Eisner' has 
 seen a leukocytosis follow injection of serum in experimental tuberculous 
 infections of rabbits and guinea-pigs, and believes that this explains the 
 good results in this particular form of therapy .„ Zinimermanu* has ex- 
 pressed a similar opinion. Other investigators assert their belief in the 
 presence of aggressins, bacteriolytic amboceptors, complements, and 
 endolysins from disintegrated leukocytes as explaining the results. It 
 is more likely that these fluids contain the bacilli or their products, and 
 constitute a form of vaccine or auto-tuberculin, stimulating body-cells 
 to produce antibodies largely in the nature of bacteriotropins and bac- 
 teriolysins. Levy, Valenzi and Ponzin,' Szurekj^" and Arnsperger^^ are 
 inclined to believe that the beneficial results are obtained independently 
 of the injections, and while the procedure is quite generally regarded as 
 perfectly safe, Jousset^- has recorded a case of opld abscess following an 
 injection. This mode of treatment seems to have failed in about 10 to 
 15 per cent, of cases. 
 
 The technic is very simple, and the injections may be given by any 
 
 ' Gaz. des Hopit., 1894, 560. = La Fresse M(5dicale, 1909, No. 71, 627. 
 
 ' Berl. klin. Wochenschr., 1909, No. 3, 97. 
 
 ' Jour. Amer. Med. Assoc, 1913, Ix, 962. 
 
 ' Wash. Med. Ann., 1914, xiii, 83. ' Per.sonal oommunication. 
 
 ' Zeitschr. f. klin. Med., 1912, Ixxvi, 34. 
 
 *St. Peters, med. Wochenschr., 1909, No. 34, 461. 
 
 » Bull. et. Mem. de la Soc. d. Hop. d. Paris, 1910, xJcvii, 265. 
 
 '» Med. Klinik, 1909, No. 44, 1665. " Therap. d. Gegenwart, 1911, lii, 495. 
 
 '2 Arch. G&. de Med., 1912, xoi, 141. 
 
782 SEEUM THERAPY 
 
 physician who can make an ordinary exploratory puncture. In all cases 
 where puncture shows the presence of a serous fluid, the needle should 
 not be withdrawn completely, but when its point has reached the sub- 
 cutaneous tissues, from 2 to 5 c.c. should be injected then and there. In 
 some patients it will be necessary to repeat the treatment several times 
 every two or three days before any effect becomes evident. Caforio' 
 has reported good results following the tapping of a bilateral hydrocele 
 and injection of a portion of the fluid into the subcutaneous tissues. It 
 would appear that this procedure may be successful in hydrocele of 
 tuberculous origin, but good results are not to be expected in cases due 
 to contusion, puncture wounds, gonorrheal epididymitis, or orchitis. 
 
 In view of the fact that the fluid may contain a sufficient number of 
 living tubercle bacilli to produce secondary infection, it would appear 
 advisable to withdraw fluid into an equal volume of 2 per cent, sodium 
 citrate in normal salt solution, heat at 60° C. for one-half to one hour, 
 and preserve in a sterile container with the addition of a few drops of 5 
 per cent, phenol until ready for subcutaneous injection in doses of from 
 2 to 5 c.c. 
 
 It would appear, therefore, that reintroduction into the body of 
 fluids obtained from the serous cavities (pleural, peritoneal) of tuber- 
 culous patients may be of value in treatment, and, as a general rule, the 
 earher in the course of the disease that the a'utoserum therapy is prac- 
 tised, the better will the results be. A similar treatment may be car- 
 ried out in the subacute or chronic forms of tuberculous meningitis. 
 Fluid is to be collected in sodium citrate solution, heated, and preserved 
 with phenol, as just described. The dose may vary from 0.5 to 2 c.c, 
 according to age and clinical conditions. 
 
 AUTOSERUM IN THE TREATMENT OF NON-TUBERCULOUS EFFUSIONS 
 
 In pleural and peritoneal effusions of renal, cardiac, or hepatic origin, 
 this method of autotherapy has generally failed. 
 
 Following the apparent success of HodenpyP in the treatment of a 
 case of cancer with injections of the patient's ascitic fluid, the autopsy 
 subsequently showing the presence of metastatic cancer not demon- 
 strable during life, Risely' treated 65 cases of cancer with ascitic fluids 
 obtained from cancer patients in all stages of the disease, and also with 
 various normal and abnormal body fluids from other than cancerous 
 conditions. None of these various transudates was found to exert any 
 
 1 Riform. M6d., 1912, xxviii, 1009. 2 y^^^ Rg^^ 1910, ixxvii, 359. 
 
 'Jour. Amer. Med. Assoc, 1911, Ivi, 1383. 
 
AUTOSERUM THERAPY 783 
 
 effect in retarding the growth of cancer in mice, and while in a small per- 
 centage of cases large doses of ascitic fiuid of cancerous origin may re- 
 lieve pain and retard the growth of the cancer for from one to five 
 months, no permanent effects, either preventing or checking the dis- 
 ease, were apparent. 
 
 Leavy and Hastings' and Carter^ have drawn attention to the ap- 
 parent improvement in marasmic infants following the daily injection, 
 for a number of doses, of an ounce of sterile ascitic fluid (the result of 
 cardiac or renal disease) into the subcutaneous tissues of the gluteal 
 region or abdominal wall. As these fluids are non-toxic, reinjection is a 
 justifiable procedure after the fluids have been subjected to the Wasser- 
 mann reaction and to careful cultural and animal injection tests to prove 
 their sterility. 
 
 1 Bost. Med. and Surg. Joiir., 1910, clxiii, 293. 
 ^Amer. Jour. Med. Sci., 1911, cxlii, 241. 
 
CHAPTER XXXII 
 CHEMOTHERAPY 
 
 From the earliest times, healers of the sick have sought specific 
 remedies in the form of drugs or methods that would always and un- 
 failingly effect the cure of a certain disease, and indeed this search has 
 been extended for a single remedy that will cure all diseases, regardless 
 of their origin and nature. To this end, throughout the ages experimen- 
 tation, conscious or otherwise, has gone hand in hand with medicine. 
 Countless substances gathered from the vegetable, animal, and mineral 
 kingdoms have been experimented with, but for the most part have been 
 discarded. Despite this persistent search for specifics, until compara- 
 tively recent times but two remedies have been found worthy of being 
 regarded as specifics, namely, cinchona bark for malaria, a remedy dis- 
 covered by the South American Indians, and mercury for syphilis. 
 
 With discoveries in bacteriology and the establishment of the etio- 
 logic relationship of various microorganisms to certain diseases, test- 
 tube experiments soon demonstrated that certain substances could 
 quickly and easily destroy these microparasit^. It was apparent, how- 
 ever, that in the animal body conditions were different; here the germi- 
 cide, even when administered in doses sufficiently large to prove dangerous 
 to life, usually failed to kill the microorganisms. The exceptions were 
 quinin and mercury, which we now know are*specifically germicidal for 
 the Plasmodium and spirochete respectively, a fact that was suspected 
 long before the parasites themselves were discovered. 
 
 In the latter part of the eighties it was (liscovered that the blood 
 possessed germicidal powers, and rapid advances were soon made in our 
 knowledge of the defensive meclianism of the animal body, and the 
 means afforded for preventing infection, and even of successfully over- 
 coming it if, by any chance, microorganisms passed the normal barriers 
 and gained a foothold on the tissues proper. Here indeed was a more 
 or less specific therapy that was not suspected until 1894, when diph- 
 theria antitoxin was discovered. It was then speedily realized that 
 body-cells could be made to produce a specifie remedy for a certain dis- 
 ease, and it was naturally assumed that this was possible for all those 
 
 784 
 
PRINCIPLES OF chemotiIerapy 785 
 
 diseases in which the specific microorganism was known and could be 
 cultivated. 
 
 In the foregoing chapters we have reviewed our knowledge of the 
 nature of these specific remedies produced by the body-cells and called 
 antibodies. In Chapter XXX we have also reviewed the application of 
 these remedies in the treatment of various infections, and have pointed 
 out their limitations and the difficulties encountered in their production. 
 Naturally, in the early days it was deemed necessaiy and advisable to 
 utilize a lower animal for the manufacture of these antibodies. With 
 the discovery of a means of attenuating or modifying a disease-producing 
 parasite it was found possible to inject these vaccines into our own 
 bodies, and thus stimulate our own cells to produce the specific anti- 
 body — a method that had been introduced empirically years before by 
 Jenner in vaccination against smallpox. 
 
 As was previously stated, scientists have- long believed that it was 
 possible to find or produce chemical substances that would not unite 
 with the blood albumin or body-cells, but wo|ild have a highly selective 
 and germicidal action on microparasites and- prove capable of killing 
 these in the living body. Curiously enough Ehrlich, to whom we are al- 
 ready indebted for much of our knowledge of the intricate problems 
 of cell life, and especially the mechanism of their defense and offense 
 against parasitic invaders, has again taken the lead and set about testing 
 hundreds of compounds in a painstaking, logKal, and scientific manner, 
 , in the hope of finding one that would prove most germicidal for various 
 trypanosomes and spirochetes, and at the same time be least toxic for 
 the body-cells. These researches finally culminated in the discovery 
 of the arsenical compound now popularly known as salvarsan. This 
 discovery constitutes a great triumph for medical science, not onlj^ be- 
 cause of the intrinsic value of the drug in the treatment and cure of 
 syphilis and frambesia, but because it demonstrates the truth of a prin- 
 ciple, and has opened up vast possibilities for future investigation. 
 
 PRINCIPLES OF CHEMOTHERAPY 
 Organotropism and Parasitotropism. — The guiding principle in 
 chemotherapy is to imitate nature's method of overcoming an infection 
 by the aid of substances that destroj^ the microorganisms, while the 
 body-cells of the host are left unharmed. To this end the chemical 
 agent employed must possess a much stronger affinity for the micro- 
 organisms than for the body-cells — to quote Ehrlich, it must be more 
 50 
 
786 CHEMOTHERAPY 
 
 parasitotropic than organotropic. If a given- chemical agent shows a 
 greater affinity for the albumins of the body-j dices and for the cells than 
 it does for the protoplasm of the microparasites, that is, if it is more 
 organotropic than parasitotropic, it is evident that it is not suitable 
 for therapeutic purposes, especially if, at the same time, the toxic dose 
 for the host should be smaller than that for the microorganism. 
 
 The object of chemotherapeutic research is, therefore, to discover chem- 
 icals that have a greater parasitotropic than organotropic activity, and the 
 greater the difference between these, the more valuable do the substances be- 
 come. 
 
 There is only one way of determining these values, and that is by 
 animal experimentation. Rats, mice, guinea-pigs, rabbits, and fowls 
 have been most generally employed for this purpose, and most work so 
 far has been done with protozoan parasites,^ such as the organism of 
 chicken spirilloses, the spirillum of recurreriit fever, various trypano- 
 spmes, and the spirochete of syphilis. These were selected because 
 they are readily found in the blood or in lesiohs, and are rapidly patho- 
 genic. 
 
 As a result of the study of several hundreds of different products by 
 Ehrlich and his collaborators and by many ot°her noted investigators, it 
 was found that there are, after all, comparatively few that exert a para- 
 sitotropic effect in animals; these are, however, well characterized chem- 
 ically, and have been classified into three main groups : 
 
 1. The group of arsenical compounds — arsenious acid (arsenic tri- 
 oxid), atoxyl, arsacetin, arsenophenylglycin, dioxydiamido-arsenobenzol 
 dichlorhydrate (popularly known as "606," or salvarsan), and various 
 antimony compounds. 
 
 2. Certain azo-dyes of the benzidin group, for example, trypan red, 
 trypan blue, and trypan violet. 
 
 3. The group of basic triphenylmethane dyes, such as parafuchsin, 
 methyl-violet, pyronin, and others. 
 
 Newly discovered drugs are administered in increasing amounts 
 to experimental animals until the toxic dose (dosis lethalis), the tolerated 
 dose {dosis tolerata), and the curative or sterilizing dose {dosis curativa 
 or sterilisana) have been determined. 
 
 While chance may succeed in giving us a.drug fulfilling the require- 
 ments, so far it has had little influence, and it is difficult to reahze the 
 tremendous amount of work done by Ehriich^ and the members of his 
 institute before salvarsan was discovered. The following table illus- 
 trates the relation of the tolerated to the curative doses of a few chem- 
 
PRINCIPLES OF CHEMOTHERAPY 
 
 787 
 
 ical substances, including salvarsan, given by' intramuscular injection, 
 in spirillosis of fowls, and illustrates the particular value of "606," as 
 dependent upon the fact that it is highly parasitotropic in an amount 
 that is far below the organotropic or toxic dose: 
 
 Chemical 
 
 Atoxyl 
 
 Arsacetin 
 
 Arsenophenylglycin 
 
 Amidophenylarsenoxid 
 
 Dioxydiamido-arsenobenzol (salvarsan) 
 
 Maximum 
 
 Tolerated 
 
 Dose 
 
 0.06 
 
 0.1 
 
 0.4 
 
 0.03 
 
 0.2 
 
 Sterilizing 
 
 ob cukative 
 
 Dose 
 
 0.03 
 
 0.03 
 
 0.12 
 
 0.0015 
 
 0.0035 
 
 PrtOfOiiT 
 
 3.3 
 
 3.3 
 20 
 
 58 
 
 Chemoreceptors. — Of considerable interest in this coiuiection is the 
 question of how substances can be modified in order to render them pro- 
 gressively more parasitotropic and less organotropic in action. When 
 ordinary germicides, such as phenol, mercury bichlorid, formalin, etc., 
 are added to suspensions of bacteria, we believe that the latter are killed 
 mainly as the result of poisoning of their protoplasm, and that the 
 chemical substance enters into chemical union with the bacterial al- 
 bumins by direct toxic action, accompanied by such physical changes as 
 that of coagulation, and alters bacterial metabolism and brings about 
 death of the cells. Such germicides do not appear to exert any selective 
 action on any particular albumin. When mercury bichlorid is added 
 to a mixture of bacteria in a serum, many of the microorganisms may 
 escape destruction through the formation of protective envelops of an 
 albuminate of mercury formed with the serum albumins; a similar 
 action may be noted with phenol. 
 
 In chemotherapeutic research, therefore, it is the aim to start with a 
 substance that primarily shows a more marked affinity for the proto- 
 plasm of the parasite than it does for the body-cells, and then, by sub- 
 tractmg or adding to the molecule or by inducing an intramolecular re- 
 arrangement, an effort is made to develop its parasitotropic action. 
 The question then arises, does the increased parasitotropism of the 
 chemical substance depend upon its higher direct and simple action 
 upon the protoplasm of the microparasite, or is this effect to be explained 
 upon its increased combining power or affinity for the molecules of the 
 parasites or other cells because the latter are provided with special groups 
 for effecting the union? Ehrlich has endeavored to answer this question 
 by maintaining that both body-cells and niicroparasites possess special 
 receptors or side-arms by which chemical substances may be bound; 
 
788 CHEMOTHERAPY 
 
 these he terms chemoreceptors. While Ehrlich originally assumed that 
 the so-called side-arms of the protoplasmic molecule served primarily for 
 the process of nutrition, he now believes that these special chemore- 
 ceptors do exist. He suggests that they may probably possess a less 
 complex structure, similar to the receptors of the first order for simple 
 toxins, that they are more firmly attached to the cell, and that, accord- 
 ingly, they are less readily cast off, thus explaining why crystalline 
 chemical substances are, as a general rule, incapal^le of eliciting the pro- 
 duction of corresponding antibodies. This theory is based upon the 
 discovery that certain strains of a microparasite develop a state of 
 "resistance" or "fastness" to a particular substance, and that this ac- 
 quired characteristic may be transmitted frorn generation to generation. 
 This subject will be further discussed elsewherfi. 
 
 According to Ehrlich's postulate, therefore, toxic agents cannot act 
 on microorganisms unless they are fixed by suitable receptors {corpora 
 nan agunt nisi fixata). This conception is similar in every way to his 
 conception of the processes of infection and immunity and the develop- 
 ment of antibodies; that is, the toxic agent must first be "fixed" or 
 "anchored" to the molecule of a cell by suitable receptors by a process 
 of chemical interaction before damage can be inflicted. Chemoreceptors 
 differ from other receptors in being more firmly attached to cells, so 
 that while the cell may become immune to the toxic effects of the agent, 
 the blood-serum may not contain the immune bodies. 
 
 When arsenic is introduced into the body, it is "fixed" by the re- 
 ceptors of certain cells; mercury in turn is fixed by other receptors, and 
 so on through the list. The basic principle of chemotherapy is that it is 
 possible to produce chemical substances that carry side-arms capable of being 
 fixed by microparasites, and to a much less extentby the body-cells. 
 
 It is not necessary that the whole molecule of a toxic substance possess 
 a combining affinity for certain receptors: if one or more atom groups 
 becomes attached, it is presumed that it carries with it the remainder 
 of the molecule. Moreover, that atom group that is anchored or is re- 
 sponsible for the anchorage of the entire molecule need not possess any 
 of the properties of the entire molecule or of any part thereof. For e::- 
 anaple, when the molecule of salvarsan is anchored to the spirochete of 
 syphilis by its OH or its NH2 side-chain, or by both, the spirochete must 
 later contend with two molecules of arsenic, which, being in a trivalent 
 condition, can exercise its toxic effects to a marked degree upon the para- 
 site. The side-arms as they exist in salvars^j;! are much more readily 
 taken up by the spirochete than by the bodj-cells, which is shown by 
 
PRINCIPLES OF CHEMOTHERAPY 789 
 
 the marked difference between the lethal and tolerated doses and the 
 curative dose. 
 
 Drug "Fastness." — In the foregoing chapters we have sought to 
 emphasize the importance of the microorganism in the processes of in- 
 fection and immunity from the standpoint of the possibilities of these 
 cells immunizing themselves against the deleterious agencies of the host, 
 and particularly the antibodies, as explained in the hypothesis of Welch. 
 (See p. 103.) It soon appeared that the problem of chemotherapy was 
 greatly complicated by these activities on the part of the parasite, so 
 that the hypothesis appears to be further supported as a result of chemo- 
 therapeutic studies. If the dose of a chemical is just small enough to 
 allow a few microorganisms to escape, these immediately fortify ("im- 
 munize") themselves against the drug and become invulnerable to its 
 effects. It was found that these microparasites were then able to multi- 
 ply, even in the presence of the drug, and, further, that this property of 
 "drug resistance" was transmitted from one generation to another. If, 
 for example, the trypanosomes in a mouse have become resistant to 
 trypan red, a quantity of these trypanosonies may be inoculated into 
 another mouse, and from this one to another, and so on, for many gen- 
 erations, and it would finally be found thai| the trypanosomes still re- 
 tained their immunity to the action of trj^jan red. 
 
 This acquired resistance or "fastness" is in a large measure specific. 
 A strain of trypanosomes resistant to the benzidin dyes is non-resistant 
 to arsenic and the triphenylmethane dyes, whereas one resistant to ar- 
 senic is not resistant to the dyes, etc. As Levaditi and Fraser have 
 shown, the antibody-resistant trypanosomes do not anchor the antibody, 
 hence it is probable that the analogy holds for the drug-resistant micro- 
 parasites. 
 
 As was previously stated, Ehrlich has explained this phenomenon 
 on the basis of chemoreceptors. He has ascertained that "fastness" 
 for a certain chemical agent does not depend on atrophy of the corres- 
 ponding receptors, but upon a modification in their structure, as is evi- 
 denced by the fact that, by changing the structure of the chemical, it 
 may still find suitable receptors and lead to the destruction of the para- 
 site. For example, mice that have been infected with arsenic-fast try- 
 panosomes may be cured by an injection of arsenophenylglycin, even 
 at a time when death is imminent. It would appear, therefore, that 
 the arsenic-fast receptors of the trypanosomes were but slightly altered, 
 and were still capable of uniting with an allied product. 
 
 This factor greatly complicates chemotherapy. If, for instance, the 
 
790 CHEMOTHERAPY 
 
 destruction of trypanosomes by an arsenical preparation has not been 
 complete, and if the antibodies produced by the body-cells do not suc- 
 ceed in destroying the remainder, there is a strong probability that a 
 new strain will now develop that will be resistant not only to the par- 
 ticular arsenical preparation used, but will also be proof against the 
 serum antibodies. This new strain may now cause a relapse of the 
 disease. Another chemical is now injected, but if this likewise fails to 
 kill all the trypanosomes, the remainder will generate still another 
 strain "resistant" to the preparation and the antibodies. This may 
 continue as long as the parasite is able to produce new receptors; when 
 this limit is reached, its nutrition will be impaired and the serum anti- 
 bodies, alone or aided by another chemical substance, may finally 
 destroy all trypanosomes, the infection "dying out," so to speak, or 
 proving completely vulnerable to a chemical agent. 
 
 These considerations have an actual experimental basis, and natural 
 examples of acquired serum-resistance or "fastness" are to be found in 
 relapsing fever, syphilis, sleeping sickness, and possibly malaria. In 
 relapsing fever the clinical course of the disease would indicate that only 
 three or four serum-fast strains can be produced, and we accordingly 
 find that, after a patient has withstood a number of relapses corre.spond- 
 ing to the number of antibody-fast strains that the spirillum may pro- 
 duce, spontaneous recovery occurs, there being then antibodies that 
 the strain cannot resist, and the infection "dies out," due in part to 
 destruction of the antibodies and in part to starvation of the parasite 
 because the number of receptors is insufficient to carry on nutrition. 
 In the mean time, however, the patient may succumb to the disease 
 before the spirillum has "played its last card." 
 
 In syphilis conditions are different, and the phenomenon of "resis- 
 tant races" further explains many conditions not previously understood. 
 The spirochete is apparently capable of repairing its receptors to a re- 
 markable degree, especially after injury received from the antibodies pro- 
 duced by body-cells, and, further, is able to maintain its nutrition with 
 a number of different food-stuffs, so that in, the untreated person re- 
 lapse follows relapse, the offensive forces of the spirochete being held in 
 abeyance by the defenses of the host over long periods of time (latent 
 periods), but the vital parts of the host being gradually damaged and 
 the defenses weakened so that death or serious symptoms may supervene 
 long before the disease has "worn itself out, " if, indeed, this ever occurs 
 in syphilis. 
 
 This phenomenon also explains why the syphilitic person cannot be 
 
PRINCIPLES OF CHEMOTHERAPY 
 
 791 
 
 reinoculated with syphilis while his disease is active. That he is vul- 
 nerable to syphilis is evidenced by the activities of the spirochetes within 
 his tissues and body-fluids. But this is so because a race is at work 
 that is resistant to the antibodies first produced, whereas these anti- 
 bodies are still potent and capable of killing a fresh, non-resistant race 
 of spirochetes. 
 
 The theory of chemoreceptors affords an explanation of this phe- 
 nomenon, just as the side-chain theory affords an explanation of the for- 
 mation of cellular antibodies and the processes of immunity. Tn adopt- 
 ing it we must be prepared to believe that the number of receptors and 
 the possibilities of their repair or modification are practically unlimited, 
 as evidenced by the large 
 number of substances capable 
 of exerting toxic action. Syph- 
 ilis, for example, is probably 
 a disease of great antiquity, 
 handed down from person to 
 person, from generation to gen- 
 eration, during which trans- 
 mission the receptors of the 
 spirochete must have under- 
 gone innumerable transforma- 
 tions and alterations, yet find- 
 ing suitable receptors in the 
 cells of practically all non- 
 syphilitic persons, although it 
 is now apparent that strains 
 have gradually been evolved 
 that possess receptors with 
 
 marked affinity for certain tissues, as, for example, those for the central 
 nervous system, cardiovascular system, etc. 
 
 Therapia Magna Sterilisans. — As the development of resistant 
 strains is thus one of the possibilities and impedunents to successful 
 specific therapy, whether with chemicals (chemotherapy) or with anti- 
 bodies (serum therapy) , our efforts should be directed toward discover- 
 ing chemical substances and a method of administration that will com- 
 pletely sterilize the individual at one time (Jilhrlich's therapia magna 
 sterilisans). This possibility has been amply demonstrated experi- 
 mentally by Ehrlich, and it has occasionally been accomplished in the 
 ' " Die Behandlungder Syphilis" (KonigsbergVersammlung), Leipzig, 1910,930. 
 
 Fig. 140. — BlcSod of a Rat Infected with 
 
 Spiboch^ta Recurrentis. 
 
 (Drawing made from dark-field illumination 
 
 just before administration of salvarsan.) 
 
792 
 
 CHEMOTHERAPY 
 
 early stages of human syphilis by means of salvarsan administered early 
 in the proper manner and in correct dosage; but, unfortunately, it is 
 not true of the majority of cases, the difficulties increasing with the 
 duration of the infection. Fortunately, the investigations of Margulies 
 have shown that while resistant races of trj^anosomes are easily evolved, 
 the evidence is entirely against the probability of the development of arsenic- 
 resistant syphilitic spirochetes as the result of prolonged treatment with non- 
 sterilizing doses of salvarsan. 
 
 With this brief discussion of the primary principles of chemotherapy, 
 which is really a very ancient method of treatment and dates from the 
 
 time, that chemicals were first 
 used for treating the sick, 
 but which has now emerged 
 from the darkness of pure em- 
 piricism into the light of a 
 science, we shall pass on to a 
 consideration of salvarsan and 
 neosalvarsan. These two sub- 
 stances were not discovered as 
 the result of accident, but 
 were the outcome of exact 
 knowledge, logical thinking, 
 and carefully planned experi- 
 mentation. It is impossible 
 to tell what these discoveries 
 presage; certainly they open 
 up vast possibilities in the 
 development of a specific therapy for all infections. One fact is cer- 
 tain: that while chance must ever play some role, future discoveries 
 will probably result only from prolonged, patient, and laborious study. 
 
 Fig. 141. — Blood of Same Rat Eigh- 
 teen Hours after Intravenous Injection 
 OF 0.0008 MG. Salvarsan. 
 
 SALVARSAN AND NEOSALVARSAN IN THE TREATMENT OF SYPHILIS 
 Historic. — The administration of arsenic in protozoan infections has 
 long been a recognized method of treatment. The organic compound 
 of arsenic known as atoxyl (the sodium salt of para-aminophenylarsenic 
 acid) was first used in the treatment of trypanosomiasis, and although 
 this drug did not produce the results that were anticipated, it formed the 
 starting-point for important researches in the preparation of organic 
 compounds of arsenic and their use in protozoan infections. On the 
 
SALVARSAN AND NEOSALVARSAN IN TREATMENT OE SYPHILIS 793 
 
 strength of Schaudinn's statement regarding the close biologic relation- 
 ship of the spirochetes of syphilis to trypanosomes, Uhlenhuth was led 
 to employ atoxyl in the treatment of experimental spirillosis in fowls. 
 These experiments, in addition to those of Weisser and Metchnikoff, 
 demonstrated beyond doubt the curative and prophylactic properties of 
 atoxyl in spirillar infections. The drug was, therefore, administered 
 in cases of syphilis in the human subject. It was found to exert a very 
 beneficial effect, especially in malignant forms of the disease. Further 
 experience, however, demonstrated that atoxyl was too toxic for use in 
 the human subject, for digestive disturbances, nephritis, and especially 
 optic atrophy could be traced to its use, even in small doses. As a 
 therapeutic agent, therefore, it has gradually come into disfavor. 
 
 Ehrlich and Bertheim then made the valuable discovery that the 
 chemical constitution of atoxyl was not, as had been supposed, that of a 
 metarsenate, but that it was in reality that of a para-amidophenyl- 
 arsenate. Working on this basis, these observers prepared the substance 
 known as arsacetin (the sodium salt of acetylpara-amidophenylarsenic 
 acid), which, although less toxic than atoxyl, did not fulfil the require- 
 ments of a remedy, and its use was discontinued because of its toxic 
 effects on human tissues. To lessen the relatively great toxicity of these 
 compounds for human tissues was a problem that Ehrlich set out to 
 ^solve. A most important advance in this direction was made when it 
 was discovered that the unsaturated trivalent arsenic in arsenobenzol 
 and arsenophenylglycin has a greater parasiticidal power relative to its 
 toxic action on the tissues of the host than the pentavalent arsenic 
 compounds, such as atoxyl and arsacetin. 
 
 Finally, after testing hundreds of these arsenical compounds, it was 
 found that in dioxydiamidoarsenobenzol we had a substance that ap- 
 proached the ideal in chemotherapy, since it is a drug that possesses a 
 maximum degree of parasitotropism and a minimum degree of organo- 
 tropism. Ehrlich and Hata designated this light yellow, readily oxidiz- 
 able powder as No. 592. Its hydrochloric acid salt was designated No. 
 606, and constitutes the well-known salvarsan. 
 
 In the published account of their researches Ehrlich and Hata^ 
 describe an extensive series of investigations in experimental relapsing 
 fever, spirillosis of fowls, and syphilis of rabbits. These go to prove the 
 high curative value of salvarsan, its relatively feeble toxicity for the 
 
 '"Die experimentelle Therapie der Spirillosen^" Berlin, 1910; Zeitschr. f. 
 Immunitatsf., Ref. 1910, 1123. Numerous publicatioiis from the Institute of Exper- 
 imental Therapy, Frankfort. 
 
794 
 
 CHEMOTHERAPY 
 
 tissues of the infected animals, and its great superiority over all other 
 substances that possess spirillicidal properties. 
 
 Clinical evidence in the treatment of human syphilis supported the 
 experimental findings. On account of the disadvantages of salvarsan, 
 due to its insolubility and the necessity for using the hydrochlorid, and 
 also in an effort still further to lessen its toxicity, Ehrlich conducted 
 further researches to discover a neutral salt that would be more soluble 
 and less toxic. As a result of these efforts neosalvarsan, or "914," has 
 been produced. This number is significant of the number of experiments 
 performed since the discovery of "592" and "606." 
 
 Properties of Salvarsan. — Salvarsan is the dihydrochlorid of dioxy- 
 diamidoarsenobenzol, and occurs as a yellow, crystalline, hygroscopic 
 powder, very unstable in air, and easily oxidized to poisonous com- 
 pounds. It is marketed commercially put up in small sealed vacuum 
 tubes. It contains 31.57 per cent, of arsenic, is readily soluble in water, 
 particularly in hot water, and yields a solution having an acid reaction. 
 If the acidity is neutralized by the addition of caustic soda solution, 
 the unsoluble base (dioxydiamidoarsenobenzql) is precipitated. If only 
 half of this amount of alkali is added, thensthe monohydrochlorid of 
 dioxydiamidoarsenobenzol is formed. If, in addition to the amount of 
 caustic soda necessary to precipitate the base, a further quantity of al- 
 kali is added, the hydrogen atoms of the phenol hydroxyls become re- 
 placed by Na and the compound goes into solution as the disodium 
 salt of dioxydiamidoarsenobenzol: 
 
 As As As As 
 
 CIH.NH: 
 
 NHnClH 
 
 NH2 
 
 OH OH 
 
 DihydrochJorid (salvarsan) 
 
 NH2 
 
 ONa ONa 
 
 Disodium salt (alkaline solution) 
 
 Test-tube experiments showed less spirillicidal power of the drug 
 than is demonstrated in the living animal. The following table shows 
 the toxicity of the preparation (Ehrlich and Hata) : 
 
 Animal 
 
 Method of Administeatio:^ 
 
 Maximum Tolerated Dose 
 
 Mouse 
 
 Mouse 
 
 Subcutaneous 
 
 Intravenous 
 
 Subcutaneous 
 
 Intramuscular 
 
 Intravenous 
 
 Intravenous 
 
 Subcutaneous 
 
 1:300 per 20 grams 
 1 : 350 per 20 grams 
 
 Rat 
 
 Fowl 
 
 Fowl 
 
 Rabbit 
 
 Rabbit 
 
 0.2 gram per kilogram 
 0.25 gram per kilogram 
 0.08 gram per kilogram 
 0.1 gram per kilogram 
 0.15 gram per kilogram 
 
SALVARSAN AND NEOSALVAESAN IN TREATMENT OF SYPHILIS 795 
 
 In experimental syphilis of rabbits the minimal dose necessary to 
 produce a complete cure was found to be between 0.01 and 0.015 gram 
 per kilogram. The tolerated dose by intravenous injection is 0.1 gram; 
 the curative dose of salvarsan in syphilis of rabbits is, therefore, only 
 from one-seventh to one-tenth of the tolerated dose. 
 
 Properties of Neosalvarsan. — This is an orange-yellow powder 
 possessing a peculiar odor. It is very unstable in the air and is readily 
 soluble in water, yielding a yellow solution that is neutral to litmus. Its 
 structure is somewhat more complex than that of salvarsan, being a 
 condensation-product of the latter and hydraldit (formaldehyd sulph- 
 oxylate of sodium), the reaction taking place according to the follow- 
 ing equation: 
 
 NHa] = NII2 + nO.CH.O.SO Na = NHj 
 
 OH OH OH 
 
 As 
 
 n. 
 
 I NH.CHjO.SO Na+ao 
 OH 
 
 
 While neosalvarsan is less toxic than salvarsan, and although it is 
 much more easily administered and largely free from irritative effects, 
 recent clinical reports would tend to show that its spirocheticidal prop- 
 erties are somewhat less than those of salvarsan. 
 
 Methods of Preparing Salvarsan for Administration. — Soon after 
 the introduction of the drug several methods of preparation and adminis- 
 tration were suggested. Many of the disadvantages attached to sal- 
 varsan treatment, and many of the bad results and complications re- 
 ported, are to be attributed to defective methods of preparation and 
 administration of the drug.' 
 
 Because of the marked stability of salvarsan and neosalvarsan, the 
 following points must be borne in mind: 
 
 1. The ampule containing the drug must be intact. 
 
 2. The powder must be of a yellow and not of a gray or brownish color. 
 Each ampule must be carefully examined before the contents are 
 
 administered. 
 
 3. The drug should be prepared for injection just prior to administra- 
 tion. 
 
796 CHEMOTHERAPY 
 
 It is not necessary to describe the earlier methods, because these are 
 now largely only of historic interest, and it is quite generally accepted, 
 from the point of view both of efficient treatment and of the comfort of 
 the patient, that the intravenous injection of q dilute solution of the di- 
 sodium salt is the best form of administration. For this reason I shall 
 briefly mention the other methods, and describe the method of intraven- 
 ous injection of the alkaline solution in greater detail further on. 
 
 The Acid Solution. — When salvarsan is dissolved in warm water or 
 warm normal saline solution, a strongly acid solution is obtained. In 
 this form the drug is most irritating and also most toxic, and when in- 
 jected subcutaneously and intramuscularly, produces severe pain and 
 necrosis. This method is seldom if ever used at the present time. 
 
 The Mono-acid Solution. — If to the watery acid solution half the 
 amount of alkali necessary to produce complete neutralization is added, 
 a solution of the mono-acid compound will be formed. This solution 
 has been given intramuscularly, but is also extremely irritating. 
 
 The Neutral Suspension. — This is the drug in the form of a precipi- 
 tate of the base, prepared by adding to the original acid solution just 
 sufficient caustic soda to neutralize it. The method was devised by 
 Michaelis and Wcchselmann, and for some time was the form of adminis- 
 tration most generally employed for subcutaneous and intramuscular 
 injection. It is probably less irritating than the acid and clear alkaline 
 solution, but occasionally there resulted encapsulation of masses of ne- 
 crotic tissue containing considerable quantities of arsenic. 
 
 Other Suspensions. — Suspensions of salvarsan in liquid paraffin, 
 sterile olive oil, oil of sesame, or almond oil are said to keep for some 
 time if placed in dark containers. This cannot, however, be always de- 
 pended upon, and the slightest decomposition of the original drug is 
 capable of producing marked toxic symptoms. These suspensions are 
 said to be comparatively non-irritating, but they may cause local necrosis 
 of tissues, and on account of their slow absorption, only small amounts 
 gain access to the general circulation at one tipie. 
 
 This method may, however, be of value, eispecially in infants and in 
 cases where slow absorption is desired in order to prolong the effect of 
 the drug. Analgesics, such as eucain, creosote", or an essential oil, may be 
 incorporated in the suspension in order to lessen the pain. 
 
 The suspension is so prepared that each cubic centimeter contains 
 0.1 gram of the drug. 
 
 Alkaline Solution of the Disodium Salt. — This is the form in which the 
 drug should be administered by intravenous injection. It is this solu- 
 
ADMINISTRATION OF SALVARSAN AND NEOSALVARSAN 797 
 
 tion which Hata used in his original experiment-s, and it was the form 
 recommended by Ehrlich. I shall, therefore, describe this method in 
 detail a little further on. 
 
 ADMINISTRATION OF SALVARSAN AND NEOSALVARSAN 
 Intravenous Injection 
 
 Dosage of Salvarsan. — Males receive in general about 0.3 to 0.6 gm.; 
 female patients are usually given from 0.25 to 0.5 gm. In the case of 
 weak and poorly nourished adult patients it in inadvisable to give more 
 than 0.3 or 0.4 gm. Since it has been generally impossible to cure a 
 patient with a single larger dose, the practice among syphilologists at 
 present is to give smaller doses frequently repeated. 
 
 For infants suffering from congenital syphilis the dose is from 0.006 
 to 0.01 gm. of salvarsan for every two pounds of body weight, so that a 
 child of eight pounds would receive from 0.024 to 0.04 gTn. of salvarsan. 
 To older children, weighing from 40 to 60 pounds, 0.2 to 0.3 gm. may be 
 given. 
 
 Dosage of Neosalvarsan. — This preparation is less toxic than sal- 
 varsan, and may be administered in larger doses, as from 0.6 to 1 gm. 
 The same general rule as to the physical condition of the patient should 
 apply here in deciding the dosage. At the present time the tendency 
 is to give adult patients about 0.6 gm. for three, four, and more injec- 
 tions at intervals of a week or so. 
 
 Frequency of Injections; Intensive Treatment. — As was just stated, 
 there is a distinct tendency among those of large experience to regard 
 salvarsan as a more potent spirocheticid than neosalvarsan. As pre- 
 viously mentioned, the original idea of sterilizing the patient with one 
 large dose of the drug has been largely abandoned, especially in the 
 treatment of syphilis in any but the earliest stages. A large number of 
 smaller doses are being given, and the results are controlled by the 
 Wassermann reaction with blood and cerebrospinal fluid, in addition to 
 the cytologic changes in the latter. Thus from 0.3 to 0.5 gm. of sal- 
 varsan or neosalvarsan is given every week or twice a week for three, 
 four, or ten doses and more, depending upon the clinical results and the 
 serologic findings. In this connection it must be remembered that a 
 negative Wassermann reaction is of little value if blood has been with- 
 drawn within two weeks of the last treatment.- (See Chapter XXIII.) 
 While it is the common practice to administer, ji number of doses of sal- 
 varsan or neosalvarsan, and to follow this with mercury and then with 
 
798 
 
 CHEMOTHERAPY 
 
 salvarsan again, this methiod must be regarded as containing an element 
 of danger, especially since Wechselmann has drawn attention to the 
 fact that mercury acts as an irritant to the kidneys. It may be stated 
 that, in general, most cases of syphilis require a number of injections 
 of salvarsan; this number depends upon the age and nature of the 
 infection, and should be controlled by the Wassermann reaction with 
 both blood and spinal fluid. 
 
 Preparation of the Patient. — In view of the fact that many of the 
 fatalities due to salvarsan have been ascribed to defective kidneys, 
 especially to kidneys damaged by the previous administration of mercury, 
 it should be a routine measure to have the urine thoroughly examined for 
 sugar, albumin, and casts previous to the administration of salvarsan 
 or neosalvarsan. 
 
 While thousands upon thousands of injections have been made with- 
 out ensuing untoward results, still the administration of this drug is not 
 without danger, and the physician should familiarize himself with the 
 contraindications, and subject his patient to a careful physical examina- 
 tion before undertaking this form of therapy. This subject will be 
 discussed further under the head of Contraindications to Salvarsan 
 Therapy. 
 
 In the majority of cases, however, no contBaindications exist and no 
 elaborate preparations are necessary. The rectum should be emptied 
 before the injection is given, and it is best to administer the drug on an 
 empty stomach. After receiving salvarsan the patient should rest over- 
 night under direct supervision, as in a hospital, the injection being given 
 during the afternoon or early evening hours. The same practice should 
 be foUowed with neosalvarsan, although in thousands of instances pa- 
 tients have received an intravenous injection, rested for an hour or so, 
 and then returned to their homes. 
 
 Preparation of Salvarsan Solution. — ^As previously mentioned, the 
 physician should examine the ampule containing the drug to convince 
 himself that it is intact. The solution should be prepared just before it is 
 injected. On account of the oxidation that occurs it is unsafe to use the 
 drug if the ampule has been open for a number of hours, and for the 
 same reason it is not good practice to prepare a bulk solution for a num- 
 ber of patients unless the physician is certain that he will be able to make 
 the injections qviickly and without interruption. 
 
 The clear alkaline solution is most generally used. It is prepared 
 as follows: 
 
 1. The diluent should consist of sterile and freshly distilled water. 
 
ADMINISTRATION OF SALVARSAN AND NEOSALVAKSAN 799 
 
 Many of the toxic and other ill effects attending salvarsan therapy have 
 been attributed to the use of raw and stale water. Experiments con- 
 ducted by Yakinoff show that the presence of the endotoxins of such 
 microorganisms as Bacillus coli, Bacillus pyocyaneus, and staphylococci 
 in the water, increases the toxicity of salvarsan from two to eight times, 
 the water alone and the salvarsan alone being without effect. In office 
 practice physicians may distil water by means of some sunple appa- 
 ratus, such as that of Muencke. The distilled water is then sterilized 
 in an Arnold sterilizer for one-half hour, or by boiling for ten or fifteen 
 minutes. 
 
 2. All glassware used should be carefully sterilized, usually by boiling, 
 as in the physician's ordinary office sterihzer. 
 
 3. The ampule containing the drug is wiped off with alcohol, the 
 neck filed across and broken off, and the contents emptied into a sterile 
 50 c.c. glass-stoppered mixing cylinder containing preferably a number of 
 smaU glass beads. From 15 to 20 c.c. of hot (about 50° C.) sterile dis- 
 tilled water are added, and with shaking the drug passes into solution. 
 A 15 per cent, solution of caustic soda is now added drop by drop to the 
 solution in the cylinder. A precipitate of the base is first deposited, 
 and on further addition of caustic soda, aid^d by shaking, this is again 
 brought into solution, the fluid being strongly alkaline. The amount of 
 alkali necessary is about four drops of a 15 per cent, solution for each 
 0.1 gm. of salvarsan; thus, for 0.6 gm., 1.14 c.c, or about from 23 to 45 
 drops of 15 per cent, solution of caustic soda, would be required. Citron 
 gives the following table: 
 
 0.2 gm. salvarsan requires 0.38 c.c. of 15 per cent, sodium hydroxid = 8 drops. 
 0.3 gm. salvarsan requires 0.54 c.c. of 15 per cent, sodium hydroxid = 12 drops. 
 0.4 gm. salvarsan requires 0.76 c.c. of 15 per cent, sodium hydroxid = 15 or 16 drops. 
 0.5 gm. salvarsan requires 0.95 c.c. of 15 per cent, sodium hydroxid = 19 or 20 drops. 
 0.6 gm. salvarsan requires 1.14 c.c. of 15 per cent, sodium hydroxid = 23 to 24 drops. 
 
 A drop more of the alkali than is just necessary to produce the clear 
 solution should be added. If this is not done, on cooling the solution 
 may show a precipitate; this can be redissplved by the addition of a 
 drop of alkali. The drug is now in a solution of about 20 c.c, and if 
 an intramuscular injection is to be given, this is the form to be preferred. 
 Intramuscular injections are, however, likely to be paiiiful, and should 
 be given only in those cases where it is practically impossil^le to give the 
 drug intravenously, as in the case of infants. For intravenous injec- 
 tion the solution of 20 c.c is diluted with warm sterile distilled water to 
 make 300 c.c. in a second sterile cylinder or beaker, which is graduated 
 
800 
 
 CHEMOTHERAPY 
 
 or bears a 300 c.o. mark; with 0.6 gm. in solution, each 50 c.c. contain 
 0.1 gm., so that the dose given may be controlled by the amount of fluid 
 injected. This flask is thoroughly shaken to insure an even diffusion 
 of the drug, and the contents poured into the cylinder from which it is 
 administered, being filtered into this cyhnder through a piece of sterile 
 gauze in order to remove any bits of broken glass from the ampule that 
 may have gained access or any other insoluble particles. (See Fig. 142.) 
 
 Fig. 142. — Intravenous Injection of Salvarsan. 
 With this apparatus the operator may give an injection without assistance. 
 Notice the three-^-ay cock, which permits the flow of salt solution or salvarsan solu- 
 tion at will. Usually a tourniquet composed of a simple rubber tubing and held in 
 position by ahemostat is better than the one shown, as the operator can quickly re- 
 lease it with least disturbance and loss of time. Note the funnels in both con- 
 tainers for straining the salvarsan solution and distilled Water or salt solution. (After 
 the apparatus of Boehm.) 
 
 Preparation of Neosalvarsan Solution. — This drug is readily soluble 
 in water, forming a clear solution which is ready for use. File the neck 
 of the ampule, cleanse it with alcohol, and break open. The contents 
 are emptied directly into a flask containing 200 c.c. of warm, sterile, 
 freshly distilled water. On gentle agitation the drug rapidly dissolves. 
 Hot water should not be used, nor should a solution be heated once it has 
 been made. From the mixing flasks the sojution is poured into the 
 cylinder through a piece of sterile gauze, which filters out any bits of 
 
ADMINISTRATION OF SALVARSAN AND NEOSALVARSAN 801 
 
 glass or other insoluble particles. For administration any simple ap- 
 paratus, such as that shown in Fig. 143, answers all purposes. 
 
 Neosalvarsan can also be given intravenously in a concentrated form 
 by dissolving the drug in 20 c.c. of sterile distilled water. It is injected 
 by means of an ordinary glass syringe or by the special syringe devised 
 for this purpose shown in Fig. 135. 
 
