LIBRARY NEW YORK STATE VETERINARY COLLEGE / ITHACA, NEW YORK Cornell University Library QR 46.P23 1914 Pathogenic microorganisms; a practical m 3 1924 000 235 329 PATHOGENIC MICROORGANISMS A PEACTICAL MANUAL FOR STUDENTS, PHYSICIANS AND HEALTH OFFICERS BY WILLIAM HALLOCK PARK, M.D. PROFESSOR OF BACTERIOLOGY AND HYGIENE, UNIVERSITY AND BELLEVUE HOSPITAL MEDICAL COLLEGE, AND DIHECTOR OP THE BUREAU OF LABORATORIES OF THE D F.PAKTMENT OF HEALTH, NEW YORK CITY AND ANNA W. WILLIAMS, M.D. ASSISTANT DIRECTOR OF THE BUREAU OF LABORATORIES; CONSULTING PATHOLOGIST TO THE NEW YORK INFIRMARY FOR WOMEN AND CHILDREN FIFTH EDITION, ENLARGED AND THOROUGHLY REVISED WITH 210 ENGRAVINGS AND 9 FULL-PAGE PLATES LEA <& FEBIGEE NEW YORK AND PHILADELPHIA Entered according to the Act of Congress, in the 3'ear 1914, bj' LEA & FEBIGER in the office of the Librarian of Congress. All rights reserved. Fa 3 PREFACE TO FIFTH EDITION. The first edition of tliis book was called Bacteriology in Medicine and Surgery. It was written to make available for others the practical kjiowledge which had been acciiiired in the work of the bacteriologic laboratories of the city of New York, and was intended more for medical practitioners than for medical students or laboratory workers. When the second edition had been exhausted the improvement in methods of cultivating and studying the protozoa had reached a point rendering it ach'antageous to include the animal as well as the vegetable germs. This was done, and the title of the third edition was altered to conform with the text, which had been broadened to give in outline practically the whole field of pathogenic microorganisms. The book in its later editions has come to be used in an ever-increasing degree by medical students, so that while its point of view has remained the same, namely, to dwell especially on the relations of microorganisms to disease in man, it has been thought wise to touch on other aspects, such as the bacteria concerned in agriculture and in some of the important fermentations. References are frecjuently gi\'en to some of the related investiga- tions, but no effort has been made to afford a complete or uniform bibliography. The question as to the practical limits of bibliographic references in such a hand-book as this is an open one. Hence the lack of uniformity in this respect. In this fifth edition we have rearranged the material in order to bring more closely together all of the pathogenic organisms. Under this arrangement Part I deals with the general characteristics and methods of study of all the microorganisms considered (molds, yeasts, bacteria, and protozoa). Part II includes the study of the individual pathogenic microorganisms and their near relatives. Part III presents certain prac- tical aspects of the subject under the title ^Ipplied Microbiology. The nine plates included in this edition have been arranged and drawn especially for this book. Owing to recent advances in the knowledge of microbiology, we have added much new material and rewritten several parts of the book. For example, the subject of complement IV PREFACE TO FIFTH EDITION fixation has increased so much in importance that a full description of its nature and its various applications has been given in a separate chapter. The whole subject of immunity has been extensivelj^ revised. A new chapter has been written on filtrable viruses. The chapter on the preparation and uses of media has been rewritten, and the recent methods for the use of anilin dyes, as well as other new procedures, have been incorporated. Other chapters which have been extensively changed are those dealing with the streptococcus, the pneumococcus, the diph- theria bacillus, the tetanus bacillus, the hemoglobinophilic bacilli, the bacillus of pertussis, the molds, the amebse, and the chapter on rabies. The chapter on complement fixation has been written by M. P. Olmstead, who for several years past has been in charge of much of the work on this subject, which is carried on in the New York City Health Department. The chapter on media has been rewritten by C. Krumwiede, one of the assistant directors of the laboratories of the Health Department. He also revised the chapters on the coli- typhoid group, on the group of acid-fast bacilli, and on the cholera spirillum and relatives. The chapter on the bacillus malleus and that on the meningococcus were revised respectively by B. Van H. Anthony, M.D.,and P. L. DuBois, M.D., both bacteriologists in our laboratories. We are further indebted to our associates in the laboratory for aid in manv different ways. W. H. P. A. W. W. New York, 1914, CONTENTS PART I. PRINCIPLES OF MICROBIOLOGY. CHAPTER I. Introdtjctort. Historical Sketch 17 CHAPTER 11. Cl.vssification and General Characteristics of Microorganisms . . 24 CHAPTER III. The Microscope and the Microscopic Examination of Microorganisms 66 CHAPTER IV. General Methods Used in the Cultivation of Microorganisms . . 86 CHAPTER V. The Use of Animals for Diagnostic and Test Purposes .... 122 CHAPTER VI. The Procuring and Handling of Material for Microbiologic Exami- nation FROM Those Suffering from Disease 125 CHAPTER VII. The Relation of Microorganisms to Disease 132 CHAPTER VIII, Immunity, Active and Passive. The Antagonism Existing between THE Living Body and Microorganisms 151 CHAPTER IX. Nature of the Protective Defences op the Body and their Manner of Action. Ehrlich's "Side Chain" and Other Theories . . . 158 CHAPTER X. Complement Fixation: The Technique of the Test and its Practical Applications 177 VI CONTENTS CHAPTER XI. The Natuee of the Substances Concerned in Agglutination . . . 193 CHAPTER XII. Opsonins. Extract of Leukocytes 208 CHAPTER XIII. Active Immunization. Vaccines 219 CHAPTER XIV. Protein Hypebsensitiveness or Anaphylaxis 224 PART II. PATHOGENIC MICROORGANISMS INDIVIDUALLY CONSIDERED. CHAPTER XV. The Pathogenic Molds (Hyphomy'cetes, Eumycetes) and Yeasts (Blastomycetes) 229 CHAPTER XVI. The Pyogenic Cocci 241 CHAPTER XVII. The Diplococcus of Pneumonia (Pneumococcus, Streptococcus Pneu- moniae. Micrococcus Lanceolatus) 262 CHAPTER XVIII. Meningococcus or Micrococcus (Intracellulabis) Meningitidis, and THE Relation of it and of Other Bacteria to Meningitis . . . 273 CHAPTER XIX. The Gonococcus or Micrococcus Gonorrhce.e. Micrococcus Melitensis 283 CHAPTER XX. The Bacillus and the Bacteriology of Diphtheria 292 CHAPTER XXI. The Bacillus and the Bacteriology- of Tetanus 320 CHAPTER XXII. Intestinal Bacteria .334 % CONTENTS VII CHAPTER XXIII, The Colon-typhoid Group of Bacilli 344 CHAPTER XXIV. The Typhoid Bacillus 355 CHAPTER XXV. Paratyphoid Group 374 CHAPTER XXVI. Dysentery Group 378 CHAPTER XXVII. B.^ciLLus Pyocyaneus (Bacillus of Green and of Blue Pus). Bacillus Proteus (Vulgaris) 383 CHAPTER XXVIII. The Bacillus and the Bacteriology of Tuberculosis 388 CHAPTER XXIX. Other Acid-fast Bacilli: Bacillus of Leprosy, Bacillus of Rat Leprosy', Bacillus of Johne's Disease in Cattle and the Group of Non-pathogenic Acid-past Bacilli 420 CHAPTER XXX. Ctl.anders B.acillus (B.acillus Mallei) i25 CHAPTER XXXI. The Group of Hemoglobinophilic B,acilli. Bordet-Gengou Bacillus 432 CHAPTER XXXII. Microorganisms Belonging to the Hemorrhagic Septicemia Group . 445 CHAPTER XXXIII. The Anthr.ax Bacillus and the Pathogenic Anaerobes 450 CHAPTER XXXIV. The Cholera Spirillum (Cholera Vibrio) and Similar Varieties . . 464 CHAPTER XXXV. Pathogenic Microorganisms Belonging to the Higher Bacteria (Trichomycetes) 476 CHAPTER XXXVI. Filtrable Viruses. Diseases of Unknown Etiology 488 CHAPTER XXXVII. Flagellata 496 viii I CONTENTS CHAPTER XXXVIII. Trypanosoma Spirocheta and Allies CHAPTER XXXIX. CHAPTER XL. BODO. POLYMASTIGIDA CHAPTER XLI. Ameba CHAPTER XLII. Sporozoa. Ci 503 515 528 532 543 CHAPTER XLIII. The Malarial Organisms. Babesia 548 CHAPTER XLIV. Smallpox (Variola) and Allied Diseases 565 CHAPTER XLV. Rabies. Yellow Fever 572 PART III. APPLIED MICROBIOLOGY. CHAPTER XLVI. The Bacteriologic Examination of Water, Air, and Soil. The Con- tamination and Purification of Water. The Disposal of Sewage 599 CHAPTER XLVII. The Bacteriology of Milk in its Relation to Disease 611 CHAPTER XLVIII. The Soil Bacteria and their Functions. Sewage Bacteria. Bacteria IN Industries g29 CHAPTER XLIX. The Destruction of Bacteria by Chemicals. Practical Use of Dis- infectants g37 CHAPTER L. Practical Disinfection and Sterilization (House, Person, Instru- ments, AND Food). Sterilization of Milk for Feeding Infants . 650 PATHOGENIC MKKOOEGANISMS. PART T. PIILXCIPLES OF MICROMOLOdY. CHAPTER I. INTRODUCTORY. HISTORICAL SKETCH. Although most of the more important discoveries in microbiology wliich place it on the footing of a science are of comparatively recent date, the foundations of its study were laid over two centuries ago. From that time the history of microorganisms has been intimately associated with that of medicine. Indeed, it is only through the inves- tigations into the life history of these minute forms that our present knowledge of the etiology, course, and prevention of the infectious diseases has been acquired. The prominent position which the study of microorganisms already holds toward medicine is, moreover, daily increasing in importance. Original discoveries are constantly adding to our knowledge of germ diseases, and the outlook is favorable for e^•entually obtaining, through serums, through attenuated cultures, or through the toxic substances produced by microorganisms themselves, means for immunizing against, if not of curing, an increased numl)er of the specific infections. Even at present, bacterial products and pro- tective serums are used successfully as preventive or curative agents in several of the most prevalent infectious diseases. Our knowledge concerning other microorganisms has enabled us largely to limit their dissemination and so to prevent disease. An acquaintance, therefore, with the main facts concerning these microorganisms is most necessary to the education of the modern physician. The vast majority of the known microorganisms which cause disease belong to the closely related groups of lowest plants and animals, i. €., the bacteria, the molds, the yeasts, and the protozoa. A few of the pathogenic metazoa in some of their stages are also microscopic (some of the parasitic worms) ; therefore, microscopic methods of study are also applicable to them ; but since they are fully presented in works on clinical microscopy they are not given here. 18 PRINCIPLES OF MICROBIOLOGY Before entering into a detailed consideration of the subject it may be interesting and instructive to review \'ery brieflj^ a few of the important steps wliich led to the de\'elopment of the science, and upon whicli its foundation rests, in which we shall sec that the results detained were gained only through long and laborious research and after many obstacles were met and overcome by accurate observation and experi- ment. Probably the first authentic observations of living microscopic organisms of which there is any record are those of Kircher, in 1659. This original investigator demonstrated the presence in putrid meat, milk, vinegar, cheese, etc., of "minute living worms," but did not describe their form or character. Not long after this, in 1675, Leeuwenhoeck observed in rain-water putrid infusions, and in his own and other sali\'a and diarrheal e^'acu- ations living, motile "animalcula" of most minute dimensions, which he described and illustrated by drawings. Leeuwenhoeck practised the art of lens-grinding, in which he eventually became so proficient that he perfected a lens superior to any magnifying glass obtainable at that day, and with which he was enabled to see objects yery much smaller than had ever been seen before. "With the greatest astonish- ment," he writes, "I observed distributed everywhere through the material which I was examining animalcules of the most microscopic size, which moved themselves about ver,y energetically." The work of this observer is conspicuous for its purely objective character and absence of speculation; and his descriptions and illustrations are done with remarkable clearness and accuracy, considering the imperfect optical instruments at his command. It was not until manj' years later, however, that any attempt was made to define the characters of these minute organisms and to classify them s\'steniatically. At that time all of the microscopic organisms seen were classed together as little animals. Indeed, all of the microorganisms first described at any length were probably protozoa, and only after further impro\'ement of lenses and a more minute study of the organisms were bacterial forms gradually recognized as a separate class. The same skepticism that is seen in the acceptance of most new dis- coveries was displa.yed by doubters of the truth of these early reports of microscopic findings. Chief among the skeptics must be placed Linnaeus, who in the first edition of his Sy sterna Natvrce (1735) absolutely denies the existence of Leeuwenhoeck's animalcula, though in the later editions he grudgingly admits them under the significant generic name of Chaos (Chaos proteus (Ameba), etc.). The first ideas of the structure of the protozoa were drawn from analogy. The early observers thought that each tiny organism possessed an internal structure made up of organs and tissues similar to those in metazoa. They could not conceive of motion without articulation, tendons, and muscles; nor of food absorj^tion without an alimentarj' tract, and they were so impressed with the ideas of what they thought they ought to see that they were con\-inced that they really saw many INTRODUCTORY 19 of the complicated structures possessed by metazoa. For example the contractile vacuole, a characteristic pulsating vesicle of the protozoa, discovered by Joblot in 1754, was thouglit by many to be lungs, other vacuoles were said to be stomachs, the mouths were often seen and the rest of the alimentary tract was supplied from the imagination; the red pigment spots of many forms were interpreted as true eyes, etc. There were many opponents to these \iews, however, and the idea of the cell being the unit of structure, which was advanced by Schleiden in 1838, helped determine the fact that j^rotozoa were single cells wath no definite organic structure. With the publications of Dujardin (1835-41) a correct idea of the struc- tural simplicity of the microorganisms gained ground. But for some time after, the contro^'ersy regarding the simple nature of protozoa was strenuously carried on. It is a most instructive bit of history in research work, showing how the lack of minute observation, the exercise of a too vivid imagination, and the close reasoning from analogy may lead one astray, while the proper use of these functions may bring out the truth. KoUiker, Biitschli, Engelmann, and Hertwig, with many others, (1870-80) finally demonstrated fully the unicellular nature of the protozoa. The most important characteristic of a protozoon, its life history, was first partially made out by Trembley in 1744-47. Biitschli helped determine the sexual activities of the members of this group, while Maupas (1889) was the first to demonstrate the conditions leading to their conjugation. From the earliest investigations into the life history and properties of germs, microorganisms have been thought to play an important part in the causation of infectious diseases. Shortly after the first investigations into this subject the opinion was advanced that puer- peral fever, measles, smallpox, typhus, pleurisy, epilepsy, gout, and many other diseases were due to contagion. In fact, so widespread became the belief in a causal relation of these minute organisms to disease that it soon amounted to a veritable craze, and all forms and kinds of diseases were said to be produced in this way upon no other foundation than that these organisms had been found in the mouth and intestinal contents of men and animals and in water. Among those who were specially conspicuous at this time for their advanced views on the germ-theory of infectious diseases was INIarcus Antonius Plenciz, a physician of Vienna. This acute obse^^•er, who published his views in 1762, maintained that not only were all infec- tious diseases caused by microorganisms, but that the infective mate- rial could be nothing else than a living organism. On these grounds he endeavored to explain the variations in the period of incubation of the different infectious diseases. He also insisted that there were special germs for each infectious disease by which the specific disease was produced. Plenciz belie\'ed, moreover, that these organisms were capable of multiplication in the body, and suggested the possibility of their being conveyed from place to place through the air. 20 PRINCIPLES OF MICROBIOLOGY These \-iews, it is true, were largely speculative, and rested upon insufficient experiment, but they were so plausible, and the arguments put forward in their support were so logical and convincing, that they continued to gain ground, in spite of considerable opposition and ridicule, and in many instances the conclusions reached have since been pro^-ed to be correct. The mode of infection, its unlimited devel- opment among large numbers of individuals, and gradual spread over wide areas — the incubation, course of, and resulting immunity in recovery from infectious diseases — all pointed to a li^•ing organism as the probable cause. Among other distinguished men of the day whose observations exerted a most powerful influence upon the doctrine of infection, may be mentioned Henle. His writings (Pathological Investigations, 1840 and Text-book of Rational Pathology, 1S53), in which he described the relation of microorganisms to infectious diseases, and defined the character and action of bacteria upon certain phases and symptoms of these affections, are remarkable for their clearness and precision. Origin of Microorganisms. — But, meanwhile, the question which most interested these investigators into the cause of infectious diseases was: Whence are these microorganisms derived which were supposed to produce them? Were they the result of spontaneous generation due to vegetative changes in the substances in which the organisms were found, or were they reproduced from similar preexisting organisms — the so-called vitalistic theory? This question is intimately connected with the in^•estigations into the origin and nature of fermentation and putrefaction. Spallanzani in 17G9 demonstrated that if putrescible infusions of organic matter were placed in hermetically sealed flasks and then boiled the liquids were sterilized; neither were living organisms found in the solutions, nor did the infusions decompose; they remained unchanged for an indefinite period. The objection was raised to these experiments that the high tem- perature to which the liquids had been subjected so altered them that spontaneous generation could no longer take place. Spallanzani met the objection by cracking one of the flasks and allowing air to enter, when living organisms and decomposition again appeared in the boiled infusions. Another objection raised b,y the believers in spontaneous generation was that, in excluding the oxygen of the air by hermetically sealing the flasks, the essential condition for the de^'elopment of fermentation, which required free admission of this gas, was interfered with. This objection was then met by Schulze, in 1836, by causing the air admitted to the boiled decomposable liquids to pass through strong sulphuric acid. Air thus robbed fif its living organisms did not produce decomposition. Schwann in 1839 obtained similar results in another way: he deprived of microorganisms the air admitted to his boiled liquids by passing it through a tube which was heated to a temperature high enough ORIGIN OF MICROORGANISMS 21 t(i destroy s't'rni^- Ti) tliis investigator is itlso (luc the credit oF having disco\'ered the spet'ifie eaiiso — tlie yeast ])iaiit, or HiiccIkudiiijicck ci'ri'tusiu' — of alcoliolie fermentation, the process hy \\ hich sugar is dccoini)osed into alcoliol and earbonit' acid. Again it was objected to these e.xptTinients tliat the heating of the air had perhaps bronght about some eliemical eliange which liindered the pr()duction of fermentation. Schroeder and von Duscii in LS54 then showed that by a simple process of filtration, which has since proved of inestimable ^•ahle in bacteriologic work, the air can be mechanically freed from germs. By placing in the mouth of the flask containing the boiled solutions a loose plug of cotton, through which the air could freely circulate, it was foimd that all suspended micro- organisms could be excluded, and that air passed through such a filter, whether hot or cold, did not cause fermentation of boiled infusions. Similar results were obtained by Hoffmann in 1860, and by Chevreul and Pasteur in 1861, without a cotton filter, by drawing out the neck of the flask to a fine tube and turning it downward, lea^dng the mouth open. In this case the force of gravity prevents the suspended bacteria from ascending, as there is no current of air to carry them upward through the tube into the flask containing the boiled infusion. These facts have since been practically confirmed on a large scale in the preservation of food by the process of sterilization. Indeed, there is scarcely any biologic problem which has been so satisfactorilj' solved or in which such uniform results have been obtained; but all through the experiments of the earlier investigators irregularities were constantly appearing. Although in the large majority of cases it was found possible to keep boiled organic liquids sterile in flasks to which the oxygen of the air had free access, the question of spontaneous generation still remained unsettled, inasmuch as occasionally, even under the most careful precautions, decomposition did occur in such boiled liquids. This fact was explained by Pasteur in 1860 by experiments showing that the temperature of boiling water was not sufficient to destro,y all living organisms, and that, especially in alkaline liquids, a higher temperature was required to insure sterilization. He showed, how- ever, that at a temperature of 110° to 112° C, which he obtained bj' boiling under a pressure of one and one-half atmospheres, all living organisms were invariably killed. Pasteur at a later date (1865) demonstrated the fact that the organ- isms which resist boiling temperature are, in fact, reproductive bodies, which are now known as spores. In 1876 the development of spores was carefully investigated and explained by Ferdinand Cohn. He, and a little later Koch, showed that certain rod-shaped organisms possess the power of passing into a resting or spore stage, and when in this stage they are much less sus- ceptible to the injurious action of higher temperatures than in their normal vegetative condition. When and how life began no one is yet able to say. That spontaneous 22 PRINCIPLES, OF MICROBIOLOGY generation may even be taking place now nnder nnknown conditions is conceivable, but all such ideas are purely hypothetical and there is no e^•idence that tuider present conditions any of the known micro- organisms have originated in an\' way except from a previous similar cell: Stimulated by the establishment of the fact, through Pasteur's investigations, that fermentation and putrefaction were due to the action of living organisms reproduced from similar preexisting forms, and that each form of fermentation was due to a special microorganism, the study of the causal relation of microorganisms to disease was taken up with renewed vigor. Reference has already been made to the opinions and h^'potheses of the earlier obser^'e^s as to the microbic origin of infectious diseases. The first positive grounds, however, for this doc- trine, founded upon actual experiment, were the investigations into the cause of certain infectious diseases in insects and plants. Thus, Bassi in 1837 demonstrated that a fatal infectious malady of the silkworm — pebrine — was due to a parasitic protozoon. Pasteur later devoted several years' study to an exhaustive investigation into the same sub- ject; and in like manner Tulasse and Kiihne showed that certain specific affections in grains, in the potato, etc., were due to the invasion of parasites. Very soon after this it Avas demonstrated that microorganisms were probably the cause of certain infectious diseases in man and the higher animals. Davaine, a famous French physician, has the honor of having first demonstrated the causal relation of a microorganism to a specific infectious disease in man and animals. The anthrax bacillus was dis- covered in the blood of animals dying from this disease by Pollender in 1S49 and by Davaine in 1850; but it was not until I860 that the last- named observer demonstrated by inoculation experiments that the bacillus was the cause of anthrax. The next discoveries made were those relating to wounds and the infections to which they are liable. Rindfleisch in 1S6G and Waldeyer and von Recklinghausen in 1871 were the first to draw attention to the minute organisms occurring in the pyemic processes resulting from in- fected wounds, and occasionally following typhoid fever. Further investigations were made by Billroth, Fehleisen, and others, in erysipe- latous inflammations secondary to injury, who agreed that in these conditions microorganisms could almost always be detected in the Ij-mph channels of the subcutaneous tissues. The brilliant results obtained by Lister in 1863-1870, in the antiseptic treatment of wounds to prevent or inhibit the action of infectiA'e organisms, exerted a powerful influence on the doctrine of bacterial infection, causing it to be recognized far and wide and gradually lessen- ing the number of its opponents. Lister's methods were suggested to him by Pasteur's investigations on putrefaction. In 1877 Weigert and Ehrlich recommended the use of the anihne dyes as staining agents and thus made possible a more exact micro- scopic examination of microorganisms in cover-glass preparations. Li the year 1880 Pasteur published his discovery of the bacillus of ORIGIN OF MICROORGANISMS 23 fowl cholera and his iiivestij^ations u]ion thr atteiitiiatioii of the virus of anthrax and of fowl t'liolera, and npon ])rot('cti\(' inoculation against these diseases. In the meantime he showed tluit ])urc cnltnres might be obtained by the dilution method. Laveran in the same year announced the tlisco\ery of parasitic bodies, the first pathogenic protozoa described, in the blood of persons sick with malarial fcAer, and thus stimulated investigations upon the immensely important unicellular animal parasites. In 1881 Koch published his fundamental researches upon pathogenic bacteria. Pie introduced solid culture media (agar had already been used by Frau Hess) and the "plate method" for obtaining pure cultures, and showed how different organisms could be isolated, cultivated independently, and, by inoculation of pure cultures into susceptible animals, could be made, in many cases, to reproduce the specific disease of which they were the cause. To him more than to any other are due the methods which have enabled us to prove absolutely, in a broad sense, the permanence of bacterial varieties. It was in the course of this work that the Abbe system of substage condensing apparatus was first used in bacteriology. In 1882 Pasteur published his first communication upon rabies. The method of treatment devised by him is still in general use. A little later came the investigations of Loffler and Roux upon the diph- theria bacillus and its toxins, and that of Kitasato upon tetanus. These researches paved the way for Behring's work on diphtheria antitoxin-, which in its turn stimulated investigation upon the whole subject of immunity. The number of investigators rapidly increased as the importance of the earlier fundamental discoveries became apparent. Additions to the science of microbiology have been made from luany sides, and the practical application of the facts learned from these investigations are steadily increasing. The most important of these are given in the following pages. CHAPTER II. CLASSIFICATION AND GENERAL CHARACTERISTICS OF MICROORGANISMS. In general the lowest forms of life are microscopic. Among these microscopic organisms are many which have in common the al)ility to produce disease in the higher animals and plants. Some of these disease-producing or pathogenic microorganisms are plants, others are animals, while some are difficult to classify. Those only that are of direct importance to man are considered in this work. CLASSIFICATION OF MICROORGANISMS. The classification of microorganisms is still in the transition stage. This is due chiefly to the difficulties encountered in studying the indi- vidual morphologic characteristics of such minute bodies and in deter- mining their limits of variation. There is no one distinctive characteristic known which separates the lowest plants from the lowest animals. While the lowest micro- organisms of all — the bacteria — are usually classed as plants, their struc- ture is so simple and their biologic characteristics are so varied that their relationship to the vegetable kingdom is not clear-cut. In their possession of more or less rigid plasmolysible bodies, in the tendency of many to grow in filaments, and in the ability of some to use simple elements as food, they resemble plants; while in the motilitj^ of many, the non-possession by all of chlorophyl, and in the necessity of many for complex food, they resemble animals. There is a similar difficulty in definitely classifying many members of the other groups of closely related microorganisms, naniel.v, the protozoa, the yeasts, and the molds, and it has been suggested that under the name Protista a third kingdom be formed consisting of all of these lowest microorganisms. As a rule, in the more minute organisms genera are based upon mor- phologic characteristics, and species upon biochemic, physiologic, or pathogenic properties. This is due to the facts that the morphology of ^'arieties may vary extremely under different conditions and that morphology may give no indication whatever of the relation of micro- organisms to disease and fermentation — the chief characteristics which gi\'e them their importance to human beings. The properties of bacteria, for example, which are fairly constant under uniform conditions and which have been more or less used in systems of classification, are those of spore and capsule formation, motility (flagella formation), reaction to staining reagents; relation to temperature, to oxygen, and to other food material, and, finally, their CLASSIFICATION OF MICROORaANISMS or^ relation to formontation and disease. 15ut any one of these ])roi)erties uniler eertain eonditions may so vary tliat, takiiifj; it as a basis for eiassi- tieation, an oru'anisin eould he dropi)ed from the liroup witli which it liad been ehissitied and be ])laee(i in an entirely dill'erent i^ronp. 'riuis, the power to proiluee spores or tiagella may be held in abeyance for a time or, in the case of the former, be totally lost; the relations to oxygen may be gradually altered, so that an anaerobic bacterium grows in the presence of oxygen; parasitic organisms may be so cultivated as to become saprophytic varieties, and those which have no power to grow in the living body may acquire pathogenic properties. The possibility of making any thoroughly satisfactory classification is rendered still more difRcult by the fact that many necessarily imper- fect attempts IvAve already been made, so that there is a great deal of confusion, which is steadily increased as new varieties are found or old ones reinvestigated and classified differently in the difi'erent systems. Until a more universally accepted classification can be determined it seems wiser to note only such a broad, simple grouping as the one given below. Nomenclature. — It is well to call attention to the fact that in naming bacterial species, especially among the bacteria, the binomial law of nomenclature has been freciuently violated. Such names as Bacillus mil communis should not be accepted; the name Bacillus cult is sufficient as well as correct. WoKKixG Classification of P.a.thogenic Micboorganisms in Ootline. Kingdom. Subkingdom. Classes in which patho- genic species occur. Genera in which chief patho- genic species occur. Plants (fungi) Animals (Proto- zoa) Molds (Hyphomycetes) Yeasts (Blastomycetes) Bacteria (Sehizomyeetes) Flagellates (Mastigophora) Amebae (Sarcodina) Sporozoa Cilia tes Mycomyoetes. Phycomycetes Unclassified (Fungi Imperfecti) Oidea Saccharomycetes Cocci (Coccacese) Bacilli (Bacteriaceffi) Spirilla (Spirillaoefe) Higher bacteria (Tricho- bacteria) Flagellata Rhizopoda Telosporidia, Neosporidia Ciliata Aspergillus, Penicillium. Mucor. Microspora, Trichophyta, Sporo- tricha, Achoria. Oidium. Saccharomyces. Micrococcus, Diplococcus, Strep- tococcus, Tetracoccus. Bacillus (Bacterium). Spirillum [Spirocheta.Treponema]. Cladothri.x, Nocardia (Strepto- thrix) . Cercomonas,Leptomonas, Herpet- omonas, Trypanosoma, Leish- mania. Trichomonas, Lamblia [Spirocheta, Treponema]. Entameba. Eimeria, Hemogregarinida, Pro- teosoma, Hemameba, Babesia, Rhinosporidium, Myxobolus, Nosema, Sarcocj'stis, etc. Balantidium. Ultramicroscopic organisms. 2G PRINCIPLES OF MICROBIOLOGY Permanence of Species. — When we come to study special varieties or groups of microbes, such as the bacilli which jjroduce typhoid fever, (liphtlieria, or tuberculosis, it is of great importance for us to deter- mine, if j)ossible, to what extent the peculiar characteristics which each of these groups of microbes possess are permanent in the generations which develop from them. We cannot believe that the multitude of varieties which now exist have always existed. The probability is very strong that with succeeding generations and changing conditions new varieties have developed with new characteristics. From time to time the changing conditions under which life pro- gresses probably expose certain animals to the invasion of varieties which never before have gained access to them. If the microorganisms find some means of transmission to other animals equally susceptible, a parasitic species becomes established which at first, perhaps, finds conditions only occasionally favorable to it. Thus in some such way a multitude of groups have arisen, some of which accustom themselves to the conditions present in living plants, others to those in fishes, others to those in birds, and others still to those in man and the higher animals. These are, however, theories. What has been actually observed in the few years during which bacteria have been studied? In this short time the pathogenic species as observed in disease have remained practically unaltered. The diphtheria bacilli are the same today as when LofBer discovered them in 1884, and the disease itself is evidently the same as history shows it to have been before the time of Christ. The same permanence of disease type is true for tuberculosis, smallpox, hydrophobia, leprosy, etc. Under practically unchanged conditions, therefore, such as exist in the bodies of men, germs which have once become established as parasites continue to reproduce new generations which retain their peculiar (specific) characteristics. It is true that among the countless organisms developed some fail to hold the parasitic characteristics. These either continue as saprophytes or cease to exist. Whether new disease varieties are coming into existence from time to time is, of course, a possibility, but not a certainty. The fact that the chief pathogenic varieties of microorganisms which excite disease in man seem to have retained for centuries their charac- teristics, in no way proves that when placed under different conditions they would remain stable. As will be shown, certain characteristics of some bacteria can be radically altered by changed conditions, such as being grown outside the natural host, either in the test-tube or in an unaccustomed host. When these new surroundings are unfavorable, the organisms, while retaining their morphology, may lose their power of developing and producing specific poisons in the original host. Such attenuation may also occur in certain organisms when retained for a long time in an apparently immune host, as is seen in the streptococci and pneumococci of the throat or in the colon bacilli of the intestines. The recovery of poison production is often brought about by developing MOLDS 27 the microorganism for a considerable lenf;tli of time under the condi- tions l)est suited for it. The reronri/ of the uhility to grow in the body of any animal s])eeies is hrou^ht about by causing tlie germ to develop in a series of animals of the same species whose resistance has been overcome by reducint;- their vitality tiiroui^h poisons, heat, cold, etc., or by giving enormous doses of microbes to produce the first infection. Another method is to accustom the microorganism to the animal's body by letting it remain surrounded by the animal fluids, but protected from i)hagocytes in a pervious capsule in the peritoneal cavity or by growing it in unheated fresh serum or blood media. Certain groups of microorganisms seem to undergo variations more readily than others. The coli group and the streptococcus group are notable examples. The above examples of \-ariations may be classed under those known as fluctuating variations. True mutations or discontinuous variations among bacteria have been from time to time recorded, but wliether or not the changed characteristics may be considered species characteristics cannot at present be decided. GENERAL CHARACTERISTICS OF EACH GROUP OF MICRO- ORGANISMS. Introduction. — The knowledge gained from the fact that each variety of microbe possible of cultivation may grow in distinctive ways upon so-called artificial culture media has been an immense aid in studying the characteristics of the more minute forms, for the individual cell of most \-arieties is so minute that even the highest magnification we ha\e may show little if any morphologic difference between organisms which produce distinctly different diseases, or between a pathogenic and a non-pathogenic form. There are, however, certain morphologic and biologic characteristics of the single cell which are pronounced, and we therefore study these before going on to the study of cultures — that is, of microorganisms in masses. Morphologic descriptions should always be accompanied by a definite statement of the habitat — if from a culture, age of the growth, the medium from which it was obtained, and the temperature at which it was developed. MOLDS (HYPHOMYCETES, EUMYCETES). The molds (see Plate I) are closely related to the higher bacteria. Like them they grow in branching filaments or threads forming a mycelium, but these filaments or hyphse, as they are called, are more definite than those of the trichobacteria, and they are multicellular; that is, the filaments are septate, in some varieties alw^ays (myco- mycetes) and in others when forming spores (phycomycetes). These two groups of molds have other differential characteristics, particularly in their fruiting organs. 28 ' PRINCIPLES OF MICROBIOLOGY The mi/CDmi/crtcs are divided into two groups: (1) Ascomyeetes, which form a si)ore sac, containing- a definite nninlier of spores, a mul- tiple of two, usually eight (ascospores) ; {2) ]5asidiomycetes, which form a definite spore-beariug cell called the basidiuui or conidiphore, from which four (usually) conidia or sterigmata protrude (mushrooms, etc.). The sexual fruiting organs are varied. Yeasts produce a definite sporangium containing a definite number of spores (two or a multiple of two), therefore they are closely related to the ascomyeetes. The phycomycetes are characterized by the formation of ?io_ definite basidium or ascus. The asexual fruiting organs are formed in three different ways: 1 . Ends of hypha? swell and are shut off by septum, forming sjjorangium in which (a) swarm spores develop. These become free by rupture of sporangium wall, swim about, and then form a new plant; (6) spores form a cell wall while in sporangium. 2. Spores produced directly by constriction at end of certain hypha?. Such spores are called conidia and the hyphaj conidophores; each conidium is able to develop into a new plant. 3. Spores produced by separation of hyphiie into short-walled seg- ments or oidia. When the spores are thick walled they are called chlamydophores. Sexual reproduction occurs in one of two ways: 1. The ends of two similar hyphse become attached, then they seg- ment from the rest of the hyphffi and become surrounded by a thick wall. The partition in the centre dissolves and the protoplasm mixes. The resulting body is called a zygospore. It produces a new plant. The members of this group are called zygomj'cetes. 2. The swollen ends of two dissimilar hypha?, one large (female) and one smaller (male), become attached, fuse, segment, and become surrounded by a thick wall. The body is called an oospore. The plants are called oomycetes. Fungi Imperfecti. — The molds that are not definitely classified are divided into two groups: 1. Known forms which do not fit in the abo-\e group. 2. Not fully known forms. To this group belong most of the molds causing human disease. EXPLANATION OF PLATE I. Partly schematic. Rearranged and drawn by Williams from the indicated authors, Fio. 1. — Aspergillus glauous. Fruiting hypha^ growing from mycelium: A, conidiopho're; B, sterygma; C, conidia; D, beginning perithecium; E, conidiophore bearing spores; F, perithecium containing rudiments in section ; G, ascus containing eight spores (De Bary) ' Fig. 2. — PeniciUium, showing formation of conidia, A. Fig. 3. — Mucor mucedo: A, .sporangium containing spores; B, spores hbcrated- C lamydospores; D, E, F, stages in the formation of a zygospore. Fig. 4. — Oidium lactis. Fig. 5, — Sporotrichum schenki, showing formation of whorlcd spores on branched mycelium. Fig. 6. — Yeast from human infection in culture showing mycelium-like growth. Fig. 7. — Saoeharomyces cerevisice (Hansen). A, budding; B, spore formation. PLATE I , WILLIAMS, DEL. YEASTS 29 YEASTS (BLASTOMYCETES). These microorganisms have been for many centuries of the greatest importance in brewing and baking. Tliey are not uncommonly present in the air and in cultures made from the throat. Certain recent experiments have shown that some Aarieties when injected are capable of producing tumor-like growths. Certain varieties are pathogenic for mice, and in recent years (since 1894) there have been reported a number of cases of human infection froni yeasts. The position which the yeasts occupy in systematic biology has not, thus far, been accurately determined. In fact, it is still a question as to whether they constitute distinct fimgi or whether they should be classed under the molds. The chief characteristic of the yeasts is their peculiar method of reproduction, which in most cases is by means of budding. For this reason these organisms go by the name of blastomycetes in contrast to the fission fungi, or schizomycetes, and the thread fungi, or hypho- mycetes. The fact was mentioned above that some yeasts form asco- spores and that they are therefore related to the ascomycetes. The soor fungus, which at one time grows to long threads, at another time (under certain conditions almost exclusively) multiplies by budding, is also a transition form. It is thus seen that no hard-and-fast line exists between yeasts and molds. The most important property of yeasts, though one not possessed by all to the same degree, is that of producing alcoholic fermentation. In brewing we distinguish between the yeasts that^ can be employed practically, "culture yeasts," and those which often act as disturbing factors, so-called "wild" yeasts. The shape of most of the culture yeasts is oval or elliptic (Plate I, Fig. 7). Round or globular forms are more often met with among the wild species which usually excite only a slight degree of fermentation. They are known as "torula" forms. But sausage-shaped and thread forms are also met with. Pathogenic forms may also be round. The individual yeast cells are strongly refractive, so that under the microscope at times they have almost the lustre of fat droplets. This is important because in examining fresh tissues the yeast cells may be hard to distinguish from fat droplets, often requiring the aid of certain reagents for their identification. The size of the individual yeast cells varies enormously, even in those of the same species or the same culture. In old colonies indi- viduals may be found no larger than cocci, 0.5m to 2/^ in diameter, while in other colonies, especially on the surface of a liquefied medium, giant yeast cells are found often attaining a diameter of 40/x or more. In spite of these wide fluctuations, howe\'cr, the various s])ecies are characterized by a fairly definite average in size and form. Fach cell contains a more or less definite nucleus, which is demonstrated by the usual chromogenic stains. During the process of budding the nucleus of the cell mo\'es toward 30 PRINCIPLES OF MICROBIOLOGY the margin, where it divides. At this point the hmiting membrane of the cell ruptures or usually a hernia-like protrusion develops which has the appearance of a button attached to the cell. The daughter-cell so formed rapidly increases in size and gradually assumes the shape and size of the mother-cell. A fact of the utmost importance for the propagation of the blasto- mycetes and continuation of the species is the formation of spores (Plate I, Fig. 7). In this also the cell nucleus takes part, dividing into several fragments, each of which becomes the centre of a new cell lying within the original cell. These new cells possess a firm membrane, a cell nucleus, and a little dense protoplasm. The number of spores developed in the yeast cells varies, but is constant for a given species. As a rule, one cell does not produce more than four endogenous spores, })ut species have been observed — e. g., Schizosaccharomyces octosporus (Beijerinck) — in which eight spores are found. Guilliermond has described conjugation in yeasts before the formation of spores. The vitality of yeasts is truly enormous. Hansen as well as Lindner were able to obtain a growth from cultures twelve years old. Busse succeeded in getting a luxuriant growth from a dry potato culture seven and a half years old, which was almost as hard as bone. BACTERIA. Definition. — Bacteria may be defined as extremely minute, simple, unicellular microorganisms, which reproduce themselves with exceeding rapidity, usuall,y by transverse division, and grow without the aid of chlorophyl. They have no morphologic nucleus, but contain nuclear material which is generally diffused throughout the cell body in the form of larger or smaller granules. Natural Habitat. — There are such wonderful dilTerences in the con- ditions of life and nutrition which suit the different varieties, that bac- teria are found all over the known world. Wherever there is sufficient moisture, one form or another will find other conditions adequate for multiplication. Thus, we meet with bacterial life between 0° and 75° C. Some live only in the tissues of men, others in lower animals, a larger number may grow in both man and lower animals, others still grow only in plants, but by far the greater number live in dead organic matter. For some free oxygen is necessary to life; for others it is a poison. Morphology (see Plates II and III).— The form and dimensions of bacterial cells at their stage of complete development must be distin- guished from those which they possess just after or just before they have divided. x\s a spherical cell develops preparatory to its division into two cells it Ix'comes elongated and appears as a short oval rod ; at the moment of its division, on the contrary, the transverse diameter of each of its two halves is greater than their long diameter. A short rod becomes in the same way, at the moment of its division, two cells, the long BACTERIA 31 diameter of each of which may be even a trifle less than its short diameter, and thus they appear on superficial examination as spheres. Size. — The dimensions of the adult individual vary greatly in the dif- ferent species as well as in mcmhers of the same species. The largest bacillus recorded is .'iO/u to (K)^' long and 4^ to r)p. wide (1>. biitachlli, see Fig. U). One of the smallest forms known (11. iiiJliicnTM) is 0.5m X 0.2^. Some pathogenic organisms (supposed to be bacteria) are so small (ultramicroscopic, see p. 70) as to be invisible with any magnification which we now possess. We know of their existence either by the fact that they may lie cultivated on artificial media, producing appearances of mass growth, and that such cultures when inoculated into susceptible animals cause the characteristic disease, or by the fact that the filtrates alone are infectious. Some filtrates are infectious after passing through the pores of the finest filter (see Filtrable Viruses). Shape. — The basic forms of the single bacterial cells are threefold — the sphere, the rod, and the segment of a spiral. Although under different conditions the type form of any one species may vary con- siderably, yet these three main divisions under similar conditions are constant; and, so far as we know, it is never possible, by any means, to bring about changes in the organisms that will result in the permanent conversion of the morphology of the members of one group into that of another — that is, micrococci always, under suitable conditions, pro- duce micrococci, bacilli produce bacilli, and spirilla produce spirilla. As bacteria multiply the cells produced from the parent cell have a greater or less tendency to remain attached. This is on account of the slimy envelope which is more or less developed in all bacteria. In some varieties this tendency is extremelj' slight, in others it is marked. This union may appear simply as an aggregation of separate bacteria or so close that the group appears as a single cell. According to the method of the cell division and the tenacity with which the cells hold together, there are different groupings of bacteria, which aid us in their differentiation and identification. Thus, in cocci we get the bacterial cell dividing into one, two, or three planes (Plate II, Figs. 1-5), while in bacilli and spirilla the division is generally in only one plane (Plate II, Figs. 6-9). 1. Spherical Form, or Coccus. — The size varies from about 0.3^ as minimum diameter to 3^ as maximum; the average size of the patho- genic cocci is O.Sfi. The single elements are, at the moment of their complete development, so far as we can determine, practically spherical ; but when seen in the process of multiplication through division the form is seldom that of a true sphere. Here we have elongated or lancet- shaped forms, as frequently seen in the diplococcus of pneumonia, or the opposite, as in the diplococcus of gonorrhea, where the cocci appear to be flattened against one another. Those cells which divide in one direction only and remain attached are found in pairs (diplococci) 1 A f, or mioromillimeter, is t,--, ,', ,, ,,- of an inch. 32 PRINCIPLES OF MICROBIOLOGY or in shorter or longer chains (streptococci). Those which divide m two (hrections, one at right angles to the other, form groups of four (tetrads). Those which divide in three directions and cling together form packets in cubes (sarcina;). Those which divide in any axis form irregularly shaped, grape-like Inuiches (stapliylococci). 2. Rod Form, ok Bacillus. — The type of this group is the cylinder. The length of the fully developed cell is always greater than its breadth. The size of the cells of different varieties varies enormously: from a length of .30m and a breadth of 4^ to a length of 0.2^ and a breadth of 0.1^. The largest bacilli met with in disease do not, however, usually develop over 3m x 1^ while the average is 2m x O.Sm- Bacilli are roughly classed, according to their form, as slender when the ratio of the long to the transverse diameter is from 1 : 4 to 1 : 10, and as thick when the proportions of the long to the short diameter is approximately 1 : 2. The characteristic form of the bacillus has a straight axis, with uniform thickness throughout, and flat ends ; but there are manj' excep- tions to this typical form. Thus frequently the motile bacteria have rounded ends, man,y of the more slender forms have the long axis, slightly bent; some few species, as for example the diphtheria bacilli, invariably produce many cells whose thickness is very unequal at difi'erent portions. Spore formation also causes an irregularity of the cell outline. The bacilli, except when they develop from spores or granules, divide only in the plane perpendicular to their long axis. A classification, therefore, of bacilli according to their manner of grouping is much simpler than in the case of the cocci. We may thus have bacilli as isolated cells, as pairs (diplobacilli), or as longer or shorter chains (streptobacilli). 3. Spiral Form, or Spirillum. — The members of the third mor- phologic group are spiral in shape, or only segments of a spiral. Here, EXPLANATION OF PLATE II. Fig. 1. — Illustrating cocci single or in irregular groups (micrococcus, staphylococcus), micrococcus from air. X 1000. Fig. 2. — Illustrating cocci in twos — Diplococcus pneumonia? from peritoneal cxuilate of rabbit. X 1000. Fig. .3. — Illustrating cocci in chains — Streptococcus pyogenes. X 1000. Fig. 4. — Illustrating cocci in fours — Micrococcus tetragenus from spleen of mouse X 1000. Fig. .5. — Illustrating cocci in packets — Sarcina lutea from air. X 1000. Fig. C. — Illustrating large single bacilli: B. subtilis (hay bacillus). X 1000. Fig. 7.— Illustrating small bacilli; mostly in twos— B, hoffmanni from human throat X 800, (Park.) Fi<:. ,S. — Illustrating bacilli in chains — Anthrax bacillus from spleen of mouse. X 500 Fig. 9.— Illustrating baoilU in bunches— Typhoid bacillus from human spleen! X .50o' Fig. 10. — Illustrating bacilli in threads — Anthrax bacilli from blood of frog. Fig. U. — Spirihum undula, single coccus, showing flagellaj. X 1000. Fig. 12. — Cholera spirill^e, gelatin culture. X 1000. Fig. 13. — Large spirillte in chains from water. X 1000. Fig. 14. — Smaller spirillge in chains — Spirillum rubrum. X 1000. Fig. 1.5. — Streptothrix Candida — Broth culture (Zettnow, from KoUe and 'Wasscrmann) Fig. 16. — Streptothrix hominis from sputum (Zettnow, from KoUe and A\'assermaiin) Unless otherwise indicated the photographs are from Friinkel and Pfeiffer. i-'ljAi Ej ii TYPES OF BACTERIA I SPHERE (COCCI OR COCCACEfE ) OO CO ocxxxx> gg 4- >'^- K n CYLINDER (bacilli OR BACTERIACErE ) ^4= 1/ I, * l^ S'^ J^.. 5 6 r * EISPIRAL (SPIRILLA OR SPIRI LLACE/t ) i^ J 11 12 /.: ^^>l "f A 13 i 7-^ IVhIGHER BACTERIA ( TRICHOBACTERIA /■S" 76 MLLIAMS, DEL. BACTERIA 33 too, we have large and small, slender and thick spirals. The twisting of the long axis, which here lies in two planes, is the chief characteristic of this group of bacteria. Under normal conditions the twisting is uniform throughout the entire length of the ceil. The spirilla, like the bacilli, di\-ide only in one direction. A single cell, a pair, or the union of two or more elements may thus present the appearance of a short segment of a spiral or a comma-shaped form, an S-shaped form, or a complete spiral or corkscrew-like form. Structure of Bacterial Cells. — When examined living in a hanging drop (see p. 72) under the microscope bacteria appear usually as color- less refractive bodies with or without spores or other more Jiighly refrac- tive areas. It is only by the use of stains that we are able to see more of their structure. Capsule. — Special staining methods (see p. 79) show that many bacteria (some investigators say all) under certain conditions possess a capsule (Plate III, Figs. 14-16), a gelatinous envelope which is supposed to be formed from the outer layer of the cell membrane. Some bacteria easily develop a much thicker capsule than others. Such forms are known as capsule bacteria. These generally produce a slimy growth on cultivation (e. g., B. mucosus). Capsules develop best in animal tissues. In cultures, with a few exceptions, they require for their development special albuminous culture media, such as milk, blood serum, bronchial mucus, etc. In ordinary nutrient media or on potatoes the capsule may be visible in the first culture generations when grown from the body, but usually it shows very indistinctly if at all. The capsule is distinguished by a diminished power of staining with ordinary aniline dyes, therefore, unlss special staining methods are used, the bacteria may appear to be lying in a clear unstained area. With certain dyes the inner portion of the capsule stains, giving the bacteria an apparent greater diameter. The demonstration of the capsule is often of help in differentiating between different but closely related bacteria; e. g., some forms of strep- tococcus and pneumococcus. Cell Membrane. — That all bacteria possess a cell membrane is shown (1) by special staining methods (e. g., flagella stains, see p. 80) and (2) by plasmolysis, demonstrated by placing the bacteria in a 1 per cent, solution of sodium chloride when the central portion (entoplasm?) con- tracts and separates in places from the membrane. In some bacteria the membrane is slightly developed, while in others (e. g., B. tuberculosis) it is well developed. It is different in composition from the membrane of higher plants in not possessing cellulose. In some forms, however, a similar carbohydrate, hemicellulose, has been demonstrated. In certain forms a sul.istance related to chitiu, found in the cyst walls of protozoa, has been found. Some observers consider the cell mem- brane merely a concentrated part of the cytoplasm, similar to the ecto- plasm of higher cells. That it is closely related to the living part of the cell is shown by the connection of the organs of locomotion (flagella) with it (Plate III, Figs. 17 and 21). 3 34 PRINCIPLES OF MICROBIOLOGY The Cell Substance.— The chief views in regard to the nature and tlie structure of the cell substance contained within the membrane may be summarized as follows: 1. Bacteria have a defmite morphologic, more or less centrally situated nucleus (Feinberg, Nakanischi, Schottelius, Swellengrebel, and others). 2. Bacteria have no nucleus or differentiated nuclear material (Fischer, Migula, Massart, and others). 3. The whole organism, except the membrane which is a delicate layer of cytoplasm, is a nucleus (Biitschh, Lowit, Boni, and others). ' 4. The nuclear material is in the form of distributed chromatin granules throughout the cj^toplasm (Hertmg, Schaudinn, Guihiermond, Zettnow, and others) . .5. A variety of the fourth view is that bacteria possess both chief elements of a cell, namely, cytoplasm and karyoplasm, but that these are so finely mixed that they cannot be morphologically differentiated (Weigert, Mitro- phanow, Gotschlich). 6. The latest view advanced, which is a variation of the views 3, 4, and 5, is that the bacterial cell is a relatively simple body — a cytode in Haeckel's sense, or the plasson of Van Beneden — which possesses both chromatin and plastin, the relative amounts of these chief substances of a ceU corresponding more to the amounts found in the nuclei of higher cells than in their cytoplasm (Ruzicka, Ambroz). These last authors call attention to the fact that both nucleus and cytoplasm in the higher cells are composed of a mixture of chromatin and plastin and that the chief difference between the two mixtures is one of amount and not of kind. EXPLANATION OF PLATE III. Partly schematic (Williams). Stained by Giemsa except where otherwise indicated. Figs. 1 to 4. — Anthrax bacilli at different stages of development. Fig. 1. — Shows chromatic staining masses (red), in plastin-staining cells (light blue). Fig. 2. — Shows same organism after one hour on fresh medium, chromatic substance distributed throughout medium and whole organism taking deeper, more homogeneous stain. Fig. 3. — Organism showing two kinds of granules according to staining powers. Fig. 4. — Same organism placed on fresh medium extrudes some granules (waste-prod- ucts) and redissolves others (chromatin granules). Figs. 5 to 9. — Similar granules in diphtheria bacilli at various staes of development. Fig. 10. — Metachromatic granules in B. pyocyaneus, Neisser stain. Fig. 11. — Metachromatic granules in Sarcinfe, Neisser stain. Fig. 12. — Metachromatic granules in B. influenzae. Fig. 13. — Metachromatic granules in gonococcce. Fig. 14. — Pneumococcus, capsule stained by Hiss method. Fig. 1.5. — Pneumococcus raucosus (Streptococcus mucosus) by Welch stain. Fig. 16. — Rhinoscleroma bacilhis by Hiss stain for capsules. Fig. 17. — Plasmolysis in cholera bacillus sho^Wng capsule. Figs. 18 to 21. — Tj'pes of flagella by Loffler stain. Fig. 18. — Monotricha, cholera spirillum. Fig. 19 — Amphitricha, water bacillus. Fig. 20. — Lophotricha, spirillum undula. Fig. 21. — Peritricha, typhoid bacillus. Fig. 22. — Formation and end germination of spores in anthrax. Fig. 23. — Lateral germination of spore in B. subtilis. I'iG. 24. — Central germination of spores in B. alvei (Wilson). Fig. 25. — Type of spores; A, central; B, eccentric; C, end. I'lG. 26. — Diphtheria bacilli in old cultures. I'iG. 27. — Plague 1)acilli in old cultures. Fni. 28. — Influenza bacilli in old cultures. PLATE in STRUCTURE OF BACTERIA NUCLEAR MATERIAL AND CELL GRANULES ' . S L ^ /•" 5 5 /o <9% • • • •» • • • « > •* /J * CAPSULES AND MEMBRANES ^ 15 FLAGELLA IT W'i f SPORES 22 •S^TtS "^El O ^O C 0» (1» ■■■■ ABC F G H 25 CS=3 CHQD c IRREGULAR FORMS IN OLD CULTURES (INVOLUTION FORMS! A. W. WILLIAMS, DEL. BACTERIA 35 Our own studies of the structure of liactcria lead us to agree with the views expressed in Nos. 4 and G of the above summary — that is, bacteria possess both chief elements of a cell, namely, chromatin and plastin, and according to the stage of growth and division (varying with species) the chromatin may be in the form of morphologic granules or may be so finely divided and mixed with the plastin as to be indis- tinguishable from it. (See Plate III, Figs. 1, 2,5, (i, 7.) MetachromaticGranules (Plate III, Figs. 1-13). — These granules appear in unstained bacteria as light-refracting, in stained preparations as deeply stained areas. They have a great affinity for dyes, and so stain readily and give up the stain with some difficulty. With complex stains they show a greater affinity than the rest of the bacillus for certain con- stituents of the stain — e. g., with poly chromic methylene blue they take up more of the azur, thus appearing red and indicating at the same time their nuclear nature. In certain bacteria, such as the diph- theria bacilli, they are especially well marked in young, vigorous cultures. Here they have diagnostic value. At least some of these granules are nuclear in character. Besides the metachromatic granules there are certain other granules which take up stains readily and others still which absorb stains with difficulty; some of these granules are of the nature of starch and some of fat or other food products. Certain saprophytic forms have sulphur, others iron granules. Organs of Motility. — The outer surface of spherical bacteria is almost always smooth and devoid of appendages; but that of the rods and spirals is frequently provided with fine, hair-like appendages, ov flagella, which are organs of motility (Plate III, Figs. 18-21). These flagella, either singly or in tufts, are sometimes distributed over the entire body of the cell, or they may only appear at one or both ends of the rod. The polar flagella appear on the bacteria shortly before division. The flagella are believed to be formed from the outer cell layer (ectoplasm) or possibly from the capsule, though they have been described by certain authors as arising in endoplasmic granules. So far as we know, the flageUa are the only means of locomotion possessed by the bacteria. They are not readily stained, special staining agents being required for this purpose (see p. 80). The envelope of the bacteria, which usually remains unstained with the ordinary dyes, then becomes colored and more distinctly visible than is commonly the case. Occasionally, how- ever, some portion of the envelope remains unstained, when the flagella present the appearance of being detached from the body of the bacteria by a narrow zone. In stained cultures of richly flagellated bacteria peculiar pleated masses sometimes are oliserved, consisting of flagella which have been detached and then matted together.. Bacteria may lose their power of producing flagella for a series of generations. Whether this power be permanently lost or not we do not know. Bacteria are named according to the number and position of the flagella they possess as follows: Monotrlcha (a single flagellum at one pole; e. g., cholera spirillum); AmpMtricha (a flagellum at each 36 PRINCIPLES OF MICROBIOLOGY pole; e. g., nian.y spirilla); lophotricha (a tuft of flagella at one pole;' e. g., Spirilhnn undulans) ; peritricha (flagella projecting from all parts of surface; e. g., B. alvei, B. typhosus, and others) (Plate III, Figs. 18-21). Fig. 1 Fig. 2 r- ..M i/ Bacilli showing one polar flagellum. Bacilli shomng multiple flagella. So far, in only a few l^acteria (the largest spirilla) have flagella been demon- strated during life, and then only under special conditions (see K. Reichert for Ijibliography) . We have, however, an organism belonging to the B. alvei group, which shows its flagella very distinctly during life when a small portion of the viscid growth in a liquefjang LofHer blood-serum tube is transferred to a hanging mass of agar (p. 73) and examined under high magnification. The flagella on this organism may also be seen with dark-field illumination. Reichert claims that all motile bacteria show their flagella Ij}^ this method.' Physiologic Characteristics of Bacteria. — The essential physiologic activities of bacteria are: motility, growth, reproduction, and spore formation. Motility. — Many bacteria when examined under the microscope are seen to exhibit active movements in fluids. The movements are of a varying character, being described as rotary, undulatory, sinuous, etc. At one time they may be slow and sluggish, at another so rapid that any detailed observation is impossible. Some bacteria are ver,y active in their movements, different individuals progressing rapidly in different directions, while w'ith many it is difficult to say positively whether there is any actual motility or whether the organism shows only molecular movements — so-called " Brownian" movements, or pedesis — a dancing, trembling motion possessed by all finely divided organic particles. In order to decide definitely with regard to the motility of any bacterial iireiiaration, it is well to make two hanging dro])s. To one, Ft ])er cent, of formalin is added, which of course kills the organism. If, now, the li\e culture shows motility, which is not shown by the killed culture, it is an indication that one is dealing with a, motile culture. Very young cultures, of but three to four hours' ' Some investigator.^ eousidor that every flagellum is essentially a tuft, composed of many small fibrils. BACTERIA 37 development, in neutral nutrient bouillon should hv examined at a temperature suitable for their best growth. Not all speeies of bacteria whieli liave Hagella exhibit at all times spontaneous mo^'ements; sueh movements may be absent in eertain eulture media and at too low or too high temperatures, or with an iiisulfieient or exeessix'e suj^ply of oxygen; hence one should examine cultures under various conditions before deciding as to the non-motility of an}- organism. The highest speed of which an organism is capable has been approxi- mately estimated with some forms, and the actual figures show an actual slow rate of movement, though, comparatively, when the size of the organism is considered, the movement is rapid. Thus, the cholera spirillum may travel for a short time at the rate of 18 cen- timeters per hour. Movement is influenced by many factors, such as chemicals (the oxygen in the air especially), heat, light, and electricity. The tactile property which enables microorganisms to take cognizance of various forces is known as taxis; when forces attract, the phenomenon is known as positi^'c taxis and when they repel it is called negative taxis. Chemo- taxis, or the effect of chemicals, is taken up in detail on page .58. Growth and Reproduction. — Under favorable conditions bacteria grow rapidly to a certain size, more or less constant for each species, and then divide by fission into approximately equal halves. The average time required for this cycle is twenty to thirty minutes. Probably in all species the nuclear material divides first. This is certainly the case in the group to which the B. dipJiiherice belongs, where division of the nuclear granules luay be observed in the living organism before the characteristic snapping of the cell body and where division into equal halves seldom occurs. Fig. 3 Successive stages in division of B, diphtheriae showing relation of line of division to metachromatic granule. Continuous observation of living bacillus drawn without camera lucida. (Williams.) According to our observations on the living cell of members of this group, division takes place at a point occupied by a metachromatic granule (Fig. 3). Before division of the cell body the metachromatic granule, which appears to contain nuclear substance, elongates and shows a darker line at or near its centre. This seems to divide and form two lines, each of which has at a point near the surface a very tiny, refractive granule, staining deeply with. chromatin stains. Between these two lines the cell body suddenly divides mth a snap, like the opening of a jackknife, division beginning at the point between the two tiny granules, and the two new cells remain for a variable time attached at opposite points, thus giving the V-shaped forms. Kurth and Hill also called attention to division by snapping in members of the diphtheria-bacillus group, though neither recognized the relation between the 38 PRINCIPLES OF MICROBIOLOGY position of the metacliromatic granules and the point of division. The tiny granules are probably similar to the cell-partition granules described by various observers. It is not often t}iat the favorable conditions mentioned abo\'e for the production of equal and rapid division obtain for any time, since even in pure cultures bacteria in their growth soon produce an environ- ment unfavorable for further multiplication. Several factors help to make this environment: First, the using up of suitable food and moisture; second, the disintegration of food substances into various injurious products, such as acids, alkalies, ferments; third, in mixed cultures the overgrowth of one or more varieties. As these unfavorable conditions are more or less constantly present, we seldom see such absolute symmetry in the growth and division of bacteria as is usually described. In fact, except under ideally favorable conditions (e. g., rapid successi^'e transfers from young cultures on the most favorable food medium), we can never see absolutely equal fission among bacteria; and in some species, notably the diphtheria group, division is extremely irregular even in our usual twenty-four cultures on favorable media. Involution and Degeneration Forms. — It follows, from the conditions considered above, that, as cultures grow older or when media unfavor- able to equal division are used, the bacteria may sho\^' extremely irregular forms, absolutely different from the J'oung forms, such as long threads or filaments with irregular thickenings, coccus forms from bacilli and spirilla which have divided without increasing in length, bacillar forms from cocci which ha^'e grown without di^'iding, and apparently branched forms from many varieties of bacilli and spirilla. These have been called involution or degeneration forms. In our study of the so-called branched forms of the diphtheria bacillus we have observed the following interesting fact: Under certain conditions, marked apparent branching appears at a definite time in the age of the culture. The conditions are: slightly disturbed growth in pellicle on nutrient broth. When such pellicles are examined every day they are found to contain, on the six-th to the twelfth day, varying chiefly with the amount of disturbance, many large intensely staining forms with one to several apparent branches and many large metachromatic granules (Figs. 4 and 5). The facts that these forms were the only ones to show active growth and division when examined on a hanging mass of agar and that in such gro\A'th the metachro- matic granules seem to fuse (Fig. 5) before fission led us to suppose that these forms represent a primitive sexual process, a sort of autogamy. Schaudinn (Fig. 6) has shown a primitive conjugation (autogamy) and a relationship between the chromatin granules, or nuclear substance, and the spores in certain bacteria. Although elongation in the greater diameter and complete division at right angles to this is the rule for the majority of bacteria, there are certain groups which, instead of becoming separated from each other as single cells, tend to produce an incomplete segmentation, the cells remaining together in masses, as the sarcinse, for example, which divide more or less regularly in three directions. The indentations upon these masses or cubes, which indicate the point of incomplete fission, give to BACTERIA 39 these bundles of cells the appearance commonly ascribed to them — that of a bale of rags. The rod-shajK'd bacteria nc\-cr di\i(lc lontritudinalh'. 1''IG. 4 Fu; B. diphtheriiie "No. 8" from 9 days' broth pellicle, showing many "branched" forms. Stained mth carbol-fuchsin. X 1500 diameters. B. diphtheriae "No. 8" from 10 days' broth pellicle, showing longitudinal fusion and position of metachromatic granules. Stained with Loffler's methylene blue. X 2000 diameters. 111 m B ki w Fig. 6 /'?\ ft n '•.V :' i i"-\"i l a 1 d 1 :./ ''-Ml f Bacillus biUschlii: a to c, incomplete division of the cell; d to/, gradual collection of chromatin granules at ends of cells; g to i, formation of end spores from these chromatin end masses. (After Schaudinn.) Spore formation must be distinguished from vegetative reproduction. This is the process by which the organisms are enabled to enter a stage in which they resist deleterious influences to a much higher degree than is possible for them to do in the growing or vegetative condition. It is true that in all non-spore-bearing cultures a certain proportion of the bacteria are more resistant than the average. No marked differ- ence in protoplasm, however, has been noted in them other than the ability to stain more intenseh' and sometimes to show strong mcta- 40 PRINCIPLES OF MICROBIOLOGY chromatic areas. The difference between these and tiie less resistant forms is nut great. Some have believed that this resistance is due to certain bodies called adhronporeff, which are abnormally large cells with, usually, a thickened cell wall and increased staining properties, formed as a rule in old cultures. Fullerton and others have described similar forms in some of the higher bacteria and consider them spores. (See Nocardia.) The true spores (endospores) of the lower bacteria are definite bodies. These are strongly refractile and glistening in appearance, oval or round in shape, and composed of concentrated protoplasm developed within the cell and surrounded by a very dense envelope (Plate III, Figs. 22-25). They are characterized by their power of resisting the injurious influences of heat, desiccation, and chemical disinfectants up to a certain limit. (See page 79 for details.) Spores also stain with great difficulty (see chapter on Disinfection). The production of endospores in the different species of bacteria, though not identical in every instance, is very similar. The conditions under which they are produced in nature are supposed to be similar to those observed in artificial cultures, but they may not always be similar, hence we must not consider a bacterium a non-spore bearer because it has not been seen to form spores in the laboratorj^. Usually the for- mation of spores in any species is best observed in a streak culture on nutrient agar or potato, which should be kept at the temperature nearest the optimum for the growth of the organism to be examined. At the end of tweh'e, eighteen, twenty-four, thirty, thirty-six hours, etc., specimens of the culture are observed, first unstained in a hang- ing drop or on an agar mass, and then, if round or o\'al, highly refractile bodies are seen, stained for spores. Each bacillus, as a rule, produces but one spore, and more than two have never been observed. Motile bacteria usually come to a state of rest or immobility previous to spore formation. Several species first become elongated. The anthrax bacillus does this, and a descriptuon of the method of its production of spores may serve as an illustration of the process in other bacteria (Plate III, Fig. 22, A-E). In the beginning, the protoplasm of the elongated filaments is homogeneous, but after a time it becomes turbid and finely granular. These fine granules are then replaced by a smaller number of coarser granules, the so-called sporogenous granules supposed to be chiefly nuclear in nature, which by coalescence finally amalgamate into a spherical or oval refractive body. This is the spore. As soon as the process is completed there may appear between each two spores a delicate partition wall. For a time the spores are retained in a linear position by the cell membranes of the bacilli, but these are later dissolved or broken up and the spores are set free. Not all the cells that make the effort to form spores, as shown by the spherical bodies contained in them, bring these to maturity; indeed, many varieties, under certain cultural conditions, lose altogether their property of forming spores. The following are the most important spore types: (a) the spore lying in the centre of the cell; which may be much distended in its central BACTERIA 41 portion, giving it a spindle shape or Clostridium, e. g., Bacillvn })utyri(:us; {!>) the spore lying at the extremity of a cell nnu-li enlarged at that end — the so-called "head spore" or pleetridiuni, e. //., the tetanus bacillus; (c) the s])ore lying eccentrically (Plate III, Fig.' 25, A-C). According to Seliaudinii and others, in certain spore-bearing bacteria tlie spore formation is part of a sexual-like process (sec under Reproduction). The germination of spores takes place as follows (Plate III, Figs. 22-24) : By the absorption of water they become swollen and pale in color, losing their shining, refractive appearance. Later, a little protuberance is seen upon one side (eciuatorial germination) or at one extremity of the spore (polar germination), and this rapidly grows out to form a rod which consists of soft-growing protoplasm enveloped in a membrane, which is formed of the endosporium or inner layer of the cellular envelope of the spore. The outer envelope, or exosporium, is cast ofi' and may be seen in the vicinity of the newly formed rod; or it may be absorbed. The chief spore formers among the pathogenic bacteria are the anaerobes (tetanus, malignant edema, intestinal bacteria). Only one distinctly pathogenic aerobe produces endospores — the anthrax bacillus. The Higher Forms of Bacteria (see end of Section II) . — Some forms grow out into true or false branching threads and thus make a group of organisms intermediate between bacteria and the molds. These have been called higher bacteria or trichobacteria (see table, p. 25). They show increased complexity of structure and function: (1) in forming irregularly segmented filaments composed of elements similar to those found in the lower forms and showing either true or false branching; (2) in developing certain portions of their substance into reproductive bodies from which the new individuals grow (Plate II, Figs. 15 and 16). The filaments seen sometimes among the lower forms have inde- pendent segments, which may easily separate and grow as tiny luiicel- Inlar forms, while in the higher forms the filaments in their growth show a certain interdependence of their parts. For example, growth often occurs from only one end of the filament while the other becomes attached to some fixed object. The members of the higher bacteria which are pathogenic for man have as yet been incompletely studied and classified. The following divisions serve as an attempt at differentiation: 1. Leptothrix grows in stifi', almost straight threads, in which division processes are seldom or never observed, and no branching has been seen. 2. Cladothrix grows in threads which rapidly fragment and produce false branching, that is, the terminal cell remains partly attached, but is pushed to one side by further growth from the parent thread; thus a Y-shaped growth is produced, and then bacillary characteristics in old cultures. 3. Actinomyces grows in threads with true branching. Spores have been observed. It is characterized by the radiating wreath-like forms which it alone produces in the living body. 42 PRINCIPLES OF MICROBIOLOGY 4. Nocardia {Streptothrix) grows in threads which produce abundant true branching; later there is fragmentation and formation of conidia. Ucprodiictioii among the Higher Bacteria. — These forms increase in length for a time and then, at the free ends, or at intervals along the filaments, they produce small rounded cells, called conidia or spores from which new individuals are formed. The terminal spores may be flagellated after their separation from the parent filament. The flagellated forms frequently resemble certain flagellata among the protozoa. PROTOZOA. Definition. — A protozoon (the lowest form of animal life) is a mor- phologically single-celled organism composed of protoplasm which is difi'erentiated into cytoplasm and nucleus (or nuclear substance) both of which show many variations throughout the more or less complicated life cycle that each individual undergoes. They are of higher grade than the bacteria because of their greater complexity in structure and life cycle (Plate IV). Their shape and size vary so widely that no general description will fit all types. Some forms are small enough to pass through a Berkefeld filter, while the largest varieties described are about two-thirds of an inch long. The cytoplasm consists of a mixture of substances, the most important of which belong to the proteids. It is more or less fluid, but, because of differences in the density and solubility of the several parts, it often presents an alveolar, linear, or granular appearance, which may come out clearly in fixed and stained specimens, but is usually not well seen in the living cells. Ectoplasm and Entoplasm. — Frequently the protozoon cytoplasm is differentiated into a concentrated, viscid, more homogeneous, or hyaline outer layer called the ectoplasm and a more fluid granular central portion called the entoplasm. These two portions have different functions. The ectoplasm helps form the various organs (organelles) of motion, contraction, and prehension such as pseudopods (false feet), flagella (whip-like threads), ciha (hair filaments), suctorial tubules (through which food passes), and myonemes (contractile fibrils found in ciliates, gregarines, and a few flagellates). The entoplasm digests the food and contains the nucleus, as well as various granules and vacuoles. Some vacuoles serve as food digesters, and hence contain digestive ferments The so-called contractile vacuoles which periodically fill and empty themselves may be considered as excretory organelles. Other substances are seen from time to time in the entoplasm, such as bacteria, red-blood cells, fatty granular pigments, bubbles of gas, crystals, etc. The Nucelus. — The simplest morphologic nucleus is a vesicular body which is differentiated from the cytoplasm by its essential constituent chromatin, so-called because it has a strong affinity for certain basic PROTOZOA 43 staining materials. Chromatin consists mostly of nuclein and appears in the form of smaller or larger granules, masses, or rods. Generally, the chromatin particles are mixed with a second less intensely staining substance with more of an affinity for acid stains, calletl plastin or paranuclein, similar to the substance from which the true nucleolus of the metazoon cell seems to be formed. This substance may appear in one or more distinct rounded bodies. Most of the chromatic substances of the nucleus in many protozoa are often massed together in an intensely staining ball-like body called the karyosome which undergoes various cyclic changes during the growth and development of the organism. The centrosome is generally imbedded in the karyosome. The chromatin and plastin lie imbedded in a third substance in the form of an achromatic network called linin, which is closely related to the cytoplasmic network. There may or may not be a definite nuclear membrane. Sometimes there is no defi- nitely structured nucleus, but the nuclear substance in the form of small chromatin masses or granules is distributed throughout the cytoplasm (the so-called "distributed nucleus") similar to that seen in bacteria. Somatic and Generative Chromatin. — Some chromatin substances of the cell have physiologic properties different from others. At times substances which have only vegetative properties are active, forming the so-called somatic or trophic chromatin; at other times, substances appear during sexual activities called generative or sexual or idiochromatin, and from these the vegetative (somatic) chromatin for the new cells is again formed. In the ciliata both these chromatin elements are present as distinct morphologic bodies during the entire life of the organism, the somatic form in the macronucleus and the generative form in the micronucleus. Chromidia. — The chromatin elements, in the form of granules, small irregu- lar masses, threads, network, etc., which at certain stages pass from the nucleus into the cytoplasm, or which at times are, possibly, formed in the cytoplasm, were named "Chromidien" by R. Hertwig, who in 1899 first de- scribed their appearance. Their function in generative processes was demon- strated in 1903 by Schaudinn. During their formation the nucleus may entirely disappear, so that morphologicaUy the cell may be considered non-nuclear. At a definite time thereafter new typical nuclei may be formed from these chromidial substances. Locomotor Nucleus {Kinetic Nucleus). — In flagellates still another definite physiologic chromatin is seen in the small body called the kinetic nucleus, which is either apart from or merged into a smaUer body, the blepharoplast, formhig the root of the flageUum. The kinetic nucleus is so called because it produces the locomotor apparatus. Both the kinetic and trophic imclei may contain somatic and generative chromatin at the same time. The Centrosome. — This is a small body which is always present in meta- zoan cells, playing an important part in cell division, but it has not been demon- strated as a morphologic entity in many varieties of protozoa; part of the karyosome, however, may take its place, or there may always be a true centro- some within the karyosome. Whenever a centrosome appears in protozoa, it has its origin in the nucleus, resembling in this the kinetic nucleus and blepharoplast. AU these four bodies, therefore, centrosome, blepharoplast, kinetic nucleus, and karyosome, may be considered as having a similar morphologic origin. Physiologic Characteristics of Protozoa. — In common with all other living organisms protozoa possess the characteristics of motility, nutri- tion, respiration and reproduction. 44 PRINCIPLES OF MICROBIOLOGY Motility. — All protozoa react in certain characteristic ways toward chcmic, mechanic, and electric stimuli. Many are att'ccted hy light, while probably none react to sound. They manifest the reaction usually by motion of some sort. Most animal parasites, especially the higher forms, e.xert a positive ta.xis for leukocytes, principally for the large mononuclears and the eosinophiles. This fact is made use of in clinical diagnosis. Nutrition. — Many protozoa, especially the pathogenic forms, absorb fluid food directly through the body wall; but the majority take in solid food, such as small animal or vegetable organisms and organic waste, some through more or less definite regions of the body, others through any part of the surface b.y extending pseudopodia and entirely surrounding the food object, forming a so-called gastric vacuole. After the food is digested the waste products are excreted, sometimes by osmosis, generally through special structures as the contractile vacuoles which regularly eject fluid substances to the outside of the organism. Respiration. — It is supposed that the contractile ^'acuole has a respi- ratory as well as an excretory function. The interchange of gases is always going on, if not through a contractile vacuole, then by osmosis through any part of the wall. Growth and Reproduction. — Under favorable conditions, new protoplasm is constructed rapidly, and the mass increases faster than the surface which, according to Spencer, initiates cell division. The changes gen- erally appear first in the nucleus. The simplest variety of reproduction is a two-celled fission which may be either longitudinal or transverse, either of which may be direct (amitotic) or indirect (mitotic). A modifi- cation of equal fission is the so-called budding which may be single or multiple. When growth occurs so that fission is for a time incomplete, one cytoplasm containing several nuclei which finally separate into as many daughter organisms, the process is called multiplicative repro- duction, or brood formation or internal budding. In the most extreme cases of multiplicative reproduction as it occurs among sporozoa the mother-cell with its nucleus separates simultaneously into large numbers of tiny daughter-cells. Such a process, when it occurs without conjuga- tion and encystment, is called schizogony and the new cells are called merozoites. When such a multiplicative division occurs (generally after fertilization) within a cyst, it is spoken of as sporogeny and the new cehs are called sporozoites. Sexual Phenomena. — Sexual phenomena (syngamy) fundamentally similar to those seen in metazoa ha^•e been obser\'ed in all groups of protozoa studied. The reproduction by the usual division or budding is interrupted at certain times in the life history of each organism and individuals come together in such a way that their nuclei fuse, usually after having undergone characteristic reduction divisions. When the union is permanent, we speak of it as copulation and liken the process to that of the fecundation of the ovum by a s])crmatozoon. Wlien the union is transient wo call it conjugation. Hero the two cells fuse for a time PROTOZOA 45 when tl^e nuclei intcrcliann-e ]H'otoplasni and then the cells separate and each one continues to grow and di\'ide independentl}'. 'When in a partly divided cell or in an apparently single cell, two nuclei, after undergoing reduction division, or its like, fuse, the process is called autogamy. The Developmeidal Cycle of a protozoon consists of all the changes which occur in its growtli from one act of fertilization to another (Plate IV, Fig. 3). ^lany protozoa carry on the sexual part of their life cycle in one host and the asexual part in another (p. g., malarial organisms). Cyst Formation. — If protozoa do not get the required amount of water or air or suitable food they cease their special movements, round out into more or less of a sphere and form a resisting membrane of chitin within which they may live for a long time, withstanding periods of desiccation, extreme heat and cold, and they may be blown about as dust until they find conditions again favorable for renewed growth; when water is absorbed the cj'st is ruptured and active life begins anew. In parasitic forms encystment plaj's an important part in the passage from the old host to the new. The majority of forms would not be able to exist outside of the body of the host without having some protective membrane. The cyst may be formed simplj^ for protection from drought, etc., when it is called a hypnocyst, from which the organism may emerge in about the same form as when it encysted; or the cyst may precede reproduction by spore formation or simple division, when it is called a sporocj^st. In either case it may consist of a simple wall or it may be formed of several walls to enable it to resist prolonged desiccation, when it is called a resting cyst. Characteristics of Each of the Four Groups of Protozoa. — Flagellata. — Flagellata are protozoa which move in the adult forms by one to several flagella or whip-Hke processes. If pseudopodia develop, they are transitory. Generally the flagella arise from the anterior part of the organism, and in motion the larger ones (primary flagella) are directed forward, while smaller ones (secondary flageUa) are directed backward, acting as rudders. Certain flagellata possess a modification of their bodies in what is called the undulating membrane, which consists of a fluted protoplasmic process attached along one side of the organism, the free edge of which is prolonged as the flagellum. It has been shown that flagella are not simple protoplasmic processes, but that they have more or less of a framework of elastic fibres as well, hence their power in locomotion can be better understood. Except wath special stains, which bring out these fibres, they appear homogeneous. The flagella arise from some definite place in the cytoplasm, some- times from a distinctly dift'erentiated chromatic body which has been given various names, such as blepharoplast, kinetic nucleus or centro- some, sometimes near this from a basal granule, microsome, diplosome, or flagellum root, sometimes directly from the nucleus. The basal granules seem to be tlerived primarily from the kinetonudeus, and may be considered from a physiologic stand-point, as a part of the motor nuclei. 40 PRINCIPLES OF MICROBIOLOGY The body of the flagellates is generally more or less elongated and, except in most primitive ones, is fixed in its outline. The latter charac- teristic is chiefly due to the fact that the organisms usually possess definite though delicate membranes containing elastic fibrils. The cytoplasm is usually not diff'erentiated into an ento- and ectoplasm. It often contains one to several contractile vacuoles, as well as food EXPLANATION OF PLATE IV. Partly schematic. Rearranged and drawn by Williams. All stained by Giemsa. I. Flagell.vtes. Fig. 1.— Illustrating one flagellum. Leishmania: ^.intracellular form; B, cultural forms. Fig. 2. — Illustrating undulating membranes: A, Trypanosoma lewisi; B, TryiJanosoma brucei; C, Trj^panosoma gambiense. Fig. 3. — Illustrating two flagella. Bode lacertie (after Prowazek) . Fig. 4. — Illustrating four flagella. Trichomonas. II. AmeBjE. Fig. 1. — Illustrating points considered differential in the two chief types of amebaj (entamebiB) described as parasitic in human beings. Fig. 1. — Entameba coli, vegetative stage. Fig. 2. — Dividing nucleus. Fig. 3. — Entameba coli cyst containing eight nuclei. Fig. 4. — Entameba histolytica, vegetative stage. Fig. 5. — Four-nucleate cyst. III. Spohozoa. A, description of Figs. 1 to 17. (After Schaudinn.) The Ufe cycle of Eimcria schubcrgi. In 1, the sporozoites, becoming free by bursting the sporocysts, pass out through an aperture in the wall of the oocyst, and are ready to enter the epithelial cells of the host. 2 to 6 represent the asexual reproduction or schizogony, commencing with infection of an epithelial cell by a merozoite or a sporozoite; the merozoite after stage 6 may start again (5) at stage 2, as indicated by the arrows, or it may go on to the formation of gametocytes (9 to 11). 9 to 11 represent the sexual generation, the line of development becoming split into two lines — male {iS) and female (9) — culminating in the highly differentiated gametes, which conjugate and become again a single line, shown in 12-14. The zygote thus formed goes on the production of spores, 15 and 16. 2 and 3 represent epithelial cells showing penetration of a merozoite or a sporozoite and its change into a schizont; 4, the nucleus of the schizont di\-ided into numerous daughter-nuclei; 6, seg- mentation of the schizont into numerous merozoites, about a central mass of residual protoplasm, which in this figure is hidden by the merozoites; 5, mero- zoites passing to reinfect host cell and repeat the process of schizogony; 7, 8, merozoites to be differentiated into male and female gametocytes; 9, the two gametocytes within a host cell; the microgametocyto (cT) has fine granulations; the macrogametocyte (5) has coarse granulations. 11, a female gametocyte undergoing maturation; 13, mature macrogaraete, freed from the host cell, and sending a cone of reception toward an approaching microgamcte. In 12 the nuclei of the last stage have become microgametes, each with two flagella. The free microgamctes are swimming to find a macrogamete. 14, the zygote (fertilized niacrogamete), surrounded by a tough membrane or oocyst, which allows no more microgametes to enter, and containing the female chromatin, which is taking the form of a spindle, and the male chromatin in a compact lump. 15, the nucleus of the zygote divided — the nuclei of the sjioroblasts. In 16 the four sporoblasts become distinct, leaving a small quantity of residual protoplasm; each sporoblast has formed a membrane, the Sjiorocyst. Within each .siiorocy.st two sporozoit(« form about a sporal residuum. /;, Babesia infecting red-blood cells: 1, pear-shaped bodies; 2, dividing forms- 3 eight pe.-ir-shaped bodies in a cell; 4, irregular ring-like ))odies; 5, large, irregular body; 6, boy/ t(%^ "fe^fr*^' PROTOZOA 47 vacuoles, and there is frequently a definite opening or cj-tostom for the entrance of food. There are usually many granules and inclusions of various kinds scattered throughout the cytoplasm, and myoneme striations are seen in some forms. The nucleus, as a rule situated ante- riorly, varies much according to different species and to different stages of development. The flagellata multiply either in the free motile condition or after encystment. In the first case, as a general thing, they divide longi- tudinally. The basal granules divide with the nuclei and the flagella of the daughter organisms are usually formed anew. Multiple division is also observed. In the second case, they may or may not conjugate before they encyst. Then they divide within the cyst. The sexual cycle varies much in different species. Isogamy has been noticed between fully grown individuals as well as between smaller forms. The union of different-sized forms, or anisogamy, has also been observed. Also autogamy is said not to be infrequent. It is claimed that certain of the flagellates pathogenic in man require a second host for the development of their sexual cycle. The flagellates are subdivided into several orders, in only two of which are forms found which are pathogenic for man. (See Part II.) The different types of pathogenic flagellates are shown in Plate IV, i. Figs. 1-4. Among the most important pathogenic forms are the trypanosomes. Trypanosoma. — The name trypanosoma (boring animal) was given by Gruby, in 184.3, to certain free-swimming hemoflagellates found by him in the blood of frogs. Much later similar flagellates were found in the blood-plasma of many different species of vertebrates and in the intestinal tract of several blood-sucking invertebrates. Some of the forms, including those found in man, are pathogenic. A number of the blood-sucking insects are carriers of the diseased species to healthy animals. Typical trypanosomes are characterized by a comparatively long, spirally twisted body, along one side of which is attached an undulating membrane having a cord-like edge that is continued forward as a free whip (flagellum). The flagellum arises near the posterior end of the organism in a small granule called the basal granule, which may be connected with the blepharoplast, a larger chromatin mass, called also the kinetonucleus because of its control over the motor apparatus. The nuclear apparatus consists of a macro- or trophonucleus, and of the above-mentioned kinetonucleus or blepharoplast, which last func- tions as a centrosome. The trophonucleus is usually situated near the middle of the organism; it is granular, thick, and egg-shaped, but varies somewhat in size and shape. The cytoplasm is faintly alveolar or granular, varying with age, environment, and possibly species Toward the straighter border of the cell the cytoplasm is more or less striated and in a few species definite myonemes are seen. Reproduction occurs usually by longitudinal, occasionally by multiple division. The life cycle is not well known. Though transmission occurs through the bites of various invertebrates, notably flies, the few sexual changes described as taking place in the intestines of some of 48 PRINCIPLES OF MICROBIOLOGY these intermediate hosts have not been fully corroborated. That an intermediate host is not necessary for the continued life of at least one species of trypanosome seems to be proved by the fact of direct trans- mission of T. equiperchtm from horse to horse through coitus. Leishmania. — Another important pathogenic flagellate is Leishmania. In humans this grows chiefly within large mononuclear cells. It shows its flagellated forms in cultures. (Plate IV, Fig. 1. See also Part II.) Amebida. — Under amebida (syn., amebfe) we include forms composed of naked, simply constructed protoplasm having the power of producing lobose pseudopodia which are used as organs of motion and of nutrition. The pseudopodia are protoplasmic processes which are projected in irregular succession from different parts of the surface of the cell, producing in this way an irregular motion. The form of the pseudopodia varies considerably in the different species. For instance, there are broad, bhmt processes or narrow, less blunted ones, and each may be short or long, single or slightly branched. The entoplasm may or may not take a share in their formation. The forms of course vary within limits according to the condition of the medium in which the amebae are living. Movements are always called forth by some physic or chemic excitant. When such an excitant is desirable for food the pseudopods flow around it, and it is subsequently absorbed in the cyto- plasm of the organism. The members of this group may possess one nucleus or several. Amelia hinuclecda has two nuclei in the young adult stage, and Pelomyxa IKilustris, living in the bottom ooze of ponds, has an enormous number of nuclei. K marked feature of the nuclear apparatus is the formation of chromidia which, as has already been noted, may play such an impor- tant part in sexual reproduction. Generally each ameba has one contrac- tile vacuole, but occasionally some are seen with several or with none. Saprophytic forms belonging to this order are common. The,y may be found wherever there are moisture and decaying vegetable matter. The pathogenic forms are not so frequent. Because of the possibility of the still unknown causes of certain diseases (see Rabies and Smallpox) being organisms related to this order, it is especially important to study both saprophytic and pathogenic varieties, since a knowledge of the former which arc more easily studied may help us imderstand obscure ])oints in the life history- of the latter. Notwithstanding the common occurrence of saprophytic forms, the full life history of few of them has been worked out, and until the full cycle of development of any so-called ameba is known it is impossible to say whether that particular form belongs among rhizopoda or whether it is a developmental form of anotiier group, as ameboid forms may occur at some time in the life history of all groups. It is (|uite possible that some of the organisms descriljed as belonging to this order are really meml)ers of entirely different orders. For instance, it is known that the fl;igelhite TrIcJtoiiioiius loses its flagella before copulation and crawls about Ijy means of short blunt pseudopods as a typical ameba. PROTOZOA 4«J Amebar reproduft- by simple fission, hy budding, and l)y l)rood for- mation. In tiie last case the reproduction is usually preceded by encyst- ment. Two forms have been described most frequently as parasitic in man. The differences between them are shown in Plate IV, ii, Figs. l-f). The Sporozoa. — The s]>orozoa are a group of exclusively j)arasitic protozoa of very widespread occurrence, living in the cells, tissues, and cavities of animals of every class. Generally they are harmless, but some varieties may produce pathologic changes and even fatal diseases severely epidemic. As their name indicates, they are all characterized by reproduction through spore formation, but they exhibit the utmost diversity of structural and developmental characteristics. As a rule, each species is parasitic on one kind of tissue of a particular species of host. They are generally taken into the system in the spore stage either (1) with the food of the host, (2) by the bites of insects, or (3) by inhalation. The spore membranes are dissolved by the fluids of the host, and thus one or more germs or sporozoites are set free to bore into the special cells of the host. Here they grow, some remaining permanently intra- cellular, others only in the young stages. The latter either pass different phases of their more or less complicated life history in different parts of the body of one and the same host or they pass some phases of their life cycle in the cells of an intermediate host. The sporozoa vary widely in size as well as in other characteristics. From the smallest, several of which can be contained in a single blood- cell, there are all gradations in size up to those that may be seen by the naked eye (Porospora giganiea, 16 mm.). Besides being characterized by the power to produce more or less resisting spores, the sporozoa are also characterized by the fact that as a class they possess none of the special organs found in other pro- tozoa for ingesting or digesting solids. Many develop flagella during sexual phases or show ameboid movement during certain stages of their life cycle, but the flagella and pseudopodia are organs of loco- motion and not of nutrition. Food vacuoles or contractile vacuoles have not been found. The life cycle of a tv'pical sporozoon is rearranged and condensed from Schaudinn in Plate IV, in. Figs. l-Ki. Ciliata. — The ciliata (Plate IV) belong to the most complex of the protozoa. They possess a definite entoplasm containing nuclei and food \'acuoles, and a definite ectoplasm containing basal granules from which arise the cilia which gi\-e the group its name. They have organijid structures which receive the food, some have definite mouth openings, indeed, and definite places for excreting waste products. The food vacuoles may contain acid or alkaline digestive products. The nuclear material is differentiated into two forms, a large macronucleus and a much smaller microiuicleus. The function of the macronucleus is sup- posed to be vegetative, and that of the micronucleus reproductive. The macronucleus varies in size and shape and is completely filled with 4 30 ■ PRINCIPLES OF MICROBIOLOGY an alveolar chromatin. The micronucleus also varies in size and shape, but unless in reproductive phases is generally vesicular in structure, with the chromatin heaped in one mass. Division of the nuclei takes place by mitosis in the case of the micronuclei, and by amitosis, as a rule, in the case of the macronuclei. Under conditions unfavorable for growth the ciliata may encyst. Conjugation is necessary to the life activity of these organisms. The phenomena of conjugation in the ciliata has been well worked out. The micronuclei play the most important part, whereas the macro- nuclei simply break up and disappear in the protoplasm. According to the arrangement of the cilia, the ciliata are divided into the four orders given in the general classification. Among these, the second, the order of the Heterotricha, interests us. In the Hetero- tricha the cilia are uniform over most of the body, while a specialized set fused into a series of firm vibratory plates is found about the mouth. Onlv one genus, Balcmtidiuvi, has been observed in man (Plate IV; also Part II). CHEMICAL COMPOSITION OF MICROORGANISMS. Qualitatively considered, the bodies of micro5rganisms consist largely of water, salts (chiefly phosphorus, potassium, chlorine, calcium and sulphur), fats, and albuminous substances. There are also present, in smaller quantities, extractive substances soluble in alcohol and in ether. Special varieties contain unusual substances, as wax and hemi- cellulose in tubercle bacilli. Microorganisms possess the capacity in a high degree of accommodating their chemical composition to the variety of soil in which they are growing. Each variety, furthermore, yields proteid substances peculiar to itself, as shown in the effects produced by animal inoculation. At present we know but little concerning the differentiation of these specific substances. This subject will be taken up in detail under Toxins, etc. According to Cramer, many bacteria contain amyloid substances wliich give a blue reaction with iodine. True cellulose has not been found in bacteria, but large quantities of a gelatinous carbohydrate similar to hemicellulose have been obtained. Nuclein is found in all microorganisms. The nuclein bases — zanthin, guanin, and adenin — have been obtained in considerable amounts. There is a group of bacteria which contain large amounts of sulphur — viz., the Beggiatoa — and another group, the Cladoihri.r, is capable of separating ferric oxide from water containing iron. The percentage of water contained in microbes grown on solid culture media, as well as the amount of residue and ash, depends largely on the coni]x>sition of tlie media. Thus, Bdcillus profligio.iiis when grown on potato contains 21.5 per cent, of dry residue and 2.7 per cent, of ash; when culti\ated on turnips it contains 12. G per cent, of dry residue and !.:> per cent, of asli. Besides the concentration of the culture, its temperature and age also influence the amount of residue and ash produced. The residue varies, moreover, qualitatively in the same EFFECTS OF SURROUNDING FORCES UPON MICROORCAN ISMS 51 species under the influence of the culture media employed. Thus it appears that an additional quantity of peptone in the culture media tends to increase the percentage of nitrogenous matter in the bacillus, while the addition of glucose decreases it. The chemical composition of the bodies of animal parasites is an almost unexplored field. The ectoplasm and the cyst sacs in general are made up principally of a substance called chitin. Glycogen has been isolated from many forms. Proteolytic enzymes and acid secre- tion in digestive \'acuoles have been demonstrated. Microchemical Reactions. — To a certain degree the chemical com- position of tlie individual organism may be studied both in the living and in the dead indi\'idual by the addition of the testing substances to a hanging drop or to a spread of such organism and the examination of it under the microscope. Of special importance in this regard is the resistance which bacteria possess to diluted alkalies. Inasmuch as the majority of animal tissues are dissolved when treated with alkalies, this method has been adopted for rendering visible unstained bacteria in tissues. (See also Principles of Staining, p. 76). EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS. 1. Food. — Naturally, the effect of food upon organisms is marked. Though the majority of pathogenic microorganisms grow easily on certain artificial foods (culture media), some of them, like most of the protozoa we have not yet been able to cultivate outside of the body of their host. Those microorganisms which seem to depend entirely upon a living host for their existence are known as strict paraMtes; those which live only upon dead organic (a few on inorganic) substances are called strict saprophytes; those which can lead a saprophytic existence, but which usually thrive only within the body of a living animal, are called facultative parasites. The strict saprophytes, whicli represent the large majority of all microorganisms are not only harmless to living organisms, but perform many exceedingly important functions in nature, such as the destruction of dead organic matter and its preparation for plant food through decomposition, putrefaction, and fermentation, while one group (see below, the Nitrifying Bacteria) are constructive in their activities. The parasites, on the contrary, may be harmful invaders of the body tissues, exciting by their growth and products many forms of disease. The substances essential for the majority of those forms which can be grown artificially are organic material, as a source of carbon and nitrogen, an abundance of water and certain salts. Either calcium or magnesium and sodium or potassium salts are usually required, also sulphur and phosphorus salts. Iron is demanded by a few varieties. The demands of microorganisms for food of a certain composition vary considerably. (See chapter on Culti^■ation of jMicro- organisms.) 52 PRINCIPLES OF MICROBIOLOGY While it is true that very wide differences in relative composition and total concentration of food media may have slight effect upon the general development of a given organism, slight changes in composition and reaction of the media often have a great effect upon morphology, rate of growtli, motility, and specific products of growth. Reaction of Media. — The reaction of the media is of very great impor- tance. Most bacteria, particularly the pathogenic forms, grow best on those media that are slightly alkaline or neutral to litmus. Yeasts and molds grow best on a slightly acid medium. An amount of acid or alkali insufficient to prevent the development of bacteria may still suffice to rob them of some of their most important functions, such as the production of poison. The difl^erent effect upon closely allied varieties of bacteria of a slight excess of acid or alkali is sometimes made use of in separating those which may be closel.y allied in many other respects. Influence of One Species upon the Growth of Another. — When one species of organism is grown in a food medium, that medium usually Irecomes less suitable for the growth of its kind and of other organisms. This is due partly to the impoverishment of the foodstuff's, but more to the production of chemical substances or enzymes. W'hen different species are grown together, the antagonistic action of one upon the other may be shown from the beginning. Some species, however, have a cooperative or symbiotic action with other species. In nature, microorganisms usually occur in mixed cultures {e. g., in water, milk, intestinal contents of all animals), and here we may see antagonistic action in the prevalence of one species over others (e. g., the lactic acid formers in the intestines), or cooperative action in the ec{ual and luxuriant growth of two or more species (p. g., pneumococcus and influenza bacillus in the lungs). Experimentally, the existence of antagonisms can be demonstrated by inoculating alternate streak cultures of various bacteria on gelatin or agar media. It is found that many species will grow not at all or only sparingly when in close proximity to some other species. This antag- onism, however, is often only one-sided in character. Again, when gelatin or agar plates are planted with a mixture of two species of bac- teria it may be observed that only one of the two grows. A third method of making this experiment is simultaneously to inoculate the same liquid medium with two species, and then to examine them later, both micro- scopically and by making plate cultures; not infrecpiently one species may take precedence of the other which after a time it may entirely overcome. Finally, it may he shown experimentally that microorganisms may oppose one another as antagonists in the animal Ijody. For instance, Emmerich has shown that animals infected with anthrax may often be cured by a secondary infection with the streptococcus. The symbiotic or cociperative action of microorganisms may be demonstrated experimentally in the following examples: (a) Pneumococci, when grown together with a bacillus obtained from the throat, produce very large, succulent colonies. The influenza Inicillus, which will not rtow aloiK' upon onlinary initriciit ;i,i;ar, wiU grow well thcrt' in the presence of certain other hacteria. Some anae- robic species grow e\x'n with the admission of air if only some aerol)ic species are present (tetanus bacilli with diphtheria bacilli). (h) Certain chemical ett'ects, as, for instance, the decomi)ositlon of nitrates, cannot be producetl b>- many s])ccies of bacteria alone, but only when two are associated. 2. Behavior toward Oxygen and other Gases.