 Injecting Apparatus. — A number of different apparatus for the ad- 
 
 Fia. 143. — Method of Making Intravenous Injection by Gravity. 
 This method is suitable for the intravenous administration of salvarsan or anti- 
 streptococcus serum, etc. The needle has been entered into a prominent vein 
 (indicated by a flow of blood); the tubing has been attached by means of a metal 
 tip which fits the needle easily and snugly; the tourniqijet has been loosened and the 
 injection is being given. 
 
 ministration of salvarsan and neosalvarsan have been invented and 
 marketed. That shown in Fig. 142 is well adapted for the purpose; one 
 cylinder carries sterile normal salt solution and the second holds the 
 salvarsan or neosalvarsan solution. By fastening the cylinders to an up- 
 right rod attached to the side of the table their height may be adjusted 
 as desired, and by means of the special cock the operator may insert the 
 needle and allow the salt solution to flow in until he is sure of having 
 satisfactorily penetrated the vein, when he may turn 6n the salvarsan 
 51 
 
802 CHEMOTHERAPY 
 
 solution. In this manner the physician is enabled to give an injection 
 without assistance. 
 
 Or, if desired, the simpler apparatus shown in Fig. 143 may be used. 
 This consists of a single cylinder to the narrow lower end of which about 
 five feet of rubber tubing are attached. A piece of glass tubing is in- 
 serted at the lower end to serve as a window, and at the end there is an 
 arrangement whereby it can easily be attached to the needle used for 
 making venipuncture. A clamp is placed on the tubing at some con- 
 venient place, where it may be operated by an assistant. 
 
 Whatever apparatus is employed, it should be sterilized before use. 
 The cylinders and needle are boiled in an office sterilizer, and the tubing 
 is cleansed by running sterile salt solution or water through it. The 
 needle should have a sharp point with a short beveled edge, as a long- 
 pointed needle may pierce the vein through and through. Sufficient 
 sterile distilled water or normal salt solution is added to fill the rubber 
 tubing, while the cylinder should contain an additional 10 or 15 c.c. 
 If the double cylinders are used (Fig. 142), one should contain from 20 to 
 30 c.c. of salt solution or water and the second the solution of the drug. 
 The solution of drug is then filtered into the second cylinder, and the 
 pinch cock opened for a minute to be sure that all air has been expelled. 
 
 2. The patient should lie on a bed or on an operating table. An arm 
 — usually the patient's left in the case of right-handed operators — is 
 prepared by placing a few towels around it and a firm tourniquet is ap- 
 plied above the elbow. Whatever material is used, whether rubber or a 
 broad muslin bandage, it should be fastened with a hemostat, for when 
 it becomes necessary to unfasten it, this may be done quickly and with 
 least disturbance by the operator, who simply unfastens the hemostat. 
 The skin over a prominent vein may be cleansed with green soap and 
 water, followed by alcohol and 1 : 100 bichlorid solution, or simply by 
 adding one or two coats of 10 per cent, tincture of iodin, which is washed 
 off with cotton and alcohol just before the needle is inserted. In fat 
 subjects a vein can frequently be felt when it cpflinot be seen. Occasion- 
 ally it may be necessary to infiltrate the skin with sterile eucain solution 
 and expose the vein by incision. 
 
 3. The operator now passes the needle into a vein. With experience, 
 considerable skill is gained in performing this little operation. A free 
 flow of blood indicates that the needle has been properly inserted, and 
 one can easily tell bj^ the sense of touch whetlwr the needle is free in the 
 lumen. The tourniquet is then quickly and deftly released by the oper- 
 ator, the clamp on the tube is opened, and while the blood is flowing from 
 
ADMINISTRATION OF SALVAESAN ANP NEOSALVARSAN 803 
 
 the vein and the saHne solution or water from the tubing the nozle of 
 the latter is fitted into the needle quickly and securely while the latter 
 is held firmly in the vein. The appearance of bulging at the side of the 
 vein and the occurrence of pain indicate that the needle has not been 
 properly inserted. In this event the clamp should be fastened, the 
 needle withdrawn, the tourniquet adjusted, and another vein punctured. 
 It is useless to attempt to enter the same vein at the same point. 
 
 4. The introduction of a full dose of salvarsan or neosalvarsan will 
 usually take from ten to twenty minutes or thereabouts. If the flow is 
 retarded, the needle may be turned gently and slightly so as to change 
 the relation of the bevel to the wall of the vein. 
 
 5. When salt solution forms the first portion of the injection, no 
 harm has been done if perivascular infiltration occurs. This method 
 gives added assurance to the operator; indeed, it should never be 
 omitted when salvarsan is being injected, and it is a good general rule to 
 have the first portion of the injection consist of normal salt solution. 
 
 6. After the requisite dose has been injected-, a few cubic centimeters 
 of salt solution are again permitted to flow into the vein, so that the tub- 
 ing and needle are washed free from salvarsan, =and at no time does the 
 drug come in contact with the tissues. 
 
 7. The needle is then quickly withdrawn, and the site of the punc- 
 ture sealed with collodion and cotton after the iodin has been removed 
 by washing with alcohol. 
 
 After-care of the Patient. — In the majority of instances the adminis- 
 tration of salvarsan is not followed by unpleasant symptoms. This 
 depends, however, to a considerable extent upon the nervous constitu- 
 tion of the patient. Many persons will complain of a feeling of fullness 
 and may perspire freely for a short time. There may be slight pain at 
 the site of injection and in the axilla of the injected side. As was pi-e- 
 viously mentioned, salvarsan should be administered at the patient's 
 home or in a hospital, followed by rest in bed until the next morning. 
 When neosalvarsan is injected, robust persons may, after resting for an 
 hour or so, travel homeward. Occasionally severer reactions follow 
 salvarsan administration, and these may be considered under the head 
 of after-effects. 
 
 After-effects of Salvarsan. — Within an hour or two after its admin- 
 istration arsenic is excreted by the kidneys and bowels and nausea may be 
 complained of. If catarrh of the stomach is present, severe vomiting may 
 ensue. The nausea is relieved by sipping a little hot water; hot applica- 
 tions over the stomach and small amounts of carbonated water or cham- 
 
804 CHEMOTHERAPY 
 
 pagne will usually control the vomiting. It may occasionally be neces- 
 sary to administer }/g grain of morphin hypodermically, especially if the 
 patient is of a neurotic temperament. Headache may occasionally de- 
 velop, and is due to a neurotic condition, constipation, anxiety, hunger, 
 or possibly to an increase in the exudate of the syphilitic process. Diar- 
 rhea may be observed in a few cases; this is readily controlled by the 
 administration of bismuth. Chills and fever are more infrequent at the 
 present time, the result probably of using freshly distilled water and 
 somewhat smaller doses of the drug. In some cases an arsenic rash, 
 in the form of an er3i;hema, may follow injection. This is not the 
 Jarisch-Herxheimer reaction, which is found, only in sjrphilis, the ar- 
 senic rash having been observed in non-syphilitic persons injected with 
 the drug. There is no evidence to show that salvarsan or neosalvarsan 
 will injure healthy kidneys, although a mild transient albuminuria may 
 follow the injections in some instances. When, however, the kidneys 
 have been damaged by mercury, salvarsan may give rise to an acute irri- 
 tation, and, indeed, Wechselmann ascribes many of the salvarsan casual- 
 ties to this condition, and issues the warning that, while mercury may 
 follow salvarsan, it should never precede it. The good general effects 
 following the administration of salvarsan are manifested, as a rule, in a 
 sense of well-being, and not infrequently patients who are anemic, 
 poorly nourished, and despondent in a short time become healthy, active, 
 and cheerful. This may be due in part to a psychic effect, but there 
 is frequently evidence of a far-reaching change in the nutrition of the 
 patient. 
 
 The Jarisch-Herxheimer Reaction. — This reaction manifests itself 
 in the development of a rash, the extension or aggravation of an existing 
 eruption, or an inflammatory reaction in any syphilitic tissue the result 
 of treatment. It has been observed in the course of mercurial treatment, 
 and before the discovery of the Spirocheta pallida and the Wassermann 
 reaction it was regarded as of considerable diagnostic importance. Any 
 aggravation of syphilitic symptoms following the administration of sal- 
 varsan or mercury has been interpreted as a Herxheimer reaction. The 
 cutaneous reaction is manifested by edema, redness, pain, and the 
 mucous patches show a similar reaction. Gummas become swollen, may 
 ulcerate, and show increased exudation. The lancinating pains of loco- 
 motor ataxia may be augmented, and various paralyses, due to pressure, 
 may follow in those nerves that traverse bony canals. These effects 
 are also known under the name of neur or elapses. They usually appear 
 two or three months or even four or five months after treatment, and 
 
ADMINISTRATION OF SALVARSAN AND NEOSALVARSAN 805 
 
 they were at first believed to be due to the contained arsenic and were 
 regarded as constituting a special danger attending the use of salvarsan. 
 Various explanations for this phenomenon have been offered: Ehrlich 
 believes that it indicates failure of the injected dose to produce complete 
 destruction of the spirochetes, with temporary stimulation of the micro- 
 organisms to increased multiplication and activity. He very perti- 
 nently compares the neurorelapse or Herxheimer reaction to the extensive 
 development of individual bacterial colonies on agar plates, when but 
 few microorganisms are present, as contrasted with their small size 
 when the number is large. The so-called provocative positive Wasser- 
 marni reaction may be considered as a part of this reaction. 
 
 Intramuscular Injection 
 
 As was previously stated, this route of adhiinistration is not gener- 
 ally employed at the present time because of the local irritative effects 
 of salvarsan especially. When slow absorptiofn and elimination are de- 
 sired, or when intravenous injections are impossible, as in the case of 
 very young children, this method may be adopted. When salvarsan is 
 to be given, the clear, concentrated alkaline solution is generally used. 
 Neosalvarsan is to be preferred, because it is less irritating than sal- 
 varsan. It may be given in a concentrated aqueous solution or sus- 
 pended in sterile oil with the addition of a local anesthetic, such as 
 eucain or creosote. The injections are made, under strict antiseptic pre- 
 cautions, in the gluteal region, into the upper and outer quadrant of the 
 muscles. The syringe and the needle should be sterilized and the in- 
 jection prepared. After cleansing the skin with alcohol the needle is 
 quickly and boldly plunged into the deep tissues. The syringe may 
 then be detached to ascertain if blood flows, which would show that 
 a vein has been punctured and require a reinsertion; if blood docs not 
 appear, the barrel should be reattached and the injection slowly given. 
 
 Intraspinous Injection of Neosalvarsan. — Owing to the fact that a 
 drug injected intravenously may not reach the tissues of the central 
 nervous system, salvarsan and neosalvarsan have not fulfilled the early 
 expectations, apparently chiefly for the reason that they cannot reach 
 the spirochetes. It would seem, therefore, th^t adequate treatment of 
 syphihs of the central nervous system consists in the direct application 
 of the remedy to the infected tissues themselves. Wechselmann ' and 
 Marinesco ^ have injected salvarsan, and Marie and Levaditi ^ and others 
 
 1 Deutsch. med. Wochensehr., 1912, 38,: 1446. 
 
 2 Zeitschr. f. phys. u. Therap., 1913, 17, 194. 
 
 = Bull, et Soc. med. d. hop., Paris, November 18, 1913. 
 
806 CHEMOTHERAPY 
 
 have given neosalvarsan, directly into the spinal canal. The results, 
 however, were cither dubious or frankly dangerous, and the injections 
 were followed by severe and alarming symptoms, due mainly to the pro- 
 duction by the drug of direct irritation upon the sensitive nervous sys- 
 tem. Ravaut^ has found that the use of hypertonic solutions of neo- 
 salvarsan are better borne and of value in the treatment of special cases. 
 Wile 2 has also employed this method, and has found it of some value in 
 cerebrospinal s5T)hilis. Tabetics presenting no bladder or rectal symp- 
 toms were found to do especially well. As is to be expected, the eariier 
 the treatment is instituted, the better are the results. This form of 
 treatment is, however, to be regarded as daiigerous, and is to be used 
 only in selected cases, and with a full understanding of the risks incurred. 
 Wile has given the following technic: 
 
 "The solution used for iBJection consists of a 6 per cent, solution of neosalvarsan 
 in distilled water. This solution is hypertonic, and made of such concentration that 
 each minim must contain 3 mg. of the drug. The dosage injected is from 3 to 12 
 mg. — that is, from one to four drops of the solution, which is made up as follows: 
 
 "An ampule containing 0.3 mg. of neosalvarsan is dissolved in 5 e.c. of freshly 
 distilled water. If the ampule contains 0.6 gm., 10 C;C,. of water are used. In both 
 solutions each drop will contain 3 mg. of the drug. The syringe employed for the 
 injection is accurately graduated in drops. The patient is placed in a position for 
 lumbar puncture, either sitting or lying, according to the choice of the operator. 
 The puncture is then made with the needle, the end of which fits the graduated 
 syringe. After a few drops of the spinal fluid have flowed out of the caimula, or a 
 greater quantity if a diagnostic puncture is desired at" this time, the syringe is fitted 
 into the needle, and the fluid is allowed to run back into the syringe barrel, thus 
 mixing with the amount of the drug in the barrel. The mixed spinal fluid and drug 
 are then gently forced into the canal, and slight suction is made on the syringe to 
 withdraw a second amount of fluid, which washes out the needle. This is then reintro- 
 duced, the needle is quickly withdrawn, and the patient placed in the Trendelenburg 
 position, in which position he is allowed to remain for at least one hour." 
 
 INTRASPINOUS mjECTION OF SALVARSANIZED SERUM 
 
 In this method the salvarsan or neosalvarsan is injected intrave- 
 nously in the usual manner, and shortly afterward blood is withdrawn, 
 the serum separated, heated to 55° C. for hajf an hour, and a portion 
 injected intraspinously. In this technic, which has been worked out 
 largely by Swift and Ellis, the drug is highly diluted, and therefore is 
 not likely to produce much irritation. In addition, some of the good 
 effects may be due to the presence of antibodies in the serum itself. 
 This method constitutes the most useful foriti of intraspinous medica- 
 
 1 Ann. de Med., 1914, 1, 49. 
 
 2 Jour. Amer. Med. Assoc, 1914, Ixii, 1165; ibid., 1914, Ixiii, 137. 
 
ADMINISTRATION OF SALVARSAN AND NEOSALVARSAN 807 
 
 tion thus far proposed. The details of this technic are given in Chapter 
 XXXI. 
 
 Mention has also been made, in the preceding chapter, of a method 
 of intraspinous medication consisting in the injection of a combination 
 of the patient's serum and salvarsan or neosalvarsan mixed in vitro. 
 This method is to be regarded as more dangerous and probably less 
 efficient than that of Swift and Ellis, and as yet in the experimental 
 stage. Recently Fordyce ^ has given the following technic (Ogilvie) : 
 
 "Fifty c.c. of blood are drawn into a centrifuge bottle and centrifuged twice. 
 It is important to have the serum clear and free from fibrin and blood-cells. To 
 obtain the requisite amount of the drug old salvarsan is mixed in the usual way, in 
 the proportion of 0.1 gm. to 40 c.c. of fluid, care being taken not to overalkalinize; 
 0.4 c.c. of this solution is the equivalent of 1 mg., and is taken as the standard for 
 measuring the dosage. For this purpose a 1 c.c. pipet graduated in hundredths should 
 be employed. The desired amovmt of salvarsan is added to from 12 to 15 c.c. of the 
 serum, shaken to and fro to mix thoroughly, and then placed in the incubator at 
 37° C. (98.6° F.) for one hour, after which it is inactivated for half an hour at 56° C. 
 (132.8° F.). The latter is the most important step in the technic; the spirocheticidal 
 properties of the serum are markedly increased by heating. 
 
 " Salvarsanized serum, prepared according to this method, must be used fresh, 
 that is, within three hours of the time that it is made up. Patients should be prepared 
 for its administration as for mtravenous injection, with a laxative the night before 
 and only a light meal two hours prior to the treatment. 
 
 "A lumbar puncture is made, and an amount of fluid equivalent to the amount 
 introduced is withdrawn. While the needle is in situ, the barrel of a Luer syringe is 
 connected by means of a piece of rubber tubing. Spinal fluid is allowed to fill this 
 to expel the air, and the serum is then permitted to flow in by gravity. After its 
 administration the patient should lie flat without any pillows, the foot of the bed 
 being kept elevated for several hours. He must be kept in bed for at least twenty- 
 four hours, in some cases forty-eight or seventy-two hours. Failure to do this may 
 result in unpleasant symptoms, as pain in the extremities, headache, anesthesia, 
 and bladder and rectal paresis." 
 
 The proper dose is still a matter of doubt, and a note of warning must 
 be sounded against employing too large amounts. Fordyce believes 
 that the limit of safety lies within 0.5 mg. It is better to begin with a 
 dose of 0.25 mg., repeating or gradvially increasing this according to the 
 tolerance of the patient. The intervals between doses should be two 
 weeks or longer. 
 
 In the treatment of syphilis of the central nervous system the in- 
 travenous method may first be tried, giving a series of six or eight in- 
 jections of salvarsan, beginning with a dose of from 0.25 to 0.3 gm., and 
 injecting it at intervals of from one to two weeks. At the end of this 
 time the blood and spinal fluid should be tested, and if it is found that 
 ' Jour. Amer. Med. Assoc, 1914, Ixiii, 552. 
 
808 CHEMOTHERAPY 
 
 no marked effect has been obtained either clinically or serologically, 
 a course of intraspinous injections may be considered. 
 
 CONTRAINDICATIONS AND PRECAUTIONS IN SALVARSAN THERAPY 
 When it is remembered that millions of doses of salvarsan and neo- 
 salvarsan have been given by thousands of different physicians, by 
 many and diverse methods, the relative safety of the drug is apparent. 
 The fact remains, nevertheless, that patients have succumbed soon 
 after receiving an injection who would not otherwise have died at that 
 time. These fatalities cannot be explained on a purely toxicologic 
 basis. Recently Wechselmann ^ has reviewed the deaths following 
 salvarsan therapy and has arrived at the conclusion that "insufficiency 
 of the kidney, and not hyper sensitiveness of the brain, is the point of the 
 entire question of salvarsan fatalities. " He is very emphatic in warning 
 against mixed mercurial and salvarsan treatment, especially if salvarsan 
 is given after a course of mercury, for the lattfer drug is a renal irritant 
 and may lead to prolonged retention of the salvarsan, which may undergo 
 a process of reduction in the tissues and form one of the poisonous 
 products that Ehrlich has called "arsenoxid."* 
 
 Contraindications to salvarsan therapy may be divided into two 
 main groups: 
 
 1. Those to whom the injection may be dangerous on account of 
 the reaction that may follow, as, for exampde, cases of early cerebral 
 lues with cranial nerve manifestations of an exudative character; also 
 cases of tabes with beginning optic atrophy. In these patients salvar- 
 san or neosalvarsan should be given with extreme caution and only in 
 small doses, and frequent examinations of the eyes should be made. 
 
 2. Those with extensive disease of the circulatory system, such as 
 severe uncompensated heart disease, coronary sclerosis, and extensive 
 aneurysm; also cases of diabetes mellitus, severe nephritis, ulceration 
 of the stomach, and advanced tuberculosis or carcinoma. 
 
 In this connection I may cite here the precautions laid down by 
 Wechselmann, director of the dermatological department of the Rudolf 
 Virchow Hospital, in whose clinic over 25,000 doses of salvarsan have 
 been given. These precautions are: 
 
 1. To use the most exact technic. 
 
 2. To employ a dose of the drug carefully adapted to the individual 
 
 case. 
 
 ' "The Pathogenesis of Salvarsan Fatalities," translated by Martin, 1913, 
 Fleming Smith Co., St. Louis. 
 
VALUE OF SALVARSAN AND NEOSALVARSAN IN SYPHILIS 809 
 
 3. To make careful observation by the most exact chemical and 
 
 microscopic examination of the urinary secretion when em- 
 ploying salvarsan. This holds good particularly when the com- 
 bined treatment is employed. 
 
 4. The conjoint use of salvarsan with heavy mercurial treatment is 
 
 dangerous! If one will use the combined treatment, then let 
 him give mercury very carefully many days after giving the 
 last salvarsan injection, but he must never reverse this rule. 
 
 5. Consider carefully every general reaction or rise of temperature 
 
 following the use of salvarsan, and make a thorough investiga- 
 tion as to its causes. 
 
 VALUE OF SALVARSAN AND NEOSALVARSAN IN THE TREATMENT OF 
 
 SYPHILIS 
 
 It is too soon to judge of the ultimate value of salvarsan in the treat- 
 ment of syphilis. It may be stated, however, that the immediate effects 
 are so beneficial that the drug is to be regarded as the best we possess 
 for the treatment of lues. As is to be expected, the best results are se- 
 cured when the remedy is administered early, and, of course, when tissue 
 changes have occurred no drug can be expected to restore a lost func- 
 tion, although it may bring about considerable improvement by causing 
 the disappearance of syphilitic tissue. In long-standing cases salvarsan 
 may, at least, hold the symptoms in abeyance, and effectively prevent 
 progress of the disease. It is especially valuable in patients who cannot 
 tolerate mercury. The remarkable efficacy of salvarsan is attested by 
 the rapidity with which spirochetes disappear from primary sores and 
 from secondary lesions, the manner in which they disappear being fre- 
 quently little short of marvelous. There can be no doubt but that sal- 
 varsan is a powerful spirocheticide, having but remarkably little toxic 
 effect upon the body-cells. It would appear that in order to rid the 
 tissues of the spirochetes it is only necessary to bring the drug into con- 
 tact with them. For this reason newer and better methods of adminis- 
 tration are bound to increase the value of the drug. Barring the well- 
 recognized contraindications, salvarsan may be used in practically all 
 stages of the disease. In the later stages of the infection, in which the 
 central nervous tissue is involved, the physician must bear in mind the 
 possible danger of a neurorelapse or a Herxheihaer reaction occurring. 
 While salvarsan may do no good in late cases, since it cannot restore lost 
 nerve tissue, it may alleviate symptoms and prevent progress of the in- 
 
810 CHEMOTHERAPY 
 
 fection. In the treatment of any case of syphilis the best results are not 
 secured from following any course of treatment according to a rule of 
 thumb; they are obtained only by a suitable combination of expert 
 clinical knowledge and training in serologic technic. All possible aid 
 should be enlisted in the treatment of the disease. 
 
 It is beyond the scope of this book to present either case reports or a 
 lengthy discussion of salvarsan in the treatment of the various stages of 
 syphilis; suffice it to say that, in the absence of contraindications, the 
 drug may be administered whenever living spirochetes are present in a 
 patient's tissues, as evidenced either by clinical symptoms or a positive 
 Wassermann reaction. The method of administration selected should 
 be that which will best bring the drug into contact with the diseased 
 tissues. 
 
 SALVARSAN IN THE TREATMENT OF NON-SYPHILITIC DISEASES 
 
 While salvarsan therapy has achieved its greatest success in the 
 treatment of syphilis, its influence on certain non-syphilitic diseases has 
 been so pronounced that it is being used in an ever-increasing number of 
 diseases, the aim being to thus enlarge its spjhere of usefulness. A re- 
 cent systematic study of the subject has been made by Best.^ Good re- 
 sults have been reported by various observers in the following diseases: 
 
 Frambesia (Yaws). — The results achieved in this disease from the 
 use of salvarsan have equaled those obtained ill syphilis. Two thousand 
 four hundred and thirty cases have been reported, a cure having been 
 effected in all but a few instances. Since the use of salvarsan has come 
 into favor hospitals for the treatment of frambesia have been closed. 
 
 Relapsing Fever. — In the treatment of this disease the drug has 
 likewise had a remarkable effect. Of 195 cases reported, in most of them 
 the microorganisms could not be found in the blood after an injection, 
 and in none of the cases were relapses observed to occur. 
 
 Filariasis. — Salvarsan has been found effective in killing the Filaria 
 sanguinis hominis and in ridding the blood of these parasites. Of 
 course, in elephantiasis it is impossible to restore the affected parts to 
 their normal appearance. 
 
 Vincent's Angina. — An immediate improvement and rapid healing 
 have been reported as following either an intravenous injection or the 
 local application of the drug in the form of a dusting-powder or in sus- 
 pension in glycerin. 
 
 ' Jour. Amcr. Med. Assoc, 1914, Ixiii, 375. 
 
CHEMOTHERAPY IN BACTERIAL DISEASES 811 
 
 Duhring's Disease (Dermatitis Herpetiformis), Scurvy, Chorea, Ma- 
 laria, Acanthosis Nigricans, Ulcus Tropicum, Variola, and Verruca Plana. 
 
 — In all these affections salvarsan has been found to exert a beneficial 
 and curative effect, either by direct action upon the microorganisms 
 present or as the result of the alterative and stimulating effect of the 
 arsenic. 
 
 In Aleppo boil, leprosy, lupus vulgaris, tuberculosis, anemia, kera- 
 tosis follicularis, hchen planus, mycosis fungoides, pellagra, and pity- 
 riasis rubra, good or indifferent results have been reported. In 
 many conditions it would appear that salvarsan exerts a direct germi- 
 cidal effect, and in others the beneficial results appear to be dependent 
 upon a certain tonic, stimulating, and alterative effect of the arsenic. 
 In chancroid, scarlet fever, Hodgkin's disease, psoriasis, trichinosis, and 
 trypanosomiasis the drug does not appear to exercise any influence, 
 which is due probably, in the last-mentioned disease, to the fact that 
 trjrpanosomes are much more likely to become arsenic-fast than are the 
 spirochetes. 
 
 CHEMOTHERAPY IN BACTERIAL DISEASES 
 While studies in chemotherapy have been largely confined to the 
 protozoan infections, similar investigations arc being made in bacterial 
 infections, especially in tuberculosis and the pyogenic disorders. 
 
 As has previously been stated in Chapter XXIX, progress in this 
 direction has been made by Lamar in the treatment of pneumococcus 
 infections with mixtures of pneumococcus immune scrum, sodium oleate, 
 and boric acid. Morgenroth has conducted similar researches in animal 
 infections with the pneumococcus, finding that, whereas quinin, hydro- 
 quinin, and hydrochlorisoquinin have no effect on the pneumococcus, 
 ethyl hydrocuprein was capable of arresting the infection in 50 per cent, 
 of the animals when given six hours after inoculation, that is, at a time 
 when the animals were severely infected. 
 
 It is to be hoped, therefore, that further researches will result in the 
 discovery of substances that have a marked bactericidal action and 
 that are yet but slightly, if at all, toxic for the body-cells, that is, sub- 
 stances in which bacteriotropism greatly exceeds organotropism. It 
 would appear that this discovery is possible and probable, but it can 
 be accomplished only as the result of persistent and prolonged research. 
 Probably the most wonderful discovery possible in medicine would be a 
 specific remedy for tuberculosis, and this may be within the realms of 
 chemotherapy. 
 
812 CHEMOTHERAPY 
 
 CHEMOTHERAPY IN MALIGNANT DISEASE 
 Brief mention may be made of certain recent advances that have 
 been made in the experimental chemotherapy! of cancer in the rat and 
 mouse. While the pathogenesis of malignant disease is still unknown, 
 specific therapeutic measures of this kind have been undertaken on the 
 assumption that the cancer-cell is different from the normal cell from 
 which it originated, and that certain substances will show a selective 
 affinity for them; in other words, that specificity may be present among 
 organotropic substances. Here, of course, the difficulties are great, 
 because the structural, biologic, and functional differences between the 
 normal cell and the cancer cell may be slight, and substances must be 
 found that possess a high affinity for the cancer-cell only. 
 
 That this condition may indeed exist has been indicated by the experi- 
 ments of Wassermann and his collaborators. These investigations 
 were based upon the discovery, by Gosio, that sodium selenate and so- 
 dium tellurate are more rapidly reduced by cancer-cells than by normal 
 cells, and that this reduction takes place within the bodies of the cells. 
 Experiments on mouse tumors showed that the injection of these sub- 
 stances into the growths may actually lead to their destruction, the 
 explanation, according to Neuberg and Caspani, being that certain 
 compounds of the heavy metals in colloidal form favor self-digestion 
 (autolysis) of the tumor cells. 
 
 The problem now resolved itself into finding some substance that 
 would carry the metals to the tumors, or, aS Wassermann said, "the 
 building of rails which would reach the tumor and by which the selenium 
 could travel," as local injection was obviously out of the question from 
 the practical standpoint. Eosin, being a substance endowed with great 
 powers of diffusion, was selected for the purpose, and a number of eosin- 
 selenium compounds were tried out. The results were encouraging, as 
 in a number of instances the tumors became soft and sloughed away or 
 their further growth was checked. "If three consecutive daily intrave- 
 nous injections of the eosin-selenium compound are given in 2.5 gm. doses 
 for 15-gram mice, a distinct softening and elasticity of the tumor are 
 noticed on the fourth day; on the fifth day a fourth injection of the 
 same dose is given, after which there is no longer the feeling of a solid 
 tumor, but rather that of a fluctuating cyst in which small, movable 
 tumor particles can be discovered. After the fifth injection on the 
 seventh day this soft mass becomes smaller, the capsule becomes lax, 
 and the configuration of a circumscribed tumor can no longer be dis- 
 
CHEMOTHERAPY IN MALIGNANT DISEASES ' 813 
 
 tinguished, but only a long edematous cord can be felt. Usually, as a 
 result of the sixth injection, in favorable cases, the absorption and 
 diminution proceed so that one gets the feeling of an empty sac. In 
 case no intercurrent disease occurs, the animal is cured in about ten 
 days, with a disappearance of all remnants of the tumor." 
 
 As these results were observed after intravenous injection of the 
 mixtures, and as no injury to the body-cells was apparent, it seems that a 
 step forward has really been taken; at least, these investigators have 
 shown that it is possible for chemical substances to pass from the blood 
 and attack tumor cells. Copper and tin have= been found to possess a 
 more marked affinity for tumor cells, and the whole subject is probably 
 just at the threshold of further discoveries that may be applied with 
 great benefit to the treatment of human malignant disease. 
 
Part V 
 
 EXPERIMENTAL INFECTION AND ZMMUNITY 
 
 INTRODUCTORY 
 
 Methods. — The exercises and experiments herein outlined are for 
 the purpose of teaching the principles of infection and immunity by 
 actual laboratory work, whereby the student performs the experiments 
 and is taught to observe the results. At the' same time a knowledge 
 of the technic of immunologic methods is obtained. The instructor in 
 charge of such an experimental course may choose certaia experiments 
 in outlining a course according to the allotme'at of time and purpose of 
 the instruction. 
 
 In all instances I have outlined the experiments according to average 
 conditions. It is readily understood that differences in the virulence of 
 a certain culture or the weight and physical condition of an experimental 
 animal will require that the doses advised be qhanged to meet the condi- 
 tions. 
 
 As a general rule, a course should be concentrated, with exercises at 
 least three times a week in order that the student may follow his work 
 closely and have an opportunity for maldng adequate and accurate ob- 
 servations. 
 
 An attempt is made to bring out the important points of an experi- 
 ment by a few pertinent questions. It should be impressed upon the 
 student that the mind should be held open for observation and that un- 
 expected and untoward results may be obtained which, however, are 
 always of interest and always instructive when the experiment is con- 
 ducted in a careful, methodical, and conscientious manner. Frequent 
 general discussions should be held for a general review of the subject and 
 correlation of facts and observations. In my experience students are 
 eager for the work and seldom fail to suggest additional work in the 
 nature of original research. 
 
 The Student. — 1. The student should work protected by an apron 
 oj gown with short sleeves; he should be careful of the hands and avoid 
 abrasions and cuts and carefully wash and disinfect the hands after the 
 
 work of each day has been completed. 
 
 814 
 
ACTIVE IMMUNIZATION OP ANIMALS 815, 
 
 2. The working table should be set in order after each day's work; 
 pipets and soiled glassware should be properly disposed of, instruments 
 thoroughly cleansed, and the table wiped off with 1 per cent, formalin 
 solution. It should be impressed upon the student that good and ac- 
 curate work is seldom done amid disorderly and dirty surroundings. 
 
 3. The student must never sacrifice accuracy for speed; painstaking 
 and accurate work is always to be desired; speed is gained only with 
 practice. 
 
 Records. — Each worker should record in writing in a suitable note- 
 book his observations of the various tests and experiments. Not infre- 
 quently unexpected results are obtained, and it is important to under- 
 stand and explain these as correctly as possible. Note-books should be 
 subject to frequent inspection; it is not necessary to write out a descrip- 
 tion of the technic, but the results of the experiment and answers to the 
 questions should be set forth clearly and with sufficient detail. 
 
 Animal Experiments and Autopsies. — In all experiments calling for 
 operative procedure an anesthetic is to be used in order that unnecessary 
 pain be avoided. Ordinarily, ether is to be employed, or in rabbits the 
 rectal injection of 0.5 to 1 gram of chloral hydrate dissolved in 5 to 10 c.c. 
 of water. Autopsies are to be carefully conducted, and the lesions 
 described in writing. After autopsy the table is to be scrubbed and 
 cleansed with a solution of formalin, and the carcass disposed of by in- 
 cineration or placing in a solution of formalin until removed and other- 
 wise disposed of. 
 
 EXERCISE I.— ACTIVE IMMUNIZATION OF ANIMALS 
 
 Experiment 1. — Peoduction of Agglutinins, Bacteriolysins, and 
 Opsonins 
 
 1. Secure a culture of Bacillus typhosus, an old laboratory culture of cholera 
 bacilli, and a culture of Staphylococcus aureus. 
 
 2. Proceed to immunize two rabbits with each culture by intravenous injections 
 according to the technic described on page 16S. 
 
 3. One week after the last injection the animals are=to be bled and the scrums 
 secured after the first method described on page 42. 
 
 ,(a) Define the meaning of antigen. 
 
 (b) What are antibodies? 
 
 (c) What is meant by active immunization? 
 
 (d) What precautions should be observed in handling these antigens? 
 
 (e) What precautions are to be observed in giving intravenous injec- 
 tions? 
 
816 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 (f) What antibodies do you suspect are present in these immune 
 serums? 
 
 Experiment 2. — Production of Precipitins 
 
 1. Immunize a rabbit with sterile horse serum by intravenous injections after the 
 first method as described on page 70; immunize a second rabbit after the second 
 method. 
 
 2. Immunize two rabbits with sterile human serum after the first method. 
 
 3. Immunize two rabbits with cow milk by intravenous injections after the third 
 method. 
 
 4. Bleed the animals from the carotid artery one week after the last injection and 
 preserve the serums in a sterile condition. 
 
 Experiment 3. — Production of Hemolysins 
 
 1. Immunize a rabbit with washed sheep corpuscles by intravenous injections 
 after the first method as described on page 72. 
 
 2. Immunize a second rabbit with sheep cells after the second method. 
 
 3. Immunize a third rabbit with washed human cells after the third method. 
 
 4. Immimize a fourth rabbit with washed human cells by intraperitoneal in- 
 jections as described on page 73. 
 
 5. Bleed the animals in four days to a week after the last injection and separate 
 the serums.' 
 
 6. Prepare a portion of human amboceptor dried on paper as described on page 
 70. 
 
 7. Preserve the balance of the serum and the other serums with equal parts of 
 neutral glycerin. 
 
 Experiment 4. — Production op Cytotoxin 
 
 1. Immunize two rabbits with dog kidney after the method described on page 73. 
 
 2. After the immunization has been completed, preserve the serum with 0.2 per 
 cent, phenol in sterOe ampules. 
 
 While these various immune serums are being -prepared the student is 
 engaged with the work on infection, or if the subject of imrnunity is taken 
 up at once, immune serums should be furnished, by the instructor. 
 
 EXERaSE 2.— INFECTION 
 
 Experiment 5. — Experimental Pneumonia 
 
 1. Grow a virulent culture of pneumococcus in glucose serum broth for forty- 
 eight to seventy-two hours at 37° C. imtil a good riob growth is secured. Prepare 
 and stain smears by Gram's method. 
 
 2. Pass a large catheter which has its tip cut off into the trachea of a dog until it 
 has passed into one of the primary bronchi. By means of a syringe inject quickly 
 15 c.c. of culture; remove the catheter and mouth-gag" Take the rectal temperature 
 and leukocyte count previous to inoculating. 
 
 3. Inject a second dog in the same manner with 2 c.c. of culture. 
 
 4. Inject a third dog with 5 c.c. of culture intravenously. 
 
TOXINS 817 
 
 5. Observe all animals for forty-eight hours, taking the rectal temperature night 
 and morning. Make leukocyte counts every four hours during the day. Make 
 physical examination of the chest. 
 
 6. Autopsy the animals under aseptic precautions and with complete anesthesia. 
 
 7. Culture the heart's blood of each in serum bouillon. 
 
 8. Culture the pulmonary lesions in serum bouilten. 
 
 9. Prepare smears of the heart's blood and lesions and stain with methylene-blue 
 and Gram's stain. 
 
 10. Remove consolidated portions of lung and place in 5 per cent, formalin. 
 After twenty-four hours, cut sections which are passed through by the paraffin 
 method and stained with hematoxylin and eosiu, methylene-blue, and Gram's stain. 
 
 (a) Did any of the animals show evidences of infection? 
 
 (b) Are there evidences of pneumonia? How do these lesions com- 
 pare with those of human pneumonia? 
 
 (c) How do you explain their production? 
 
 (d) Does the animal receiving the smaller dose of pneumococci intra- 
 bronchially show evidences of pneumonia? If not, why not? Does 
 this show a numerical relationship of bacteria to infection? 
 
 (e) Were the temperature changes similar to those observed in 
 human lobar pneumonia? 
 
 (f) Did leukocytosis occur and if so, why? 
 
 (g) Are there any evidences of pleuritis and if so, how do you explain 
 its production? 
 
 (h) Did the dog receiving the pneumococci intravenously show evi- 
 dences of pneumonia? Does this bear any relation to the question of 
 the route of introduction of bacteria to infection? 
 
 (i) Are the pneumococci seen in the smears of the lesions and blood 
 encapsulated? If so, what is the significance of these capsules? Com- 
 pare these cocci with those shown in the smear of the culture before in- 
 jection? Are the capsules lost in the artificial culture-media? 
 
 (j) Discuss the question of the possible modes of infection in human 
 lobar pneumonia, especially inspiratory pneumonia. 
 
 EXERCISE 3.— TOXINS 
 
 Experiment 6. — Diphtheria Toxin 
 
 1. Inoculate tubes of neutral or slightly alkaline bouillon with a virulent culture 
 of diphtheria bacilli (Park- Williams Bacillus No. 8 being especially desirable). 
 
 2. Grow at 35° C. for five days and filter the culture through a Berkefeld filter. 
 
 3. Inject a 250- to 300-gram guinea-pig in the median abdominal line with 0.5 
 CO. of the filtrate (toxin). 
 
 4. Heat 1 c.c. of toxin at 60° C. for an hour and inject 0.5 c.c. into a second guinea- 
 pig subcutaneously. 
 
 52 
 
818 EXPERIMENTAL INFECTION AN© IMMUNITY 
 
 5. Observe the animals for at least four days, especially for the development of a 
 characteristic edema about the site of injection. 
 
 6. After death perform a careful autopsy, paying particular attention to the 
 bloody edema at the site of injection and marked hyperemia of the suprarenal glands. 
 Make cultures on LoefHer's blood-serum medium of ed'einatous area, peritoneum, and 
 heart blood. 
 
 (a) To what has death been due? 
 
 (b) Has diphtheria toxin a selective affinity for any particular tissue? 
 
 (c) Has heat any effect upon diphtheria toxin? 
 
 (d) Is there a period of incubation before syinptoms develop and why? 
 
 Experiment 7. — Method of Testing the Yirulence and Toxicity 
 OF Diphtheria Bacilli 
 
 1. Make a culture of a patient harboring the bacilli on a tube of LoefHer's serum 
 medium. Inoculate at 35° C. for from eighteen to twenty-four hours; prepare a 
 smear and stain with LoefHer's methylene-blue. If diphtheria bacilli are present, 
 they must be isolated in pure culture. Never attempt a guinea-pig test with an impure 
 culture. 
 
 2. Isolate by the "streak" method, on plates of blood-serum. 
 
 3. Inoculate a tube of I per cent, glucose bouillon, which is neutral or slightly 
 alkaline, with several different colonies. 
 
 4. Incubate at 35° C. for three days, keeping the tube in a slanted position in 
 order to give the culture as much oxygen as possible. If a good growth does not 
 appear in twenty-four hours, transplant to another tube of bouillon until the bacilli 
 have been "educated" to grow on the medium. 
 
 5. Examine for purity. Select a 250- to 300-gram guinea-pig and inject 2 c.c. of 
 the unfiltered culture in the median abdominal line. Animals over the weight speci- 
 fied are more resistant and less reliable for test. The unfiltered culture is used, since 
 toxin is but one element of the disease-producing power of diphtheria bacilli, and 
 toxin production in bouillon may not be a true index of the toxin production in mucous 
 membranes. 
 
 6. Carefully observe the animal for at least four: days. Even slight toxemia, 
 especially if accompanied by edema at the site of injection, should be regarded as a 
 positive result. 
 
 7. After death perform a careful autopsy. Make cultures of the edematous area, 
 peritoneum, and heart blood. Observe whether acute hyperemia of the suprarenal 
 glands is present. 
 
 8. Not infrequently animals showing mild or even an absence of the symptoms 
 of toxemia developparalysis of the hindquarters two or three weeks later. According 
 to Ehrlich, this paralysis is due to the action of "toxdn," a toxic substance secreted 
 by the bacillus or, as believed by others, a modified form of toxin. 
 
 9. To prove that diphtheria was the cause of the toxemia or death, mix 2 c.c. of 
 the culture in a test-tube with 1 c.c. of diphtheria antitoxin (500 units). After 
 standing aside for an hour at room temperature, inject the mixture subcutaneously 
 in the median abdominal line of a 250- to 300-gram guinea-pig. 
 
 (a) Is the diphtheria bacillus aggressive? 
 
 (b) What evidence have you that the lesiofis and death are due to a 
 toxin? 
 
TOXINS 819 
 
 (c) Why does the use of diphtheria antitoxin make the test con- 
 clusive? Would tetanus antitoxin be capable of neutralizing diphtheria 
 toxin? 
 
 Experiment 8. — Tetanus Toxin 
 
 Tetanus toxin is composed of two distinct poisons of different prop- 
 erties. One, tetanospasmin, has a great affinity for the central nervous 
 system, and is largely responsible for the symptoms of tetanus infec- 
 tion (neurotoxic) ; the second, tetanolysin, is thermolabile and is hema- 
 toxic. Tetanus toxin is very labile, and when in solution soon becomes 
 attenuated. For these experiments it is necessary to use either fresh 
 toxin or that which has been recently precipitated and dried. 
 
 1. Secure some dried tetanus toxin and dissolve in sterile salt solution. The 
 toxin may be secured from an antitoxin laboratory and preserved indefinitely in the 
 refrigerator. Since the strength varies with different products, the lethal dose for a 
 300-gram guinea-pig should be ascertained from the laboratory furnishing the toxin. 
 
 2. Secure 2 grams of fresh normal guinea-pig brain and liver. Crush in separate 
 sterile mortars. 
 
 3. Add a lethal dose of fresh tetanus toxin and 5 c.c. sterile salt solution to each. 
 Mix thoroughly and place in the incubator for an hour. Remove and carefully trans- 
 fer to sterile centrifuge tubes. Centrifuge thoroughly. 
 
 4. Inject two 300-gram guinea-pigs subcutaneously in the median abdominal 
 line with the supernatant fluids. 
 
 5. Inject a third guinea-pig with a similar dose of toxin and 5 c.c. .salt solution 
 (control) . 
 
 6. Mark pigs carefully and observe for several days. 
 
 (a) What are the symptoms of tetanus? 
 
 (b) To what constituents of tetanus toxin are the chief symptoms due? 
 
 (c) Does the pig which received the toxin-brain mixture show symp- 
 toms? How do you explain the result? 
 
 (d) Does the pig which received the toxin-liver mixture show symp- 
 toms? How do you account for the result? 
 
 (e) Is the tetanus bacillus characterized by its toxicity or aggressive- 
 ness? 
 
 (f) Is there a period of incubation before symptoms develop and 
 why? 
 
 (g) Autopsy the animal. Are there any definite lesions of the tissues 
 and internal organs? 
 
 Experiment 9. — Tetanus Toxin 
 
 1, Secure some fresh tetanus toxin. 
 
 2. Prepare a 1 per cent, emulsion of washed sheep corpuscles (washed three times 
 in an excess of normal salt solution). 
 
820 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 3. Into a series of sLx test-tubes place 1 c.c. of the corpuscle emulsion and in- 
 creasing doses of toxin: 0.1 c.c, 0.2 c.c, 0.4 c.c, O.S c.c, 1 c.c, 2 c.c Add 
 sufficient normal salt solution to bring the total volume to 3 c.c. 
 
 4. Place 1 c.c of the corpuscle suspension in 2 do. salt solution as a control to 
 make sure that the salt solution is isotonic. 
 
 5. Heat a portion of toxin at 60° C. for an hour in a water-bath and set up a 
 similar set of tubes. 
 
 6. Incubate all tubes for two hours at 37° C. 
 
 7. Read results at the end of this time and again after the tubes have settled in 
 the refrigerator overnight. 
 
 (a) Has hemolysis occurred in any of the tubes? 
 
 (b) What is meant by hemolysis? 
 
 (c) What constituent of tetanus toxin is responsible for hemolyzing 
 these cells? 
 
 (d) Docs this experiment show the selective af&nity of a toxin for 
 certain cells? 
 
 (e) How is tetanus toxin produced? 
 
 (f) Does heat destroy the hemotoxic ageifl?? 
 
 EXERCISE 4.— TOXINS (Continued) 
 Experiment 10. — Botulism Toxin 
 
 1. Prepare toxin by cultivating the Bacillus botulinus in an alkaline bouillon 
 made in the form of an infusion from ham with the addition of 1 per cent, of glucose, 
 1 per cent, of peptone, and 1 per cent, of sodium chlorid. Strict anaerobic cultures 
 should be grown for four weeks and filtered through a Bcrkofeld filter. The toxin 
 may be preserved in brown, sealed vials, and kept on ice, or in a dried form in vacuum. 
 
 2. Dilute 0.2 c.c. of the toxin with 3 or 4 c.c. salt solution and administer, per os, 
 to a cat by means of a small catheter passed into the stomach. 
 
 3. Inject 0.1 c.c. of the toxin subcutaneously in Uie abdominal wall of a rabbit. 
 
 4. Observe animals closely for several hours following administration of the toxin, 
 for some products are so highly toxic that symptoms may appear within a few hours, 
 
 (a) What are the symptoms of botulism infection? 
 
 (b) Do these symptoms show any selective action of botulism toxin 
 for certain tissues? 
 
 (c) Autopsy the animals. Are there any gross tissue changes? 
 
 Experiment 11. — Dysentery Toxin 
 
 1. Inoculate a flask of moderately alkaline bouillon with a culture of dysentery 
 baciUi, preferably of the Shiga-Kruse type. Inoculate at 37° C. for two weeks and 
 pass it through a bacterial filter. 
 
 2. Inoculate three rabbits intravenously with 1, 2, Jmd 5 c.c. of the filtered toxin. 
 
 3. Give a fourth rabbit 2 c.c of the toxin by mouth through a small catheter 
 passed into the stomach. 
 
 4. Observe the animals very closely. 
 
TOXINS 821 
 
 (a) What are the symptoms of dysentery intoxication? 
 
 (b) Observe the temperature at frequent intervals. Are there any 
 changes? 
 
 (c) Does the animal which received the toxin by mouth show symp- 
 toms? How do you explain the result? 
 
 (d) Autopsy the animals with particular attention to the gastro- 
 intestinal tract. Are there any evidences of a selective action of the 
 toxin? 
 
 Experiment 12. — Staphylotoxin 
 
 Inject 2 c.c. of a twenty-four-hour bouillon culture of Staphylococcus aureus in 
 the ear vein of a rabbit. Take the temperature before and every twelve hours 
 after injection. Autopsy the animal seventy-two hours later under aseptic precau- 
 tions. 
 
 (a) What lesions are found? 
 
 (b) Where are these chiefly situated and why? 
 
 (c) Culture the heart's blood on tubes of agar, also several of the 
 lesions. 
 
 (d) After twenty-four hours' incubation examine your cultures. 
 
 (e) What was death due to? 
 
 (f) Define bacteremia. 
 
 (g) Prepare and stain sections of the kidney, including a lesion. 
 What are the histologic changes? How do you explain the production 
 of these tissue changes? 
 
 Experiment 13. — Staphylotoxin 
 
 1. Inoculate a flask of neutral bouillon with a virulent culture of Staphylococcus 
 aureus and grow for two weeks at 37° C. Filter through a Berkefeld filter. 
 
 2. Prepare a 1 per cent, suspension of washed rabbit erythrocytes in normal salt 
 solution. 
 
 3. Into a series of six test-tubes place 1 c.c. of the, corpuscle suspension and in- 
 creasing amounts of staphylococcus filtrate: 0.1 c.c, 0.2 c.c, 0.4 c.c. 0.8 c.c, 1 c.c, 
 and 2 c.c. Add normal salt solution to bring the total volume to 3 c.c 
 
 4. Prepare a control by placing 1 c.c. of corpuscle suspension in 2 c.c salt solution. 
 
 5. Incubate tubes for two hours at 37° C. and read results. 
 
 6. This test will be referred to again in the technic for determining the antilysin 
 in blood-serum. 
 
 (a) Has hemolysis occurred in any of the tubes? 
 
 (b) To what constituent of the filtrate are these results due? 
 
 (c) Does this experiment show the selective action of a toxin? 
 
 (d) Do the results have any bearing upon" the anemia and jaundice 
 of severe staphylococcus infections? 
 
822 EXPERIMENTAL INFECTION AN6 IMMUNITY 
 
 EXERCISE 5.— TOXINS (Continued)— PLANT AND ANIMAL TOXINS 
 Experiment 14. — Streptotoxin 
 
 1. Cultivate a strain of virulent streptococci in tubes of slightly alkaline serum 
 or ascites bouillon for forty-eight hours at 37° C. 
 
 2. Inoculate a guinea-pig intraperitoneally with 1 or 2 c.o. of the unfiltered cul- 
 ture. 
 
 3. Autopsy aseptioally eighteen or twenty-four hours later. 
 
 (a) What are the gross features of the exudate? 
 
 (b) Prepare smears of the exudate and stain with methylene-blue. 
 Are there many cells present? How do you explain the cellular con- 
 tent? 
 
 (c) Are streptococci present? Are these inclosed by any of the 
 cells? How do you explain the condition? 
 
 (d) Is the exudate bloody? If so, why? 
 
 (e) Examine your blood-agar plates. Are there any peculiar 
 changes around the colonies? To what are these changes due? Does 
 this have any connection with the bloody character of the exudate? 
 
 (f) Why are streptococcus infections so virulent and spreading in 
 character? 
 
 (g) Is the streptococcus an aggressive miclfoorganism? 
 (h) Do streptococci contain an endotoxin? 
 
 Experiment 15. — Phytotoxins 
 
 1. Secure 0.01 gm. ricin or abrin and dissolve in 10 c.c. normal salt solution or 
 distilled water. 
 
 2. Inject 1 c.c. intravenously into a rabbit. Place the animal in a metabolic 
 cage and collect urine, or catheterize every twelve hours or at death. 
 
 3. Examine the urine for hemoglobin and erythrocytes. 
 
 4. Prepare a 1 per cent, suspension of washed rabbit and guinea-pig corpuscles. 
 
 5. Into a series of six small test-tubes place increasing doses of the ricin or abrin 
 solution as follows: 0.1, 0.2, 0.3, 0.4, 0..5, and 0.8 c.c. Add 1 c.c. of rabbit-cell emul- 
 sion to each and sufficient normal salt solution to make the total volume in each tube 
 equal to 2 c.c. A seventh tube is the corpuscle control and contains 1 c.c. of the ery- 
 throcyte suspension and 1 c.c. of salt solution. 
 
 6. Prepare a similar series of tubes with the guiiiea-pig erythrocyte suspension. 
 
 7. Shake the tubes gently and incubate for two hours. 
 
 (a) Do any of the tubes show hemolysis or hemagglutination? 
 
 (b) Is the action the same with both bloods? 
 
 (c) Does the plant toxin show a selective affinity? 
 
 (d) Does the rabbit show any evidenc^ of hemolytic jaundice? 
 Are there blood elements in the urine? 
 
ENDOTOXINS AND AGGRESSINS 823 
 
 (e) Autopsy the animal, paying particular attention to the kidneys 
 and gastro-intestinal tract? What changes have occurred? 
 
 Experiment 16. — Zootoxin (Cobra Venom) 
 
 1. Collect about 2 c.c. of normal human blood and place in 5 c.c. of 2 per cent, 
 sodium citrate in 0.85 per cent, sodium chlorid solution. This mixture must not be 
 shaken and the cells should be washed at least four times with normal salt solution, 
 after which they are made up to a 4 per cent, suspension. 
 
 2. Prepare a 4 per cent, suspension of sheep corpuscles in the same manner. 
 
 3. Prepare a stock dilution of cobra venom by dissolving 0.005 gm. dried venom 
 in 10 c.c. of normal salt solution. Tliis solution will keep about one week in the re- 
 frigerator. 
 
 4. Prepare a solution of venom 1 ; 10,000 by placing 2 c.c. of the stock solution in 
 8 c.c. salt solution. Prepare a 1 : 20,000 dilution by placing 2 c.c. of dilution 1 : 10,000 
 in 2 c.c. salt solution. 
 
 5. Place 1 c.c. of each corpuscle suspension into" two small test-tubes and add 1 
 c.c. and 0..5 c.c. each venom dilution. Shake the tubes gently and place in the incu- 
 bator for an hour and then in the refrigerator overnight. 
 
 6. Inject 2 c.c. of the 1: 10,000 dilution intravenously into a rabbit and 1 c.c. 
 intravenously into a guinea-pig. 
 
 7. Observe the animals closely, particularly the' urine. 
 
 (a) Does the venom show a hemotoxic action? Does it act the 
 same on rabbit and guinea-pig corpuscles? If not, why not? 
 
 (b) How do you explain venom hemolysis? 
 
 (c) What are the evidences of venom hemolysis in vivof 
 
 (d) Is the hemotoxic poison the most dangerous constituent of venom? 
 
 EXERCISE 6.— ENDOTOXINS AN1> AGGRESSINS 
 
 Experiment 17. — Endotoxins 
 
 1. Incubate eight agar slants with Bacillus typhosus and cultivate at 37° C. for 
 seventy-two hours. 
 
 2. Wash off the growths with 5 c.c. distilled water for each tube. 
 
 .3. Place the emulsion in a sterile bottle with glass beads and shake for twenty- 
 four hours at room temperature. At the same time inoculate si-x more slants of agar 
 and wash off the growths at the end of twenty-four hours with 3 c.c. of normal salt 
 solution for each tube. 
 
 4. Filter the emulsion, which has been shaken, through a Berkefeld filter. 
 
 .5. Inject two rabbits intravenously with 2 c.c. each of the filtrate (endotoxins) 
 and unfiltered culture. 
 
 6. Observe influence on body-weight and temperature and for symptoms of 
 toxemia. 
 
 7. Inject three pigs intraperitoneally as follows: 
 
 1st pig: 2 c.c. of emulsion of typhoid bacilli. 
 2d pig: 2 c.o. of filtrate. 
 ■ 3d pig: 1 c.o. each of emulsion and filtrate. 
 
824 EXPERIMENTAL INEECTION AND IMMUNITY 
 
 (a) Are there symptoms of toxemia in the rabbits? Are these symp- 
 toms alike in both animals? Which is more toxic, the filtrate or the 
 whole culture? 
 
 (b) Does the filtrate enhance the effect of the whole culture? 
 
 (c) If any of the animals succumb, autopsy under aseptic precau- 
 tions. Prepare cultures of the peritoneum and heart's blood on agar 
 slants or in neutral bouillon. Is the typhoid bacillus markedly ag- 
 gressive? 
 
 (d) If there is a peritoneal exudate, prepare smears and stain with 
 carbolfuchsin (1 :10). What type of cell predominates? Are any of 
 the bacilli engulfed in the cells? 
 
 (e) Do any of the bacilli show signs of disintegration? If so, do you 
 know what these changes are due to? 
 
 ExPEKiMENT 18. — Natural AoGREgfeiNS 
 
 1. Inject a guinea-pig intraperitoneally with 3 c.c. of twenty-four-hour bouillon 
 culture of Bacillus typhosus. 
 
 2. After twenty-four hours, anesthetize the anircial and remove the exudate 
 aseptically into 5 c.c. of a 2 per cent, solution of sodium citrate to prevent coagula^ 
 tion. 
 
 3. Filter. 
 
 4. Inject three guinea-pigs intraperitoneally as follows: 
 
 1st pig: 3 c.c. of filtrate. 
 
 2d pig: 2 c.c. of twenty-four-hour bouillon culture of 
 
 typhoid bacilh. 
 3d pig: 1 c.c. each of culture and filtrate. 
 
 5. Observe animals for several days, particularly the temperature reaction and 
 weights. 
 
 (a) Is the filtrate alone toxic? 
 
 (b) Does the filtrate appear to enhance the effects of the unfiltered 
 culture? 
 
 (c) If the animals succumb, autopsy aseptically. Make cultures 
 of the peritoneum and heart's blood on agar slants or in bouillon. Pre- 
 pare smears of the exudate and stain with 1 : 10 carbolfuchsin. Do any 
 of the animals show a typhoid bacteremia? Does the filtrate appear to 
 prevent phagocytosis? 
 
 EXERCISE 7.— BACTERIAL PROTEIN; PTOMAMS; MECHANICAL ACnON 
 
 OF BACTERIA 
 
 Experiment 19. — Bacterial Protein (Vaughan) 
 
 1. Inoculate 10 slants of 2 per cent, neutral agar in large bottles with a culture 
 of Bacillus coli and grow at 37° C. for four days. 
 
 2. Add about 10 c.c. of distilled water to each Bottle and gently remove the 
 
BACTERIAL PROTEIN; PTOMAINS 825 
 
 growth with a sterilized platinum wire, being particularly careful not to remove frag- 
 ments of agar. 
 
 3. Pipet the heavy emulsion to a large centrifuge tube. Another cubic centi- 
 meter or two of water is added to each bottle to remove the balance of the growth. 
 
 4. Centrifuge at high speed until the baciUi are thoroughly settled. Decant off 
 the supernatant fluid and add 50 per cent, alcohol; mix and centrifuge. Decant and 
 add 95 per cent, alcohol; mix and centrifuge. 
 
 5. Remove the sediment of bacteria to a small flask with 50 c.c. of absolute 
 alcohol and set aside at room temperature for a day. Decant off the alcohol and add 
 50 c.c. of ether. Mix and set aside for another twenty-four hours. Decant off the 
 ether that remains and remove sediment to a porcela,in or agate mortar. Place in 
 the incubator for a few hours until thoroughly dry. 
 
 6. Grind the dry mass very thoroughly, the operator wearing a mask, until a 
 fine powder is produced. Place the powder of bacteria;l substance in a wide-mouthed 
 dark-glass bottle and preserve in a dark closet. A portion will be needed later for 
 experiments in anaphylaxis. 
 
 7. Mix 0.02 gm. of the dry powder with 10 c.c. salt solution. 
 
 8. Inject 2 c.c. of this emulsion intraperitoneally in a 300-gram guinea-pig. 
 
 9. Heat 2 c.c. of the emulsion at 60° C. for two hours and inject intraperitoneally 
 in a guinea-pig. 
 
 10. Inject a third pig intraperitoneally with 2 c.c. of a four-day bouillon culture 
 of Bacillus coli. 
 
 (a) Could endotoxins withstand these various manipulations? 
 
 (b) Does the heated extract kill the animal more quickly than the 
 unheated, and if so, why? 
 
 (c) What are the symptoms produced? 
 
 (d) Autopsy the animals. Are there evidences of peritonitis? Are 
 there differences in the lesions of the three animals? If so, how do you 
 explain them? 
 
 (e) The bacterial split portion will be studied later under Anaphylaxis. 
 
 Experiment 20. — Ptomains 
 
 1. Procure 4 ounces of beef and mince. 
 
 2. Place in a flask with 250 c.c. tap water and inoculate with a culture of Bacillus 
 coU. Fit the flask with a rubber stopper with a glass tube to carry off gases. 
 
 3. Inoculate a flask of neutral bouillon at the same time with the same culture. 
 
 4. Cultivate both at 37° C. for two weeks. 
 
 5. Filter both through a coarse Berkefeld filter. 
 
 6. Concentrate both filtrates to one-half their volume at a low temperature on a 
 water-bath. 
 
 7. Inject two guinea-pigs with each preparation, giving 1 c.c. subcutaneously 
 and 0.5 c.c. intraperitoneally. 
 
 8. Observe the four animals closely. 
 
 (a) What symptoms develop in both sets of animals? 
 
 (b) What are ptomains? "What role do they play in the production 
 of disease? 
 
 (c) Can antibodies be produced for true ptomains? 
 
826 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 Experiment 21. — Mechanical Action of Bacteria 
 
 1. Inject a 300-gram pig intraperitoneally with 2 c.c, of a forty-eight-liour-old 
 bouillon culture of Bacillus anthraeis. Great care must be exercised in handling and 
 injecting this culture. 
 
 2. Autopsy the animal at the end of forty-eight hours and make cultures on agar 
 slants of the heart's blood, liver, and spleen. Remove the heart, lungs, liver, spleen, 
 and kidneys, and place in 2 per cent, formalin. Prepare smears of the blood and 
 stain with Gram's method. 
 
 3. After fLxing for twenty-four hours, sections of these organs are to be prepared 
 and stained with methylene-blue and with Gram's stain. 
 
 4. Examine the cultures at the end of twenty-four hours. 
 
 5. Examine the sections. 
 
 (a) Is the anthrax bacillus aggressive? 
 
 (b) Does the anthrax bacillus produce much toxin? 
 
 (c) Did the animal show any symptoms of its infection? 
 
 (d) Are there evidences of peritonitis? 
 
 (e) How do you explain the probable causes of death in human an- 
 thrax? 
 
 EXERaSE 8,— KINDS OF IMMUNITY 
 
 Experiment 22. — Phagocytosis in Natural Immunity 
 
 1. Inject a healthy guinea-pig intraperitoneally w^ith 1 c.c. of a twenty-four- 
 hour or forty-eight-hour glucose bouillon culture of streptococci of low or moderate 
 virulence. A culture of staphylococci may be used instead, although the former is 
 preferable. 
 
 2. At the end of eighteen hours study the peritoneal fluid. Prepare smears and 
 stain with methylene-blue or other suitable stain. Prepare cultures in glucose 
 bouillon of the heart's blood. 
 
 (a) Did the animal show any evidences of infection? 
 
 (b) Has phagocytosis occurred in the peritoneal cavity? 
 
 (c) What constituent of normal serum aids phagocytosis? 
 
 (d) Did a bacteremia develop? 
 
 Experiment 23. — Natural Antibacterial Immunity 
 
 1. Inject a 250-gram guinea-pig subcutaneously with 1 c.c. of a twenty-four-hour 
 bouillon culture of Bacillus anthraeis. 
 
 2. Inject a large albino rat with the same dose and in the same manner. 
 
 3. Observe the animals for twenty-four to forty-eight hours. At autopsy pre- 
 pare smears and cultures of the heart blood of each. Stain the smears with methylene- 
 blue and according to the method of Gram. 
 
 Do the animals present symptoms of infection? Are there any 
 differences between the two? 
 
ACQUIRED IMMUNITY 827 
 
 ExPiJKiMENT 24. — Relative Factors in Natural Immunity 
 
 1. Place a frog in a shallow vessel of warm water in the incubator at 37° C 
 
 2. After twenty-four hours give a subcutaneous injection of tetanus toxin equal 
 to one-tenth the fatal dose for a 300-gram guinea-pig. 
 
 3. Inject a second frog with an equal amount of toxin and keep it at ordinary 
 room temperature in cold water. 
 
 4. Observe both animals. 
 
 (a) Do symptoms of tetanus develop in any of the animals? 
 
 (b) Why does heat favor tetanus infection? 
 
 (c) Give further examples of relative natural immunity. 
 
 (d) Are anthrax bacilli found in the smears and cultures of both ani- 
 mals? Which animal is immune? Could phagocytosis play a role in 
 this immunity? 
 
 Experiment 25. — Influence op Temperature Upon Natural Im- 
 munity 
 
 1. Procure dried tetanus toxin and dissolve sufficient in 6 c.c. normal salt solu- 
 tion equivalent to 6 lethal doses for a 350-gram guinea-pig. 
 
 2. Inject 2 c.c. into the pectoral muscles of a young hen. Take the rectal tem- 
 perature. 
 
 3. Inject 2 c.c. into a second hen. Take the rectal temperature and place her 
 in cold water until the temperature has dropped several degrees. 
 
 4. Place hen No. I in a cage and keep her at ordinary laboratory temperature. 
 
 5. Place hen No. 2 in a cage and keep her in a cold place or renew the bath 
 several times in order to keep her temperature subnormal. 
 
 (a) What is the normal temperature of the hen? 
 
 (b) Do both animals present symptoms of tetanus? 
 
 (c) How do you explain the difference? 
 
 6. If hen No. 1 does not show symptoms of tetanus by the second day, reinject 
 her with an amount of toxin equal to 10 lethal doses for a guinea-pig. 
 
 (a) Does this hen present evidences of tetanus? 
 
 (b) If so, how do you explain the result? 
 
 EXERCISE 9.— ACQUIRED IMMUNITY 
 
 Experiment 26. — Acquired Active (Antibacterial) Immunity 
 
 1. Immunize a rabbit with typhoid bacilli as described in the chapter on jVctive 
 Immunization. 
 
 2. Inject this immune rabbit intravenously with 6 loopfuls of a twenty-four- 
 hour culture of Bacillus typhosus thoroughly emulsified in 2 c.c. sterile salt solution. 
 
 3. Inject a normal rabbit of equal weight with an equal dose and in the same 
 manner. 
 
 4. Both animals are weighed and their temperatures recorded before the injec- 
 
828 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 tions are given. If possible, take temperature every four hours. Observe both 
 animals for symptoms of infection. If one or both succumb, autopsy aseptically and 
 culture the heart blood in neutral bouillon or bile. 
 
 (a) Are there differences in the chnical condition of the two animals? 
 
 (b) To what do you ascribe these differences? 
 
 (c) What chief antibody is responsible for the destruction of the 
 bacilli in the immune animal? 
 
 (d) Has typhoid bacteremia developed? 
 
 Passive Acquired Immunity 
 Experiment 27. — Acquired Passive (Antitoxic) Immunity 
 
 1. Inject a 250- to 300-gram guinea-pig subcutaneously with 1 o.c. (500 units) of 
 diphtheria antitoxin (pig No. 1). 
 
 2. Secure diphtheria toxin in which the lethal dose (L. D.) for a 250- to 300-gram 
 guinea-pig is known. 
 
 3. Place this dose of toxin in a small test-tube or, better, in the barrel of a pre- 
 cision syringe (Kitchens), and add 1 c.c. antitoxin (500 units). Mix thoroughly and 
 keep at room temperature for an hour. 
 
 4. Inject pig No. 1 with an amount of toxin equal to the L. D. dose. 
 
 5. Inject the toxin and antitoxin mixture into a second pig of 250 to 300 grams. 
 
 6. Inject the same amount of toxin into a third pig of equal weight as a control. 
 
 7. Observe animals closely for at least four or five days. 
 
 (a) What are the chief symptoms of diphtheria intoxication of the 
 guinea-pig? 
 
 (b) Do all animals show these symptoms? 
 
 (c) How do you explain the absence of symptoms in pig No. 1? 
 How in pig No. 2 ? Is the mechanism of protection the same in both? 
 
 (d) What bearing has this experiment upon the treatment of diph- 
 theria? 
 
 Experiment 28. — Acquired Passive (Antitoxic) Immunity 
 
 The above experiment may be conducted in exactly the same maimer, using 
 tetanus toxin and antitoxin. 
 
 Experiment 29. — Acquired Passive (Antibacterial) Immunity 
 
 1. Secure a culture of pneumococcus, and if its lethal dose for white mice is un- 
 known, determine this by injecting a series of mice with decreasing doses of a forty- 
 eight-hour serum bouillon culture. 
 
 2. Secure a few cubic centimeters of antipneumocOccus serum, especially a serum 
 prepared with the culture being used, or at least one that is polyvalent. 
 
 3. Inject throe mice subcutaneously with 0.1, 0.5, and I c.c. of the serum. Then 
 inject them subcutaneously with double the lethal dose of pneumococci. Inject a 
 fourth mouse with culture alone (control). 
 
 4. Obser\re the animals for several days, and at autopsy culture the heart blood in 
 tubes of serum bouillon and prepare smears which are to be stained according to 
 Gram. 
 
PHAGOCYTOSIS 829 
 
 (a) Has the serum served to protect any of the luice? 
 
 (b) Is this protection due to bacteriolysins, bacteriotropins, or both? 
 Do antitoxins play any part? 
 
 (c) Why is it preferable to use homologous culture and immune 
 serum? What bearing has this upon the treatment of human pneumo- 
 coccus infections? 
 
 (d) Do you know a method for quickly isolating and identifying 
 pneumococci from sputum? 
 
 Note. — This experiment may be conducted with rabbits; also a cul- 
 ture of streptococcus and its immune serum may be used. 
 
 EXERaSE 10.— PHAGOCYTOSIS 
 
 Experiment 30. — Phagocytosis (Macrophages) 
 
 1. Secure pigeon.?' blood and defibrinate. Wash the corpuscles several times and 
 prepare a 5 per cent, suspension. 
 
 2. Inject a guinea-pig intraperitoneally with 3 c.c. of this corpuscle suspension. 
 
 3. After three hours withdraw a small amount of peritoneal exudate by means 
 of a capillary pipet. Examine with hanging-drop preparations and prepare smears 
 and stain with Wright's stain. 
 
 4. Make similar preparations twelve, eighteen, twenty-four, and forty-eight 
 hours after injection. 
 
 (a) Has phagocytosis occurred? 
 
 (b) Which cells have become phagocytes? 
 
 (c) Do the pigeon cells appear as if undergoing digestion? 
 
 (d) Which portion of the pigeon cell resists digestion? 
 (c) Explain mechanism of intracellular digestion. 
 
 Experiment 31. — Phagocytosis (Microphagbs) 
 
 1. Place a drop of blood in the hollow cell of a ground-out slide such as is used for 
 hanging-drop preparations. Cover with a clean slide, seal with a ring of vaselin, and 
 place in a large Petri dish containing pieces of filter-paper moistened with water 
 (moist chamber). Place in the incubator for fifteen minutes. 
 
 2. Remove the cover-glass and carefully wash cover-glass and cell with normal 
 salt solution. The erythrocytes are removed and the leukocytes left adherent to the 
 cell and cover-glass. 
 
 3. Fill the cell with fresh serum and add a quantity of culture, preferably a loop- 
 ful of a twenty-four-hour bouillon culture of non- virulent anthrax bacilli. Apply the 
 cover-glass and vaselin the margins to prevent evaporation. 
 
 4. If at all possible, employ a warm stage and watch the process under the micro- 
 scope. 
 
 (a) Does phagocytosis occur? 
 
 (b) Which cells are acting as phagocytes? 
 
830 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 (c) Can you make out the phase of fixation and phase of ingestion? 
 
 (d) By what agencies do leukocytes kill engulfed bacteria? 
 
 Experiment 32. — Phagocytosis 
 
 I. Inject a guinea-pig intraperitoneally with 5 o.c. of finely divided cinnabar 
 suspension and I c.c. subcutaneously on each side in the region of the inguinal lymph- 
 
 2. Autopsy the animal at the end of twenty-four hours. Prepare hanging-drop 
 preparations and smears of the peritoneal exudate (stained lightly with methylene- 
 blue). Prepare sections of the inguinal and abdominal lymphatic glands. 
 
 (a) Has phagocytosis occurred in the peritoneal cavity? Which 
 cells have become phagocji;ic? 
 
 (b) Do you find phagocytes in the lymph-glands? Where are the 
 leukocytes situated? Which cells have become phagocytes? 
 
 (c) How did the material reach the glands'? 
 
 (d) How do you explain the presence of cinnabar in the endothelial 
 cells of the gland? 
 
 EXERCISE II.— CHEMOTAXIS 
 
 Experiment 33. — Positive Chbmotaxis 
 
 1. Inject a guinea-pig intraperitoneally with 1 or 2 c.c. of a twenty-four-hour 
 culture of Staphylococcus aureus. 
 
 2. Autopsy the animal eighteen hours later and prepare smears of the exudate. 
 Fix with methyl alcohol for five minutes, dry, and stain with carbol-thionin, Wright's 
 or Loeffler's methylene-blue, counterstained with eosin. 
 
 3. Prepare cultures of the peritoneal exudate. 
 
 (a) Describe the exudate. 
 
 (b) Has phagocytosis occurred? 
 
 (c) Which cells are actively phagocytic? 
 
 (d) Are all the cocci engulfed? 
 
 (e) Are there any evidences of the cocci undergoing intracellular 
 digestion? 
 
 (f) Could a sterile substance call forth an exudation of leukocytes 
 when injected into a serous cavity? 
 
 Experiment 34. — Negative Chemotaxis 
 
 1. Inject a guinea-pig intraperitoneally with 0.5 to 1 c.c. of a forty-eight-hour 
 serum bouillon culture of virulent streptococci. 
 
 2. Autopsy at the end of eighteen to twenty-four hoius if death has not already 
 occurred . 
 
 3. Prepare cultures in senun bouillon and a number of smears. Staia the latter 
 with methylene-blue or according to Gram. 
 
OPSONINS 831 
 
 (a) Describe the exudate. How does it differ from that found in 
 the preceding experiment? 
 
 (b) How do you explain the serous character of the exudate? 
 
 (c) Has phagocytosis occurred? 
 
 (d) Do you think tlie streptococci have multiplied in the peritoneal 
 cavity? 
 
 (e) What means could you suggest for overcoming this action of 
 virulent streptococci? 
 
 EXERCISE 12.— OPSONINS 
 
 .Experiment 35. — Normal Opsonins 
 
 1. Prick the finger and secure 1 c.c. of blood in a ginall tesHube. Also 1 c.c. in a 
 centrifuge tube containing 2 c.c. of sodium citrate solution. After coagulation re- 
 move the serum from the first tube. 
 
 2. Divide the serum into two portions and heat one portion at 56° C. for thirty 
 minutes. 
 
 3. Prepare an emulsion of leukocytes by centrifuging the blood collected in the 
 citrate solution, removing the supernatant fluid, adding normal salt solution, and 
 centrifuging again. Repeat this step once more in order to wash the cells thoroughly 
 and after the last centrifuging remove the supernatant fluid and add sufficient salt 
 solution to make the total volume 1 c.c. and mix thoroughly. 
 
 4. Prepare an emulsion of staphylococci which is homogeneous and free of clumps. 
 
 5. Mark two capillary tubes with a wax pencil about an inch from the tip; fit 
 rubber teats to the other end. 
 
 6. With pipet No. 1 take up a volume of blood^cclls; allow a bubble of air to 
 enter and then an equal volume of bacterial emulsion; bubble of air and .in equal 
 volume of the fresh unheatcd serum. Mix well by alternate expulsion and aspiration 
 on a clean slide. Then draw the whole into the stem of the pipet and seal the tip in 
 a flame. 
 
 7. Repeat with pipet No. 2, using the h&ated serum. 
 
 8. Incubate both pipets at 37° C. for half an hour. 
 
 9. Remove the pipets from the incubator, break off the tips, mix the contents, 
 and prepare smears. 
 
 10. Fix the smears with a saturated solution of bichlorid of mercurj' for one 
 minute; wash in water and stain with carbol-thionin for two minutes; wash in water 
 and dry. 
 
 11. Examine with oil-immersion lens. 
 
 (a) What are the requisites of a satisfactory opsonic preparation? 
 
 (b) Is there any difference in the amount of phagocytosis between 
 the heated and unheated serums? If so, how do you explain the result? 
 
 (c) Do normal opsonins play any role in natural immunity? 
 
 (d) Give the properties of normal opsonins. 
 
832 EXPERIMENTAL INFECTION Alvf© IMMUNITY 
 
 Experiment 36. — Immune 6psonins (Bacteeiotropin) 
 
 1. Secure 1 c.c. of serum from a rabbit immunized with staphylococci and heat 
 0.5 c.c. at 56° C. for thirty minutes. 
 
 2. Using the same blood and bacterial emulsion= as prepared in the preceding 
 experiment, prepare two opsonic preparations with the heated and unheated immune 
 serum. 
 
 (a) Is phagocytosis more marked than in the preceding experiment? 
 If so, why? 
 
 (b) Is there any difference in the degree and extent of phagocytosis 
 with the heated and unheated serum? 
 
 (c) Give the properties of immune opsonin or bacteriotropin. 
 
 Experiment 37. — Hemopsonin 
 
 1. Secure 0.5 c.c. serum from a rabbit immunized with sheep cells and heat at 
 56° C. for half an hour to destroy hemolytic complement. 
 
 2. Prepare a 5 per cent, suspension of sheep erythrocytes in normal salt solution 
 after washing them three times. 
 
 3. Prepare an emulsion of rabbit leukocytes, or:the emulsion of human cells 
 in the preceding experiments may be used. 
 
 4. Take a capillary pipet; make a mark about an inch from the tip and fit the 
 other end with a rubber teat. 
 
 5. Draw up equal volumes of leukocytic emulsion, sheep cell suspension, and 
 serum. Mix well, seal the pipet, and inoculate for an-hour at 37° C. 
 
 6. Prepare smears and stain with Wright's blood-stain. 
 
 (a) Has phagocytosis occurred? 
 
 (b) Which cells have become phagocytes? 
 
 (c) Has hemolysis occurred in the mixtures and if not, why not? 
 
 EXERQSE 13.— OPSONINS (Continued) 
 
 Experiment 38. — Mechanism of Action of Opsonins 
 
 1. Secure serum from a rabbit immunized with Staphylococcus pyogenes aureus. 
 
 2. Prepare a leukocytic suspension from normal r|ibbit blood. 
 
 3. Prepare an emulsion of eigh teen-hour culture of Staphylococcus aureus. 
 
 4. Make the following mixtures in capillary pipet«: 
 
 No. 1 : Equal parts of leukocytes, serum, and bacterial emulsion. 
 No. 2: Equal parts of leukocytes and bacterial suspension. 
 
 5. In two small test-tubes mix: 
 
 No. 3: 0.5 c.c. each of leukocytic suspension and serum. 
 No. 4; 0.5 c.c. each of serum and bacterial emylsion. 
 
 6. Incubate pipets 1 and 2 and tubes 3 and 4 for fifteen minutes at 37° C. 
 
 7. Prepare smears of capillary tubes 1 and 2 and stain with oarbol-thionin. 
 
 S. To tubes 3 and 4 add 15 c.c. normal salt solution, mix, and centrifuge thor- 
 oughly. Decant off the supernatant fluid and restore volume in each tube to 1 c.c. 
 9. Prepare capUlary pipets as follows: 
 
OPSONINS 833 
 
 No. 3: Equal parts of contents of tube 3 and bacterial emulsion. 
 No. 4: Equal parts of contents of tube 4 and leukocytic mixture. 
 10. Incubate both pipets for fifteen minutes, prepare smears, and stain with car- 
 bol-thionin. 
 
 (a) Carefully compare the degree and extent of phagocytosis in the 
 four mixtures. 
 
 (b) Has leukocytosis occurred in mixture No. 2 ? If so, what special 
 term is applied to phagocytosis in the absence of serum? 
 
 (c) What r61e does serum play in phagocytosis? Explain the 
 mechanism involved. 
 
 (d) Discuss the question of "stimulin" and opsonin as demonstrated 
 by the results in preparations Nos. 3 and 4. 
 
 Experiment 39. — Specificity of Opsonins 
 
 1. Secure 1 c.c. of the serum of a rabbit immunized with staphylococci and heat 
 at 56° C. for thirty minutes. Divide into two portions in separate small test-tubes. 
 
 2. To No. 1 add 1 o.o. of normal salt solution and 4 or 5 loopfuls of a twenty- 
 four-hour agar slant culture of staphylococci. Incubate for thirty minutes; centri- 
 fuge thoroughly and remove the supernatant fluid (diluted seruhi) to a separate tube. 
 Call this "treated" serum. 
 
 3. To the untreated serum add 1 c.c. salt solution, so that both are diluted 
 equally. 
 
 4. Prepare an emulsion of rabbit or human leukocytes. 
 
 5. Prepare an emulsion of staphylococci, homogeneous and free of clumps. 
 
 6. Prepare two opsonic mixtures: No. 1 containing equal parts of blood suspen- 
 sion, bacterial emulsion, and untreated serum; No. 2 containing equal parts of blood, 
 bacterial emulsion, and treated serum. 
 
 7. Incubate for thirty minutes. During this interval proceed as follows: 
 
 8. Prepare an emulsion of typhoid bacilli, homogeneous p,nd free of clumps. 
 
 9. Secure 0.5 c.c. of serum from a rabbit immunized with typhoid bacilli and 
 heat at 36° C. for thirty minutes. 
 
 10. Prepare the following mixtures in capillary pipets-: 
 
 No. 3 blood-cells +typhoid serum +typhoid bacterial emulsion. 
 
 No. 4 blood-cells4- typhoid serum -[-staphylococcus bacterial emulsion. 
 
 No. 5 blood-cel!s-l-staphylococcus serum+typhoid emulsion. 
 
 11. Incubate for fifteen minutes. 
 
 12. Prepare smears of all and stain with carbol-thionin. 
 
 (a) Is there any difference in the degree of phagocytosis in mixtures 
 No. 1 and 2? If so, why? 
 
 (b) Are opsonins specific? 
 
 (c) Has phagocytosis occurred in the cross-mixtures of staphylococci 
 with typhoid serum, and typhoid bacilli with staphylococcus serum? If 
 so, how do you explain this apparent lack of specificity? 
 
 (d) What may happen in a mixture of fresh unheated typhoid serum, 
 typhoid bacilli, and leukocytic emulsion? 
 
 53 
 
834 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE J 4.— OPSONIC INDEX 
 
 Experiment 40. — Determining the Opsonic Index 
 
 1. Secure a small quantity of blood from a guineji-pig or rabbit which has been 
 immunized with Staphylococcus pyogenes aureus. Also two specimens from normal 
 animals to serve as control serums. Remove the serums, being careful to keep them 
 marked and separate. The two normal serums may, be mixed in a watch-glass or 
 small test-tube (pooled). 
 
 2. Prepare a leukocyte mixture, using normal guinea-pig or rabbit blood according 
 to the immune animal used. 
 
 3. Prepare a bacterial emulsion of an eighteen-hour culture of Staphylococcus 
 pyogenes aureus. 
 
 4. Proceed to determine the phagocytic and opsonic index as per technic given in 
 the text (page 195). 
 
 (a) What constitutes a satisfactory bacterial emulsion? 
 
 (b) What constitutes a satisfactory leukocytic suspension? Name 
 several methods of obtaining leukocytes for this technic. 
 
 (c) What constitutes a satisfactory phagocytic film? 
 
 (d) Should the serum be fresh? 
 
 (e) How do you determine the phagocytic index? 
 
 (f) How do you determine the opsonic index? 
 
 (g) What is the relation between the opsonic and phagocytic indices? 
 (h) Give the practical value of the opsonic index in disease. 
 
 (i) Give the practical value of the opsonic index as a measure of 
 immunity. 
 
 Note.-'lf any of the students have received typhoid vaccine, the 
 opsonic index with his serum may be determined. 
 
 Experiment 41. — Quantitative Estimation of Bacteri ©tropins 
 
 1. Secure a culture of pneumococcus. 
 
 2. Secure a few cubic centimeters of antipneumococcus serum, especially a serum 
 corresponding to the culture. 
 
 3. Prepare a leukocytic emulsion. 
 
 4. Conduct the test after the technic given in the text (page 200). 
 
 (a) Give the value of a bacteriotropic estiiiiation of a serum. 
 
 (b) What relation do bacteriotropins bear to immunity? 
 
 (c) In what class of diseases are bacteriotropins of most value? 
 
 EXERCISE 15.— BACTERIAL VACCINES 
 
 Experiment 42. — Preparation of Typhoid Vaccine 
 
 1. Prepare two to six agar slant cultures of Bacillus typhosus and grow for forty- 
 eight hours at 37° C 
 
 2. Remove cultures, prepare a suspension count: by the method of Wright, 
 sterilize, dilute, and prepare vaccine for administration as given in the text. 
 
BACTERIAL VACCIN=ES 835 
 
 3. Prepare two emulsions: one to contain about 500 million bacilli in each cubic 
 centimeter (first dose), and the second 1000 million per cubic centimeter (second and 
 third doses). 
 
 Experiment 43. — Preparation of Staphylococcus Vaccine 
 
 1. Prepare two to six agar slant cultures of Staphylococcus aureus and grow for 
 twenty-four hours at 37° C. If a patient with f urunculosis is available, make cultures 
 of pus and secure staphylococcus, of which a vaccine is prepared. 
 
 2. Proceed in the preparation of the vaccine as given in the text, placing each 
 dose in separate ampules, and so diluting that each dose is of one cubic centimeter 
 and contains 1000 million of cocci. Count by the method of Wright and by the 
 counting chamber method. 
 
 EXERCISE 16— ANTITOXINS 
 
 Experiment 44. — Standardizing Diphtheria Antitoxin 
 
 1. Prepare a strong diphtheria toxin with the Park-Williams Bacillus No. 8, after 
 the technic given in the text. 
 
 2. Secure some of the dried Standard Antitoxin and dilute so that 1 c.c. is equal 
 to one immunily unit. 
 
 3. With this antitoxin determine the L-f- dose of the toxin prepared accordin to 
 the technic given, using six guinea-pigs and the following doses of toxin: 0.1 c.c, 
 0.12 c.c, 0.15 cc, 0.18 c.c, 0.2 cc, 0.25 cc 
 
 4. Secure a sample of diphtheria antitoxin in the open market containing about 
 4 c.c serum and 2000 units of antitoxin. If the titration given on the label is still 
 correct, one would expect about 500 units of antitoxin per cubic centimeter of senim. 
 Carefully remove 1 cc. of serum and dilute with 19 cc salt solution (1:20). From 
 this stock dilution prepare the following dilutions (taken from Bulletin No. 21, 
 Hygienic Laboratory, M. J. Rosenau): 
 
 1 c.c. -1-11 c.c. NaCl solution 1 c.c. = .00333 Or yjjf = 300 units per c.c. 
 
 1 CC.-1-16 c.c. NaCl solution 1 c.c. = .00294 er ^J^ = 340 units per c.c. 
 
 1 C.C.-I-18 c.c. NaCl solution 1 c.c. = .00263 or jj^ =380 units per c.c. 
 
 1 c.c. -1-20 c.c. NaCl .solution 1 c.c. = .00238 or 3-!^ = 420 units per c.c. 
 
 1 c.c. +22 c.c. NaCl solution 1 c.c. = .00217 or ^^g = 460 units per c.c. 
 
 1 c.c. -|- 24 c.c. NaCl .solution 1 c.c. =.002 or -5^^ = 500 units per c.c. 
 
 5. Mix 1 c.c. of these various dilutions with the L | dose of toxin; stand aside 
 for an hour and inject subcutaneously in median abdominal line of 250- to 300-gram 
 guinea-pig as per the technic already given. 
 
 6. Carefully observe all animals for a period of four days at least. .Autopsy 
 those that succumb, paying particular attention to the condition of the abdominal 
 waU and suprarenal glands. If the serum should contain less than 300 imits of anti- 
 toxin per cubic centimeter of serum, the te.st should be repeated with lower dilutions. 
 
 7. Inject 1 c.c. of the serum subcutaneously into a white mouse to test for excess 
 of preservative. It requires 1 c.c. of a 0.5 per cent, solution of tricresol or 0.5 c.c. of 
 a 0.5 per cent, phenol solution to kill a medium-sized mouse. If the mouse shows 
 trembling, it would indicate that the serum contains nearly this percentage of tricresol 
 (Bulletin No. 21, Hygienic Laboratory). 
 
 8. Inoculate 1 c.c. of the serum in a flask containing 100 c.c. sterile neutral 
 bouillon. Incubate at 37° C. for-at least four days. This will test the sterility of the 
 product. 
 
836 EXPERIMENTAL INFECTION ANU IMMUNITY 
 
 (a) Define the unit of diphtheria antitoxin*. 
 
 (b) Of what practical value is the measurement of diphtheria anti- 
 toxin? 
 
 (c) Is there any practical method of determining the quantity of 
 toxin in the blood of a diphtheric patient? 
 
 (d) How would you determine the amount of natural antitoxin in 
 the blood of a person? 
 
 Experiment 45. — Standardizing Tetanus Antitoxin 
 
 The technic of standardizing tetanus antitoxin may be carried out in a similar 
 maimer with dried Standard Toxin and an antitoxin purchased in the open market. 
 
 EXERCISE J7.— ANTITOXINS (Continued) 
 Experiment 46. — Specificity of Antitoxins 
 
 1. Secure small quantities of fresh diphtheria and' tetanus toxins; the L+dose 
 of each should be known. 
 
 2. Secure small quantities of diphtheria and tetanus antitoxins; the number of 
 units per cubic centimeter of serum should be known. 
 
 3. Into four precision syringes place the following mixtures. After standing an 
 hour at room temperature, inject subcutaneously into. 300-gram guinea-pigs. 
 
 No. 1: L-l-dose of diphtheria toxin -|- 100 units of diphtheria antitoxin. Inject 
 into pig No. 1. 
 
 No. 2: L-|-dose of diphtheria toxin -f- 100 units of tetanus antitoxin. Inject into 
 pig No. 2. 
 
 No. 3: L-|-dose of tetanus toxin-)- 100 units of tetanus antitoxin. Inject into 
 pig No. 3. 
 
 No. 4: L-|-dose of tetanus toxin + 100 units of diphtheria antitoxin. Inject into 
 pig No. 4. 
 
 (a) What do you observe regarding the specificity of antitoxins? 
 
 (b) What are the main symptoms of diphtheria, and tetanus in the 
 guinea-pig? 
 
 (c) Even though a pig injected with diphtheria toxin shows no gen- 
 eral symptoms of intoxication, what local sign may be present? 
 
 (d) What is the nature of the toxin-antitoxin reaction? 
 
 Experiment 47. — Nature of the Toxin-antitoxin Reaction. 
 Action op Anti-tetanolysin 
 
 1. Secure fresh tetanus toxin and determine the ddse producing complete hemoly- 
 sis of 1 c.c. of a 1 per cent, suspension of rabbit corpuscles in two hours at 37° C. 
 
 2. Place double this dose of toxin in a series of six small test-tubes and add in- 
 creasing doses of fresh tetanus antitoxin: 0.001 c.c, 0.005 c.c, 0.01 c.c, 0.05 c.c, 
 0.1 CO., 0.2 c.c. Add salt solution to bring the total volume to 1 c.c; incubate at 37° C. 
 for an hour. Add 1 c.c of 1 per cent, suspension of rabbit corpuscles to each tube. 
 Prepare two controls, one with the dose of toxin and corpuscles but to which no serum 
 
BACTERIAL VACCINES 837 
 
 is added, the second with 0.2 c.c. serum and dose of corpuscles. Shake tubes and 
 incubate for two hours. Make a preliminary reading and again after tubes have 
 settled twenty-four hours in the refrigerator. 
 
 3. The first control should be completely hemolyzed, indicating that a sufficient 
 lytic dose of toxin was employed. 
 
 (a) What constituent of tetanus toxin has a marked affinity for 
 erythrocytes? 
 
 (b) Is this agent thermostabile? How can you determine this? 
 
 (c) What role does it play in tetanus intoxication? 
 
 (d) How is this hemotoxic agent neutralized by tetanus antitoxin? 
 
 (e) Would anti-tetanospasmin neutralize the hemotoxic activity 
 of tetanus toxin? 
 
 (f) Would diphtheria antitoxin neutralize this hemotoxic agent? 
 If not, why not? 
 
 (g) Explain the mechanism of neutralization of a toxin by antitoxin 
 in vitro? Is it the same as that occurring in vivo? 
 
 Experiment 48. — Antistaphyloltsin 
 
 1. Prepare a staphylolysin by growing a culture of Staphylococcus aureus in 
 bouillon for two or three weeks. Pass through a Berkefeld filter and preserve the 
 filtrate with 0.5 phenol. Determine the lytic dose for 1 c.c. of a 1 per cent, sus- 
 pension of rabbit corpuscles. 
 
 2. .Secure serum from a rabbit immunized with staphylococci. Heat at 56° C 
 for thirty minutes. 
 
 3. Secure normal horse serum. Heat it at 56° C. for thirty minutes. 
 
 4. Secure normal rabbit serum. Heat at 56° C. for thirty minutes. 
 
 5. Into a series of six small test-tubes place the lytic dose of staphylolysin and 
 increasing amounts of rabbit immune serum as follows: 0.001, 0.005, 0.01, 0.05, 0.1, 
 0.2 c.c. Arr.ange a similar series, using normal horse serum and normal rabbit serum. 
 Prepare a control containing the lytic dose of toxin. Add 1 c.c. of a 1 per cent, sus- 
 pension of rabbit cells to each tube and suiScient normal salt solution to make the 
 total volume equal 2 c.c. Shake each tube gently and incubate at 37° C. for two 
 hours. 
 