— The majority of micro- organisms absolutely require free oxygen for their growth, but a con- siderable minority fail to grow unless it is excludecl. This latter fact, noted first by Pasteur, led him to divide germs into aerobic and anae- robic forms. Between these two groiips we ha^'e those that can grow either with or without the presence of oxygen, called respectively facultative anaerobic and facultative aerobic organisms. (a) Aerobic Organisms. — Growth only in the presence of free oxygen. The slightest restriction of air inhibits development. Spore formation, especially, requires the free admission of air. (6) Anaerobic Organisms. — Growth and spore formation only on the total exclusion of free oxygen. Among this class of organisms are the bacillus of malignant edema, the tetanus bacillus, the bacillus of symp- tomatic anthrax, and many soil bacteria. Exposed to the action of oxygen, the vegetative forms of these bacteria are readily destroyed; the spores, on the contrary, are very resistant. Anaerobic germs being unable to use, deprived the air of oxygen — the chief source of energy used by the aerobic species to oxidize the nutriti\'e substances in the culture media — are dependent for their oxygen upon decomposable substances, such as grape-sugar. (c) Facultative Aerobic and Facultative Anaerobic Organisms. — The greater number of aerobic germs, including most of the pathogenic species, are capable of withstanding, without being seriously affected, some restriction in the amount of oxygen admitted, and many, indeed, grow equall}' luxuriantly with the partial exclusion of oxygen. Life in the animal body, for example, as in the intestines, necessitates existence with diminished supply of oxygen. If in any given variety of bacteria, the amount of oxygen present is unfavorable, there w'ill be more or less restriction in some of the life processes of this variety, such as pigment and toxin production, spore formation, etc. Pigment forma- tion almost always ceases with the exclusion of oxygen, but poisonous products of decomposition may be more abundantly produced. It has been observed not infreciuently that certain species which on their isolation at first show more or less anaerobic development — that is, a preference to grow in the depth of an agar-stick culture, for instance — after a while seem to become strict aerobes, growing only on the surface of the medium. Those organisms that grow best or grow exclusively when the oxygen is only partly removed are called mkro-aerophilic organisms. Other Gases. — While all facultative organisms as well as strict anae- robes grow well in nitrogen and hydrogen, they beha\'e \'ery differently^ 54 PRINCIPLES OF MICROBIOLOGY toward carbonic acid gas. A large number of these species clo not grow at all, being completely inhibited in their development until oxygen is again admitted— for example, B. anthracis and B. .siibtilis and other allied species. It has been found in some species, as glanders and cholera, that the majority of the organisms are cjuickly killed l>y CO2, while few, such as staphylococci, offer a great resistance, rendering impossible complete sterilization by means of this gas. The streptococcus as well. as the staphylococcus exhibits a scanty growth. A mixture of one- fourth air to three-fourths carbonic acid gas seems to ha^'e no injurious effect on bacteria which cannot grow in an atmosphere of pure CO2. Under pressure CO2 is more effective (page 57). Sulphureted hydrogen in large quantity is a strong bacterial poison. Even in small amounts it kills some bacteria. 3. Effect of Temperature. — Some form of microbic life is possible within the limits of 0° and 70° C. The maximum and minimum tem- perature for each individual species ordinarily lies from 10° to 30° C. apart, and the optimum covers about 5°. Usually the temperature of the soil in which the germs are deposited is the controlling factor in deciding whether growth will or will not take place. Thus, nearly all parasitic microorganisms require a temperature near that of the body of their host for their development, while many saprophytic forms can grow only at much lower temperatures. Microbes when exposed to lower temperature than suffices for their growth, while having their activities decreased, may not be otherwise injured unless actually frozen for a certain time ; while exposure to higher temperatures than allows of growth more or less quickly destroys the lifeof the organism. Microorganisms have been classified according to the temperatures at which they develop, as follows: Psychrophilic Microbes. — Minimum at 0° C, optimum at 15° to 20° C, maximum at about 30° C. To this class belong many of the water microorganisms, such as the phosphorescent bacteria in sea-water; and many molds and yeasts. Mesojjhilic Micwhes. — Minimum at 5° to 25° C, optimum about 37° C, maximum at about 43° C. To this class belong all pathogenic bacteria, most parasitic and many saprophytic forms. Thermophilic Microbes. — Minimum at 25° to 45° C, optimum at 50° to 55° C, maximum at 60° to 70° C. This class includes a number of soil bacteria which are almost exclusively spore-bearing bacilli. They are also found widely distributed in feces. By carefully elevating or reducing the temperature the limits within which a species will grow can be altered. Thus, the anthrax bacillus may be gradually made to accommodate itself to a temperature of 42° C, and pigeons, which are comparati-s-ely immime to anthrax, partly on account of their high body temperature (42° C), when inoculated with this anthrax succumb to the infection. Another culture accustomed to a temperature of 12° C. kills frogs kept at 12° C. We have culti- vated a very virulent diphtheria bacillus so that it will grow at 43° C. and produce strong toxin. EFFECTS OF SURROUNDING FORCES UPON MTCROORCANTSMS Sf) Effect of Low Temperature. — Temperatures even far under (1° C. are ouly slowly injurious to nucrocirg-anisms, ditt'ereut species heiiig all'eeted with varying rapidity. This has heeu demonstrated by numerous experiments in which tiiey lia\-e l)een e.\i)osed for weeks in a refrijicr- ating mixture at — l.S° ('. If a culture of typhoid l)acilli is frozen, ahout 50 to 70 per cent, of the organisms are killed at the time. At the end of one week not more than 10 per cent, survive, and at four weeks not over 1 per cent. After six months none survive. More resistant bac- teria live longer and spores may survive in ice for years. Bacteria have even been subjected to a temperature of — 175° C. by immersing them in liquid air kept in an open tube for two hours, and 15 to 80 per cent, were found still to grow when placed in favorable conditions. We found about 10 per cent, of typhoid bacilli alive after thirty minutes' exposure to this low temperature. Staphylococci were more resistant. Spores were scarcely aft'ected at all. Effect of High Temperatures. — Prolonged temperatures from 5° to 10° C. over the optimum affect microorganisms injuriously in several respects. For instance, varieties may be produced of diminished activity of growth, the \-irulence and the property of causing fermentation may be decreased, and the power of spore formation may be gradually lost. If the maximum temperature is exceeded, the organism dies. The thermal death point for the psychrophilic species is about o7° C, for the mesophilic species about 45° to 55° C, and for the thermophilic species about 75° C. There are no non-spore-bearing bacteria which when moist are able to withstand a temperature of 100° C. even for a few minutes. A long exposure to temperatures between 00° and 80° C. has the same result as a shorter one at the higher temperatures. Ten to thirty minutes' exposure to moist heat will at 60° C. kill the cholera spirillum, the streptococcus, the typhoid bacillus, and the gonococeus, and at 70° C. the staphylococcus, the latter being among the most resistant of the pathogenic organisms which show no spores. A much shorter exposure will kill a large percentage of any mass of these bacteria. Effect of Dry Heat. — When microorganisms in a desiccated condi- tion are exposed to the action of heated dry air the temperature required for their destruction is much above that required when they are in a moist condition or when they are exposed to the action of hot water or steam. A large number of pathogenic and non-pathogenic species are able occasionally to resist a temperature of over 100° C. dry heat for from ten minutes to one hour. In any large number of bacteria a few are always more resistant then the majority. A temperature of 120° to 130° C. dry heat maintained for one and a half hours will destroy all bacteria, in the absence of spores. Resistance of Spores to Heat. — Spores possess a great power of resist- ance to both moist and dry heat. Dry heat is comparatively well- borne, many bacterial spores resisting a temperature of over 130° C. for as long as three hours. Exposed to 150° C. for one hour, practically all spores are killed. Moist heat at a temperature of 100° C, either boiling water or free-flowing steam, destroys the spores of most varieties 56 PRINCIPLES OF MICROBIOLOGY of bacteria witliiii fifteen minutes; certain i)ath()genic and non-patlio- genic species, however, resist this temperature for hours. The spores of a bacilkis from the soil reciuired fi\'e and a half to six hours' exposure to streaming steam for their destruction. They were destroyed, however, by exposure for twenty-five minutes in steam at llo° to 116° C. and in two minutes at 127° C. The spores from tetanus bacilli may require longer than fifteen minutes' exposure to kill them. The resistance of spores to moist heat is tested by suspending threads, upon which the spores have been dried, in boiling water or steam. The threads are removed from minute to minute and laid upon agar or in broth and kept at a suitable temperature for the germination of any living spores. 4. Influence of Light. — A large number of microorganisms are inhibited in growth by the action of bright daylight, more are affected by direct sunlight, and when the action of the sun's rays is prolonged they lose their power of developing when later placed in the dark. Some motile organisms move toward the point of greatest luminosity, others away from it. Light-seeking protozoa have green or yellow chromatophores, and usuallj- at the anterior end a red pigment spot. The violet and blue rays are more active than other parts of the spectrum in determining motion. The susceptibility of bacteria to light may be tested, according to H. Buchner, by suspending a large number of bacteria in nutrient gelatin or agar and pouring the media, while still fluid in a Petri dish, upon which has been pasted a strip of black paper on the side exposed to the light. The action of heat may be excluded by allowing the ray of light to pass through a layer of water or alum of several centimeters' thickness. After the plate has been exposed to the light for one-half, one, one and a half, two hours, etc., it is taken into a dark room and allowed to stand at 20° or 35° C. a sufficient length of time to allow of growth, and then examined to see whether there are colonies anywhere except on the spot covered by the paper; when the colonies exposed to the light have been completely destroyed there is lying in a clear sterile field a sharply defined region of the shape of the paper strip crowded with colonies. Protected by ordinary non-colored glass the sun's rays act very slowly. Only the ultraviolet, violet, and blue rays of the spectrum seem to possess bactericidal action; green light has very much less; red and yellow light none at all. The action of light is apparently assisted by the admission of air; aneorbic species, like the tetanus bacillus, and facultative anaerobic species, such as the colon bacillus, are able to withstand quite well the action of intense, direct sunlight in the absence of oxygen, for four hours. According to Richardson and Dieudonne, the mechanism of the action of light may be at least partially explained by the fact that in agar plates exposed to light for a short time (even after ten minutes' exposure to direct sunlight) hydrogen peroxide (H2O2) is formed. EFFECTS OF SURROUNDING FORCES UPON MICROORGANISMS r-,7 Tliis is demonstrated l)y exposin;;' an a,t;ar ]>late lialf coveri'd with blaek j)aper, upon winch a weak sohition oC iochde of starch is ponred, and over this ao-ain a diinte solntion of snlpliate of iron; the side exposed to the hght tnrns hlne-hlack. In gases contuininj;- no oxygen, hydrogen peroxide is not prodnced, and the light has no injnrions ett'ect. x\ecess of oxygen also explains the ett'eet which light prodnces on culture media which have been exposed to the action of sunlight, as standing in the sun for a time, when afterward used for inoculation. The bacteria subsequently introduced into such media grow badly — far worse than in fresh culture media which are kept in the shade. Influence of Radium. — Radio-active fluids have a slight inhibiting effect on microbal growth, but nothing decided enough to be used for therapeutic purposes has been evolved up to the present time. Influence of X-rays. — These rays have a slight inhibiting effect on micToorganisms when they are directly exposed to them. 5. Influence of Electricity. — The majority of the observations hereto- fore made on this subject would seem to indicate that there is no direct action of the gahanic current on bacteria ; but the effect of heat and the electrolytic changes in the culture liquid resulting from the electrolysis may destroy them. Protozoa may be contracted by moderate induction shocks and killed by strong ones. When a current of electricity is passed through a liquid medium most active protozoa swim with their long diameters in the direction of the lines of force to assemble behind the cathode. Most flagellates and a few ciliates, however, mo\'e toward the anode. The direction of motion has been shown by Dale to \'ary with the nature and concen- tration of the medium. This whole question has been little studied. (). Influence of Agitation. — Meltzer has shown that the vitality of bacteria is destroyed by protracted and violent shaking, which causes a disintegration of the cells. Many species are more quickly autolyzed after violent shaking. 7. Influence of Pressure. — Microorganisms in fluids which are sub- jected to great pressure are for a time inhibited in their growth. When oxygen or nitrogen are used the same moderate inhibition occurs. Influence of Carbonic Acid Under Pressure. — D'Arsonval and Charrin submitted a culture of Bacillus pyocyaneus to a pressure of fifty atmos- pheres under carbonic acid. At the end of four hours cultures could still be obtained, but the bacillus had lost its power of pigment produc- tion. A few colonies were developed after six hours' exposure to this pressure, but after twenty-four hours no development occurred. Other bacteria subjected to pressure have exhibited more resistance. We have subjected broth and milk containing typhoid, dysentery, diphtheria, and colon bacilli to the gas under a pressure of seventy-five and one hundred and fifty pounds. Within twenty-four hours 99 per cent, of those in the broth and 98 per cent, of those in the milk were destroyed. Within one week the broth was sterile and within four weeks the milk was sterile. Tuberde bacilli and staphjdococci were much more resistant, but little 58 PRINCIPLES OF MICROBIOLOGY effect lieing noticed in twenty-four hours. The results were the same whether the cultures were kept at 10° or 25°. Bottled waters charged with carl)oiiic acid are usually sterile. 8. Effect of Drying. — For growth, microorganisms require much moisture. Want of water ati'ects them in different ways. Upon dried culture media development soon ceases; but on media dried gradually at the room temperature (nutrient agar, gelatin, potato) they live often for a long time, even when there are no spores to account for their lon- gevity. A shrunken residue of such cultures placed in bouillon has often been foimd, after a year or more, to yield living bacteria. The yeasts and molds are still more resistant. The question as to how long the non-spore-bearing forms are capable of retaining their vitality when dried on a co\-er-glass or silk threads has been variously answered. We know now that there are many factors which influence the retention of vitality; spores, of course, being more resistant than vegetative forms. The varying results sometimes reported by different observers may be explained by the fact that the conditions under which thej^ were made were different, depending upon the desiccator used, the medium upon which the cultures were grown, and the use of silk threads or cover- glasses. In all these experiments, of course, it should be previously determined that in spore-bearing species there are no spores present. Even when a dried culture lives for a long time the majority of the organisms die in a few hours after drying. We have found 1,500,000 colon bacilli to be reduced to 100,000 after three hours' drying. In pathogenic tissues or exudates they resist drying much longer than when unprotected. Encysted protozoa withstand long periods of desiccation. IMost forms when dried quickly remain viable much longer than when dried slowly. Duration of Life in Pure Water. — When microorganisms which require much organic food for their development (and these include most of the pathogenic species) are placed in distilled water they soon die — that is, within a few days; even in sterilized well-water or surface water their life duration does not usually exceed eight to fourteen days, and they rarely multiply. Instances, however, of much more extended life under certain conditions are recorded. 9. Tactic Effect of Chemicals.— CAe?7(0!'o.rw.— The deleterious effect of chemicals, especially those used as germicides, will be considered under Disinfection. Sorae chemical substances exert a peculiar attraction for micro- organisms, known as positive chemotaxis, while others repel them — negative chemotaxis. Moreover, all varieties are not affected alike. Oxygen, for example, attracts aerobic and repels anaerobic bacteria, and for each variety there is a definite proportion of oxygen, which most strongly attracts. The chemotactic properties of substances are tested by pushing the open end of a fine capillary tube, filled with the substance to be tested, into the edge of a drop of fluid containing the organisms and examining under the microscope. We are able thus to watch the PRODUCTS OF MICROBAL GROWTH 59 action of the microbes and note whctlier they crowd al)out the tiil)e opening; or are repelled from it. Among substances showing positive chemotaxis for nearly all microorganisms are peptone and urea, wiiile among- those showing llegati^•e chemotaxis are alcohol and many of the metallic salts. Such experiments are of course rough. The diffusion of the substances from the tube into the surrounding medium must play an extremely active role in the final result. PRODUCTS OF MICROBAL GROWTH. Microorganisms not only are acted upon by their surroundings, as has already been shown, but they themselves act, often markedly, upon these surroundings. We have spoken, under the effect of food (p. 51) of the great changes which may be produced in growths by slight changes in the food medium. So, many of the products, as noted below, are influenced to a greater or a less extent by environment. Production of Light. — Microorganisms which have the property of emitting light are quite widely distributed in nature, particularly in media rich in salt, as in sea-water. Many of these have been accurately studied. The emission of light is a property of the living protoplasm of the organism, and is not usually due to the oxidation of any photo- genic substance given off by them; at least only in two instances has such substance been claimed to have been isolated. While these organisms cannot emit light except during life, they can live without emitting light. They are best grown under free access of oxygen in a culture medium prepared by boiling fish in sea-water (or water containing 3 per cent, sea-salt), to which 1 per cent, peptone, 1 per cent, glycerin, and 0.5 per cent, asparagin are added. The power of emitting hght is soon lost unless the organism is constantly transplanted to fresh media. Thermic Effects. — The production of heat by microorganisms does not attract attention in our usual cultures because of its slight amount, and even fermenting culture liquids with abundance of bacteria cause no sensation of warmth when touched by the hand. Careful tests, however, show that heat is produced. The increase of temperature in organic substances when stored in a moist condition, as tobacco, hay, manure, etc., is due, partly at least, to the action of bacteria. Chemical Effects. — The chemical changes which take place in sub- stances as they are split up by microorganisms depend on the nature of the substances involved and the conditions under which they exist, and on the varieties of germs present. Chemists can as yet only enumerate some of the substances evolved, and describe, in a few cases, the manner in which they were produced. The chemical activity may be divided into the following four types; (1) Production of substances which help in some way the life of the cell. These substances maj' be secreted and retained within the cell, or liberated from it; e. g., ferments or enzymes; true toxins (?). (2) Production of substances liberated by the bacteria as waste products. (.3) Production of substances by the breaking down of the food media; e. g., putrefactive products, due fifl PRINCIPLES OF MICROBIOLOGY lars>fly to enzyme actioii. (4) The ijnxluction of siihstaiiees whieli lielp form the protoplasm of the bacterial cell itself. Fermentation. — Fermentation may be defined broadly as a chemical decomposition of an organic compound, induced by the life processes of livino- organisms (organized ferments), or by chemical substances thrown off from the organisms (unorganized or chemical ferments or enzymes). It has been shown by Buchner anfl others that, in those cases of fermentation in which formerly it was believed the organized cell itself was necessarily concerned, ferments causing the same changes as the organized cells may be separated by various methods, such as crushing, filtering and so on. This brings fermentation by unorganized and organized ferments very closely together, the one being a substance thrown off from the cell, the other a substance ordinarily retained within the cell. The elaboration of both ceases with the death of the bacteria producing them. Fermentation, therefore, requires the living agent or its enzyme. It furthermore demands the proper nutriment, tempera- ture, moisture, and the absence of deleterious substances. Fermentation yields products that are poisonous to the ferment; hence fermentation ceases when the nutriment is exhausted or the fermentation is in excess. That the process will often begin again after diluting the fermented medium, shows that the concentration of the harmful products plays an important part in the inhibitory action. Characteristics of Ferments or Enzymes. — Ferments are non-dialyz- able. They withstand moderate dry heat, but are usually destroyed in watery solutions on exposure of ten to thirty minutes to a tempera- ture of ()0° to 70° C. They are injured by acids, but are resistant to all alkalies. They, even when present in the most minute quantities, are able partly to split up or decompose complex organic compounds and thus to render the foodstuff suitable for bacterial growth. A simple example of bacterial fermentation of carbohydrates produced by an enzyme is that of grape-sugar. Or, Or, Far less common is oxidizing fermentation, such as occurs, for example, in the production of acetic acid from alcohol. Here the energy is acquired not from the decomposition, but by the oxidation of "the alcohol. The Proteolytic Ferments. —The proteolytic ferments which are somewhat analogous to trypsin— being capable of changing albumin- ous bodies into soluble and diffusible substances — are very widely distributed. The liquefaction of gelatin, which is chemically allied CeHi.Oc = 2C2H6O Grape-sugar 2 Alcohol. + 2CO2 2 Carbon dioxide. CcHijOs Grape-sugar. = 2C3H„03 2 Lactic acid. CcH,.,Oe Grape-sugar. = 3 Acetic acid. PRODUCTS OF MICROBAL GROWTH Gl to albumin, is due to the presence of a proteolytic ferment or trypsin. The production of proteolytic ferments by ditt'erent cultures of the same variety of bacteria \-aries considerably — far more than is generally supposed. Bitter-tasting- products of decomposition ma\' be formed by cer- tain liquefying bacteria in media containing proteid, as, for example, in milk. Diastatic FeDiwut-s. — Diastatic ferments con\-ert starch into sugar. This action is demonstrated by mixing starch paste with suitable cultures to the resulting mixture of which thymol has been added, and keeping the digestion for six to eight hours in the incubating oven ; then, on the addition of Fehling's solution and heating, the reaction for sugar appears — the reddish-yellow precipitate due to the reduction of the copper. Inverting Feniient.-i. — Inverting ferments (that is, those which convert polysaccharides into monosaccharides) are of very frequent occurrence. Bacterial invertin withstands a temperature of 100° C. for more than an hour, and is produced in culture media free from proteid. Renn in-like Ferments. — Rennin-like ferments (substances having the power of coagulating milk with neutral reaction, independent of acids) are found not infreciuently among bacteria. Alkaline Products and the Fermentation of Urea. — Aerobic bacteria always produce alkaline products from albimiinous substances. Many species also produce acids from sugars, which explain the fact that neutral or slightly alkaline broth often becomes acid at first from the fermentation of the sugar contained in the meat used for making the media. When the sugar is used up the reaction often becomes alkaline, as the production of alkalies continues. The substances producing the alkalinity in cultures are chiefly ammonia, amine, and the ammonium bases. The con\'ersion of urea into carbonate of ammonia by Micrococcus iirece affords an example of the production of alkaline substances by bacteria : C0(NH2), + 2H2O = C03(NH4)2 Urea. 2 Water. Ammonium carbonate. The poisonous products of microorganisms are considered in Chap- ter VII, pages 145-150. Pigment Production. — Pigments ha^'e no known importance in con- nection with disease, but are of interest and ha\'e value in identifying bacteria. Their chemical composition is not generally known. Red and Yellow Pigments. — Of the twenty-seven red and yellow chromogenic bacteria studied by Schneider, almost all produce pig- ments soluble in alcohol and insoluble in water. The large majority of these pigments possess in common the property of being colored blue green by sulphuric acid and red or orange by a solution of potash. Though varying considerably in their chemical composition and in their spectra, they may be classified, for the most part, among that large (32 PRINCIPLES OF MICROBIOLOGY group of pigments common to both the animal and vegetable kingdoms known as lipochromes, and to which belong the pigments of fat, yolk of eggs, the carotin of carrots, turnips, etc. Violet Pigments. — Certain bacteria produce violet pigments, also insoluble in water and soluble in alcohol, but insoluble in ether, benzol, and chloroform. These are colored yellow when treated in a dry state with sulphuric acid, and emerald green with potash solution. Blue Pigments. — Blue pigments, such as the blue pyocyanin produced by B. pyocyaneus; and the fluorescent pigment common to many so-called fluorescent bacteria (bacteriofluorescence) are examples. In cultures the pigment is at first blue; later, as the cultures become alkaline, it is green. Numerous investigations have been made to determine the cause of the variation in the chromogenic function of bacteria. All condi- tions which are unfavorable to the growth of the bacteria decrease the production of pigment, as cultivation in unsuitable media or at too low or too high a temperature, etc. The B. prodigiosus seldom makes pigment at 37° C, and when transplanted at this temperature, even into favorable media, the power of pigment production is gradually lost. B. pyocyaneus does not produce pigment under anaerobic conditions. Occasionally colored and uncolored colonies of the same species of bac- teria may be seen to occur side by side in one plate culture, as, for example, in the case of Staphylococcus pyogenes. Reduction Processes. — The following processes depend wholly or in part upon the reducing action of nascent hydrogen: 1. Sulphuretted Hydrogen (H2S). All bacteria, according to Petri and Maassen, possess the power of forming sulphuretted hydrogen, particularly in liquid culture media containing much peptone (5 to 10 per cent.); only a few bacteria form H2S in bouillon in the absence of peptone, while about 50 per cent, in media containing 1 per cent, peptone possess the property of converting sulphur into sulphuretted hydrogen, for which purpose is required the presence of nascent hydrogen. (For demonstration see page 120.) 2. The reduction of blue litmus pigments, methylene blue, and indigo to colorless substances. The superficial layer of cultures in contact with the air shows often no reduction, only the deeper layers being afl'ected. By agitation with access of air the colors may be again restored, but at the same time, if acid has been formed, the litmus pigment is turned red. 3. The reduction of nitrates to nitrites, ammonia, and free nitrogen. The first of these properties seems to pertain to a great many bacteria. Aromatic Products of Decomposition. — Many microbes produce- aromatic substances as the result of their growth. The best known of these are indol, skatol, phenol and tyrosin. Systematic investigations have only been made with regard to the occurrence of indol and phenol. Decomposition of Fats.—Pure melted butter is not a suitable culture medium for microl)es. The rancidity of butter is brought aljout (1) PRODUCTS OF MICROBAL GROWTH 03 as the result of a purely chemical decomposition of the butter hy the oxygeu of the air under the influence of sunlight, and (2) through tlie formation of lactic acid from the milk-sugar left in the butter. Fats are, however, attacked by bacteria with the consequent production of acid when mixed with gelatin and used as culture media. Putrefaction. — By putrefaction is understood in common jjarlancc every kind of decomposition due to microbes which results in the pro- duction of malodorous substances. Scientifically considered, putre- faction depends upon the decomposition of albuminous substances, which are frequently first peptonized and then further decomposed. Typical putrefaction occurs only when oxygen is absent or scanty; the free passage of air through a culture of putrefactive bacteria — an event which does not take place in natural putrefaction — very much modifies the process: first, biologically, as the anaerobic bacteria are inhibited, ancl then by the action of the oxygen on the products or by-products of the aerobic and facultative anaerobic bacteria. As putrefactive products we have peptone, ammonia, and amines, leucin, tyrosin, and other amino substances; oxyfatty acids, indol, skatol, phenol, ptomains, toxins, and, finally, sulphuretted hydrogen, mercaptans, carbonic acid, hydrogen, and possibly marsh-gas. Nitrifying Bacteria. — According to recent observations, nitrification is produced by a special group of bacteria, cultivated in the laboratory with difficulty, w^hich do not grow on our usual culture media. From the investigations of Winogradsky it would appear that there are two common microorganisms present in the soil, one of which con- verts ammonia into nitrites and the other converts nitrites into nitrates. Conversion of Nitrous and Nitric Acids into Free Nitrogen. — This process is performed by a number of bacteria. The practical importance of these organisms is that by their action large quantities of nitrates in the soil, and especially in manure, may become lost as plant food by being converted into nitrogen. By the aid of certain root bacteria, which gain entrance to the roots of legumes and there produce nodular formations, the leguminous plants are enabled to assimilate nitrogen from the atmosphere. It is not known exactly how this assimilation of nitrogen occurs, but it is assumed that the zooglea-like bacteria, called hacteroids, constantly observed in the nodules, either alone or in a special degree, possess the property of assimilating and combining nitrogen. It seems, moreover, to have been recently established that, independently of the assistance of the legumes, certain bacteria exist free in the soil, which accumulate nitrogen by absorbing it from the air. These various nitrifying, denitrifying, and nitrogen-fixing bacteria are described in detail in tlic special chapter upon bacteria in nature. Formation of Acids from Carbohydrates. — Free acids are formed by many microbes in culture media containing some form of sugar or other fermentable carbohydrates, such as the alcohol mannite; the production of acid in ordinary bouillon takes place on account of the 64 PRINCIPLES OF MICROBIOLOGY presence of sugar, which is usually found in small quantities in the meat. If after the sugar is consumed, not enough acid has been formed to kill the bacteria, the acid is neutralized gradually and in the end the reaction becomes less acid or even alkaline. Among the acids produced the most important is lactic acid; also traces of formic acid, acetic acid, propionic acid, and butyric acid, and not infrequently some ethyl alcohol and aldehyde or acetone are formed. Occasionally no lactic acid is present. Formation of Gas from Carbohydrates and Other Fermentable Substances of the Fatty Series. — The only gas produced in visible quantity in sugar-free culture media is nitrogen. If sugar is vigorously decomposed by bacteria, as long as pure lactic acid or acetic acid is produced there may be no development of gas, as, for instance, with the B. typhosus on grape-sugar, but frequently there is much gas devel- oped, especially in the absence of air. About one-third of the acid- producing species also develop gas abundantly, this consisting chiefly of CO2, which is alwaj's mixed with H. Marsh-gas is seldom formed by bacteria, with the exception of those decomposing cellulose. (For demonstration see page 119.) Formation of Acids from Alcohol and Other Organic Acids. — It has long been known that Bacillus aceti and allied bacteria convert dilute solutions of ethyl alcohol into acetic acid by oxidization: CHs + 0, = CH;, + H..0. I I CH.OH COOH. The higher alcohols — glycerin, dulcit, mannite, etc. — are also con- verted into acids. Finally, numerous results have been obtained from the conversion of the fatty acids and their salts into other fattv acids bv bacteria. References. Ambroz. Entwickclungszyklus des B. nilri n. sp., etc., Centralbl. f. Bakt., etc., I. .\bt., orig., 1909, 51, 193 (with bibliography on structure and development of bacteria). Buchner. Berichto d. Deutsch. chem. Gesellsch., xxx, 117-124 and 1110-1113. Buschke. Die Sprosspilze in Kolle und Wassermann's Die Mikroorganismen Jena 2d edition, 1913. Calkins. The Protozoa, 1st edition, New York, 1901. Also article entitled. The Protozoa, in Osier's Modern Medicine, Philadelphia, 1907, vol. i- also Protozo61o"V Now York and Philadelphia, 1909. ' " ' Doflein. Lehrbuch der Protozoenkunde, 2d edition, Jena, 1909; Handbuch dcr pathogeuen Mikroorganismen, Kolle und Wassermann, 2d edition, Jena, 1913, .feiinings. Behavior in Lower Organisms, New York. Macmillan & Co., 1906. Lniifi. Protozoa in Vergleichende Anatomie der wierbellosen Thiere, new'edition 1909 Laid-eslvr. Treatise on Zoology, 1st edition, London, Part I, first and second fascicles, Mc.i/rr. Flora, 190S, p, 9.5. Miuula. System dcr Baktcrien, Jena, 1S97, Moore. The Pathology of Infectious Diseases of Animals, 1st edition, Ithaca, 1902. PRODUCTS OF MICROBAL GROWTH 65 Pelruschky. Die pathogenen Mychomyccton, in Kollc und Wasscrniann's Die Milcro- organismen, Jena, 2d edition, 1913. Plant. Die Hyphenpilze in KoUe und Wasscrmann's Die Mikro-organismen, Jena, 2d edition, 1913. Ruzicka. Cytologie der sporenbildcnden Balvterion, etc., Centralbl. f. Balct., 1909, II, Abt., vol. xxvii. Schaudinn. Beitrage zur Kenntnis der Balcterien, etc.. Arch. f. Protistenk, 1902, i, 300, and 1903, ii, 416. Zettnow. Romanowski's Farliung bei Bakterieii. Zeitschr. f. Hyg., etc., 1899, xxx, 1; and Centralbl. f. Bakt., 1900, Abt. I, xxvii, 803. CHAPTER III. THE MICROSCOPE AND THE MICROSCOPIC EXAMINATION OF INIICROORGANISMS. THE MICROSCOPE. If lenses were capable of refracting all light equally, and bringing to a focus in one plane all rays proceeding from one plane in the object the microscope would be a comparatively simple instrument. • But simple lenses have several serious optical defects. 1 . Spherical Aberration. — Points in the plane of the object are imaged on the curved surface of the spherical lens. This defect may be some- what diminished by combining conve.x and concave lenses, and by restricting the size of the field. Objectives corrected in this way are called aplanatic. 2. Chromatic Aberration. — This defect is due t,o the fact that the rays of light vary in their refraction according to their w^ave lengths (colors) , e. g., the red rays have the longest focus and the violet the shortest. This is considerably corrected by combining planoconcave lenses of flint glass with bi-convex lenses of crown glass — achromatic objectives. Still more of a correction is made by combining several different kinds of lenses with a lens of fluorite — apochromatic objectives. ^lonochromatic light may be employed and thus chromatic aberra- tion may be entirely avoided. 3. Diffraction. — Less luminous secondary images about the primary image, due to scratches or foreign particles or other defects may occa- sionally occur in the very best lenses. In order to understand fully the principle of the microscope, works on optics should be consulted. Different Parts of the Microscope (Figs. 7 and S). — A complete instrument usually has four oculars, or eye-pieces, .1, which are num- bered from 1 to 4, according to the amount of magnification which they yield. Nos. 2 and 4 are most useful for bacteriologic work. The objective — the lens, B, at the distal end of the barrel — serves to give the main magnification, of the object. For stained bacteria, the ^2 achromatic oil-immersion lens is regularly employed; for photographic purposes the apochromatic lenses are needed, although even here they are not indispensable. A y\; lens may at tiiues be useful, but hardly necessar.y; a .\o. 4 ocular and a rV h'ns give a magnification of about lOOO diameters (Fig. !)). For unstained bacteria we employ either the yV immersion or j dry lens, according to the purpose for which we study the bacteria; for the examination of colonies where, as a rule THE MICROSCOPE 07 we do not wish to see iii(li\-i(hial bacteria, but only the general appear- ance of whole groups, we use lenses of much lower magnification (Fig. 10). The stage C— the platform upon which the object rests— should be large enough to support the Petri plates if culture work is to be done. The distance between the oj^tical axis of the instrument and the pillar must be great enough to permit one to examine rather more than half the surface of the Petri dish without re\oh-ing it. The iris diaphragm Fiii. 7 > of the Ocular Draw Tube Diapliragra with Society Screw 1 ^Y _L >f Sodctj --ISIount Je^ ^ Back-Ieiis J.Ikldle-leus CoverAlas^ 'Cj^ Working Distance of the Oijjoctive Mir njscope. Internal structure of the microscope. D opens and closes, and, as the iris of the eye controls, the amount of light. Its opening is diminished or increased by moving a small arm underneath the stage. The reflector or mirror E placed beneath the stage serves to direct the light to the object to be examined. It has two surfaces — one concave and one plane. The concave surface must not be employed when the substage condenser is being used, otherwise the ravs of light reaching the stage from the condenser will not be G8 PRINCIPLES OF MICROBIOLOGY correctly focused. The concave surface may be used when unstained objects, such as colonies, or hanging drops are examined. At the same time the Abbe condenser should be lowered and the iris blender regulated. The coarse adjustment F is the rack-and-pinion arrangement by which the barrel of the microscope can be quickly raised or lowered. It is used to bring the bacteria roughly into focus. If the bearings become loose tighten the little screws at the back of the pinion box. _ Keep the teeth clean. If the bearings need oiling use an acid-free lubricant, such as paraffin oil. The fine adjustment G serves to raise and lower the barrel very slowly and evenly, and is used for the exact study of the bacteria when high-power lenses are used. It is necessarily of limited range and delicate in its mechanism. If, when looking into the eye-piece, no change of focus is noticed by turning the micrometer head, or if the micrometer head ceases to turn, the adjustment has reached its limit. Raise the barrel of the microscope by means of the coarse adjustment, Fig. 9 Fig. 10 Anthrax bacilli and blood cells X 1000 diameters. Colonies of diphtheria bacilli. X 200 diameters. then turn the micrometer back to bring the fine adjustment midway within its range. When the fine adjustment head stops do not force it. For the microscopic study of microbes it is essential that we magnify the organisms as much as possible and still have their definition clear and sharp. For this purpose the microscope should be provided with an oil-immersion system and a substage condensing apparatus. In using the oil-immersion lens a drop of oil (oil of cedar) of the same index of refraction as the glass is placed upon the face of the lens, so as to connect it with the cover-glass when the bacteria are in focus. There is thus no loss of light through deflection, as is the case in the dry system. If the lenses become dirty they should be wiped gently with Japanese lens paper or a clean, soft, old-linen handkerchief. If necessary lireathe on the lens before wiping, and if this does not succeed use a little xylol or chloroform. These substances are not to be used unless necessary. An immersion objective should always be cleaned imme- THE MICROSCOPE 69 diately after using. Tlie objective should always lie ke])t co\'ere(l so as to prevent dust dropping in. Light. — The best light is obtained IVdni white clouds or a blue sky with a northern exposure. Avoid direct sunlight. If necessary use white shades to modify the sunlight. iVrtificial light has one advantage over daylight in that it is constant in quality and quantity. The Welsbacli burner and a whitened incandescent bulb give a good light. A blue glass between the artificial light and the lens is often of value. An eye-shade may be helpful. Substage condensing apparatus H is a, system of lenses situated beneath the central opening of the stage. It serves to condense the light passing through the reflector to the object in such a way that it is focused upon the object, thus furnishing the greatest amount of luminosity. Between the condenser and the reflector is placed the iris diaphragm. Focusing. — Focus the body tube down by means of the coarse adjust- ment until the objective approaches very near to the cover-glass, being careful not to touch it. Then with the eye at the eye-piece focus up carefully with the coarse adjustment until the specimen comes plainly into view. Be careful not to pass this focal point. It is easily unnoticed if the light be too intense and the specimen thin and transparent. If the sliding tube coarse adjustment is used, focus carefully by giving the tube a spiral movement. When the object is brought fairly well into focus by means of the coarse adjustment, use the fine adjustment to focus on the particular spot desired, for if this spot is in the centre of the field of the low power it should be somewhere in the field of the higher power. It is too much to ask of the maker that the lenses be made absolutely parfocal and centred. The delicacy of the centring can be appreciated when the magnification and the extremely small portion examined are considered. When the objectives are not thus fitted to the nose-piece, refocusing and again hunting up the object are necessary. In so doing we repeat the caution always to focus up before turning the nose-piece. When no revolving nose-piece is used the change of objectives means the unscrewing of one and the screwing of the other into its place, and refocusing. The beginner should ahvays use the low-power objectives and oculars first. The low-power objectives have longer working distances and are not so apt to be injured. They always show a larger portion of the specimen and thus give one a better idea of the general contour. After obtaining this general idea the higher powers can be used to bring out greater detail in any particular part. Generally speaking, it is best to use a high-power objective and low-power eye-piece in preference to a low-power objective and high-power eye-piece. In the latter case any imperfections in the objective are magnified unduly by the eye-piece, giving, as a rule, poor definition. Tube Length and Cover-glass. — All objectives are corrected to a certain tube length (160 mm. by most makers— Leitz, 170 mm.) and 70 PRINCIPLES OF MICROBIOLOOY all ol)jecti\-es in fixed mounts of over 0.7 X. A. are correeted to a definite thickness of cover-fi'lass as well (Zeiss, 0.15 mm., 0.2 mm.; Leitz, 0.17 mm.; Bauseh & Lomb and Spencer, O.IN mm.). These objecti\'es give their best results only when used with the co\'er-glass and tube length for which they are corrected. As indicated in Fig. 8 the tube length extends from the eye lens of the eye-piece to the end of the tube into which the objective or nose-piece is screwed. If a nose- piece is used the draw tube must be correspondingly shortened. If the cover-glass is thinner than that for which the objective is corrected, the tube must be lengthened to obtain best results; if thicker, shortened. The more expensive objectives are provided with adjustable mounts by which the distances between the lens systems may be changed to compensate for difference of thickness of cover. They are successively used only in the hands of an expert. One of them out of adjustment is worse than an ordinary objective. Fig. 11 Virulent diphtheria bacilli. Cultures two davs old. Unstained X 2400 (After Siebert.) Dark Ground Illumination and the Examination of Ultramicro- scopic Particles.— The apparatus constructed by Siedentopf and Zsigmondy makes visible, even in solutions otherwise apparently homogeneous, very minute particles, which heretofore could not be seen even with the highest magnifications. Particles l/z^ (a milli- micron = one-millionth of a millimeter) are thus rendered visible. This increased power in microscopic analysis is made possible by intense (electric arc lamp) focal lateral illumination of the objects examined, and by shutting off the rays passing in the usual way through MICROSCOPIC METHODS 71 the tube of the microscope. The greater the rliiference lictween the refractive index of the objects colloi(lail\' iHssoInciI or otiierwisc held in suspension and the tiuid which surrounds tlieni, tiic Ijriglitcr will he the aijpearancc of the objects, and therefore the more readily visible. The microscopic field, as will be seen by the photogram herewith (Fig. 11), is dark; the objects which refract the light show as l)rightly illuminated, sharply defined pictures, in which the black margin cor- responds to the contour of the object. The illuminated portion is sur- rounded by a fine dark zone, this in turn by alternate bright and dark zones, in which the illumination rapidly decreases. Reichert, of Vienna, has recently simplified this apparatus by devising a new condenser. The light which illuminates the object has a greater refraction than the cone of light entering the objecti\'e which produces the image. Its advantages over the first method are: (1) it utilizes the source of light better; (2) any dry objective can be used without alterations; (3) small particles are seen without the disturbing refraction rings. With this apparatus such living organisms as the Spirocheia paUida, and the flagella on certain bacteria, which can scarcely be seen by ordinary microscopes on account of their low refractive indices, may be demonstrated with great clearness. The use of microphotography with ultraviolet light (according to A. Kohler) makes visible particles that cannot be seen by ordinary hght, because of the inabihty of the violet rays to pass through certain substances, e. g., chromatin. This method is said to increase 40,000 times our present limit of vision. The few discoveries claimed by these means for diseases of unknown origin have so far lacked sufficient corroboration to constitute them proved. Double Microscopes. — These have been devised by Metz and others for the purpose of making comparative studies of two objects. MICROSCOPIC METHODS. The direct microscopic examination of suspected substances for microorganisms can be made either with or without staining. Unstained, the microbes are examined living in a hanging drop or on transparent solid media, under daylight, or better, artificial light, to note their number, their motility, their size, form, and spore formation, their general arrangement, their reactions to specific serums and to vital stains, etc. But for more exact study of their structure they must be stained in a dried film preparation on a glass slide or a cover-glass or when in tissues, in sections. Elimination of Foreign Organisms from Preparations. — Since germs are present in the air, in dust, in tap-water, on our bodies, clothes, and on all surrounding objects, it follows that when we begin to examine substances for microbes the first requisite is, that the materials we use, such as staining fluids, cover-glasses, etc., should be practically free from organisms, both living and dead, otherwise we may not he able to 72 PRINCIPLES OF MICROBIOLOGY tell whether those we detect belong originally in the substances examined or only in the materials we have used in the in\-estigation. Therefore all solutions are filtered and all apparatus tiioroughly cleaned and when necessary sterilized. Examination of Bacteria in the Hanging Drop. — For this examination special slides and methods are desirable. The slide used is one in which there is ground out on one surface a hollow having a diameter of about half an inch (Fig. 12). According to the purpose for which the hanging drop is to be studied, sterilization of the slide and cover-glass may or may not be necessary. Fig. 12 pg|il|iililii[lll|iiillillllll|i|lllllH||i|>ii;; Hollow slide mth cover-glass. The technique of preparing and studjdng the hanging drop is as follows: The surface of the glass around the hollow in the slide is smeared mth a little vaselin or other inert oil. This has for its purpose both the sticking of the cover-glass to the slide and the prevention of evaporation in the drop placed in the httle chamber, which is to be formed between the cover-glass when placed over the hollow, and the slide. If the bacteria to be studied are in a fluid we place a large platinum loopful upon the centre of the cover-glass and, to avoid drying, immediately invert it by means of a slender pair of forceps over the hollow in the slide, being verj' careful to have the drop over the centre of the cover- glass. The cover-glass is then pressed on the slide so as to spread the vaselin and make a perfect seal. If the bacteria are growing on solid media, or are obtained from thick pus or tissues from organs, they are mixed with a suitable amount of bouillon or sterile physiologic salt solution' either before or after being placed upon the cover-glass. In studying living bacteria to determine only their grouping and motion we may use less magnification than for studying other charac- teristics. In studying unstained bacteria and tissues we shut off as large a portion of the light with our diaphragm as is compatible with distinct vision, and thus favor contrasts wdiich appear as lights and shadows, due to the differences in light transmission of the different materials under examination. It is necessary to remember that they are seen with difficulty, and that we are very apt, unless extremely careful in focusing, to allow the lens to go too far, and so come upon the cover-glass, break it, destroy our preparation, and, if examining patho- genic bacteria, infect the lens. This may be avoided by first finding the hanging drop with a low-power lens and then centring it. The edge of the drop is focused more easily than the centre. The lens of higher magnification is now very gradually lowered, while at the same time gently moving the slide back and forth to the slightest extent possible with the left hand. If any resistance is felt the lens should be raised, for it has gone beyond the point of focus and is touching the cover-glass. I Physiologic salt solution is usually 0.8 per cent. NaCl in distilled water. MICROSCOPIC METHODS 73 Hanging Mass or Hanging Block Cultures.— In order to study the morphology ami manner of multiplication of individual microorganisms to better advantage than in the hanging drop, we ha^■e used hanging masses of agar, made by phicing a. large ]ihi.tinum loop full of melted agar on a sterile co\er-glass and allowing it to harden, protected from tlust. The organisms are placed on the free surface of this mass which is then inverted over a hollow slide and studied as in a hanging drop. Hill devised the following procedure: Melted nutrient agar is poured into a Petri dish to a depth of about one-eighth to one-quarter of an inch. When cool a block is cut out about one-quarter of an inch square. The block is placed, under surface do-\\ai, on a slide and protected from dust. A very dilute sus- pension of the growth to be examined is then made in sterile bouillon and spread over the upper surface of the block. The slide and block are then put in the incubator for ten mmutes to drj^ slightly. A clean cover-slip is now placed on the agar block in such a way as to avoid large air-bubbles. The slide is then removed. "With the aid of a platinum loop a chop or two of melted agar is rmi along each side of the block to fill any angles between it and the cover-glass. After dryhig in the incubator for five minutes it is placed over a hollow slide and sealed with paraffin. We consider the hanging-mass method better than that of the hanging block in many mstances, because in the former method no pressure is exerted on the bacteria, and more oxj-gen is allowed them. Film Preparation (spread, smear). — Film preparation is made as follows : A very small amount of the blood, pus, discharges from mucous membranes, cultures from fluid media, or other material to be examined is removed, usually by means of a sterile swab or platinum loop, and smeared undiluted in an even, thin film over a perfectly clean, ^ thin cover-glass or slide. From cultures on solid media, however, on account of the abundance of organisms in the material, a little of the growth is diluted by adding it to a small loopful of filtered or distilled water, which has been previously placed on the glass slide. It is best to add to the drop just enough of the culture to make a perceptible cloudiness. Blood films may be made either by the cover-glass or the slide method. To make a cover-glass preparation, two square, very thin (hence flexilDle) cover-glasses are cleaned. Holding one with thumb and index fingers by oppo- 1 To render new cover-slipa clean and free from grease, the method recommended by Gage is useful: Place in following solution over night. Bichromate of potash (K2Cr207) 200 grams Water, tap or distilled 800 o.c. Sulphuric acid 1200 c.c. The bichromate is dissolved in the water by heating in an agate kettle; the sulphurous acid is added very slowly and carefully on account of great heat developed. After cool- ing, it is kept in a glass vessel. It may be used more than once. Glasses are removed the next morning and cleansed in running tap-water until the yellow color disappears. They are then placed in ammonia alcohol until used. When used wipe with soft, clean hnen or cotton cloth. If old cover-shps are used, boil first in 5 per cent, sodium carbonate solution. Another procedure is, after washing with soap and water and rinsing in water, to soak the cover-glasses in alcohol, then wipe with soft linen, then place in a Petri dish, and heat in the dry sterilizer for one hour at 200° C. to burn oS fatty substances. The heating may be done by holding the cover-glass in the flame sufficiently to heat thoroughly with- out softening. A cover-glass is not clean when a drop of water spread over it does not remain evenly distributed, but gathers in droplets. 74 PRINCIPLES OF MICROBIOLOGY •site corners, the tip of a drop of blood obtained by needle i)uiicture of finger or lobe of ear is made to touclr the centre of the cover-glass, and the second clean co^'er-glass held similarly is allowed to fall upon the first one in such a manner that the corners do not coincide. The blood droplet spreads by capil- larity into a thin film, which is a sign to pull the two covers apart in the plane in which they lie; good results depend upon cleanliness, rapidity, and success in sliding the two covers apart. To make a slide film the tip of the exuded blood drop is made to touch one slide near one end and the eclge of the second slide, held at an acute angle to the first one, is made to bisect the drop, which will spread at the point of con- tact by capillarity across the slide. Upon pulling the second or spreading slide over the first slide, never changing the angle and applying gentle pressure, a thin laj^er of blood suitable for examination will be formed. A slide made in this manner should be dried immediatelj' by agitation in the air. It may then be fixed and stained in various waj^s. Milk films, after fixation, are cleared of fat by mean.s of ether or alkaline solutions.