 6. Inspect the tubes. The smallest amount of normal horse serum inhibiting 
 hemolysis is taken as 1, or the unit. Compare the values of the immune and normal 
 rabbit serums with this unit. 
 
 (a) Why is normal horse serum adopted as the standard? 
 
 (b) What is antistaphylolysin? 
 
 (c) What are the various agents produced by staphylococci and re- 
 sponsible for the lesions and symptoms of staphylococcus infections? 
 
 (d) Would the antilysin neutralize the leukocidin? 
 
 (e) Explain the mechanism of lysin-antilysin action. 
 
 (f) Which r61e does the lysin play in staphylococcus infections? 
 
 (g) Of what value would be the titration of antistaphylolysin in the 
 serum of the patient? 
 
838 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 18.— FERMENTS AND ANTIFERMENTS 
 Experiment 49. — Tryptic Ferment of Leukocytes 
 
 1. Collect 0.5 CO. of blood in each of two small test-tubes by puncture of a finger; 
 set aside untO coagulation has occurred. Add several changes of warm distilled water 
 until the red corpuscles are hemolyzed and colorless clots of leukoc3^es, fibrin plate- 
 lets, and detritus are secured. 
 
 2. Place one clot in the bottom of a tube of sterile and slanted Loeffler blood- 
 serum medium which is fairly dry and firm. 
 
 3. Place the second clot in a second tube of this medium and add 0.2 to 0.4 c.c. of 
 fresh serum. 
 
 4. Plug these tubes firmly, paraffin the stoppers to prevent evaporation, and 
 incubate along with a non-inoculated control at 50° C. for two days. 
 
 (a) In which tube do you note evidences of digestion? 
 
 (b) Describe the appearance of the digested medium. 
 
 (c) Does the tube containing serum sho-w* digestion? How do you 
 explain the result? 
 
 (d) What role may this ferment play in infection? 
 
 (e) Why do not the leukocytes digest themselves? 
 
 (f) What is the nature of the ferment? 
 
 (g) May this ferment play a role in the disposal of old and dead 
 leukocytes? 
 
 Experiment 50. — Testing the Antitryptic Power of Blood- 
 Serum (After the Marcus Modification of the Method of 
 
 MtJLLER AND JoCHMANn) 
 
 1. Prepare a solution of trypsin by thoroughly shaking 0.1 gm. of Kahlbaiun's 
 trypsin with 5 c.c. glycerin and 5 c.c. distilled water. Incubate at 55° C. for halt an 
 hour, shake thoroughly, filter, and preserve in the refrigerator. 
 
 2. Secure six Petri dishes of Loeffler blood-serum culture-media which have been 
 sufficiently dried to drive off the water of condensation and with a firm elastic surface. 
 
 3. Collect 1 c.c. of blood from a patient three hours after last meal; separate the 
 serum. 
 
 4. In a watch-glass or hanging-drop slide mix one drop of serum with an equal 
 sized drop of trypsin solution. Mix well and transfer five loopfuls to an area on a 
 Petri dish of medium. With a blue-wax pencil draw a circle on the cover of the plate 
 to include the site of planting and mark No. 1. 
 
 5. Prepare serial dilutions with one drop of serum with two, three, four, five, six, 
 seven, eight, nine, and ten drops of trypsin solution. Mix well and plant five loop- 
 fuls of each dilution on the medium in five dishes. Two plantings may be made in each 
 plate and with care they will not become confluent. Mark each plate. 
 
 6. Inoculate the sixth plate with five drops of trypsin solution without serum 
 (control) . 
 
 7. Incubate all tubes at 37° C. for six to twelve hours until the control shows 
 well-marked digestion. 
 
 8. Wherever the trypsin has not been neutralized :by the serum, shallow liquefied 
 
FERMENTS 839 
 
 dimples appear on the surface of the Loeffler medium. The greater the amount of 
 trypsin required to cause digestion, the higher the titer of antitrypsin in the blood. 
 By conducting a control series with the two normal pooled serums one may determine 
 whether in a given case the antitryptic power of the blood is normal, increased, or 
 decreased. 
 
 (a) Does this test possess any practical value? 
 
 (b) Discuss the presence of antitrypsin in the blood-serum. 
 
 EXERCISE 19.— FERMENTS (Continued) 
 
 Experiment 51. — Abderhalden Seho-enzyme Reaction in Preg- 
 nancy 
 
 The technic of this test is very exact. The glassware should be 
 sterile and all manipulations carried out carefully and exactly as laid 
 down_ by Abderhalden. Consult the text for the technic (Chapter 
 
 XV).' 
 
 1. Test five Schleichter and Schull shells No. 579a for permeability to albumin, 
 using 5 per cent, egg-albumen water and the biuret or ninhydrm reaction. 
 
 2. Those shells which prove impermeable to albumin are now tested with a 1 
 per cent, solution of silk peptone (Ilochst), using ninhydrin as the indicator. The 
 Shells impermeable to albumin and permeable to peptone are suitable for the main 
 test. 
 
 3. Secure a fresh human placenta; wash thoroughly to remove blood; cut into 
 pieces about the sisse of a dime; wash and rewash until perfectly white; search care- 
 fully for tiny blood-clots; boil repeatedly rmtil the water reacts negatively to nin- 
 hydrin. Consult the text-book for the exact technic. Abderhalden lays a great 
 deal of stress upon the proper preparation of the substratum. 
 
 4. Secure blood from a patient in advanced pregnancy; also a specimen from a 
 male. After a few hours, separate the serums and centrifuge thoroughly until free 
 of cells. The serums should not be over twelve hours old. 
 
 5. Conduct a reaction after the technic given in the text-book, including the 
 controls. 
 
 (a) Describe the biuret and ninhydrin resfctions. 
 
 (b) Why must the shells be so carefully tested? 
 
 (c) Why must the placenta be free of blood and boiled previous to 
 the test? 
 
 (d) Why should an aseptic technic be employed? 
 
 (e) What are the prevailing views regarding the specificity of the 
 ferments? 
 
 (f) Has the pregnancy test a practical value? 
 
 (g) Give the principles of the optical method, 
 (h) Why is the serum control so important? 
 
 (i) Enumerate the principal sources of error in the technic of the 
 dialysation method. 
 
840 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 20.— AGGLUTININS 
 
 Experiment 52, — The Gruber-Widal Reaction in Typhoid Fever 
 ("Wet" Method). Microscopic Reaction 
 
 1. Collect blood in a Wright capsule or small test-tube from a typhoid conva- 
 lescent patient. Rabbit immune serum may be used instead. Separate theserum 
 from the clot. 
 
 2. Prepare two dilutions with hanging-drop slides, 1 : 50 and 1 : 100, and a culture 
 control. Use a twenty-four-hour bouillon culture of 'Bacillus typhosus — one free of 
 clumps and in which the bacilli are long and motile. ' 
 
 3. It is well to prepare a similar test with a known positive typhoid serum and 
 a normal negative serum. 
 
 4. Dilution and slides are prepared according to the technic given in the text- 
 book. A second set of tests in dilutions of 1 : 40 and 1 : 80 may be prepared with the 
 aid of the white corpuscle pipet. 
 
 5. Place slides away from direct sunlight and examine at the end of an hour. 
 Examinations are readily made with the J^ objective, the Ught being well cut off. 
 Examine the culture control first, then the higher and lower dilutions. 
 
 (a) Describe the phenomenon of agglutinailon. 
 
 (b) Is it necessary for all bacilli to be agglutinated to constitute a 
 positive reaction? 
 
 (c) What are the features of a doubtful reaction? Of a negative re- 
 action? 
 
 (d) Does the normal serum contain agglutinin for the typhoid ba- 
 cillus? 
 
 (e) Why is it necessary to use high dilutions? What dilution is the 
 lower limit of practical safety? 
 
 (f) Does the control show agglutination? If so, by what term is 
 this agglutination known? 
 
 (g) What are thp characteristics of a satisfactory culture for the 
 microscopic agglutination test? 
 
 (h) Would a dead culture be serviceable in this reaction? 
 
 (i) What practical value has the Widal reaction in the diagnosis of 
 typhoid fever? 
 
 (j) What value has the agglutination reaction in determining the 
 degree of immunity following typhoid immunization? 
 
 Note. — If the students are immunized with typhoid vaccine, they 
 may use one another's serum in this and following experiments. 
 
 Experiment 53. — Grubler- Widal Reaction in Typhoid Fever 
 ("Dry" Method) 
 
 1. Prepare smears of blood of a typhoid convalieecent patient on clean glass 
 shdes or collect a few drops on partially glazed paper, ^s prescription blanks. Allow 
 blood to dry and do not apply heat. 
 
AGGLUTININS 841 
 
 2. Using a good twenty-four-hour-old culture of Bacillus typhosus, prepare an 
 agglutination test after the technic given in the text-book. Re particularly careful 
 not to transfer paper fiber to the slide — apply the salt solution and dissolve the blood 
 by gently rubbing with a small platinum loop (2 mm.). Mix the blood in the loop- 
 ful of culture with the slide held or placed over a white surface so that the proper 
 delicate orange tint is secured. 
 
 3. Prepare culture control as usual; also similar tests with a known positive and 
 negative serum. 
 
 4. Examine at the end of an hour. 
 
 (a) What special precautions are to be observed in this method? 
 
 (b) What is the particular practical value of this reaction? 
 
 (c) Are accurate dilutions possible and if so, by what technic? 
 
 (d) Are agglutinins resistant to deleterious influences? 
 
 (e) Compare the value of this method with the one used in the pre- 
 ceding experiment. 
 
 EXERCISE 21.— AGGLUXmiNS (Continued) 
 Experiment 54. — Macroscopic Agglutination Reaction 
 
 1. Secure serum from a rabbit which has been immunized with Bacillus typhosus. 
 Serum of a typhoid couvulescent may be used instead. 
 
 2. Prepare a series of serum dilutions in small narrow test tubes in amounts of 
 1 c.c, ranging from 1: 10 up to 1: 640. 
 
 3. Add to each 1 c c. of the baoUlary emulsion of a good twenty-four- to forty- 
 eight-hour bouillon culture of Bacillus typhosus or an emulsion prepared by washing 
 twenty-four-hour growths from agar slant cultures with normal salt solution, accord- 
 ing to the technic given in the text. Tliis doubles the dilutions, wliich now range 
 from 1: 20 up to 1: 1280. Prepare the culture control. 
 
 4. Shake gently and incubate for two hours at 37° C. and then record results after 
 tubes have been standing at room temperature for six hours. Reexamine tubes with 
 the agglutinoscope and note the higher delicacy of such readings. 
 
 5. If a typhoid immune serum of unknown titer is used and agglutination is 
 complete in the highest dilution, the test must be repeated with still higher dilutions 
 in order to determine the agglutinin titer of the serum. 
 
 (a) Has agglutination occurred in the control tube? Why is this 
 control so important in this and all agglutination reactions? 
 
 (b) Is agglutination as complete in the lower as in the higher reac- 
 tion? If not, how do you explain this result? Of what practical im- 
 port is this phenomenon? 
 
 (c) Describe the appearance of macroscopic agglutination. 
 
 (d) Are the agglutinated bacilli dead? To determine this, pipet off 
 the supernatant fluids of several tubes into a germicidal solution; then 
 add an excess of sterile normal salt solution to the sediment of aggluti- 
 nated bacilli, stir up the sediment, and transfer with a sterile pipet to a 
 sterile centrifuge tube; centrifuge thoroughly; remove the supernatant 
 
842 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 fluid with a sterile pipet and plant several loopfuls of bacteria on slants of 
 agar and in neutral bouillon. Why is it advisable to wash the sediment? 
 (e) What advantages has the macroscopic over the microscopic 
 method? 
 
 Experiment 55. — Macroscopic Agglutination Reaction (Kolle) 
 
 1. Prepare dilutions in amounts of 1 c.c. of a tyjfhoid immune serum in proper 
 test-tubes, ranging from 1 : 20 up to 1 : 1280. 
 
 2. Add one loopful of a twenty-four-hour culture of Bacillus typhosus lo each 
 tube, being careful to emulsify thoroughly on the side of the test-tube according to the 
 technic given. This does not materially alter the degree of dilution. Prepare the 
 culture control as usual. 
 
 3. Incubate and examine tubes as in the previous, experiment. 
 
 What are the advantages and disadvantages of this method? 
 
 Experiment 56. — Macroscopic Agglutination Reaction (Killed 
 Culture) 
 
 1. Inoculate a flask containing 200 c.c. of neutral broth with Bacillus typhosus 
 and incubate for forty-eight hours. At the end of this time a good rich growth is 
 usually secured. Shake gently to stir and break up any clumps of bacilli and heat 
 at 60° C. for one hour on a water-bath, gently shaking the flask once or twice during 
 this time. Add 5 c.c. formalin; shake, and place in the refrigerator for three days; 
 stopper the flask with a rubber stopper, and before using shake well in order thor- 
 oughly to mix the dead bacUh which drop to the bottom of the flask. 
 
 2. Prepare a series of dilutions of typhoid immune serum and conduct this 
 experiment according to the macroscopic technic, using the dead instead of a living 
 bacillary emulsion. 
 
 (a) What are the advantages of using a killed culture? 
 
 (b) Is this method as delicate as when living cultures are used? 
 
 (c) Is spontaneous agglutination likely to;occur? 
 
 (d) What constitutes a satisfactory killed culture for this reaction? 
 
 EXERCISE 22.— AGGLUTININS (Continued) 
 Experiment 57. — Group Agglutination 
 
 1. Prepare five series of test-tubes containing 1 c.c> of dilutions ranging from 1:20 
 to 1 : 640 of a typhoid immune serum. 
 
 2. To the first series add 1 c.c. of a thirty-six-hqiur boufllon culture of Bacillus 
 typhosus; to the second series, Bacillus para typhosus "B"; to the third. Bacillus 
 paratyphosus "A"; to the fourth, BacUlus enteritidis (Gartner); to the fifth. Bacillus 
 coh. The dilutions are thus doubled. Prepare culture controls of all five cultures, 
 and be careful that tubes are properly labeled. 
 
 3. Incubate for two hours and record reactions at the end of further six hours at 
 room temperature. 
 
 4. This experiment may be repeated with the serum of a typhoid convalescent 
 patient. 
 
AGGLUTININS 843 
 
 (a) In which series of tubes is agglutination most complete? 
 
 (b) Discuss the question of specificity of the agglutinins. 
 
 (c) How do you explain group agglutination? 
 
 (d) Are group agglutinins present to the same degree as the main 
 agglutinin? How may the group agglutinins be eliminated? 
 
 (e) Of what value is the agglutination reaction in showing the bio- 
 logic relationship of bacteria? 
 
 (f) How would the agglutination reaction be used in the diagnosis 
 of an unknown microorganism? 
 
 Experiment 58. — Pro-agglutination — Agglutinoids 
 
 1. This very important phase of agglutination may have been noted in the pre- 
 vious experiments, especially if old immune serums were used, when there is a possi- 
 biUty that agglutinin has degenerated in agglutinoids. Agglutinoids having a stronger 
 affinity than agglutinin for the agglutinogen, and having lost the agglutinophore 
 group, produce little or no agglutination and prevent the action of agglutinin until 
 diluted out of action in the higher senim dilutions. In this way agglutination may be 
 poor or absent in low and present in higher dilutions, an important practical fact 
 to be remembered. 
 
 2. The action of agglutinoids may be seen by conducting a macroscopic test with 
 an old immune serum. 
 
 3. Otherwise some agglutinoid may be produced by heating an immune serum 
 to 60° C. for an hour on the water-bath and conducting the test with dilutions of the 
 heated serum. 
 
 4. Repeat the tests with old and heated immune serums in dilutions of 1:40, 
 1:80, 1:160, 1:320, using the macroscopic and microscopic technic, as this phe- 
 nomenon of pro-agglutination is not infrequently found in the routine Widal reaction 
 in typhoid fever. 
 
 Experiment 59. — The Absorption or Sattjratton Aggluttnatton 
 Reaction (Castellani) 
 
 1. Immunize a rabbit with three intravenous injections of a heated emulsion of 
 Bacillus typhosus and three of Bacillus paratyphosus "B" or Bacillus coli, according 
 to the technic given under Active Immunization and Methods of Animal Inoculation. 
 
 2. With this immune serum conduct a test according to the technic given in the 
 text. 
 
 (a) Of what practical value is this method? 
 
 (b) What are "major" and "minor" agglutinins? 
 
 EXERCISE 23.— AGGLUTININS (Continoed) 
 -Experiment 60. — Hemagglutinins 
 
 1. Secure 0.1 c.c. of antihuman hemolysin prepared by giving a rabbit a series 
 of injections of washed human erythrocytes. Inactivate the serum by heating to 
 55° C. for half an hour. Dilute 1 : 100 by adding 9.9 c.c. salt solution. 
 
 2. Prepare 10 c.c. of a 1 per cent, suspension of washed human erythrocytes. 
 
844 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 3. Into a series of six small test-tubes place 0.1 c.c.j 0.2 c.c, 0.4 c.c, 0.6 c.c, 0.8 
 c.c, 1 c.c. of the diluted immune serum; add 1 c.c. of suspension of blood-cells and 
 1 c.c. of salt solution to each tube. As a control, place 1 c.c. of corpuscle suspension 
 and 1 c.c. of normal salt solution into a seventh tube. 
 
 4. Shake gently and place in the incubator for an hour. 
 
 (a) Describe agglutination of red corpuscles. 
 
 (b) Are the clumps easily broken up? 
 
 (c) What would have occurred if complement were present? 
 
 (d) Why was it necessary to heat the fr^h immune serum? Is it 
 necessary to heat an old immune serum? 
 
 (e) Examine a loopful of the sedimented corpuscles microscopically. 
 
 Experiment 61. — Blood Transfusion Tests 
 
 Several students may contribute a few cubic centimeters of their blood and 
 agglutination and hemolysin tests made as described in the text (Chapter XVI). 
 
 Of what value are agglutination and hem8l3rtic tests preliminary to 
 blood transfusion? 
 
 EXERCISE 24.— PRECIPITINS 
 Experiment 62. — Titration op a Precipitin Serum 
 
 1. Secure 1 c.c. of antihorse immune serum prepared by immunizing a rabbit 
 with normal horse serum. 
 
 2. Secure 1 c.c. of normal horse serum and place 2 c.c. of the following dilutions 
 made with normal salt solution into a series of six narrow test-tubes: 1: 100, 1:500, 
 1 : 1000, 1 : 2000, 1 : 5000, and 1 : 10,000. 
 
 3. To each tube add 0.1 c.c. of the immune serum. The tubes must not be 
 shaken. 
 
 4. Place tubes in an incubator at 37° C. and observe every thirty minutes for 
 two hours and again after standing in a refrigerator overnight. 
 
 (a) Describe the phenomenon of precipitation. 
 
 (b) What are the requisites of a satisfactory precipitin serum? 
 
 (c) What are the requisites of a satisfactory preparation of the 
 antigen for this reaction? 
 
 (d) Discuss the delicacy of this reaction. 
 
 Experiment 63. — Titration op a Precipitin Serum 
 
 Repeat this titration, using antihuman immvme serum and normal human 
 
 Experiment 64. — Specificity of Precipitins 
 
 1. Secure 1 c.c. each of clear antihorse and antihuman serums. 
 
 2. Secure 1 c.c. each of normal horse, normal human, and normal guinea-pig 
 
PRECIPITINS 845 
 
 serum. Prepare 1;100 dilutions with normal salt solution; set up the following in 
 long narrow test-tubes: 
 
 Tube 1: 2 c.c. of normal horse serum (1: 100) +0.1 c.c. antihorse serum. 
 Tube 2: 2 c.c. of normal horse serum (1: 100) +0.1 c.c. antihuman serum. 
 Tube 3: 2 c.c. of normal human serum (1: 100)+0;1 c.c. antihuman serum. 
 Tube 4: 2 c.c. of normal human serum (1:100) +0.1 c.c. antihorse serum. 
 Tube 5: 2 c.c. of normal guinea-pig serum (1:100) +0.1 c.c. antihorse serum. 
 Tube 6: 2 c.c. of normal giiinea-pig serum (1: 100).+0.1 c.c. antihuman serum. 
 Tube 7: 2 c.c. of normal salt solution+0.1 c.c. antihorse serum (control). 
 Tube 8: 2 c.c. of normal salt solution+0.1 c.c. antihuman serum (control). 
 
 3. Do not shake the tubes; place them in the incubator at 37° C; inspect every 
 thirty minutes for two hours. 
 
 (a) Discuss the question of specificity of precipitins. 
 
 (b) Of what practical value are precipitin reactions? 
 
 (c) Enumerate the chief points in the technic of a precipitin reac- 
 tion in the differentiation of proteins. 
 
 EXERCISE 25— PRECIPITINS (C-ontinoed) 
 Experiment 65. — Forensic Blood Test 
 
 1. Secure from the instructor two pieces of muslin or gauze containing respec- 
 tively a stain of human and horse blood. These are to be numbered and the source of 
 each stain known only to the instructor. Secure 1 c.c each of normal human and 
 horse serum and dilute 1 : 1000. 
 
 2. Prepare extracts of each stain as described in the text (Chapter XVII). 
 
 3. Secure 1 c.c. of clear antihuman and antihorse immune serum. 
 
 4. Set up the following in long narrow test-tubes:: 
 
 Tube 1 : 2 c.c. of extract No. 1 in dilution of 1 : 1000+0.1 c.c. of antihuman serum. 
 
 Tube 2: 2 c.c. of extract No. 1 in dilution of 1 : 1000+0.1 c.c. of antihorse serum. 
 
 Tube 3: 2 c.c. of extract No. 2 in dilution of 1: 1000+0.1 c.c. of antihorse serum. 
 
 Tube 4: 2 c.c. of extract No. 2 in dilution of 1 : 1000+0.1 c.c. of antihuman serum. 
 
 Tube 5: 2 c.c. of normal human serum in dilution 1:1000+0.1 c.c. antihuman 
 serum (control). 
 
 Tube 6: 2 c.c. of normal horse serum in dilution, of 1:1000+0.1 c.c. antihorse 
 serum (control). 
 
 Tube 7: 2 c.c. of normal salt solution+0.1 c.c. of- antihuman serum. 
 
 Tube 8: 1 c.c. of normal salt solution+0.1 c.c. of antihorse serum. 
 
 5. Do not shake tubes; place in the incubator at 37° 0. and inspect every thirty 
 minutes for two hours and after standing in the refrigerator overnight. 
 
 (a) Inspect the controls. Are the antiserums potent and satis- 
 factory? Why is it advisable to have these controls? 
 
 (b) Are you able to diagnose the source of each stain? 
 
 (c) In a medicolegal test, if these reactions were negative, how would 
 you proceed further in your efforts to establish the identity of a particu- 
 lar stain? 
 
846 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 26.— PRECIPITINS (Continaed) 
 
 Experiment 66. — Milk-precipitin (Lactoserum) 
 
 1. Secure a cubic centimeter of anticow serum previously prepared by immuniz- 
 ing rabbits with injections of fresh cow's milk. 
 
 2. Prepare a 1:50 dilution of cow's milk (precipitinogen) by mixing 0.2 c.c. milk 
 with 9.8 c.c. normal salt solution. Prepare a similar dilution of human or goat milk 
 for control tests. 
 
 3. Into a series of six small test-tubes place 2 c.c. of the following dilutions of 
 cow's milk: 1:50, 1:100, 1:200, 1:400, 1:800, and 1:1600. 
 
 4. Into a second series of three tubes place 2 c.c, of the following dilutions of 
 control milk; 1:50, 1:100, 1:200. 
 
 5. Into the nine tubes of these series add 0.1 c.c. cow lactoserum. 
 
 6. A third series of six tubes containing 2 c.c. of the above dilutions of cow's 
 milk plus 0.1 CO. of normal rabbit serum may be set ftp as further controls. 
 
 7. Keep the tubes at room temperature and inspect at the end of fifteen min- 
 utes; half an hour; one hour, and two hours. 
 
 (a) Describe a positive reaction. 
 
 (b) Discuss the specificity of lactoserums, 
 
 (c) Of what practical value are lactoserum reactions? 
 
 Experiment 67. — Bacterial Precipitins 
 
 1. Inoculate two flasks each containing 50 c.c. of sterile neutral bouillon with 
 cultures of Bacillus typhosus and Bacillus coli and cultivate at 37° C. for three weeks. 
 Filter cultures through a sterile Berkefeld filter untU clear. 
 
 2. Into a series of four small test-tubes place 2 c.c of the following dilutions of 
 the typhoid filtrate (precipitinogen) undiluted: 1:2, 1:4, and 1:10. Prepare a 
 second series of tubes with similar amounts of the same dilutions of BaciUus coli filtrate. 
 
 3. Add 0.1 c.c. of potent typhoid immune serum to all tubes of both series. A 
 serum with high agglutinin titer will be satisfactory. 
 
 4. Place 2 c.c of the undiluted tj^phoid and coli filtrates in separate tubes as 
 controls. Prepare an additional control by placing (J-l c.c. of the typhoid immune 
 serum in 2 c.c. normal salt solution. 
 
 5. Observe the tubes at the end of half an hour^ and after one, two, and six 
 hours. 
 
 (a) Describe a positive bacterial precipitin reaction. 
 
 (b) Has a precipitate formed with the Bacillus coli filtrate? With 
 what microorganism would typhoid immune serum be likely to produce 
 a precipitate? 
 
 (c) Discuss the relative delicacy of precipitation and agglutination 
 reactions in the differentiation of bacteria. 
 
 Experiment 68. — Noguchi Globulin Reaction 
 
 1. Secure 1 c.c. each of several cerebrospinal fluids»from cases of paresis, tubercu- 
 lous meningitis, serous meningitis, and normal persons. 
 
 2. Conduct this floccule-forming reaction after the teclmic given in the text. 
 
 3. Make total cell counts on each fluid (see text)., 
 
AMBOCEPTORS AND COMPLEMENTS. — HEMOLYSINS 847 
 
 (a) Explain the appearance of a positive reaction. 
 
 (b) What constitutes a negative reaction? 
 
 (c) What is the diagnostic value of this reaction in syphilis? 
 
 (d) What other value has this reaction in diagnosis? 
 
 (e) What relation does this reaction bear" to total cell counts? 
 
 (f) Explain the mechanism of the reaction. 
 
 EXERaSE 27.— AMBOCEPTORS AND COMPLEMENTS.— HEMOLYSINS 
 
 ExPEniMENT 69. — Resistance of Red Blood-corpuscles to Salt 
 Solution of Various Tonicities. Non-specific Hemolysis 
 
 1. Arrange a series of twenty-three small sterile test-tubes in a rack; to each of 
 the first twenty-one tubes add 3 c.c. of various dilutions of salt solutions ranging 
 from 0.6 per cent, to 0.2 per cent, in steps of 0.02 per cent. Large amounts of these 
 solutions should be at hand, preserved in bottles fitted with tight cork stoppers to 
 prevent evaporation, or prepared at the time according to the technic given in the 
 text (page 380). To tube No. 22 add 3 c.c. distilled- water and to No. 23 the same 
 amount of normal salt solution (0.85 per cent.). 
 
 2. Secure blood-corpuscles after the method given in the text. Dog blood may 
 be substituted for human blood. After the final washing, add 0.05 c.c. to each tube. 
 Shake gently. 
 
 3. Make a prehminary reading at once; the final reading is made after the tubes 
 have been standing in the refrigerator overnight. 
 
 4. Note the appearance of hemolyzed blood. Prepare a color scale by placing 
 1 c.c. blood-corpuscles in 30 c.c. distilled water, which represents a standard of 100 
 per cent, hemolysis. From this solution prepare dilutions with distilled water to 
 represent 80, 60, 40 and 20 per cent, hemolysis. F6r example, 4 c.c. of the stock 
 solution plus 1 c.c. distilled water equals 5 c.c. of an-80 percent, solution; 3 c.c. of 
 the stock plus 2 c.c. water equals a 60 per cent, solution; 2 c.c. of stock plus 3 c.c. of 
 water equals a 40 per cent, solution, and so on. Carefully compare the volume of 
 non-hemolyzed cells in the bottom of some of the test-tubes witli the amount of color 
 in the supernatant fluid. A Duboscq colorimeter may be used for making com- 
 parisons with the controls. 
 
 5. Determine the minimal and maximal resistanee of the corpuscle employed. 
 
 (a) AVhat is the appearance of hemolyzed blood? 
 
 (b) What is the meaning of the term hemolysis? 
 
 (c) Why is this called non-specific hemolysis? 
 
 (d) What does a normal salt solution mean? 
 
 (e) What is the importance of using a normal salt solution in hemo- 
 lytic experiments? 
 
 (f) How is hemolysis produced by hypofonic solutions? 
 
 (g) What would be the objections of using a hypertonic salt solution? 
 (h) How is an isotonic salt solution prepared? 
 
 (i) What is the meaning of the terms minimal and maximal re- 
 sistance of red blood-corpuscles? 
 
 (j) Of what practical value is this test? 
 
848 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 Experiment 70. — Serum Hemolysis in Vitro 
 
 1. Secure 2 c.c. of blood from the ear of a rabbit which has received at least two 
 intravenous injections of sheep cells. Separate the serum and divide into two 
 portions. Inactivate one portion (A) by heating in a water-bath for halt an hour at 
 66° C. 
 
 2. Place 0.1 c.c. and 0.2 c.c. of the fresh unheated serum of portion (B) in two 
 test-tubes. Likewise the same amounts of the heated serum (A) in two more tubes. 
 Add I c.c. of a I per cent, suspension of washed sheep cells to each tube and sufficient 
 salt solution to bring the total volume to 4 c.c. As a control, place I c.c. of corpuscle 
 suspension and 3 c.c. salt solution in a tube. Shake gently, and incubate for an 
 hour. 
 
 (a) Which tubes show hemolysis? 
 
 (b) What substances are concerned in serum hemolysis? 
 
 (c) Why did hemolysis occur with the unheated and not with the 
 heated serum? What is the meaning of inactivation of a serum? 
 
 (d) Why is this called serum hemolysis? 
 
 (e) What is the appearance of the control tube? Why is this con- 
 trol included? What would have happened if a hypotonic salt solution 
 had been used? 
 
 Experiment 71. — Sebum Hemolysis in Vivo 
 
 1. Immunize a rabbit with three intravenous injections of 5 c.c. each of a 10 per 
 cent, suspension of washed oat erythrocytes in sterile salt solution. Give the in- 
 jections each day; four days after the last injection the rabbit blood is titrated and 
 usually contains a fair amount of anticat hemolysin. 
 
 2. Heat 2 c.c. of the immune serum at 56° C. for thirty minutes and inject into 
 the external jugular vein of a cat. 
 
 3. Place the animal in a metabolic cage and collect the urine. 
 
 4. After two or three days, autopsy, removing the spleen, liver, and kidneys. 
 Place in 5 per cent, formalin and prepare and stain sections with hematoxylin and 
 eosin and Giemsa solution. 
 
 (a) Has blood destruction occurred? What are the evidences? 
 
 (b) Would hemolysis occur in the test-tube with a heated immune 
 serum? If not, why not? 
 
 (c) How then do you explain hemolysis imvivo with a heated serum? 
 
 (d) Examine sections of the spleen, liver,^ and kidneys. Are there 
 any evidences of phagocytosis of blood-cells, focal necrosis, and nephritis? 
 Explain the probable mechanism of the production of these changes. 
 
 EXERaSE 28.— AMBOCEPTORS AND COMPLEMENTS. HEMOLYSINS 
 
 Experiment 72. — Titration op a Hemolytic Amboceptor 
 
 1. Secure 1 or 2 c.c. of blood from the ear of a rabbit which has been immunized 
 with injections of sheep cells. Separate the serum and inactivate by heating to 56° 
 
AMBOCEPTORS AND COMPLEMENTS 849 
 
 C. for half an hour. Dilute 1: 100 by adding 0.1 c.o. serum to 9.9 c.c. normal salt 
 solution. 
 
 2. Prepare 40 c.c. of a 5 per cent, dilution of fresh guinea-pig serum to be used for 
 complement. Prepare 40 c.c. of a 2J^ per cent, suspension of washed sheep cells by 
 adding 1 c.c. of corpuscles to 39 c.c. of normal salt solution. 
 
 3. Proceed with the titration as given in the text 6n page 375. If the smallest 
 dose, viz., 0.1 c.c. of the 1 : 100 dilution, completely hemolyzes the corpuscles, it will be 
 necessary to retitrate with a dilution of 1 : 1000. 
 
 (a) Is it necessary to use exactly the same' amounts of complement 
 and corpuscles in all tubes and if so, why? 
 
 (b) If hemolysis did not occur at all in this experiment, what fac- 
 tors may be at fault? 
 
 (c) If the corpuscle control were completely hemolyzed, what de- 
 duction would you draw? 
 
 (d) What is the amboceptor unit of this serum? 
 
 Experiment 73. — Quantitative Factors in Serum Hemolysis 
 
 1. Having determined the amboceptor unit of the above antisheep immune serum, 
 proceed as follows: 
 
 2. To a series of four test-tubes add an amount of immune serum equaling one, 
 two, three, and five amboceptor units respectively. To each tube add 0.5 c.c. of the 
 1:20 dilution of complement serum. This amount is just haK the dose of comple- 
 ment used in titrating the amboceptor. Add 1 c.c. of a 2J^ per cent, suspension of 
 sheep cells to each tube and sufficient salt solution. 
 
 3. In a second series of four test-tubes place one half an amboceptor unit and the 
 following amounts of diluted complement serum: 1 c.c:, 2 c.c, 3 c.c, and 4 c.c. Add 
 
 1 c.c. of the suspension of sheep corpuscles and sufficient salt solution to make the 
 total volume in each tube about equal. Incubate for one hour and read the results. 
 
 4. In a third series of four test-tubes place one amboceptor unit, 1 c.c. of com- 
 plement serum (1:20), and the following amounts of corpuscle suspension: 1 c.c, 
 
 2 c.c, 3 c.c, and 4 c.c. Add sufficient salt solution to make the total volume in each 
 tube about equal. 
 
 5. Prepare a corpuscle control with 1 c.c of suspension and 4 c.c. normal salt 
 solution. 
 
 6. Shake all tubes gently and incubate for two hours at 37° C. 
 
 (a) Can an excess of amboceptor make up for a deficiency in comple- 
 ment? 
 
 (b) Can an excess of complement make up for a deficiency of ambo- 
 ceptor? 
 
 (c) What happens when an excess of corpuscle suspension is used? 
 
 (d) Discuss the importance of quantitative factors in serum hem- 
 olysis. 
 
 54 
 
850 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 29.— AMBOCEPTORS AND COMPLEMENTS (Continued) 
 
 Experiment 74. — Role of Amboceptor ai^d Complement in Hem- 
 olysis 
 
 1. Prepare a 2}2 per cent, suspension of washed sheep corpuscles. Bleed a 
 healthy guinea-pig under ether anesthesia, separate tKe serum, and dilute 1:20 with 
 normal salt solution (complement). Secure antisheep hemolytic serum (inactivated) 
 whose hemolytic titer is known (consult instructor). 
 
 2. Proceed to set up a series of four test-tubes as follows: 
 
 Tube 1: 1 c.c. corpuscle suspension -|-sufficient normal salt solution to make the 
 
 total volume about 4 c.c. 
 Tube 2: 1 c.c. corpuscle suspension -|-1 c.c. complement serum (l:20)-|-sufiicient 
 
 salt solution. 
 Tube 3: 1 c.c. corpuscle suspension -|- two hemolytic doses of antisheep hemolysin 
 
 -|- salt solution. 
 Tube 4: 1 c.c. corpuscle suspension -1-1 c.c. complement serum-|-two doses of 
 
 hemolysin -fsalt solution. 
 
 3. Shake each tube gently and incubate at 37° C. for one or two hours. 
 
 (a) In which tube has hemolysis occurred? Explain the results. 
 
 (b) What does inactivation of a serum mean? 
 
 (c) Could some hemolysis occur, using the complement serum and 
 corpuscle,s without immune hemol\sin? 
 
 Experiment 75. — Specificity of Amboceptors 
 
 1. Prepare a. 2}^ per cent, suspension of washed sheep corpuscles and a 1 per 
 cent, suspension of washed human corpuscles. Bleed a guinea-pig under ether, 
 separate serum, and dOute 1:20 with normal salt solution. Secure antisheep and 
 antihuman hemolytic serums (inactivated) whose hemolytic titers are known. 
 
 2. Proceed as follows : 
 
 Tube 1: 1 c.c. sheep corpuscles4-l c.c. complement (l:20)-|-two doses of anti- 
 sheep hemolysin -|-salt solution up to 4 c.c. 
 
 Tube 2: 1 c.c. sheep corpuscles -|-1 c.c. complement (1 : 20) -)-five doses of anti- 
 human hemolysin. 
 
 Tube 3: 1 c.c. human corpuscles -|- 1 c.c. complement (l:20)-l-two doses of anti- 
 human hemolysin. 
 
 Tube 4: 1 c.c. human corpuscles -|-1 c.c. complement (l:20)-|-five doses of anti- 
 sheep hemolysin. 
 
 Tube 5: 1 c.c. sheep corpuscles-(-3 c.c. salt solution (control). 
 
 Tube 6: 1 c.c. human corpuscles -f 1 c.c. salt solution (control). 
 
 3. Shake tubes gently and incubate for an hour or two. 
 
 (a) In which tubes has hemolysis occurred? 
 
 (b) What does this experiment teach as to the specificity of these 
 amboceptors? 
 
 (c) Discuss the specificity of hemolytic and bacteriolj-tic ambo- 
 ceptors. 
 
amboceptors and complements 851 
 
 Experiment 76. — General Properties of Amboceptors 
 
 1. Secure antisheep amboceptor so diluted that 1 c.e. contains 1 hemolytic unit. 
 
 2. Prepare a 2}^ per cent, suspension of sheep cofpuscles. 
 
 3. Secure fresh guinea-pig complement serum and dilute 1 : 20. 
 
 4. Place two units of amboceptor into each of six" test-tubes. Place these in a 
 water-bath and heat at 65° C. 
 
 5. Remove a tube from the water-bath after fifteen minutes, thirty minutes, 
 forty-five minutes, one hour, one and one-half hours, and two hours. After allowing 
 them to cool, add 1 c.e. corpuscle suspension and 1 c.c. complement serum (1:20). 
 
 6. Set up a tube containing two units of amboceptor (not heated) + 1 c.c. cor- 
 puscle suspension+1 c.c. complement serum (control).- Shake each tube gently and 
 incubate for an hour at 37° C. 
 
 (a) Has the amboceptor deteriorated by diposure to this degree of 
 heat? 
 
 (b) Discuss tlie stability of amboceptors to temperature^ age, germi- 
 cides, and drying. 
 
 (c) Discuss the relative stability of amboceptors and complements. 
 
 EXERCISE 30.— AMBOCEPTORS AND COMPLEMENTS.— HEMOLYSINS 
 
 (Continued) 
 
 Experiment 77. — Mechanism or Amboceptor Action 
 
 1. Secure antisheep amboceptor so diluted that 1 c.c. contains one hemolytic 
 unit. 
 
 2. Prepare a 2}^ per cent, suspension of sheep corpuscles. 
 
 3. Secure fresh guinea-pig serum and dilute 1 : 20. 
 
 4. Into two centrifuge tubes place two units of amboceptor and 1 c.c. of cor- 
 puscle suspension. Mix, and place one tube in a glass of cracked ice, leaving the 
 other at room temperature. After one hour centrifuge both. Pipet the supernatant 
 fluids into two separate test-tubes. 
 
 5. To the sedimented corpuscles in both centrifuge tubes add 1 c.c. corpuscle 
 suspension and 1 c.c. diluted complement serum. Mix well. Incubate tubes for 
 one hour at 37° C. 
 
 6. To the supernatant fluid of each tube add 1 c.c. corpuscle suspension and 1 
 c.c. of diluted complement serum. Mix. Incubate tubes for an hovi. 
 
 (a) In which tubes has hemolysis occurred? 
 
 (b) How do you explain the results? 
 
 (c) Discuss the prevailing views of amboceptor action. 
 
 Experiment 78. — A Further Study of the Mechanism op Ambo- 
 ceptors 
 
 1. In a centrifuge tube place six hemolytic doses of antisheep hemolysin and 2 c.c. 
 of fresh guinea-pig complement diluted 1 ; 20. Place in a glass of cracked ice until the 
 mixture is thoroughly chilled. Then add 2 c.c. of a 2}^ per cent, suspension of sheep 
 cells (also chilled). Mix and keep at a low temperature for an hour, centrifuge 
 thoroughly, and pipet the supernatant fluid to a separate test-tube. 
 
852 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 2. In a second centrifuge tube place 2 c.c. of diluted complement and 2 c.c. cor- 
 puscle suspension. Keep at room temperature for one hour, centrifuge, and pipet the 
 supernatant fluid into a test-tube. 
 
 3. Proceed as follows: To 2 c.c. of the supernatant fluid from the first centrifuge 
 tube add 1 c.c. corpuscle suspension; to the remaining 2 c.c, add 1 c.c. corpuscle sus- 
 pension and two units of amboceptor; to the sedimented corpuscles add 2 c.c. of 
 diluted complement serum. 
 
 4. To 2 c.c. of the supernatant fluid from the second centrifuge tube add 1 c.c. 
 corpuscle suspension; to the remaining 2 c.c. add 1 c.c.. corpuscle suspension and two 
 units of hemolytic amboceptor. 
 
 5. To the sedimented corpuscles in both centrifuge tubes add 1 c.c. complement 
 serum and 1 c.c. salt solution. Mix well. 
 
 6. Shake all tubes gently and incubate for one hour at 37° C. 
 
 (a) In which tubes has hemolysis occurred? 
 
 (b) Does complement unite directly with corpuscles? 
 
 (c) What evidence have you that amboceptors unite directly with 
 corpuscles? 
 
 (d) What is meant by the term "sensitizing corpuscles"? 
 
 (e) Is complement-amboceptor activity apparent at low temperature? 
 
 (f) What temperature best favors complement-amboceptor activity? 
 
 EXERaSE 31.— AMBOCEPTORS AND COMPLEMENTS.— HEMOLYSINS 
 
 (Continued) 
 
 Experiment 79. — Natural Hemolysins. Removal of Natural 
 Hemolysins 
 
 1. Secure I c.c. of serum from the blood of five djfferent persons and inactivate 
 in the water-bath. 
 
 2. Remove 0.5 c.c. of serum from each to five separate centrifuge tubes and add 
 4.5 c.c. of 2J^ per cent, suspension of sheep cells to each. Shake gently and after 
 half an hour at room temperature centrifuge thoroughly and pipet the supernatant 
 dilute serum to separate tubes. Do not discard the corpuscles in the centrifuge 
 tubes. 
 
 3. To each of the remaining 0.5 c.c. amounts of serum add 4.5 c.c. salt solution 
 and place 1 c.c. and 2 c.c. into two test-tubes respectively (0.1 c.c. and 0.2 c.c. undi- 
 luted serum). 
 
 4. Into two more test-tubes place 1 and 2 c.c. respectively of the diluted serum 
 which has been treated with corpuscles. 
 
 5. Add to each tube I c.c. of guinea-pig complement (1:20), 1 c.c. of 2J^ per 
 cent, suspension of sheep cells, and sufficient salt solution. Shake gently and incu- 
 bate for one hour. 
 
 6. To the sedimented corpuscles in the centrifuge tubes add 2 c.c. of diluted com- 
 plement serum and sufficient salt solution. Shake gently and incubate for one hour. 
 
 (a) Has hemolysis occurred with the untreated serums? 
 
 (b) How do you explain the results? 
 
 (c) Has hemolysis occurred with the treat^tl serums and if not, why 
 not? 
 
AMBOCEPTORS AND COMPLEMENTS 853 
 
 (d) Has hemolysis occurred with the corpuscles used in treating 
 the serums and why? 
 
 (e) Of what practical importance are natural hemolysins? 
 
 (f) Is natural antihuman hemolysin ever found in human serums? 
 What are they called? 
 
 Note. — The quantity of natural amboceptor in any of these serums 
 may be determined by the method of titration given in the text. In 
 choosing five serums at random it may be possible that some will not 
 show an appreciable amount of natural antisheep amboceptor. 
 
 EXERCISE 32.— AMBOCEPTORS AND COMPLEMENTS (Continued) 
 ExPEmMENT 80. — Hemolytic Complement 
 
 1. After giving a rabbit three intravenous injections of 5 c.c. of a 10 per cent, 
 suspension of washed sheep cells at intervals of three days, remove three cubic centi- 
 meters of blood from an ear and separate the serum. Dilute the serum 1:10 with 
 salt solution. 
 
 2. Into four test-tubes place 1 c.c. of a 23^ per cent, suspension of sheep corpuscles 
 and increasing amounts of the above dilution of rabbit serum (must be fresh — not 
 over twelve hours old) as follows: 0.4, 0.8, 1, and 2 c.c; add suiBcient salt solution. 
 Shake and incubate for one hour. 
 
 (a) Has hemolysis occurred? 
 
 (b) How do you explain the reaction? 
 
 (c) From where was the complement derived? 
 
 (d) Are complements found in the bloods of all animals? 
 
 (e) Discuss the question of the multiplicity of complements. 
 
 Experiment 81. — Inactivation and Reactivation of Complement 
 
 1. Heat 1 CO. of the 1:10 dilution of fresh rabbit immune serum used in the pre- 
 ceding experiment to 56° C. for half an hour (in a water-bath). 
 
 2. In a test-tube place a cubic centimeter of this heated serum and 1 c.c. of a 2J^ 
 per cent, suspension of sheep cells and sufficient salt solution. Shake gently and 
 incubate for one hour. 
 
 (a) Does hemolysis occur? 
 
 (b) How do you explain the result? 
 
 (c) What is meant by inactivation of complement? 
 
 (d) Is complement thermostabile? 
 
 3. Add to the tube 1 c.c. of fresh guinea-pig serum diluted 1: 10 and reinoubate 
 for an hour. 
 
 (e) Has hemolysis occurred? 
 
 (f) How do you explain the result? 
 
 (g) What is meant by reinactivation? 
 
854 experimental infection alsfd immunity 
 
 Experiment 82. — General Properties of Complement 
 
 1. Secure antisheep amboceptor the hemolytic unit of which is known by pre- 
 vious titration. 
 
 2. Prepare a 23^ per cent, suspension of washed sheep corpuscles. 
 
 3. Into a series of four test-tubes place the following: 
 Tube 1 : 1 c.c. of fresh guinea-pig serum diluted 1 : 20. 
 
 Tube 2: 1 c.c. of guinea-pig serum (1:20) which Has been kept at room tempera- 
 ture for two days. 
 
 Tube 3: 1 c.c. of guinea-pig serum (1:20) three days old. 
 
 Tube 4: I c.c. of guinea-pig serum (1:20) five days old. 
 
 Add 1 c.c. of a 2J4 per cent, suspension of sheep corpuscles, two hemolytic units 
 of antisheep amboceptor, and sufficient normal salt solution to each tube. Shake 
 gently and incubate at 37° C. for one hour. 
 
 4. Into a series of six test-tubes place 1 c.c. of fresh =guinea-pig complement serum 
 diluted 1 : 20. Place these in a water-bath at 56° C. At intervals of ten, twenty, 
 thirty, forty, fifty, and sixty minutes remove a tube, aidd I c.c. corpuscle suspension, 
 two units of amboceptor, and sufficient salt solution. Shake gently each tube and 
 incubate for one hour at 37° C. 
 
 (a) Record the results. Does hemolytic complement deteriorate 
 readily at room teniperature? 
 
 (b) What are complementoids? 
 
 (c) What practical significance has this experiment? Should a 
 complement serum be fresh when used in hemolytic work? 
 
 (d) Is complement thermolabile? How long does it take to destroy 
 a diluted complement at 56° C? 
 
 (e) In inactivating a serum why do we not use a higher temperature? 
 
 EXERCISE 33.— AMBOCEPTORS AND COMPLEMENTS (Continacd) 
 
 Experiment 83. — Titration of Hemolytic Complement 
 
 1. Prepare 20 c.c. of complement 1:20; prepare a 2|-2 per cent, suspension of 
 sheep cells. 
 
 2. To a series of 10 test-tubes add increasing doses of diluted complement serum: 
 0.1, 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, and 1.5 c.c; add 1}^ units of hemolytic ambo- 
 ceptor (determined by previous titration) ; 1 c.c. of corpuscle suspension and sufficient 
 salt solution to bring the total volume in each tube to 3»c.c. 
 
 3. Shake gently and incubate for one hour at 37° C. 
 
 (a) Record the results. What is the complement unit of this serum? 
 
 (b) What animal serum is best adapted ioj> complement in hemolytic 
 work? 
 
 (c) Is the complement content of the serums of different animals 
 constant? Does it vary in animals of the same species? In the same 
 animal at different times? 
 
AMBOCEPTORS AND COMPLEMENTS 855 
 
 ExPEEiMENT 84. — Phenomenon of CoMPLEMENfr Fixation 
 
 This experiment is introduced here to show the Bordet-Gengou 
 phenomenon of complement fixation. The exact technic of complement- 
 fixation reactions as conducted for the diagnosis of syphilis and other 
 infections will be given in subsequent exercises. 
 
 1. Use the same complement serum, amboceptor and corpuscle suspension as 
 used in the preceding experiment. The unit of complement is now known. 
 
 2. Secure 1 c.c. of an antigonococcus and a normal serum and heat at 56° C. for 
 thirty minutes. 
 
 3. Secure an emulsion of gonooocci which is now called the antigen; for the proper 
 dose to use consult the instructor. 
 
 4. Proceed as follows: 
 
 Tube 1: 0.2 c.c. antigonococcus serum + dose of antigen + 2 units of comple- 
 ment + normal salt solution. 
 
 Tube 2: 0.2 c.c. antigonococcus serum + 2 units of complement + normal salt 
 solution (serum control). 
 
 Tube 3: 0.2 c.c. normal serum + doses of antigen + 2 units of complement + 
 normal salt solution. 
 
 Tube 4: 0.2 c.c. normal serum + 2 units of complement -|- normal salt solution 
 (serum control). 
 
 Tube 5 : dose of antigen + 2 units of complement -1- normal salt solution (anti- 
 gen control). 
 
 Tube 6: 2 units of complement + normal salt solution (hemolytic control). 
 
 Tube 7: 1 c.c. corpuscle suspension -|- normal salt solution (corpuscle control). 
 
 Plug tube with cotton as it is finished. 
 
 5. Shake all tubes gently and incubate for one hour at 37° C. 
 
 6. Add 1.^2 units of amboceptor and 1 c.c. corpuscle suspension to all tubes 
 except corpuscle control. Shake gently and reincubate for one hour. 
 
 (a) Examine tubes and record your results. 
 
 (b) Why was the serum control on each serum necessary? 
 
 (c) Why was the hemolytic system control jiecessary? 
 
 (d) Why was the antigen control necessary?" 
 
 (e) What is meant by non-specific complement fixation? 
 
 (f) What is meant by specific complement- fixation? Explain the 
 phenomenon. Which tube of this series shows specific complement fixa- 
 tion and why? Is there any evidence of non-specific fixation in any of 
 the tubes? If so, what bearing would this have on the results of this 
 test? 
 
 (g) What is meant by complement deviation? 
 
 (h) Upon what does the specificity of complement fixation depend? 
 
856 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 34.— ANTIGENS FOR THE WASSERMANN REACTIONS 
 
 ExPBHiMENT 85. — Preparation of Antigens for the Wassermann 
 Reaction 
 
 1. Prepare 50 c.c. of an alcoholic extract of syphilitic liver (see page 420). 
 
 2. Prepare an extract of acetone-insoluble lipoids from 20 grams of beef heart 
 (see page 422) . 
 
 3. Prepare 50 c.c. of a cholestcrinized alcoholic extract of human heart (see 
 page 421). 
 
 (a) Are antigens for the Wassermann syphilis reaction specific? 
 
 (b) Upon what does the high specificity of the Wassermann reac- 
 tion depend? 
 
 (c) What constitutes a specific antigen for the Wassermann reac- 
 tion? 
 
 (d) Discuss the important relation of antigen to the Wassermann re- 
 action. 
 
 EXERCISE 35.— ANTIGENS (Continced) 
 Experiment 86. — Methods of Titration of "Antigens 
 
 While the above extracts are in the course -of preparation, stock ex- 
 tracts may be used for titration. After the" student has finished the 
 above three extracts, they are titrated in a similar manner. 
 
 1. Dilute 1 c.c. of an alcoholic extract of syphOitie liver in a test-tube 1:10 by 
 slowly adding 9 c.c. of normal saline solution. Dilute a cholesterinized extract (1 : 20) 
 by slowly adding 9.5 c.c. salt solution to 0.5 c.c. of extract. 
 
 2. Conduct the anticomplementary titration of each (page 428). 
 
 3. Conduct the antigenic titration of each (page 431). 
 
 (a) What is meant by the anticomplementary dose of an antigen? 
 
 (b) What is meant by the antigenic dose of antigen? 
 
 (c) Why are these titrations so important? 
 
 (d) In a complement-fixation test what 'v^rould be the result if the 
 ahtigen were used in the anticomplementary dose? What would be 
 the result if the antigen were used in less than the antigenic dose? 
 
 (e) What constitutes a satisfactory antigen for any complement-fix- 
 ation test? 
 
 (f ) In conducting a diagnostic reaction, should the antigen be used 
 in exactly one antigenic dose or double or treble this amount? Why 
 and under what conditions would this be a safe procedure? 
 
 (g) Why should the antigen be diluted slowly with salt solution? 
 (h) Why is a serum control so important m these titrations? What 
 
 other controls are used and why? 
 
WASSERMANN REACTION 857 
 
 (i) Which is more important, the anticoinplementary or antigenic 
 titration and why? 
 
 EXERaSE 36.— WASSERMANN REACTION 
 Experiment 87. — Anticomplementary Action of Serums 
 
 1. Secure 1 c.c. of five different specimens of human serums which have been 
 standing in the laboratory for one, two, four, six, an"d ten days respectively. The 
 last specimen should be intentionally infected with a culture of staphylococcus. 
 
 2. Divide each serum into two portions and heat portion "B" to 56° C. for half 
 an hour. 
 
 3. Place 0.2 c.c. of each specimen (unheated) in a-series of five test-tubes. 
 
 4. Place 0.2 c.c. of each specimen (heated) in a second series of five test-tubes. 
 
 5. To each tube add 1 c.c. of complement 1:20 and sufficient salt solution to 
 bring the total volume to 3 c.c. Shake gently and incubate for half an hour. Add 
 1}4 units of amboceptor and 1 c.c. corpuscle suspension to each tube and incubate 
 for one hour. 
 
 6. A hemolytic system control (complement, corpuscles, and amboceptor) should 
 be included; also a corpuscle control. 
 
 (a) Record your results. Are any of the serums anticomplementary? 
 How do you determine this? 
 
 (b) What is meant by the terms anticomplementary action of a serum? 
 
 (c) What significance has this phenomenon in complement-fixation 
 work? 
 
 (d) Are anticomplementary bodies thermolabile or thermostabile? 
 May both exist? Under what conditions are each most likely to be 
 present? 
 
 (e) How are thermolabile anticomplementary bodies removed or 
 their influence overcome? When a serum is^ three or more days old, 
 what is the chief object of heating it before it is used in a complement- 
 fixation test? 
 
 (f) Does complement keep for three days? 
 
 (g) Is it possible to remove the thermostabile anticomplementary 
 action of a serum? Could such a serum be used in a complement-fixa- 
 tion reaction? How is this condition of the serum detected? 
 
 (h) Under what conditions is a serum likely to become anticomple- 
 mentary in action? Is a sterile serum likely to become anticomple- 
 mentary? 
 
 EXERCISE 37.— "WASSERMANN REACTION (Continued) 
 
 Experiment 88. — Wassbkmann Reaction (First Method) 
 
 1. Secure four specimens of blood: one from a known syphilitic person; the 
 second from a known normal person, the third and fourth specimens for diagnosis. 
 Also a specimen of cerebrospinal fluid. 
 
858 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 2. Conduct a \A'assermaiin reaction with each serum after the first method, 
 using an antigen of alcohohc extract of syphiHtic liver. ' 
 
 (a) Record your results. In case the hemolytic system control was 
 not completely hemolyzed, what may be the reasons and what influence 
 would this result have on interpreting the reactions? 
 
 (b) In case the antigen control was not completely hemolyzed, what 
 influence would this result have on the reactisns? 
 
 (c) If a serum control were incompletely hemolyzed, what influence 
 would this have on the results of the test? 
 
 (d) Is this method a quantitative reaction? 
 
 (e) What is the nature of the antibody in a syphilitic serum? 
 
 (f) How are these reactions recorded? How should they be re- 
 ported to a clinician? 
 
 (g) Discuss the value of the Wassermann reaction in the various 
 stages of syphilis as a guide to treatment. 
 
 EXERCISE 38.— WASSERMANN REACTION (Continued) 
 
 Experiment 89. — Wassermann Reaction (Second Method) 
 
 1. Conduct a Wassermann reaction with each of- five specimens of serum after 
 the second method, using an alcohohc extract of syphilitic liver, an extract of acetone- 
 insoluble lipoids, and an alcoholic extract of heart reenforced with cholesterin. One 
 of these serums should be from a syphilitic person (positive control) and one from a 
 normal person (negative control). 
 
 (a) What are the advantages of using more than one antigen? 
 
 (b) Discuss the relative value of these three antigens. 
 
 (c) Under what conditions may a cholesterinized extract be used? 
 
 EXERCISE 39.— WASSERMANN REACTION (Continoed) 
 
 Experiment 90. — Wassermann Reaction (Third Method) 
 
 Conduct a Wassermann reaction with each of four specimens of serum after 
 the third method, using an extract of acetone-insoluble lipoids. One of these serums 
 should be a positive control and one a normal or negative control. 
 
 What is the advantage of using a quantitative test? 
 
 EXERCISE 40.— WASSERMANN REACTION (Continued) 
 
 Experiment 91.- Wassermann Reaction (Fourth Method) 
 
 Conduct a Wassermann reaction with a knowri positive, a known negative, 
 and two unknown serums after the fourth method, using an alcoholic extract of 
 syphilitic liver. 
 
 What are the main advantages of this technic? 
 
WASSBRMANN AND NOGUCHI REACTIONS 859 
 
 EXERCISE 4 J. —NOGUCHI MODIFICATION OF THE WASSERMANN RE- 
 ACTION 
 
 Experiment 92. — Titration of Antihuman Hemolysin 
 
 1. Secure 0.1 c.c. of inactivated antihuman rabbit amboceptor serum and dilute 
 1:100 by adding 9.9 c.c. normal saline solution. 
 
 2. To a series of six small test-tubes add increasing amounts of diluted ambo- 
 ceptor as follows; 0.1, 0.2, 0.4, 0.6, 0.8, and 1 c.c, add 0.1 c.c. of 40 per cent, com- 
 plement, 1 c.c. of 1 per cent, human corpuscle suspension, and sufBciont saline solu- 
 tion to bring the total volume to 2 c.c. 
 
 3. Incubate for one to two hours, shaking the- tubes once or twice during this 
 time. 
 
 (a) Are there any evidences of hemagglutination? 
 
 (b) What constitutes the unit of hemolytic amboceptor? 
 
 (c) Is an antihuman hemolj^tic system more delicate than an anti- 
 sheep system in complement-fixation work? 
 
 (d) What are the advantages of using ah antihuman hemolj'tic sys- 
 tem? 
 
 Experiment 93. — Technic of the Noguchi Modification 
 
 1. Secure five specimens of blood not over twenty-four hours old, including at 
 least one positive and one negative serum. 
 
 2. Conduct the Noguchi reaction with each serum in the unheated or active 
 state, using an anligen of acetone-insoluble lipoids. (See page 450.) 
 
 - 3. Conduct the reactions with the same antigen, using the serums after heating 
 to 56° C. for half an hour. 
 
 (a) Record your results. Are they equal in both series? 
 
 (b) What is meant by proteotropic reaction? 
 
 (c) Wliat effect has heat upon syphilis reagin? 
 
 (d) Does an active serum react more delicately than an inactivated 
 
 one 
 
 ? 
 
 (e) In performing this test with unheated serum what precautions 
 are to be observed? 
 
 EXERCISE 42.— WASSERMANN AND NOGUCHI REACTIONS 
 Experiment 94. — Comparison of Methods 
 
 1. Secure five serums and a cerebrospinal fluid. 
 
 2. Conduct a Wassermann reaction with each after the second method. 
 
 3. Conduct a Noguchi test with each serum injictivated and using an extract of 
 acetone-insoluble lipoids. 
 
 (a) How do the results compare? 
 
 (b) Which reactions are more easily read? 
 
 (c) Discuss the relative delicacy and value of the Wassermann and 
 Noguchi reactions with both active and inactivated serum in the latter. 
 
860 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 43.— GONOCOCCUS COMPLEMENT-FIXATION REACTION 
 
 ExPEKiMENT 95. — Titration of Gonococcus Antigen 
 
 1. Secure I c.c. of gonococcus antigen and dilute 1:10 by adding 9 c.c. normal 
 saline solution. 
 
 2. Secure I c.c. of an antigonococous serum or the serum of a person who reacts 
 positively and inactivate. 
 
 3. Conduct an anticomplementary and antigenic -t'itration as described on page 
 479. 
 
 4. A similar titration may be conducted with glanders antigen and antisermn. 
 
 (a) Give three methods of preparing a bacterial antigen. 
 
 (b) Is it advisable to make poljrvalent antigens and whj'-? 
 
 (c) Which is of more practical value, the anticompleraentary or anti- 
 genic titration and why? 
 
 (d) What part of the anticomplementary dose of an antigen may be 
 safely used in a diagnostic test? 
 
 (e) Is it advisable to titrate a bacterial antjgen at frequent intervals? 
 
 EXERCISE 44.— GONOCOCCUS COMPLEMENT-FIXATION REACTION 
 ExPEKiMENT 96. — Technic of the Gonococcus Reaction 
 
 1. Secure six specimens of serum from a genito-urinary clinic, particularly of 
 men suffering with chronic gonococcus infections. Also a known positive and a 
 known normal serum for controls. 
 
 2. Conduct the reactions after the method described on page 478. 
 
 (a) Is the gonococcus reaction specific? 
 
 (b) Is complement fixation in bacterial inffections likely to be as well 
 marked as in syphilis? 
 
 (c) In what cases of gonococcus infection is the reaction likely to be 
 negative? Likely to be positive? 
 
 (d) How soon after infection is the reaction likely to become posi- 
 tive? 
 
 (e) Discuss the practical value of the gonococcus complement-fixation 
 test. 
 
 EXERCISE 45.— GONOCOCCUS COMPLEMENT-FIXATION TEST 
 
 Experiment 97. — Technic of the Gonococcus Reaction 
 
 I. Using a similar set of serums as in the preceding experiment, conduct the 
 reactions with the one-tenth technic as described on page 481. 
 
 (a) What is the main advantage of this technic? 
 
 (b) By which method are the reactions more easily read and re- 
 corded? 
 
VENOM HEMOLYSIS 861 
 
 EXERCISE 46.— COMPLEMENT FIXATION IN THE DIFFERENTIATION OF 
 
 PROTEINS 
 
 ExPEKiMENT 98. — Titration of Immune Serums 
 
 1. Secure 1 o.c. each of antihuman and antihorse serum. Inactivate both. 
 
 2. Secure 0.1 c.c. each of fresh human and horse;Serum and dilute 1: 1000. 
 
 3. Conduct an antigenic titration of each immune serum aa described on page 495. 
 
 (a) Explain the mechanism of this reaction. 
 
 (b) Are protein amboceptors specific? 
 
 (c) Discuss the question of inhibition of hemolysis due to a precipitin 
 reaction. 
 
 EXERCISE 47.— COMPLEMENT FIXATION IN THE DIFFERENTIATION OF 
 
 PROTEINS 
 
 Experiment 99. — Technic of the Forensic Blood Test 
 
 1. Secure from the instructor two pieces of gauze stained respectively with human 
 and horse blood. These are to be numbered and their identity known only to in- 
 structor. 
 
 2. Secure 1 o.c. each of antihuman and antihorse serum. 
 
 3. Secure 0.1 c.c. of known human and horse serum for controls. 
 
 4. Proceed with the test for identifying these bloods as described on page 494. 
 
 (a) Discuss the specificity of this test. 
 
 (b) Is this test more delicate than the precipitin reaction? 
 
 (c) Which is more liable to error in technic? 
 
 (d) Discuss the applicability of the cornplement-fixation technic to 
 the differentiation of proteins in general, as animal, vegetable, and bac- 
 terial proteins. 
 
 EXERCISE 48.— VENOM HEMOLYSIS 
 Experiment 100. — Venom Hemolysis in Syphilis 
 
 1. Secure 1 c.c. of venom solution 1: 1000. 
 
 2. Secure four specimens of blood (for technic see page 386) from cases of late 
 secondary or tertiary syphilis and one specimen of normal blood. Also a known 
 normal blood for a control. 
 
 3. Prepare the subdilutions of venom and conduct the tests after the technic 
 described on page 387. 
 
 (a) Discuss the prevailing views regarding the mechanism of venom 
 hemolysis. 
 
 (b) Discuss the value of the venom test in the diagnosis of syphilis. 
 
 (c) Discuss the main points in the technic. 
 
862 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 EXERCISE 49.— BACTERICa-YSIS 
 Experiment 101. — Pfeiffer Bacteriolytic Test 
 
 1. Secure 1 ex. of serum from a rabbit which has. been immunized with typhoid 
 bacilh. By working with cholera and a highly potent serum better results are secured, 
 but as there is probably more danger connected with the handlmg of cholera cultures, 
 typhoid may be substituted with fairly good results. Inactivate by heating to 56° 
 C. for half an hour. 
 
 2. Prepare a twenty-four-hour-old agar culture oj a suitable strain of Bacillus 
 typhosus. 
 
 3. Prepare a 1 : 100 dilution of the immune serum -and place 3 c.c. in a small test- 
 tube. Add three loopfuls of culture and emulsify thoroughly. Inject a guinea-pig 
 intraperitoneally with 2 c.c. of the emulsion. 
 
 4. At intervals of ten, twenty, forty, and sixty minutes withdraw small amounts 
 of exudate and study bacteriolysis; prepare hanging-drop preparations which may be 
 compared with a similar control on the culture; prepare smears of the culture and 
 peritoneal exudate and stain with carbol-thionin or carbolfuchsin. 
 
 5. If desirable, the bacteriolytic titer of the serum- may be determined after the 
 method given in the text. 
 
 (a) Describe the phenomenon of bacteriolysis. 
 
 (b) Are bacteriolysis and hemolysis similar processes? Give the 
 source of complement in this reaction. 
 
 (c) Discuss the specificity of bacteriolytic reactions. 
 
 (d) Discuss the practical value of the bacteriolytic test in the diag- 
 nosis of a microorganism. 
 
 EXERCISE 50.— BACTERIOLYSIS 
 
 Experiment 102. — Microscopic Method of Measuring the Bac- 
 teriolytic Power of the Blood 
 
 This method may be employed for a rapid estimation of the bac- 
 teriolysin produced in the blood in response to an inoculation of typhoid 
 
 1. Secvire a small quantity of the patient's serum by collecting blood aseptically 
 in a Wright capsule. About 1 c.c. of serum will be sufficient. Secure a control 
 serum, preferably a "pooled serum," in the same mariner. Prepare dilutions of the 
 patient's serum in the following manner; Place a series of six small test-tubes (sterile) 
 in a rack; add I c.c. sterile broth to each tube; into the first tube place 1 c.c. of the 
 fresh serum from the patient (1:2 dilution) ; mix well and transfer 1 c.c. to the second 
 tube; mix and transfer 1 c.c. to the third, and so on to the last tube, when 1 c.c. is 
 discarded. 
 
 2. Secure a twenty-four-hour culture of typhoid bacilli in neutral bouillon. 
 
 3. Take a simple capillary pipet with a long stem, plugged with cotton and steril- 
 ized, and make a mark about 3 cm. from the end. Draw up the highest dilution of 
 serum to the pencil mark, then a bubble of air, and finally an equal volume of culture. 
 Thoroughly mix by carefully aspirating and driving out the contents on a hollow 
 
BACTERIOLYSIS CYTOTOXINS 863 
 
 ground slide or in a small tubule. Aspirate the mixture into the middle portion of 
 the stem and seal the pipet in a flame. Label with the final dilution (1: 128). 
 
 4. Proceed in the same manner with the remaining five dilutions of the patient's 
 serum, which then, mixed with an equal quantity of culture, equals 1 : 64, 1 : 32, 1 : 16, 
 1 : 8, and 1 : 4. Place these pipets in a large test-tube and label with the patient's 
 name and time when placed in the incubator. 
 
 5. Prepare a similar set of pipets using the control serum. 
 
 6. Prepare a culture control by mixing equal volumes of culttu'e and sterile broth. 
 
 7. Place all pipets in the incubator at 37° C. for two hours. 
 
 8. Prepare hanging-drop preparations of each pipet by breaking off the tip and 
 placing a drop of the contents (after mixing) on a cover slide and suspending in the 
 usual manner. First examine the culture control and then each of the various dilu- 
 tions, noting in each case the dilution in which there is the first bacteriolytic effect, 
 and the dilution in which there is a complete effect ; arrive at the bacieriolylic index 
 by comparing the patient's and the control blood just as the opsonic index is stated. 
 
 (a) What are the first evidences of bacteriolysis? 
 
 (b) Are endotoxins neutrahzed when bacteriolysis occurs? 
 
 EXERCISE 51.— BACTERIOLYSIS 
 
 Experiment 103. — Method of Measuring the Bactericidal Ac- 
 tivity OF the Blood in Vitro (Method of Stern and Korte) 
 
 With a culture of typhoid bacilli, rabbit typhoid immune serum, and rabbit 
 complement serum, a test may be carried out after the method given in the text. 
 
 (a) What main precautions are to be followed in this method? 
 
 (b) Discuss complement deviation. 
 
 (c) Discuss the practical value of this method. 
 
 EXERCISE 52.— CYTOTOXINS 
 Experiment 104. — Action of Nephrotoxin 
 
 1. Prepare an emulsion of dog kidney as described on page 73 and immunize 
 a rabbit. 
 
 2. Inject a dog intravenously with 2 c.c. of rabbit immune serum per kilo of body 
 weight. 
 
 3. Place the animal in a metabolism cage, collect all urine, and carefully examine 
 for albumin, casts, and hemoglobin; make quantitative albuiuiii determinations, 
 
 4. After three days autopsy and examine the kidneys and liver histologically. 
 
 5. Heat the immune serum to 56° C. for thirty minutes and place increasing 
 amounts in a series of test-tubes as follows: 0.01, 0.0.5, 0.08, 0.1, and 0.2 c.c; add 
 1 c.c. of fresh guinea-pig complement serum 1 : 20, and 1 c.c. of 23^2 per cent, suspension 
 of washed dog erythrocytes. Incubate for two hours. 
 
 (a) Describe the changes occurring in the liver and kidneys. Give 
 the prevailing views regarding the mechanism of their production. 
 
 (b) Are there any evidences of a hemolytic action of the serum in 
 vivo? In vitrof 
 
864 EXPERIMENTAL INFECTION AlflS IMMUNITY 
 
 (c) How do you explain the presence o*^ hemolysin in this immune 
 serum? 
 
 (d) Discuss the specificity of cytotoxins in general. 
 
 (e) How may a hemolysin be removed from a nephrocytotoxic 
 serum? 
 
 (f) Have the cytotoxins any practical value in the treatment of dis- 
 ease? 
 
 EXERCISE 53.— MIOSTAGMIN REACTION 
 Experiment 105. — Technic of the Miostagmin Reaction 
 
 1. Secure a portion of dry pulverized cancer tissue and prepare an antigen after 
 the technic described in the text. 
 
 2. Titrate this antigen. 
 
 3. Secure four specimens of serum: one from a known case of cancer; one from 
 a normal person, and two for diagnosis. 
 
 4. Proceed with the reaction as described in the text. 
 
 (a) Discuss the principles of this reaction.. 
 
 (b) Discuss the practical value of this reaction in the diagnosis of 
 cancer. 
 
 EXERCISE 54.— ANAPHYLAXIS 
 For experiments in anaphylaxis sensitize a series of animals as follows: 
 
 (a) Give seven young guinea-pigs an intraperitoneal injection of 0.01 
 c.c. horse serum (1 c.c. of a 1 : 100 dilution). 
 
 (b) Give three young guinea-pigs an intraperitoneal injection of 0.01 
 c.c. of human serum. 
 
 (c) Give two young guinea-pigs an intraperitoneal injection of 0.1 
 ■c.c. egg-albumen (1 c.c. of 1 : 10 dilution). 
 
 (d) Give two rabbits 1 c.c. of horse serum intravenously every three 
 days for .three doses. 
 
 (e) Give a dog 10 c.c. of horse serum subcutaneously. 
 
 Experiment 106. — Anaphylaxis in the Guinea-pig. Specificity 
 OF Anaphylaxis 
 1. Two weeks after the sensitizing injections proceed as follows: 
 
 (a) Inject a guinea-pig sensitized to horse serum with.l c.c. horse serum intra- 
 peritoneally. 
 
 (b) Inject a guinea-pig sensitized to human serum with 0.1 c.c. of human serum 
 intravenously. 
 
 (c) Inject a guinea-pig sensitized to egg-albumeii with 1 c.c. of diluted (1: 4) 
 albumin intraperitoneally. 
 
ANAPHYLAXIS 865 
 
 (d) Inject a guinea-pig sensitized to horse serum with 1 c.o. of diluted (1:4) egg- 
 albumen. 
 
 (e) Inject a guinea-pig sensitized to human serum with 0.1 c.c. horse serum 
 intravenously. 
 
 (f) Inject a guinea-pig sensitized to horse serum with 1 c.c. human serum intra- 
 peritoneally. 
 
 2, Carefully study the symptoms of anaphylaxis. After death autopsy the 
 animals. 
 
 (a) Describe acute anaphylactic shock in the guinea-pig. Does 
 death always occur? 
 
 (b) How soon after the intraperitoneal injection of the intoxicating 
 dose do symptoms develop? After the intravenous? Why the differ- 
 ence? 
 
 (c) Is anaphylaxis a specific reaction? 
 
 (d) What are anaphylactogens? 
 
 (e) Discuss the prevailing views regarding the mechanism of the 
 anaphylactic reaction. 
 
 (f) Describe the anatomic changes occurring in acute and fatal 
 anaphylaxis in the guinea-pig. Discuss the cause and nature of these 
 changes. 
 
 EXERCISE 55.— ANAPHYLAXIS (Continued) 
 
 Experiment 107. — Nature of the Anaphylatoxin 
 
 1. Place 15 grams of coli bacterial substance prepared as described on page 126 
 in a distilling flask and add 2-50 c.c. of a 2 per cent, caustic soda in absolute ethyl 
 alcohol. Place on a water-bath attached to a reflux condenser and collect the dis- 
 tillate. 
 
 2. Evaporate the distillate to dryness and for each 10 c.c. of the distillate add 
 
 5 c.c. of water. Neutralize with ^ hydrochloric acid. 
 
 3. Inject 5 c.c. intraperitoneally into young guinei-pigs. Inject 1 or 2 c.c. intra- 
 venously. 
 
 (a) Do anaphylactic symptoms develop? 
 
 (b) Are the anatomic changes in the lungsssimilar to those observed 
 in serum anaphylaxis? 
 
 (c) Discuss the prevailing views of the nature of anaphylatoxuis. 
 
 (d) Discuss the prevailing views of the anaphylaxis antibody, toxo- 
 gen, or anaphylactin. 
 
 EXERCISE 56.— ANAPHYLAXIS (Continued) 
 Experiment 108. — Anaphylaxis in the Dog 
 
 1. Five weeks after sensitization anesthetize a dog with ether and connect the 
 carotid or femoral artery with a mercurial manometer and arrange for a kymographic 
 tracing. 
 
 55 
 
866 EXPERIMENTAL INFECTION AND IMMUNITY 
 
 2. After recording the normal blood-pressure tracing, inject 5 c.c. of homologous 
 serum (horse) into a jugular vein. 
 
 3. Record the blood-pressure. 
 
 4. Study the coagulation time of the blood. 
 
 5. Autopsy. 
 
 (a) Describe the blood-pressure changes. To what may they be 
 ascribed? 
 
 (b) Is blood coagulation delayed? Give a reason for this change. 
 
 (c) Do anatomic changes occur in anaphylaxis of the dog? 
 
 EXERCISE 57.— ANAPHYLAXIS (Continoed) 
 Experiment 109. — Passive Anaphylaxis 
 
 1. Secure 1 c.c. of serum from a rabbit sensitized to horse serum five weeks previ- 
 ously and inject 0.5 c.c. intraperitoneally into each of two guinea-pigs. 
 
 2. Twenty-four and forty-eight hours later inject both animals intravenously 
 with 0.2 c.c. of horse serum. 
 
 (a) Are anaphylactic symptoms in evidence? 
 
 (b) Discuss the nature of passive anaphylaxis. 
 
 (c) If allowed to live, would these animals become sensitized to rab- 
 bit serum? 
 
 EXERCISE 58.— ANAPHYLAXIS (Continued) 
 Experiment 110. — Anti -anaphylaxis 
 
 1. Eight days after sensitizing a guinea-pig with horse serum, inject 1 c.c. of 
 hprse serum subcutaneously. 
 
 2. Fifteen days after sensitizing a guinea-pig with horse serum, inject 2 c.c. of 
 serum into the rcctmn. 
 
 .3. Fifteen days after sensitizing a guinea-pig with.horse serum, inject 0.0001 c.c. 
 serum subcutaneously every fifteen minutes for six dcfees. 
 
 4. On the sixteenth day after sensitizing, inject a guinea-pig with 1 c.c. of horse 
 serum. If this animal develops anaphylactic shock, inject the other three guinea- 
 pigs in a similar manner. 
 
 (a) Do anaphylactic symptoms develop? If not, why not? 
 
 (b) Discuss the importance of quantitative factors in producing 
 anti-anaphylaxis. 
 
 (c) Discuss the importance of anaphylaxis and anti-anaphylaxis 
 in serum therapy. 
 
 (d) What method is generally employed in the effort to induce an 
 anti-anaphylactic state in serum therapy? 
 
 (e) What drug has been found experimentally of value in ameliorat- 
 ing or preventing the pulmonary changes of anaphylaxis? 
 
ANAPHYLAXIS CHEMOTHERAPY 867 
 
 (f) Do anesthetics prevent anaphylaxis? 
 
 (g) Under what conditions would you expect anaphylaxis in persons? 
 
 EXERCISE 59.— ANAPHYLAXIS (Continued) 
 Experiment 111. — Local Anaphylactic Reactions 
 
 1. Shave the abdomen of the rabbit sensitized to horse serum five weeks after 
 the time of sensitization. 
 
 2. Make two superficial abrasions over the upper portion of the abdomen; into 
 one rub horse serum, and in the other, normal salt= solution. 
 
 (a) Does a local reaction occur? If so, describe the lesion. 
 
 3. Then inject 0.01 c.c. of serum subcutaneously. If acute anaphylactic death 
 does not follow, observe the animal during the next twenty-four hours. 
 
 (a) Does a local Arthus reaction follow this injection of serum? If 
 so, describe the reaction. 
 
 (b) Explain the mechanism of a local anaphylactic reaction. 
 
 (c) Are the tuberculin, luetin, and mallein reactions anaphylactic? 
 Explain their mechanism and discuss their practical value in diagnosis. 
 
 4. Inject 1 c.c. horse serum intravenously. If anaphylaxis occurs, record the 
 symptoms. Autopsy with particular attention to the heart. 
 
 EXERCISE 60.— CHEMOTHERAPY 
 Experiment 112. — Salvaesan 
 
 1. Secure a white rat infected with Sp. recurrens (Sp. Obermayer) and one with T. 
 equiperdum. Also two normal rats. 
 
 2. Examine a drop of blood from the tail of each infected rat and form a rough 
 estimate of the number of parasites per microscopic field. 
 
 3. Inject both rats intravenously with 0.001 gm. salvarsan freshly prepared. 
 
 4. Examine the blood at the end of two hours; after forty-eight, seventy-two, 
 and ninety-six hours. 
 
 5. Inject a control rat intravenously with 0.01 gm. salvarsan and the second with 
 0.05 gm. 
 
 (a) Do the control rats survive? 
 
 (b) Are the infected rats sterilized? 
 
 (c) What is meant by the dosis lethaKs? By the dosis curativa 
 By the dosis toleraiaf 
 
 (d) Discuss organotropism and parasitotropism. 
 
 (e) Discuss "drug fastness." 
 
 (f) Discuss the question of chemoreceptors. 
 
INDEX 
 
 Abdebhalden's serodiagnosis of preg- 
 nancy, 252 
 dialyzation method, 252, 253 
 blood-serum, 258 
 glassware, 253 
 mnhydi'in, 254 
 precautions, 254 
 preparation of placental tis- 
 sue, 257 
 reading reaction, 259 
 reagents, 254 
 sources of error, 260 
 testing dialyzing shell, 253 
 shell for non-permeabihty 
 to albumin, 255 
 for permeability to pep- 
 tone, 256 
 ejqjerimental work, 839 
 optical method, 252, 261 
 placental peptone, 261 
 polariscope, 262 
 reading reaction, 262 
 testing peptone, 262 
 practical value, 263 
 principles, 252 
 Abortion, contagious, complement-fixa- 
 tion test in, 486 
 Abrin, 222 
 
 toxin, 118 
 Absorption agglutination reaction, ex- 
 perimental work, 843 
 in mixed infection, 288 
 by colloids, 515 
 
 methods for differentiating between 
 mixed and single infection, 272 
 Abwehrfermcnte, 249 
 Acanthosis nigricans, salvarsan in, 811 
 Acetone-insoluble hpoids for Wasser- 
 
 mann reaction, 422 
 Acid-fast bacilli, fixing and staining, 197 
 Acne, vaccine therapy, 657 
 Actionocongestin, 533 
 Adenitis, tuberculous, tubercuhn treat- 
 ment, 679 
 Adulteration, meat, detection of, bio- 
 logic blood test for, 310 
 technio, 312 
 Agglutinating power of serum, variation, 
 274 
 serums, 68 
 Agglutination, 266 
 
 group, experimental work, 842 
 mechanism of, 270 
 
 AgglutiOation, mechanism of, Bordet's 
 theory, 271 
 Grubcr theory, 270 
 Paltauf's theory, 270 
 test, 278 
 
 absorption, experimental work, 843 
 
 in mixed infection, 288 
 in cerebrospinal meningitis, 277 
 in cholera, 277 
 in diagnosis of disease, 275 
 in dysentery, 276 
 in glanders, 277 
 in identification of microorganism, 
 
 277 
 in Malta fever, 277 
 in measuring immunizing response, 
 
 278 
 in plague, 277 
 in pneumonia, 277 
 in single or mixed infection, 278 
 in tuberculosis, 277 
 in typhoid fever, 275, 279, 282 
 macroscopic, 279 
 
 experimental work, 841, 842 
 Kolle and Pfeiffer's method, 288 
 tBchnic, 284 
 microscopic, 279 
 
 dry method, technic, 283 
 experimental work, 840 
 wet method, technic, 282 
 practical apphcations, 275 
 precautions, 281 
 requisites for, 279 
 Agglutinins, 152, 266 
 
 absorption methods for differentiating 
 between a mixed and single infec- 
 tion, 272 
 action of, based on colloidal reactions, 
 
 517 
 defini1;ion, 266 
 experimental work, 815, 840 
 formation, 268 
 group, 271 
 history, 266 
 immune, 268 
 in immunity, role of, 274 
 nature, 270 
 normal, 268 
 origin,. 269 
 partial, 271 
 
 preservation of, in dried paper form, 79 
 production of, 68 
 
 intraperitoneal method, 69 
 
870 
 
 INDEX 
 
 Agglutinins, [production of, intravenous 
 method, 68 
 properties, 270 
 speoifioity, 271 
 Agglutinogen, 269 
 
 experimental work, 843 
 Agglutinoids, 268 
 Agglutinoscope, 288 
 Aggressins, 108, 122, 182 
 anti-, 126 
 artificial, 103, 125 
 in relation to infection, 103 
 natural, 125 
 
 experimental work, 824 
 nature of, 124 
 Albuminolysin, 553 
 
 Alcoholic extracts of normal organs 
 for Wassermann reaction, 
 420 
 reenforced with cholesterin for 
 Wassermann reaction, 421 
 of syphilitic livers for Wassermann 
 reaction, 420 
 Aleppo boil, salvarsan in, 811 
 Aleuronat, preparation of, 339 
 Alexin. See Com-plement. 
 Allergen, 536, 543 
 Allergic reactions, 582. See also Ana- 
 
 . phylactic reaction. 
 Allergin, 552 
 AUergy, 534, 536 
 Amboceptoids, 321 
 
 Amboceptor, 154, 157, 185, 316, 319, 326, 
 337, 338 
 and complement, quantitative rela- 
 tionship, 374 
 anti-, 325 
 . bacteriolytic, 155, 319 
 
 titration of, 325 
 formation, 322 
 hemolytic, 319, 362 
 
 and complement, quantitali\'e re- 
 lationship, 374 
 for Noguchi's modification of Was- 
 sermann reaction, 451 
 for Wassermann reaction, 415 
 preservation of, 79 
 titration of, 325 
 
 ex-perimeutal work, 848 
 history, 319 
 mechanism of action, 321 
 
 experimental work, 851 
 native, 324 
 natural, 324 
 properties, 321 
 
 experimental work, 851 
 quantitative estimation, 325 
 role of, in hemolysis, experimental 
 
 work, 850 
 specificity, 323 
 
 experimental work, 850 
 structure, 320 
 unit of serum, 374 
 Amebadiastase, 183 
 Amorphous colloids, 512 
 
 Ampules, vaccine, conversion of test- 
 tub^ into, 24 
 
 makiijg of, 24 
 Anaphylactic reactions, 582 
 
 as m^sure of immunity, 610 
 
 experimental work, 867 
 
 in diphtheria, 609 
 
 in gonococcus infections, 609 
 
 in leprosy, 610 
 
 in pregnancy, 610 
 
 in spQrotrichosis, 610 
 
 in typhoid fever, 607 
 ■prognostic value, 610 
 AnaphylaCtin, 552, 557, 563 
 nature, 553 
 terminology, 552 
 Anaphylactogens, 536, 542, 543 
 bacterial, 547 
 endotoxins as, 548 
 non-protein, 543 
 physical state, 546 
 protein,, chemistry of, 544 
 Anaphylac£otoxins, 542 
 Anaphylatoxin, 536, 548 
 Anaphylaxis, 531 
 
 accelerated, in infectious diseases, 568 
 
 antibody, 552, 563 
 
 Arthus piienomenon, 533 
 
 Besredka's theory, 557 
 
 cellular *t*heory, 555 
 
 chronic, 540 
 
 definition, 535 
 
 experimental work, 864 
 
 fall in blOod-pressure in, 541, 542 
 
 Friedberger's theory, 558 
 
 Gay and Southard's theory, 557 
 
 Hamburger and Moro's theory, 556 
 
 heterologous, 559 
 
 history, 532 
 
 homologous, 559 
 
 immediate, in infectious diseases, 568 
 
 in cats, 539 
 
 in cold-blooded animals, 541 
 
 in cows, 541 
 
 in dogs,»540 
 
 experimental work, 865 
 in guinea-pig, 537 
 
 experimental work, 864 
 in hens,; 541 
 in horse, 541 
 in man, S37 
 in pigedns, 541 
 in rabbits, 539 
 in relation to immunity, 565 
 
 to infection, 565 
 in serum treatment, 686 
 in sheep, 541 
 in white mice, 540 
 
 rats, 540 
 indirect, 543 
 hpoid, 544 
 mechanism, 541, 561 
 nature of, experimental work, 865 
 Nolf's theory, 558 
 passive, 548, 559 
 
INDEX 
 
 871 
 
 Anaphylaxis, passive, production of, 560, 
 866 
 pliysical ttieory, 558 
 relation of, to infectious diseases, 566 
 
 to non-infectious diseases, 570 
 Richet's studies on, 533 
 
 theory, 556 
 Smith's phenomenon, 535 
 specificity, 563 
 terminology, 536 
 theories, 556 
 
 torpid early, in infectious diseases, 568 
 Vaughan and Wheeler's theory, 557 
 von Pirquet's studies on, 533 
 Anemia, salvarsan in, 811 
 Anesthesia for subdural inoculation of 
 serum, 697 
 in lumbar puncture, 39 
 Wassermann reaction after, 466 
 Angina, Vincent's, salvarsan in, 810 
 Animal blood. See Blood, Animal. 
 cold-blooded, anaphylaxis in, 541 
 conjunctival tuberculin test in, 600 
 cutaneous tubercuUn test in, 601 
 experiments, 815 
 
 immunization, active, general technic, 
 66 
 methods for effecting, 65 
 inoculation, 53 
 intracardial, 62 
 
 of guinea-pigs, 62 
 intramuscular, 56 
 intraperitoneal, 64 
 of guinea-pig, 64 
 of rabbit, 64 
 intravenous, 56 
 of dog, 62 
 of goats, 62 
 of guinea-pig, 57 
 of horse, 61 
 of mice, 60 
 of rabbit, 56 
 of rats, 60 
 of sheep, 62 
 subcutaneous, 54 
 
 with fluid inoculum, 54 
 with solid inoculum, 55 
 technic, 53 
 
 general rules, 53 
 intracutaneous tubercuUn test in, 601 
 parasites, aggressiveness, 133 
 infection with, 132 
 
 mode, 132 
 production of disease by, 133 
 preparation of, for vaccination, 627 
 protective immunization, 653 
 subcutaneous tubercuhn test in, 600 
 tubcrcuhn reaction in, 600 
 vaccination of, 627 
 preparation for, 627 
 Anopheles maculipennis, 166 
 
 punctipennis, 166 
 Antenatal infection, 90 
 Tinthrax, internal, salvarsan in, 768 
 serum treatment, 767, 768 
 
 Anthrax, vaccination, 653 
 Antiaggressins, 126 
 Antiamboceptors, 325 
 Antianaphylaxis, 561, 687 
 
 experimental production, 562, 866 
 mechanism, 563 
 Anti-anthrax serum, 767 
 administration, 767 
 Antibacterial immunity, 684 
 acquired, 171 
 experimental work, 826 
 immunization, 684, 735 
 Antibody, 65, 66, 148, 149, 161, 317 
 anaphylaxis, 552, 563 
 and antiferments, similarity between, 
 
 247 
 of first order, 149 
 of second order, 152 
 of third order, 154 
 specificity of, 162 
 Anticholera serum, 770 
 
 Kraus', 770 
 Anticomplementary action of serums, 
 experimental work, 857 
 titration of antigens in Wassermann 
 reaction, 428 
 Anticomplements, 327 
 
 auto-, 327 
 Anticytbtoxic serums, 506 
 Antidiphtheric serum, production of, 227 
 Antidysenteric serum, collecting and 
 tesling, 238 
 production of, 236 
 cultvu-e, 236 
 
 immunizing animals, 237 
 Antiferments, 246 
 
 and antibodies, similarity between, 
 
 247 
 experimental work, 838 
 in disease, 247 
 simple, 149 
 Antigen,= 65, 66, 67, 148, 159 
 
 bacterial, identification of, comple- 
 ment-fixation test for, 498 
 preparation of, in complement fixa- 
 tion, 473 
 principles of complement fixation 
 
 with, 476 
 standardizing, in complement fixa- 
 tion, 475 
 determination of, by complement 
 
 fixation, 494 
 for gonococcus complement-fixation 
 
 test^ 47S ^ 
 for Noguchi's modification of Wasser- 
 mann reaction, 453 
 titration of, 453 
 for Wassermann reaction, 417 
 
 anticomplementary titration, 428 
 antigenic titration, 431 
 experimental work, 856 
 hemolytic titration, 430 
 method of diluting, 427 
 
 of titrating, 428 
 preparation, 419 
 
872 
 
 INDEX 
 
 Antigen, gonocooous, titration of, experi- 
 mental work, 860 
 non-protein, 159 
 Antigenic dose of syphilitic serum, 418 
 titration in Wassermann reaction, 431 
 values of various extracts in serum 
 
 diagnosis of syphilis, 424 
 Antigonoooecus serum, 765 
 
 action, 766 
 
 administration, 766 
 
 preparation, 766 
 Antihemolysins, 372 
 Anti-influenza serum, 750 
 
 administration, 751 
 Antilactase, 246 
 Antilysin test for antistaphylococcus 
 
 serum, 239 
 Antimeningococous serum, 736 
 
 action, 741 
 
 administration, 742 
 
 dosage, 74 
 repeating, 744 
 
 Flexner and Jobling's method of 
 preparing, 739 
 
 intravenous injection, 743 
 
 Kolle's method of preparing, 740 
 
 preparation, 739 
 
 repeating doses, 744 
 
 serum sickness from, 746 
 
 standardization, 740 
 
 subdural inoculation, 742 
 Antipepsin, 246 
 Antiplague serum, 769 
 Antipneumococcus serum, 757 
 
 action, 759 
 
 administrationj 759 
 
 intravenous injection, 759 
 
 KoUe's method of preparing, 758 
 
 preparation, 758 
 
 results from, 759 
 
 standardization, 758 
 Antiprecipitin, 296 
 Antirennin, 246 
 AntisensibiUsin, 557 
 
 Antiscptioa, preservation of serum by, 76 
 Antistaphylococcus serum, 238, 766 
 
 antilysin test for, 239 
 
 preparation, 239 
 Antistaphylolysin, experimental work^ 
 837 
 method of titrating, in serum, 240 
 Antisteapsin, 246 
 Antistreptococcus serum, 760 
 
 action, 761 
 
 administration, 763 
 
 in bronchopneumonia, 764 
 
 in diphtheria, 764 
 
 in endocarditis, 764 
 
 in erysipelas, 764 
 
 in puerperal sepsis, 764 
 
 in scarlet fever, 764 
 
 in smallpox, 764 
 
 in tuberculosis, 764 
 
 in wound infections, 764 
 
 intravenous injection, 763 
 
 Antistreptococcus serum, Kolle's method 
 of preparing, 763 
 preparation, 762 
 standardization, 763 
 value, 764 
 Antitetanic serum, production of, 234 
 Antitetanolysin, action of, experimental 
 
 work, 836 
 Antitoxic immunity, 684 
 acquired, 171 
 passive, 173 
 immunization, 684, 702 
 Antitoxin,* 149, 220 
 
 action of, based on colloidal reactions, 
 516 
 on toSin, 224 
 antidysenteric, 236 
 antistaphylococcus, 238 
 botulinus, 236 
 definition, 220 
 diphtheria, 702. See also Diphtheria 
 
 antitofin. 
 dysentery, 730 
 
 admiijistration and uses, 730 
 experimental work, 835 
 formation, 221 
 hay-fever, 242, 734 
 history, 220 
 immunity, natural, 169 
 measure of, 242 
 natural,- 224 
 poUen, 342, 734 
 practical application, 243 
 production of, 68 
 
 for therapeutic purposes, 226 
 properties, 223 
 relationjof, to proteids, 223 
 specificity, 224 
 
 experimental work, 836 
 structure, 223 
 
 tetanus,.719. See also Tetanus anti- 
 toxin. 
 unit,242, 
 
 Behring-Ehrlich, 231 
 Antitrypsin, 246 
 test, 250 
 
 BergE?iann and Meyer's, 250 
 Antituberculosis serum, 771 
 Antityphoid serum, 768 
 Antityrosinase, 246 
 Antiurease, 246 
 Antivenene, Calmette's, 734 
 Antivenin, preparation of, 241 
 
 production of, 241 
 Apotoxin, 556 
 
 Aqueous extract of pallidum culture for 
 Wassermann reaction, 424 
 of syphilitic livers for Wassermann 
 reaction, 419 
 Arsenoxid, 808 
 
 Arthritis, gonorrheal, autoserum treat- 
 ment, 776 
 vaociije therapy, 658 
 Arthus phenomenon of anaphylaxis, 533 
 Artificial aggressins, 103, 125 
 
INDEX 
 
 873 
 
 Ascoli and Izar's miostagmin test, 526. 
 