^ The film either is dried thoroughly in the air and then fixed with heat or any chemical fixative, or it may be placed in any of the fixatives while still moist. The usual fixatives are methyl alcohol, absolute ethyl alcohol, Zenker's solution, etc. (see p. 84). When fixed with heat the glass is held b\' any one of the several kinds of forceps commonly used, and is passed three times by a rather slow movement through the Bunsen or alcohol flame. The film thus prepared is usually stained either by the simple addi- tion of a solution of an aniline dye, for from a few seconds to five minutes, or by one of the more complicated special stains described later. When the stain is to be hastened or made more intense the dye is used warm. The cover-glass or slide, with the charged side uppermost, may either rest on the table or be held by some modification of Cornet's forceps. When the solution is to he warmed the cover-glass may be floated, smeared side down, upon the fluid contained in a porcelain dish resting on a wire mat, supported on a stand ; or the solution may be poured on the glass which may then be held over the flame in the Cornet forceps. If a slide is used it is simply inserted in the fluid or covered by it. The fluid both in the dish and on the slide should be carefully warmed so as to steam without actually boiling. The slide should be kept completely covered with fluid. After staining the film, the cover-glass or sHde is grasped in the forceps and thoroughly but gently washed in clean water and then dried, first between layers of filter paper and then in the air or high over a flame. If a cover-glass has been used a drop of balsam or water is placed on a glass slide and the cover-glass put upon it with the film side down. Films made on slides are usually unmounted. Cedar oil is added at the time of examination with the oil-immersion lens and washed oft' with xylol immediately after. Bvrrifi India-ink tnethod of demonstrating bacteria. In 1907, 1908, and 1909 Burri recommended the following method for isolating and studying single bacterial cells. A solution of India ink (fliissige Perl- ' One-half to 1 per cent, sorlium hydrate. MICROSCOPIC METHODS 75 tusche) in water 1 to 10 [better 1 to 4] is sterilized in test-tubes in the auto- clave for fifteen minutes. A small drop of this ink is mixed carefully with a drop of the Huid to be examined. If cultures from isolated cells are desired the fluid should first be diluted so that a drop contains presumably a single organism; then tlrops of the mixture are placed in rows upon nutrient agar plates. If the bacteria are to be examined immediately a drop of the mixture (ink plus undiluted bacterial fluid) is allowed to dry upon a glass slide and then examined under an oil- immersion lens. The bacteria appear a brilliant white upon a dark field, particles of the ink surrounding the organisms like a capsule. This method is especially applicable for the demonstration of such organisms as the Tr. pallidum which have poor staining qualities and a low index of refraction. Stains and Staining Methods for Microorganisms. — Protozoa stain in general as do animal cells. The protoplasm of mature bacteria reacts to stains much as does the nuclear chromatin of animal cells, though the intensity of the staining varies somewhat with the condition of growth, such as the age, the species, the media, and so on. The best bacterial stains are the basic aniline dyes, which are com- pounds derived from the coal-tar product aniline (CeHsNHo).^ Aniline Dyes. — The aniline dyes which are employed for staining pur- poses are divided into two groups. In one the basic part of the molecule acts and the stains are spoken of as nuclear stains, since they color the nuclear chromatin of both cells and bacteria. In the other the staining act depends upon the acid part of the molecule, and the bacteria and cytoplasm of the higher cells are stained faintly. The stains in the latter group are used chiefly for contrast coloring. The basic dyes are usually employed as salts of hydrochloric acid, while the acid dyes occur as sodium or potassium salts. The following are the most commonly used basic aniline stains: Blue stains — methylene blue, thionin blue \ (give the best differentiation ; Red stains — basic fuchsin, safranin / difficult to overstain) . Brown stain — Bismarck brown (weak, may be used as counter-stain). Green stain — methyl green. Pink stain — eosin (weak, may be used as counter-stain). Violet stains — ^methyl violet, gentian violet, crystal violet (most intense stains; may overstain) . These dyes are all more or less crystalline powders, and while some arc definite chemical compounds, others are mixtures. For this reason various brands are met vnth on the market and the exact duplication of stains is not always possible. Dyes should be obtained from reliable houses only. It is advisable to keep on hand stock saturated alcoholic solutions from which the staining solutions are made. These stock solutions are made by pouring into a bottle enough of the dye in substance to fill it to about one-quarter of its capacity. The bottle should then be filled -with 95 per cent, ethyl alcohol, tightly corked, well shaken, and allowed to stand twenty-four hours. If at the end of this time all the staining material has been dissolved, more should be added, the bottle being again shaken and allowed to stand for another twenty- 1 For a good description of the composition and action of the various stains, see A. R. Lee's Microtomist's Vade-Mecum, 7th edition, 1913. 76 PRINCIPLES OF MICROBIOLOGY four hours. This must be rei^eated until a permanent sediment of undissolved coloring matter is seen upon the Ijottom of the bottle. Tliis bottle will then be labelled "saturaterl alcoholic solution," of whatever dye has been employed. Tlie dilution for use in staining is made by filling a small bottle three-fourths with distilled water, and then adding the concentrated alcoholic solution of the dye, little by little, until one can just see through the solution. It is some- times desirable to use a more concentrated solution wth dyes such as methylene blue. Care must be taken tliat the color does not become too dense; usually about one part to ten is sufficient. General Observations on the Principles of Staining Bacteria. — The staining of bacteria is not to be considered simply as a mechanical saturation of the cell body with the dye, in which the latter is dissolved in the plasma. It is rather a chemical combination between the dye substance and the plasma. The dependence of the staining process upon the solvent condition of the dye is shown in the following observations : 1. Entirely water-free, pure alcoholic dye solutions do not stain well. 2. Absolute alcohol does not decolorize bacteria, while diluted alcohol is an active decolorizing agent. The compound of dye substance and plasma is therefore insoluble in pure alcohol. .3. The more completely a d3re is dissolved the weaker is its staining power. For this reason pure alcoholic solutions are inactive; and the so-called weak dye solutions to which strong dye solvents have been added are limited in their action on certain bacteria in which the dye substance is closely united. This is the principle of Neisser's stain for diphtheria bacilli, viz., acetic- acid- methylene-blue solution. On the other hand, the addition of alkalies to the dye mixture renders the solvent action less complete and the staining power more intense. According to Michaels, however, in Loffler's methylene-lslue solution the role of the alkali is purely of a chemical nature, by which it converts the methylene blue into methylene azure (azure II). The dependence of the staining process upon the nature of the microbe is exhibited in the following facts : Certain microbes stain easily, others with difficulty. To the latter belong, for example, the tubercle bacillus and lepra bacillus. Spores and flageUa also stain with difficulty. The easily stained objects require but a minimum of time to be immersed in a watery solution, while the others must be stained by special dyes with or without the aid of outside influences (heat, mordants, etc.). The difficultly stained objects areat the same time not easily decolorized. The explanation of the resistance which these bacteria show to staining as well as to decolorizing agents is to be sought in two ways: either on the assump- tion that they possess a difficultly permeable or a resisting envelope, or that they have a special chemical constitution. The latter hypothesis holds good only, if at all, in regard to flageUa and spores; while the assumption of the resisting envelope has reference more particularly to the tubercle bacillus, and is probably correct. The presence of fatty and waxy bodies in the envelope of these microorganisms is capable of demonstration. Moreover, after extrac- tion of these bodies by ether the tubercle bacillus loses its power of resisting acids, which peculiar resistance can also be artificially produced in other bacteria having normally no such resisting power. In many instances, doubtless, both of these causes, viz., resistant envelope and chemically different constitution work together to produce the above-mentioned results. ' Elective staining properties, whereby certain species of organisms are exclu- sively or rapidly and intensely stained by certain dyes, have repeatedly been observed. Of the greatest practical importance in this respect is the Gram stain (see p. 78, and Chapter VI), used for the differential diagnosis of many species MICROSCOPIC METHODS 77 of bacteria; although a distinct classification of bacteria into those which arc stained and those which are not stained by Gram's solution has licen shown to be impracticable. There are some bacteria, however, which act uniformly toward Gram under most conditions; as, for example, the anthrax bacillus and the pyogenic cocci are always positive, the cholera and plague bacilli and gonococci are always negative to Gram. Other species again are at one time stained and at another decolorized by Gram; thus pyocyaneus is stained only in young indi^aduals. Previous heating or extraction with ether does not prevent the action of Gram's stain, but treatment with acids or alkalies renders it impossible. Bacteria so treated, however, after one hour's immersion in LofHer's mordant regain their property of staining with Gram. As to the nature of Gram's staining solution, it may be mentioned that only the pararosanilines (gentian violet, methyl violet, and Victoria blue) are suit- able for the purpose, whereas the rosanilines (fuchsin and methylene blue) give negative results. The reason for this is that the iodine compounds with the pararosanilines are fast colors, while those with the rosanilines are unstable. These latter compounds when treated with alcohol break up into their con- stituents, the iodine is washed out, and the dye substance remaining in the tissues stain them uniformly; that is, mthout differentiation. But iodine- pararosaniline compounds are not thus broken up and consequently stain those portions of the tissue more or less, according to the aiEiiitj^ which they have for the dye substance. The parts stained by Gram are thus distinguished from those stained violet, not only quantitatively, but qualitatively; it is not a gentian ^^olet, but an iodine-pararosaniline staining which occurs. Mordants and Decolorizing Agents. — We have already noted that the protoplasm of unrelated microbes may respond differently to the several dyes. There is, however, seldom any difficulty in selecting a dye which will stain sufficiently to make microbal cells in pure cultures distinctly visible. When the microbes are imbedded in tissue or mixed in a film with blood or pus, it is frequently difficult to prevent the stain from so acting on the tissue or pus elements as to obscure the organisms. Various methods are then employed to stain the germs more intensely than the tissues or to decolorize the tissue more than the organisms. Heating, the addition of alkali to the staining fluid and prolonging the action of the dyes increase the staining properties. We regulate these so as to give the best results. We also use mordants; that is, substances which fix the dye to the bacterial cell, such as aniline oil or solutions of carbolic acid on metallic salts. The decolorizing agents used chiefly are mineral acids, vegetable acids, diluted alcohols, various oils, and hot w^ater. Formulae of the More Generally Used Staining Mixtures. — Loffler's Methylene-blue Solution. — Concentrated alcoholic solution of methylene blue, 30 c.c; caustic potash in a 0.01 per cent, solution, 100 c.c. (The alkali not only makes the cell more permeable, but also increases the staining power by liberating the free base from the dj^e.) Films stained two to five mirmtes, licated if more intense stain is desired. Sections stained one-quarter to several hours and decolorized mitil faint blue; contrast stain eosin; washed, dehydrated, cleared, and mounted. Cakbol-Fuchsin, or Ziehl-Neelsen Solution. — Distilled water, 100 c.c; carbolic acid (crystalline), !> gm.; alcohol, 10 c.c; fuchsin, 1 gni.; or it may be prepared by adding to a .5 per-cent. watery solution of carbolic acid the saturated alcoholic solution of fuchsin until a metallic lustre appears on the surface of the fluid. The carbolic acid, like the alkali, favors the penetration of the stain. 78 PRINCIPLES OF MICROBIOLOGY The last two methods, combined with heating, are used to stain spores and certain resistant bacteria as the tubercle bacilli and other "acid resisters," so that they retain their color when exposed to decolorizing agents (see below). CarhoWentian Violet. — One part of saturated alcoholic solution of gentian violet to 10 parts of a 5 per-cent. solution of carbolic acid. Carbol-methylene blue, first used by Kiihne, consists of 1.5 gm. of methylene blue, 10 gm. of absolute alcohol, and 100 c.c. of a 5 per-cent. solution of carbolic acid. Carbol-thionin consists of 10 parts of a saturated alcoholic solution of thionin and 100 parts of a 1 per-cent. solution of carbolic acid. Polychrome Methylene Blue (Goldhorn). — To prepare the stain dissolve 1 gm. lithium carbonate in 200 c.c. clean water and add 1 gm. methylene blue. Shake and dissolve. Pour into porcelain dish over water-bath, stirring fre- quently until blue color changes to a rich purple. Run through cotton in funnel; make up to 200 c.c. To 100 c.c. add 5 per cent, acetic acid until a faint pink is just visible on litmus paper above level of point discolored by the dye. Now add the remaining 100 c.c. of dye and allow to stand in open dish for fortj^-eight hours. Run once more through cotton into clean bottle. It is not necessary to use distilled water, and satisfactory results are ob- tained with all the different forms of methylene blue tried. B-X (Griiebler) is preferable. Fix the smear by immersion in commercial wood alcohol for fifteen to thirty seconds; wash well and stain for about ten to fifteen seconds in poly- chrome; wash and stain for from fifteen to sixty seconds in ^V P^r cent, aqueous eosin. Wash again in water and dry in air without heat. Body of parasites blue; chromatin is red to purple. Results may be varied by using polychrome or eosin for different lengths of time. Admirable preparations may be obtained, even when there is pre- cipitation, by just rinsing the smear a little in 50 per cent, ethyl alcohol. This will remove anjr precipitation. Koch-Ehrlich aniline-water solution op puchsin oe gentian violet is prepared as follows: To 98 c.c. of distilled water add 2 c.c. aniline oil, or, more roughly but wdth equally good results, pour a few cubic centimeters of saturated aniline oil into a test-tube, then add sufficient water nearly to fill it. In either case the mixtures are thoroughly shaken and then filtered into a beaker through moistened filter paper until the filtrate is perfectly clear. To 75 c.c. of the filtrate (aniline-oil water) add 25 c.c. of the concentrated alcoholic solu- tion of either fuchsin, methylene blue, or gentian violet, or add the alcoholic solution until the aniline water becomes opaque and a film begins to form on the surface. Gram's Stain. — Another differential method of staining which is employed is that known as Gram's method. In this method the objects to be stained are floated on or covered with the aniline or carbolic gentian-violet solution described above. After remaining in this for a few minutes they are rinsed in water and then immersed in an iodine solution (Lugol's), composed of iodine, 1 gm.; potassium iodide, 2 gm.; distilled water, 300 c.c. In this they remain for from one to three minutes and are again rinsed in water. They are then placed in strong alcohol until most of the dye has been washed out. If the cover-glass as a whole still shows a violet color, it is again treated mth the iodine solution, followed by alcohol, and this is continued until no trace of violet color is visible to the naked aye. It may then be washed in water and examined, or before examination it may be counter-stained for a few minutes by a weak solution of a contrasting dye, such as eosin, fuchsin, carmine, or Bismarck brown. This method is useful in demonstrating the capsule which is seen to suiTound some bacteria — particularly the pneumococcus — and also in differentiating between varieties of liacteria; for some do and others do not retain their stain when put in the iodine solution for a suitable time (see C'hapter VI, for further remarks upon Gram's stain; see also pp. 76-77). The MndificaUonfi of Gram's Slain are Many. — One only is given here. MICROSCOPIC METHODS 79 Nicolle's Modification. — Stain cold in carhol-geiitiiui violet one minute; wash in tap-water; stain cold in the iodine mixture 1 minute; wash in tap-water; decolorize ten seconds in acetone (1 part) and alcohol (3 parts); wash in tap- water; counter-stain ten seconds in dilute carbol-fuehsin (1 to 10). Staining of Capsules. — ^lany methods of demonstrating the cap- sule has been devised. Two only will be given here. Welch's glacial acetic acid method is as follows: (1) Cover the prepara- tion with glacial acetic acid for a few seconds; (2) drain off and replace with aniline gentian- violet solution; this is to be repeatedly added until all the acid is replaced; (3) wash in 1 to 2 per cent, solution of sodium chloride and mount in the same. Do not use water at any stage. The capsule stains a pale violet. (See Plate III, Fig. 15.) Fig. 13 Capsule stain by Hiss' method. Rhinosclcroma bacillus. X 1000. (Thro.) Hiss' Copper Sulphate Method (Fig. 12). — The organisms are grown, if possible, on ascitic fluid or serum media. If not, the organisms should be spread on the cover-glass mixed with a drop of serum, or better, with a drop of one of the diluted serum media. Dry in the air and fix by heat. The capsules are stained as follows: A 5 per cent, or 10 per cent, aqueous solution of gentian -snolet or fuchsin (5 c.c. saturated alcoholic solution gentian violet to 9.5 c.c. distilled water) is used. This is placed on the dried and fixed cover-glass preparation and gently heated for a few seconds until steam arises. The dye is washed off with a 20 per cent, solution of copper sulphate (crystals). The preparation is then placed between filter paper and thoroughly dried (Plate III, Figs. 14 and 16). Staining Spores and Acid-fast Bacteria. — We have already noted that during certain stages in the growth of a number of bacteria spores are formed which refuse to take up color when the bacteria are stained in the ordinary manner. Special methods have been devised for causing the color to penetrate through the resistant spore membrane. In the simplest method a cover-slip after having been prepared in the usual way is covered wnth Ziehl's carbol-fuehsin solution and held over the Bunsen flame until the fluid steams. This is continued for one or two minutes. It is then washed and dipped in a decolorizing acid solution, such as a 2 per cent, alcoholic solution of nitric acid, or a 1 per cent, solution of sulphuric acid in water, until all visible color has disappeared, then it is washed and dipped for 80 PRINCIPLES OF MICROBIOLOGY onc-lialf minute in a saturated waterj^ solution of methylene blue. The bodies of the bacilli are blue and the spores red. This same method is used for stain- ing acid-fast bacilli. Sometimes the spores refuse to take the stain in this manner. We then can adopt Moeller's method, which is designed still further to favor the penetration of the coloring matter through the spore membrane. The prepared cover-slip is held for two minutes in chloroform, then washed off in water, and placed from one-half to three minutes in a 5 per cent, solution of chromic acid, again washed off in water, and now stained by carbolic fuchsin, which is steamed for several minutes. The staining fluid is then washed off and the preparation decolorized in a 3 per cent, solution of hydrochloric acid or a 5 per cent, solution of sulphuric acid. The preparation is finally stained for a minute in methylene-blue solution. The spores wU be red and the body of the cells blue. The different spores vary greatly in the readiness with which they take up the dyes, and we have, therefore, to experiment with each variety as to the length of time it should be exposed to the maceration of the chromic acid. Even under the best conditions it is almost impossible to stain some spores. Plate III, Fig. 22, show stained spores. Staining Flagella. — For the demonstration of flagella, which are possessed by all motile bacteria, we are indebted first to Loffler. The staining of flagella satisfactorily is one of the most difficult of bacterio- logical procedures. In all methods young (twelve-to-eighteen-hour) cultures of agar should be chosen. Enough of the culture to produce slight cloudiness is placed in a few cubic centimeters of filtered tap- water in a test-tube. This may be used immediately, or allowed to stand in the thermostat at blood heat for from one to two hours to permit slight development. A tiny drop of this rather thin emulsion is allowed to spread with as little manipulation as possible over the cover-glass so that it may dry quickly. This latter point seems to be the important one since slow drying allows the bacteria to shed their flagella. Bunge's modification of Loffler^ s method is carried out as follows: Cover- glasses which have been most carefully cleaned are covered by a very thin smear. After drying in the air and passing three times through the flame the smear is treated mth a mordant solution, which is prepared as follows: To 3 parts of saturated watery solution of tannin add 1 part of a 25 per cent, solu- tion of ferric chloride. This mordant should be allowed to stand for several weeks before using. After preparing the cover-slip wdth all precautions neces- sary to cleanliness, the filtered mordant is allowed to act cold for five minutes, after which it is warmed and then in one minute washed off. After drying, the smear is stained with the carbol-fuchsin or carbol-gentian violet solution, and then washed, dried, and mounted. (Plate III, Figs. 18-2f .) Frecjuently the flagella appear well stained, but often the process has to be repeated a number of times. Overheating of the film prevents the staining of the flagella. The cell membrane may also show by this method. Van Ermengen's method gives good results. It is as follows: The films are placed for one hour at room temperature, or are heated for fi^^c minutes o\'er a water-bath at 100° C. in the following solution: Solution A. Osniic acid, 2 per ceut. solution 1 part Tannin, 10 to 25 per cent, solution 2 parts Wash successively with water, absolule alcohol, and water, then place in the following solution for a few seconds: SOIAITION B. 0.5 per cent, solution of AgNOs in distilled water. MICROSCOPIC METHODS 81 Without wasliiiig transfer tht'in to a third solution: Solution C. Gallic acid S grams Tannin 3 grams Fused potassium acetate 10 grams Distilled water 350 e.c. After Iveeping in this for a few seconds, place again in Solution B until film begins to turn black. Then wash and examine. Eosin-methylene-blue Stains. — These are polychrome staining mixtures introduced by Nocht, Romanowsky and others chiefly for the staining of animal cells, but they are also useful in differentiating bacteria and other germs in tissues and exudates. They are fine differential stains for chromatin. Many modifications have been proposed. In a study of the essential constituents of the Romanowsky stain, MacXeal says, both methylene azure and methylene violet are present and participating in the nuclear staining. The preparation of solutions directly from the pure dyes, methylene azure, methylene violet, methylene blue and eosin, has been recommended as the best manner of preparing these staining solutions as the proportion of the various constituents may be varied at will to obtain various kinds of dift'erentiation. As a routine blood-stain for study of leukocytes and staining of hematozoa, the following is recommended by MacNeal : Solution A. Methylene azure 0.3 Methj'lene violet (Bernthsen's, insoluble in water) 0.1 Methylene blue 2.4 Methyl alcohol, pure 500.0 Solution B. Eosin, yellowish, water soluble 2.5 Methyl alcohol, pure 500.0 These solutions keep for at least a year. The}' are mixed in equal parts and diluted by the addition of 2.5 c.c. of methyl alcohol to each f 00 c.o. of the mixture. This final mixture is employed in the same manner as Leislmian's stain. It keeps for a few months. Giemsa's Method. — Smears are fixed in neutralized methyl alcohol for one minute. The staining solution is one of several reconuuended by Giemsa. Azur II — Eosin ' . . . . 3.0 grams. Azur II 0.8 gram. Glycerin (Merck, chem. pure) 250.0 c.c. Methyl alcohol (chem. pure) 250.0 c.c. Both glycerin and alcohol are heated to 60° C. The dyes are put into the alcohol and the glycerin is added slowly, stirring. The mixture is allowed to stand at room tem- perature overnight, and after filtration is ready for use. The solution is prepared ready for use by Griibler, Leipzig. 1 drop of the stain to every c.c. of distilled water made alkaline liy the ijrevious addition of 1 drop of a 1 per cent, solution of potassium carbonate to 10 c.c. of the water is poured over the slide and allowed to stand for one-half to three hours. The longer time brings out the structure better, and in twenty-four hours well-made smears are not overstained. After the stain is poured off, the smear is washed in running tap-water for one to tlu'ee minutes, and dried 82 PRINCIPLES OF MICROBIOLOGY with filter paper. If the smear is thick, the organisms may come out a little more clearly by dipping in 50 per cent, methyl alcohol before washing in water; then tlie washing need not be as thorough. By this method of staining, the cj'toplasm of protozoa stains blue and the nuclear substance a blue red or azure. Wright's Stain. — One per cent, methylene blue (alcohol rectified), and 0.5 per cent, sodium carbonate are mixed and placed in a steam sterilizer for one hour. When cold 0.1 per cent, solution of extra B-A eosin (500 c.c. eosin to 600 c.c. methylene-blue solution), is added until the mixture becomes purplish, and a finely granular black precipitate appears. This precipitate is filtered off and dried without being washed. A saturated solution of this is made in pure methyl alcohol. This is filtered and then diluted by adding to 40 c.c. of it 10 c.c. of methyl alcohol. In using, a few drops are placed on the film for a minute; then water is dropped on till a greenish irridescence appears. The stain then remains on for two minutes; then is washed off with distilled water, allowing a little to remain on until differentiation is complete. Dried with filter paper. Leishman's Stain — This is a modification of Jenner's stain which is simply a solution of eosin and methylene blue in methyl alcohol. Instead of ordinary niethjdene blue Leishman used the acti^'e constituents formed in this stain. Solution A. — To a 1 per cent, solution of medicinally pure methjdene blue in distilled water add 0.5 per cent, sodium carbonate and heat at 65° C. for twelve hours, then allow it to stand ten days at room temperature. Solution B. — Eosin extra B-A (Griibler) 0.1 per cent, solution in distilled water. Mix Solution A and B in equal amounts and allow to stand six to twelve hours, stirring at intervals. Filter and wash the precipitate thoroughly. Collect, dry, and powder it. 0.15 gram is dissolved in 100 c.c. of pure methyl alcohol to form the staining solution. It keeps perfectly for at least five months. To stain, cover the dried but unfixed film of blood with the staining solution. After thirty to sixty seconds add about an equal amount of distilled water. Allow this mixture to act for five minutes. Wash in distilled water for about one minute, examining the specimen mounted in water under the microscope. Blot, dry thoroughly, mount in balsam, or preserve the specimen as an un- mounted film. Goldhorn's Method of Staining Spirochetes.— Dye; w-ater, 200 cm.; lithium carbonate, 2 grams; methylene blue, 2 grams. (Merck's medicinal or a similar preparation.) This mixture is heated in a rice boiler vni\\ a moderate amount of heat until a rich polychrome has formed. This is determined by examining a sample against artificial light and noting the appearance of a distinctly red color. The solution is allowed to cool and the residue is removed by filtering through cotton. To one-half of this filtrate 5 per cent, acetic acid is'gradually added until a .strip of litmus paper shows aboA-e the line of discoloration a distinct acid reaction. The remaining half of the dye is now added, so as to carry the reaction back to a low degree of alkalinity. A one-half per cent. French eosin solution is now added gradually, while the mixture is being stirrecl until a filtered sample shows a pale bluish P(.lor with slight fluorescence. The mixture is allowed to stand for one day and filtered. The precipitate is collected on a double filter ])aper and dried at a temperature not exceeding 40° C. It is then removed from the filter paper and dissolved in commercial wood alcohol. It is allowed to stand for one day in an open vessel and then filtered. To use the stain on smears sufficient dye to cover tlie smear is dropped on an unfixed preparation and allowed to remain for three or four seconds- the excess is then jioured off. The slide is now introduced slowly into clean water with the film side down, is held there for four or five seconds and is then shaken in the water to wash off the excess of dye. It is then allowed to dry and is ready for examination. The pallidum stains violet. Until recently the demonstration in smears of the syphilis spirochete by MICROSCOPIC METHODS ' 83 the silver impregnation method, so successfully used by I.evaditi in section, has been unsatisfactory. Stern, however, and Flexncr corroborating him, have gotten beautiful results by the following simple method : (a) Air-dried into 37° incubator for some hours. (h) Ten per cent, aqueous silver nitrate for some hours (Flexner thinks three to foiu' days' exposure better) in diffuse daylight. (c) When the brownish color reaches a certain tone (easily recognized after experience) and when a metallic sheen develops the slide is waslied well in water, dried, and mounted. The blood cells are well preser\'ed, they have a delicate dark brown contour, and contain fine light brown granules. The spirochetes are deep black on a pale bro^^ai and in places a colorless background. Other spirochetal organisms may be silvered by this method, but as they may be differentiated with greater difficulty than with Giemsa's .stain, the latter should always be used as well. These organisms may also be demonstrated by the India-ink method (see p. 74). The flagella are brought out by Loffler's method or by the stain recom- mended by Goldhorn. Sections are prepared by the silver iriipregnation method of Levaditi as follows : Fix small pieces of tissue one-half mm. in thickness for twenty-four to forty- eight hours in formalin, 10 per cent. AVash in 95 per cent, alcohol twelve to sixteen hours. Wash in distilled water till the pieces sink. Impregnate two to three hours at room temperature and four to six hours at 50° C. in the follow- ing fluid: Nitrate of silver, 1; pyridine, 10 (added just before using); aq. dest., 100. Wash rapidly in 10 per cent, pyridine. Reduce the silver by placing in the following mixture for several hours: Pyrogallic acid, 4; acetone, 10 (added just before using); pwidine, 15; aq. dest., 100. Harden in alcohol; xylol; paraffin. Levaditi's first method is longer but more reliable. Fix small pieces in formalin, 10 per cent. Harden in 95 per cent, alcohol. Wash in distilled water se^'eral minutes. Impregnate three to five days at 37° C. in 1.5 per cent, solution silver nitrate. Reduce twenty-four hours in: Pyrogallic acid, 4; formalin, 5; water, 100. Embed in paraffin. Bj^ these method.s the spirochetes appear clenseh' black. Giemsa's method gives excellent results. Ross' Method of Examining a Large Quantity of Malarial Blood in One Film. — A large drop of blood (about 20 c.mm.) is placed on a glass slide and is slightly spread o^'er an area which can be covered by an ordinary cover- glass. This is allowed to dry in the air or it is warmed over a flame without heating it more than enough to fix the hemoglobin. The dry film is then covered with an aqueous solution of eosin (10 per cent.) and allowed to remain about fifteen minutes. This is then gently washed off and a weak alkaline methylene-blue solution is run over the film and left for a few seconds, when the ]ireparation is again gently washed. After drying it is readj' for examina- tion. The Eosin-methylene-blue Method Recommended by Mallory for Tissues may be Used for Smears as Follows : — The smears are fixed in Zenker's solution for one-half hour ; after being rinsed in tap-water they are placed successively in 95 per cent, alcohol + iodine, one-quarter hour; 95 per cent, alcohol, one- half hour; absolute alcohol, one-half hour; eosin solution, twenty minutes, rinsed in tap-water; methylene-blue solution, fifteen minutes, differentiated in 95 per cent, alcohol from one to five minutes; and dried with filter paper. Staming Method for Negri Bodies {Williams' Modification of Van Giesons Method). — Smears partially air-ilried are fixed for ten seconds in neutral methyl alcohol to which 0.1 per cent, picric acid has Ijeen added. Excess of fixative removed by filter paper. Smears then stained in following solution : Saturated alcohol solution fuchsin, 0.5 c.c; saturated alcohol solution methyene blue, 10 c.c; distilled water, 30 c.c. The stain is poured on the smear and held over 84 PRINCIPLES OF MICROBIOLOGY the flame until it steams. The smear is then washed in tap-water and blotted with fine filter paper. The Negri bodies are magenta, the nerve cells blue, and the red-blood cells j^ellow, or salmon color (Plate X, Fig. 1). Heidenhain's Iron-hematoxylin Stain. — (a) Mordant and differentiating, fluid: Iron o.xjTlammonium sulphate, 2.5 grams; distilled water, 100 c.c. (6) Staining fluid: Hematoxyhn, 1 gram; alcohol, 10 c.c; distilled H2O, 90 c.c. (To be kept in a red bottle and allowed to stand for about four weeks before using.) For use see chapter on Amebae. Preservation of Specimens. — Dry-stained preparations of bacteria keep indefinitely, but if mounted in Canada balsam, cedar oil, or dammar lac they tend gradually to fade, although many preparations may be preserved for many months or years. Dry unstained spreads should be kept in the ice-box until stained. Examination of Microorganisms in Tissues. — Occasionally it is of imjwr- tance to exannne the organisms as they occur in the tissues themselves. The tissues should be oljtained soon after death, so as to prevent as much as pos- sible postmortem changes, with consequent increase or decrease in the number of microbes. Selected pieces of tissues can be frozen by ether or carbon dioxide and sections cut, but the best results are obtained when the material is imbedded in paraffin or in celloidin. Fixing and Hardening Tissues. — From the properly selected spots small portions, not larger than one-quarter inch by one-eighth inch, are removed and placed in one of the following fixatives : 1. Absolute alcohol for from four to eight hours, and longer if thicker. For the larger pieces it is better to change the alcohol after eight hours. The pieces of tissue should be kept from falling to the bottom as the higher laj'ers of alcohol remain nearer absolute. If along with the microorganisms one TOshes to study the finer structure of the tissue, it is better to employ one of the other fixatives. 2. Formalin. — For fixing in formalin the tissue is put in 4 to 10 per cent, formalin solution for three to twenty-four hours, and then in successive strengths of alcohol. 3. Corrosive sublimate (saturated solution in 0.75 per cent, sodium chloride solution) is an excellent fixing agent. Dissolve the sublimate in the salt solu- tion by heat, allow it to cool; the separation of crystals will show that satura- tion is complete. For pieces of tissue one-eighth inch in thickness four hours' immersion is sufficient; for larger, twenty-four hours maj^ be necessary. They should then be placed in pieces of gauze and left in running water for from twelve to twenty-four hours, according to the size of the pieces, to wash out the excess of sublimate. 4. Hot sublimate alcohol (50° C.) (Schaudinn), or saturate sublimate, to which 5 per cent, glacial acetic acid may be added. The preparation should remain in it a few seconds, then should be washed for one-half hour in 60 per cent, iodine-alcohol, and then placed in 70 per cent, alcohol. They may remain here for an indefinite time, until they are to be stained, when they are rinsed in distilled water and then placed in the staining fluid. 5. Two per cent, osmic acid (to be kept in a red glass with a ground-glass stopper). Moist smears are exposed to its fumes for a few seconds, small pieces for sections, four to eight hours, then carried through the various alcohols and xylol and mounted or imbedded in the usual way. 6. Hertiiaiiii's Fluid. — Platinum chloride, 15 c.c; a 1 per cent, solution osnfie acid, 4 c.c; a 2 per cent, solution glacial acetic acid, 1 c.c Moist spreads may be fixed for several minutes; very small pieces of tissue for twenty-four hovu's. 7. Zenker's Fluid.— Add to a solution of Muller (bichromate of potash, 2 to2| parts; sulphate of soda, 1 part; water, 100 parts) 5 per cent, of saturated MICROSCOPIC METHODS 85 sublimate solution and, when ready to use, .5 per cent, of glacial acetic acid. Moist s])reails are lixed for (uie to li\'e minutes, small )iieces of (issue for threes to tweh'e hours. They are then washed with water or ))ut immediately' into suceessix'e alcohols. To linnk'ii they are ])laced successi\'ely for twenty-four hours each in the followin"- strengths of etliyl alcohol: 30 per cent., 00 per cent., and 90 |)er cent. Finally they are placed in absolute alcoliol for twenty-four lioiu's wliich de- hydrates them and they are then ready to be imbetkled in paraffin. To imbed in paraffin, the pieces are put in (1) cedar oil until translucent, (2) cedar oil and paraffin, equal parts, at 52° C. for two hours, (3) jaaraffin 52° C. two hours in each of two baths. They are then boxed ready for sec- tions. Sections are cut at 3 to 6/j, and are dried at 36° C. for about twenty- four hours, protected from dust. The paraffin sections of tissue having been prepared and cut, they are read}' for staining after the paraffin is removed. If all of the sublimate has not been removetl liy the water the sections may be immersed in iodine-alcohol for ten minutes. Loffler's St.wning Method for Sections. — The section is placed in Loftier 's alkaline methylene-blue solution for five to thirty minutes, decolor- ized for a few seconds in f per cent, acetic acid. It is tlien placed in absolute alcohol, xylol, and Canada balsam. The number of seconds during which the preparation remains in the acetic acid must be tested by trials. References. Burri. Das Tuscheverfaliren als Einfaclies Mittol, etc., Jena, 1909. Giemsn. Deutsclie med. Wocli., 1905, xxxi, 1026. Kohler. Ztsch, f. wiss. Milcrosc, 1904, xxi, 129. Lea. Microtomist's Vade-mecum, 191.3, 7th ed. Leishman. Brit. Med. Jour., 1901, p. 6.35- and 1902, p. 7.57. Meiz. Ztschr. f. wiss. iVIiliroskop., 1913, xxx, 188. Reichert. Jour. Roy. Micr. Soc, 1907, 364. Wright. Jour. Med. Res., 1902, ii (New Series), 138. MacNeil. Jour, Infect. Dis., 1906, iii, 412. Williams. Am. Jour. Pub. Hyg., 1908, xviii. No. 1. CHAPTER IV. GENERAL METHODS USED IN THE CULTIVATION OF MICROORGANISMS. CULTURE MEDIA, METHODS OF ISOLATION AND CULTIVATION, CULTURAL CHARACTERISTICS. The methods employed for the artificial cultivation of microorganisms are of fundamental importance. Bj' their use we can obtain one variety growing apart from all others, namely, in pure culture. This pure culture may be planted on various media and the morphologic, bio- chemic, and cultural characteristics studied for classification and identi- fication. It is evident that all glassware and instruments used must be free from other microorganisms; that is, they must be sterile. CULTURE MEDIA. Preparation of Glassware. — Various type.s of glassware, such as test-tubes, flasks, bottles, beakers, pipettes, etc., are used (see Figs. 14 to 22.) New glass- ware, as a rule, only requires mechanical cleansing with soap and water, loosen- ing adherent dirt with test-tube or bottle brushes. Old glassware containing cultures should be sterilized either in the autoclave or in a covered boiler. In the latter case the tubes are covered with water, about .5 per cent, of washing soda or soap added, and boiled for one hour. Any solid medium present is melted and the glassware can then be washed according to the directions already given. After washing, drain and allow to dry. Neutralization of Glassware. — Where slight changes of reaction are important the glassware should be neutralized after washing. Soak in 1 per cent, hydro- chloric acid for several hours or boil for one-half hour, wash free of acid, and rinse in distilled water. New glassware, especially the cheaper grades, is most likely to give off free alkali. The clean glassware is plugged loosely with ordinary non-absorbent cotton. The cotton should not be twisted in, as creases will form along the glass, leaving channels for contamination. Either fold into a plug or take a square of cotton, fold one corner, place a rod against the fold, and push the cotton into the neck of the container or tube. A sufficient amount of cotton should project for handling and the plug should be just tight enough to allow one to lift the con- tainer by means of the plug. Several thicknesses of filter paper may be used to cover beakers and other wide-mouth containers. Sterilization of Glassware. — All glassware is sterilized by dry heat. This is done after plugging. Some type of hot-air sterilizer is used (Fig. 24). Heat ■ to 160° C. for one hour. Leave the sterilizer closed when the sterilization is finished, so that cooling is gradual or the glassware may crack. This heating not only sterilizes but sets the shape of the cotton plugs. Composition of Culture Media. — Most microorganisms causing disease require complex foodstuffs similar in constitution to those in the animal body. The general basis of media for these types is an CULTURE MEDIA 87 extract or infusion of meat. To this may be added peptone and sodium cliloride. Some may retjuire uncoagulateil proteins, such as serum or Fig. M Vm. 1.0 Test-tubes can be also used mthout lip. Average size, 6 x | inches. Beakers. blood, or even fresh tissues. Carbohydrates may lie added. The non- pathogenic microorganisms vary in their abiUty to grow on these more complex media, and some will only grow on simple media containing inorganic salts. Media may be either fluid or solid. In the latter case Fig. le Fig. 17 Fig. 18 Globe flask. Volumetric flask. Erlenmeyer flask. there is added some jelly substance such as gelatin or agar or an albumin which is coagulated by heating. ss PRINCIPLES OF MICROBIOLOGY Certain technical methods are employed in the preparation of nearly all culture media, such as adjustment of reaction, clearing, filtering, KiG. I'J Flu. 20 Fig. 21 Type of bottle com- monly used tor dilutions, etc., or as substitutes for flasks. Blake bottle. Laid on its flat side it gives a large surface of broth or agar. Petri dish. Commonest size is 10 e.o, in diameter. etc. These details must be understood before the actual preparation of media is given. Fig. 22 Types of fermentation tubes. Reaction of Media. — Titration and Adjustment. — A moderately alkaline reac- tion to litmus is satisfaetorjr for the gro\vth of most pathogenic microorganisms. For ordinary work the iLsual acid reaction of the media in preparation may be reduced by the addition of a 4 per cent, sodium hydrate solution until red litmus paper is turned slightly but distinctly blue and blue litmus paper remains the same color. Litmus, however, is not a delicate indicator and phenolphthalein is generally used, giving us more accurate knowledge of the reaction. The neutral points of the two indicators are different, so that media which are alkaline to litmus are still acid to phenolphthalein. The materials necessary for titration and adjustments of the reaction to phenolphthalein are normal and twentieth normal solutions of sodium hydrate and of hydrochloric acid, CULTURE MEDIA 89 ;i 0.5 per cent, solution of plR'iiolplitlialcin in 50 per cent, alcohol, hiircilcs, I'usserole, and stirriiis;; rod. Five c.c. of mediuin to be tested, 45 e.c. of distilled water, and 1 c.c. of the l)lieiiolplitlialeiii solution are nii.xed in the c-a-sserole and hoileil for two minutes. If no color is present the medium is aeid and while hot the twentieth normal solution of sodium h>;drate (N NaUH solution) is added from a burette until a faint but distinct pink color appears. This ct)lor must remain on reheating, otherwise moi'e alkali is needed. From the amount added we determine the acidity of the medium and estimate how much normal (N) solution of NaOH must be added to obtain the reaction desired, for example : Fig. 23 Hot-air sterilizer. Lautenschlagcr form. Five c.c. required 2.4 c.c. of |^ NaOH to neutralize, therefore 100 c.c. (twenty times as much) would require 2.4 c.c. of ? NaOH (twenty times as strong), in other words, the medium is 2.4 per cent, acid to phenolphthalein (+2.4 per cent.). Assuming we desire a reaction of +1 per cent., we must then add 2.4 c.c. —1 c.c. or 1.4 c.c. of ^ NaOH to every 100 c.c. of medium or 14 c.c. to a liter. Should, on the other hand, the mixture in the casserole show a pink color then the medium is alkaline and 7]^ HCl is used, heating as above, until onlj' a faint pink color persists. If we use 0.5 c.c. then the medium is 0.5 per cent, alkaline ( — 0.5 per cent.), and it will require 0.5 c.c. of ^- HCl for every 100 c.c, or 5 c.c. for every liter to bring it to the neutral point. But we want the reaction 90 PRINCIPLES OF MICROBIOLOGY to be +1 per cent, or 1 per cent, acid, we therefore add 5 c.c. plus 10 c.c. oi 1,5 c.c. of ■? HCl to each liter. ,. . r i ii-oi; .„. After the fir.st coi'rcetion of the reaction liy the addition of iiormal alkali oi acid the medium should be again titrated and further correction wll usua;lly he necessary. This may have to be repeated several times before the reaction desired is obtained. Various combinations, precipitation, etc., occur wtiicn make this necessar3^ Fig, 24 cc 20°C = 2 =23 1=24 =I5 OUTFLOW 6?. Set 44:^ DELIVERS 50 CC Fig. 2i' Graduated Volumetric pipette. pipette. Obtainable in various sizes and graduations. Burettes. Most convenient type has a blue line against a white background, on back giving sharper readings. The reaction of media should alwaj^s be adjusted before filtration, and after adjustment the medium must be heated for three to five minutes or precipita- tion will occur on subsequent sterilization, especially if sterilized in the auto- clave, due to the fact that the temperature is higher than that emploj'ed during the adjustment of the reaction. Titration of mecUa maj^ be done with the mixture in the casserole at about 4.5° G. instead of the standard method given. The method used is not so impor- tant as the reproduction of a reaction whicli is satisfactorj'- for the purpose in view. Where reactions are given it is assumed that they have been obtained according to the standard method. Any variations in procedure should be stated. CULTURE MEDIA 91 Fig. 26 Clearing Media. — This is done by coagulation of an albumin, which, as it (■(lagtilatos, enmeshes the fine particles. In certain methods the coagulation of the solulile albumins in the watery extract of the meat clears tlie medium when heated. ITnder other conditions an albumin such a.seggsis added. 11' 1hc medium is hot it must iirst be cooled to below ()0° C. One or two eggs are usefore coagulation such as sera or tissue extracts and tlie tem]3eratiue selected should be l>elow the coagulation-point of the material, usually aliout 60° C. Coagulated media, where overheating will spoil the medium or render it too opaque, may be sterilized in the same way. All material to be treated in this manner should be as free from contamination as possible. The method has its 94 PRINCIPLES OF MICROBIOLOGY best application to sterile material which, because of necessary manipulation, may Ijecome slightly contaminated. The heating may be done in a water bath or in a water-jacket oven. Fig. 34 Fig. 35 tlirit'lit. typr. Horizontal type. .\ii(orlavfs may be liratccl by direct application of heat, or liy steam under pressure, when aA'ailablc. Intermittent Sterilization. — This term is applied to the heating of material to or close to the boiliiig-iioint of water on three successive days. The reasons for the repetitions are the same as in fractional sterilization. lii this metlaod, how- CULTURE MEDIA 95 over, only the very resistant spores escape. An Arnold sterilizer is usually employed (Figs. 32, 33) . The material is heated in the flowing steam. The time of heating will depend on the size of the container employed; test-tubes of media usually require twenty minutes, whereas liter flasks of media would require forty-five minutes at least to allow the heat to penetrate sufficiently. ^ Sterilization by Steam under Pressure. — This is done by means of an autoclave. \'arious forms are available which may be heated by gas or by steam (Figs. 34, 35) . All air should be displaced by the steam before closing the vent. Too rapid reduction of pressure will cause the media to boil up and wet or even blow out the plugs. The time and pressure employed will depend on conditions. As a rule, fifteen pounds' pressure is employed and twenty minutes are sufficient for media in test-tubes. Flasks should be heated one-half to one hour, depending on their size. Lower pressures are at times desirable where o^'erheating is a factor, and the heating may be repeated on two successive days. The heating should Ije timed from the time the pressure desired is reached. The temperature will vary vnth the pressure thus : 5 pounds' pressure = temperature 10S.8° C. 10 15 = .. 11.5.6° 121.3° C. C. Storage of Media. — Media after sterilization may deteriorate for two reasons, namely, contamination (especiaUy by molds penetrating the cotton stoppers), and by drying. The former may be avoided by keeping the media at a low temperature, 40° to 45° C, in a dry refrigerator. Where media are used in small amounts it is best kept in flasks and tubed as needed. Evaporation of the tubed media can l^e lessened b)' dipping the plugs in paraffin or ]')y the use of rubber caps made for this purpose. The stock flasks after removal from the sterilizer may be sealed with paraffin or sealmg wax. The plug should be cut off and pushed in slightly and melted paraffin poured on it, care being taken that it does not run through the plug into the media. This can be avoided if the paraffin is nearly cold enough to set. Sealing wax may be applied in the same wa^y or rubber caps or rubber tissue tied over the neck. If the stock flasks are capped a small pledget of cotton moistened wth l)ichloride of mercury solution may be placed between the plug and the cap to discourage the development of molds which maj' adhere during manipulation. The neck of the flask should be mped free of the bichloride Ijefore pouring out the media. This precaution is specially useful for media stored" in flasks at room temperature. The necks of all con- tainers may be protected from dust by covering with paper before sterilization or inverting tumblers over the necks of flasks. Preparation of Meat Infusion. — One pound (500 grams) of finely chopjied meat, usuallj^ beef or veal, is macerated with 1000 c.c. of water and placed in the ice-chest for eighteen to twenty-four hours, or it may be extracted by heating to a temperature not exceeding 60° C. for one hour. The infusion is then strained through cheese-cloth and the meat squeezed out by twisting the cloth or by means of a meat press. The fluid contains the soluble albumins, extractives, salts, carbohydrates, and coloring matter of the meat. This forms the basis for the various media to be described. If this infusion is to be stored as such it must be boiled to coagulate the albumins (which also clears it), filtered, placed in flasks, and sterilized. Many advise the neutralization to phenol- phthalein' before heating, on the ground that less of the foodstuff' is precipitated than l)y heating the verjr acid infusion. As a sul)stitute for the meat 2 to 5 grams of Liebig's extract of Ix'ef may be used for each liter of water. When media are made with this basis they are spoken of as meat-extract media in contradistinction to meat-infusion media. Nutrient Bouillon. — Meat infusion 1000 c.c, peptone 10 grams, and sodium chloride 5 grams. Warm the meat infusion to about 50° C, add the peptone ' See Reaction of Media. 96 PRINCIPLES OF MICROBIOLOGY and salt and stir until dissolved. A convenient way is to use one's hand, as in this way the fluid will not get too hot, and the lumps of wet peptone are easily crushed. The mixture is then boiled over the free flame to coagulate the albu- mins, preferably after neutralizing the excess of acid. Evaporation should be made good by the addition of water. Correct the reaction and boil again for five minutes, filter, and place in the appropriate containers for sterilization. This medium may also be made from meat extract. Gelatin Media. — Meat infusion 1000 c.c, peptone 10 grams, sodium chloride 5 grams, and gelatin ("gold label") 100' grams. Dissolve as for nutrient broth. The acidity may then be reduced and the mixture then boiled or heated in the Arnold sterilizer for a half-hour. The reaction is now set and the media again heated for fifteen minutes and filtered. The clearing is done in this way b}' the albumins in the meat infusion. It should be tubed at once and sterilized in the Arnold sterilizer for twenty minutes on three successive daj^s. Reheating and resterilization is to be avoided as the media toU not set after too much lieating. If the gelatin media is made from meat extract or mth meat infusion which has been heated it must be cleared with eggs before filtration. In this case the ingredients are dissolved by heat and the reaction set. The mixture is then cooled below 60° C. and the eggs added; and then it is heated as above and filtered. Agar Media. — Two methods may be employed. The simplest method is to take 1000 c.c. of infusion or extract broth and add 1.5 per cent, of shredded agar. This is then dissolved by boiling over the free flame for one-half to three- (luarters of an hour, making up the volume lost l5j' evaporation. A better way is to place the mixture in the autoclave and heat for one-half to one hour at fifteen pounds' pressure, depending on the bulk. The reaction is then set, and the mixture cooled to below 60° C. and the eggs added. The mixture is again heated to coagulate the egg, either over the free flame or in the autoclave, and filtered. Another method of prejiaring agar is to make a double strength meat infusion, in which is dissolved double the amounts of peptone and salt and the excess acidity' neutralized. To an equal quantitj^ of water add 3 per cent, of agar and dissolve. Cool the agar below 60° C. and mix the two solutions. Set the reaction. Then heat for one-half to three-quarters of an hour to coagulate the meat albumin, which also clears the medium, correct the reaction if necessarj', and filter. Peptone Solution (Dunham's).— Water 1000 c.c, peptone 10 grams, and sodium chloride 5 grams. Dissoh'e by heating and filter. The reaction neecls no adjust- ment for ordinarj' use. Nitrate Broth. — Water 1000 c.c, peptone 1 gram, and nitrate-free potassium nitrate 0.2 gram. Milk. — The milk used should be as fresh as possible and preferably the best grade obtainable. Steam the milk in the Arnold sterilizer for a half-hour and place on ice for several hours or overnight to allow the cream to rise. By means of a siphon remove the milk from below the creanr laj^er. This maj'^ be tubed or litmus solution added {litmus milk). If a good grade of milk is used no change of reaction is necessary; if acid, sufficient sodium hydrate solution sliouid be added to render it slightly alkaline to litmus; if very acid, the milk sliould be discarded. Potato Media. — Large potatoes should be used and scrubl)ed with a nail brush under running water. Cylinders are then cut by means of an ap])le corer. The skin is cut off the ends of the potato cylinder and by an oljlique cut, wedge- shaped pieces are obtained. A good butt should be left or tlie pieces will coil. While preparing the ])otatoes they should be kept submerged in water or the cut sui-faces will disef)lor. As the jwtato is acid the reaction must Ije changed. This is done by soaking in running water overnight or by soaking several hours in a 1 to 1000 solution of sodium carbonate. The pieces are then placed in test- ' Use 120 granjp in warm weather. CULTURE MEDIA 97 tubes, a little water addetl to avoid drying, and sterilized. The test-tubes used shoTild be one incli in diameter or over. If the pieces of potato are too small and thin they will curl on sterilizing. Glycerin potato for the cultivation of tubercle bacilli should be cut as above, soaked overnight in 1 to 1000 sodium carbonate solution, drained, and covered with a 5 per cent, glycerin solution for twenty-four hours. When tubed the glycerin solution or water is added for maintaining the moisture. P"iG. 36 Fig. 37 Fig. 38 Potato borer. Cylinder of potato, line of cut. Finished medium. Glycerin-potato Agar. — To extract agar add 5 per cent, potato juice and 5 per cent, glj'cerin. (Potato juice: 1 pound of grated potato to 1000 c.c. of water. Soak overnight, strain, and boil. Clear with eggs and filter.) Semisolid Media. — Various types of semisolids are in use. The amount of agar wall vary according to the use to which it is put. For slants, 0.75 per cent, to 1 per cent, of agar is necessary; for stab cultures, 0.5 per cent, or even less is sufficient. If the agar is to be diluted by the addition of serum or other enriching substance this must be allowed for in preparing the medium. The following is an example: Meat infusion (1 pound of meat to 500 c.c. of water) is warmed and 20 grams of gelatin and 20 grams of peptone dissolved in it. In 500 c.c. of water dissolve 10 grams of agar and cool to below 50° C. Mix the two, adjust the reaction, and heat to coagulate the albtmiin and clear the medium. Readjust the reaction and reheat if necessary; filter. A very satisfactory medium can be made by simply using 0.5 per cent, of agar instead of the usual 1.5 per cent, employed. This can l)e diluted l)y tlic addition of one-third of its liulk of an enrichment fluid and will still set sufficicntlj' for stab cultiu'cs. Carbohydrates in Media. — C'arjjohydrates are added to media to determine whether acid or gas or l)oth are produced or because of its utilization as a food- stuff to cause a more abundant growt-h on the part of the inoculated organisms. 