 See also Miostagmin reaction. 
 Asthma, horse, 578 
 
 in serum treatment, 687 
 ..Athrepsia, 170 
 Athreptic immmiity, 170 
 Atmosphere, bacteria in, 85 
 Atrophy, receptoric, SO, 171 
 Atropin sulphate as preventive of serum 
 
 disease, 576 
 Auto-anticomplements, 327 
 Autocytotoxins, 505 
 .Autogenous bacterial vaccines, 620 
 Autolysins, 363 
 Autonephrotoxins, 505 
 Autopsies, 815 
 Autoserum treatment, 775 
 
 of acute infectious diseases, 775 
 
 of cancer, 782 
 
 of erysipelas, 776 
 
 of gonorrheal arthritis, 776 
 
 of hydrocele, 782 
 
 of influenza, 776 
 
 of leprosy, 776 
 
 of Malta fever, 776 
 
 of marasmus, 783 
 
 of non-tuberculous effusions, 782 
 
 of pneumonia, 776 
 
 of scarlet fever, 775 
 
 of skin diseases, 775 
 
 of smallpox, 776 
 
 of tuberculosis of serous membranes, 
 
 781 
 of tuberculous meningitis, 782 
 
 plevuisy, 781 
 of typhoid fever, 776 
 salvarsanized, in syphilis of brain, 
 776 
 after-treatment, 780 
 repeating dose, 780 
 serobiologie findings in cere- 
 brospinal fluid, 780 
 technic, 778 
 Autumnal catarrh, 242 
 Ayer and Gay's method of titration of 
 bacteriolytic complement, 334 
 
 Bacillen emulsion, tuberculin, prepara- 
 tion, 666 
 Bacillus, acid-fast, fixing and staining, 
 197 
 botuUnus, 116 
 diphtheria, 112 
 
 virulence and toxicity, method of 
 testing, 818 
 dysentery, 116 
 tetanus, 115 
 
 tubercle, Uving, in treatment of tuber- 
 culosis, 669 
 Bacteremia, 93 
 Bacteria, 83 
 
 agglutination reaction in identification 
 
 of, 277 
 aggressiveness of, 95 
 
 Bacteria, Bail's classification, 124 
 defensive mechanism of, in relation 
 
 to infection, 102 
 effect of opsonins on, 189 
 fixing and staining, 197 
 in atmosphere, 85 
 in foods, 85 
 in milk, 85 
 in placfenta, 86, 90 
 in water, 85 
 mechanical action, 108, 131 
 
 exp.erimental action, 826 
 morphologic changes, in relation to 
 
 infection, 102 
 non-agglutinable species, 274 
 non-pathogenic, 84 
 numeric relationship, to infection, 99 
 on skin, 85, 87 
 pathogenic, 83, 84 
 physiologic changes, in relation to 
 
 infection, 102 
 recovered from feces, water-suppHes, 
 
 etc., -bacteriolytic test for identifi- 
 cation, 343 
 toxicity of, 94, 95 
 
 transmission of, by suctorial insects, 86 
 virulence of, 94 
 
 decrease, 95 
 
 increase, 96 
 
 by addition of animal fluids to 
 
 culture-medium, 97 
 by passage through animals, 96 
 by, use of collodion sacs, 97 
 Bacteria-free filtration, preservation of 
 
 scrum by, 76 
 Bacterial anaphylactogens, 547 
 
 antigens, identification of, comple- 
 ment -fixation test for, 498 
 
 preparation, in complement fixa- 
 tion, 473 
 
 principles of complement fixation 
 with, 476 
 
 standardizing, for complement fixa- 
 tion, 475 
 diseases, chemotherapy in, 811 
 
 complement fixation in, 473 
 emulsion in opsonic index, 194 
 ferments, 244 
 invasion, factors preventing, 90 
 
 mechanism, 91 
 
 normal defenses against, 90 
 precipitinogens, preparation, 313 
 precipitins, experimental work, 846 
 
 preci*pitin test with, 298 
 technic, 313 
 
 production of, 71 
 proteins, 108, 126 
 
 action, 128 
 
 experimental work, 824 
 
 nature, 127 
 
 Vaughan's theory, 128 
 split proteins, 126 
 toxins, 108. See also Tozins. 
 vaccines, 206, 613. See also Vaccina, 
 
 badei-ial. 
 
874 
 
 INDEX 
 
 Bactericidal power of blood, capillary 
 
 pipet method of measuring, 355 
 
 Neisser and Wechsberg's method 
 
 of measm'ing, 349 
 plate culture method for measur- 
 ing, 349 
 teohnic, 350 
 Stern and Kortc's method of 
 measuring, 349 
 experimental work, 863 
 Topfer and Jaffe's method of 
 
 measuring, 3.52 
 Wright's capillary pipet method 
 of measuring, 355 
 Bactericidins, 337 
 Bacterin, 613 
 
 therapy, 611, 614, 617 
 history, 611 
 Bacteriolysins, 154, 318, 336 
 
 and hemolysins, analogy between, 367 
 definition, 337 
 experimental work, 815 
 history, 336 
 
 influence of, on endotoxins, 340 
 normal, 341 
 origin, 338 
 
 practical applications, 341 
 production of, 69 
 properties, 341 
 specificity, 341 
 Bacteriolysis, 146, 336 
 
 and hemolysis, analogy between, 367 
 experimental work, 862 
 mechanism, 340 
 Bacteriolytic amboceptor, 155, 319 
 titration of, 325 
 complement, titration of, 334 
 
 Gay and Ayer's method, 334 
 power of blood, microscopic method of 
 
 measuring, 862 
 serum in treatment of disease, 359 
 method of titrating, 344 
 production of, 69 
 test for identification of bacteria re- 
 covered from feces, water-sup- 
 pUes, etc., 343 
 method of testing virulence of cul- 
 ture, 343 
 of titrating bacteriolytic serum, 
 
 Pfeiffer's, 342 
 
 experimental work, 862 
 in diagnosis of disease, 348 
 technic of, 347 
 preparing immune serum, 343 
 technic, 342 
 Baoteriotropins, 145, 185, 188 
 experimental work, 832 
 ..quantitative estimation, experimental 
 work, S34 
 Neufeld's technic, 220. See also 
 Neiifeld's quantitative estima- 
 tion. 
 Simon's method, 203 
 Bail's classification of bacteria, 124 
 
 Bail's hypothesis, 123 
 
 Bandi anci Term's plague vaccine, 648 
 
 Bauer's modification of Wassermann 
 
 reaction, 456 
 B. E. tuberculin, preparation of, 666 
 Behring-Bhrlich antitoxin unit, 231 
 Behring's method of immunization 
 
 against diphtheria, 717 
 Beraneck'sltubercufin, dose of, 675 
 
 preparation of, 666 
 Bergmann' and Meyer's antitrypsin test, 
 
 250 
 Berkefeld filter, 77 
 
 Besredka's theory of anaphylaxis, 557 
 B. F. tuberculin, preparation of, 666 
 Biologic blood test for detection of blood- 
 stains, 303 
 technic, 308 
 oT meat adulteration, 310 
 technic, 312 
 Blackfan's apparatus for collecting blood, 
 
 36 
 Blackleg vaccination, 655 
 Blood, animal and human, differentia- 
 tion, precipitin test for, 303 
 obtaining large amounts, from dog, 
 51 
 from guinea-pig, 46 
 from hog, 50 
 from horse, 52 
 from monkey, 50 
 from rabbit, 42-46 
 
 Nuttall's method, 42 
 from rats, 46 
 from sheep, 48 
 small amounts, 41 
 from dog, 51 
 from guinea-pig, 41 
 from horse, 52 
 from monkey, 50 
 from rabbit, 41 
 from rats, 46 
 from sheep, 42, 49 
 bactericidal power, capillary pipet 
 method of measuring, 355 
 Neisser and Wechsberg's method 
 
 of measuring, 349 
 plate culture method for measur- 
 ing, 349 
 . technic^ 350 
 Stetn and KOrte's method of 
 measuring, 349 
 experimental work, 863 
 Topfer and Jaffe's method of 
 
 ineasuring, 352 
 Wright's capillary pipet method 
 of measuring, 355 
 bacteriolytic power, microscopic 
 method of measuring, experimental 
 work, S62 
 Blackfan's apparatus for collecting, 
 
 36 
 capsuled, Wright's, making of, 23 
 method of sealing, 34 
 removing serum from, 34 
 
INDEX 
 
 875 
 
 Blood, collecting of, for Wassermann re- 
 action, 408 
 New York Board of Health 
 outfit, 410 
 films for phagocytic counts, 199 
 
 method of preparing, 198 
 human and animal, differentiation, 
 precipitin test for, 303 
 obtaining large amounts, 33 
 phlebotomy, 33 
 wet cupping, 36 
 small amounts, 32 
 
 from infants and children, 
 33 
 Keidel tube for collecting, 35, 36 
 methods of obtaining, 28 
 placental, method of obtaining, 37 
 plasma, obtaining, 30 
 precipitin tost for, 303 
 technic, 308 
 Blood-corpuscles and blood-serum, ob- 
 taining, 30 
 obtaining, 28 
 red. See Erythrocytes. 
 test, biologic, for detection of blood- 
 stains, 303 
 technic, 308 
 of meat adulteration, 310 
 technic, 312 
 Teichmann's, 303 
 transfusion, 273 
 
 experimental work, 844 
 tests before, for isohemagglutinins 
 and isohemolysins, 290 
 Blood-pressure as guide in administering 
 serum subdurally, 695 
 fall in, in anaphylaxis, 541, 542 
 Blood-serum. See Serum. 
 Blood-stains, biologic test for, 303 
 technic, 308 
 experimental work, 845, 861 
 identification of, complement-fixation 
 test for, 494 
 Body-fluids, relation of, to phagocj'tosis, 
 
 184 
 Boil, Aleppo, salvarsan in, 811 
 Bones, tuberculosis of, tuberculin test in 
 diagnosis of, 590 
 treatment, 679 
 Bordet-Gengou phenomenon of com- 
 plement fixation, 332, 391 
 as colloidal reaction, 519 
 determination of antigen bv, 
 
 494 
 experimental work, 855 
 for identification of bacterial 
 antigens, 491S 
 of blood-stains, 494 
 of meats, 498 
 in bacterial diseases, 473 
 in cancer, 499 
 in contagious abortion, 486 
 in dourine, 487 
 in echinococcus disease, 492 
 in glanders, 484 
 
 Bordet-Gengou phenomenon of comple- 
 ment fixation in gonococcus 
 infections, 477. Sec also Gono- 
 coccus complement-fixation test. 
 in horse syphilis, 487 
 in standardization of immune 
 
 serums, 491 
 in tuberculosis, 490 
 in typhoid fever, 489 
 mechanism, 395 
 non-specific, 396 
 original, 394 
 
 practical application, 399 
 preparation of bacterial anti- 
 gens, 473 
 principles, 391 
 
 with bacterial antigens, 476 
 protein differentiation by, 494 
 quantitative factors in, .397 
 standardizing bacterial anti- 
 gens, 475 
 technic, 401, 473 
 Bordet's theory of mechanism of agglu- 
 tination, 271 
 Botuhnus antitoxin, 236 
 Botulism toxin, 116 
 
 experimental work, 820 
 Bouillon filtrate, preparation of, 666 
 Brain, syphihs of, salvarsanized auto- 
 serum in, 776 
 after-treatment, 780 
 repeating dose, 780 
 serobiologic findings in cere- 
 brospinal fluid, 780 
 technic, 778 
 Brezovsky and Detre's modification of 
 
 Wassermann reaction, 459 
 Bronchitis, vaccine therapy, 659 
 Bronchopneumonia, antistreptococcus 
 
 serum in^ 764 
 Browning and Mackenzie's modification 
 of Wassermann reaction, 459 
 test for estimating amount of com- 
 plement absorbed in ^A'assermann 
 reaction, 434 
 Bubonic plague, serum treatment, 769 
 
 vaccination, 647 
 Buckwheat idiosyncrasy, 578 
 Butyric-acid test, Xoguchi's, for pro- 
 tein, 300 
 
 C.4DAVER serums, testing, in Wasser- 
 mann rea,ction, 411 
 
 Calf cholera serum, 733 
 
 Calmette's antivenene, 734 
 
 conjunctival tuberculin test, 587, 598 
 dangers, 592 
 in aiiimals, 600 
 
 Cancer, autoserum treatment, 782 
 chemothefepy in, 812 
 complement-fixation test in, 499 
 contraindications to vaccines in, 623 
 eosin-selenium compound in, 812 
 epiphanin reaction in, 526 
 
876 
 
 INDEX 
 
 Cancer, Freund and Kaminer's cytolytic 
 test for, 509 
 miostagmin reaction in, 528, 530 
 precipitin test in, 314 
 
 Freund and Kaminer's, 314 
 sero-enzymes in, 264 
 venom hemolysis in, 390 
 von Dungern's test in, 500 
 Capillary pipet for counting bacterial 
 vaccine, 210 
 for opsonic index determination, 196 
 making of, 18 
 method of sealing, 198 
 rubber teats for, 20 
 Capsules, blood, Wright's making of, 23 
 method of seaUng, 34 
 removing serum from, 34 
 Carbuncle, antistaphylococcus serum in, 
 766 
 vaccine therapy, 656 
 Castellani's agglutination reaction, ex- 
 perimental work, 843 
 Cataphoresis, 513 
 Catarrh, autumnal, 242 
 Cats, anaphylaxis in, 539 
 Cells, functions of, 147 
 
 heart failure, 178 
 Cellular theory of anaphylaxis, 555 
 
 of immunity, 143 
 Centrifuge, 17 
 care of, 17 
 electric, 18 
 Cerebral syphilis, Wassermann reaction 
 
 -in, 462 
 Cerebrospinal fluid for Wassermann re- 
 action, 411 
 method of securing, 37 
 meningitis, agglutination test in, 277 
 precipitin test in diagnosis, 298 
 serum treatment, 736 
 vaccination, 652 
 Chancroid, salvarsan in, 811 
 Chantemesse's serum, 768 
 Chemoreceptors, 787 
 Chemotaxis, 179 
 
 experimental work, 830 
 negative, 145, 179, 182 
 
 experimental work, 830 
 positive, 145, 179 
 
 experimental work, 830 
 Chemotherapy, 784 
 experimental work, 867 
 in bacterial diseases, 81 1 
 in cancer, 812 
 in malignant disease, 812 
 . principles, 785 
 Chills and fever after intravenous in- 
 jection of salvarsan, 804 
 Cholera, agglutination test in, 277 
 hog, serum treatment, 732 
 serum, calf, 733 
 
 hog, production of, 732 
 
 standardization of, 732 
 treatment, 770 
 vaccination, 650 
 
 Cholera, vaccination, dosage of vaccine, 
 651 
 Haffkine's vaccine, 650 
 Kolle's vaccine, 650 
 preparation of vaccine, 650 
 results, 651 
 Stron'g's vaccine, 651 
 Cholesterin and lecithin for Wassermann 
 
 reaction, 423 
 Chorea, salvarsan in, 811 
 Cobra hemotoxin, 120 
 lecithid,= 330 
 
 venom, experimental work, 823 
 preparation, 385 
 
 tests, ;383. See also Venom hemoly- 
 sis. 
 Cobralecithinase, 384 
 Cold-blood animals, anaphylaxis in, 541 
 Cole's metliod of determining type of 
 
 pneumococcus, 757 
 CoUapse in subdural inoculation of 
 serum, 694, 696 
 symptoms, 696 
 treatment, 696 
 Colles' law", Wassermann reaction and, 
 
 464 
 Collodion sacs for increasing virulence of 
 
 bacteria, 97 
 Colloidal f eactions, action of agglutinins 
 based on, 517 
 of aatitoxins based on, 516 
 of hemolysins based on, 518 
 of precipitins based on, 517 
 complement-fixation test as, 519 
 immimity reactions and, analogy 
 
 between, 515 
 Wassermann test as, 520 
 solutions, 512 
 suspensions, 512 
 Colloids, a:bsorption by, 515 
 amorphous, 512 
 nature, 511 
 osmotic ;pressure, 512 
 physical structure, 513 
 precipitation, 513 
 properties, 511 
 relation.of, to immunity, 511 
 surface tension, 512 
 varieties, 511 
 Comer's automatic pipet, 215 
 Complemfet, 146, 154, 157, 185, 316, 
 318, 326, 337, 362 
 action, 329 
 
 and amboceptor, quantitative rela- 
 tionship, 374 
 bacteriolytic, titration of, 334 
 
 Gay"' and Ayer's method, 334 
 definitio*a, 326 
 deflection of, 333 
 deviation of, 333 
 dominant, 321, 366 
 endoceltjilar, 330 
 
 fixation, Bordet-Gengou phenomenon, 
 332, 391. Sec also Bordet-Gengou 
 pheno'rAenon. 
 
INDEX 
 
 877 
 
 Complement fixation reactions, experi- 
 mental work, 855 
 in differentiation of proteins, 
 
 experimental work, 861 
 non-specific, 396 
 practical applications, 399 
 principles of, 391 
 quantitative factors in, 397 
 technic, 401 
 for Noguchi's modification of Wasser- 
 
 mann reaction, 450 
 for Wassermann reaction, 411 
 
 titration, 413 
 hemolytic, experimental work, 853 
 titration of, 334 
 
 experimental work, 854 
 history, 326 
 inaotivation of, experimental work, 
 
 853 
 multiphcity, 328 
 nature, 329 
 non-dominant, 321 
 origin, 328 
 properties, 326 
 
 experimental work, 854 
 reactivation of, experimental work, 853 
 r61e of, in hemolysis, experimental 
 
 work, 850 
 spUtting, 331 
 structure, 326 
 
 titration of, quantitative, 334 
 Complementoid, 327 
 Congenital mental deficiency, Wasser- 
 mann reaction in, 465 
 syphilis, Wassermann reaction in, 463, 
 464 
 Congestion, 556 
 
 Conjunctival tubercuhn test of Cal- 
 mette, 587, 598 
 dangers, 592 
 in animals, 600 
 of Wolff-Eisner, 587, 598 
 dangers, 592 
 in animals, 600 
 Contagious abortion, complement-fixa- 
 tion test in, 486 
 diseases, 84 
 Contamination, 81 
 Cow-disease, 613 
 Cowpox, 613 
 
 vaccine, preparation, 626 
 animals, 627 
 
 collection of virus, 628 
 seed virus, 626 
 testing virus, 629 
 Cows, anaphylaxis in, 541 
 Crotin toxin, 118 
 Cryptogenic infections, 92 
 Crystalloids, 511 
 Culex, 166 
 Cupping, wet, 36 
 
 Cutaneous reaction in typhoid fever, 608 
 tubercuhn reaction of von Pirquet, 
 587, 596 
 in animals, 601 
 
 Cyst, echinococcus, complement-fixation 
 
 test in, 492 
 Cystitis, vaccine therapy, 657 
 Cytase, 144, 157, 318, 337, 338 
 Cytolysins, 316, 502 
 
 definition, 317 
 
 nomenclature, 318 
 
 varieties,. 318 
 Cytotoxic reactions, 509 
 in diagnosis, 509 
 
 of Freund and Kaminer for cancer, 
 509 
 Cytotoxins, 154, 318, 502 
 
 experimental production, 816 
 
 in diagnosis, 509 
 
 in immunity, role of, 508 
 
 in treatment, 508 
 
 methods of studying, 503 
 
 nature, 502 
 
 nomenclature, 502 
 
 practical apphcations, 508 
 
 preparation, 503 
 
 production of, 73 
 Pearce's method, 73 
 
 properties, 502 
 
 specificity, 504 
 
 varieties, 506 
 
 Dementia, paralytic, Wassermann re- 
 action in, 462 
 Dermatitis herpetiformis, salvarsan in, 
 
 811 
 Detre and Brezovsky's modification of 
 
 Wassermann reaction, 459 
 Deuterotoxin, 115 
 Deviation of complement, 333 
 Diabetes, contraindications to vaccines 
 
 in, 623 
 Diarrhea after intravenous injection of 
 
 salvarsah, 804 
 Diet as prgjiisposing to infection, 101 
 Digestive tract as portal of entrance for 
 
 bacteriaj 88 
 Diphtheria, allergic reaction in, 609 
 antistreptococcus serum in, 764 
 antitoxlA, 702 
 action, 705 
 administration, 706 
 dosage, 709 
 
 in diphtheria of eye, 711 
 of vulva, 711 
 of wounds, 711 
 in nasal diphtheria, 710 
 in tonsillar diphtheria, 710 
 influence of age, 711 
 repeating, 711 
 total, 712 
 early use, importance, 707 
 intramuscular injection, 707 
 intravenous injection, 707 
 mortaUty after use, 714 
 
 before use, 713 
 oral method of administering, 707 
 preparation, 703 
 
878 
 
 INDEX 
 
 Diphtheria antitoxin, production of, 227 
 collecting serum, 230 
 immunizing animals, 228 
 rectal method of administering, 707 
 sequels, 712 
 
 serum sickness from, 712 
 standardizing, 231 
 
 experimental work, 835 
 subcutaneous injection, 707 
 total amount to be administered, 712 
 treatment of relapses with, 712 
 unit of, 242 
 value, 713 
 bacillus, 112 
 
 virulence and toxicity, method of 
 testing, 818 
 Behring's method of immunization 
 
 against, 717 
 nasal, dosage of antitoxin in, 710 
 nature of, 704 
 
 of eye, dosage of antitoxin in, 711 
 of vulva, dosage of antitoxin in, 711 
 of wounds, dosage of antitoxin in, 711 
 prophylactic immunization against, 
 
 715 
 serum treatment, 702. See also Diph- 
 theria antitoxin. 
 tonsillar, dosage of antitoxin in, 710 
 toxin, 112 
 
 experimental work, 817 
 hmes death dose, 232 
 
 zero dose, 232 
 method of testing virulence, 113 
 production of, 227 
 standardizing, 114 
 testing, 228 
 toxoid, 115 
 
 treatment of, 704. See also Diph- 
 theria antitoxin. 
 Diplococcus pneumoniae, 75.5 
 Disease, antiferments in, 247 
 
 bacteriolytic serums in treatment of, 
 
 359 
 diagnosis of, Pfeiffer bacteriolytic test, 
 
 348 
 ferments in, 248 
 Pfeiffer's bacteriolytic test in diagnosis 
 
 of, 348 
 production of, 107 
 sero-enzymes in, 264 
 Dixon's tubercuhn, dose of, 675 
 
 preparation, 667 
 Dog, anaphylaxis in, 540 
 intravenous inoculation, 62 
 obtaining large amounts of blood from, 
 51 
 small amount of blood from, 51 
 Donath and Landsteiner's method of 
 serum diagnosis of paroxysmal hemo- 
 globinuria, 379 
 Dose, intoxicating, 536 
 Domine, complement-fixation test in, 
 
 487 
 Drug fastness, 789 
 idiosyncrasy, 578 
 
 Duhring's disease, salvarsan in, 811 
 Dungern's complement-fixation test in 
 canc(5r, 500 
 modification of Wassermann reaction, 
 459 
 DysenterJ-, agglutination test in, 276 
 antitoxin, 730 
 
 administration and uses, 730 
 serum treatment, 730 
 
 results, 731 
 toxin, 116 
 
 experimental work, 820 
 vaccination, 652 
 
 Eae, tuberculosis of, tuberculin test in 
 diagnosis of, 590 
 treatment, 679 
 Ecliinococcus disease, complement-fixa- 
 tion'test in, 492 
 miostagmin reaction in, 530 
 Effusions,; non-tuberculous, autoserum 
 
 treatment, 782 
 Egg-albumen idiosyncrasy, 578 
 EhrUch's inethod of serima diagnosis of 
 paroxysmal hemoglobinuria, 379 
 theory of immunity, 146 
 
 and Metchnikoff's theory, com- 
 patibiUty, 155 
 therapia magna sterihsans, 791 
 Electric ciitrifuge, 18 
 Emulsion, bacterial, in opsonic index, 194 
 Emulsiona; 611 
 Endemic infection, 82 
 Endocarditis, antistreptococcus serum in, 
 764 
 ulcerative, vaccine therapy, 661 
 Endocellular complement, 330 
 Endocomplement, 330 
 Endogenous infection, 85, 86 
 Endolysins, 183, 339 
 Endotoxic isubstances, method of ob- 
 taining, 121 
 Endotoxins, 108, 120, 182 
 as anaphylactogens, 548 
 experimental work, 823 
 influence of bacteriolysins on, 340 
 method of obtaining, 121 
 nature of, 121 
 Enteritis anaphylactica, 540 
 Enzootic infection, 82 
 Enzymes, 244 
 
 sero-, in disease, 264. See also Sero- 
 enzymes. 
 Eosin-solenium compound in cancer, 812 
 Epidemic cerebrospinal meningitis, treat- 
 ment, 738 
 infection, 82 
 Epiphanin reaction, 522 
 in cancer, 526 
 in mali|,nant disease, 526 
 in pregnancy, 526 
 in sarcoma, 626 
 in syphilis, 526 
 principle, 522 
 
INDEX 
 
 879 
 
 Bpiphanin reaction, reading results, 525 
 
 specificity, 523 
 Epitheliotoxin, 606 
 Epizootic infection, 82 
 Erysipelas, antistreptococcus serum in, 
 764 
 autoserum treatment, 776 
 vaccine therapy, 657 
 Erythrocytes for Wassermann reaction, 
 416 
 method of determining resistance, 380 
 obtaining of, 28 
 
 resistance of, to salt solution, experi- 
 mental work, 847 
 washing of, 28 
 Exhaustion theory of immunity, 144 
 Exogenous infection, 85 
 Experimental immunity, 814 
 
 infection, 814 
 Experiments, animal, 815 
 Exposure as predisposing to infection, 
 
 102 
 Eye, diphtheria of, dosage of antitoxin 
 in, 711 
 tuberculosis of, tubercuUn test in 
 diagnosis of, 590 
 treatment, 679 
 
 Familial susceptibility, 100 
 
 Fastigium period of infection, 135 
 
 Fastness, drug, 789 
 
 Feces, bacteria recovered from, bacterio- 
 lytic test for identification of, 343 
 
 Ferment reactions, 230 
 
 Ferments, 244 
 
 and toxins, similarity between, 244 
 bacterial, 244 
 experimental work, 838 
 in disease, 248 
 in pregnancy, 248 
 
 Fever and chills after intravenous in- 
 jection of salvarsan, 804 
 of infection, 137 
 protein, 137 
 
 Filariasis, salvarsan in, 810 
 
 Film, blood, for phagocytic counts, 199 
 method of preparing, 198 
 
 Filter, 78 
 Berkefeld, 77 
 Uhlenhuth, 305 
 
 Filtration, bacteria-free, preservation of 
 serum by, 76 
 
 Finger pricking, method of, 31 
 
 Fixateur. See Amboceptor. 
 
 Fixator, 322 
 
 Flexner and Jobling's method of pre- 
 paring antimeningococcus serum, 739 
 
 Flocoule-forming precipitin tests, 299 
 
 Fomites, 84 
 
 Foods, bacteria in, 85 
 idiosyncrasy, 578 
 
 Fordyce's technic of intraspinous in- 
 jection of salvarsanized serum, 807 
 
 Forensic blood test, 498 
 
 Fornet's ring test for syphiHs, 299 
 Frambesia, luetin reaction in, 605 
 
 salvarsan in, 810 
 
 Wassermann reaction in, 465, 466 
 Freezing serum, preservation by, 78 
 Freund and Kaminer's cytolytic cancer 
 diagnosis, 509 
 precipitin test in cancer, 314 
 Friedberger on anaphylatoxin, 550 
 
 theory of anaphylaxis, 558 
 Friedmann's treatment of tuberculosis, 
 
 670 
 Frigo, 79 
 
 Fruits, idiosyncrasy, 578 
 Fulminating infection, 136 
 Furunculosis, antistaphylococcus serura 
 in, 766" 
 
 vaccine therapy, 656 
 
 Galeotti and Lustig's plague vaccine, 
 
 648 
 Gastrotoxin, 507 
 Gay and Ayer's method of titration of 
 
 bacteriolytic complement, 334 
 Gay and Southard's theory of anaphy- 
 laxis, 557 
 Genital organs as portal of entrance foi 
 
 bacteria, 89 
 Genito-urinary diseases, vaccine therapy, 
 657 
 tubercidosis, tubercuhn test in diag- 
 nosis of, 590 
 treatment, 680 
 Glanders, agglutination reaction in, 277 
 complement-fixation test in, 484 
 maUein reaction for, 606. See also 
 Mallein reaction. 
 Glandular tuberculosis, tubercuhn test 
 
 in diagnosis of, 590 
 Globuhns, Noguchi's butyric-acid test 
 
 for, 300 
 Goats, intravenous inoculation, 62 
 Gonococcal infections, serum treatment, 
 
 765 
 Gonococcus antigen, titration of, ex- 
 perimental work, 860 
 complement-fixation test, 477 
 antigen for, 478 
 experimental work, 800 
 hemoly tic systems, 478 
 practical value, 482 
 specificity, 482 
 technic, 478 
 
 using one-tenth usual amounts, 48] 
 infections, allergic reaction in, 609 
 complement fixation in, 477 
 Gonorrheal arthi-itis, autoserum treat- 
 ment, 776 
 vaccine therapy, 658 
 Graduated pipets, 21 
 Gravity method of intravenous injecliot 
 of salvarsan, 801 
 of serum, 691 
 of subdural injection of serum, 697 
 
880 
 
 INDEX 
 
 Group immune bodies, 323 
 
 precipitins, 293, 295 
 
 Gruber-Widal reaction in typhoid fever, 
 
 268, 275, 279, 282 
 
 experimental work, 840 
 
 Griiber's theory of mechanism of 
 
 agglutination, 270 
 Guinea-pig, anaphylaxis in, 537 
 
 complement serum for Wassermann 
 
 reaction, 412 
 intracardial inoculation, 62 
 intraperitoneal inoculation, 64 
 intravenous inoculation, 57 
 obtaining large amount of blood from, 
 46 
 small amount of blood from, 41 
 
 Hapfkinb's plague vaccine, dosage, 649 
 effects, 649 
 preparation, 648, 650 
 results, 649 
 Hamburger and Moro's theory of anaphy- 
 laxis, 556 
 Hamman and Wolman's method of 
 preparing tubercuUn for sub- 
 cutaneous tuberculin test, 592 
 plan of dosage for tubercuUn in sub- 
 cutaneous test, 594 
 Haptines, 147 
 
 Haptophore group of toxin, 110 
 Hay-fever, 242, 578 
 ■ antitoxin, 734 
 .serum treatment, 734 
 toxin of, 119 
 Headache after injection of salvarsan, 
 
 804 
 Heart failure cells, 178 
 Hecht-Weinberg modification of Was- 
 sermann reaction, 457 
 Hemagglutinins, 272 
 
 experimental work, 843 
 Hemocytometer chamber, counting bac- 
 terial vaccines with, 211 
 Hemoglobinuria, paroxysmal, serum 
 
 diagnosis, 379 
 Hemolysins, 154, 318, 361 • 
 
 action of, based on colloidal reaction, 
 
 518 
 and bacteriolysins, analogy between, 
 
 367 
 definition, 362 
 
 experimental production, 816 
 history, 361 
 immune, 363 
 
 method of titration, 375 
 production of, 371 
 methods for removing, from serum, 
 
 378 
 natural, 368 
 
 experimental work, 852 
 
 method of determining, in serum, 
 
 378 
 removal of, experimental work, 852 
 nature, 363 
 
 Hemolysiiis", nomenclature, 363 
 ■ normal, 368 
 
 practical sappUcations, 373 
 preservation of, in dried paper form, 79 
 production of , 71 
 
 intraperitoneal method, 72 
 intravenous method, 72 
 properties, 371 
 sources, -372 
 specific, 363 
 specificity, 368 
 Hemolysis, 361 
 
 and bacteriolysis, analogy between, 
 
 367 
 non-specific, 380 
 relation. of lipoids to, 521 
 role of amboceptor and complement 
 
 in, experimental work, 850 
 serum, experimental work, 848 
 
 quantitative factors, experimental 
 work, 849 
 venom, 383, 521. See also Venom 
 hemolysis. 
 Hemolytic amboceptor, 319, 362 
 
 and complement, quantitative re- 
 lationship, 374 
 for Noguchi's modification of Was- 
 sermann reaction, 451 
 for Wassermann reaction, 415 
 preservation of, 79 
 titration of, 325 
 
 exjferimental work, 848 
 complement, experimental work, 853 
 titration of, 334 
 
 experimental work, 854 
 jaundice, 372 
 
 titration of antigens in Wassermann 
 reaction, 430 
 Hemophiha, normal serum treatment, 
 
 772 
 Hemopsonins, 188 
 
 experimental work, 832 
 Hemorrhage, normal serum treatment, 
 
 772 
 Hemorrhagin, 734 
 Hemotoxin, 117 
 
 cobra, 120 
 Hens, anaphylaxis in, 541 
 Hepatotoxin, 507 
 
 Herman-Eerutz test for syphilis, 299 
 HerxheimeE-Jarisch reaction, 804 
 Heterolysins, 363 
 Histamin,.552 
 Hitchens syringe, 233 
 Hodgkin's disease, salvarsan in, 811 
 Hog cholera serum, production, 732 
 standardization, 732 
 treatment, 732 
 obtaining large amount of blood from, 
 50 
 Hopkins' inethod of counting bacterial 
 vaccines, 212 
 tube tor standardizing bacterial vac- 
 cine, 213 
 Horse, anaphylaxis in, 541 
 
INDEX 
 
 881 
 
 Horse asthma, 578 
 
 intravenous inoculation, 61 
 
 obtaining large amount of blood from, 
 52 
 small amount of blood from, 52 
 
 syphilis, complement-fixation test in, 
 487 
 Humoral theory of immunity, 143, 146 
 Hydatid disease, complement-fixation 
 
 test in, 492 
 Hydrocele, autoserum treatment, 782 
 Hydrocephalus in meningococcus menin- 
 gitis, effect of serum treatment on, 746 
 Hydrophobia, 636. See also Rabies. 
 Hypersensitiveness, 531, 577. See also 
 
 Idiosyncrasy. 
 Hypothesis of Bail, 123 
 
 of Welch, 103 
 
 Idiosyncrasy, 531, 577 
 buckwheat, 578 
 drugs, 578 
 egg-albumin, 578 
 foods, 578 
 fruits, 578 
 hay-fever, 578 
 horse asthma, 578 
 iodoform, 578 
 porlc, 578 
 strawberries, 578 
 vegetables, 578 
 Immune agglutinins, 268 
 bodies, 319, 362 
 group, 323 
 partial, 233 
 hemolysins, 363 
 
 method of titration, 375 
 production of, 371 
 opsonins, experimental work, 832 
 
 production of, 69 
 precipitins, 294 
 
 serums and normal serums, difference 
 between, 324 
 methods for making, 66 
 precipitin, 70 
 preservation of, 76 
 
 in dried paper form, 79 
 in fluid form, by bacteria-free fil- 
 tration, 76 
 by freezing, 78 
 with antiseptics, 76 
 in living animal, 80 
 in powder form, 79 
 standardization of, complement-fix- 
 ation test in, 491 
 Immunity, 138 
 acquired, 170 
 active, 170 
 
 by vaccination, 171 
 causes of, 170 
 experimental work, 827 
 antibacterial, 171 
 antitoxic, 171 
 experimental work, 827 
 56 
 
 Immunity, acquired, passive, 172 
 antitoxic, 173 
 experimental work, 828 
 Vaughan's theory, 174 
 active, 65 
 
 agglutinins in, role of, 274 
 anaphylaxis in relation to, 565 
 antibacterial, 684 
 
 experimental work, 826 
 antitoxic, 684 
 antitoxin, natural, 169 
 athreptic, 170 
 cellular theory, 143 
 cytotoxins in, role of, 508 
 definition of, 140 
 Ehrlich's theory, 146 
 
 and' Metchnikoff's theory, com- 
 patibiUty, 155 
 exhaustion theory, 144 
 experimental, 814 
 history of, 140 
 humoral theory, 143, 146 
 individual, 166 
 Metchnikoff's theory, 144 
 
 and Ehrlich's theory, compati- 
 biUty, 155 
 natural, 165 
 antitoxin, 169 
 causes of, 167 
 
 influence of temperature on, ex- 
 perimental work, 827 
 phagocytosis in, 168 
 
 experimental work, 826 
 relative factors in, experimental 
 
 work, 827 
 Vaughan's theory, 174 
 opsonins in, 190 
 passive, 682 
 
 varieties of, 683 
 phagocytosis theory, 144 
 
 and- side-chain theory, compati- 
 bUity, 155 
 precipitins in, role of, 297 
 racial, 166 
 reaction, 569 
 
 colloidal reaction and, analogy be- 
 tween, 515 
 relation of coUoids to, 511 
 of infection to, 83 
 of lipoids to, 520 
 of phytotoxins to, 118 
 retention theory, 144 
 side-chain theory, 146 
 
 and phagocytic theory, compati- 
 bjUty, 155 
 species, 166 
 theories of, 138, 144 
 types of, 165 
 Vaughan's theory, 173 
 Immunization, active, 611 
 contraindications to, 622 
 for prophylaxis, 616 
 for therapeutic purposes, 655 
 for treatment of disease, 617 
 history of, 611 
 
882 
 
 INDEX 
 
 Immunization, active, mechanism of, 616 
 
 negative phase, 621 
 
 of animals, 653 
 general technic, 66 
 methods for effecting, 65 
 antibacterial, 684, 735 
 antitoxic, 684, 702 
 Behring's method, against diphtheria, 
 
 717 
 curative, 682, 683 
 passive, 681 
 
 contraindications to, 686 
 
 indications for, 684 
 
 purposes of, 682 
 prophylactic, 580, 623, 682. See also 
 vaccination. 
 
 therapeutic, 580 
 Incubation period of infection, 134 
 Index, opsonic, 191. See also Opsonic 
 
 index. 
 phagocytic, 200 
 Individual immunity, 166 
 
 predisposition, 100 
 Infection, 81 
 acute, 136 
 
 sero-enzymes in, 265 
 
 vaccine therapy, 660 
 aggressins in relation to, 103 
 anaphylaxis in relation to, 565 
 antenatal, 90 
 
 avenue of, tissue susceptibility and, 97 
 avenues of, 86 
 chronic, 136 
 complications, 136 
 course, 134 
 cryptogenic, 92 
 
 defensive mechanism of microorgan- 
 ism in relation to, 102 
 definition of, 81 
 diet as predisposing to, 101 
 endemic, 82 
 endogenous, 85, 86 
 enzootic, 82 
 epidemic, 82 
 epizootic, 82 
 exogenous, 85 
 experimental, 814, 816 
 exposure as predisposing to, 102 
 fever of, 137 
 fulminating, 136 
 
 general susceptibility in relation to, 99 
 gonococcus, allergic reaction in, 609 
 
 serum treatment, 765 
 grades of^ 136 
 
 intoxications as predisposing to, 101 
 malignant, 136 
 
 malnutrition as predisposing to, 101 
 mechanism of, 94 
 mixed, 105 
 
 absorption agglutination test in, 288 
 morbid conditions as predisposing to, 
 
 102 
 morphologic changes of microorgan- 
 isms in relation to, 102 
 numeric relationship of bacteria to, 99 
 
 Infection, pandemic, 82 
 period of convalescence, 136 
 of decline, 135 
 of fastigium, 135 
 of higli fever, 135 
 of incpbation, 134 
 of prodromal symptoms, 135 
 physiologic changes of micro6rgan.sms 
 
 in relation to, 102 
 pneumofpccus, serum treatment, 754 
 previous,* as predisposing to infection, 
 
 101 
 relapse pf, 136 
 relation_of, to immunity, 83 
 remittent, 136 
 sequels of, 136 
 sources oT, 83 
 sporadia, 82 
 stages of, 134 
 
 staphylecoccus, serum treatment, 766 
 streptococcus, serum treatment, 760 
 systemic reaction to, 137 
 trauma as predisposing to, 102 
 with animal parasites, 132 
 
 modes, 132 
 wound, antistreptococcus serum in, 
 764 
 Infectious; diseases, 84 
 
 accelerated anaphylaxis reaction in, 
 
 568' 
 acute, autoserum treatment, 775 
 anapMylaxis, torpid, early reaction in, 
 
 568 
 immediate anaphylaxis reaction in, 
 
 568 
 production of, 107 
 relation of anaphylaxis to, 566 
 InfestatioUj definition of, 81 
 Infestment, definition of, 81 
 Inflammation, behavior of leukocytes in, 
 
 180 
 Influenza, autoserum treatment, 776 
 Influenzal, meningitis, serum treatment, 
 
 749 
 Inoculation, animal, 53. See also 
 
 Animal inoculation. 
 Insects, suctorial, transmission of bac- 
 teria by, 86 
 Interbody, 154, 319 
 Intestine, tuberculosis of, tuberculin 
 
 treatment, 679 
 Intoxicating dose, 536 
 Intoxicatiolis as predisposing to infec- 
 tion, 101 
 Intrabronehial route for tuberculin treat- 
 ment, 677 
 Intracardi^l inoculation of animals, 62 
 
 of guinea-pigs, 62 
 Intracutaneous tuberculin test of Man- 
 tbux, 587, 595 
 of Mendel, 587, 595 
 in animals, 601 
 IntrafocaLroute for tuberculin treatment, 
 
 677 
 Intramuscular injection of animals, 56 
 
INDEX 
 
 883 
 
 Intramuscular injection of diphtheria 
 antitoxin, 707 
 of neosalvarsan in syphilis, 805 
 of salvarsan in syphilis, 805 
 of serum, treatment, 690 
 of tetanus antitoxin, 722 
 Intraneural injection of tetanus anti- 
 toxin, 723 
 Intraperitoneal inoculation of animals, 64 
 of guinea-pig, 64 
 of rabbit, 64 
 method of production of agglutinins, 
 69 
 of hemolysins, 72 
 Intravenous injection of animals, 56 
 of antimeningococcuB serum, 743 
 of antipneumococcus serum, 759 
 of antistreptococcus serum, 763 
 of diphtheria antitoxin, 707 
 of dog, 62 
 of goats, 62 
 of guinea-pig, 57 
 of horse, 61 
 of mice, 60 
 
 of neosalvarsan in sypliilis, 797 
 after-care, 803 
 apparatus, 801 
 dosage, 797 
 frequency, 797 
 gravity method, 801 
 intensive treatment, 797 
 preparation of patient, 798 
 of solution, 800 
 of rabbit, 56 
 of rat, 00 
 
 of salvarsan in sypliilis, 797 
 after-care, 803 
 after-effects, 803 
 apparatus, 801 
 chills and fever after, 804 
 diarrhea after, 804 
 dosage, 797 
 frequency, 797 
 gravity method, 801 
 headache after, 804 
 Herxheimer reaction after, 805 
 intensive treatment, 797 
 Jarisch-Herxlieimer reaction 
 
 after, 804 
 preparation of patient, 798 
 of solution, 798 
 of serum, gravity method, 691 
 syringe method, 690 
 treatment, 090 
 of sheep, 02 
 
 of tetanus antitoxin, 722 
 method of producing hemolysins, 72 
 
 serum precipitins, 70 
 route for tuberculin treatment, 677 
 Invasion, 82 
 
 Iodoform idiosyncrasy, 578 
 Isocytotoxins, 506 
 Isohemagglutinins, 273 
 
 tests for, before transfusion of blood, 
 290 
 
 Isohemolysins, 370 
 
 tests for, before transfusion of blood, 
 290 
 Isolysins, 363 
 Isoprecipitin, 295 
 Izar and AscoU's miostagmin test, 526, 
 
 See alsa Miostagmin reaction. 
 