98 PRINCIPLES OF MICROBIOLOGY Oiily the purest carbohj'drates obtainable should be used. The following is a partial list of substances which may be used: Mono-hexoses — destrose, levulose, galactose, and mannose. Bi-hexoses — saccharose, maltose, and lactose. Tri-hexoses — raffinose . Polyhydric-alcohols—erythnte (tetra), adonite (penta), and mannite, sorbite, dulcite, and glycerin (hexa). Pentoses — arabinose, xylose, and rhamnose. Polysaccharids — glj'cogen, dextrin, and inulin. Carbohydrates are usually added to media in 1 per cent, amounts, with the exception that 5 per cent, of glycerin is used. Besides the true carbo- hydrates, various glucosides, such as salicin, coniferin, etc., are used. They may be used in any medium, either soUd or fluid. Where meat extract or meat infusion is used as a basis it must be remembered that this contains fermentable sugar in muscle. If the media is to be used to determine whether a sugar is acted upon or not the muscle sugar must be removed before preparing the media. The meat extract or infusion to be used should be neutralized to phenolphthalein, and for each liter add a broth culture of B. coli or one of its allies. This is incubated for forty-eight hours. The bacillus ferments the sugar present. The infusion is now sterilized and used in the prepa- ration of media. Media made in this way are called "sugar-free media." To the sugar-free media the sugars may then be added. Serum water (Hiss) (see Serum Media) may also be used as a basis for carbohydrate media. Many of the carbohydrates are very susceptible to heat. They are split into simpler compounds which may be fermented by an organism unable to ferment the unaltered sugar and thus lead to error. The usual method is to add sufficient sugar to sterile water to make a 10 per cent, or 20 per cent, solu- tion and heat this in small containers in the Arnold sterilizer. This solution is then added to the media in sufficient amounts to give a final 1 per cent, content. Certain sugars, especially some samples of maltose, are so easily split by heat that they must be sterilized by filtration. In routine work wth glucose, lactose, saccharose, mannite, and dulcite it is usually sufficient to add the sugar to the media and sterilize by intermittent sterilization. Although there may be a slight destruction of the glucose it is not enough to Ije important. Inulin is an exception. Because of the resistant spores commonly present the inulin solution should be sterilized in the autoclave. Fluid sugar media, with the exception of serum-water media, are usually filled into fermentation tubes, where gas production may be determined. Three types are used (Fig. 22, o, 6, c, p. 88). The tj^pe (c) can only be used where sterilization is done in the Arnold sterilizer or autoclave. To fill these tubes, the small tube is first filled, and vnih as little inversion as possible, slipped mouth down into the large tube. Sufficient media is then added to allow the small tube to fill after expulsion of the air contained and leave the proper amount of media in the outside tube. The air in the under tube is driven out during sterilization. An indicator may be added to show the changes in reaction. If litmus is used the media should be very slightly alkaline to this indicator, or slight acid production will not become evident. In careful work, titration is the best method of determining the changes in reaction. Litmus and Other Indicators. — The ordinarj^ litmus of commerce is not a very delicate indicator because of the impurities present which give it a reddish color. A purified litmus (Merck or Kahlbaum), or a solution such as that known as "Kubel and Thiemann," should be used. The purified htmus comes in the dry form and sliould be pulverized and added to distilled water in 5 per cent, amounts. This is steamed in the Arnold sterilizer for two hours, shaking the mixture every twenty minutes. The solution is then filtered and the filtrate sterilized. The solution must be kept sterile. Usually 5 per cent, of the litmus solution is added to the media. This may be varied to suit CULTURE MEDIA 99 personal preferences. Media to which litmus is added should be carefully ad- justed to a slightly alkaline reaction to litmus or slight changes \vill be obscured. (In place of litmus a 1 per cent, solution of Kahlbaum's azo-litmus may be employed.) Litmus media may be decolorized by the grow^th of liacteria which rob it of its oxygen and reduce it to the colorless leukobase. The color will return on exposure to the air. Where stab cultures are used it may be necessary to melt the medium and pour it into Petri dishes to get sufficient air exposure to cause the color to reappear. Where the color change of individual colonies is to be observed, the diffusion of any acid produced may be prevented by using 3 per cent, agar instead of 1.5 per cent. Various aniline dyes which are reduced to their colorless leukobase by the action of sodium sulphite may be used as indicators. When the sugar is split liy the bacteria, aldehydes are produced, which cause the color to return. Free acids do not make any change. The decolorized dj'e must be freshly prepared and the medium prepared with it used at once as exposure to the air causes the color to reappear. An example of such an indicator is the use of decolorized fuchsin in Endo's media. (See Tj^phoid Media.) Media to be used with this indicator should be slightly alkaline to litmus. Powdered insoluble carbonates may be used in solid plating media. If the powder is evenly distributed throughout the media, acid-producing colonies \vill be surrounded by a clear area. Phenolphthalein is also employed. The medium should be faintly alkaline and sufficient 1 per cent, alcoholic solution added to give a distinct pink color. The color is discharged when acid is produced. Media containing indicators are preferably sterilized in the Arnold sterilizer. The autoclave may be used in emergencies, but the indicator is somewhat injured. Use of Insoluble Carbonates. — Either crushed marble or calcium carbonate powder may be used. About 1 per cent, by bulk is sufficient. Their main application is in fluid media, especially as carbonate broth for the streptococcus- pneumococcus group. The value of the carbonates is that they neutralize the acids that may be produced by the growth of the bacteria, and which might prevent further devel- opment or even injure the viability of the culture. Sugars may be used, there- fc>re, in media containing carbonates, and the gro-\\rth and viability of the cul- ture thus increased. The exact manner in which the calcium helps is not known, but it probably has some value. Where the medium is used in large amounts the containers should be shaken from time to time. Fig. .39 Serum inspissator (Park). Eingstands are usp(1 for support, and the tilt is easily variefl as needed. Media Solidified by Coagulation of Albumin.— L(#er'.s Blood-scrum.— Mix 3 ]iarts of calf or sheep serum and 1 part of neutral nutrient broth to which 1 per cent, of dextrose has been added. The mixture is then run into tubes and the tubes slanted in an apparatus where the temperature can be slowly raised to between 80° and 90° C. The heating in any case should not exceed 95° C. 100 PRINCIPLES OF MICROBIOLOGY until the medium is coagulated or bubbling will occur and the surface will be spoiled. A Koch serum coagulator may be used for this purpose or a small water-oven or even in an Arnold sterilizer, covering the top with a cloth instead of the usual cover. A convenient inspissator we have devised is shown (Fig. 39). It has the advantage that the temperature can be controlled and each tube equally heated. After coagulation the tubes should be sterilized in the Arnold sterilizer for twenty minutes on two successive daj^s. The medium maj^ be coagulated and sterilized in the autoclave by allo\ving the temperature to rise very slowly until 110° C. is reached. This is rapid and convenient, but it has seemed to us that the high temperature injured the medium somewhat. The serum is obtained at the slaughter-house in tall cjdindrical vessels. These should be disturbed as little as possible till coagulation is complete. If the coagulum adheres to the sides of the vessel, it should be loosened with a glass rod. After twentj'-four hours on ice the serum is pipetted or siphoned off. If bloody it can be placed on ice to allow the corpuscles to settle out. Coagulated Blood-serum Media. — Serum from the cow, horse, sheep, or dog may be coagulated in the form of slants. This type of medium has been used with and without the addition of glj^cerin for cultivation of tubercle bacilli, but a more satisfactory medium can be prepared from eggs. The serum used should be sterile and, after tubing, heated to 70° C. for one hour. If a clear medium is desired the best wa}' is to remove a tube from time to time for observation and cease heating when set sufficiently. Higher temperatures or too long an exposure render serum opaque. Serum may be coagulated in high columns in test-tubes especially for culti- vation of spirochetes. The tubes should be warmed to 65° C. and a few at a time placecl in water at 75° C. These are tilted every few seconds and removed as soon as they start to set. The heat retained in the tube T\all complete the coagulation. In this way a soft almost transparent coagulum is formed. The serum ma}' be diluted with saline broth, etc., for special purposes and treated in the same way. Satisfactory media of the above types require serum free from red cells or dissolved hemoglobin. Fig. 40 77/^ pie plate "Clteese eloth Fr/?ine2 , - Hvbber tubhio __,,- PuicJi cock (rlass tube Perforated cork Glass tube J a r^e enoufftf to aetmit test tube Straining and filling apparatus. (Krumwiede) The lower tul^e is plugged with cotton and the top wrapped. It is necessary to loo.sen the pinch-cook before heating. The whole is sterilized in the autoclave. Egg y¥eJto.— Freshly laid eggs are sterile and should be employed for the lircjiaration of media. Egg media are usually coagulated. Plain Egg {Dorset) .^Thc eggs are thoroughly cleansed with water of any adherent dirt and then washed with 5 per cent, carbolic solution and allowed CULTURE MEDIA 101 to partially dry. Tlie ends are then gently dried in (lie flame a,iid pierced with a burned sharp foreejis. The hole at one end sliould he about three-eighllis ineh in diameter and the membrane broken; Ihe other which is lo be blown into should be smaller and the membrane left unbroken if possible. The eggs ai'e then blown into a sterile l']rlenmeyer flask, the blowing being done from the cheeks, which will help avoitl spilling saliva and leakage of air around the out- side of the egg. To the egg is then added 10 per cent, of water liy volume of the weight of the eggs. This is mixed by twirling tlie flask or l)y gently stirring wdth a glass rod. Bubbling nuist be avoided. The mixture is then filtereil through I'heese-cloth by gravity and tubed. (See Apparatus, Fig. 40.) The tubes are then slanted and coagulated by heating to 70° C. for two to two and a quarter hours on two successive days (see Fig. 36 for apparatus). No further sterilization is employed. The medium is incubated to test its sterility. Glycerin Egg (Lubenau). — Ten egg's are blown into a flask and 200 c.c. of glycerin broth (5 per cent, glycerin, neutral or slightly alkaline to litmus) added. The further preparation is the same as the preceding. If the media requires it a drop or two of water can be added after coagulation to supply the necessary moisture. To conserve the moisture the plugs should be paraffined or cut ofi^ and burned and a charred cork used to seal the tube. Addition of Seram or Other Enriching Substances. — Serum, blood, albuminous body fluids, or tissue extracts containing uncoagulated albumins may be added to media. Usually 1 part to 2 or 3 parts of medium are used. If agar is used it must be cooled to 50° C. after melting before mixing. If slants are to be made they should be allowed to set thoroughly preferably over night befoi'e raising them or they may slip down. For certain work it may be necessary to use a 2 ])er cent, agar to get sufficient stiffness so that the surface is not easily broken; this holds especially for media for plating where surface inoculation is done. Media thus prepared is spoken of as blood-agar or broth, ascitic agar or brotli, serum-agar or broth, etc. Watery extracts of tissues, especially of placenta, are used in the same manner; 500 grams of tissue to a liter of water are used and exti'acted as for meat infusion, but no heat applied. After preliminary filtration through paper or sand, the fluid is sterilizecl by filtration through a filter candle. Ahdrose may be used in media in 1 per cent, amounts to aid growth. Serum Media. — For special purposes serum or other albuminous fluids, such as ascitic fluid, are used. They may be used as such or diluted with saline or by the addition of nutrient media. For these special purposes, sterilization by heat or even filtration should be avoided. Serum-water Media. — If serum is dialyzed to remove the salts it can be heated to 100° C. without coagulation. Serum diluted with two to three times its volume of distilled water can be sterilized in the Arnold sterilizer. This is so-called "serum-water medium" (Hiss) to which 1 per cent, of sugar is added for fermentation tests. Acid production is shown by the change in the litmus added and by coagulation of the serum. Gas production is shown by the bubbles in the coagulum. Inulin serum-water medium is the most commonly used preparation of this type. (See under Carbohydrates.) A similar medium is prepared as follows : Inulin (or other carbohydrate) 4 grama Peptone 4 grams Water 200 c.c. Litmus 5 per cent, solution. Sufficient to give a deep blue color. Dissolve and tube (2 to 3 c.c. to a tube) and sterilize. The method depending on the sugar used and precautions already given. After cooling add to each tube an equal quantity of sterile ascitic fluid or serum. Blood-media. — Blood for media is most easily obtained from larger animals by introducing a trocar into the jugular. From smaUer animals anesthesia 102 PRINCIPLES OF MICROBIOLOGY is used and the carotid is dissected out for the introduction of the trocar or cannula. Small quantities of blood may he obtained liy heart puncture with a syringe. The steps nmst be aseptic as the lilood cannot l)e sterilized. Tiie fluid lilood ma>' be mixed witli the medium at once or it is defi-il)rinated oi- mixed witli citrate solution when it i.s to be stored. One part of a 10 per cent, sodium citrate solution is diluted to nine parts of blood. The blood may be mixed with solid or fluid media as already' described. For streptococci and its allies a good medium is obtained by placing a drop of blood on the surface of an agar slant. For certain purposes the agar is used in Petri dishes with a drop of blood on the surface. The blood is streaked out during inoculation. Hemoglobin Media. — Certain bacteria, like the influenza bacillus, require hemoglobin for their gro^vth. This ra&y be supplied hy the addition of whole blood or by dilutions of the blood which has been laked in distilled water. A final dilution of 1 to 500 still gives a good growth. For agar slants the blood should be mixed with the agar at 80° to 90° C. For plate work the mixing should be below 55° C. Pancreatin Broth. — Hottinger makes the statement that the greater part of the nutritive elements of meat are lost in the usual process of making broth for laboratory use. To avoid this a process of slow digestion is recommended, with the addition of pancreatin, and it is claimed that in this way a much more favorable medium can be obtained for the growth of bacteria, with so much peptone as a result of the meat digestion that no addition of commercial peptone is required. Sodium chloride is also ondtted. As carried out in this laboratory the slightly modified Hottinger process is as follows: Meat, 750 gm. Water, 1500 c.c. Meat, carefully freed from fat and fascia, cut in finger-thick pieces. Water heated to boiling. Meat dropped in piece by piece, with constant stirring. Boil up stronglj^ and take from fire. Take out meat and put through cutting machine. Cool water to 370° C. Add sodium carbonate 1^ gm. per liter to water. Put chopped meat in flasks (2-liter Erlenmeyer), 550 gm. per flask. Flasks filled with water up to narrow necks. Add pancreatin siccum, 3 gm. per flask; chloroform, 10 c.c. per flask; toluol, 10 c.c. per flask. Cork tightly and shake weU. Incubate at 37° C. overnight. Shake next day and add more pancreatin unless the fluid shows a yellow color and particles of meat look smaller, sho-ndng that digestion is taking place. The process of digestion should continue for forty-five days at room temjiera- ture or for two to tliree days in incubator, shaking weU each day. At the end of this time the meat has become a finely divided mass, giving off a very offensive odor. According to Hottinger the medium may be stored in the ice-box at this point without again heating; after testing with litmus paper and acidifying with a slight amount of dilute HCl, if found to be alkaline. In our experience this plan has not worked well, and we have found it best to proceed at once as follows : Decant liquid through cheese-cloth. Add an equal amount of water to the residue in flask. Shake weU. Allow meat to settle and again decant. Finally place meat on cheese-cloth and allow to drain. Boil tlie filtrate for a few minutes, then filter thi-ougli absorbent cotton and paper imtil clear. CULTURE MEDIA 103 Store in flasks as pancreatin stock broth after aiitoclaving at 115 pounds' pressure for one-half hour. ITse diluted to desired strength. Diluted one-half (one part stock hroth, one jiart water) this broth gave excel- lent results with ty])hoid, paratyphoid, coh staphylococcus, dysentery Flcxner and dysentery Mt. Desert. Diphtheria did not grow well in this medium. After successive transfers in different varieties of broth, cultures of typhoid, paratyphoid, and staphylococcus plated out showed a generally higher percent- age of colonies from the pancreatin broth one-,half strength' than from the regular veal broth. Pancreatin Agar. — Add sufficient water to pancreating stock broth to malce the required dilution. Add agar 1.5 per cent. Heat in autoclave at 15 pounds' pressure for one hour. Cool to 55° C. and add eggs well beaten. Heat in autoclave one-Iialf hour. Filter through cotton in hot-water funnel until clear. Titrate at a temperature as near as possible to 40° C. not above 50° C. Adjust to desired reaction and store in tubes or flasks after again autoclaving. Agar made with pancreatin broth of the 1 to 1 dilution used for successive slant agar cultures with typhoid, paratyphoid, coli, dysentery Flexner, dysentery Mt. Desert, staphjdococcus, streptococcus, pneumococcus, and meningococcus gave equaUy good results as ordinary veal agar, but not as good growth with diphtheria and gonococcus. Dilutions of Media in Milk Examinations. — Berry has found that a steadily increasing ratio of colonies is obtained in miUc plates made from Ijoth pancreatin and plain agars up to a broth dilution of 1 to 15. The size of the colonies varies, being on the whole slightly smaller mth the higher degree of dilution, but the difference is not marked. Beyond 1 to 15 dilution of broth up to 1 to 30, nearly as many colonies are obtained, but these are increasingly smaller and less distinct. Used Agar. — Tests have been made mth used agar in order to ascertain whether it would be possible to utilize such agar a second time for culture pm'poses. The tests have been made with veal agar which had been used in Blake bottles for the preparation of various antigens and vaccines. After the surface growth is removed as far as possible, by scraping, or by wash- ing with salt solution, or with distilled water, the agar is melted and the different lots poured together as they come. The whole amount is then made neutral to phenolphthalein, tubed, sterilized, and again planted with various organisms. Except for a loss of transparency which can probably be restored by fresh clearing and filtering, the previous use of the agar seems m no way to affect its value as a culture medium. Typhoid, paratyphoid, coli, staphylococcus, strep- tococcus, pneumococcus, gonococcus, diphtheria, and other varieties of the ordinary laboratory organisms grow as vigorously as on new agar, and in some cases the growth is heavier on the old than on the new medium. Abundant cultures of amebEe are also obtained on used agar. Special Media. — Bordet-Gengou. — Water 1000 c.c, glycerin 40 c.c, potatoes (sliced) 500 grams. Heat in an autoclave at 15 pounds' pressure for a haff- hour. Pour off the liquid. To 500 c.c. of potato extract add salt solution (0.6 per cent.) 1500 c.c, and agar 60 grams. Autoclave to dissolve, filter and tube. When used an equal quantity of defirbrinated blood is added. Use of Fresh Tissue in Media. — Sterile fresh tissue may be added to media. This not onlj^ adds nutrient material but aids the production of anaerobic conditions. Smith used it in fermentation tubes for the growth of anaerobes, Williams uses it on agar for the growth of pure cultures of amebffi, and Noguchi uses it in special media for spirochetes and other feeble-growing organisms. In the latter medium rabbit's kidney is most commonly used. Small pieces are added to fluid media or placed in test-tubes and serum or ascitic agar added. In the case of coagulated serum the tissue is pushed to the iDottom of the tube. 104 PRINCIPLES OF MICROBIOLOGY Synthetic Media. — For certain work it is an advantage to know the exact chemical constitutiun of the medium. Then, too, some bacteria, especially certain species in soil, refuse to grow on the more complex media. Pigment pro- duction is ^'ery easily observed on these media. Ucliinaky's {FrankcVs Modification). — Water 1000 c.c, asparagin 4 grams, ammonium lactate 6 grams, Na2HP04 2 grams, NaCl 5 grams. Modified Formula for Pijocyaneus Used in Disinfection Tests. — Water 1000 c.c, asparagin 6 grams, sodium phosphate (ortho) 2 grams, sodium chloride 5 grams. Dissolve and if necessary render alkaline to litmus by addition of NaOH. Sterilize in small tubes and test for color production with B. pyocyaneus. Ringer's Solution. — Sodium chloride 10 grams, potassium chloride 0.2 gr.im, calcium chloride 0.2 gram, sodium bicarbonate 0.1 gram, glucose 1 gram, water 1000 c.c. For broth add 1 per cent, or 2 per cent, peptone. For agar add 1.5 to 2 per cent, agar and 1 to 2 per cent, peptone. Dissolve, clear, and filter. Use of Aniline Dyes in Media. — Various basic aniline dyes show a differential restraming action on the growth of bacteria. Gentian violet and allied dj^es when present in a dilution of 1 to 100,000, preferably in agar, inhibits the growth of the Gram-positive group of bacteria but has no effect on the gro\\'th of the Gram-negative bacteria. This is a general rule. Exceptions are found and variations are noted with different dyes. Gentian violet may be employed to prevent growth of Gram-positive germs or for isolation of Gram-negative germs from contaminated material where the contamination is Gram-positive. An example of this is the use of crystal ^dolet in Conradi medium. Certain dyes have a differential action on closely allied bacteria inhibiting completely or partialljf the growth of one, but allowing another to grow freel}'. An example of this is the action of brilliant green on the typhoid, paratyphoid, and colon bacilli. The colon tj'pes are completely or nearly completely restrained, tj'phoid less so, and paratyphoid least of all. This is applied in isolation of typhoid or paratyphoid from stools. The use of dyes as indicators has been already noted. Special Media for Typhoid, Paratyphoid, Dysentery, and Colon. — Numerous media are used. Tlie most commonly employed plating media are Endo and Conradi-Drigalsky. Endo-medium {KendaU's Modification). — The basis is ordinary meat-extract agar, slightly alkaline to litmus. This should be sterilized in bottles in 100 c.c. amounts. When needed 1 gram of lactose is poured into a bottle and the agar melted which dissolves and sterilizes the lactose. To each bottle, after melting, is added 1 c.c. of decolorized fuchsin prepared as follows: To 10 c.c. of a 10 per cent, watery solution of sodium sulphite add 1 c.c. of a saturated alcoholic solution of fuchsin and heat in the Arnold sterilizer for twenty minutes. Plates are then poured and allowed to harden without the covers, and dried in the incubator for thirty minutes, protecting the plates from dust. The medium must be mixed each time when needed and the plates used. The color returns gradually and the plates are then useless. Conradi-Drigalsky Medium. — Water 1000 c.c, agar 20 grams, sodium chloride 5 grams, peptone 20 grams, nutrose 10 grams, beef extract (Liebig's) 4 grams, normal NaOH 50 c.c. Dissolve the ingredients in an autoclave, cool, and clear with eggs. Adjust reaction to a moderate but distinct alkalinity to litmus. To each liter of agar thus prepared are added 130 c.c of Kubel and Thiemann litmus solution, crystal violet (1 to 1000 solution) 10 c.c, and 15 grams of lactose. Heat in an Arnold sterilizer ten minutes to obtain thorough mixing and fill in tubes or bottles and sterilize in Arnold sterilizer. Omit the crj'stal -s-iolet if to be used for dj'senterjr. Russell's Double Sugar Medium. — To ordinary extract agar adjusted neutral to litmus add 1 per cent, of lactose and 0.1 per cent, of glucose and sufficient litmus to give a good color. Tube and slant, leaving a generous "butt" at bot- tf)m of tube for stab inoculation. CULTURE MEDIA 105 Brilliant Green Broth.— -To extract broth neutral to plienol])litlialoin add 1 jier eciit. of shieose and sufficient of a 0.1 per eenf. .solution of Ihc dye to give dilutions of 1 to 500,000 down. The nii.xing- is done immediately before use. Neutnil Red Agar.— To extract agar add 1 i^er cent, of glucose and 1 per cent, of a saturateil aqueous solution of neutral red. Neutral Red Laetose Peptone. — To peptone-water add 1 i)cr cent, of lactose and 1 per cent, of a saturated aqueous solution of neutral red. Tube in fei-men- tation tubes. Liver Broth. — Beef liver 500 grams, peptone 10 grams, dextrose 10 grams, di-potassium phosphate (KoHP04) 1 gram, water 1000 c.c. Tiie liver is boiled for two hours for extraction and strained. Then dissolve the otlier ingredients and adjust the reaction and sterilize. Bile Media. — For Typhoid Blood-cultures. — To ox bile add 10 per cent, glyc- erin and 2 per cent, of peptone. For Water Analysis. To ox bile add 1 per cent, of peptone and 1 per cent, of lactose. Tube in fermentation tube, and sterilize. In place of ox bile a 10 per cent, solution dried fresh ox gall can be used. Special Media for Cholera. — Peptone Solution (see p. 96). Saccharose Peptone Water {Bendick). — To 1000 c.c. peptone solution, neu- tralized to phenolphthalein add 1 gram of anhydrous sodium carbonate. Boil and filter. Add 5 grams of saccharose and 5 c.c. of a saturated solution of phenolphthalein in 50 per cent, alcohol. Tube and sterilize in Arnold sterilizer. Diendonne's Medium. — Mix equal parts of defibrinated beef -blood and normal sodium or potassium hydrate solution and steam in the Arnold sterilizer for a half- hour ; 3 parts of this are added to 7 parts of 3 per cent, agar (neutral to litmus) and poured into Petri dishes (15 c.c. to a 10 cm. dish). Allow to harden uncovered, but protected b}' paper. Place strips of filter paper between the dish and cover to aid in the absorption of the moisture and anuuonia and place in the incubator for twelve to fifteen hours. Nothing will grow on the medium when first made. The plates are good for about ten to foui'teen days. Various modifications of this medium have been suggested. Pilon. — Substitute 12 per cent, sodium carbonate (crystalline) solution for the sochum hydrate. The plates can be used after thirty minutes' drying. Alkaline Egg Medium (Krumwiede). — Make an egg-water mixture, using equal parts of egg and water. Mix equal parts of the egg- water and 12 per cent, sodium carbonate (crj^stalline) and steam in the Arnold sterilizer for twenty minutes. Mix while hot 30 parts to 70 parts of 3 per cent, agar (the meat extract may be omitted) and pour on plates and drj^ for tw^ent}' minutes. If the meat extract is omitted the colonies are small but very characteristic. The colonies are much increased in size when extract is present or if 0.5 per cent, of glucose or saccharose is added. The reaction of the agar need not be corrected. Goldberger claims that the alkalinity is unnecessarily liigh, and makes the egg mixture as given with a 6.5 per cent, solution of anhydrous sochum carbonate and mixes 1 part of the egg with 5 parts of glucose extract agar. Special Media for Toxin Production, etc. — For Diphtheria Toxin {Banzhnf). — Lean veal free from fibrous tissues, minced, 1 pound to the liter of water. Soak overnight and then heat to between 45° and 55° C. for one hour. Then bring to a boil. Strain. To fluid add peptone 2 per cent, and sodium chloride 0.5 per cent. Boil for a half-hour. Adjust reaction to 1.2 per cent, acid to phenol- phtalein. Place about 800 c.c. in 2-liter Erlenmeyer flasks and add 3 grams of calcium carbonate. Sterilize in an autoclave at 5 pounds' pressure for a half-hour on two successive daj'S. For Tetanus Toxin. — (1) One pound of lean veal per liter of water. Soak over- night at 37° C. If meat rises to top it is ready for use; if not, allow to stand another day. Heat to 45° to 55° C. for one hour, then boil. Strain, add peptone 1 per cent., salt 0.5 per cent. Boil for half an hour, adjust reaction to -|-1.6 per cent. Sterilize in globe flasks, medium reaching into neck, in Arnold sterilizer 106 PRINCIPLES OF MICROBIOLOGY Fig. 41 for one hour at a time for two days. Immediately after sterilization fill the neck of the flask with boiling paraffin or albolenc. The Hygienic Lnhoratory {Washington, D. C.) Method for Making Tetanus Toxiii..^Mcdia.—V\\o\ixieA round steak, 500 gi-ams; tap-water, 1000 c.c. Mix. I'lace in it-e-box overnight, strain, and iiress out juice through cheese-clotli until 1000 c.c. are obtained. Boil to coagulate albumins, filter through paper, making up loss of evaporation. Add 1 per cent, peptone (Witte's), 0.5 per cent, sodium chloride, and 1 per cent, anhydrous glucose. Heat untU peptone is in solution; titrate and adjust reaction to 1. Filter through paper and put m one liter flask. Sterilize in Arnold for ninety mmutes. Preliminary Cidtivation.—Fill some 7 x 1 inch test-tubes with glucose broth to within 2 inches of top of tube and sterilize. When stiU warm (about 35° to 40° C.) inoculate and place in thermostat at 37° C. From the first tube inocu- late a' second in twentj^-four hours and so on for five to six days. Inoculation for Toxin. — From the last tube the 1000- c-.c. flask of glucose broth (previously heated in the Arnold for one hour and cooled to 35° or 40° C.) is uioculated and placed in the thermostat at 37° C. . for fifteen days. The culture is now passed thi'ough a Berkefeld filter and 750 grams of ammonium sulphate are added per 1000 c.c. of filtrate. The brownish scum is skinimed off, placed between hard filter papers and the moisture pressed out, dried over sulphuric acid in vacuum for al30ut tlii-ee da}fs, pulverized, and stored in U-shaped tubes with P^Os at 5° C. Broth for Production of Mullein. — Meat uif usion 2000 c.c, potato juice (see under Potato) 2000 c.c. Mix and add peptone SO grams and sodium chloride 20 grams. Boil ten minutes, adjust reaction to -|-1 per cent, to plienolphtalein. Boil, filter, add 4.5 per cent, glycerin. Put about 250 c.c. to flask and sterilize. For ej^e mal- lein concentrate to one-tenth volume. Special Media for Yeasts and Molds. — Most of the pathogenic j^easts and molds can be cultivated on the media already described. Growth is much unproved if glucose or maltose is added. The reaction should, however, be acid or at most slightly alkaline to litmus. For molds, especiaUy those uifecting the skin, an agar containing no meat but 1 to 2 per cent, peptone and 2 per cent, of glucose or maltose and 0.5 per cent, of glycerin witli no change of reaction gives a very favorable medium. For yeasts, beerwort media are especially favorable. Hopped l^eerwort is obtained at a brewery. Autoclave, cool, and filter, and then tube (this avoids subsequent precipitation). For solid media add 10 per cent, gelatin or 1.5 per cent. agar. Various carbohydrates may be added in 2 per cent, amounts. Special Media for Protozoa. — For Blood Flagellcdes (Novy and MaoNeal). — Equal parts of nutrient agar and fresh defibrinated blood (rabbit or rat). The medium is allowed to stiffen slanted, so more water of condensation may gather at bottom of slanted surface. The medium should be planted while fresh with blood or other infected material containing the living flagellates (p. g., trypanosomes, Leishmania) . Medium for Malarial Organisms (Bass and Johns). — 10 c.c. of blood drawn from a malarial patient, and carefully defibrinated. Tliis is placed in smaller test-tubes in 1 c.c. amounts. To each small test-tube before adcUng the blood, 1 per cent, of a 50 per cent, solution of dextrose is added. The red corpuscles settle so tliat 5 cm. layer of serum is left about them. The parasites grow in a i Platinum needle and loop. For most purpo.=ies finer wire is used. CULTIVATION OF MICROORGANISMS 107 thill layer near the top of the cell seilimeiits. Beneath tliis zone tlie parasites die. Bass says leukocytes should be gotton rid of bcx'ause they destroy the parasites, but the Thompsons say tins is unnecessary. Most favorabh^ tem- perature 30° to 40° c;. Medium for Pure CuUures of Aiiidiw {Willkiiiiti). — Fresh sterile brain, h\ei', or kidney is cut in small pieces and [ilaced on the surface of alkaline, slightly- nutrient tissue agar. CULTIVATION OF MICROORGANISMS. jNIIcroorganisms can seldom be identified by their microscopic and staining characteristics alone. By these methods only their individnal form, arrangement, and motility or lack of motility can be studied. To go beyond this we grow the organism in pure culture, that is apart from all other organisms, and determine its cultural characteristics on various media, and in some instances its immunity reactions and its action in the animal body. In cultivating microorganisms it is not only necessary to supply the appropriate foodstuff, but also to have the appropriate conditions as to temperature, moisture, access of oxygen, etc. When we make cultures of any material we are apt to find that instead of only one variety of microorganism, there are a number present. If we grow the material in fluid media we find that as the different varieties develop they spread through the medium and become hopelessly mixed; furthermore, the more vigorous varieties outgrow the feebler ones, which are thus lost. If, however, the microorganisms in the material be scattered through or on a solid medium, each individual will develop at the spot where it lights and produce in its growth a colony which is visible to the naked eye or can be seen with a low-power lens. This is an example of how pure cultures are obtained without which the study of microorganisms would be impossible. Methods of Isolating Pure Cultures. — Plating Methods. — Two plating methods are employed, pom- plates or streak plates. Agar or gelatin media are used. The medium is first rendered fluid: gelatin, by placing the tubes in warm water (35° to 40° C); agar, by immersing the tubes in boilmg water until fluid and then coohng the tubes to between 40° and 42° C. Agar only melts completely when heated to the boiling-point of water or thereabouts and hardens as the temperature falls below 40° C. The coohng of the agar is neces- sary, otherwise the organisms would be injured by the heat. If enriching fluids are to be used they are added to the agar when between 45° and 50° C. The material from which we wish to obtain pure cultures is then inoculated into the fluidified media. The method of inoculation is in general as follows: A loop of the material is carried into a tube of the medium and thoroughly mixed with the medium by tilting and rolling the tube. Agitation sufficient to produce bubbles must be avoided. From this tube three loops are carried over to a second tube which is mixed in the same mamier. From the second tube five loops are carried to a third tube and mixed. The lips of the tubes are flamed and the contents of each poured into a Petri dish, which is if necessary tilted to spread the agar evenly over the bottom of the dish. In pouring, the lid of the dish is raised just sufficiently to pour the contents of the tube into the dish, the lid actiiig as a protection against falling dust. In diluting the material with culture media, it is our object to separate the organisms so that when colonies develop from them they will be well separated, that is, discrete. If they were not well sepa- rated, we would touch several colonies in attempting to transfer the growth of one ins PRINCIPLES OF MICROBIOLOGY ami thus fail to obtain pure cultures. As we have no wa.v of telling what dilution will give us well-sepai-ateil colonies a series of dilutions is necessary. The method will have to vary with the probable number of organisms in the material. If they arc in great mimbers further dilutions of the material will be necessary, as the first tulie will gi\'e overcrowded plates. A preliminary dilution or two may be made in saline solution or broth. Where the material contains very few organisms more material must be inoculated into the first tube. The dilu- tions may be made with a sterile pipette instead of a loop. The tip of the pipette is touched to the material and carried over to the first tube of medium, mi.xing being done by sucking the medium up and down in the pipette; about to c.c. of the contents of this tube is carried to a second tube, to c.c. of this to a third, etc. Streak Plates. — In this method the medium (agar) is poured into Petri dishes and allowed to harden. The material is then streaked over the surface of the medium cither with a platinum loop or spatulum or a bent-glass rod. Two or more jilates are inoculated in succession without further sterilization of the loop or spatulum. This dilutes the material so that discrete colonies are obtained. A diflSculty that is apt to arise in the use of streak plates, and to a less degree with pour plates, is collection of water in condensation, which finding its way to the surface of the plate will cause a spreading of the growth over the whole surface. This can be avoided to a large extent by cooling the agar before pouring on the plates. Special covers made of porous earthenware can be obtained, which will absorb the moisture. Incubating the plates upside down is a good precau- tion. Motile bacteria are most likel.v to spread over the surface of media. The special media used for the isolation of fecal bacteria must, because of the great numbers of motile bacteria in the stool, be comparatively dry. As most of these media restrain the ordinary contaminating bacteria from growing they may be allowed to harden and dry in the Petri dishes with the covers left off, and the drying continued by placing them in the incubator partly open. Fishing Colonies. — This is the term used for the process of transferring the microorganisms from the colony to a tube of medium which after growth has taken place is our pure culture. This is usually done with a straight platinum wire, which is less likely to touch any colony other than the one we wish to fish. Fishing is done either by selecting the colonj'' with the unaided eye or by the aid of a microscope. The advantage gained by the use of the microscope is that we can be sure that the colony to be fished has no microscopic colonies in the immediate neighborhood likely to he touched by the platinum wire. I'urthermore, if the fishing is done under the microscope the fact that one and only one colony has been touched is visually proved. Opaque media are fished by eye. In fishing under the microscope the colony to be fished is centred under a No. 2 or AA objective and the wire introduced" between the objective and the agar until the tip of the wire touches the colony. The progress of the wire is watched through the microscope to be sure that nothing is touched but the colony, and to observe by the broken appearance of the colony that it has been touched. The sense of touch is relied on to tell whether the objective is touched. In fishing by eye the plate is held against the light and the colony touched with the wire. In either case the organism is transferred to a tube of medium— usually an agar slant— by rubbing the tip of the wire over the surface of the agar. Possible Sources of Error in Plating Methods. — Theoretically each colony is the progeny of one single organism, or in the case of organisms that adhere to form pairs, chains, etc., the progeny of such an aggregate of one variety of organism. Practically it not infreciuently happens that dissimilar varieties "cling together and a colony develops containing both. A second plating from such a colony is usually sufficient to yield pure cultures. In exceptional instances there is the greatest difficulty in sei)arating such "dingers" and it is sometimes impossible. Special Methods of Isolation.— Di/w/ion Method.~lt is possible by gradual dilution of material to reach a point where some of the loops of the material may only contain one organism which when carried to tuljes of medi\un will CULTIVATION OF MICROORGANISMS 109 .vickl pure cultures. This method was eiuiiloyed before the introduction of solid metUa. The uncertainty of the method is evident. For certain purposes modified dilutions are still emjiloyed, stai'tinR usually with cultures obtained from colonies on plates, the method bein,a; eniployetl not so much to obtain pure cultures as to obtain cultures known to lie tlie progeny of one cell. Hanson's Method for Yenstx. — Cover-slips are given a thin coating of paraffin, through wliich lines are drami dividing the cover into small squares and each numbered. The marking is done with a, needle. The covers are now exposed to hydroflouric acid to etch the markings into the glass. They are then cleaned and sterilized. Dilute the yeast-containing material with fluidified gelatin until one loop averages one yeast cell as determinetl by microscopic examination. The gelatin is then spread over the surface of the sterile cover-slip, hardened, and inverted over a Bottcher moist chamber, sealing with vaselin. This is placed under the microscope and the ])osition of the single cells determined, making a sketch for later reference. Incubate at room temperature and when colonies have developed fish those developing from single cells. The advantage of this method over plating is that on plates a proportion of the colonies will develop from adhering cells. The pm-ity of yeasts in industries is of enormous financial importance. India-ink Method (Burri). — The use of india-ink for microscopic examination has already been described. By making successive dilutions of small amounts of culture in drops of ink it is possible to make small spots of ink containing only one organism. This is determined microscopically. This is the starting- point for the pure culture from one cell. The reader is referred to the article by Burri for the technical details. The method has only a vei'y limited application (see page 74). Capillaiij Method (Bnrlier and others). — Reference is made to the original articles for the details of this method for isolating one cell. Preliminary Enrichment Methods. — Very often it is impossible to isolate certain organisms from material because they are too few in number. With certain organisms it is possible to inoculate the material in special media which will allow free multiplication of this organism, but be unfavorable for the others contained in the material. Examples of this are the use of LofHer's medium for mixed cultures from the throat to find diphtheria, or the peptone method for the enrichment of cholera and other vibrios. Another method is the use of ascitic broth for the isolation of diphtheria from mixed material. The last two methods take advantage of the .surface growth of the Ijacteria. The same end is attained by the use of the following factors: blood-media for hemo- globinophilic bacilli; increase of the alkalinity, as in selective media for vibrios; increased acidity, as with tartaric acid, for yeasts; dyestuffs, gentian violet, to inhibit Gram-positive bacteria; brilliant green to inhibit the colon types in mixed cultures from stools; use of heat, spore-containing mixed cultures being heated to 80° C, to kill the non-spore-bearing types; motility, the motile organism spreading from the point of inoculation of the mixed material, as with amebse and spirochete-containing material; filtration through filters, spiro- chetes passing filters, most l)acteria being held back (see Filtrablc Viruses); germicidal agents; antiformin for separation of acid-fasts from other bacteria; finally, the use of animals (see p. 122). Special Methods for Amierobes. — (See Anaerobic Methods.) Applications of the Plating Methods. — Study of Colony. — The appearance of the colony is one of the points used in identifying bacteria, or the colony appear- ance being known the isolation of a certain organism is greatly simplified. In plates showing two or more types of colonies it is an aid in determining the rela- tive propoi'tion of the subsequently identified varieties in the material examined. In examining colonics both the macroscopic and microscopic characteristics are determined. The age at which a colony will be most characteristic dejiends on the ra]iidity of growth of the organism. Daih' oliservations are most suited for full information. As a rule young colonies arc most characteristic, although no PRINCIPLES OF MICROBIOLOGY such characteristics as pigment production and gelatin liquefaction maj' only appear on longer incubation. With the naked eye the following points should be observed : moist or dry transparent, translucent or opaque, edge sharply defined or indefinite, regular or irregular outline, fringe-like margin or thread- like outgrowths, color, flat, raised, or umljUicated, etc. On microscopic exami- nation the presence or absence of granules, whether fine or coarse, their arrange- FiG. 42 Fig. 43 Irregular fringed colony (B. malignant edema). (From KoUe and Wassermann.) Colonies of typhoid and colon bacilli in rather soft gelatin. mcnt and distribution, should be observed, as well as the finer details of the characteristics mentioned under the naked-eye examination. The observations are usuallj' made with a low-power objective, No. 2 or No. 3. The fine details may be obser^-ed with a higher lens, and the arrangement of the bacteria espe- ciall.y at the edge of the colony can be observed. The accompanying figures illustrate the colony characteristics mentioned. Fig. 44 Fig. 45 Colony of colon bacilli grown in soft gelatin. One large irregular colony of colon and two smaller colonies of typhoid baeill in soft gelatin. (Figs. 41 to 43 from photo- graphs by Dunham.) De-term inalioii. of ihe Niimher of Bacteria.— If a measiu'ed amount of fluid is mixed with a plating medium we shoukV theoretically be alile to determine tlie numl)er of Iiactcria l)y counting the numlicr of colonies that develop. This woulfl he true if each bacterium was completely separate, if each was alile to multiply, if the medium was satisfactory for the development of each variety contamed in the material, and if the oxygen access, moisture, and temperature CULTIVATION OF MICROORGANISMS 111 of inc'uliatiun were favorable for the growth for all tlu; varieties. This is an impossibility. Most iiearlj' accurate results are obtaiueil where only one or at Fig. 46 Fig. 47 Fig. 46. — Moist raised colonies with no visible strueturo, locjking like a drop of wa(er Fig. 47. — Deep colonies, usually cither light brown, gray, or yellow in color, opaque with little marking. (Figs. 46-5.3 from Lehman and Neumann.) Fig. 48 Fig. 49 Fig. 50 "^"^ M p- " '"" Fig. 48. — The colony very finely granular. Fig. 49. — Colonies opaque in centre with lighter borders. The margin is coarsely granular, or has twisted threads. Fig. 50. — Colony in gelatin. The centre is coarsely granular in partly fluid gelatin. The borders are formed of wavy bands of threads. Fig. 51 Fig Fig, 53 Fig. 51. — Colonies circular in form, compo.sed of radiating threads. Fir. 52. — Colonies with opaque centres, with a tliin border fringe. Fig. 53. — Colony showing a network of threads which is thicker in centre most several varieties are pre.sent and the conditions for their growth main- tained. Although the method gives an incorrect result when mixed material 112 PRINCIPLES OF MICROBIOLOGY is used the enumeration of the colonies that develop is of a great deal of practical value, as in the examination of milk and water. If the methods employed are always the same, the comparison of the results gives us valuable information as to the bacterial content. The method employed is in general as follows: To 9 c.c. of water is added 1 c.c. of the material to be examined; this is thoroughly shaken to separate the organisms; 1 c.c. of this is then added to 9 c.c. of water thoroughly shaken, and 1 c.c. of this added to 9 c.c. of water and so on. This gives a dilution of 1 in 10, 1 in 100, 1 in 1000, etc. How far to dilute the material must be determined from the character and probable bacterial content. One c.c. of each of these dilutions is placed in a Petri dish and the melted plating medium poured into the dish, the two being mixed Ij}' tilting the dish. Mixing can be done by adding the dilutions to the tubes of jslating medium and pouring the content into the dishes. Though in this way all the medium cannot be poured out of the tube and some bacteria are left behind, it is easier to obtain even distriljution of the colonies in this way and for comparative work the method gives reliable results. The counts bj^ this method average about 10 per cent, lower than where mixing is done in the dish. Fig. .54 Photograph of a large number ot colonies de-\'eloping in a layer of gelatin contained in a small Petri dish. Some colonies are only pinpoint in size; some as large as the end of a pencil. The colonies here appear in their actual size. In place of dilutions, made by pipettes, loops or rings on a platinum needle can be employed which hold a known quantity (small) of the material to be examined. To obtain a satisfactory count the colonies should Ijc about 100 to 200 to a plate. Lower numbers than this are too few to give a fair average, higher numbers are difficult to count because the colonies have not sufficient space for full development, some of the colonies become confluent, and some of the feebler Ijacteria are checked in their development Ijy the crowding and do not develop colonies. The plate that shows about 100 colonies is eho.sen, therefore, for count- ing. Where no one plate is completely satisfactory in numljer and even distrilni- tioii of the colonies, two of the nearest satisfactory plates are counted and the results averaged. Wherever possible all the colonies on a plate should Ijc counted. WIku'c the colonies are crowded it is necessary to divide the jilate into sections to facilitate counting. This is best done by jilaciiig the Petri dish on a Wolff- hiigel counting plate, which is a glass plate ruled in square centimeters some of tlic squares being still further divided into 9 small squares. A row of squares CULTIVATION OF MICROORGANISMS 113 is counted ami then a row at right angles to the first, to obtain a fair average. The niuuber oi colonies is di^'ided by the number of squares counted. If the usual 10 c.c. Petri dish is used the nunilier per square centimeter is multiplied by 63, the number of square centimeters on the dish to give the total number. This is multiplied by the dilution used in this dish which gives the colonies devel- oping from 1 c.c. of the material used for plating. If dishes of another size are used the area in square centimeters is determined by multiplying the diameter by 3.1416. Fig. Fiu. 56 A\'('U-distributed colonies in agar in portion of Petri dish. Wolffhilgel's apparatus for counting colonies. Methods of Inoculating Culture Media. — The inoculation of plating media has been described under the methods of isolating pure cultures. Platinum wire is usualh' employed for transferring the growth from one medium to another. The wire is either attached to the end of a glass rod or to special aluminum holders which are made for this purpose. Straight wire or wire with the ends bent into a loop is used. The wire is sterilized bj^ heating it in the flame of a Bunsen burner until red-hot. Care must be taken that the wire is cool before it is used. The straight wire is used for fishing colonies or where tubed solid media is to be inoculated by long puncture through the media (stab culture). The loop is used for ordinary transfers. In transferring from solid media the loop is drawn upward over the medium filling the loop with the growth; this is rubbed over the surface of solid media or in the case of fluid media against the glass at upper le^'el of the fluid. In transferring fluid cultures a loop of the fluid is used. Where the growth on fluid media is in the form of a pellicle it may be necessary to inoculate this pellicle so that it floats or growth will not take place. In trans- ferring the tubes or other containers it must not be kept open any longer than necessary or contamination is likely to occur. In transferring from and to tubed medium the tubes are held by placing them against the palm and fin- gers of the left hand, grasping the tubes by the butt between the thumb and the palm, low enough so that the contents of the tube are not covered by the thumb. The plugs, if dust^', are passed quickly through the flame and if necessary the resulting flame blown out. The right hand holding the sterilized platinum wire is used to remove the cotton plugs, one being grasped between the small finger and the other between the fourth and fifth finger. After drawing out the plugs the necks of the tubes are flamed and the growtli then transferred and the plugs replaced; the platinum wire is then sterilized. Apparatus and Methods of Incubation. — The selection of the temperature for incubation depends on the organism to be grown and the medium employed. Although the optimum temperature for growth may varjr between 5° and 50° C. or more, the great majority of organisms will grow at either 22° or 37° C. These are the temperatures usually employed. For general purposes the former 8 114 PRINCIPLES OF MICROBIOLOGY is obtained by inculcation at room temperature. Where a moi'e exact and uni- form temperature is necessary an inculcator must be employed. This is a double- walled oven, the space between the walls being filled with water; heat is supplied by gas or electrieitj' and cooling by the addition of cold water. The latter is only necessary where the outside temperature rises above 22° C. The heating or the addition of water is controlled bj' an automatic regulator. To maintain a temperature of 37° C. a similar incubator is employed, the heating, however, being continuous, an automatic regulator controls the temperature. The Inculoator. heat-controllmg de^dccs are of varied construction, the expansion of a fluid or the change m shape of a bar of two unequally expanding metals is used to make and break the electric contact or open and close the gas vent. In the former mercury is used alone or at the jwint of control. Where a large incubating- room IS required a room can be heated with a large gas stove, a funnel being placed oyer the stove and connected with this a radiator-like arrangement of four- to six-mch pipes ending in a chimncy-likc outlet for the products of combus- tion. The rachator distributes the heat more evenly through the room. In CULTIVATION OF MICROORGANISMS 115 spite of this there will be a \'ariatiun in temperature in different parts of the room, the shelves near the radiator or nearest the ceiling Ijeing the warmer. This is an advantage rather than a disadA-antage, as a range of about .5° is available for special purjioses. In using gas, all connections arc best made of metal; flexible metal tubing is available. Snudl burners are jweferably of the safety- type, which, if extinguished, turn off the gas autcjmatically. In the case of gas stoves there should be an inilependcnt pilot flame not influenced by the regulator, which will relight the stove if turned off. Fig. 58 Fig. 59 Safety-burner. Heat regulator. (Dunham.) Heat regulator. (Rou.