 Jaffe and Topfer's method of measuring 
 bactericidal power of blood, 352 
 
 Jarisch-Herxheimer reaction, 804 
 
 Jaundice, hemolytic, 372 
 
 Jenner on vaccination, 141, 624 
 
 Jennerian vaccination, 171 
 
 .Tobling and Flexner's method of prepar- 
 ing antrmeningococcus serum, 739 
 
 Joints, tuberculosis of, tubercuhn test 
 in diagnosis of, 590 
 treatment, 679 
 
 KA:\tTNER and Freund's cytolytic cancer 
 diagnosis, 509 
 precipitin test in cancer, 314 
 Keidel tubp for collecting blood, 35, 36 
 Keratosis fpllicularis, salvarsan in, 811 
 Koch's subcutaneous tubercuhn test, 
 587, 592 
 in animals, 600 
 KoUe and "Pfeiffer's macroscopic agglu- 
 tination test, 288 
 and Strong's plague vaccine, 648 
 KoUe's cholera vaccine, preparation, 650 
 method of counting bacterial vaccines, 
 212 
 of preparing antimeningococcus se- 
 rum, 740 
 antipneumococcus serum, 758 
 antistreptococcus serum, 763 
 plague serum, 769 
 plague vaccine, preparation, 648 
 Kolmer's method of testing virulence and 
 
 toxicity»of diphtheria bacilli, 113 
 Korte and Stern's method of measuring 
 bactericidal power of blood, 
 349 
 experimental work, 863 
 Kraus' anticholera serum, 770 
 Kretz, paradox of, 548 
 
 Lactoserum, 293 
 
 experimental work, 846 
 production of, 71 
 
 Landsteiner and Donath's method of 
 serum diagnosis of paroxysmal hemo- 
 globinuria, 379 
 
 Larynx, tuberculosis of, tuberculin test 
 in diagnosis of, 590 
 
 Law, Colles', Wassermann reaction and, 
 464 ' 
 Profeta'S, Wassermann reaction and, 
 464 
 
 Lecithid, 521 
 
884 
 
 INDEX 
 
 Lecithid, cobra, 330 
 Lecithin, 384, 521 
 
 and cholesterin for Wassermann re- 
 action, 423 
 mono-fatty-acid-, 384 
 Leistenkern, 147 
 Leitenketter, 147 
 Leprosy, allergic reactions in, 610 
 autoserum treatment, 776 
 luetin reaction in, 605 
 salvarsan in, 811 
 
 Wassermann reaction in, 465, 466 
 Leukins, 338 
 Leukocidin, 117 
 
 Leukocytes, behavior of, in inflammation, 
 180 
 in quantitative estimation of bacterio- 
 
 tropins, 201 
 obtaining of, 28 
 washed, in opsonic index, 195 
 Leukocytic extracts, 338 
 
 preparation, 339 
 Leukotoxins, 506 
 Lichen planus, salvarsan in, 811 
 Limes death dose of diphtheria toxin, 232 
 
 zero dose of diphtheria toxin, 232 
 Lipoid anaphylaxis, 544 
 Lipoids, 160, 404 
 
 acetone-insoluble, tor Wassermann re- 
 action, 422 
 relation of, to hemolysis, 521 
 to immunity, 520 
 to Wassermann reaction, 522 
 Livers, syphilitic, alcoholic extracts, for 
 Wassermann reaction, 420 
 aqueous extracts, for Wassermann 
 reaction, 419 
 Lobar pneumonia, nature, 755 
 
 serum treatment, 754 
 Locomotor ataxia, Wassermann reaction 
 
 in, 462 
 Looped pipots, 20 
 making of, 20 
 Luetin, preparation of, 602 
 reaction, 601 
 
 in frambesia, 605 
 in leprosy, 605 
 in yaws, 605 
 
 method of application, 502 
 negative, 603 
 normal, 603 
 papular form, 603 
 positive, 603 
 practical value, 605 
 preparation of luetin, 602 
 pustular form, 604 
 results, 605 
 torpid form, 604 
 Lumbar puncture, 37 
 
 after-treatment of patient, 41 
 anesthesia in, 39 
 contraindications, 37 
 disposal of fluid, 41 
 injection of antimeningococcus se- 
 rum by, 742 
 
 Lumbar puncture, injection of neosal- 
 varsan by, 805 
 of serum by, 694 
 of tetanus antitoxin by, 723 
 preparation of patient, 38 
 technic, 39 
 valuCj 37 
 Lupus vulgaris, salvarsan in, 811 
 Lustig and Galeotti's plague vaccine, 648 
 Lustig's method of preparing plague 
 
 serum, 769 
 Lymphocytosis, 178 
 Lysins, 73, 154 
 
 Mackenzie and Browning's modification 
 of Wassermann reaction, 459 
 test for estimating amount of com- 
 plement absorbed in Wassermann 
 reaction, 434 
 Macrooytase, 157, 183 
 Macrophages, 145, 177 
 
 experimental work, 829 
 Malaria, salvarsan in, 811 
 
 Wassermann reaction in, 465 
 Malignant disease, chemotherapy in, 812 
 epiphanin reaction in, 526 
 infection,* 136 
 Malleiu reaction, 606 
 ophthalmic, 607 
 preparation of mallein, 606 
 subcutaneous, 607 
 Malnutrition as predisposing to infec- 
 tion, 101 
 Malta fever, agglutination test in, 277 
 
 autoserum treatment, 776 
 Mantoux's intracutaneous tuberculin 
 
 test, 587^ 595 
 Maraglianb's tuberculosis serum, 771 
 Marasmus, autoserum treatment, 783 
 Marcus modification of MtiUer and Joch- 
 mann's method of testing blood-serum, 
 experimental work, 838 
 Markl's iriethod of preparing plague 
 
 serum, 769 
 Marmorek's tuberculosis serum, 771 
 Measles, effect of, on tubercuhn reaction, 
 
 586 
 Meat adulteration, detection, biologic 
 blood test for, 310 
 technic, 312 
 Meats, identification of, complement- 
 fixation test for, 498 
 Mendel's intracutaneous tuberculin test, 
 ^87, 595 
 iBi^nimals, 601 
 Meningitis, cerebrospinal, agglutination 
 test in, 277 
 precipjitin test in diagnosis, 298 
 serum treatment, 736 
 vaocidation against, 652 
 influenzal, serum treatment, 749 
 meningococcus, epidemic, treatment 
 of, 738 
 nature of, 738 
 
INDEX 
 
 885 
 
 Meningitis, meningococcus, prophylactic 
 immunization in, 748, 749 
 serum treatment, 730 
 
 cases of posterior basal menin- 
 gitis, 745 
 with dry canal, 745 
 with tliick elastic exudate, 745 
 chronic cases, 746 
 results, 746 
 
 serum sickness in, 746 
 subacute cases, 746 
 pneumococcus, 752 
 
 serum treatment, 751, 752 
 tuberculous, autoserum treatment, 782 
 tuberculin test in diagnosis of, 591 
 treatment, 679 
 Meningococcus meningitis, epidemic, 
 treatment of, 73S 
 nature, 738 
 prophylactic immunization in, 748, 
 
 749 
 serum treatment, 736 
 
 cases of posterior basal menin- 
 gitis, 745 
 with dry canal, 745 
 with thick elastic exudate, 
 745 
 chronic cases, 746 
 results, 746 
 serum sickness in, 746 
 subacute cases, 745 
 Mental deficiency, congenital, Wasser- 
 mann reaction in, 465 
 diseases, sero-enzymes in, 265 
 Mercury treatment of syphiUs, effect 
 
 of, on Wassermann reaction, 466 
 Mesenteric glands, tuberculosis of, tu- 
 berculin treatment, 679 
 Metchnikoff's theory of immunity, 144 
 
 and Ehrlich's theory, compatibil- 
 ity, 155 
 of phagocytosis, 176 
 Meyer and Bergmann's antitrypsin test, 
 
 250 
 Mice, intravenous inoculation, 60 
 
 wWte, anaphylaxis in, 540 
 Microc}rt,ase, 157, 183 
 Microorganisms, 83. See also Bacierla. 
 Microphages, 144, 177 
 
 experimental work, 829 
 Milk, bacteria in, 85 
 
 precipitins, experimental work, 846 
 production of, 71 
 Miostagmin reaction, 526 
 experimental work, 864 
 in cancer, 528, 530 
 in echinococcus disease, 530 
 in paratyphoid fever, 530 
 in syphiUs, 530 
 in tuberculosis, 530 
 in typhoid fever, 530 
 practical value, 529 
 principles, 526 
 technic, 527 
 Mixed infection, 105 
 
 Monkey, obtaining large amount of blood> 
 from, 50 '^ 
 
 small amount of blood from, 50 
 
 Mono-fatty-acid-lecithin, 384 
 
 Morbid conditions as predisposing to 
 infection, 102 
 
 Moro and Hamburger's theory of ana- 
 phylaxis, 556 
 
 Moro's percutaneous tuberculin test, 
 587, ,^99 
 
 Much's. psycho-reaction, 388 
 technic, 388 
 
 MuUer and Jochmann's method of test- 
 ing Blood-serum, Marcus modifica- 
 tion, experimental work, 838 
 
 Multiform rashes in serum disease, 574 
 
 Mycosis fungoides, salvarsan in, 811 
 
 Nasal diphtheria, dosage of antitoxin 
 
 in, 710 
 Nastin, 160 ^ 
 Negri bodies in rabies, 637 
 Neisser and ^A'echsberg's method of 
 measuring bactericidal power of blood, 
 349 
 Neosalv-arsan in syphilis, 792 
 administration, 797 
 effect of, on Wassermann reaction, 
 
 469 
 history, 792 
 
 intramuscular injection, 805 
 intravenous injection, 797. See also 
 Intravenous injection of neosalvar- 
 san. 
 subdural injection, 805 
 Wile's teclmic, 806 
 value, 809 
 properties of, 795 _ 
 Nephritis, contraindications to vaccines 
 in, "623 
 normal serum treatment, 774 
 Nephrotoxic serum, production of, 73 
 Nephrotoxin, 506 
 
 action of, experimental work, 863 
 Neufeld's quantitative estimation of 
 bacteriotropins, 200 
 controls, 202 
 
 cultu 
 
 202 
 
 leukocytes, 201 
 precautions, 203 
 readings, 202 
 serum, 201 
 Neurorrhyctes hydrophobic, 637 
 Neurotoxin, 508 
 
 New York Board of Health outfit for 
 collecting blood for Wassermann re- 
 action, 410 
 Ninhydrin, 254 
 
 Noguchi's butyric-acid test for protein, 
 300 
 globulin reaction, experimental work, 
 
 846 
 luetin reaction, 601. See also Luetin 
 reaction. 
 
INDEX 
 
 Noguchi's modification of Wassermann 
 reaction, 440 
 antigen for, 453 
 
 titration of, 453 
 complement for, 450 
 experimental work, 859 
 fluid to be tested, 455 
 hemolytic amboceptor for, 451 
 human corpuscles for, 451 
 teohnio, 450 
 test, 455 
 NoK's theory of anaphylaxis, 558 
 Nucleoproteins, production of, 74 
 Kuttall's method of obtaining large 
 amount of blood from rabbit, 42 
 
 Ophthalmic mallein, preparation, 607 
 reaction, 607 
 reaction in typhoid fever, 608 
 Opsonic index, 191 
 
 as diagnostic procedure, 204 
 
 as guide to size and frequi^ncy of 
 
 doses of bacterial vaccines, 205 
 bacterial emulsion in, 194 
 collection of patient's and control 
 
 serum, 193 
 definition, 191 
 
 determining, experimental work, 834 
 in prognosis, 204 
 limitation, 192 
 practical value, 203 
 precautions in technie, 193 
 principle, 191 
 purpose, 192 
 technie, 193 
 
 precautions in, 193 
 washed leukocytes in, 195 
 Opsonification, susceptibiUty to, 189 
 Oi)Konins, 145, 1S5, 187 
 
 action of, mechanism, experimental 
 
 work, 832 
 definition of, 188 
 effect of, on bacteria, 189 
 experimental work, 815, 831 
 history of, 187 
 -immune, experimental work, 832 
 
 production of, 69 
 in inununity, 190 
 -nature of, 188 
 
 normal, experimental work, 831 
 properties of, 188 
 source of, 189 
 
 .specificity of, experimental work, 833 
 Oral method of administering diphtheria 
 antitoxin, 707 
 route for tubercuUn treatment, 676 
 Organotropism, 785 
 Osmotic pressure of colloids, 512 
 0..T. tubercuhn, dose of, 674 
 
 preparation of, 664 
 Otitis media, vaccine therapy, 660 
 Overproduitioii theory of Wiiigert, 148 
 Overwork as predisposing to disease, 100 
 
 Paltauf'sj tlieory of mechanism of 
 agglutiilation, 270 
 
 Pandemic infect ion, 82 
 
 Paradox of'Kretsi, 548 
 
 Paralysis, general, Wassermann reaction 
 in, 462 
 
 Paralytic dementia, Wassermann reac- 
 tion in, 462 
 
 Parasites, .S3 
 
 animal, .aggressiveness of, 133 
 infection with, 132 
 
 modes, 132 
 production of disease by, 133 
 half, ^2■i 
 partial, 124 
 true, 124, 
 
 Parasitotrppism, 7S5 
 
 Parasyphilitic diseases, W'assermann re- 
 action in, 462 
 
 Paratyphoid fever, miostagmin reaction 
 in, 530 
 
 Paroxysmal henioglobinuria, serum diag- 
 nosis, 379 
 
 Pasteur on immunity, 141, 142 
 treatmf'ht of rabies, intensive, 642 
 mil(% 642 
 prJMtiijle, 640 
 results, 642 
 
 Pasteurian vaccination, 171 ^ 
 
 Pc/arce's method of proilucing nephro- 
 toxic sesum, 73 
 
 Pellagra, fSilvarsan in, 811 
 Wassei-mann reaction in, 466 
 
 Pelvic tuberculosis, tubercuUn test in 
 diagnosis of, 590 
 
 Percutan(;6us tuberculin test of Moro, 
 587, 599 ' 
 
 Peritonitis', tuberculous, tuberculin [test 
 in diagnosis of, 591 
 
 Pertussis, vaccine therapy, 659 
 
 Pfaundler's reaction, 267 
 
 Pfeiffer ai)d KoUe's macroscopic agglu- 
 tination' test, 288 
 
 Pfeiffer's Bacteriolytic test, 342 
 experimental work, 862 
 in diiignosis of disease, 348 
 technie, 347 
 
 Phagocytes, 144, 170 
 varieties,. 177 
 
 Phagocyti?: index, 200 
 
 Phagocytosis, 145, 175 
 (•xprrimetital work, 829, 830 
 history, 175 
 in natural inununity, 168 
 
 experimental work, 826 
 Metchnjkoff's theory, 176 
 relat,ion,0f body-fluids to, 184 
 of cell types to infection, 178 
 results of, 182 
 revised theory of, 186 
 spontaneous, 187 
 theory cif immunity, 144 
 
 and side-ffjain theory, compatibil- 
 ity, 155 
 
 Phenomenon, yVrlhus, of anaphylaxis, 533 
 
INDEX 
 
 887 
 
 Phenomenon, Bordet-Gengou, 332, 391. 
 See also Bordet-Gengou -phenomenon. 
 Smith's, of anaphylaxis, 535 
 Phlebotomy, 33 
 
 ..in children, 33 
 Phogosin, ISO 
 
 Physical theory of anaphylaxis, 558 
 Phytoprecipitin, 292 
 Phytotoxins, 109, 118 
 experimental work, 822 
 general properties, 118 
 relation to immunity, 118 
 Pigeons, anaphylaxis in, 541 
 Pipets, 18 
 
 capillary, for counting bacterial vac- 
 cine, 210 
 for opsonic index determination, 196 
 making of, 18 
 method of sealing, 198 
 rubber teats for, 20 
 Comer's automatic, 215 
 for Wassermarm reaction, 407 
 graduated, 21 
 looped, 20 
 
 making of, 20 
 sterihzation of, 22 
 Piroplasma bigeminum, 171 
 Pirquet's eutaneoios tuberculin test, 587, 
 596 
 in animals, 601 
 studies on anaphylaxis, 533 
 Pityriasis rubra, salvarsan in, 811 
 Placenta as portal of entrance for bac- 
 teria, 90 
 bacteria in, 86, 90 
 Placental blood, method of obtaining, 37 
 
 serum, method of obtaining, 773 
 Plague, agglutination test in, 277 
 serum treatment, 769 
 vaccination, 647 
 
 dosage of vaccine, 649 
 duration, 649 
 effects, 649 
 
 Haffkine's vaccine, 648 
 dosage, 649 
 effects, 649 
 results, 649 
 Kolle and Strong's vaccine, 648 
 Kolle's vaccine, 648 
 Lustig and Galeotti's vaccine, 648 
 preparation of vaccine, 648 
 results, 649 
 
 Terni and Bandi's vaccine, 648 
 Plants, higher, toxins of, 118 
 Plasma, blood, obtaining of, 30 
 Pleurisy, tuberculous, autoserum treat- 
 ment, 781 
 tuberculin test in diagnosis of, 591 
 Pneumococcus, Cole's method of deter- 
 mining type, 757 
 infections, serum treatment, 754 
 meningitis, 752 
 
 serum treatment, 751, 752 
 Pneumonia, agglutination test in, 277 
 autoserum treatment, 776 
 
 Pneumonia, bacterial vaccines in, 660, 
 661 
 experimental, .^16 
 nature af, 755 
 serum tftatment, 754 
 results, 759 
 Poison, protein, 548 
 Polariscope, 262 
 Pollen antitoxin, 224, 734 
 
 toxin, 119 
 Polyceptor, 320 
 
 Porges-AIeier test for syphilis, 299 
 Pork idiosj'ncrasy, 578 
 Precipitate, 294 
 Precipitation of coUoids, 513. See Pre- 
 
 cipitin test. 
 Precipitin, 152, 292 
 
 action of, based on colloidal reactions, 
 
 517 
 bacterial,, experimental work, 846 
 precipitin test with, 298 
 
 technic, 313 
 produjjtion of, 71 
 definition, 292 
 experimental production, 816 
 
 work,, 844 
 formation, 295 
 group, 293, 295 
 history, 292 
 immune, 294 
 in immunity, role of, 297 
 milk, experimental work, 846 
 
 production of, 71 
 nomenclature, 294 
 normal, 294 
 production of, 70 
 
 intrav.enous method, 70 
 properties, 294 
 
 protein, jirecipitin test with, 301 
 senmi, |;-oduction of, 70 
 
 titration, experimental work, 844 
 specificity, 296 
 
 experimental work, 844 
 structurfe, 294 
 test, floccule-forming, 299 
 for blood-stain, 303 
 
 tectinic, 308 
 for detection of meat adulteration, 
 310 
 . technic, 312 
 for differentiation of human and 
 
 animal blood, 303 
 Fomet's, for syphiUs, 299 
 Herman-Perutz, for syphilis, 299 
 in cancer, 314 
 
 Fre\{nd and Kaminer's, 314 
 mechanism, 294 
 Porges-Meier, for syphilis, 299 
 practical appMcations, 298 
 technic, 303 
 
 with bacterial precipitins, in diag- 
 nosis of cerebrospinal menin- 
 gitis, 298 
 practical appUcation, 298 
 technic, 313 
 
INDEX 
 
 Precipitin test with protein precipitins, 
 
 practical application, 301 
 Precipitinogen, 294 
 
 bacterial, preparation of, 313 
 Precipitoid, 294, 295 
 Predisposition, 100. See also Suscepti- 
 
 ■ bility. 
 Pregnancy, Abderhalden's serodiagnosis 
 of, 252. See also Abderhalden's 
 serodiagnosis of pregnancy. 
 allergic reaction in, 610 
 epiphanin reaction in, 526 
 ' ferments in, 248 
 toxicoses of, normal, serum treatment, 
 
 773 
 vomiting of, normal, serum treatment, 
 773 
 Preparator, 319 
 Pricking finger, method, 31 
 Pro-agglutination, 288 
 
 experimental work, 843 
 Pro-agglutinoids, 268 
 Prodromal symptoms of infection, 135 
 Profeta's law, Wassermann reaction and, 
 
 464 
 Prophylactic immunization, 580, 623, 
 
 682. See also Vaccination. 
 Protective substances, 65 
 Proteids, relation of antitoxins to, 223 
 Protein anaphylactogens, chemistry, 544 
 bacterial, 108, 126 
 action of, 12S 
 experimental work, 824 
 nature of, 127 
 split, 126 
 
 Vaughan's theory, 128 
 complement fixation in differentiation 
 
 of, experimental work, 861 
 differentiation by complement fixa- 
 tion, 494 
 fever, 137 
 
 increased amount, Noguchi's butyric- 
 acid test for detection, 300 
 poison, 548 
 
 precipitins, precipitin test with, 301 
 Protoxin, 115 
 Provocatory stimulation of Wassermann 
 
 reaction, 469 
 Psoriasis, salvarsan in, 811 
 Psycho-reaction of Much, 388 
 
 technic, 388 
 Ptomains, 108, 129 
 
 experimental work, 825 
 Puerperal sepsis, antistreptococcus se- 
 rum in, 764 
 vaccine therapy, 661 
 Pulmonary tuberculosis, tuberculin test 
 
 in diagnosis of, 589 
 Pump, suction, 29 
 
 Puncture, lumbar, 37. See also Lumbar 
 puncture. 
 spinal, 37. See also Lumbar puncture. 
 Pyemia, 93 
 Pyocyanase, 244 
 
 Rabbit, aaiaphylaxis in, 539 
 intrapeifi'toneal inoculation, 64 
 
 method of production of agglutin- 
 ins in, 69 
 intravenous inoculation, 56 
 obtainiiig large amounts of blood from, 
 42-46 
 Nuttall's method, 42 
 small amounts of blood from, 41 
 Rabies, 636 
 diagnosis, 638 
 incubation period, 638 
 managetnent, 638 
 nature, 637 
 Negri bodies, 637 
 Pasteui* treatment, intensive, 642 
 mild, 642 
 principle, 640 
 results, 642 
 vaccinej preparation, 640, 641 
 virus fix<; of, 640 
 Eachicentesis, 37. See also Lumbar 
 
 puncturff. 
 Racial immunity, 166 
 
 susceptibility, 100 
 Rashes in serum disease, 573-575 
 multiform, 574 
 scarlatiniform, 575 
 urticarial, 574 
 Rats, intrafvenous inoculation, 60 
 
 obtaining large amount of blood from, 
 46^ 
 small amount of blood from, 46 
 white, aiiaphylaxis in, 540 
 Reaction, Abderhalden's pregnancy, 252. 
 See also Abderhalden's serodiagnosis 
 of pregnancy. 
 agglutisation, 278. See also Aggluti- 
 nation test. 
 aUergiCj 582. See Anaphylactic reac- 
 tion. 
 anaphylactic, 582. See Anaphylactic 
 
 reactOm. 
 antilysih, for antistaphylococous se- 
 rum, 239 
 antitrypsin, 250 
 
 Bergmann and Meyer's, 250 
 Ascoh and Izar's miostagmin, 526. 
 
 See stlso Miostagmin reaction. 
 bacteriolytic, 342. See also Bacterio- 
 lytic te.'it. 
 Bauer's modification of Wassermann, 
 
 456 
 Bergman and Meyer's antitrypsin, 
 
 250 
 biologie blood, for detection of blood- 
 stains, 303 
 technic, 308 
 of meat adulteration, 310 
 technic, 312 
 blood, biologic, for detection of blood- 
 stains, 303 
 technic, 308 
 of meat adulteration, 310 
 technic, 312 
 
INDEX 
 
 889 
 
 Reaction, blood, Teichmann's, 303 
 body, 553 
 
 Bordet-Gengou, 332, 391. See also 
 Bordet-Gengou phenomenon of com- 
 plement fixation. 
 Browning and JNIackenzie's modifica- 
 tion of Wasserniann, 459 
 Calmette tuberculin, 587, 598 
 dangers, 592 
 in animals, 600 
 Castellani's saturation, 288 
 cobra venom, 383. See also Venom 
 
 hemolysis. 
 complement-fixation, 332, 391. See 
 also Bordet-Gengou phenomenon of 
 complement fixation. 
 cutaneous, in typlioid fever, 608 
 cytotoxic, 509 
 in diagnosis, 509 
 
 of Freund and Kaminer, for cancer, 
 509 
 Detre and Brezovsky's modification 
 
 of Wassermann, 459 
 epiphanin, 522. See also Epiphanin 
 
 reaction. 
 for seminal stain, 304 
 Fornet's ring, for syphilis, 299 
 Freund and Kaminer's cytolj^tio can- 
 cer, 509 
 precipitin, for cancer, 314 
 gonococcus complement-fixation, 477. 
 See also Gonococcus complermmt-fixa- 
 tion test. 
 Grviber-Widal, in typhoid fever, 268, 
 
 275, 279, 282 
 Hecht- Weinberg modification of Was- 
 sermann, 457 
 Herman-Perutz, for syphilis, 299 
 immunity, 569 
 Jarisch-Herxheimer, 804 
 Koch's tubercuUn, 587, 592 
 
 in animals, 600 
 KoUe and Pfeiffer's macroscopic ag- 
 glutination, 288 
 luetin, 601. See also Luelin reaction. 
 mallein, 606. See also Mullein reac- 
 tion. 
 Mantoux tuberculin, 587, 595 
 Mendel tuberculin, 587, 595 
 
 in animals, 601 
 miostagmin, 526. See also Miostag- 
 
 min reactio7i. 
 Moro tubercuUn, 587, 599 
 Much's psycho-, 388 
 Noguchi's butyric-acid, for protein, 
 300 
 luetin, 601. See also Luetin reac- 
 tion. 
 modification of Wassermann, 449. 
 See also Noguchi's modification. 
 ophthalmic, in typhoid fever, 608 
 Pfeiffer's bacteriolytic, 342 
 experimental work, 862 
 in diagnosis of disease, 348 
 technic, 347 
 
 Reaction, Porges-Meier, for s3T)hilis, 299 
 precipiti,n. See Precipitin test. 
 psychoi,- of Much, 388 
 saturation, of Castellani, 288 
 Seifert's' epiphanin, 526 
 Stern's: modification of Wassermann, 
 
 458 
 Tchernogubou's modification of Was- 
 sermann, 458 
 Teichmann's blood, 303 
 toxin-antitoxin, nature of experimental 
 
 reaclsion, 836 
 tuberculin, 582. See also Tuberculin 
 
 reaction. 
 typhoidin, 608, 609 
 vaccinoid, 569 
 von Dungern's, in cancer, 500 
 
 modification of Wassermann, 459 
 von Pirquet's tubercuUn, 587, 596 
 
 in animals, 601 
 Wassermann, in syphiUs, 401. See 
 
 also Wassermann reaction. 
 Weichardt's epiphanin, 522. See also 
 
 EpipEanin reaction. 
 Widal, in typhoid fever, experimental 
 
 work, 840 
 Wolff-Eisner tubercuUn, 587, 698 
 dangers, 592 
 in animals, 600 
 Reagin, sypfulis, 405 
 Receptoric atrophy, 80, 171 
 Receptors, 147 
 
 of first order-, 149, 223 
 of third order, 317 
 three orders, 149 
 Rectal injections of serum as preventive 
 of serum disease, 576 
 method of administering diphtheria 
 antitoxin, 707 
 Relapsing fever, salvarsan in, 810 
 Wassermann reaction in, 465 
 Remittent infection, 136 
 Resistance, drug, 789 
 Resistant races, 790 
 
 Respiratory diseases, vaccine therapy, 
 659 
 organs as portal of entrance for bac- 
 teria, 88 
 Retention theory of immunity, 144 
 Revaccination, smallpox, 634 
 Rhinitis, vaccine therapy, 659 
 Richet's studies on anaphylaxis, 533 
 
 theory of anaphylaxis, 556 
 Picin toxin, 118 
 
 Ring test, Fornet's, for syphiUs, 299 
 Rubber teats for capiUary pipets, 20 
 Ruck's tubercuUn, preparation of, 666 
 
 Salt solution, 27 
 
 resistance of erythrocytes to, ex- 
 perimental work, 847 
 Salvarsan, 785 
 
 experimental work, 867 
 in acanthosis nigricans, 811 
 
890 
 
 INDEX 
 
 Salvarsan in Aleppo boil, Sll 
 in anemia, 811 
 in chancroid, 811 
 in chorea, 811 
 
 in dermatitis herpetiformis, 811 
 in Duhring's disease, 811 
 in filariasis, 810 
 in frambesia, 810 
 in Hodgkin's disease, 811 
 in internal anthrax, 768 
 in keratosis folhcularis, 811 
 in leprosy, 811 
 in lichen planus, 811 
 in lupus ■s'ulgaris, 811 
 in malaria, 811 
 in mycosis fungoides, 811 
 in non-syphihtic diseases, 810 
 in pellagra, 811 
 in pityriasis rubra, 811 
 in psoriasis, Sll 
 in relapsing fever, 810 
 in scarlet fever, 811 
 in scurvy, 811 
 in smallpox, 811 
 in syphilis, 792 
 acid solution, 796 
 administration, 797 
 alkaline solution of disodium salt, 
 
 796 
 contraindications, 808 
 effect of, on Wassermann reaction, 
 
 469 
 history, 792 _ 
 intramuscular injection, 805 
 intravenous injection, 797. See 
 also Intrauenous injection of sal- 
 varsan, 
 methods of preparing, for adminis- 
 tration, 795 
 mono-acid solution, 796 
 neutral suspension, 796 
 precautions, 803 
 value, 809 
 in trichinosis, 811 
 in tropical ulcer, 811 
 in trypanosomiasis, 811 
 in tuberculosis, 811 
 in verruca plana, 811 
 in Vincent's angina, 810 
 in yaws, 810 
 properties of, 794 
 Salvarsanized autoserum in syphihs of 
 brain and spinal cord, 
 776 
 after-treatment, 780 
 repeating dose, 780 
 serobiologic findings in 
 
 cerebrospinal fluid, 780 
 technic, 778 
 serum, intraspinous injection, 806 
 Fordyce's technic, 807 
 Saponin, 521 
 Sapremia, 93 
 Saprophytes, 83, 124 
 Sarcoma, epiphanin reaction in, 526 
 
 Saturatioii agglutination reaction, ex- 
 periniental work, 843 
 test of ©astellam, 288 
 Scar in smallpox vaccination, 633 
 Scarlatiniform rashes in serum disease, 
 
 575 
 Scarlet fever, antistreptococcus serum 
 in, 764 
 autosprum treatment, 775 
 salvarsan in, 811 
 vaccination, 652 
 Wassermann reaction in, 465 
 Sclavo's serimi, 767 
 Scurvy, salvarsan in, 811 
 Seifert's epiphanin reaction, 526 
 Selenium-eosin compound in cancer, 812 
 Seminal stains, test for, 304 
 SensibiUsin, 553, 557 
 Sensibilisinogen, 557 
 Sensitization, 536 
 Sensitized vaccines, 620 
 
 bacterial, preparation of, 216 
 Sensitizers, 322 
 Sensitizing substance, 146 
 Sepsis, puerperal, antistreptococcus 
 serum in, 764 
 vaccine therapy, 661 
 Septicemia, 94 
 Serobacterin, 620 
 
 Serodiagnosis of pregnancy, Abderhal- 
 deu's, 252. See also Abderhalden' s 
 serodiagnosis of pregnancy. 
 Sero-cnzymes in acute infections, 265 
 in cancer, 264 
 in disease, 264 
 in mental diseases, 265 
 in syphihs, 265 
 in tubeftulosis, 265 
 Serous mernbranes, tuberculosis of, auto- 
 serum treatment, 781 
 tuitercuUn test in diagnosis of, 
 591 
 Serum, active, 328 
 agglutinating, 68 
 
 powei:, variation in, 274 
 amboceptor unit, 374 
 and corpuscles, obtaining of, 30 
 anti-anthrax, 767 
 
 admi^stration of, 767 
 antichojera, 770 
 
 anticomplementary action, experi- 
 mental work, 857 
 anticytotoxic, 506 
 antidiphthoric, production of, 227 
 antidysenteric, collecting and testing, 
 238 
 production of, 236 
 cultm-e, 236 
 
 immunizing animals, 237 
 antigonococcus, 765 
 actioH of, 766 
 administration of, 766 
 preparation of, 766 
 anti-influenza, 750 
 administration of, 751 
 
INDEX 
 
 891 
 
 Serum, antimeningococcus, 736. See also 
 Antimerdngococciis serum. 
 antiplague, 769 
 antipneumococcus, 757. See also 
 
 Antipneumococcus serum. 
 antistaphylococcus, 238, 766 
 antilysin test for, 239 
 preparation of, 239 
 antistreptococcus, 760. See also Anti- 
 streptococcus serum. 
 antitetanic, production of, 234 
 antitryptic power, testing, experi- 
 mental work, 838 
 antituberculosis, 771 
 antit3rphoid, 768 
 auto-, treatment with, 775. See also 
 
 Autoserum treatment. 
 bacteriolytic, in treatment of disease, 
 359 
 method of titrating, 344 
 production of, 69 
 cadaver, testing of, in Wasscrmann 
 
 reaction, 411 
 calf cholera, 733 
 ..Chantemesse's, 768 
 cytotoxic, production of, 73 
 diagnosis of paroxysmal hemoglobin- 
 uria, 379 
 disease, 534, 537, 571 
 
 accelerated reaction in, 573 
 atropin sulphate as preventive, 576 
 from antimeningococcus serum, 746 
 from diphtheria antitoxin, 712 
 immediate reaction in, 573 
 multiform rashes in, 574 
 nature, 572 
 prevention, 576 
 rashes in, 573-575 
 rectal injections of serum as pre- 
 ventive, 576 
 scarlatiniform rashes in, 575 
 severe, 575 
 symptoms, 573 
 treatment, 577 
 urticarial rashes in, 574 
 doses of, in Wasscrmaim reaction, 411 
 hemolysis, experimental work, 848 
 quantitative factors, experimental 
 work, 849 
 hemolytic, production of, 71 
 hog cholera, production of, 732 
 
 standardization of, 732 
 immune, and normal serum, difference 
 between, 324 
 methods for making, 66 
 preservation of, 76 
 
 in dried paper form, 79 
 in fluid form, by bacteria-free 
 filtration, 76 
 by freezing, 78 
 with antiseptics, 76 
 in living animal, 80 
 in powder form, 79 
 standardization of, complement- 
 fixation test in, 491 
 
 Serum, inactivated, 327, 328 
 
 intramuscular inoculation, technic, 690 
 intravenous inoculation, gravity 
 method, 691 
 syringe method, 690 
 technic, 690 
 Kraus' anticholera, 770 
 MaragUano's tuberculosis, 771 
 Marmorok's tuberculosis, 771 
 methods of inoculation with, 687 
 nephrotoxic, production of, 73 
 normal, 772. Sec also Serum treat- 
 ment, normal. 
 and immune serum, difference be- 
 tween, 324 
 preservation of, 75 
 obtaining of, 30 
 
 patient's own, 775. See also Auto- 
 serum treat7nent. 
 placental, method of obtaining, 773 
 precipitins, 70 
 
 experimental work, 844 
 production of, 70 
 preservation of, 75 
 reactivated, 328 
 rectal injections, as preventive of 
 
 serum disease, 576 
 salvarsanized, intraspinous injection, 
 806 
 Fordyce's technic, 807 
 Sclavo'Sr 767 
 
 sickness, 534, 537, 571. See also Se- 
 rum disease. 
 subcutaneous inoculation, technic, 688 
 subdural; inoculation, technic, 694. 
 
 See also Subdural inoculation. 
 treatment, 614, 681 
 anaphylaxis in, 686 
 asthma in, 687 
 contraindications to, 686 
 indications, 684 
 intramuscular inoculation, technic, 
 
 690 
 intravenous inoculation, gravity 
 method, 691 
 syringe method, 690 
 technic, 690 
 methods of inoculation, 687 
 normal, 772 
 in hemorrhage, 772 
 in nephritis, 774 
 in skin diseases, 774 
 in toxicoses of pregnancy, 773 
 in vomiting of pregnancy, 773 
 of anthrax, 767 
 of bubonic plague, 769 
 of cerebrospinal meningitis, 736 
 of cholera, 770 
 
 of diphtheria, 702. See also Diph- 
 theria antitoxin. 
 of dysentery, 730 
 
 resuHs, 731 
 of gonococcal infections, 765 
 of hay-fever, 734 
 of hog; cholera, 732 
 
892 
 
 INDEX 
 
 Serum treatment of influenzal meain- 
 gitis, 749 
 of internal antlirax, 768 
 of memngococcus meningitis, 736 
 cases of posterior basal menin- 
 gitis, 745 
 with dry canal, 745 
 with thick elastic exudate, 745 
 chronic cases, 746 
 results, 746 
 serum sickness in, 646 
 subacute cases, 746 
 of plague, 769 
 of pneumococcus infections, 754 
 
 meningitis, 751, 752 
 of pneumonia, 754 
 
 results, 759 
 of snake-bites, 733 
 of staphylococcus infections, 766 
 of streptococcus infections, 760 
 of tetanus, 719. See also Tetanus 
 
 antitoxin. 
 of tuberculosis, 771 
 of typhoid fever, 768 
 purposes, 682 
 status lymphaticus in, 687 
 subcutaneous inoculation, technic, 
 
 688 
 subdural inoculation, technic, 694. 
 See also Subdural inoculation. 
 Sheep, anaphylaxis in, 541 
 intravenous inoculation, 62 
 obtaining large amount of blood from, 
 48 
 small amount of blood from, 42, 49 
 Side-chain theory of immunity, 146 
 
 and phagocytic theory, compati- 
 biUty, 155 
 Simon and Lamar's modification of 
 Wright's method for opsonic index, 200 
 Skin as portal of entrance for bacteria, 87 
 , bacteria on, 85, 87 
 diseases, autoserum treatment, 775 
 normal serum treatment, 774 
 salvarsan in, 811 
 vaccine therapy, 656 
 tuberculosis of, tuberculin test in diag- 
 nosis of, 590 
 treatment, 679 
 Smallpox, antistreptooooous senmi in, 
 764 
 autoserum treatment, 776 
 revaccination, 634 
 salvarsan in, 811 
 vaccination, 623. See also Vaccination, 
 
 smallpox. 
 vaccinia and, relationsliip, 625 
 Smith's phenomenon of anaphylaxis, 535 
 Snake venoms, 119, 241 
 nature of, 120 
 properties of, 119 
 Snake-bites, serum treatment, 733 
 Sodium citrate, 27 
 Solutions, 27 
 colloidal, 512 
 
 Southard and Gay's theory of anaphy- 
 laxis, 557 
 Species immunity, 166 
 
 susceptibiUty, 100 
 SpermatotQxin, 506 
 
 Spinal cord, syphihs of, salvarsanized 
 autoserum in, 776 
 after-treatment, 780 
 repeating dose, 780 
 serobiologic findings in cere- 
 brospinal fluid, 780 
 technic, 778 
 puncture, 37. See also Lumbar punc- 
 ture. 
 Sphtting (Jf complement, 331 
 Spontaneous phagoc;>tosiB, 187 
 Sporadic infection, 82 
 Sporotrichosis, allergic reactions in, 610 
 Stain, bloo^, biologic test for, 303 
 technic, 308 
 experimental work, S45 
 identification of, complement-fixa- 
 
 tioij test for, 494 
 test f6r, experimental work, 861 
 seminal, test for, 304 
 Staining acid-fast bacilli, 197 
 
 bacteria; 197 
 Stalagmometer, Traube's, 527 
 Standardization of antimeningococeus 
 serum, 740 
 of antipneumococcus serum, 758 
 of antistfeptococcus serum, 763 
 of bacterial antigens in complement 
 fixation, 475 
 vaccines, 210 
 of diphtneria antitoxin, 231 
 experimental work, 835 
 toxin, 114 
 of hog cholera serum, 732 
 of immune serums, complement-fixa- 
 tion test in, 491 
 of tetanus antitoxin, 234, 720 
 experimental work, 836 
 Staphylococcus, 117 
 
 infections, serum treatment, 766 
 vaccine,; preparation, experimental 
 work, §35 
 Staphyloljssin, 117 
 
 method of titrating, 240 
 preparation of, 239 
 Staphylotoxin, experimental work, 821 
 Status Ijrnjphaticus in scrum treatment, 
 
 687 , 
 
 SteriUzatioil of bacterial vaccines, 213 
 testing, 213 
 of pipets, 22 
 of syringes, 27 
 of test-tubes, 25 
 Stern and Korte's method of measuring 
 bactericidal power of blood, 
 349 
 experimental work, 863 
 Stern's modification of Wassermann re- 
 action, 4S8 
 Stichreaktion, 586 
 
INDEX 
 
 893 
 
 Stimulation, provocatory, of Wasser- 
 
 mann reaction, 469 
 Stimulins, 145, 187 
 Stocl?: bacterial vaccines, 620 
 Strawberries, idiosyncrasy, 578 
 Streptococcus, 117 
 
 infections, serum treatment, 760 
 Streptolysin, 117 
 
 Streptotoxin, experimental work, 822 
 Strong and KoUe's plague vaccine, 648 
 Strong's cholera vaccine, preparation, 651 
 Subcutaneous injection of animals, 54 
 with fluid inoculum, 54 
 with solid inoculum, 55 
 of diphtheria antitoxin, 707 
 of serum, technic, 688 
 of tetanus antitoxin, 722 
 of tuberculin, 671, 675 
 mallein reaction^ 607 
 tuberculin reaction, 587, 592 
 in animals, 600 
 Subdural injection of antimeningococcus 
 serum, 742 
 of neosalvarsan in syphilis, 805 
 
 Wile's technic, 806 
 of salvarsanized serum, 806 
 of serum, anesthesia for, 697 
 blood-pressure as guide, 695 
 coUapse in, 694, 696 
 symptoms, 696 
 treatment, 696 
 gravity method, 697 
 syringe method, 700 
 technic, 694 
 of tetanus antitoxin, 723 
 Subinfection, 81, 89 
 Substance sensibilisatrice, 185, 319, 322, 
 
 337, 362 
 Suction pump, 29 
 
 Suctorial insects, transmission of bac- 
 teria by, 86 
 -Susceptibility, acquired, 100 
 familial, 100 
 
 general, in relation to infection, 99 
 individual, 100 
 inherited, 100 
 racial, 100 
 species, 100 
 Suspensions, 511 
 
 coUoidal, 512 
 Sycosis, vaccine therapy, 657 
 Synocytotoxin, 507 
 
 S3T)hiii3 after smallpox vaccination, 635 
 Bauer's modification of Wassermann 
 
 reaction, 456 
 Browning and Mackenzie's modifica- 
 tion of Wassermann reaction, 459 
 complement fixation in, 401. Sec also 
 
 Wassermann reaction. 
 Detre and Brezovsky's modification 
 
 of Wassermann reaction, 459 
 epiphanin reaction in, 526 
 Fornet's ring test for, 299 
 Hecht-Weinberg modification of Was- 
 sermann reaction, 457 
 
 Syphilis, Herman-Perutz test for, 299 
 horse, complement-fixation test in, 487 
 luetin reaction for, 601. See also 
 
 Luelin reaction. 
 mercury treatment, effect of, on Was- 
 sermann reaction, 466 
 miostagmin reaction in, 530 
 neosalvarsan in, 792. See also Neo- 
 salvarsan in syphilis. 
 Noguchi's modification of Wasser- 
 maim reaction, 449. See also No- 
 guchi's modification. 
 of brain, salvarsanized autoserum in, 
 776 
 after-treatment, 780 
 repeating dose, 780 
 serobiologic findings in cere- 
 brospinal fluid, 780 
 technic, 778 
 of spinal cord, salvarsanized auto- 
 serum, 776 
 after-treatment, 780 
 repeating dose, 780 
 serobiologic findings in cere- 
 brospinal fluid, 780 
 technic, 778 
 Porges-Meier test for, 299 
 reagin, 405 
 salvafsau in, 792. See also Salvarsan 
 
 in syphilis. 
 sero-enzymes in, 265 
 Stern's modification of Wassermann 
 
 reaction, 458 
 Tchernogubou's modification of Was- 
 sermann reaction, 458 
 venom hemolysis in, 385 
 experimental work, 861 
 practical value, 388 
 technic, 385 
 von BTungern's modification of Was- 
 sermann reaction, 459 
 Wassermann reaction in, 401. See 
 also Wassermann reaction. 
 Syphifitic Uvers, alcohoUo extracts, for 
 Wassermann reaction, 420 
 aqueous extracts, for Wassermann 
 reaction, 419 
 Syringe,' 26 
 
 for administration of bacterial vac- 
 cines, 217 
 Kitchens', 233 
 
 method of intravenous inoculation of 
 selum, 690 
 of subdural inoculation of serum, 
 700 
 steriUzation of, 27 
 Systemic reaction to infection, 137 
 
 Tabes dorsalis, Wassermann reaction in, 
 462 
 
 T. B. tu>erculin, 666 
 
 Tcherndgubou's modification of Wasser- 
 mann. reaction, 458 
 
 Teats, rubber, for capillary pipets, 20 
 
894 
 
 INDEX 
 
 Technic, general, 17 
 Teichmann's blood test, 303 
 Temperature, influence of, on natural 
 
 immuiiity, experimental work, 827 
 Terni-Bandi's method of preparing 
 plague serum, 769 
 plague vaccine, 648 
 Tertiary syphilis, Wassermann reaction 
 
 in, 641 
 Test. See Reaction. 
 