\.) Bimetallic. Oxygen Requirement Methods. — Where aerobic conditions are required the access air to the growth is all that is required. For anaerobic conditions the oxygen must be removed. This is done (1) by exhaustion of the air, (2) by chem- ical absorjition of the oxygen, (3) by displacement of the an as by hjalrogen, or (4) l33' growing in the depths of solid media, or in fluid media under a layer of alboleneto prevent the rcab.sorption of air driven off during sterilization; (.5) or, lastl.y, by a combination of these methods. The addition of fresh animal tissues to media for the enhancement of the growth of anaerobic organisms has been mentioned under Media. Occasionally reducing sul^stances are added directly to the medium, or the organism is grown in symbiosis with an aerobic bacillus to absorb the oxygen. A group of organisms spoken of as " micro-aerophilic " will only grow when the oxygen is partly removed. This is done by exhaustion of the air to measured degree, by growing the organism in a sealed jar containing also a stated niunber of cultint's of an organism that absorbs oxygen in its growth, as B. suhtilis, or by mixing the material with melted agar in tubes and allowing the agar to set, the development taking jjlace at the depth that has the favorable oxygen content. In the second method the optimum number of square centimeters of B. siibtiUs culture per liter must be known. IIG PRINCIPLES OF MICROBIOLOGY Fig. 62 Anaerobic Methods. — For exhaustion a pump is required; the filter pumps for attacliment to the faucet are the most convenient where water under pressure is available. Any type of pump, however, may be employed. The extent exhausted can be measured by a manometer. Any t.ype of jar with a tightly fitting cover and an opening with a stop-cock and of appro- priate dimensions for purpose can be used. The Novy jar is a satisfactory type. Museum jars with a side opening are very satisfactory substitutes. The jar must be absolutely tight or the vacuum will not be held. Absorption can be added to exhaus- tion by placing pyrogallic acid and a stick of sodium hydrate in the bottom of the jar. After exhaustion is completed a small amount of water is allowed to be sucked back into the jar. This dissolves the sodium hydrate and the resulting reaction of the sodium hydrate with the pjTogallic acid is accompanied by absorption of the oxj^gen from the air remaining in the jar. Hydrogen may be used to replace the exhausted air. After exhaustion, hydrogen from a Kipp apparatus is sucked back into the jar diluting the remaining air; the jar is again exhausted and then filled with hydro- gen, again exhausted and filled. Only traces of air will remain, and if aljsolute anaerol)ia- sis is required ]iyrogallic acid ma}' be used to absorb this last trace as above, allowing the water to be sucked back before the negative pressure is comjjletely relieved by the hydrogen. In relieving the negative pressure it may be necessary to use a pneumatic trough to collect the hydrogen, as the generation of hydrogen will be too Fig. 61 Novy jai fui imc lohic (ulturcs Buchner's anaerobic tube. The fluid consists of pyrogallic acid dis- .solvcd in 10 per cent. NaOH solution. By Wilson's method the tubes are charged with pieces of caustic potash covered with pyrogallic acid. .slow and air will Ije drawn through the Kijip apparatus. It is advisable not to relieve the negative pressure completely, as this heljw to hold the lid tightly 111 place, and furthermore the subsequent expansion when the jar is CULTIVATION OF MICROdRGANISMS 117 ineubatod will tend to loosen the lid and allow inward diffusion of air. When the pyrogalUc method is used the tubes or plates must be raised above the level of the Huid.^ Of the methods given the seeond and third are the most satis- factory. Tiio absorption method may lie used alone, either usinK a jar to hold the tubes or plates, or indi^-idual |)lates oi' lubes may be handled as in the following: Biirliiicr'.'i Mdhiid. — The eulture tube is jilaeed in a larger tulie, at tlie bottom of which is placed the pyrogallic acid, on wliieh some sodium hyilrate is jjoured and the tulie quickly closed with a ruliber stopjier, or the NaOH may be added dr\' ami water addetl. \ynght's Method. — The tubes are plugged with absorbent cotton; after inocu- lation of the medium the plug is pushed into the tube, pyrogallic placed on the stopper, sodium hydrate solution added, and the tube quickly corked. Zinsser's Method for Plates. — The dishes employed must lie deeper than the ordinary Petri dishes. The agar is poured in the smaller dish in the ordinary manner, and as soon as hard inverted over moistened filter paper to protect it from contamination. In the larger dish is placed the pyrogallic acid and the smaller dish placed in it. One side of the smaller dish is raised and the hydrate solution poured in the space between the two dishes and the inner dish dropped back. As rapidly as possible albolene is run in the space between the dishes from a pipette previously filled so that no time is lost. The methods for e.rclii^'iion of oxygen are simple and can be, in most instances, substituted for the more cumbersome methods described. Plating may be done with the ordinary Petri dishes as follows: The bottom part of the dish, open side up, is placed in the co^-er and sterilized in this shape and protected from dust until used. The plating medium is inoculated as though pour plates were to be made in the ordinary way. The inoculated medium is then poured into the covers and the bottom of the dish laid on the fluid medium, tilting it so that the air can escape as it falls into place. With a little practice this can be done so that no bubbles are present. After the medium has set the edge is painted over with melted paraffin to prevent drying and contamination. If any of the medium flows over the edge it is wiped away by carbolized cotton (see Fig. 63). After incubation the parts of the dish are carefully pulled apart Fig. 03 r Bottom of dish. Agar. -.- Cover of dish. Anaerobic plating method. and the colonies may be fished. As a rule the agar remains in the cover, if it should adhere to the other part of the dish this can be laid in the co\'er to allow of fishing under the microscope. If gas-producing bacteria are present fishing should be done as soon as sufficient growth has taken place or the gas will disrupt the medium and cause the growth to spread over the plate. If an actively motile organism is present the method may fail because of spreading between the medium and the glass. Another method for the isolation of pure cultures is the shake culture. The material is diluted with a plating medium in the usual way, although more medium is used in the tubes. Instead of pouring the medium into plates the medium is allowed to solidify in the tubes. The colony desired can be obtained by scratching the tube with a file at the level of the colony, then wet the tube with alcohol, and burn this off and press on the scratch with a very hot jjiece of metal or glass rod. The tube will crack and the column of agar can lie cut through to allow the separation of the two parts of the tube and the colony fished. This method has a limited application, but is especially useful where the organism 118 PRINCIPLES OF MICROBIOLOGY to be isolated is a partial anaerobe and grows at a certain level in the agar. Pure cultures of these organisms niaj' not be obtained at tlie fii'st attempt, but the level of thickest gro\v1h may then ))C used, the first culture serving in tliis way as an enrichment. For the cultivation of jiuro cultures the use of stall cultures in agar or gelatin may be used. The most satisfactory meilium for this i)(n-pose is a semisolid agar. Because of the softness of tlie medium the material for subinoculation is easily obtained; the stab inoculation closes better than if made in stiffer medium, and the medium does not dry out and split so readily. For subinoculation a platinum -vvire bent like a corkscrew is a help in obtaining the material from the depths of the media. With spirochetes it is much easier to obtain the growth with the capillary pipette from aloout the tissue at the liottoni of the tube than where the usual 1| per cent, is employed. The use of the capillar}' pipette for the transfer of cultures is open to much wider appli- cation witli semisolid media, because if the medium has a satisfactory density the ordinary rubber teat exerts sufficient suction to draw u]i tlie medium and a syringe is not needed. The exclusion of oxygen for fluid medium is most simpl)' carried out by steriliz- ing the medium under a litjer of albolene. This can be done either in tubes or flasks, preferably the globe flasks. In the latter the medium is filled to the neck and albolene added, leaving room for expansion, after sterilization and con- traction of the medium more sterile albolene is aclded if necessary. Occasionally anaerobes are grown in mixed culture with an aerobic organism to absorb the oxj'gen. One of the best organisms for this purpose is the hay bacillus. As a general rule the addition of a fermentable carljohydrate to the medium gives much better growi;h of anaerobic organisms. The Study of Pure Cultures. — The study of pure cultures is the basis for classification and identification. The characteristics studied are: the morphology, staining reactions, and motility when grown on or in both solid and fliuid media (see Microscopic Study) ; the cultural charac- teristics when grown on various media, its food, oxygen, and temperature reciuirements, its reaction to the various immunity tests (see chapters on Immunity), and the action of the organism or its products on experi- mental animals (see Use of Animals). The following is a short summary of the more important cultural reactions and the methods of eliciting them. Cultural Characteristics. — Colony Morphology. — (See Application of Plating Methods.) Growth on Agar and Other Solid Media (Surface Growth) .—Moist or dry, flat or raised, flaky or easily emulsified, adherent to medium or not, mucoicl or slimy, smooth, irregular, or threaded margin, color or pigment production (see below), extension into medium, etc. Growth in Solid Media. — In stab culture, depth to which growi^h extends, character of gro\vth around the surface puncture, the spreading of the growth into the medium (an index of motility best shown in semisolid media), thread- like extensions from the line of puncture, in gelatin, liquefaction, non-lique- faction, rate, area and character if present. In sliake cultures, the character of the colonies. Fluid Media. — Pellicle, tenacious or easily broken, spreatliug up the side of the tube or not, thick or thin, clear or cloudy, and degree of clouding, sedi- ment or deposit on sides of tube, granular, flaky, gelatinous, nuicoid, or stringy, stalactite formation, more densely clouded at one level than another. CULTIVATION OF MICROORGANISM!? 119 Food Requirements. — Gi-owth on simple or complex media, albumins such as serum required, blootl or lienuifiloliin necessary, caii)olij'dra(e needed for full development (example glycerin for tubercle bacillus). Oxygen. Requircinenl^. — Growth only in free oxygen obligate tu'robe; growth only where oxygen is absent (tlepths of media, etc.), obligate anaerobe; growth under both conditions, facultative anaerobe (an available carbohydrate is neces- sary to elicit the facultati\-e character); growth with a definite but lessened amount of oxygen (development only in one level of solid media), micro-aero- philic. This last characteristic may be lost after cultivation for some time on artificial culture media. Optimum Temperature. — Not often used, but may furnish absolute evidence of tlie dissimilarity of two organisms. Not only the optimum but also the range, especially lower, should be determined. Pigment Production. — The color and shade, the optimum medium and tem- perature for its production, limited to the area of growth or diffused through the medium; its solubility in extracting agents, as alcohol, ether, chloroform, etc.; more than one pigment present, as showai bj' use of different extractives. In the case of the coccaceaj, especially the presence of pigment and its color and shade majr be determined with ordmary agar; the gi'owtli is taken up with a platinum loop and spread on a piece of white glazed paper. Although the presence of pigment may be completely obscured on the agar, its presence is immediately evident on the paper. Ferment Action (Cultural Evidence of). — The most important culturally are those causing fermentation of carbohydrates. Less important are proteo- lytic, diastatic, inverting, and rennin-like ferments. Fig. 64 Stab cultures of three cholera spn-illa in gelatm. showing ni upper portion of growth considerable liquefaction of nutrient gelatin. Fermentation of Carbohydrates. — The sugars selected will depend on the organism to be identified as well as the medium selected as a basis for the addi- tion of the sugar. The preparation and the use of indicators or titration to determine the production of acid and the use of fermentation tubes to demon- strate gas production have been described. The amount of gas is expressed in terms of percentage, thus if the closed arm is haU'-full of gas, 50 per cent., etc. To determine roughly proportion of carbon-dioxide and of hydrogen, mark on tube quantity of gas produced, then fill the bulb with sodium hydroxide solution (10 per cent.) and insert a rubber stopper; the tube is inverted several times to mix and the gas again collected in the closed arm. The gas absorbed is carbon-dioxide; the remainder is usually hydrogen. In the case of media containing coagulable proteins acid production if sufficient is followed by 120 PRINCIPLES OF MICROBIOLOGY coagulation, as milk, serum water, serum broth; the coagulation is shown in solid media liy pred]3itation or opacity which develops. The ability to attack a carliohvdi-ate may differ under aerobic and anaerobic conditions. I'riiltofylic (icUon. is shown by gelatin liquefaction, liquefaction of LiifHci' s blood-serum, digestion of milk. Prolonged incubation even for a month may Ix' needed. The ))rofluction of jieptone may also be used for dctennuung the digestion of albuminous media. Note odor and, if present, its character. Alkali Prorfitc^jon.— Determined by indicators or titration; note m milk cultures especially. An initial acidity may be noted due to traces of a fer- mentable carbohydrate. The cultures may have to be incul:)ated for one to three weeks. Milk on prolonged incubation may become translucent whether due to alkali production or to a proteolytic enzyme not determined. Sulphuretted hydrogen, to demonstrate, use peptone water cultures to whicli is added 1 per cent, of a 1 per cent, solution of lead acetate or ferric tartrate; the precipitate which forms on mixing these turns black if sulphuretted hydrogen is produced. Nitrate Product ion. ^Giwv in nitrate broth. Two solutions are necessary for the test. 1. Naphthylamin 0.1 gram Distilled water 20.0 c.c. Acetic acid 2.5 per cent 150.0 c.c. Dissolve the naphthylamin in the water by means of heat, cool, filter, and add the acetic acid. 2. Sulphanilic acid 0.5 gram Acetic acid dil. 1 to 16 150.0 c.c. Keep them separate and mix in equal parts as needed. To about 4 c.c. of the culture add 2 c.c. of the mixed solutions. The develop- ment of a pink color shows the presence of nitrates, the intensity of the color being proportional to amount present. Similar reduction processes are seen in the decolorization of litmus and some of the aniline dyes. The bacteria utilize the oxygen of the d3^es, reducing them to their leuco-bases. Aromatic Products (Indol Production). — The usual medium is peptone water. Two methods are available for testing: (1) Salkowski Method. To the culture add several drops of concentrated sulphuric acid or 1 c.c. of a 10 per cent, solution and then add 1 c.c. of a 1 to 10,000 of sodium sulphite solution. A pink color develops at the point of contact with the acid, which on shaking diffuses throughout. (2) Ehrlich Method. This test is more constant and reliable. To the culture add 1 c.c. of a 2 per cent, solution of paradimethylamino- benzaldehyde in 95 per cent, alcohol and then add, drop by drop, concentrated liydrochloric acid until a red zone at the point of contact of the alcohol and the peptone. Not more than 0.5 c.c. of the acid is required. On standing the zone deepens and widens. The color is soluble and the test should be confirmed by shaking with chloroform, which dissolves out the color. The tests for indol are made after four to six days' incubation. Cholera-red Reaction. — The nitrate is produced by some bacteria from the nitrate present as an impurity in the peptone, and the red color appears on the addition of acid alone. Both of the above tests are interfered with by the presence of a fermentalile carbohydrate. Voges-Proshauer Reaction. — Grow for three days in 2 per cent, glucose-peptone solution in fermentation tubes. Add 2 to 3 c.c. of a strong solution of potassium hydrate. A pink color develops on exposure to tlie air. CULTIVATION OF MICROORGANISMS 121 To.vin Prodiiclioii. — Tlio presence of a toxin is not unusually elicited by cul- lui-al melliods; one exception is the demonstration of lieiuolysiiis hy plaliii^; with hlood-anar. The hemolysis is shown by a. cleariuf;; about the eohiny. I'or oilier melhods of denionstratiu};; the production of toxin, and the use of iluinuu()k)Kical reactions and aniin;d ti'sis se(> the appropriale sections of this work. The accompanying chart is given as an example of a. complete (uillural study of an organism and the methods of using cultural reaction for classiHcation. The characteristics to be elicited will depend on the groui) of organism studied. In the case of many of the organisms pathogenic for man and animal, the cultural characteristics only give us a jiresumptive identification, the filial identification depending on animal and hnmunological tests. References. Bass and Johtis. Jour. Exper. Med., 1912, xvi. HnitingcT. Centinll)!. f. Bakt., December 4, 1912. Krumwiede and Fralt. Jour. Inf. Dis., 19V.i, xii, 199. Williams. Jour. Med. Res., 1911, xx, 263. CHAPTER V. THE USE OF ANIMALS FOR DIAGNOSTIC AND TEST PURPOSES. Suitable animals are necessarily employed for many microbiologic purposes. (1) To obtain a growth of varieties that for any reason grow with difficulty on artificial culture media, as in the case of tubercle bacilli: hence material suspected to contain tubercle bacilli is injected into guinea-pigs, with the knowledge that, if present, although in too small numbers to be detected by microscopic or culture methods, they will develop in the animals' bodies, and thus reveal themselves. The same may be true of glanders, tetanus, and anthrax bacilh, of pneumococci, of other bacteria, and of protozoa. Certain microorganisms have not yet been grown on artificial media. This is true of few bacteria, of most protozoa, of many of the spirochetes, and of certain unknown infectious agents such as produce smallpox and Rocky Mountain spotted fever. (2) To cause an increase of one variety of organisms in a mixture and thus obtain a pure culture: An injection of sputum subcutaneously in rabbits may give rise to a pure pneumococcus septicemia or a pure tuberculosis. (3) To test virulence: Animals are used to test the virulence or toxin production of organisms, where, as in the case of diphtheria, we have very virulent, attenuated, and non-virulent bacilli of, so far as we know, identical cultural characteristics. Here the injec- tion of a susceptible animal, such as the guinea-pig, is the only way that we can differentiate between those capable of producing diseases from those that are harmless. Still another use of the animals is to differentiate between two virulent organisms, which, though entirely different in their specific disease poisons, are yet so closely allied mor- phologically and in culture characteristics that they cannot always be separated except by studying their action in the animal body both with and without the influence of specific serums. In this way the typhoid and colon bacilli may be separated, or the pneumococcus and streptococcus. (4) To test the antitoxic or microbicidal strength of sera : Diphtheria antitoxin is added to diphtheria toxin and injected into guinea-pigs, and streptococcus immunizing serum is mixed with living streptococci and injected into the vein of a rabbit. (5) To produce antitoxic, bacteri- cidal, or agglutinating sera. The Inoculation of Animals. — The inoculation of animals may be made either through natural channels or through artificial ones: 1. Cutaneous. The material is rubbed into the abraded skin. 2. Intracutaneous. The material is injected into the skin. Im- portant specific local reactions may be obtained by this method. THE INOCULATION OF ANIMALS 123 3. Subcutaneous. The sul)stauces are injected hy means of a hypo- dermic needle under the skin, or are introduced hy a platinum loop into a pocket made by an incision. 4. Intraxenous. The substances arc injV'ctcd by means oF a h.ypo- dermic needle into the vein. This is usually carried out in the ear vein of the rabbit. If rabbits are placed iu a holder, so that the animal remains quiet and only the head projects, it is usually easy to pass a small needle directly into one of the ear veins, especially those running along the edge of the ear. If the ear is first moistened with a 3 per cent, carbolic acid solution, and then supported between the finger inside and the thumb outside, the vein is usually clearly seen and entered with ease, if a small, sharp needle is held almost parallel with the ear surface and gently pushed into it. When no holder is present, the rabbit can be held by an assistant seizing the forelegs in one hand and the hind in another and holding the rabbit head downward, or the animal may be held between the knees of the operator, its body resting on the operator's apron. 5. Into the anterior chamber of the eye, 6. Into the body cavities. The peritoneal and less often the pleural cavities are used for microbal injection. The hypodermic needle is usually employed, less often a glass tube drawn out to a fine point. The needle or the pointed glass tube is gently pushed through the abdominal wall, moved about to be certain that the intestines have not been perforated and the fluid injected. 7. By inhalation. This method is carried out by forcing the animal to inhale an infected spray or dust. S. By the trachea. This method is carried out by making an incision in the trachea and then inoculating the mucous meml)rane or injecting substances into the trachea and bronchi. 9. Through the intestinal tract by swallowing or by the passage of a rubber tube. Morphine may be given to prevent peristalsis. 10. Into the brain substance or ventricles after trephining, or when the parietal bones are thin, as in the guinea-pig and the rabbit, after making a tiny opening with the point of a small, heavy scalpel. Mice, which are usually inoculated subcutaneously in the body or at the root of the tail, are best placed in a mouse holder, but can be inoculated by grasping the tail in a pair of forceps, and then, while allowing the mouse to hang head downward in a jar, a glass plate is pushed across the top until only space for its tail is left. Monkeys and apes are used for certain infections, such as sj^philis and smallpox, where only they and man are markedly susceptible. All these methods must be carried out with the greatest care as to cleanliness, the hair being clipped and the skin partially, at least, disinfected. The operator must be careful not to infect himself or his surroundings. After the inoculations the animals should be given the required food and kept in appropriate quarters. For food, rabbits and guinea-pigs usually require only carrots and hay. When possible, all animals should be anesthetized during painful experiments. 124 PRINCIPLES OF MICROBIOLOGY If animals die, autopsies should be made at the earliest moment pos- sible, for soon after death some of the speeies of the bacteria in the intestines are alile to penetrate throui,di the intestinal walls and infeet the body tissues. If delay is unavoidable, the animals shouM be put immediately in a place where the temperature is near tiie freezing;- ])oint. In making cultures from the dead bodies the greatest care should be taken to avoid contamination. The skin should be disinfected, and any dust prevented by wetting with a 5 per cent, solution of carbolic acid. All instruments are sterilized by boiling in 3 per cent, washing-soda solution for five minutes. Changes of knives, scissors, and forceps should be made as frequently as the old ones become infected. When organs are examined the portion of the surface through which an incision is to be made must be sterilized, if there is danger that the surrounding cavity is infected, by searing with the flat blade of an iron spatula which has been heated to a dull-red heat. Tissues if removed should be immediatelj' placed under cover so as not to become infected. Sterile deep Petri plates are useful for this purpose. When it is necessary to transport tissues from a distance they should be wrapped in sterile cloths and sent to the point of destination as soon as possible. In warm weather they may be kept cool by sur- rounding the vessels which contain them with ice. Animals rarely show the same gross lesions as man when both suft'er from the same infection. The cell changes, however, are similar, and, also, so far as we can test them, the curative or immunizing effects of protective serums. Leukocytes for Testing Phagocytosis. — Inoculate into the pleural cavity of a rabbit 5 c.c. of a thick suspension of aleuronat powder in a boiled starch solution. The solution should be thick enough to hold the aleuronat in suspension. A 20 to 25 per cent, solution of peptone gives good results. The fluid is withdrawn eighteen to twenty-four hours after the injection. For purposes of obtaining the opsonic index the whole blood is taken. For description of the method see chapter on opsonins. Leukocytes from the horse can be readily obtained by mixing the blood with 1 per cent, of sodium citrate and allowing the mixture to stand. The red cells rapidly sink and leave the leukocytes in the super- natant fluid. CHAPTER VI. THE rUOCURIXG AND HANDLING OF MATERIAL FOR ^IICROBIOLOGIC EXAMINATION FROM THOSE SUFFERING FROM DISEASE. A LONG experience has taught us that physicians very frequently take a great amount of trouble, and yet, on account of not carrying Qut certain simple but necessary precautions, make worthless cultures or send material almost useless for microbiologic study. In making cultures from diseased tissues various procedures may l)e carried out, according to the facilities which the physician has and the kind of information that he desires to obtain. From the dead body culture material should be removed at the first moment pos- sible after death. Every hour's delay makes the results less reliable. From both dead and living tissues, the less the alteration that occurs in any substance between its removal from the body and its examination and inoculation upon or in culture media or animals, the more exact will be the information obtained. If the material is allowed to drj- many microbes will be destroyed in the process, and certain forms which were present will be obliterated or, at least, entirely altered in the proportion which they bear to others. If possible, therefore, smears should be made and culture media should be inoculated directly from the patient or dead body. For the latter purpose a microbiologist should take the most suitable of the culture media to the bedside or autopsy table. Such a list of media, if fairly complete, would com- prise nutrient bouillon alone and mixed with one-third its ciuantity of ascitic fluid, slanted nutrient agar, slanted agar streaked with rabbit or human blood, firmly solidified slanted blood-serum and slanted ascitic glucose agar. Additional media will be necessary for special purposes, such as the isolation of typhoid or tetanus bacilli. If only one variety of media is to be used the solidified blood-serum is most useful for parasitic bacteria, and this can be easily carried by the physician and inoculated by him, even if he is not very familiar with bacteriologic technique. In the first place some of the infected material should always be smeared on a couple of clean slides or co^'er-glasses' and fixed moist in methyl alcohol or allowed to dry in the air. These can be stained and examined later, and may gi\'e much valuable information. The material must be obtained in different waj-s, according to the nature of the infection. 12G PRINCIPLES OF MICROBIOLOGY For the detection of the bacteria causing septicemia we are met with the difficulty that there are apt to be very few organisms^ present in the blood until shortly before death. It will, therefore, be almost useless to take only a drop of blood for cultures, as even when present there may not be more than eight or ten organisms in a cubic centi- meter. If cultures are to be made at all, it is, therefore, best to make them correctly by taking from 5 to 20 c.c. of blood by means of a sterile hypodermic needle or a suitable glass tube armed with a hypodermic needle, from the. vein of the arm after disinfecting the skin with tincture of iodine. To each of five different tubes containing bouillon we add 1 c.c. of blood, and to a flask containing 100 c.c. we add 5 c.c. We ha\'e made by this mixture of blood and bouillon a most suitable medium for the growth of all bacteria which produce septicemia, and, at the same time, have added a sufficient quantity of blood to insure us the best possible chance of having added some of the bacteria pro- ducing the disease. We also add to each of several tubes of melted nutrient agar, at 40° C, 1 c.c. of blood and pour the mixture into Petri plates, so as to indicate roughly the number of organisms present by the number of colonies developing. When blood must be carried to a distance, clotting should be pre\-ented by having in the test-tube suffi- cient 10 per cent, solution of sodium citrate, bile, or ammonium oxalate to prevent clotting. From wounds, abscesses, cellulitis, etc., the substance for microbio- logic examination can, as a rule, best be obtained by means of a syringe, or when the lesion is opened, by small rods armed with a little absorbent cotton. A number of these swabs can be sterihzed in a test-tube and so carried. The swab is inserted in the wound, then streaked gently over the oblique surface of the nutrient agar in one tube, over the blood serum in another, and then inserted in the bouillon. Finally, either at the bedside or in the laboratory, material is thinly streaked over the surface of nutrient agar contained in several Petri dishes. We inoculate several varieties of media, with the hope that one at least will pro^'e a suitable soil for the growth of the organisms present. From surface infections of mucous membranes, as in the nose, throat, vagina, etc., the swab, again, is probably the most useful instrument for obtaining the material for examination. The greatest care, of course, must be used in all cases to remove the material for study without contaminating it in any way by other material which does not belong to it. Thus, for instance, if we wish to obtain material from an abscess of the liver, where the organ lies in a peritoneal cavity infected with microbes, one must first absolutely sterilize the surface of the \i\er by pressing on it the blade of a hot-iron spatula before cutting into the abscess, so that we may not attribute the infection which caused the abscess to the germs which we obtained frona the infected surface of the liver. From such an organ as the uterus it is only with the greatest care that we can avoid outside contamination, and only an expert microbiologist familiar with such material will be able to eliminate the vaginal from the uterine microbes. ROUTINE TECHNIQUE CARRIED OUT AT LABORATORY 127 A statement of the conditions under whic'li materials are obtained should always accompany them when sent to the laboratory for examina- tion, even if the examination is to be made by the one who made the cultures. These facts should be noted, or otherwise at some future date they may be forgotten and misleading- information sent out. The work of obtaining material for examination without contamination is at times one of extreme difficulty. It simply must be remembered that if contamination does take place our results may become entirely vitiated, and if the difficulties are so great that we cannot avoid it, it may simply mean that under such conditions no suitable examination can be made. AYhere the substance to be studied cannot be imme- diately subjected to cultures or animal inoculations, it should be trans- ferred in a sterile bottle as soon as possible to a location where the cultures can be made. If for any reason delay must take place, the material should at least be put in a refrigerator where cold will both . prevent any further growth of some varieties of microbes and lessen the danger of the death of others. In obtaining samples of fluid, such as urine, feces, etc., the bottles in which they are placed should always be sterile, and, of course, no antiseptic should be added. It is necessary clearly to explain this to the nurse, for she has probably been instructed to add disinfectants to all discharges. Disinfected material is, of course, entirely useless for complete microbiologic investigations. It cannot be too much emphasized that materials which are not immediately used should be sent to the laboratory as quickly as possible, for in such substances as feces, where enormous numbers of various kinds of microbes are present, those which we seek most, such as the typhoid bacilli, frequently suc- cumb to the deleterious products of the other microbes present. Even when abundantly present, living typhoid bacilli ma,y entirely disappear from the feces in the course of tweh^e hours, while at other times they may remain for weeks. These differences depend on the associated organisms present, the chemical constitution of the feces or urine, and the conditions under which the material is obtained. Water and milk rapidly change in their bacterial content if not kept under 40° F. For obtaining fluid for agglutination and other purposes, blister fluid is valuable. A blister can be raised quickly by placing a piece of blotting paper moistened with a little strong ammonia on the skin and covering with a watch-glass, or one may be more slowly formed by a cantharides plaster. Routine Technique Carried Out at Laboratory when Thorough Examination Required. — As has just been indicated, the microbio- logic examination proceeds somewhat difl'erently according to the information needed. When, as is the case with most clinical material, definite knowledge in regard to the presence or absence of a particular organism is desired, the culture media are used which are known to be most suitable for the organisms sought, such as Loffler's blood- medium for the diphtheria bacillus. These media have been already partly given in the chapter on Culture Media. Others are later 128 PRINCIPLES OF MICROBIOLOGY described under each microorganism. When, as is generally the case \\\t\\ autops}' material and sometimes with clinical, a comj)lete ex- amination is needed in order to determine unknown organisms, the procedure may be as follows : 1. At the autopsy table the routine cultures and smears are made as described above. 2. Material from the different parts is secured under aseptic precau- tions in sterile receptacles and taken to the bacteriologic laboratory. The receptacles should be surrounded b,y ice if the laboratory' is at a distance. 3. A smear from each part is stained and examined in order to deter- mine in some measure the kind and number of microbes present, so we may more wisely select suitable culture media if other than those already used be needed, and may make the right culture dilutions if these be necessary. Gram's stain (see p. 78) gives more information, especially in regard to the first point, than any other one stain, so when possible this stain should be used. Other stains, however, may help, if for any reason Gram's is not at hand; and smears made from blood or from sus- pected syphilitic material should be stained by Giemsa's method or an equivalent (see Staining Methods). A Gram-stained smear may show all Gram-negative or all Gram- positive microbes or a mixture of the two, or it may show a number only partially stained (gram amphophile). The following points must l)e remembered in usmg this stain and in inter- preting the results: (a) The smears should be thin and e^'enly spread. (h) The staining solutions should be fresh (aniline- water- gentian-violet lasts about three weeks). (c) Controls, fresh cultures (about twenty-four hours old) of a Gram-negative and a Gram-positive bacterium, should be used on the same slide with the smear to be examined. (f/) If there is much albumin in the suspected material less heat should be used in fixing. (c) If the urine is very acid the results may not be good. (/) Mix urinary sediment with egg albumen, better to fix it, and wash out urinary salts with tap-water and stain. (,17) Too nmeh dependence sliould not tie placed ujion the finding of Gram- negative microbes in tissues, since organisms which in jnu'c >'oung cultures may }:)C positive to Gram, show forms as they grow older Ijoth in tissues and in cultures, intermediate between the positive and negative, as well as a varying number of negative forms. If the smears show only Gram-negative organisms, the material probably contains one or more of the following: ROUTINE TECHNIQUE CARRIED OUT AT LABORATORY 129 Gram- negative bacilli. J Graiu- iiegati\-e cocci. Gram- negative spirilla. B. coli group. B. typhosus group. B. dysenteria' group. B. proteus. B. mucosas (capsulatus). B. pyocyaneus. B. influenza* group. B. pertussis group. B. tusiformis. B. mallei. B. edematis (malignant edema). B. of symptomatic anthrax. B. pestis. B. of Morax-Axenfeld. ^Micrococcus intracellularis. Micrococcus catarrhalis. Micrococcus gonorrheas. Micrococcus melitensis. S. cholera* antl allied forms. Most fi'cqucntly from irttestinal tract. Most from tents. frequently chest con- Most frequently found, and some indication of their pres- ence in history. Less frequently found, and gen- erally a marked indication of their presence in history. I Generalh' marketl indication of I their presence in history. Marked indication of presence of first form in history. Mouth spirals. f Unimportant, unless indicated in history, Tr. pallidum. \ when Tr. pallidum or Sp. recurrentis, Sp. recurrentis. [ respectively, should be looked for. Old forms of any of the Gram-positive or Gram-amphophile organisms. If only Gram-positive organisms are demonstrated, the material may contain one or more of the following: B. diphtheria group. B. tetani (not often demon- strated in smears from lesion). B. tuberculosis group. B. leprffi group. B. anthracis group. B. wclchii and some other in- testinal anaerobes. Staphylococcus group. Streptococcus group (including pneumococcus and its variety, pneumococcus mucosus). Micrococcus tetragcnus. Gram- positive bacilli. Gram- positive cocci. Generally marked indication of their j^resence in history. Some indication of their pres- ence in history. Gram- positive spirilla. Higher bacteria. None. Nocardia. Actinomycosis. Yeasts and molds, certain forms. If organisms, partially stained by Gram's method are demonstrated, the material may contain one or more of the following: f Molds. Amplio- J Yeasts, phile. 1 Protozoa. I Slightly known cocci and bacilli. Older forms of Gram-positive organisms. 9 130 PRINCIPLES OF MICROBIOLOGY Certain slightly known streptococci and bacilli, older forms of any of the Gram-positive organisms. Micrococcus intra- cellularis. Fluid generally Streptococcus (in- cloudy with many Meningeal cluding pneumo- leukocjdes. (Cerebrospinal). coccus) group. B. influenzfE group. B. tuberculosis . Fluid generally clear. Fluids. group. J from Streptococcus (in- serous cluding pneumo- mem- coccus) group. Fluiil niaj' be cloudy. branes. Pericardial and B. mucosus (capsu- latus) group. B. influenza group. pleural. B. tuberculosis I Fluid generally clear. group. J B. cob group. Streptococcus group. Peritoneal. B. tuberculosis, group. ^ Yeast, molds, amebae. Streptococcus (including pncumococcus) group. Micrococcus (including staphjdococcus and diplococcu.s) group. B. mucosus (capsulatus). Lungs. B. influenza group. B. tuberculosis group. B. pertussis group. Yeasts, molds, ameb89. B. diphtheriiB group. B. influenzffi group. Nose and Streptococcus group. throat. B. mucosus group. B. tuberculosis. Yeasts, molds, amebae. . Tr. pallidum. Streptococcus (including pneumococcus) group. Group of hemiglobinopliilic bacilli. Gonococcus group. Eye. B. Diphtheria} group. B. Morax Axenfeld group. B. tuberculosis group. Yeasts and molds. B. coli group (includuig B. fecalis alcaligenes antl B. acidi lactici). B. typhosus group. Feces. B. dysenteriee group. Gram-positive anaerobes. Yeasts, molds, amebte. Many forms whose importance has not been worked out. ; B. coli group. Streptococcus (kidney). Urine. M. gonorrhere. B. typhosus. B. tuliereulosis. Molds, flagellates. • ROUTINE TECHNIQUE CARRIED OUT AT LABORATORY 131 Pelvic organs. Blood. Brain. M. gonorrheae. Streptococcus. B. tuberculosis. Treponema pallidum . Certain anaerobes. Many other forms probaljly nnich less ini]jortant. Streptococcus (including pneumococcus) group. B. typhosus group. Trypanosome group. Group of malarial organisms. Sp. recurrentis. Meningococcus group. Streptopneumococcus group. Group of hemiglobinophilic bacilli. B. tuberculosis group. Tr. pallidum. Malarial organisms. Rabies organisms. Yeasts, molds, etc. The following media should be used for the accompanying reasons: Nutrient liroth, for motility, morphology, and arrangement (chains, groups, etc.). Potato for color and abundance of growth. Peptone liroth for indol. Fermentation tube for anaeroljes, aciditj' and gas. Nutrient agar and gelatin. (a) Poured plates for isolated colonies (dilutions accord- ing to the numljer of organisms seen in smears). (Blood- agar if pneumococcus or streptococcus indicated.) (6) Streaked plates for surface colonies. (Blood-agar if influenza bacilli are indicated.) Special media according to the kinds of organisms demonstrated in smears or indicated in histories. Such special media are described in the chapter on Media, and are indicated under the individual organisms. CHAPTER VII. THE RELATION OF .AIICROORGANISMS TO DISEASE. In preceding chapters we have considered the growth of microorgan- isms for the most part in dead organic substances. Now we have to consider their growth and the production of their poisons in the living host and the results of such development. While it is true that there is a great difference between living and dead matter, and that, therefore, the living animal cannot be looked upon as merely a, quantity of special . material to be used for food for growth, still, in a very real sense, we are warranted in considering the infected living body as a food mass more or less favorable for the growth of microorganisms. The difference is that besides the chemical substances, temperature, and conditions inherent in the fluids and cells of the living body, microorganisms have also to reckon with the constant production of new substances by the living cells of the invaded organism, which may be antagonistic to them. In the production of lesions there are four main factors involved — viz., on the part of micoorganisms, the power to elaborate poison or proteins that may be split by ferment to yield poisons, and the abihty to multiply; on the part of the cells of the body the degree of sensi- tiveness to the poisons of the microbe and the tendency of the cells which have absorbed the poisons or proteins of the invading germs to develop substances which neutralize the poisons or attack the invading bacteria or protozoa. No known variety of microorganisms has, in small numbers, the ability to produce enough poison to do appreciable injury in the body, nor is it probable that there is any variety which, if it multiplied in the body to the extent that some pathogenic bacteria are capable of, would not produce disease. To understand the germ factor in the production of disease we must recognize that both the body invaded and the parasitic cells which invade are living organisms, and that the products of the cellular activity of the body act on the microorganisms at the same time their products act upon the human cells. Just as there are different races and species of animals having dissimilar characteristics, there are dif- ferent races and species among bacteria and protozoa. The descendants of both under changing conditions gradually become diverse. In fact, the rapidity of the dc^•el()pment of new generations of the unicellular organisms allow in tliem of much quicker changes under new conditions than arc possible in the higher animals and plants. Considering these and other facts, we can readily understand how the different types of the microtirganisms do not grow equally well in every variety of animal, and after discovering that there are variations in the properties of the blood from day to day we are not surprised that they do not find the THE RELATION OF MICROORGANISM!^ TO DISEASE 133 body of the same animal ahvays equally suitable. The study of bacteria in the more simple and known eouditions of artiKcial culture media has shown us how extremely sensiti\e many bacteria are to sli{>-ht chemical and otiier changes. We ha\e al.so found that conditions which are favorable to multiplit'ation may still be mifavorable for the ]>roduc- tion of toxins. If we take specimens of diphtheria bacilli from three different cases of diphtheria, we sometimes find that on growing them for several days iu suitable bouillon one culture will have produced poison in the fluid to such a degree that a single drop suffices to kill a large guinea- pig; the second, grown in a similar manner, will kill another animal of the same size with half a drop; while the third will kill with one-tenth of a drop. This illustrates the important fact that different varieties of the same bacillus have different toxin-producing powers under prac- tically the same conditions. The cultivation of the tetanus bacillus also furnishes some inter- esting facts which illustrate the complicated ways in which the growth of varieties of bacteria are hindered or assisted. The tetanus bacillus, when placed in suitable media, will not grow except in the absence of oxygen; but place it under the same conditions, together with a micro- organism which actively assimilates oxygen, and the two in association will grow in the presence of air. As a rule, when tetanus bacilli are driven into the flesh by a dirty nail or blank cartridge plug, aerobic bacteria are driven in also and so help to further infection by using up the free oxygen, thus introducing an anaerobic environment. The influenza bacillus is a striking example of the special require- ments of certain bacteria. On media it will thrive in pure culture only in the presence of hemoglobin. It is evident, therefore, that for each variety of organism there are special conditions requisite for growth, and that a temperature, degree of acidity, kind of food, supply of oxygen, etc., suitable for one may be utterly unsuitable for another; that, still further, when two organisms grow together one may so alter some of these conditions as to render unsuitable ones suitable, and vice versa. The pathogenic protozoa, indeed, the parasitic forms, are few in numbers compared with the total number of protozoa. They exert their harmful action mainly mechanically or by the direct destruction of the special host tissue which they find suitable for food. That they may produce specific toxic substances has been demonstrated in only one or two instances, the most marked of which is that of the poison obtained in the aqueous or glycerin extracts and the dried powder from mutton sarcosporidia which will be spoken of later. Though in general no specific toxins have been shown to exist in protozoan forms or to be excreted by them, the fact that there is spontaneous recovery from various protozoan infections and that a reinfection does not take place soon after, indicates that some specific toxins or substances are formed which help to produce immunity. Rossle has stated that he has obtained immune sera against infusoria; 134 PRINCIPLES OF MICROBIOLOGY and antibodies have been demonstrated in animals which iiave re- ceived non-lethal doses of trypanosomes and of amebse. Infection throngh protozoa is often iiccomplished by means of some of the lower animals acting either as intermediary hosts or as direct carriers of the virus (see Germ Carriers). Progress of Poisoning in an Infecting Disease. — When a fatal dose of a hving virulent organism is injected into the peritoneal cavity of a guinea-pig Vaughan states the following effects result: For a period of time which usually varies from eight to twelve hours the animal remains apparently normal. Its temperature may fluctuate slightly, but not beyond the normal limits. This is the period of incubation and varies within certain time limits, but within these it is fairly constant. During this time, the bacteria are multiplying enormously in the animal body. They are converting animal proteins into bacterial proteins. This is largely a synthetic or con- structive process. The soluble proteins of blood and lymph are built into the cellular proteins of the bacteria. There is no liberation of the protein poison and consequently no disturbance in the well-being of the host. During the period of incubation of an infectious disease the invading organism supplies the ferment, the soluble proteins of the animal body constitute the substrate, the process is constructive, simple proteins are built into more complex ones, no protein poison is liberated, and no recognizable symptoms mark the progress of the infection. Still, in the development of the phenomena of infection, the period of incubation is critical, and the rate at which the infecting virus multi- plies during this time is an important factor in determining the final outcome. The more virulent the virus, the more rapidly does it mul- tiply and this means a larger amount of animal protein converted into bacterial protein. Somewhat abruptly there is a change in the behavior of our inoculated guinea-pig. The hairs behind the ears begin to stand out and soon the entire coat becomes rough. It no longer eats, but retires to one corner of the cage and seems to be in distress. Slight pressure over the abdomen elicits evidence of pain and the temperature begins to fall and continues to do so until death. In case of recover j^ a rise of temperature is the first evidence of improvement. This somewhat abrupt change in the condition of the animal marks the end of the period of incubation and the beginning of the active dis- ease. The animal cells have become sensitized and are now pouring out a specific ferment which digests the bacterial proteins. In the active stage of the disease, the animal cells supply the ferment, the bacterial proteins constitute the substrate, complex cellular proteins are split into simpler bodies, the process is analytic and destructive, the protein poison is liberated, the sjanptoms of disease develop, and life is placed in jeopardy. Adaptation of Pathogenic Bacteria and Protozoa to Certain Tissues. — Parasitic microorganisms have gradually adapted themselves not only to certain species of animals, but to certain circumscribed areas of the LOCAL EFFECTS PRODUCED BY MTCROORGANrShrS 135 body. Thus the diphtheria bacilli grow chiefly upon the mucous membraues of the respiratory tract, but cannot develop in the Itlood or in the subcutaneous tissues. The cholera si)irilla develop in the inHamed intestinal mucous membrane, but cannot grow in the respi- ratory tract, blood, or tissues. The tetanus bacilli develop in wounds of the subcutaneous tissues, but cannot grow on the intestinal mucous membranes or in the blood. The malarial parasites develop in the lumian body chiefly in or on the red-blood cells. Other microbes find, indeed, certain regions especially suitable for their growth, but under conditions favorable for them are capable of developing in other locations. Thus, the typhoid bacillus grows most luxuriantly in the Peyer patches and mesenteric glands, but also invades the blood, spleen, and other regions. The tubercle bacillus often remains localized in the apex of a lung or a gland for years, but maj' at any time invade many tissues of the body. The gonococcus finds the mucous membrane of the genito-urinary tract most suitable for its development, but also frequently is capable of growth in the eye and peritoneum and sometimes in the general circulation. The pneumococcus develops most readily in the lungs, but also invades the connective tissues, serous membranes, and the blood. The malarial protozoa grow not only in human red cells, but also in the cells of the stomach and the salivary glands of the mosquito. Most of these germs, although ordinarily increasing only in the body of man,''can be grown on suitable dead material. There are a few unicellular organisms which, in so far as we know, find the bodies of human beings or animals the only fit soil for their growth. These are called strict parasites. Adaptation of Microbes to the Soil upon which They are Grown. — Those organisms which grow both in living and dead substances vary from time to time as to their readiness to develop in either the one or the other. As a general rule, bacteria grown in any one medium become more and more accustomed to that and other media more or less analogous to it, while, on the other hand, they are less easfly culti- vated on media widely different from that in which they have developed. Thus we had a culture of tubercle bacilli which, after having grown for three years in the bodies of guinea-pigs, would grow only with great difficulty on dead organic matter, while a bacillus which was obtained from the same stock, but grown since on bouillon, will usually no longer increase in the animal body. From the same stock, therefore, two varieties have developed, the one having lost and the other gained in ability to develop as a parasite. Local Effects Produced by Microorganisms and Their Products.— The local effects of the microbal poisons and proteins upon the cells give rise to the various kinds of inflammation, such as serous, fibrinous, purulent, croupous, hemorrhagic, necrotic, gangrenous, and, finally, proliferative. Some organisms incite specific forms of inflammation along with those common to many organisms; others produce, so far as we can detect, no peculiar form of lesions. 13fi PRINCIPLES OF MICROBIOLOGY Thus inflammation and serous exudation into the subcutaneous tissues follow injections of the pneumocoecus or anthrax bac'ilhis. The development of the streptococcus or pneumocoecus in the endo- cardium or pleural cavity is followed by a serous exudation, frequently with more or less fibrin production. The fcjrmation of pus results more especially from the streptococcus, pneumocoecus, and staphylo- coccus; but nearly all forms of bacteria, when they accumulate in one locality, may produce purulent inflammation. The colon, typhoid, and influenza bacilli frequently cause the formation of abscesses. Catarrhal inflammation, with or without pus, follows the absorp- tion of the products of many bacteria, such as the gonococcus, pneu- mocoecus, streptococcus, and influenza baciflus, etc. The hemor- rhagic exudation seen in pneumonia is usually due to the pneumo- coecus; it is observed also in other infections. Cell necrosis is pro- duced frequently by the products of the diphtheria and of the typhoid bacilli and by those of other bacteria. Specific proliferative inflam- mation follows the localization of the products derived from the tubercle bacillus and the leprosy bacillus. Not only can the poisons of one species of organism- s, according to the tissues attacked, produce several forms of inflammation, but the same organism will vary as to its mode and extent of invasion, this depending, first, upon its own characteristics, at the time, as to viru- lence, etc., and, second, upon the conditions in the infected animal, such as its health and power of resistance, the location of infection, and the circumstances under which the animal is kept. Such ^'aria- tions, therefore, are in no case specific, for dift'erent poisons will pro- duce changes which appear identical. Manner in which Microorganisms Produce Injury. — They produce serious mechanical injury only when they exist in such enormous numbers or bunched together as to interfere mechanically with the circulation or, together with fibrin, cause minute thrombi, and later emboli, which finally produce infarction and abscesses in different parts of the body. The eifects are almost wholly due to the chemical substances contained in the cells or excreted by them, which are directly poisonous or after being spht by ferments. Some portion of the protoplasm of almost every variety of bacteria acts as an irritant to tissues and combines with some of the substance of some of the Ijody cells, and the protoplasm of most exerts a positive chemotaxis. The poisonous products can often be separated from the fluid in which the organisms have grown, or the,y can be extracted from the microbes. Injected into animals these products cause essentiall,y the same cellular lesions as are produced by the organisms when they develop in the animal body. The substances contained in or produced by the organ- isms, with few exceptions, attract the leukocytes, and when great masses of bacteria die suppuration usually follows. The same properties are undoubtedly true of the poisons derived from the protozoa. VARIATION IN DEGREE OF VIRULENCE IN MICROBES IM? General Symptoms Caused by Microbal Poisons Absorbed into the Circulation.— Fi'MT is i)rti(liRr(l, uikIci- r;i\c>ral)l(' tdjiilitioiis, hy all mitrohal prott'ids. A requisite is that sufficient proteid be al)S()rl)e(l; on tlie other iianil, they must not be absorbed with such rapidity as to o^•er^^•llelm the infected host, for a moderate dose may raise the temperature, while a very large dose lowers it, as occurs sometimes when a very large surface, such as the peritoneum, is suddenly involved. As Yanghan and others have shown, these foreign proteids have to be acted upon by the body ferments before they exert their specific effect. With few exceptions the microbal proteids produce an increase in the number of leukocytes and a lessening in the amount of hemoglobin in the blood. In uncomplicated infection with typhoid bacilli there is a leucopemia. The polynuclear leukocytes are usually increased in varying proportions in bacterial infections. The red-blood cells are directly injured by a number of bacterial substances. The deleterious effects on the nutrition are partly due to the direct effect of the poison and partly to the diseased conditions of the organs of the body, such as the spleen, kidney, and liver. Degeneration of the nerve cells is frec^uently noticed after infectious diseases; especially is this true of diphtheria. Several bacterial poisons have been found to produce convulsions; the best example of this is the tetanus toxin. In protozoan infections the mononuclear leukocytes and the eosino- philes are chiefly increased. Influence of Quantity in Infection. — With pathogenic microbes the number introduced has an immense influence upon the probability of infection taking place. If we introduce a few bacteria into a culture medium containing some fresh human blood or serum it is probalile that they will all die because of the presence of sufRcient bactericidal substance in the blood to destroy them; whereas if a greater number are introduced there will be at first a great diminution of these, those that die having com- bined with the bactericidal substances in the serum which neutralizes them; then those bacteria which survive begin to increase, and soon they multiply enormously. The same is true for parasitic bacteria in the body. A few only gaining entrance, they may die; a larger number being introduced, some may or may not survive; but if a still greater quantity is injected it is almost certain unless the animal is immune that there will be some surviving members, which will begin to proliferate and excite disease. Variation in Degree of Virulence Possessed by Microbes. — ^licrobes differ, as has already been stated, as to the ease and rapidity with which they grow in any nutritive substance and the amount of poison they produce. Both of these properties not only vary greatly in different members of the same species, but each variety of organism may to a large extent be increased or diminished in virulence. The septicemic class of bacteria when grown in the body fluids seem to gradually develop the power to elaborate protective substances in their 138 PRINCIPLED OF MICROBIOLOGY own bodies or produce cells with less substance having affinity for the bactericidal bo(hes of the blood, and thus become less vulnerable. With those bacteria whose virulence is great a very few organisms will produce disease almost as quickly as a million, allowance only being made for the short time required for the few to become equal in number to the million. At the other extreme of virulence, however, many millions may have to be introduced to permit of the development of any of the organisms in the body. With these bacteria we are thus able to produce either no effect whatever, or a local effect, or in some cases a general septicemia, by regulating the amount of infection introduced. Somewhat distinct, again, from that class of microorganisms which multiply rapidly are those like the tubercle and leprosy bacilli, which, while surely developing infection, increase more slowly. Here increase of virulence is shown, as before, by the production of disease through the introduction of very small numbers into the body, but increase in rapidity of development cannot progress except to within certain limits. A single streptococcus may, through its rapid multiplication, produce death in eighteen hours; a single tubercle bacillus, on the other hand, cannot produce sufficient numbers in less than two weeks. The virulence of the septicemic class of bacteria is not at all the same when measured in different animals, and it is largely for this reason that the virulence in test animals does not usually correspond with the severity of the case from which the organism was derived. Experimental Increase and Decrease in Toxicity and Virulence. — The power to produce toxin can be taken from microbes by growing them under adverse circumstances, such as cultivation at the maximum temperature at which they are capable of development. Some microbes are easily attenuated; others are robbed of their virulence only with great difficulty. Increase of toxin production is more difficult, and it is only possible to obtain it to a certain extent. The means usually employed are the frequent replanting of cultures. But with all our efforts we are usually only able to restore approximately the degree of toxin formation which the cultures originally possessed. The adap- tation of microbes to any nutritive substance, living or dead, so that they will grow more readily, is more easily brought about, provided they will grow at all. The streptococcus from erysipelas and the pneumococcus from pneumonia are typical of this class of organisms. Inoculate a rabbit with a few streptococci obtained from a case of human sepsis, and, as a rule, no result follows; inject a few million, and usually a local induration or abscess appears; but if one hundred million are administered septicemia develops. From this rabbit now inoculate another, and we find that a dose slightly smaller suffices to produce the same effect; in the next animal inoculated from this, still less is required, and so on, until in time, with some cultures, a very minute number will surely develop and produce death. With other cultures increase in virulence does not take place. The same increase in virulence can be noted when septic infection is carried in surgery or MIXED INFECTION 139 obstetrics from one human case to another. By allowing niicruhes to continue to de\ek>p under certain fixed conditions they Ijcconic accais- tonied to these conditions, and less a(lai)ted to all that dill'cr. Mixed Infection.— The combined ell'ects upon the tissues of the products of two or more varieties of pathogenic microbes, iuid also of the influence of these different forms on each other, are of great im- portance in the production of disease. Tlie infection from several different organisms may occur at the same time, or one may follow the other or others — so-called secondary infection. Thus, an abscess is often due to several forms of pyogenic cocci. If a fresh wound is infected from such a source the inflammation produced will probably be caused by all the varieties present in the original infection. Peri- tonitis following intestinal injuries must necessarily be due to more than one variety of organism. Thus, whenever two or more varieties of bacteria are transferred to a new soil, mixed infection takes place if more than one is capable of developing in that locality. Forms of infection which are allied to both mixed and secondary infection are those occurring in the mucous membranes of the respi- ratory and digestive tract. In these situations pathogenic microbes of slight virulence are always present even in health. Thus, in the upper air passages there are usually found streptococci, staphylococci, and pneumococci. When through the invasion of one or several infective agents, as the diphtheria bacillus or the virus of smallpox or scarlet fever, the epithelium of the mucous membrane of the throat is injured or destroyed, the pyogenic cocci already present are now enabled in this diseased membrane to grow, produce their poison, and even invade deeper tissues. The intestinal mucous membrane is invaded in a similar way by the colon bacilli and other organisms after injury by the typhoid or dysentery bacilli or cholera spirilla. Generally speaking, all inflammations of the mucous membranes and skin contain some of the elements of mixed infection. Blood-infection, on the other hand, is usually due to one form of microbe, as even when several varieties are introduced, only one, as a rule, is capable of development. The same is true to a somewhat less extent of inflam- mation of the connective tissue. The additional poison given off by the associated microbes aid infection by the primary invaders by- causing a lowering of the vital resistance of the body. In some cases the secondary infection is a greater danger than the primary one, as pneumococcic bronchopneumonia in laryngeal diphtheria or strep- tococcic septicemia in scarlet fever and smallpox. Microorganisms are also at times directly influenced by the products of associated organisms. These may affect them injuriously, as, for example, the pyogenic cocci in anthrax; or they may be necessary to their development, as in the case of anaerobic bacteria. Not infre- quently the tetanus bacilli or spores would not be able to develop in wounds were it not for the presence of aerobic bacteria introduced with them. This is shown outside the body, where tetanus bacilli will not grow in the presence of oxygen unless aerobic bacteria are 140 PRINCIPLES OF MICROBIOLOGY associated with them. Again, it is found that the association of one variety with another may increase its \'iriilence. Streptococci are stated to increase the virulence of di]jhtheria bacilli, but here it is probal)ly the loss of resistance of the tissues l)ecause of the strepto- coccic poison. On the other hand, the absorption of the products of certain bacteria immunizes the body against the invasion of other bacteria, as shown by Pasteur that attenuated chicken-cholera cultures l^roduce immunity against anthrax. In intestinal putrefaction harm- less varieties of bacteria may be made to crowd out dangerous ones. Tissue Characteristics Influencing the Entrance and Growth of Microbes. — The Skin. — The skin is a poor soil for bacteria and is a great protection against the penetration of microorganisms. When they do penetrate, it is through the diseased glands, or more often through some unobserved wound. The bacterial toxins are, when at all, absorbed to a slight extent through the skin. There is an apparent exception to the above statements in the fact that the pyogenic staphylococci and sometimes the streptococci exist upon the skin or in it between its superficial horny cells, some excep- tional circumstances, such as wounds or burns, being required to allow the organisms to penetrate deeper. The cutaneous sweat glands, and the hair follicles with their appended sebaceous glands, may allow entrance of infection, as various incidents may lead to the introduction and retention of virulent microorganisms. When this occurs the retained products may lead to necrosis of the epithelium and thus allow the bacteria to penetrate to the deeper tissues. The secretion of the sebaceous glands appears to be little, if at all, bac- tericidal, but the perspiration, on account of the acidity, is slightly so. A species of protozoa (Leishmania) is found frecpiently in certain skin lesions in the tropics. Subcutaneous Connective Tissues. — Many microbes cannot develop in the connective tissues and others produce a milder infection there than elsewhere. Others develop readily. The tissue fluids have micro- bicidal properties similar to but less in amount than the blood. The Mucous Membranes. — The moist condition of the surface of the membranes and their frequent contact with irritating substances render them liable to microbal infection. Organisms, such as the pneu- mococci and streptococci, reproducing themselves in it become some- what attenuated. The mucous membranes are protected by the cleansing produced by the flow of the secretion and by its slight germ- icidal action. In infancy the membranes are readily infected by gonococci and later by pneumoeocci, by the Koch-Weeks bacillus and others. The mucous membranes of the nasal cavity are somewhat cleansed by the nasal secretion. The deeper portions of the nasal cavity are usually the seat of streptococci and other bacteria, while the extreme anterior portion contains saprophytic bacteria from the air. The mouth in a person in health is cleansed by the feebly bac- tericidal saliva. When the teeth are decayed many varieties of microbes abound. Many of these are difficult to cultivate. The bacteria, LOCATION OF POINT OF ENTRY OF ORGANISMS 141 such as the diphtheria bacilh, streptococci, etc., invade the mucous membrane of the tongue or mouth comparatively selik^m. The tonsils with their crypts are usually the seat of the pyogeuic cocci and are readily infected by the diphtheria bacilli and others. Whether the absolutely intact epithelium allows the passage of these bacteria is disputed, but the probability is that it does. With the slight pathologic lesions usually present it undoubtedly does. The Lungs. — ]Most inhaled organisms which pass the larynx are caught in the bronchi. Many of these are gradually remo\'ed by the ciliated epithelium. Both the alveolar epithelial cells and the leukocytes which enter the air sacs and bronchioles \mxe been shown to take up bacteria. The normal lung is, therefore, rapidly freed of saprophytic and many parasitic bacteria. When subjected to deleterious influences, such as exposure to cold, the lung tissues seem to lose their protective defences and become subject to infection. The Stomach. — The pure gastric juice, through the hydrochloric acid it contains, is able to kill most non-spore-bearing organisms in a short time, but because of neutralization through food, or because the microbes are protected in the food, many of them pass into the intes- tines. Tubercle, typhoid, colon, and dysentery bacilli, when fed by the mouth with food, readily pass beyond the stomach. Certain acidophilic germs, as well as yeasts and torula^, seem to grow in the gastric secretion; these are largely non-pathogenic. Perforation of the stomach is usually followed by peritonitis, because of the irritant effect of the gastric juice and the bacteria which were temporarily' pres- ent in the stomach. The gastric juice alters tetanus and diphtheria toxins. The toxicity of some poisons, such as occur in decayed meat, are not destroyed. The stomach is exceptionally free from bacterial inflammations. Intestines. — The bile is feebly germicidal for some organisms, but, on the whole, the intestinal secretions have little or no germicidal power. The number of microbes increases steadily from the duode- num to the head of the colon, and diminishes slightly from the upper to the lower end of the colon. The pancreatic juice destroj's many of the toxic microbal products. The presence of the bacilli of the colon group, of streptococci, etc., does not often lead to any inflamma- tory condition in the normal intestines of healthy persons. In children suffering from the prostrating eft'ects of heat they are apt to excite inflammatory changes. Even pathogenic bacteria, such as the typhoid, dysentery, and tubercle bacilli, may pass through the whole length of the healthy intestines without inciting inflammations. Slight lesions aid the passage of bacteria to the deeper structures. Tubercle bacilh and other pathogenic bacteria may pass through the intestinal wall to the lymph and cause distant infection without lea\'ing any trace of their passage. Non-pathogenic protozoa are frc(|ucntly foiuid in the intestines, and, in tropical countries, ])athogenic forms are also found. Importance of Location of Point of Entry .of Organisms. I\ lost microbes cause infection only when they gain access to special tissues 142 PRINCIPLES OF MICROBIOLOGY and must, therefore, enter through certain portals. This fact is of immense importance in the transmission or prevention of disease. Thus, for example, let us rub very virulent streptococci, typhoid bacilli, and diphtheria bacilli into an abrasion on the hand. The typhoid bacillus produces no lesion, the diphtheria bacillus but a very minute infected area, but the streptococcus may give rise to a severe cellulitis or fatal septicemia. Now place the same bacteria on an abrasion in the throat. The typhoid bacillus is again harmless; the diphtheria bacillus produces inflammation, a pseudomembrane, and toxemia, and the streptococcus causes an exudate, an abscess, or a septicemia. Finally, introduce the same bacteria into the intestines, and now it is the typhoid bacillus which produces its characteristic lesions, while the streptococcus and diphtheria bacillus are usually innocuous. If we tried in this way all the parasitic organisms we would find that certain varieties are capable of developing, and thereby exciting disease only on the mucous membrane of the throat, others of the intestine, others of the urethra; some develop only in the connective tissues or in the blood; while others, again, under favorable conditions, seem able to grow in or upon most regions of the body. The Dissemination of Disease. — The spread of infection is influ- enced by: 1. The number of species of animals subject to infection. Many human infectious diseases do not occur in animals, and many animal infections are not found in man. Thus, so far as we know, gonorrhea, syphilis, measles, smallpox, typhoid fever, etc., do not occur in animals under ordinary conditions; while tuberculosis, an- thrax, glanders hydrophobia, and some other diseases are common to both man and animals. 2. The quantity of the infectious material and the manner in which it is thrown off from the body. In diphtheria, typhoid fever, cholera, pulmonary tuberculosis, septic endometritis, influenza, and gonorrhea, enormous numbers of infec- tious bacteria are cast off through the discharges from the mouth, intestines, and genito-urinary secretions, causing great danger of infec- tion. On the other hand, in tuberculous peritonitis, streptococcic meningitis and endocarditis, gonorrheal rheumatism, and the like there is little or no danger of infecting others, as few or no bacteria are cast off. 3. The resistance of the infectious organisms to the deleterious effects of drying, light, heat, etc. In this case the presence or absence of spores is of the greatest importance. The spore-bearing bacilli such as tetanus and anthrax, being able to withstand destruction for a long time, retain their power of producing infection for months or even years after elimination from the body. The bacteria which form no spores show great varia- tion in their resistance to outside influences. Some of these, such as the influenza bacilli and the gonococci, the virus of syphilis and hydrophobia, are extremely sensitive; the pneumococci, cholera spirilla, glanders bacilli, etc., are a little hardier; then follow the diphtheria GERM CARRIERS 143 bacilli, and after them the typhoid and tubercle bacilli and the staphylococci. Yeasts, molds, and protozoa produce resisting sj)ores, which, however, are not as highly resistant as most bacterial spores. 4. The ability or the lack of ability to grow outside of the infected tissues. Such bacteria as the pneumococcus, tubercle, influenza, diphtheria, glanders, and leprosy bacilli do not develop, as far as we know, outside of the body under ordinary conditions. Under exceptional circum- stances, as in milk, some may develop. Others, again, such as the streptococcus and staphylococcus, typhoid and anthrax bacillus, the cholera spirillum, and some anaerobes, may develop under peculiar conditions existing in water or soil. While for the pathogenic bacteria, as a rule, the saprophytes met with in the soil and water are antagonistic, yet in some cases — and especially is this true of the anaerobic bacteria — they are helpful. 5. Germ Carriers. — In human carriers microorganisms develop in or upon some portion of the skin or mucous membrane, either after or be- fore disease, and without causing infection. As complete a knowledge as possible of this saprophytic development in man of parasitic microbes is necessary if we are to combat the spread of infection. In the super- ficial layers of the epithelium and on the surface of the skin we find the different pyogenic cocci, which are capable of infecting a wounded or injured part or causing inflammation in the glands. Acne, the pustules in smallpox, the pus on a burned surface, boils, etc., all come from these pyogenic cocci. In surgical cases the skin has to be as thoroughly disinfected as possible, to prevent the formation of stitch-hole abscesses and wound suppuration. In the secretion of the mucous membrane covering the pharynx and nasopharynx there is always an abundance of microbes. In throats examined in New York City, streptococci, staphylococci, and pneu- mococci are found in almost every instance, and even in the country they are often present. In the anterior nares there are fewer parasitic bacteria than in the posterior portions. Many other varieties of bacteria, such as the meningococci and the influenza bacilli, are probably often present in small numbers. In those constantly in contact with cases of diphtheria, and in those convalescent from diphtheria, virulent diphtheria bacilli are frequently found in the throat. After convalescence from typhoid fever, from one to three per cent, remain bacillus carriers for months or years. The bacilli continue to develop in the bile passages and are passed with the feces. Certain pathogenic protozoa may be carried for some time in the intestines of man. Lower yhiimals. — The lower animals, as a rule, do not retain in their bodies bacteria pathogenic for human beings, but, as direct carriers of bacterial infection, they are important factors. Flies and other insects may convey organisms which are simply attached to their feet or other surfaces of their bodies. Biting insects, especially, such as fleas, ticks, bed-bugs, lice, flies, and mosquitoes, are a source of danger in certain 144 PRINCIPLES OF MICROBIOLOGY protozoan infections, since these insects act as intermediate hosts in these cases. (See Malaria and Other Protozoan Diseases.) Microbal Auto-infection. — When the intestinal canal is injured, or its circulation hindered by strangulation, etc., Bacilhi^ coli and some other bacteria may penetrate through the injured walls and cause peritonitis or general infection. Under certain conditions, as during the debility due to hot weather, the bacteria in the intestines cause, through their products, irritation, and in children even serious intes- tinal inflammation. Long after an acute gonorrhea has passed gonococci may remain in sufficient numbers to cause a new inflam- mation or produce infection in others. A cystitis may run on chron- ically for years, and then suddenly become acute or spread infection to the kidneys. A persistent gonorrheal vaginal infection may lead to a gonorrheal endometritis, or peritonitis or salpingitis, under suitable conditions. The staphylococci in the skin and the colon bacilli and pyogenic cocci in the fecal discharges may also be carried into the bladder and uterus and produce septic infection. Persons carrying diphtheria bacilli in their throats or typhoid bacilli in their gall-bladder may, under predisposing conditions, develop diphtheria or typhoid fever. In nearly all cases of infection the products of bacterial growth are absorbed into the lilood, and along with them a few bacteria also, even when they do not reproduce themselves in it. The greater the extent of the infection and the more deep-seated it is, the greater is the amount of absorption. The bacteria enter the blood, according to Kruse, by (!) passive entrance through the stomata of the capillary walls; (2) carriage into the blood in the bodies of leukocytes; (3) growth of the bacteria through the walls of the vessels; (4) transmission of the bac- teria through the lymph glands placed between the lymph- and blood- vessels. When bacteria are abundant in the blood they become fixed in the capillaries of one or all of the organs, especially of the liver, kidneys, spleen, and lungs, and then directly or by means of the leukocytes, which penetrate the capillary walls, they pass into the tissues and substance of the organs. They thus reach the lymph channels and glands, or gain entrance into the gall-bladder, saliva, etc., or press through the epithelium, as in the alveoli of the lungs; more rarely they pass through the kidney tissue into the urine, as in typhoid fever, though some deny that this can happen unless there is a pre- vious inflammation of the kidneys. Microbes: Their Elimination through the Milk. — The passage of bac- teria through tlie breast is important, from the fact that milk is so largely used as food. ()l)servers ha^'e reported the finding of tubercle bacilli in cows' milk when the gland itself was intact and the ani- mal tuberculous. Some authorities have put its presence in milk, under these circumstances, as high as 50 per cent, of the cases. This, in our experience, is undoubtedly too high. The fact that tubercle bacilli swallowed with the sputum are passed alive in the feces ex- EXTRACELLULAR TOXINS 115 plains the frequent occurrence of bacilli in the milk of cows without udder tuberculosis because of the contamination of the milk with manure. They are undoubtedly present, however, in the milk of some animals in which tuberculous disease of the gland could not be demonstrated. In these cases lymph glands adjacent to the udder are usually infected. The finding of streptococci and staphylococci is due probably in the majority of cases to the infections taking place as the milk is voided, for the epithelium at the outlet of the lacteal ducts is always infected with staphylococci, and frequently with streptococci, which have often been received from the mouth of the sucking infant. Elimination of Microbes by the Skin and Mucous Membranes. — Whether bacteria pass from the blood by the sweat is a mooted point. The skin is always the seat of the staphylococcus and frequently of other bacteria, so that it is difficult to determine in any given case the origin of the bacteria found in the sweat. Many observers have reported the passage of bacteria from the blood through the mucous membrane. These, as shown by Hess, are few in number, however. Bacteria are sometimes eliminated through the urine, but here, as a rule, when great numbers of organisms are found, it is due to devel- opment in the bladder. The removal of the poisonous products of bacteria by the kidneys, intestines, etc., on the contrary, is of great advantage to the organism. Some of the More Important Microbal Poisons. — Toxins. — Any poisonous substance formed in the growth of bacteria or other micro- organism is loosely called a toxin, but in the strict sense this term should be restricted to the extracellular poisons, as these alone have the important characteristic of causing the cells of the body affected by them to produce antitoxins. The different bacterial poisons vary greatly in their characteristics. As little is known concerning their chemical nature, except that they are proteins, we are not able to classify them. There are certain known differences among them which are important and which may be made use of for purposes of study to divide the bacteria into two groups: 1. Those varieties of bacteria that excrete in ordinary culture media water-soluble, very specific, toxic products, extracellular toxins. Type: diphtheria, tetanus. These alone produce poisons. 2. Those varieties which possess toxic protein substances, which are more or less closely bound to the living cell, and which are only in a small degree separable in unchanged condition outside of the body. On death of the cell they partly become free, partly remain united, or become secondary poisonous modifications. Type: cholera, typhoid, pneumococcus. These are frequently called endotoxins. Some of these substances are only poisonous when split up by specific ferments present in the serum or cell fluids. Extracellular Toxins. — Among the properties of the extracellular toxins are the following: They are, so far as known, uncrystallizable, and thus differ from ptomains; they are soluble in water and they 10 140 PRINCIPLES OF MICROBIOLOGY are slowly dialyzable, through thin membranes, but not through thick membranes such as are used in refining antitoxic globulins; they are precipitated along with peptones by alcohol, and also by ammonium sul- phate; if they are proteins they are either albumoses or allied to the albumoses; tliey are relativelj' unstable, having their toxicity diminished or destroyed by heat and freezing as well as by chemical manipulation (the degree of heat, etc., which is destructive varies much in different cases). Their potency is often altered in the precipitations practised to obtain them in a pure or concentrated condition, but among the precipitants ammonium sulphate has but moderate harmful effect. A remarkable characteristic of the group is that the.y are highly specific in their properties and have the power in the infected body to excite the production of antitoxins. The diphtheria and tetanus bacilli are the best known extracellular toxin producers. Precipitation of the Extracellular Toxins. — Ammonium sulphate crys- tals are added to the fluid containing the toxin until it is saturated. A large excess of ammonium sulphate crystals is then added and the whole kept at about 37° C. for twelve to eighteen hours. The toxin is precipitated along with the albumoses and peptones and rises to the surface. This is skimmed off and dried in a ^•acuum or in an exsiccator containing strong sulphuric acid. The dried powder is placed in vacuum tubes and stored in the flark. lender these conditions the toxins deteri- orate very slowly. During the process there may be a considerable loss of toxin, even when e\'ery care is taken. Tetanus toxin is espe- cially liable to deterioration. Banzhaf has obtained a diphtheria toxin that is practically free from the meat extractions in the broth. He adds alcohol up to 65 per cent, to the slightly acidified toxin broth. The slight flocculent precipitate that is formed, after standing about one hour, is filtered off and dried in a vacuum. This driefl toxin does not deteriorate. One gram of the powder contains 40,000 m. L. D., whereas the original toxin broth contained 5000 m. L. D. per gram of solids. Ferment Characteristics of Toxins. — The comjjarison of the action of bacteria in the tissues in the production of these toxins to what takes place in the gastric digestion has raised the question of the possibility of the elaboration by these bacteria of ferments, by which the process may be started. It would not be prudent to dogmatize as to whether the toxins do or do not belong to such an ill-defined group of substances as the ferments. It may be pointed out, however, that the essential concept of ferments is that of a body which can originate change without itself being appreciably changed, and no evidence has been adduced that toxins fulfil this condition. Another ])roperty of ferments is that, so long as the products of fermentation are removed, the action of a given amount of ferment is indefinite. In the case of toxins no evidence of such an occurrence has been found. A certain amount of a toxin is always associated with a given amount of disease eft'ect. Intracellular Poisons, — Regarding the intracellular poisons or toxins which are more intimately associated with the bacterial cell and are SIMILAR VEGETABLE AND ANIMAL POISONS 147 produced by all bacteria we know much less. They are ai)pareutly proteins, and it is probable that their chemical nature is somewhat similar. They do not produce antibodies. These bacterial proteins may be poisonous originally, or only when split by a ferment. They have been studied by Yaughan, Friedberg anil others. Nature of the Microbal Proteins. — Vaughan in his recent Herter lecture describes his theories of their nature in the following words: "(a) " The infective bacteria, taking the colon, typhoid, tubercle, and the pneumococcus as types, contain an intracellular poison. This is not a toxin; it is not specific; it produces no antibody when injected into animals. (6) These bacteria elaborate no soluble toxin or poison. In old cultures there may be a trace of poison, but this results from the autolysis of the cells and is not a cellular secretion, (c) This poison is a group in the protein molecule and can be obtained in soluble form only after cleavage of the cellular proteins, which may be accomplished by superheated steam, dilute acids, or alkalis, (d) It exists in all true proteins, in patho- genic and non-pathogenic bacteria, and in vegetable and animal proteins. (e) It may result from the cleavage action of proteolytic ferments. (/) In most vegetable and animal proteins the poisonous group is neutral- ized by combination with non-poisonous groups; consequently such proteins have no poisonous action until they undergo molecular dis- ruption, (g) This primary group is poisonous because of the avidity with which it combines with secondary groups in the proteins of the animal body, (h) The specificity of proteins lies in their secondary, non-poisonous groups. It is in these that one protein chemically and biologically differs from another. (i) Biologic relationship among proteins is determined bj' the chemical structure of their molecules. There are as many kinds of proteins as there are kinds of cells, {k) The symptoms of the infections differ chiefly on account of the organ or tissue in which the virus accumulates and where it is split up and its poison liberated. (/) The ferment which causes the cleavage of the bacterial proteins in the different infectious diseases is specific." Similar Vegetable and Animal Poisons. — Substances similar to those classed as bacterial endotoxins and as soluble toxins are formed by many varieties of cells other than bacteria. The ricin and abrin poisons obtained from the seeds of the Ricimis communis- and the ^Ibrus pre- catorius have a number of properties similar to those of the diphtheria and tetanus poisons. The actiA^e poisons contained in ricin and abrin have not yet been isolated, but the impure substances are extremely poisonous. When injected into suitable animals anti-poisons are pro- duced and accumulate in the serum. These neutralize the poisons wherever they come in contact with them. They resemble the toxins in a general Avay in the manner in which they react to heat and chemicals. They are precipitated by alcohol. Through animal membranes they are less dialyzable than albumoses. Substances having these characteristics are called toxalbumins. Poisonous snakes secrete poisons which ha^'e many of the character- istics of the bacterial albumoses. The venom contains some substances 14S PRINCIPLES OF MICROBIOLOGY similar to peptone and others similar to globulin. The former cause general nervous symptoms and paralysis of the respiratory centre, while the latter cause intense local reaction with hemorrhages around the point of injection. The injection of venins into animals is followed by the production of antivenins which neutralize the venins. When the serum containing abundant suitable antivenins is injected into an infected person it has considerable therapeutic value. The pyogenic action of their proteins is common to all microbes, this depending principally upon their being extraneous albuminous substances. Pyogenic effects may be produced in like manner by extraneous albumins of non-microbal origin. That every extraneous albuminous substance is harmful to the organism which seeks to resist its action is shown by those specific precipitating ferments, the precipitins, which are produced in the organisms after the introduction of ever}' extraneous albumin. Ehrlich's Theories as to the Nature of Extracellular Toxins. — From a large number of most carefully conducted experiments with the toxin and antitoxin of diphtheria, Ehrlich has formulated a theory concerning the former. This theory has undergone several modifi- cations since it was first proposed, and it is difficult to give an exact statement of its present status. Generally speaking, however, in con- densed form its essential points are as follows: Toxins and antitoxins neutralize one another after the manner of chemical reagents. The chief reasons for this belief lie in the ob- served facts: (a) that neutralization takes place more rapidly in con- centrated than in dilute solutions, and (6) that warmth hastens and cold retards neutralization. From these observations Ehrlich con- cludes that toxins and antitoxins act as chemical reagents do in the formation of double salts. A molecule of the poison requires an exact and constant quantity of the antitoxin in order to produce a neutral or harmless substance. This implies that a specific atomic group in the toxin molecule combines with a certain atomic group in the anti- toxin molecule. The toxins, however, are not simple bodies, but easily split into other substances which differ from one another in the avidity with which they combine with antitoxin. These derivatives Ehrlich calls prototoxins, deuterotoxins, and tritotoxins. All forms of toxins are supposed to consist of two modifications, which combine in an equally energetic manner with antitoxin or with suitable substance in the cells, Init differ in their resistance to heat and other destructive agents. The less resistant form passes readily into a substance called tox- oid, which has tlie same affinity for the antitoxin as tiie original toxin, hut is not poisonous. The facts observed, Ehrlich thinks, are best explained on the supposition that the toxic molecule contains two independent groups of atoms, one of which may be designated as the haptophorous and the other as the toxophorous group. It is by the PTOMAINS 149 action of the former that toxin unites witli antitoxin or cell molecule and allows the latter to exert its poisonous ett'eet. The toxophorous j^roup is unstable, hut after its destruction the molecule still imites with the antitoxin or tiie sensiti\'e molecule through its retained haptoi)horous group. Bordet has shown that toxin unites in different multiples with anti- toxin, so that the toxin molecule may have its affinity slightly, partly, or wholly satisfied by antitoxin. Slightly satisfied, it is still feebly toxic; combined with a larger amount of antitoxin, it is not toxic; but still may, when absorbed into the system, lead to the production of antitoxin. Fully saturated, it has no poisonous properties and no ability to stimulate the production of antitoxin. Ptomains. — Nencki, and later, Brieger, \'aughan, and others, suc- ceeded in isolating organic bases of a definite chemical composition out of putrefying fluids — meat, fish, old cheese, and milk — as well as from pure bacterial cultures. Some of these were found to exert a poisonous effect, while others were harmless. The poisons may be present in the decomposing cadaver (hence the name ptomain, from ZT (.011.1/., putrefaction), and, in consequence, have to be taken into consideration in questions of legal medicine. They may be formed also in the living human body, and, if not made harmless by oxida- tion, may come to act therein as self-poisous or leukomains. They possess the characteristics of alkaloid bodies and are different from the specific poisonous toxins. Man,y ptomains are known already and among them are some whose exact chemical constitution is established. Especially interesting is the substance cadaverin, which was separated by Brieger from portions of decomposing dead bodies and from cholera cultures, by reason of the fact that Ladenburg prepared it synthetically and showed it to be pentamethylenediamin [(NH2)2(CH2).>i]. The cholin group is particularly interesting. Cholin itself (CsHuNOa) arises from the hydrolytic breaking-up of lecithin, the fat-like substance found in considerable amounts in the brain and other nervous tissue. By the oxidation of cholin there can be produced the highly toxic muscarin, found by Schmiedeberg in a poisonous toadstool and isolated by Brieger in certain decomposing substances: C5H15NO2 -i- = CsH.sNOa Cholin. Muscarin. The ptomain tyrotoxicon was obtained from cheese, milk, and ice- cream by Vaughan. Pyocyanin (CUH14N2O), which produces the color of blue or bhie- green pus, is a ptomainic pigment. Similar bodies of a basic nature may be found in the intestinal contents as the products of bacterial decomposition. Some of these are poisons and can be absorbed into the body. Since the name ptomain was given to the poisonous products of bacterial growth before these products were chemically understood 150 PRINCIPLES OF MICROBIOLOGY it is l:)y many wrongly applied to all poisons found in food. Such poisoning may he due to true toxins or even living germs. The isolation of these substances can here be only brief!.)' referre. THE ANTACONISM EXISTING BETWEEN THE LIVIN(i BODY AND MICROOKdANISMS. That certain races of animals and men, and certain individnals among these, are more refractory to disease than others is a fact which has long been known. Experience and observation have taught us, further, that the same individuals are at one time more resistant to disease than at another. This inborn or spontaneous refractory condition to an infectious disease is termed natural im- munity, in contradistinction to that acquired by recovery from infection. It is not always easy to separate these two forms of immunity. In regard to \'ariations in susceptibility, certain known facts have been accumulated. Thus, cold-blooded animals are generally insus- ceptible to infection from those bacteria which produce disease in warm-blooded animals, and vice versa. This is partly explained by the inability of the bacteria which grow at the temperature of warm- blooded animals to thrive at the temperature commonly existing in cold-blooded animals. But differences are observed not only between warm-blooded and cold-blooded animals, but also between the several races of warm-blooded animals. The anthrax bacillus is very infec- tious for the mouse and guinea-pig, while the rat is not susceptible to it unless its body resistance is reduced by disease and the amount of infection is great. The inability of the microorganism to grow in the body of an animal does not usually indicate, however, an insus- ceptibihty to its poison; thus, for instance, rabbits are less suscep- tible than dogs to the effects of the poison elaborated by the pneumo- cocci, but these bacteria develop much better in the former than in the latter. In animals, as a whole, it is noticed experimentally that the young ones are less resistant to infection than the older and larger ones, except in those still young enough to have immunizing sub- stances acquired from the mother before or shortly after birth. Decrease of Resistance. — The difficulty experienced by the majority of microbes in developing in the healthy body can be to a great extent removed by any cause which lowers the general or local vitality of the tissues. Among the causes which bring about such lessened resistance of the body are hunger and starvation, bad ventilation and heating, exhaustion from overexertion, exposure to cold, the delete- rious effects of poisons, bacterial or other, acute and chronic diseases. 152 PRINCIPLE!^ OF MICROBIOLOGY vicious liabits, drunkenness, etc. Purely local injuries, such as wounds, contusions, etc., give a point of entrance for infection, and tissue of less resistance, where the bacteria may develop and through their poison produce adjacent injury and so jjredispose to further bacterial invasion in much the same way as the heat of the forest fire dries the green trees in front of it and so prepares them to ignite. Local affections, such as endocarditis, may also afford an area of lessened resistance. The presence of foreign bodies in the tissues in like manner predisposes them to bacterial invasion. Interference with free circulation of blood and retention in the body of poisonous sub- stances which should be eliminated also tend to lessen the vitality. In these and other similar ways animals which are otherwise refractory may acquire a susceptibility to disease. Increase of Resistance by Non-specific Means. — All conditions which are favorable to the health of the body increase its resistance, and thus aid in preventing and overcoming infection. The internal use of antiseptics against bacteria is so far unsuccessful, for the reason that an amount still too small to inhibit bacterial growth is found to be poisonous to the tissue cells. The efficacy of mercury in syphilis is, possibly, an exception to the rule. In dealing with animal para- sites, and with certain molds there are a number of notable exceptions to this rule; e. g., quinine in malaria, emetin in amebiasis, trypan red in trypanosomiasis, and iodides in sporotrichosis. Such substances as leukocytic extract, nuclein, and similar organic substances contained in blood-serum, when introduced into the body in considerable quan- tity, aid somewhat in inhibiting or preventing the growth of many microbes. Even bouillon, salt solution, and small amounts of urine have a slight inliibitory action. The hastening of elimination of the microbal poisons by free intestinal evacuation and encouragement of the functions of the skin and kidneys are also of some avail. The enzymes formed by certain bacteria have been found to exert a slight bactericidal action not only on the germs which have directly or indi- rectly produced them in the body, but also on other varieties. None of these enzymes are sufficiently protective to be of practical value, nor are they equal in power to the protective substances formed by the tissues from the bacterial products. Use of Local Treatment in Limiting Microbal Invasion. — The total extirpation of the infected area by surgical means, if thoroughly carried out, removes the microbes entirely; but, unfortunately, this procedure is rarely possible. When incomplete it is frequently help- ful; but it may be harmful, for by creating tissue injury and expos- ing fresh wounded surfaces to infection it may lead to the further development of the disease. In some cases, however, like anthrax and infection from bites of rabid animals, almost complete removal of the virus, either by the knife or thorough cauterization, will prevent a general infection or so lessen the number of organisms in the body as to allow the germicidal element of its fluids to exterminate them. So also in tetanus, the invasion being limited, surgical interference may be SPECIFIC IMMUNITY 153 of g-reat vise by removing not only the bacilli themselves, bnt also that portion of their poison which has not as yet been absorlwd from the tissnes. The beneficial effects of oi)ening an abscess, or cleansing and tlraining' the pleural, peritoneal or uterine cax'itics are well known. The retention of the poisonous products of the organisms leads to their absorption, and then through their combining with some of the tissue cells and with the protective substances of the adjacent fluids the tone of the tissues is lowered at the same time that germicidal substances have been neutralized. This enables the germs to penetrate into tissues which would otherwise resist them. The mechanical effect of pressure on the walls of an abscess by its contents also aids absorption of toxins and microbal progress. Local bleeding and the application of cold probably act by lessening absorption. The ap- plication of warmth increases the blood flow to the part, and so, when the general blood-supply is germicidal, as it often is, it acts favorably on the inflammation. A similar effect of operative interference is noticed in the frequently observed beneficial result of laparotomy in tuberculous peritonitis. Antiseptic solutions have the power of cleansing and rendering sterile the surfaces of a wound — that is, of lessening the introduction of infection. After infection has taken place, however, it is doubt- ful whether antiseptic washing has much more direct influence than simple cleansing, and it certainly can have no germicidal effect at any distance from the surface, either direct or indirect. Certain infectious diseases which are comparatively superficial are probably benefited by antiseptic solutions; such are gonorrhea, diphtheria, and other inflammations of the mucous membranes. Even here, how- ever, it is impossible to do more than disinfect superficially, and in some cases any irritation of the tissues is apt to do more harm than good. In the superficial lesions of syphilis, tuberculosis, and other chronic processes, the local use of antiseptics is sometimes of great value. In these diseases the irritant effects of the antiseptics which stimulate the tissues may also be beneficial. Specific Immunity, or a Condition of the Body which Prevents the Development in it of One Variety of Microorganisms or Renders it Unaffected by their Specific Poisons. — The invasion of the body by almost every variety of microorganism is followed, if death does not quickly ensue, by conditions which for a variable period and to a variable degree are deleterious to the further growth of that variety. This more or less pronounced specific immunity may be created in various ways: 1. Through recovery from disease naturally contracted or from infection artificially produced. According to the nature of the invading microorganism this immunity may be slight, as after recovery from erysipelas or pneumonia, marked for a limited period of time, as in diphtheria and typhoid fever, or prolonged, as after scarlet fever or syphilis. The extent of this protection also varies in different persons after the same infection. ]r)4 PRINCIPLES OF MICROBIOLOGY 2. By inoculation •with microorganisms attenuated by heat, chem- icals, or other means. In this case an infection of the animal is jjro- (luced, of moderate severity, as a rule, and the immunity is not cjuite as marked ;ind lasting' as after reco^'e^y from more serious attack; but it is, nevertheless, considerable. The inoculation of sheep with the atteimated anthrax bacillus and the use of vaccination with cow-pox in man are examples of this method. o. By the injection of the living organisms into tissues where develop- ment will not take place, as the injection of diphtheria bacilli, typhoid bacilli or cholera spirilla into the subcutaneous tissues. Here the de- struction of the bacteria with the absorption of their products causes mild chemical poisoning, with considerable resulting immimity. 4. By the injection of the dead bodies of bacteria or of the chemical products which they elaborate and discharge into the surrounding culture media during their life. This produces a less marked immunity than when the living culture is used, but the method is a safer one. 5. By the injection of the blood-serum of animals which have pre- viously passed thrcjugh a specific disease or have been inoculated with the microbal products. The first, probably, to think of the possibility of effecting this was Kaynaud, who in 1877 showed that the injection of large quantities of serum derived from a vaccinated calf into an animal prevented its successful vaccination. The results obtained bj' V. Behring and Kitasato upon diphtheria and tetanus, where the serum neutralized the poisons rather than the direct development of the bac- teria, gave a still greater impetus to these investigations. Suitable animals after repeated infections gradualh' accumulate in their blood considerable amounts of these protecti^'e substances, so that very small amounts of serum inserted in another animal will inhibit the growth of the microbes or neutralize their products. Thus, 0.1 c.c. of a serum from a horse frequently infected by the pneumococcus will prevent the develr)pment in the body of a rabbit of many thousand times the fatal dose of very virulent pneumococci, and a few times a fatal dose of less virulent ones, the actual number as well as the virulence of the bacteria affecting the protective value of the serum. These protective substances are found also in other ffuids of the body than in the blood; they occur, indeed, in the substance of many cells to a greater or less extent. The immunity produced by these five methods affects the entire body, as is natural, since the blood and lymph which contain the pro- tec ti\'e substances are distributed everywhere. These protective sub- stances pass from the blood through the walls of the capillaries and finally find their way to the lymph and back to the blood. When the immunity is but slight, infection may take place in the more sensitive regions (.)r wliere a large number of organisms have gained access, and still be impossible in those tissues having more natural resistance or slighter infection. Passive as Contrasted with Active Immunity. — After the immune serum is injected into man the immunity is greatest at the time of its reception into the blood. This, of course, is instantaneous after an PRACTICAL THERAPEUTIC VALUE OF BACTERICIDAL SERA 155 intravenous injection, but only after thirty-six to forty-eight hours when given suheutaneously (.Fig. 05), und then lieelines, being lost rather quickly (in several months or weeks, according as to Avliether nr not the serum is injectetl into the same species of animal as the one from which it was draw n or into another species, when repeated injections are re([uired tv maintain the innnnnit.y)- This passive imnumity is distinctly in contrast to the active imnumity acquired after the intro- duction of microlies or microbal products, where the tissues of the organism, in ways to us unknown, throw out, in response to the microbal stimidus, inhibitory or antitoxic substances. Here immunity is actually lessened for one or two days, and then is increased, and reaches its height a week or ten days after the injection, and then continues for a week or two, when it slowly declines again and is lost after several nn)nths or years. Fig. 65 Units 9 8 7 6 5 4 3 2 I ^ -^ rrrr ,^ / --''' >;:. ^ / / / / / // / 7 / / A ] Mi ba( pel the in poi ] ser on ani 6 12 l8 24 30 36 42 48 54 60 -66 verage antitoxin content of one c.c. of the blood of goats re injection of 10,000 units of antitoxin. [.imitation of Curative Power of Serums which A croorganisms. — As a rule, the serum has to b jteria introduced into the body have multiplied j iod has elapsed the serum usually fails to act. Tl germicidal and antitoxic substances of the sei amount and partly because suitable antibodies -tion of the varied types of poison produced by ni 'ractical Therapeutic Value of Bactericidal £ ums having specific protective properties has be a large scale in man as a preventive of infecti mals injections of some of the very virulent ba ?2 ceivi ctr e gi ;rea1 lis is um dev icro Sera en t on. cter 78 ng a )irec ven :iy- par are elop orga — T ried In ia, £ '4 subc tly J befc Aft tly I insi for nisn he pra susc s p 30 < Litane 4.gai )re 1 er tl ieca ffici onlj as. use ctica epti leun 6 hrs. ous nst the lat use int ' a of iiy ble 10- 15G PRINCIPLES OF MICROBIOLOGY cocci, streptococci, meningococci, and typlioid bacilli, can l^e robbed of all danger if small doses of their respective serums are given before the bacteria have increased to any great extent in the bod}r. If gi\'en later they are usually ineffective. For some bacteria, such as tubercle bacilli, no serum has been obtained of sufficient power surely to prevent infection. Through bactericidal serums, therefore, we can immunize against many infections, and even stop some just commencing; but as yet we cannot cure an infection which is already fully developed, though even here there is reason to believe that we may possibly pre- vent an invasion of the general system from a diseased organ, as by the pneumococcus from an infected lung in pneumonia. On the whole, the serums which simply inhibit the growth of microbes without neu- tralizing the toxins ha\'e not given conclusive evidence, as obser^'ed in practice, of great value in already developed disease, unless the serum can be brought in considerable concentration in contact with the infected tissues as is done by intraspinal injections in cerebrospinal meningitis. Relative Development of Antitoxins and Bactericidal Substances in the Different Infections. — Although the serum of animals which have been infected with any one of many varieties of bacteria is usually both antitoxic and bactericidal, still, one form of these protective sub- stances is usually present almost alone; thus antitoxic substances are present almost exclusively in animals injected with two species of bacteria which produce powerful specific poisons — viz., the bacilli of diphtheria and tetanus. When the toxins of either of these are injected in small amounts the animals after complete recovery are able to bear a larger dose without deleterious effects. To v. Behring and Kitasato we owe the discovery that this protecting substance accumu- lates to such an extent in the blood that very small amounts of serum are sufficient to protect other animals from the effects of the true extra- cellular toxins. Except the diphtheria and tetanus bacilli, a few only of the impor- tant parasitic bacteria attacking man produce extracellular toxins in any considerable amount, thus being capable of causing the production in the body of antitoxins, and even these bacteria do it to a far less extent than those of tetanus and diphtheria. Following them are the dysentery and plague bacilli, and then the cholera spirilla, the typhoid bacilli, the gonococci, meningococci, streptococci, etc. These latter bacteria when injected excite more of the substances which inhibit bac- terial growth than of those which neutralize their toxins. The bacillus of symptomatic anthrax and of botulismus and the vegetable poisons ricin, crotin, and abrin also produce specific antitoxins. So far, neither protozoa nor molds have been shown to produce specific antitoxin, though with a few species agglutinins and other antibodies have been demonstrated. Antitoxin a Preventive. — Antitoxin prevents the poisonous action of toxin. It does not restore the cells after they have been injured by the toxin; it is, therefore, like the bactericidal substances, a pre- METHOD OF ADMINISTRATION 157 ventive rather than a cure. We find, experimentally, that a very mueh smaller amount of antitoxin will neutralize a fatal dose of toxin in an animal, if given before or at the same time, than if given only shortly after it. An animal already fatally poisoned by the toxin is unaffected by any amount of antitoxin. Method of Administration. — Antitoxins and germicidal substances are absorbed by the gastro-intestinal tract to only a very slight extent — certainly less than 2 per cent. They must, therefore, be introduced subcutaneously, intraspinally, or intravenously to enter the body in appreciable amounts. CHAPTER IX. NATUJIE OF THE PROTECTIVE DEFENCES OF THE BODY AND THEIR MANNER OF ACTION— EHRLICH'S "SIDE CHAIN" AND OTHER THEORIES. The fluids and tissues of the animal hotly under the normal con- ditions of life are, as we have seen, not only unsuitable for the growth of the great majority of the \'arieties of organisms, but even germicidal to the living organisms. In seeking to account for the germicidal property of the blood, which to a greater or less extent affects all microbes, we cannot find it either in the insufficient or excessive concentration of the nutritive substances, or in the temperature, or in the reaction. We are thus driven to the conclusion that the body fluids and cells contain substances which are deleterious to microorganisms. Germicidal Properties of the Blood. — The bactericidal effect upon most bacteria of the blood serum, noted by Nuttall in ISSfi, is now undisputed, and is readily shown by the fact that moderate numbers of bacteria when inoculated into freshly drawn blood usually die soon, and this destruction may be so rapid that in a few hours none of millions remains alive. Even when some of the bacteria survive there is for a time a decrease in the number living. Buchner in 1S89 showed that serum heated to 55° lost its destructi\'e power. He believed that in serum there was but a single bactericidal substance and called it alexin. In 1S94-95 Pfeift'er showed that when cultures containing dead or living cholera spirilla or typhoid bacilli are injected subcutaneously into animals or man, specific protective substances are formed in the blood of the individuals thus treated. These substances confer a more or less complete immunity against the invasion of the living germs of the respective diseases. He also described the occurrence of a peculiar phenomenon when some fresh culture of the typhoid bacillus on agar is added to a small quantity of serum from an animal immunized against typhoid bacilli and the mixture injected into the peritoneal cavity of a non-immunized guinea-pig. After this procedure, if from time to time minute drops of the liquid be withdrawn in a capil- lary tube and examined microscopically, it is found that the bacteria previously motile and vigorous, and which remain so in control animals inoculated without the specific serum, rapidly lose their motility and die. They are first immobilized, then they become somewhat swollen and agglomerated into balls or clumps, which gradually become paler and paler, until finally they are dissolved in the peritoneal fluid. This GERMICIDAL PROPERTIES OF THE liLOOD 150 process usually takes place in about twenty minutes, provided a suf- ficient degree of immunity be present in the animals from which the serum was obtained. The animals injected with the mixture of the serum of immunized animals and typhoid cultures remain unaffected, while control animals treated with a fluid containing only the serum of non-immunized animals mixed with typhoid cultures die. Pfeiffer claimed that the reaction of the serum thus employed is so distinctly specific that it could serve for the difl^erential diagnosis of the cholera vibrio or typhoitl bacillus from other vibrios or allied bacilli, such as Finkler's and Prior's or those of the colon group respectively. He thus showed that there was a great increase in the bactericidal power of a serimi after immunization for the species of bacteria used in immu- nization. Metchnikoff then showed that the immunized serum added to peritoneal fluid in the test-tube would have the same effect on the spirilla. In March, 1896, Pfeiffer and Kolle published an article entitled "The Differential Diagnosis of Typhoid Fevers by Means of the Serum of Animals Immunized against Typhoid Infection," in which they claimed that by the presence or absence of this reaction in the serum of convalescents from suspected typhoid fever the nature of the disease could be determined. Bordet in 1895 reported that defibrinated blood filtered free of blood cells could be used instead of the peritoneal fluid and that if to a serum from an immunized animal, which had lost its bactericidal power through age, fresh serum from an untreated animal was added, the serum re- gained its destructive powers, i. e., it was activated, although the fresh serum by itself had almost no effect. These observations of Pfeiffer and Bordet indicated clearly that two types of substances were required to destroy cells. Both of these were present in fresh immune serum, one of which was stable and more or less specific, and the other un- stable and non-specific. The latter was proven to be present in all blood, while the former existed, except in minute amount, only in the blood of the immunized. The number of microbes introduced in a germicidal test is of great importance, for the serum with its contained substances is capable of destroying only a certain number, and after that it has lost its germicidal properties. Thus the following test illustrates this: Approximate number alive after being liept at 37^ C. ^0. of bacteria Amount of in 1 c.c. fluid serum added 30,000 100,000 1,000,000 0.1 c.c. 0. 