 Test-tubes, conversion of, into ampules 
 for holding vaccines, etc., 24 
 for immunologic work, 25 
 for Wassermann reaction, 407 
 sterihzation of, 25 
 Totanolysin, 111, 115, 819 
 Tetanospasmin, 111, 115, 819 
 Tetanus after smallpox vaccination, 634 
 antitoxin, 719, 726 
 action, 721 
 
 intramuscular injection, 722 
 intraneural injection, 723 
 intravenous injection, 722 
 method of titrating, 235 
 methods of administering, 722 
 preparation, 720 
 production of, 234 
 collecting serum, 234 
 immunizing animals, 234 
 prophylactic admimst ration, 723, 
 
 725 
 results of treatment, 728 
 standardization, 234, 720 
 experimental work, 836 
 subcutaneous injection, 722 
 subdural injection, 723 
 unit of, 242 
 bacillus, 115 
 prophylaxis, 723 
 serum treatment, 719. See also 
 
 Tetanus anliloxin. 
 surgical treatment, 725, 726 
 toxin, 115 
 action, 720 
 
 experimental work, 819 
 production of, 234 
 -treatment of, 719, 726. See also 
 Tetanus antitoxin. 
 Therapeutic immunization, 580 
 Therapia magna sterihsans, 791 
 Thyrotoxins, 508 
 Tissue susocptibihty, avenue of infection 
 
 and, 97 
 T. O. tuberculin, 665 
 Tonsillar diphtheria, dosage of antitoxin 
 
 in, 710 
 Topfer and Jaffe's method of measuring 
 
 bactericidal power of blood, 352 
 Toxemia, 94 
 
 Toxicity of bacteria, 94, 95 
 Toxicoses of pregnancy, normal serum 
 
 treatment, 773 
 Toxin, 73, 108 
 abrin, 118 
 and ferment, similarity between, 244 
 
 Toxin, bottllism, 116 
 
 experiinental work, 820 
 crotin, 118 
 diphtheria, 112 
 
 expeiamental work, 817 
 Umes death dose, 232 
 
 zero dose, 232 
 
 production of, 227 
 
 standardizing, 114 
 
 testing, 228 
 
 virulence, 113 
 
 dysentery, 116 
 
 experimental work, 820 
 exogenous, 108 
 extracelltilar, 108, 109 
 chemical properties, 110 
 general properties, 109 
 haptophore group, 110 
 nature of, 110 
 nomencjatuie, 108 
 of hay-f'ever, 119 
 of higher plants and animals, 118 
 pollen, 119 
 
 principal, special properties, 112 
 ricin, if 8 
 
 selective action. 111 
 soluble,^ 108 
 
 chemical properties, 110 
 general properties, 109 
 structmie of, 110 
 tetanus, 115 
 action of, 720 
 experimental work, 819 
 production of, 234 
 toxophore group, 110 
 Toxin-antitoxin reaction, nature of, 224 
 
 experimental work, 836 
 Toxogen, 553 
 Toxogenin, 556 
 Toxoid, 110 
 
 diphtheria, 115 
 Toxon, 111 
 
 Toxophore group of toxin, 110 
 T. R. tubercuUn, dose of, 674 
 
 preparation of, 665 
 Transfusion, blood, 273 
 
 experimental work, 844 
 tests before, for isohemagglutinins 
 and isohemolysins, 290 
 Traube's ^talagmometer, 527 
 Trauma as predisposing to infection, 102 
 Treatment, cytotoxins in, 508 
 
 effect of, on Wassermann reaction, 466 
 Treponema paUidum, aqueous extract 
 of cultdre, for Wassermann reaction, 
 424 
 Trichina spiralis, 167 
 Tricliinosis, salvarsan in, 811 
 Tritotoxin,115 
 
 Tropical ulcer, salvarsan in, 811 
 Trypanosomiasis, salvarsan in, 811 
 Tubercle baciUi, Uving, in treatment of 
 
 tuberculosis, 669 
 Tuberculin, action of, 668 
 
 administration of, methods, 671, 676 
 
INDEX 
 
 895 
 
 Tuberculin, bacillen emulsion, prepara- 
 tion of, 666 
 B. E., preparation of, 666 
 Beraneck's, dose of, 675 
 
 preparation of, 666 
 B. F., preparation of, 666 
 bouillon filtrate, preparation of, 666 
 delirium, 672 
 Dixon's, dose of, 675 
 prepa^tion of, 667 
 new, dose of, 674 
 
 preparation of, 665 
 old, dose of, 674 
 
 preparation of, 664 
 O. T., dose of, 674 
 
 preparation of, 664 
 preparation of, 664 
 reaction, 582 
 
 Calmette's, 587, 598 
 dangers, 592 
 in animals, 600 
 comparative delicacy and relation, 
 
 587 
 conjunctival, of Calmette, 587, 598 
 dangers, 592 
 in animals, 600 
 of Wolff-Eisner, 587, 598 
 . dangers, 592 
 in animals, 600 
 cutaneous, of von Pirquet, 587, 596 
 
 in animals, 601 
 dangers, 591 
 delicacy, 587 
 effect of measles on, 586 
 error in, sources, 585 
 false negative, 585 
 
 positive, 585 
 in animals, 600 
 
 in diagnoBis of pelvic tuberculosis, 
 590 
 of pulmonary tuberculosis, 589 
 of tuberculosis of bones, joints, 
 and glands, 590 
 of eye, ear, and larynx, 590 
 of genito-urinary system, 590 
 of serum membrane, 591 
 of skin, 590 
 of tuberculous meningitis, 591 
 peritonitis, 591 
 pleurisy, 591 
 value, 589 
 in prognosis, value, 591 
 intracutaneous, of Mantoux, 587, 
 595 
 of Mendel, 587, 595 
 in animals, 601 
 Mantoux's, 587, 595 
 Mendel's, 587, 595 
 in animals, 601 
 methods of conducting, 587 
 Moro's, 587, 599 
 nature, 583 
 
 percutaneous, of Moro, 587, 599 
 relation, 587 
 Bpeoificity, 583 
 
 Tuberculin reaction, subcutaneous, 587, 
 592 
 in animals, 600 
 von Pirquet's, 587, 596 
 
 in animals, 601 
 Wolff-Eisner's, 587, 598 
 dangers, 592 
 in animals, 600 
 residue, preparation, 665 
 subcutaneous injection, 671 
 T. 0., 0G5 
 T. R., d6se of, 674 
 
 preparation of, 605 
 treatmeMt, 661 
 
 action of tuberculin, 668 
 contraindications, 671 
 dosage,- 674 
 history, 661 
 
 in tuberculosis of bones and joints, 
 679 
 of ear, 679 
 of eye, 679 
 
 of genito-urinary organs, 680 
 of intestine, 679 
 of mesenteric glands, 679 
 of skin, 679 
 in tuberculous adenitis, 679 
 
 meningitis, 679 
 intrabronchially, 677 
 intrafocal route, 677 
 intravenous, 677 
 living tubercle bacilli, 669 
 methods of administration, 671, 676 
 oral route, 676 
 patients suitable, 670 
 preparation of tuberculin, 664 
 reaction in, 672 
 
 constitutional symptoms, 672 
 focafl'signs, 673 
 local signs, 673 
 results, 678 
 
 subcutaneous injection, 671, 675 
 von Ruck's, preparation of, 666 
 Tuberculonastin, 160 
 Tuberculosis, agglutination test in, 277 
 antistreptococcus serum in, 764 
 complement-fixation test in, 490 
 contraindications to vaccines in, 623 
 Friedmanji's treatment, 670 
 genito-urinary, tuberculin test in diag- 
 nosis of, 590 
 living tubercle baciUi in treatment of, 
 
 669 
 miostagmin reaction in, 530 
 of bones and joints, tuberculin treat- 
 ment, 679 
 tuberculin test in diagnosis of, 590 
 of ear, tubercuUn test in diagnosis of, 
 590 
 treatment, 679 
 of eye, tubercuUn test in diagnosis of, 
 590 
 treatment, 679 
 of genito-urinary organs, tuberculin 
 treatment, 680 
 
896 
 
 INDEX 
 
 Tuberculosis of glands, tuberculin test in 
 diagnosis of, 590 
 of intestine, tuberculin treatment, 679 
 of joints, tuberculin test in diagnosis of , 
 
 590 
 of larynx, tuberculin test in diagnosis 
 
 of, 590 
 of mesenteric glands, tuberculin treat- 
 ment, 679 
 of serous membranes, autoserum treat- 
 ment, 781 
 tuberculin test in diagnosis of, 591 
 of skin, tuberculin test in diagnosis of, 
 590 
 treatment, 679 
 pelvic, tuberculin test in diagnosis of, 
 
 590 
 pulmonary, tuberculin reaction in 
 
 diagnosis of, 589 
 salvarsan in, 811 
 sero-enzymes in, 265 
 serum treatment, 771 
 tuberculin treatment, 661. See also 
 
 Tuberculin treatment. 
 venom hemolysis in, 390 
 Tuberculous adenitis, tuberculin treat- 
 ment, 679 
 meningitis, autoserum treatment, 782 
 tuberculin test in diagnosis of, 591 
 treatment, 679 
 peritonitis, tuberculin test in diagnosis 
 
 of, 591 
 pleurisy, autoserum treatment, 781 
 tuberculin test in diagnosis of, 591 
 Typhoid fever, agglutination test in, 275, 
 279, 282 
 allergic reactions in, 607 
 autoserum treatment, 776 
 complement-fixation test in, 489 
 cutaneous reaction in, 608 
 Gruber-TVidal reaction in, 268, 275, 
 
 279, 282 
 miostagmin reaction in, 530 
 ophthalmic reaction in, 608 
 serum treatment, 768 
 vaccination, 643 
 
 dosage of vaccine, 645 
 duration and degree, 646 
 method of inoculation, 644 
 preparation of vaccine, 643 
 reactions, 645 
 recommendations, 647 
 results, 646 
 vaccine therapy, 660 
 Widal reaction in, experimental 
 work, 840 
 vaccine, preparation, experimental 
 work, 834 
 Tjrphoidin reaction, 608, 609 
 Typho-protein, 608 
 
 Upfenheimer's method of determining 
 
 presence of diphtheria toxin, 114 
 Uhlenhuth filter, 305 
 
 Ulcer, tro{>ical, salvarsan in, 811 
 Ulcerative endocarditis, vaccine therapy, 
 
 661 
 Umstimmung, 601 
 Unit, amboceptor, of serum, 374 
 
 antitoxin, 242 
 
 of diphtheria antitoxin, 242 
 
 of tetanus antitoxin, 242 
 Urethritis, Vaccine therapy, 658 
 Urticarial 'rashes in serum disease, 574 
 
 Vaccination, 65, 611, 613, 614, 623, 
 
 682 
 active acfluired immunity by, 171 
 anthrax; 653 
 blackleg', 655 
 bubonic plague, 647 
 cerebrospinal meningitis, 652 
 cholera, 650 
 
 dosage of vaccine, 651 
 
 Haffkine's vaccine, 650 
 
 Kolle's vaccine, 650 
 
 preparation of vaccine, 650 
 
 results of, 651 
 
 Stronf!.'s vaccine, 651 
 diphtheria, 715 
 dysentery, 652 
 history of, 611 
 Jennerian, 171 
 
 meningococcus meningitis, 748, 749 
 of animals, 627 
 
 preparation for, 627 
 Pasteurian, 171 
 plague, 647 
 
 dosage of vaccine, 649 
 
 duration of, 649 
 
 effects of, 649 
 
 Haffkine's vaccine, 648 
 dosage, 649 
 e|fects, 649 
 results, 649 
 
 KoUe and Strong's vaccine, 648 
 
 Kolle's vaccine, 648 
 
 Lustig and Galeotti's vaccine, 648 
 
 preparation of vaccine, 648 
 
 results of, 649 
 
 Terni and Bandi's vaccine, 648 
 preparation of animals for, 627 
 rabies, 630. See also Rabies. 
 scarlet f^ver, 652 
 smallpox' 623 
 
 history of, 623 
 
 phenomena of, 632 
 
 preparation of vaccine, 626 
 
 protective value, 635 
 
 revaccination, 634 
 
 risks of, 634 
 
 scar in, 633 
 
 subsequent care of wound, 632 
 
 syphilis after, 635 
 
 tcchnic of, 630 
 
 tetanus after, 634 
 typhoidi fever, 643. See also Typhoid 
 
 fever iaccination. 
 
INDEX 
 
 897 
 
 Vaccine, 613 
 
 ampules, conversion of test-tubes into, 
 24 
 making of, 24 
 anthrax, 653 
 autogenous, 620 
 bacterial, 206, 613 
 administration of, 217 
 frequency, 218 
 syringe for, 217 
 autogenous, 620 
 counting of, 210 
 
 Hopkins' method, 212 
 
 KoUe's method, 212 
 
 with hemocytometcr chamber, 
 
 211 
 Wright's method, 210 
 definition of, 206 
 dosage of, 218 
 
 opsonic index as guide to, 206 
 experimental work, 834 
 in acne, 657 
 in acute infections, 660 
 in bronchitis, 659 
 in carbuncles, 656 
 in cystitis, 657 
 in erysipelas, 657 
 in furunculosis, 656 
 in genito-urinary diseases, 657 
 in gonorrheal arthritis, 658 
 in otitis media, 660 
 in pertussis, 659 
 in pneumonia, 660, 661 
 in puerperal sepsis, 661 
 in respiratory diseases, 659 
 in rhinitis, 659 
 in skin diseases, 656 
 in sycosis, 657 
 in typhoid fever, 660 
 in ulcerative endocarditis, 661 
 in urethritis, 658 
 in vulvovaginitis of children, 659 
 in whooping-cough, 659 
 inoculation with, dosage, 218 
 effects of, 218 
 method of making, 217 
 sensitized, preparation of, 216 
 standardization of, 210 
 stock, 620 
 technic for preparing, 206 
 
 diluting and adding preserva- 
 tive, 214 
 emulsion, 208 
 
 procuring infected material, 206 
 pure cultures, 207 
 sterilization, 213 
 testing, 213 
 treatment by, 655 
 blackleg, 655 
 cerebrospinal, 652 
 cholera, preparation of, 650 
 contraindications to, 622 
 in cancer, 623 
 in diabetes, 625 
 in nephritis, 623 
 57 
 
 Vaccine, contraindications to, in tubercu- 
 losis, 623 
 cowpox, preparation of, 626 
 animals, 627 
 collection of virus, 628 
 seed virus, 626 
 testing virus, 629 
 dead, 629 
 dysentery, 652 
 
 Haffkine's cholera, preparation, 650 
 plague, 648 
 dosage, 649 
 effects, 649 
 results, 649 
 in prophylaxis of disease, 611 
 KoUe and Strong's plague, 648 
 KoUe's cholera, preparation, 650 
 
 plague, 648 
 living, 620 
 
 Lustig and Galeotti's plague, 648 
 nomenclature, 613 
 plague, preparation of, 648 
 preparation of, method, 614 
 rabies, administration of, 641 
 
 preparation of, 640 
 scarlet fever, 652 
 sensitized, 620 
 staphylococcus, experimental work, 
 
 835 
 stock, 620 
 
 Strong's- cholera, preparation, 651 
 Terni and Bandi's plague, 648 
 treatment, 611, 614, 617 
 
 history, 611 
 typhoid, experimental work, 834 
 preparation of, 643 
 Vaccinia, 612, 613, 632 
 
 smallpox and, relationship, 625 
 ^'aeeinoid, 633 
 reaction,, 569 
 Variola. Sec Smallpox. 
 Varioloid, 634 
 Vaughan and Wheeler on anaphylatoxin, 
 
 548, 549 
 Vaughan and 'WTieeler's theory of ana- 
 phylaxis, 557 
 Vaughan's theory of bacterial proteins, 
 128 
 of immunity, 173 
 Vegetables, idiosyncrasy, 578 
 Venesection, 33 
 
 in children, 33 
 Venom, cobra, experimental work, 823 
 preparation of, 385 
 hemolysis, 383, 521 
 in cancer, 390 
 in syphihs, 385 
 
 experimental work, 861 
 practical value, 388 
 technic, 385 
 in tuberculosis, 390 
 nature, 383 
 snake, 119, 241 
 properties of, 119 
 nature of, 120 
 
898 
 
 INDEX 
 
 Verruca plana, salvarsan in, 811 
 Vincent's angina, sal\-arsau in, SIO 
 Virulence of bacteria, 94 
 decrease, 95 
 increase, 96 
 
 by addition of animal fluids to 
 
 culture-medium, 97 
 by passage through animals, 96 
 by use of collodion sacs, 97 
 Mrus fixe of rabies, 143, 640 
 ^"Qmiting of pregnane}', normal sertmi 
 
 treatment, 773 
 von Dungern's complement-fixation test 
 in cancer, 500 
 modification of VVasserman reaction, 
 459 
 von Pirquet's cutaneous tubcrcuUn test, 
 587, 596 
 in animals, 601 
 studies on anaphylaxis, 533 
 von Ruck's tuberculin, preparation, 666 
 Vulva, diphtheria of, dosage of antitoxin 
 
 in, 711 
 Vulvo^'aginitis of cliildren, vaccine 
 therapy, 659 
 
 Washed leukocytes in opsonic index, 195 
 Washing erythrocytes, 28 
 Wassermann reaction after anesthesia, 
 466 
 CoUes' law and, 464 
 in cerebral syphilis, 462 
 in congenital mental deficiency, 465 
 
 syphihs, 463, 464 
 in frambesia, 465, 466 
 in general paralysis, 462 
 in latent syphihs, 462 
 in leprosy, 465, 466 
 in malaria, 465 
 in pai'alytic dementia, 462 
 in parasyphilitic diseases, 462 
 in pellagra, 400 
 in primary sypliilis, 459 
 in relapsing fever, 465 
 in scarlet fever, 465 
 in secondary syphihs, 460 
 in S3rphilis, 401 
 
 acetone-insoluble hpoids for, 422 
 aloohoUc extracts of normal or- 
 gans for, 420 
 reenforced with cholesterin 
 for, 421 
 of syphihtio hvers for, 420 
 antigen for, 417 
 
 anticomplementary titration, 
 
 428 
 antigenic titration, 431 
 experimental work, 856 
 hemolytic titration, 430 
 method of diluting, 427 
 
 titrating, 428 
 preparation, 419 
 antigenic dose of serum, 41 8 
 antisheep amboceptor for, 415 
 
 Wassermarm reaction in syphilis, aque- 
 ous ex-tract of pallidum cul- 
 ture for, 424 
 of sypliilitic livers for, 419 
 as colloidal reaction, 520 
 Bauer's modification, 456 
 Browning and Mackenzie's modi- 
 fication, 459 
 cerebrospinal fluid for, 411 
 colfe'cting blood for, 408 
 
 Xew York Board of Health 
 outfit, 410 
 Colles' law and, 464 
 comparative antigenic values of 
 
 various extracts, 424 
 complement for, 411 
 
 titration, 413 
 congenital, 463, 464 
 Detre and Brezovsky's modifica- 
 tion, 459 
 doses of serum in, 411 
 eifeict of mercury treatment on, 
 400 
 of neosaharsan on, 469 
 of salvarsa.n on, 469 
 oj treatment on, 466 
 erythrocytes for, 416 
 ex-pel-imental work, 857, 858 
 first method, 432 
 technic, 435 
 flui^ to be tested, 408 
 foujth method, 434 
 
 reading results, 449 
 technic, 446 
 glassware for, 407 
 guinea-pig complement serum 
 
 fo«, 412 
 Hecht-A\ einberg modification, 457 
 herflolytic amboceptor for, 415 
 history, 401 
 latent, 462 
 
 lecitliin and cholesterin for, 423 
 methods for conducting, 432 
 modifications, 449 
 Noguchi's modification, 449. See 
 
 also Noguchi's modification. 
 origmal, 432, 435 
 technic of, 435 
 pipete for, 407 
 praetical value, 469 
 primary, 459 
 
 principles and theories, 404 
 Profeta's law and, 464 
 provocatory stimulation, 469 
 reading and recording, 439 
 red blood-corpuscles for, 416 
 relation of hpoids to, 522 
 second method, 433 
 
 reading restdts, 442 
 teclmic, 441 
 secondary, 460 
 serum for, 408 
 specificitjr, 465 
 Stem's modification, 458 
 Tclte'rnogubou's modification, 458 
 
INDEX 
 
 Wassermann reaction in syphilis, technic, 
 407 
 tertiary, 461 
 
 testing cadaver serums, 411 
 test-tubes for, 407 
 third method, 434 
 
 reading results, 444 
 technic, 443 
 various stages, 459 
 von Dungern's modification, 459 
 in tabes dorsalis, 462 
 in tertiary sypliilis, 461 
 in various stages o£ sypliiliis, 459 
 in yaws, 465 
 Profeta's law and, 464 
 Water, bacteria in, y5 
 Water-supplies, bacteria recovered from, 
 bacteriolytic test for identification of, 
 343 
 Wechsberg and Neisser's method of 
 measuring bactericidal power of blood, 
 349 
 ■Wechseknann's method of converting 
 negative sypliilitic serums to positive 
 syphilitic serums, 410 
 Weichardt's epiphanin reaction, ,522. 
 
 See also Epiphanin reaction. 
 Weigert's overproduction theory, 14S 
 Welch, hjrpothesis of, 103 
 Wet cupping, 36 
 
 Wheeler and Vaughan's theory of ana- 
 phylaxis, 557 
 White mice, anaphylaxis in, 540 
 
 rats, anaphylaxis in, 540 
 Whooping-cough, vaccine therapy, 659 
 Widal reaction in typhoid fever, 268, 275, 
 279, 282 
 
 Widal reaction in typhoid fever, experi- 
 mental work, 840 
 Wile's technic of intraspinous injection 
 
 of neosalvarsan, 806 
 Wolff-Eisner conjunctival tuberculin 
 test, 587, 598 
 dangers, 592 
 in animals, 600 
 Wolman and Hamman's method of pre- 
 paring tuberculin for subcutane- 
 ous tuberculin test, 592 
 plan of dosage for tuberculin in (sub- 
 cutaneous test, 594 
 Wounds, diphtheria of, dosage of anti- 
 toxin in, 711 
 infection, antistreptococcus serum in, 
 764 
 Wright's blood capsules, making of, 23 
 method of sealing, 34 
 removing serum from, 34 
 capillaFy pipet method of measuring 
 
 bactericidal power of blood, 355 
 method of counting bacterial vaccines, 
 210 
 
 Yaws, luetin reaction in, 605 
 
 salvarsan in, 810 
 
 Wassermann reaction in, 465 
 Yersin's plague serum, 769 
 
 ZOOPBBOIPITIN, 292 
 
 Zootoxins, 109, 119 
 
 experimental work, 823 
 
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 you need this new book because it gives you just the information you want. 
 
SAUNDERS' BOOKS ON 
 
 Sisson's Anatomy 
 of Domestic Animals 
 
 Anatomy of the Domestic Animals. By Septimus Stsson, S. B., 
 V. S., Professor of Comparative Anatomy in Ohio State University. 
 Octavo volume of 930 pages, with 725 illustrations, mostly original 
 and many in colors. Cloth, ;^7.00 net ; Half Morocco, $i.^O net. 
 
 JUST OUT— NEW (2d) EDITION, RESET 
 
 Dr. Sisson's work has been entirely reset. Moje than 300 new and original 
 illustrations have been introduced, the book now containing 725 illustrations, 100 
 of them in colors. This makes Dr. Sisson's work an atlas of comparative anatomy 
 as well as a descriptive text-book. The nomenclature- throughout has been changed 
 to' conform to that adopted by the American Veterinary Medical Association. The 
 work takes up the horse, the cow, the ox, the pig, the dog, the sheep — really a 
 comparative anatomy of the domesticated animals. 
 
 Boston Medical and Surgical Journal 
 
 " It is not amiss to say that the work ranks with the best,, A marked advance in English 
 veterinary literature, upon which student and practitioner may well congratulate themselves 
 and no medical school can afford to be without," 
 
 Gant on Constipation and 
 Intestinal Obstruction 
 
 Constipation and Intestinal Obstruction. By Samuel G. Gant, 
 M. D., Professor of Diseases of the Rectunt and Anus, New York 
 Post-Graduate Medical School and Hospital. Octavo of 559 pages, 
 with 250 original illustrations. Cloth, $6.00 net ; Half Morocco, f 7.50 net. 
 
 INCLUDING RECTUM AND ANUS 
 
 In this work the consideration given to the medical treatment of constipation 
 is unusually extensive. The practitioner will find of great assistance the chapter 
 devoted to formulas. The descriptions of the operative procedures are concise, 
 yet fully explicit. 
 
 The Proctologist 
 
 ' ' Were the profession better posted on the contents of this book there would 
 be less suffering from the ill effects of constipation.. We congratulate the author 
 on this most complete book." 
 
SURGERY AND ANATl^MY 
 
 Kelly 6 Noble's Gynecology 
 and Abdominal Surgery 
 
 Gynecology and Abdominal Surgery. Edited by Howard A. 
 Kelly, M.D., Professor of Gynecology in Johns Hopkins University; 
 and Charles P. Noble, M.D., formerly Cluiical Professor of Gyne- 
 cology in the Woman's Medical College, Philadelphia. Two imperial 
 octavo volumes of 950 pages each, containing 880 original illustrations, 
 some in colors. Per volume: Cloth, gS.oo net; Half Morocco, $9.50 
 net. 
 
 WITH 880 ILLUSTRATIONS— TRANSLATED INTO SPANISH 
 
 This work possesses a number of valuable features not to be found in any 
 other publication covering the same fields. It contains a chapter upon the bac- 
 teriology and one upon the pathology of gynecology, and a large chapter devoted 
 entirely to mfdical gynecology, written especially for the physician engaged in 
 general practice. Abdominal surgery proper, as distinct from gynecology, is 
 fully treated, embracing operations upon the stomach, intestines, liver, bile-ducts, 
 pancreas, spleen, kidneys, ureter, bladder, and peritoneum. 
 
 American Journal of Medical Sciences 
 
 " It is needless to say that the work has been thoroughly 4one ; the names of the authors 
 and editors would guarantee this, but much may be said in praise of the method of presentation, 
 and attention may be called to the inclusion of matter not to be found elsewhere.*' 
 
 Bickham's Operative Surgery 
 
 A Text-Book of Operative Surgery. By Warren Stone Bickham, 
 M.D., of New York. Octavo of 1200 pages, with 854 original illustra- 
 tions. Cloth, ^6.50 net ; Half Morocco, g8.o6=net. 
 
 THE NEW (3d) EDITION 
 This work completely covers the surgical anatomy and operative technic in- 
 volved in the operations of general surgery. The practicability of the work is 
 particularly emphasized in the 854 magnificent illustrations. 
 
 Boston Medical and Surgical Journal 
 
 "The book is a valuable contribution to the literature of operative surgery. It represents 
 g vast amount of careful work and technical knowledge on the part of the author. For the sur- 
 geon in active practice or the instructor of surgery it is an unusually good review of the subject." 
 
SAUNDERS' BOOKS ON 
 
 Eisendrath's 
 Surgical Diag'nosis 
 
 A Text-Book of Surgical Diagnosis. By Daniel N. Eisendrath, 
 M.D., Professor of Surgery in the College of Physicians and Surgeons, 
 Chicago. Octavo of 885 pages, with 574 entirely new and original 
 text-illustrations and some colored plates. Cloth, #6.50 net; Half 
 Morocco, ^8.00 net. 
 
 THE NEW (2d) EDITION 
 
 Of first importance in every surgical condition i| a correct diagnosis, for upon 
 this depends the treatment to be pursued ; and the two — diagnosis and treatment — 
 constitute the most practical part of practical surgery. Dr. Eisendrath takes up 
 each disease and injury amenable to surgical treatment, and sets forth the means 
 of correct diagnosis in a systematic and comprehensive way. Definite directions 
 as to methods of examination are presented clearly and concisely, providing for 
 all contingencies that might arise in any given case. Each illustration indi- 
 cates precisely how to diagnose the condition considered. 
 
 Surgery, Gynecolo^, and Obstetrics 
 
 "The book is one which is well adapted to the uses of the practising surgeon who desires 
 information concisely and accurately given. . . . Nothing of xiiagnostic importance is omitted, 
 yet the author does not run into endless detail." 
 
 £isendrath's Clinical Anatomy 
 
 A Text-Book of Clinical Anatomy. By Daniel N. Eisendrath, 
 A.B., M.D., Professor of Surgery in the College of Physicians and 
 Surgeons, Chicago. Octavo of 53S pages, illustrated. Cloth, ^5.00 
 net; Half Morocco, $(i.^O net. 
 
 THE NEW (2d) EDITION 
 
 This new anatomy discusses the subject from the cHnical standpoint. A por- 
 tion of each chapter is devoted to the examination of the living through palpation 
 and marking of surface outlines of landmarks, vessels, nerves, thoracic and 
 abdominal viscera. The illustrations are from new and original drawings and 
 photographs. This edition has been carefully revised, 
 
 Medic&l Record, New York 
 
 " A special recommendation for the figures is that th,fey are mostly original and were 
 made for the purpose in view. The sections of joints and trunks are those of formalinized 
 cadavers and are unimpeachable in accuracy." 
 
SURGERY AND ANA TOMY 
 
 Fenger Memorial Volumes 
 
 Fenger Memorial Volumes. Edited by Ludvig Hektoen, M. D., 
 Professor of Pathology, Rush Medical College, Chicago. Two octavos 
 of 5 25 pages each. Per set : Cloth, $15.00 net ; Half Morocco, g 18.00 net. 
 
 LIMITED EDITION 
 
 These handsome volumes consist of all the important papers written by the late 
 Christian Fenger, for many years professor of surgery at Rush Medical College, 
 Chicago. Not only the papers published in English are included, but also those 
 which originally appeared in Danish, German, and French. 
 
 The name of Christian Fenger typifies thoroughness, extreme care, deep re- 
 search, and sound judgment. His contributions to the advancement of the world's 
 surgical knowledge are indeed as valuable and interesting reading to-day as at 
 the time of their original publication. They are pregnant with suggestions. 
 Fenger' s literary prolificacy may be judged from this memorial volume — over 
 1000 pages. 
 
 Sobotta and McMurrich's 
 Human Anatomy 
 
 Atlas and Text-Book of Human Anatomy. In Three Volumes. By 
 J, Sobotta, M.D., of Wlirzburg. Edited, with additions, by J. Playfair 
 McMuRRicH, A. M., Ph. D., Professor of Anatomy, University of 
 Toronto, Canada. Three large quartos, each containing about 250 
 pages of text and over 300 illustrations, mostly in colors. Per volume: 
 Cloth, $6.00 net ; Half Morocco, ^7.50 net. 
 
 Edward Martin, M.D., Professor of Clinical Surgery, University of Pennsylvania 
 
 " This is a piece of bookmaking which is truly admirable, with plates and text so well 
 chosen and so clear that the work is most useful to the practfsing surgeon." 
 
 Campbell's Surgical Anatomy 
 
 A Text-Book of Surgical Anatomy. By William Francis Camp- 
 bell, M. D., Professor of Anatomy, Long Island College Hospital. 
 Octavo of 67s pages, with 319 original illustrations. Cloth, ^^5.00 net. 
 
 SECOND EDITION 
 
 This is in the fullest sense an applied anatomy^— an anatomy that will be of 
 inestimable value to the surgeon because only those facts are discussed and only 
 those structures and regions emphasized that have a peculiar interest to him. 
 
 Boston Medical and Surgical Journal 
 
 " The author has an excellent command of his subject, and treats it with the freedom and 
 the conviction of the experienced anatomist. He is also an admirable clinician." 
 
SAUNDERS' BOOKS ON 
 
 Moynihan's 
 Abdominal Operations 
 
 Abdominal Operations. By Sir Berkeley Moynihan, M. S. 
 (London), F. R. C. S., of Leeds, England. Two octavos, totaling 
 nearly looo pages, with 385 illustrations. Per set: Cloth, $10.00 net; 
 Half Morocco, $13.00 net. 
 
 JUST OUT— NEW (3d) EDITION,. ENLARGED 
 
 This new ( jff ) edition was so thoroughly revised that the work had to be reset from 
 cover to cover. Over 150 pages of new matter and some*85 new illustrations were added, 
 making 385 illustrations, 5 of them in colors — really an atlai of abdominal surgery. This 
 work is a personal record of Moynihan' s operative work. You get his own successful methods 
 of diagnosis. You get his own technic, in every case fuUy illustrated with handsome pic- 
 tures. You get the bacteriology of the stomach and intestines, sterilization and preparation 
 of patient and operator. You get complications, sequels, and after-care. Then the various 
 operations are detailed with forceful clearness, discussing first gastric operations, following 
 with intestinal operations, operations upon the liver, the pancreas, the spleen. Two new 
 chapters added in this edition are excision of gastric ulcer and complete gastrectomy, giving 
 the latest developments in these operative measures. 
 
 Moynihan's Duodenal Ulcer Sn 
 
 Duodenal Ulcer. By Sir Berkeley Moynihan, M. S. (London), F. R. C. S., 
 Leeds, England. Octavo of 486 pages, illustrated. Cloth, Jfs.oo net ; Half 
 Morocco, J!6.5o net. 
 
 For the practitioner, who first meets with these cases, Mr. Moynihan fixes the diagnosis 
 with precision, so that the case, if desired, may be referred in the early stage to the sur- 
 geon. The surgeon finds here the newest and best technic as used by one of the leaders 
 in this field. 
 
 " Easily the best work on the subject ; coming, as it docs, from the pen of one of the mas- 
 ters of surgery of the upper abdomen, it may be accepted as authoritative." — London Lancet. 
 
 Moynihan on Gall-stones l^^^on 
 
 Gall-stones and Their Surgical Treatment. By Sir Berkeley Moyni- 
 han, M. S. (London), F. R. C. S., Leeds, England. Octavo of 458 pages, 
 illustrated. Cloth, $5.00 net ; Half Morocco, J6. 50 net. 
 
 This work gives special attention to the early symptoifls in cholelithiasis, enabling you to 
 diagnose the case in the early stages. 
 
 " We can heartily recommend this work as most satisfactory and of the greatest prac- 
 tical value." — American Journal of the Medical Sciences. 
 
SURGER V AND ANA TOMY »3 
 
 Schultze and Stewart's Topographic Anatomy 
 
 Atlas and Text-Book of Topographic and Applied Anatomy. By Frof. 
 Dr. O. Schultze, of Wiirzburg. Edited, with additions, by George D. 
 Stewart, M. D., Professor of Anatomy and Clinical Surgery, University 
 and Bellevue Hospital Medical College, N. Y.. Large quarto of 189 pages, 
 with 25 colored figures on 22 colored lithographic plates, and 89 text-cuts, 60 
 in colors. Cloth, gj, 50 net. 
 
 Griffith's Hand-Book of Surgery 
 
 A Manual of Surgery. By Frederic R. Griffith, M. D., Surgeon to the 
 Bellevue Dispensary, New York City. i2mo of 579 pages, with 417 illus- 
 trations. Flexible leather, g2.oo net. 
 
 Keen's Addresses and Other Papers 
 
 Addresses and Other Papers. Delivered by William W. Keen, M. D., 
 LL.D., F. R. C. S. (Hon.), Professor of the Principles of Surgery and of Clin- 
 ical Surgery, Jefferson Medical College, Philadelphia. Octavo volume of 
 441 pages, illustrated. . Cloth, iSj.75 net. 
 
 Keen on the Surgery of Typhoid 
 
 The Surgical Complications and Sequels of Typhoid Fever. By Wm. W o 
 
 Keen, M.D., LL.D., F.R.C.S. (Hon.), Professor of the Principles of Surgery 
 and of Clinical Surgery, Jefferson Medical, College, Philadelphia, etc. 
 Octavo volume of 386 pages, illustrated. Cloth, $3.00 net. 
 
 Dannreuther's Minor and Emergency Surgery 
 
 Minor and Emergency Surgery. By Walter T. Dannreuther, M. D., Sur- 
 geon to St. Elizabeth's Hospital and to St. Bartholomew's Clinic, New York 
 City. i2mo of 225 pages, illustrated. Cloth, j?l.25 net. 
 
 Bier's Hyperemia second Edition 
 
 Bier's Hyperemic Treatment in Surgery, Medicine, and the Specialties : 
 A Manual of its Practical Application. By Willy Meyer, M. D., Professor 
 of Surgery at the New York Post-Graduate Medical School and Hospital ; and 
 Prof. Dr. Victor Schmieden, Assistant to Prof. Bier, University of Berlin, 
 Germany. Octavo of 280 pages, with original illustrations. Cloth, $3.00 net. 
 
 " We commend this work to all those who are interested in the treatment of infections, either acute or 
 chonic, for it is the only authoritative treatise we have in the English language. "^ — New York State 
 Journal of Medicine. 
 
 Morris' Dawn of the Fourth Era in Surgery 
 
 Dawn of the Fourth Era in Surgery and Other Articles. By 
 
 Robert T. Morris, M. D., Professor of Surgerj-, New York Post-Graduate 
 Medical School and Hospital. i2mo of 145 pkges, illustrated. S1.25 net. 
 
H SAUNDERS' BOOKS ON 
 
 — ^ — u 
 
 Haynes* Anatomy 
 
 A Manual of Anatomy. By Irving S. Haynes, M.D., Professor of Prac- 
 tical Anatomy, Cornell University Medical College. Octavo, 680 pages, 
 with 42 diagrams and 134 full-page half-tones. Cloth, ^2.50 net 
 
 American Pocket Dictionary New (sthi Edition 
 
 The American Pocket Medical Dictionary. Edited by W. A. Newman 
 
 Borland, A. M., M. D., Editor "American Illustrated Medical Dictionary." 
 677 pages. Full leather, limp, with gold edges, Jfi.oo net; with patent 
 thumb index, $1.25 net. 
 
 Fowler's Surgery in i wo voiumea 
 
 A Treatise on Surgery. By George R. FcTwler, M. D., Emeritus Pro- 
 fessor of Surgery, New York Polyclinic. Two itnperial octavos of 725 pages 
 each, with 888 original text-illustrations and 4 colored plates. Per set : 
 Cloth, $15.00 net ; Half Morocco, J 18. 00 net. 
 
 International Text-Book of Surgery second Edition 
 
 Tlie International Text=Book of Surgery. In two volumes. By Ameri- 
 can and British authors. Edited by J. Collins Warren, M. D., LL. D.. 
 F. R. C. S. (Hon.), Professor of Surgery, Harvard Medical School ; and A. 
 Pearce Gould, M. S., F. R. C. S., of London, England. Vol. 1. : General 
 and Operative Surgery. Royal octavo, 975 pages, 461 illustrations, 9 full- 
 page colored plates. Vol. II. : Special or Regional Surgery. Royal octavo, 
 1 1 22 pages, 499 illustrations, and 8 full-page colored plates. Per volume : 
 Cloth, JS5.00 net ; Half Morocco, td.^o net. 
 
 American Text-Book of Surgery Fourth Edition 
 
 American Text-Book of Surgery. Editec! by W. W. Keen, M. D., 
 LL. D., Hon. F. R. C. S., Exg. and Edin;, and J. William White, 
 M. D., Ph. D. Octavo, 1363 pages, 551 text-cuts and 39 colored and 
 half-tone plates. Cloth, $7.00 net ; Half Morocco, {$8.50 net. 
 
 Robson and Cammidge on the Pancreas 
 
 The Pancreas : Its Surgery and Pathology. By A. W. Mayo Robson, 
 F. R. C. S., of London, England; and P. J. Cammidge, F. R. C. S., of 
 London, England. Octavo of 546 pages, illustrated. Cloth, ^S.oo net; 
 Half Morocco, $6.50 net. 
 
SURGERY AND ANATOM\. it 
 
 American Illustrated Dictionary The New 7th) Edition 
 
 The American Illustrated Medical Dictionary. With tables 
 of Arteries, Muscles, Nerves, Veins, etc. ; of Bacilli, Bacteria, etc. ; 
 Eponymic Tables of Diseases, Operations, Stains, Tests, etc. By W. A. 
 Newman Borland, M.D. Large octavo, 1107 pages. Flexible leather, 
 ^4.50 net; with thumb index, ^5.00 net. 
 
 Howard A.Kelly, M.D., Professor of Gynecology, Johns Hopkins Universiiy, Baltimore. 
 
 "Dr. Dorland's dictionary is admirable. It is so well gotten up and of such con- 
 venient size. No errors have been found in my use of it." 
 
 Golebiewski and Bailey's Accident Diseases 
 
 Atlas and Epitome of Diseases Caused by Accidents. By Dr, 
 
 Ed. Golebiewski, of Berlin. Edited, with additions, by Pearce Bailey, 
 M.D. Consulting Neurologist to St. Luke's Hospital, New York City. 
 With 71 colored figures on 40 plates, 143 text-cuts, and 549 pages of 
 text. Cloth, §4.00 net. In Saunders^ Hand-Atlas Series. 
 
 Helferich and Bloodgood on Fractures 
 
 Atlas and Epitome of Traumatic Fractures and Dislocations 
 
 By Prof. Dr. H. Helferich, of Greifswald, Prussia. Edited, with ad- 
 ditions, by Joseph C. Bloodgood, M. D., Associate in Surgery, Johns 
 Hopkins University, Baltimore. 216 colored figures on 64 lithographic 
 plates, 190 text-cuts, and 353 pages of text. Cloth, ;J3. 00 net. In Saun- 
 ders^ Atlas Series. 
 
 Sultan and Coley on Abdominal Hernias 
 
 Atlas and Epitome of Abdominal Hernias. By Pr. Dr. G. Sul- 
 tan, of Gottingen. Edited, with additions,, by Wm. B. Coley, M. D., 
 Clinical Lecturer and Instructor in Surgery, Columbia University, New 
 York. 119 illustrations, 36 in colors, and 277 pages of text. Cloth, 
 ^3.00 net. In Saunders' Hand-Atlas Series. 
 
 Warren's Surgical Pathology ^^^^^ 
 
 Surgical Pathology and Therapeutics. By J. Collins Warren, 
 M.D., LL.D., F.R.C.S. (Hon.), Professor of Surgery, Harvard Medical 
 School. Octavo, 873 pages ; 136 illustrations, 33 in colors. Cloth, 
 ^5.00 net; Half Morocco, $6.50 net. 
 
 Zuckerkandl and DaCosta's Surgery IdWon 
 
 Atlas and Epitome of Operative Surgery. By Dr. O. Zucker- 
 kandl, of Vienna. Edited, with additions, by J. Chalmers DaCosta, 
 M.D., Samuel D. Gross Professor of Surgery, Jefferson Medical Col- 
 lege, Philadelphia. 40 colored plates, 278 .text-cuts, and 410 pages of 
 text. Cloth, ^3.50 net. In Saunders^ Atlas Series. 
 
1 6 SURGER Y AND ANA TOMY 
 
 Moore's Orthopedic Surgery 
 
 A Manual of Orthopedic Surgery. By James E. Moore, M.D., Professoit 
 of Clinical Surgery, University of Minnesota, College of Medicine and Surgery. 
 Octavo of 356 pages, handsomely illustrated. Cloth, S2.50 net. 
 
 " The b09k is eminently practical. It is a safe guide in the understanding and treatment of 
 Should be owned by every surgenn zxA^x&a\'Cvyi\e.t."— Annals of Surgery-. 
 
 orthopedic cases. 
 
 Fowler's Operating Room (,ew (3d) Edition. Reset 
 
 The Operating Room and the Patient. By Russell S. Fowler, M. D., 
 
 Surgeon to the German Hospital, Brooklyn, New York. Octavo of 6il 
 
 pages, illustrated. Cloth, $3.50 net. 
 
 Dr. Fowler has written his book for surgeons, nurses assisting at an operation, internes, 
 and all others whose duties bring them into the operating room. It contains explicit 
 directions for the preparation of material, instruments needed, position of patient, etc., 
 all beautifully illustrated. 
 
 Nancrede's Principles of Surgery New (2d) Edition 
 
 Lectures on the Principles of Surgery. By Chas. B. Nancrede, M.D., 
 LL. D., Professor of Surgery and of Clinical Surgery, University of Michigan, 
 Ann Arbor. Octavo, 407 pages, illustrated. Cloth, ^2.5^ net. 
 
 *' We can strongly recommend this book to all students and those who would see something 
 of the scientific foundation upon which the art of surgery is built." — Quarterly Medical Journal^ 
 Sheffield, England. 
 
 Nancrede's Essentials of Anatomy. ^^^^^^ Edition 
 
 Essentials of Anatomy, including the Anatomy of the Viscera. By Chas, 
 
 B. Nancrede, M.D., Professor of Surgery and of Chnical Surgery, University 
 
 of Michigan, Ann Arbor. Crown octavo, 388- pages ; 1 80 cuts. With an 
 
 Appendix containing over 60 illustrations of the osteology of the body. Based 
 
 on Cray s Anaiomy. Cloth, jfi.oonet. In SaU7tders' Question Compends. 
 
 ** The questions have been wisely selected, and the answers accurately and concisely given." — 
 University Medical Magazine. 
 
 Martin's Essentials of Surgery. ^^"'^RLvifed*"'" 
 
 Essentials of Surgery. Containing also Venereal Diseases, Surgical Land- 
 marks, Minor and Operative Surgery, and a complete description, with illus- 
 trations, of the Handkerchief and Roller Bandages. By Edward Martin, 
 A.M., M.D., Professor of Clinical Surgery, University of Pennsylvania, etc. 
 Crown octavo, 338 pages, illustrated. With an Appendix on Antiseptic Sur- 
 gery, etc. Cloth, gi.oo net. In Saunders' Question Compends. 
 
 " Written to assist the student, it will be of undoubted value to the practitioner, containing as it 
 does the essence of surgical work." — Boston Medical and Surgical Journal. 
 
 Metheny's Dissection Methods Just issued 
 
 Dissection Methods and Guides. By Davfd Gregg Metheny, M. D., 
 
 L. R. C. P., L. R. C. S. (Edin.), I.. F. P. S. (Glas.), Associate in Anatomy, 
 
 Jefferson Medical College, Philadelphia. Octavo of 131 pages, illustrated. 
 
 Cloth, $1.25 net. 
 
 This work bridges the gap heretofore existing between the descriptive text-book and the 
 dissecting table. In order that it may be used in connection with any text-book or atlas 
 both the old and the new anatomic names are given. Everything the student could be ex- 
 pected to do is carefully explained in detail.