1 c.c.c 0.1 c.c. One hour Two hours Four hours 400 2 .5,000 1,000 2,000 400,000 1,000,000 5,000,000 Haas found that the circulating blood is not always bactericidal for any given variety of bacteria to the same extent the serum is. During the testing of the bactericidal power of the serum on dif- ferent bacteria it was discovered that numerous \'arieties were not destroyed by the serum alone, but only when exposed to both serum and leukocytes. 160 PRINCIPLES OF MICROBIOLOGY During these earlier years Metchnikoff perceived that the infected host was too httle considered, and he drew attention to the role of the leukocytes. He noted that in inflammation there is an active mi- gration of leukocytes through the walls of the vessels toward the infect- ing bacteria. If the bacteria are very virulent they continue to increase, destroying the leukocytes. If the bacteria are not sufficiently virulent to set up a progressive inflammation they are themselves disintegrated. Later it was discovered that bacteria were much more susceptible to the leukocytes if they had been acted upon by a serum from a body that had had an infection. (See chapter on Opsonins.) Buchner made many experiments on the nature of the process. He showed that bacteria absorbed these bactericidal substances. Later, Bordet, Ehrlich, and others established that the alexin of Buchner was really a mixture of two types of substances of which one, named "immune body," "sensitizer," or "opsonin," is developed as the re- sult of the injection of foreign-cell substance, and the other, named "complement" or "alexin," is present in the blood of normal animals, and is not increased by injection. Neither one of these types of sub- stances alone destroys bacteria, while together they destroy certain varieties. Other bacteria require the action of the complement-like ferment in the leukocytes also. The Nature of Bacteriolytic, Hemolytic, Cytolytic Sera. — Bordet, through his own researches and those of Gruber and Durham, was able to show that the same type of reaction took place in the animal body when cells of any kind were injected. He showed, for instance, that there was a close similarity between bacteria and the cells of the blood. By immunizing an animal, species A, with red-blood cells of animal, species B, he found that the blood of A became hemolytic for the cells of B, just as if immunized with cholera spirilla it would have been bacteriolytic for cholera spirilla. Since then truths obtained from investigation with any type of cells have been applied equally to all others. This made it possible for Ehrlich, Bordet, and others to study the nature of these processes upon blood cells instead of bacteria. Experiments Devised by Ehrlich to Show the Nature of Cytolytic (Bacterio- lytic, Hemolytic, etc.) Substances in the Blood. — Ehrlich usked himself two ([uestions: (1) \\^hat relation does the hemolytic serum or its two acti^'c com- jionents, immune body and complement, bear to the cell to be dissolved? (2) On what does the specificity of this hemolytic process depend? He made his ex)ieriments with a hemolytic serum that had been derived from a goat treated with the red cells of a sheep. This serum, therefore, was hemolytic specifically for sheep-blood cells — i. e., it possessed increased solvent projjerties exclusively for sheep-blood cells. Ehrlich argued as follows: "If tlie hemolysin is able to exert a specific solvent action on sheep-blood cells, then either of its two factors, the immune Ijody or the alexin (complement) of norm;il serum, nuist possess a specifics affinity for these I'cd cells." To show this lie devised in conjunction with IMorgenroUi tlu^ following series of experiments: Experiment 1. — The .serum that was specifically hemolytic for sheep-lilood cells was made inactive by heating to .55° C., so that then it contained only the lieat-resistant substance (immune body). To this was then added a sufficient ciuantity of sheep red-blood cells, and after a time the mixture was centrifuged. NAMES ATTACHED TO SUBSTANCES PHUDUCfNG CYTOLYSIS Ull lOhrlich and Morgenroth wrre now able to show that the red cells liatl combined with all the heat-resistant substances, and that the supernatant clear liquid was free from the same. In order to prove that such was the ease they proceeded thus: To some of the clear centrifuged fluid they added more sheep red cells; and, ui order to reactivate the serum, a sufficient amount of alexin in the form of normal serum was also added. The red cells, however, did not dissolve — there was no sensitizing substance. The next point to prove was that immune body had actually comliined with red cells. The red cells which had Ijeen separated by the cei\trifuge were mixed with a little normal salt solution after freeing them as much as possible from fluid. Then a little alexin in the form of normal serum was added. After remining thus for two hours at 37° C. these cells had all given up their hemoglobin to the surrounding fluid. In this experiment, therefore, the red cells had comljined with all the sensitizing substance, entirely freeing the serum of the same. The second important ciuestion solved by these authors was this: What relation does the alexin bear to the retl cells? They studied this by means of a series of experiments similar to the preceding. ExPERiiMENT 2. — Sheep red-blood cells were mixed with normal — i. e., not hemolytic goat serum. After a time the mixture was centrifuged and the washed red cells tested with the addition of sensitizing substance to determine the pres- ence of alexin. It was found that in this case the red cells, in direct contrast to their behavior toward the sensitizing substance in the first experiment, did not combine with even the smallest portion of alexin, and remained unchanged. This experiment showed that the sensitizing substance first combined with the cell and then only could the alexin unite with the combined cell-immune bodj^ complex. Experiment 3. — The third series of experiments was undertaken to show what relations existed between the blood cells on the one hand and the sensitizing substance and the alexin on the other, when Ijoth were present at the same time, and not, as in the other experiments, when they were present separately. This investigation was complicated by the fact that the specific immune serum very rapidly dissolves the red cells for which it is specific, and that any pro- longed contact between the cells and the serum at ordinary temperatures, in order to effect union, is out of the question. Ehrlich and Morgenroth found that at 0° C. no solution of the red cells by the hemolytic serum takes place. They therefore mixed some of their specific hemolytic serum with sheep-blood cells, and kept this mixture at 0° to 3° C. for several hours. No solution took place. They now centrifuged and tested both the sedimented red cells and the clear supernatant serum. It was found that at the temperature 0° to 3° C. the red cells had combined with all of the sensitizing substance, but had left the alexin practicallj^ untouched. The addition of red cells in the experiments was always in the form of a 5 per cent, mixture or suspension in 0.85 per cent. — i. e., isotonic-salt solution. The significance of the last of the above-cited experiments is, according to Ehrlich, at once apparent. It is that the sensitizing substance possesses one combining group with an intense affinity (active even at 0° C.) for the red cell, and a second group possessing a weaker affinity (one requiring a higher tem- perature) for the alexin. Names Attached to Substances Producing Cytolysis. — Different investigators have applied to them different names. The one which is resistant to heat, which attaches itself directly to the cells, even at low temperatures, and is increased during immunization, is called sensitizing substance, interbody, amboceptor, or immune body. The other, which is sensitive to heat, is present in the healthy normal serum, is not increased during immunization, and 11 I(i2 PRINCIPLES OF MICROBIOLOGY which unites with the cells' protoplasm only at temperatures above the freezing-point, is called alexin, or complement. The immune body attaches itself to the cellular substance, but does not appreciably harm the cells. The complement destroys the cells after the immune body has made the cell vulnerable. According to Ehrlich, the immune body first unites with the proto- plasm of the cell and this develops in the immune body an affinity for the complement and the two unite. (See Fig. (id.) He believes that it is through the immune body that the complement exerts its action on the cell. Very similar to the immune body is the substance called opsonin. Thisuniteswith the cell, but instead Fio. 66 of making it sensitive to the complement it \x^ Wi/ makes it sensitive to some ferment contained ■I jl|| in the leukocytes. The destruction of organ- ^B Hf ""'■* isms by the opsonins and leukocytes will be rWn f*\---K considered in detail in a special chapter. Bordet's Theory. — Bordet supposes that, instead of the tissue cell receptors which have combined with the toxin or foreign cell substance (antigen, haptine) producing an excess of similar receptors, the body of the animal that is immunized, instead of repro- ducing old receptors in large amount without changing them, builds up substances which in their character resemble but are not iden- tical with preexistent principles. These new substances ha\-e become endowed with a more marked affinity for the specific antigen in question. Bordet considers that Ehrlich, in offering explanations which seem definitive, has caused certain problems which ha\'e scarcely been touched upon to be regarded as settled. According to Bordet, Ehrlich is wrong in attributing such special properties to the immune body alone rather than to both antigen and immune body to an equal degree. He states that, "as a matter of fact, these pheno- mena should be related, not as regards antigen or antibody considered separately, but as regards the complexes which result from their union, and it is evident that the special properties of the antigen must affect markedly and perhaps to a preponderating degree the qualities of such complexes. Just as the union of agglutinins with microbes produces in them a remarkable sensitivity to the agglutinating effect of electrolytes by modifying their ])roperty of molecular adhesion, in a similar way sensitizers confer on their antigens a similar modified property of adhesion, namely, alexin absorption." In his opinion, antibodies, whatever their nature, act \'ery much alike; but the effects which they l)roduce differ with the antigen in question. Aluir has shown that when cells are saturated with both immune bod\' ^■Y-D Graphic representation of amboceptor or receptors of the third order and of complement, showing on left the immune body uniting complement to foreign cell and on right the action of anticomplement, binding complement: A, com- plement ; B, intermediary body : C, receptor; D, cell; E, anti- complement. ORIGIN OF COMPLEMENT 163 ami complement, the addition of fresh cells causes a splitting' off of im- mune body, but not of complement. This throws further doubt upon the direct union of immime body and complement. Tliere ai-e exceptional normal sera, the comijlenient of which may be fixed liy certain cells without the presence of an immune scrum. Malvoz showed that this is the ease with dog serum mixed with B. authracis. This serum acts, moreover, as if it contained a true sensitizer, Ijecause, in tlie presence of this organism it will cause the fixation of the complement of the sera of rabbits and guinea-pigs. Most of the experiments which ha^'e been made with the purpose of clearing up these difficult problems have been made upon red-blood cells. Here the absorption of the immune bodies at low temperatures and the lack of noticeable injury until the complement is added at a suitably high temperature is very striking. Multiplicity of Immune Bodies and Complements. — The immune bodies are very numerous and fairly specific in their action. The complement substance is much less specific and, although probably multiple, when chemically considered, each \'ariety acts upon widely different bacteria and cells after they have united with the immune body. There is little reason to think that the complement of one animal is any more capable of attacking cells prepared by immune bodies developed in its blood than by immune bodies developed in some other species. Relation between Virulence and the Building of Immune Bodies. — It is believed by most to take place the more rapidly the more viru- lent the infecting organisms. In our experiments this has not been e^•ident. It must be remembered that increase of virulence for one species of animal does not mean increase for all animals; so that in order to draw conclusions, the animal upon which the ^'irulence is tested must be the same variety as the one being immunized. Origin of Immune Bodies. — Their source must undoubtedly be at- tributed to the cells, but probably only certain cells produce them. The red-blood cells, for instance, seem rather to destroy than to increase them. Injections into the lung and into the subcutaneous tissues of toxins and microbal substances give rise to the formation of antibodies which are certainly formed partly, if not wholly, locally, and later find their way to the blood. The nuclein derived from the cells, although it has a general germicidal action, and may enter into the complement (alexin), has different properties, and so cannot itself be one of these bodies. Origin of Complement (Alexin). — The cells which have abundant nuclear substance, such as the leukocytes and lymph cells, seem espe- cially to be a source, and MetchnikofF asserts their preeminent role as the producers of both complements and immune bodies. Buchner and others have found that after injection of bacterial filtrates the leukocytes were attracted in great numbers to the region of injection, 1G4 PRINCIPLES OF MICROBIOLOGY and that the fluid here, which was rich in leukocytes, was more bac- tericidal than that of the blood-serum elsewhere. Some claim to have demonstrated that along with increased leukocytosis there is a general increase in the complement in the blood; still, it has not yet been positively established that the complement is derived solely from the leukocytes, nor from all leukocytes, and a mere increase in them does not always mean an increase in the complement. Multipartial or Polyvalent Sera. — Microbes are not homogeneous masses of protoplasm, but are made up of various molecules which differ biologically from one another. Conforming to this, the anti- substances, immune bodies (antitoxins, opsonins, etc.), which appear in a serum are made up of the sum of the antibodies which correspond to these partial elements in the microorganism. These separate groups are called "partial groups." An immune serum, therefore, consists of the partial groups which correspond to the separate partial elements of the microbal body. We are further able to show that these partial elements in one and the same microbal species are not the same for all individuals of that species. Thus one culture of streptococci or of Bacillus coli may have a few partial elements which differ from those of another culture. What is the consequence of this? The consequence will be that when we immunize with a culture a of such bacteria we shall obtain a serum which acts completely on this culture, for in this serum all the partial elements present in culture a are represented. If, however, we employ culture b, c, or d, which perhaps possesses other partial elements, we shall find that the serum does not completely affect these cultures. As already stated, such a condition of things is met with in inflammations due to streptococci and other bacteria, and is, therefore, of considerable practical importance. It is because of this fact that a serum from an animal immunized to one culture acts best only in a certain percentage of cases. In order to overcome this difficulty in persons infected with these bacterial species we have no choice but to make sera, not by means of one culture, but by means of a number of different strains of the same species. .The result of this will be that, corresponding to the various partial elements in these different cultures, we shall obtain a serum containing a large number of the partial groups. Such a serum will then exert a specific action on a large number of different cultures, but not quite as great an influence on any one as if only that variety had been injected. In other words, the development and the closer analysis of the problem of immunity, especially during the past few years, have shown us that we must make use, more than heretofore, of so-called polyvnleiit or multipartial sera. In the serum therapy of streptococcus infections, of dysentery, etc., the profluction of such multipartial sera is an advantage in practice. Owing to these partial groups also, a serum — e. (]., antityphoid serum — can specifically affect to a very slight degree a closely allied species of bacterium, like Bacilhis coli, for example. For it is kn(^wn that closely related species of bacteria possess certain partial groups in common, and a serum is thus produced ANTITOXIN, NATURE AND AFFINITY 165 which to a certain extent acts on such alUed species. Tliis constitutes what is known as the "group reaction." Antitoxin, Nature and Affinity. — During the investigations on the bactericidal power of the blood the discovery of the antitoxins which combine with the toxins, but leave iuitt)uched the bacteria, was made by V. Behring and Kitasato, and the nature of the union was investigated by Bordet, Ehrlich, and others. The pecidiar facts developed by these extensive studies became the basis for Ehrlich's side-chain theory. During the earlier investigations on diphtheria toxin the filtered or sterilized bouillon, in which the diphtheria bacillus had grown and produced its "toxin," was supposed to require for its neutralization an amount of antitoxin directly proportional to its toxicity as tested in guinea-pigs. Thus, if from one bouillon culture ten fatal doses of "toxin" were required to neutralize a certain quantity of antitoxin, it was believed that ten fatal doses from every culture, without regard to the way in which it had been produced or preserved, would also neutralize the same amount of antitoxin. Upon this belief was founded the original v. Behring-Ehrlich definition of an antitoxin unit, viz., that it was ten times the amount of antitoxin which neutralized ten fatal doses of toxin. The results of tests by different experimenters with the same anti- toxic serum, but with different diphtheria toxins, proved this opinion to be incorrect. Ehrlich^ deserves the credit for first clearly perceiving this. He obtained from various sources twelve toxins and compared their neutralizing value upon antitoxin; these tests gave most interesting and important information. The results in four toxins, which are repre- sentative of the twelve, are as shown in the following table : Smallest number of fatal doses of Fatal doses required toxic bouillon re- to "completely^ Toxin Estimated quired to kill a neutralize one anti- L+ — Lo Data upon "toxin" specimen "minimal" 250-gm. guinea- toxin unit" as de- = fatal specimen given number of fatal dose pig within 5 days, termined by the doses. by Ehrlich. Ehrlich. for 250-gm. when mixed with health of the guinea- guinea-pigs. one antitoxin unit, "L+ Ehr- lich." pig remaining unaf- fected "Lo" Ehrlich. 4 0.009 39.4 33.4 6 Old, deteriorated from 0.003 to 0.009. 7 0.0165 76.3 54.4 22 Fresh toxin, pre- served with tri- cresol. 9 0.039 123 . 108.0 15 A number of fresh cultures grown at 37° C. 4 and 8 days. Tested immediate- 12 0.0025 100.0 50.0 50 ly after its with- drawal. From the facts set forth in the table, Ehrlich believed that the diphtheria bacilli in their growth produce a toxin which, so long as 1 Die Wertbemessung des Diphtherieheilsenims und deren theoretische Grundlagen. Klinisches Jahrbuch, 1897. 166 PRINCIPLES OF MICROBIOLOGY it remains clieinically unaltered, has a definite poist)n the prodtidiim of diphtheria antitoxin: The horses used should be young, vigorous, of fair size, and abso- lutely healthy. The horses are severally injected with 1(),0()() units of antitoxin so as to allow giving a much larger dose of toxin' than would otherwise be safe and thus gain several weeks in time. The following figures give the actual injections in a horse which produced an luiusually high grade of serum. Injections of toxin were given at first every two days and then later every tliree days in the following amounts: First day, 12 c.c. toxin (^Lj c.c. fatal dose), the 10,000 units of diphtheria antitoxin having been given the previous day. Second and later injections of toxin without antitoxin at three-day intervals as follows: 15 cc, 20 c.c, 30 e.c, 40 c.c, 50 c.c, 60 c.c, 80 cc, 100 c.c, 125 c.c, 150 c.c, 170 c.c, 205 c.c, 250 c.c, 300 cc (fortieth day). The injections were gradually increased until, on the sixtieth day, 675 c.c. were given. The whole injection should not be in one place, but divided into six or eight jjortions. The antitoxic strength of the serum was on the twenty-eighth day, 225 units; on the fortieth day, 850 imits; and on the sixtieth day, 1000 units. Regular bleedings were made weekly for the next four months, when the serum had fallen to 600 units in spite of weekly, gradually increasing doses of toxin. If the antitoxin is not given we begin with 10 fatal doses of toxin diluted to 10 c.c. and increase the amount each time about 25 per cent. There is absolutely no way of judging which horses will produce the highest grades of antitoxin. Very roughly, those horses which are extremely sensitive and those which react hardly at all are the poorest, but even here there are exceptions. The only way, therefore, is to bleed the horses and test then serum at the end of six weeks or two months. If only high-grade serum is wanted all horses that give less than 150 units per cc. are discarded. The retained horses receive steadily increasing doses, the rapiditj^ of the increase and the interval of time between the doses (two days to one week) depending somewhat on the reaction following the injection, an elevation of temperature of more than 3° F. being undesu-able. At the end of three months the antitoxin 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 none so far has given as much as 2000 units per c.c. The very best horses if pushed to their hmit continue to furnish blood containing the maximum amount of antitoxin for several months, and then, in spite of increas- ing injections of toxin, begin to furnish blood of gradually decreasing strength ' The culture, after a week's growth, is removed, aud having been tested for purity by microscopic and culture tests is rendered sterile by the addition of 10 per cent, of a 5 per cent, solution of carbolic aeid. After forty-eight hours the dead bacilli have set- tled on the bottom of the jar and the clear fiuid above is siphoned off, filtered, and stored in full bottles in a cold place until needed. Its strength is then tested by giving a series of guinea-pigs carefully measured amounts. Less than 0.005 c.c., when injected hypodermically, should kill a 250-gram guinea-pig. 172 PRINCIPLES OF MICROBIOLOGY If an interval of three months' freedom from inoculations is given every nine months, the liest horses furnish high-grade serum during their periods of treat- ment for from two to four years. Tetanus Antitoxin Production. — This is carried on exactly as in tlie case of diphtheria antitoxin except that one proceeds more slowly. Good horses yield a serum containing 200 or 300 units per c.c. For the collection and preservation of the antitoxin the blood is withdrawn from the jugular vein by means of a sharp-pointed cannula, which is plunged through the vein wall, a slit having been made in the skin. The blood is carried by a sterile rubber tube into large Erlenmeyer flasks, held slanted, or into cylindrical jars, and allowed to clot. The serum is drawn off after four days by means of sterile glass and rubber tubing, and is stored in large flasks. When the globulins are to be separated the blood may be added directly to one-tenth of its volume of a 10 per cent, solution of sodium citrate. This prevents clotting of the blood. Small phials are filled with the serum or globulin solu- tion. The phials and their stoppers, as indeed all the utensils used for holding the serum, must be absolutely sterile, and every possible pre- caution must be taken to avoid contamination. An antiseptic may be added to the serum as a j^reservative, but it is not necessary except when the serum is to be sent to great distances, where it cannot be kept under supervision. Kept from access of air and light and in a cold place it is fairly stable, deteriorating not more than 30 per cent., and often much less within a year. Diphtheria antitoxin, when stored in phials and kept under the above conditions, contains within 10 per cent, of its original strength for at least two months; after that it can be used bj' allowing for a maximum deterioration of 2 per cent, for each month. The antitoxin in old serum is just as effective as in that freshly bottled, only there is less of it. The serum itself is less apt to produce rashes. All producers put more units in the phials than the label calls for, so as to allow for gradual loss of strength. Standardizing of Antitoxin Testing. — Ehrlich, with the knowledge gained by his investigations, called attention to the necessity of all laboratories using the same toxin so that a unit would become a definite amount. The German Government undertook to supply this need by testing all sera produced in Germany and by supplying a carefully tested serum to be used by the producers as a standard to test their toxins and antitoxins by. In this way smaller testing stations can make their results correspond with those of the central station. The United States Marine Hospital Laboratory has also distributed to laboratories in the United States an equally carefully standardized serum for diphtheria and a toxin for tetanus. It is seen that the old definition of v. Behring and Ehrlich, that a diph- theria antitoxin unit contains the amount of antitoxin which will protect the life of a guinea-pig from one hundred fatal doses of toxin, is true only in a general sense and for freshly prepared toxins. The important point to remember is that for fourteen years the German, United States, THE SEPARATION OF ANTITOXIN FROM SERUM 173 and other governments have kept carefully preserved a serum which is supplied to all testing stations, so that the unit measure is the same in all countries and from year to year. The actual test to estimate the number of units in one c.c. of an antitoxin serum is, therefore, carried out as follows: Six guinea-pigs are injected with mixtures of tested toxin and the serum to be tested. In each of the mixtures there is the amount of toxin sufficient to just neutralize 1 unit of antitoxin. This has been standardized by testing with the serum from Washington. In each of the mixtures the amount of the unknown serum varies; for instance, No. 1 would contain 0.002 c.c. serum; No. 2, 0.003 c.c; No. 3, 0.004 c.c; No. 4, 0.005 c.c, etc If at the end of four days, Nos. 1, 2, and 3 were dead and Nos. 4, 5, and 6 were alive we would consider the serum to contain over 200 units per c.c. and less than 250 units because 0.004 (.j^y^ c.c) c.c of the serum did not protect the guinea-pig for a full four days, while -jiin c.c protected the guinea-pig for longer than four days. We would then test again between these limits. When we test for experimental purposes sera with very little antitoxin, we often use only one-tenth the above amount of toxin. In this case the resistance of the guinea-pig must be considered so that the guinea-pig must not only not die, but must remain well. The mixed toxin and antitoxin must remain together for fifteen minutes before injecting, so that complete union may occur. Doerr showed that very small amounts of the mixture could be injected intracutaneously. Tetanus antitoxin is tested in the same way except that the serum is tested against 1000 fatal doses of standard tetanus toxin instead of 100 of diphtheria toxin. The Separation of Antitoxin from Serum. — There have already been many attempts to accomplish this in the case of the antitoxins. Those interested in the chemical side of these investigations are re- ferred to the recent article by Banzhaf, already mentioned. In 1900, Atkinson, working in the Research Laboratory of the Health Depart- ment, eliminated all but the globulin from the antitoxic serum, and we tried this partially refined serum in 36 cases. The results were so nearly identical with an equal number of cases treated with the whole serum from the same horse that it did not seem to be worth while to go to the expense of preparing such an antitoxic solution. The idea that a practical separation of the antitoxin from much of the protein non- antitoxic portion of the serum was possible was not given up. In August of 1905 we began trials with an antitoxic preparation which offered grounds for hoping for better success. Dr. R. B. Gibson, chemist in the Research Laboratory, placed the ammonium sulphate precipitate from the antitoxic serum in saturated sodium chloride so- lution and found that the portion of the globulin soluble in this con- tained all the antitoxin. In this way the nucleoproteins and the insoluble globulins present in the Atkinson preparation were elimi- nated, as jthe following summary shows : _^Ordinary_j^anti toxic serum contains serum globulins (antitoxic), 174 PRINCIPLES OF MICROBIOLOGY serum globulins (non-antitoxic), serum albumins (non-antitoxic), serum nucleoproteins (non-antitoxic), cholesterin, lecithin, traces of bile-coloring matter, traces of bile salts and acids, traces of inorganic blood-salts and other non-protein compounds. Refined serum con- tains serum globulins (antitoxic), traces of serum globulins (non- antitoxic), dissolved in dilute saline solution. Later, Dr. E. J. Banzhaf, who had succeeded Gibson, discovered that if the antitoxic serum or plasma was heated to 57° for 18 hours there was a change of a con- siderable portion of the soluble globulins into insoluble globulins. The antitoxin remained unchanged. This permitted a greater elimination of the non-antitoxic proteins. Method of Concentration. — The material we use is blood-plasma instead of blood-serum. This is obtained by allowing the blood to flow directly from the jugular vein of the immunized horse into 10 per cent, sodium citrate solution, which prevents it from clotting and allows the red corpuscles to settle out. This plasma is used, in place of serum, merely as a matter of convenience and economy. Isolating the Antitoxin Globulins. — From year to year Banzhaf has succeeded in discovering methods for further purifying the anti- toxin. These methods have greatly lessened the early rashes and subse- ciuent "serum sickness." Briefly his latest method is as follows: The citrated plasma is diluted with half its volume of water and saturated ainmoniimi sulphate solution is added up to 30 per cent, saturated solution. This mixture is heated up to 60° centigrade and held there for one hour. Then filtered while hot. The precipitate contains the native non-antitoxic proteins and a large amount of non-antitoxic pro- teins newly formed by the above method of heating. This precipitate is discarded. The filtrate is brought up to 50 per cent, saturated ammonium sulphate solution. The resulting precipitate contains only pseudoglobulin and antitoxin. The albumin remains in solutions and is discarded. The pseudoglobulins and antitoxin precipitate is pressed to remove excess of fluid and then dialyzed until free from salts. After dialysis is completed 0.8 per cent, sodium chloride is added for iso- tonicity and O.o per cent, trikresol for preservation. It is then filtered through paper pulp to remove extraneous matter, then through a Berkefeld clay filter to remove bacteria, tested for sterility and potency and filled in sterile syringes. The above method gives a concentration of about six times the original potency. Aggressins. — A further contribution has recently been made to the problems of virulence and immunity in the form of the "aggres- sin theory" of Bail. Apparently it grew out of an attempt to explain the so-called "phenomenon of Koch" — an observation made years ago by Koch— to the efi'ect that tuberculous animals when inoculated intni])eritoncall\' with a fresh culture of tubercle bacilli succumb qnickly to an acute attack of the disease, the resulting exudate contain- ing almost exclusively lymphocytes. Bail found that if tul)ercle bacilli, together with sterilized tuberculous exudate, were injected into healthy guinea-pigs, the animal died very suddenly — i. e., in twenty-four hours AGGHESSINS 175 or tliereal)oiits. The exudate alone had no ai)preeia,l)1e eil'ect on the animal, while inoculation with tubercle bacilli alone jjroduced death in a number of weeks. He therefore concludes that there is something in the exudate that allows the bacilli to become more aggressive, and hence has called this hypothetical substance "aggressin." He thinks it is an endotoxin liberated from the Inicteria as a residt of bacteriolysis and that it acts by paralyzing the polynuclear leukocyte, thereby pre\'enting phagocytosis. Heating the exudate to ()0° ('. increases rather than diminishes its aggressive properties and small doses act relatively more strongly than larger ones. These facts he explains by assuming the presence of two properties, one that prevents rapid death, is thermolabile and acts feebly in small doses, and one that favors rapid death and is thermostabile. He assumes that in a tuber- culous animal the tissues are saturated wdth the aggressin and when fluid collects in the body cavities, as it does on injection of tubercle Ijacilli, it contains large quantities of aggressin, which prevents migra- tion of the polynuclear leukocytes, but not of the lymphocytes, and hence allow's the bacilli to develop freely, producing acute symptoms. In the peritoneal cavity of the normal animal injected with tubercle bacilli, on the other hand, are large numbers of polynuclear leukocytes which engulf the bacilli, thus inhibiting their rapid development, there being here no aggressin to prevent phagocytosis. This theory has been applied to a number of infections, including typhoid, cholera, dysentery, chicken cholera, pneumonia, and staphylo- coccus infections. In all, similar results have been obtained as with tubercle bacilli. When exudates, produced by virulent cultures of these various organisms and properly sterilized, are injected with fresh cultures into an animal, death occurs in much shorter time than when the organisms alone are injected. Moreover, it has been possible to immunize animals against these various infections by repeated injections of the aggressin in the form of exudates. This results in the formation of an "antiaggressin," wdiich opposes the action of the aggressin, thereby enabling the leuko- cytes to take up the bacteria and thus protect the animal. This has been done in staphylococcus, dysentery, typhoid, cholera, pneu- mococcus, and chicken-cholera infection in animals. In addition, a ^•ery marked agglutinative property of the blood is acquired for the bacteria in the animals so immunized. The present view is that the body fluids contain both the split products and the unchanged protoplasm of the bacteria. This accounts both for their aiding the virulence of the bacteria and for the production of antibodies from their injection. References. Bail. Wioner klin. Wnch., 190.5, No. i1. Ibid., 190.5, N..s. 14, 10, 17. Bcrliiur kliii. AVooh., 1905, No. 15. Zeit. f, Hyg., 1905, vol. i, No. :i. .\ich. I'. Hy-., vol. Hi, |.|i. 1!T-' and 411. BanzluiJ. Collci'ted studies, Dopartmoiit of Health, City of New Voik, 1912, \-ol. \ii, p. 114. 176 PRINCIPLES OF MICROBIOLOGY Bordet Resume of Immunity from 1898 to 1909, trans, by Gay. Buchner. Cent. f. Bakt., 1889. Ehrlich and Morgenroth. Berl. klin. wooh., 1899, 1900. Bhrlich and Sachs. Collected Studies on Immunity, trans, by Bolduan. Mahoz. Annales de I'lnstitut Pasteur, Aug., 1902. Metchnikoff. Ann. de I'lnst. Past., 1889. Nultall. Zeit. f. Hyg., 1886. Park and Atkinson. Journal of Experimental Medicine, vol. iii, No. 4. Pfeiffer u. Isaeff. Ztschr. f. Hyg., 1894. Vaughan and Vaughan. Protein Split Products, etc., 1913, Phila. and New York. Vaughan. Herter Lectures, 1913-14, New York. CHAPTER X. COMPLEMENT-FIXATION: THE TECHNIQUE OF THE TEST AND ITS PRACTICAL APPLICATIONS. BoRDET^ and Gengoii showed that the existence of a sensitizer or specific immune body in an antimicrobal serum, by uniting with its specific antigen (bacterium or other cell or proteid material), absorbs alexin (complement). This experiment is usually spoken of as the "Bordet-Gengou phe- nomenon." Wassermann and others applied this method in measuring the am- boceptor content of specific sera, the first important practical applica- tion of its use being in the diagnosis of syphilis. In order to demonstrate fixation of complement the phenomenon of hemolysis is brought into use. Hemolysis. — Hemolysis consists of the breaking up of the red-blood cell with the consequent passing of hemoglobin into the outer medium (serum or physiologic salt solution), the originally opaque fluid becoming transparent. If an animal (A) lie inoculated with the erj^hrocytes (red-blood cells) of an animal (B) of a different species, the blood-serum of A acquires the property of hemolyzing the red-blood cells of B. The immune serum of A, through heating at 55° to 56° C. for 30 minutes or through standing for over 24 hours, loses its hemolyzing power, but the latter may be restored l)y the adtlition of fresh (not over 24 hours old) serum. The hemolyzing power of an immune serum depends on the pres- ence in the serum of two components, one of which is thermostabile and specific, the other thermolabile and non-specific. The thermostabile component is, according to Ehrlich's side-chain theory, an antibody of the third order, the amboceptor type, and is known as hemolysin, hemolytic imrmine body, hemolytic amboceptor, substance senstbilatrice (Bordet), or sensitizer. Erythrocytes to which sufficient hemolysin has been added to prepare them for complete dissolution by comple- ment are called sensitized erythrocytes. The thermolabile activating complement of serum is a normal constituent of all fresh serum and is known as complement, alexin (Buchner and Bordet), or cytase (Metchnikoff). The hemolytic system consists of three substances: erythrocyte suspension, homologous immune serum, and complement; and the presence of all three in definite proportions is necessary for hemolysis. 1 Aiiiiales dc I'lustitut Pasteur, 19U1, xv, 29U. 12 178 PRINCIPLES OF MICROBIOLOGY Complement Fixation Defined.— If an animal be immunized, naturally or artificially, against a species (C) of bacterial or other protem the serum (D) of that animal acquires, through the development of certam antibodies of amboceptor type, the property of fixing or absorbing complement when mixed in proper proportion with the antigen (C). As this phenomenon, termed complement fixation, complement binding, or complement deviation, is invisible, sensitized erythrocytes are used as an indicator of the reaction. If complement is bound by the mter- action of antigen^ (C) and amboceptor (D) it cannot join sensitized erythrocytes to form the hemolytic system ; hence the latter is incomplete and hemolysis does not occur (Fig. 69). If antigen (C) is mixed with Fig. 69 DIAGRAM OF POSITIVE COMPLEMENT FIXATION TEST BACTERIAL ANTIGEN PLUS HOMOLOGOUS IMMUNE SERUM BACTERIAL ANTIGEN PLUS HETEROLOGOUS IMMUNE SERUM ERYTHROCYTES PLUS HEMOLYSIN ERYTHROCYTES PLUS HEMOLYSIN DIAGRAM OF NEGATIVE COMPLEMENT FIXATION TEST an immune serum (E) that is heterologous instead of homologous, /. c., the serum from an animal innnunized with an antigen unrelated to C, then complement instead of being bound is still free and sensitized erythrtjcytes are hemolyzed (Fig. 69). The complement-fixation test has been developed from the following classical experiment of Bordet and Gengou (1901): Tyjihoid bacteria (antigen), inactivated typhoid immune serum (amboceptor), and normal .serum (complement) were mixed in a test-tube; after an interval washed erythrocytes and inactivated liomologous immune serum (hemolysin) ' In complement-fixation the term antigen is confined to substances capaljle of pro- ducing, after one or more administration to a suitable animal, antibodies of tlie ambo- ceptor type. TECHNIQUE 179 wore added. No hemolysis occurred, indicating that the complement had Ijccn fixed by the interaction of antigen and amboceptor, whereby the hemolytic system was left incomplete. The Bordet-Gengou phenomenon may be applied to the identification of an unknown organism or other proteid material that is tested as antigen against known immune serum, or it may be applied to the identification of unknown amboceptor in an immune serum that is tested against known antigen, inhibition of hemolysis showing that the antigen and immune serum are homologous. Complement-fixation is used in the diagnosis of various infectious diseases, e. g., syphilis, gonococcus infections, and glanders, in the differentiation of proteids, in the standardization of some immune sera (for example, antigono- coccus and antistreptococcus horse sera), and in establishing the etiology of infectious diseases. Technique. — Preparation of Reagents. — The reaction of glassivare and reagents has been found to have an important bearing on the accuracy of the test, acidity or alkalinity giving rise to false reactions, positive or negative according to the degree of acidity or alkalinity. All new glassware should be neutralized by standing overnight in 1 per cent, hydrochloric acid. Then, like previously neutralized glass- ware, it is washed in tap-water, thoroughly rinsed in hot distilled water, and sterilized. The distilled water and salt solution used in the prepara- tion of reagents and in the performance of the test are tested for neu- trality to phenolphthalein. Physiologic salt solution, 0.85 per cent, to 0.9 per cent., is used in blood-washing, in diluting other reagents, and in the performance of the test. Immune Serum. — The method of withdrawing immune serum from an animal depends on the animal and the purpose of bleeding. In the case of a horse, the bleeding is made from the jugular vein. Rabbits are bled from the marginal ear-vein if only a few cubic centimeters (less than 5 c.c.) of serum are required. If a large amount is required, the rabbit is etherized, tied on a board made for that purpose, and under aseptic precautions bled to death from the carotid artery. A cannula may be inserted in the artery and the blood allowed to flow into one or more tubes. To obtain the maximum amount of serum, the tube of blood should be slanted at room temperature until coagulation has taken place. In obtaining blood for complement-fixation tests on human beings venous puncture is the most satisfactory method. The drawing of sufficient blood from finger or ear-lobe is a tedious process and the blood-cells are apt to be somewhat broken, so that the serum is tinged with hemoglobin and rendered unfit for testing. Blood is most easily obtained from the median basilic vein of the elbow. The arm should be rendered aseptic by the usual method of scrubbing with soap and water and the application of a 1 to 2000 bichloride of mercury pack for ten minutes. The site of puncture should then be rubbed with alcohol and ether. A ligature is placed above the elbow sufficiently tight to fill the vein, but not tight enough to impede the arterial circulation. A sterile 180 PRINCIPLES OF MICROBIOLOGY needle is then introduced and 5 to 10 c.c. of blood allowed to flow into a sterile test-tulje, which is corked and left slanted at room temperature until the blood is firmlv clotted. The tube should then be placed in the ice-box until serum has separated. If it is necessary to mail a blood specimen, serum only should be sent, as lysis of the blood-cells would be caused by the heat and shaking to which the specimen would be exposed in transit. The serum should be mailed in a sealed ampoule or tightly corked tube. All immune serum is separated from the red-blood cells and fibrin of the blood by centrifugalization before coagulation has taken place, or, better, b>' pipettmg or pouring from the clot after coagulation. Serum thus removed may be entirely freed from erythrocytes by centrifugaliza- tion. Serum should be removed from the clot before spontaneous lysis of the blood-cells occurs, as hemoglobin has the power of fixing com- plement in itself and a hemolyzed specimen of serum, one containing hemoglobin, is anficumplementary, i. e., it inhihils hemolysis without the presence of a specific antigen, hence cannot be tested for a specific ambo- ceptor. Serum, both before and after removal from the clot, should be kept in a cool place, at a temperature not higher than 0° C, as the antibodj' content weakens more rapidly at a high temperature than at a low. For practical purposes serum is best preserved by freezing.^ The addi- tion of a preservative is not advisable, as the accuracy of the test may thereby be invalidated. Contamination of an immune serum should be avoided, as it may result in an anticomplementary and non-specific action of the serum. .1// immune serum before use iti tests should be inactivated, i. e., heated for one-half hour at 56° C. to destroy complement and serum components that might give rise to a non-specific fixation. Antigen. — The method of preparing antigen depends on the nature of the test to be made. Each antigen is described in detail below. A bacterial antigen may be prepared as in the original method of Bordet- Gengou, by suspending in physiologic salt solution a 24-hour agar growth of the bacteria, making a rather concentrated emulsion. Bacterial extracts give more specific results than emulsions. The best method of extraction depends on the organism. Complement. — Not all fresh serum has the power of reactivating the serum of an alien species, but that of the guinea-pig has unusual power in this respect; hence guinea-pig serum is generally used for complement in hemolytic work. There is a wide ^•a^iation in the activating power of guinea-pig serum, and also in its power of combining with antigen and immune serum in the complement-fixation reaction. It is advisable to use the pooled serum of at least three pigs, in order to obtain comple- ment of average activating and combining properties. Guinea-pigs tliat have been u.setl for antitoxin tests anil other purposes may be of use in comi^lement-fixation work after a rest of three months, but their scrum is less cipi than that of unused pigs to be of normal activating ]wwer. In the complement-fixation test for glanders pigs that have been inoculated ' StTunj is ijeifectly prr-sfrvcd by evaporating to dryness iu a vacuum desiccator, but tlie procedure is complicated. ANTIGEN ISl with horse serum must ne\'er be used, as sul)st:ui('es are formed that eauNe a reaction with tlie horse serum that is beiiiK testc-d for Khuiders and the lest is unsatisfactory; serum eontrols in themsehes inhihil hemolysin and no icadinj;' can he made of a specific reaction. (lra\id piu;s should not he used for comple- ment, as their serum is apt to he weak in acti\atin};' power. To obtain eoinplement, guinea-pigs may he bled from the heart by aspiration and used again after several weeks' rest, or they may be bled to death. In this laboratory the pigs, after being stunned by a blow at the base of the skull, are bled from the throat' into Petri dishes, whieh are left at room temperature until the serum begins to separate and then set in the ice-box for several hours. The serum is drawn off with a capillary pipette and centrifuged if not free from blood-cells. Before being pooled, the serum from each pig should be tested separately for natural hemolysin, whidi is occasionally present, and for activating power. Serum containing natural hemolysin or senmi of weak acti\'ating power should be discarded. Complement deteriorates rapidly if exposed to sunlight or to a warm temperature (over 70° F.) ; if kept in the ice-box (at a temperature below 17° C.) it is good for at least twenty-four hours. Complement may be preserved for several weeks by freezing. Its activating power is retained, but its capacity for being bound may weaken, so that it is not considered rehable. In our experience complement frozen for one week is as good as perfectly fresh complement. Complement is used in a 10 per cent, dihdiun made with physiologic salt solidion. Erythrocytes from sheep, goat, man, ox, or other animal may be used, and they must be washed free from serum, with physiologic salt solution. In our laboratory sheep's cells, washed six times, are used, in a 5 per cent, suspension. A sheep is bled from the jugular vein into a sterile bottle or flask containing glass beads, and before the blood coagulates it is thoroughly shaken for the purpose of defibrination. Blood is washed in the following manner: In washing a small amount of blood it is convenient to put 2 to 4 c.c. in a 15- c.e. graduated centrifuge tube. The tube is filled witli ph3'siologic salt solu- tion and a mixture of blood and saline is effected by means of a pipette. The volume of saline should be three times that of the blood, otherwise the washing is not thorough. The blood is rapidly centrifugalized long enough for the ary- throcytes to fall to the bottom of the tube. The supernatant fluid is decanted or draNvn off with a pipette attached to a pressure pump or fitted with a rubber bulb. Phj'siologic salt solution is again mixed with the cells and the tube centrifugalized. This process is repeated until the blood-cells are entirely free from serum. In most laboratories three washings arc considered sufficient, but we have found that after even four washings the serum is not always com- pleteb^ removed. Hence we make it a ride to umsk six times. After the last washing the level of the erythrocytes is marked on the tulje, Ijefore the cells have been disturbed bj' the removal of the supernatant fluid. The amount ' Care must be taken not to cut the esophagus, as the stomach contents might render the serum unfit for use in complement-fixation work. 182 PRINCIPLES OF MICROBIOLOGY of blood per centrifuge tube, the speed and duration of centrifugalization should always be the same, at least for the last washing, m order that the packnig of the cells be uniform day by daJ^ Blood that has been drawn for over two days in suiinner or three days in winter is unsuitable for hemol.ytic work, as the resistance of the erythrocytes weakens on standing and hemolysis occurs rapidly, so that perfect balance of the hemolj'tic system cannot be obtained. In places where it is inconvenient to obtain sheep's cells, human erythrocytes are frequently used. An advantage in the human system arises from the fact that by its use one error in the test is avoided. Human serum frequently contains natural antisheep amboceptor and the excess of hemolysin introduced into the hemolytic system when such a serum is being tested may, according to some investigators, cause a w^eakening or loss of a positive reaction. In our experience this error rarely or never occurs in the Wassermann reaction, owing to our practice of reading the reaction as soon as controls are completely hemolyzed; hence the use of a hemolytic system other than sheep or the absorption of the natural hemolysin from the serum is not necessary in Wassermann work. In tests with bacterial antigens where the fixation of com- plement is less firm than in the Wassermann reaction the native anti- sheep ambpceptor in human serum may be a source of error. Hemolysin is obtained by successive inoculations of a rabbit (or other experimental animal) with the type of erythrocytes to be used in tests. Inoculations may be made intraperitoneally or intravenously. Owing to the frequency of abscesses after intraperitoneal inoculations the intravenous method is preferable. In this laboratory, rabbits are inoculated intravenously at intervals of 2 days with fresh, thoroughly washed sheep's cells in a 50 per cent, suspension in physiologic salt solution, the doses being 2 c.c, 4 c.c, and 6 c.c. On the ninth day after the last inoculation the rabbit is bled to death from the carotid. The serum is put up in small bottles or ampoules and heated for one-half hour at 56° C. on three successive daj^s, to destroy the complement and to insure sterihzation. Standardization of Reagents. — An accurate daily standardization of the hemolytic system is very important for reliable results, and this may be accomplished by means of a hemolysin or a complement titra- tion. In the former, varying amounts of hemolysin are incubated with a constant amount of complement and erythrocyte suspension. In a complement titration the amounts of complement are varied and hemolysin is constant. In most laboratories it is considered lyrejerabk to follow a hemolysin titration. In this laboratory preliminary titra- tions of new hemolytic immune serum are made in dilutions of 1 to 100, 1 to 1000, etc., to determine the dilution of hemolysin in which 0.05 c.c. is one unit. By a unit of hemolysin is meant the smallest amount that gives complete hemolysis of 0.1 c.c. of a 5 per cent, sheep's cell suspension in the presence of an excess of complement after one hour's incubation in a .37° C. ivater bath. STANDARDIZATION OF REAGENTS 183 Once standardized the henu)l^•sin is nsed in the same dilntion every T) le volume day. The technique oi the titration is given helo that of all our complement-fixation tests, is one-tenth the volume of the classical Wassermaun — i.e., 01) c.c. instead of f) c.c. After thorough shaking the titration is incubated one hour in the water-bath at o7° C. and examined frequently to determine the rapidity of the reaction, according to which from two to three units are used in the test. As hemolysin is stable and fresh, and erythrocytes from a healthy sheep vary little in resistance, such a titration indicates sufficiently for practical work the value of the complement. If the complement is normally active and free from hemolysin, the erythrocytes fresh and thoroughly washed, the saline solution isotonic and all reagents accurately diluted, hemolysis at the end of the hour's incubation is complete (Plate V, e) in the first six tubes, nearly complete (Plate V, d) in the seventh, partial (Plate V, c) in the eighth, slight in the ninth, and lacking (Plate V, a and b) in the last four tubes. Then since -0.05 c.c. is the unit of hemolysis, 0.1 c.c. is, in general, used to sensitize 0.1 c.c. of erythrocytes. The interpreta- tion of a hemolysin titration can be learned by experience only. Hemolysin Titration Number of tube. Hemolysin in standard dilution. C.c. 10 per cent. complenient. C.c. .^ per cent. erythrocyte suspension. C.c. 0.85 per cent saline. Co. 1 0.1 0.1 0. 1 0.0 2 0.09 0.1 0, 1 0.21 3 0.08 0.1 0,1 0,22 4 0.07 0,1 0,1 0,23 5 0.06 0. 1 0, 1 0,24 6 0.05 0. 1 0, 1 . 2.5 7 0.04 0. 1 0, 1 . 26 8 0.03 0, 1 0,1 0,27 9 0.02 (1. 1 0,1 0,28 111 0.01 0. 1 0,1 , 29 11 0.1 (1,0 0.1 0,3 12 0.0 0, 1 0. 1 0,3 i;-; 0.0 0,0 0,1 0,4 3 ° o t- .C CO J2 ? Complement Titration umber of t\d)e. 10 per cent. complement. C.c. Hemolysin in standard dilution. C.c. 5 per cent. er.vthroc>'te suspension. C.c. 0..S5 per cen saline. C.c. 1 0.1 , 05 0.1 , 25 2 0.09 0.05 0.1 . 26 3 0.08 0.05 0.1 0,27 4 0.07 0.05 0.1 , 28 5 0.06 0.05 0.1 0,29 6 0.05 0.05 0.1 0,3- 7 0.04 0.05 0.1 0.31 8 0.03 0.05 0.1 0.32 9 0.02 0.05 0.1 0.33 10 0.01 0.05 0.1 , 34 11 0.1 0.0 0.1 0.3 12 0.0 , 05 0.1 0,35 13 0.0 0.0 0.1 0,4 1S4 PRINCIPLES- OF MICROBIOLOGY Antigen Tituaticin. Nun.berof 10 per cent. 0.85 per o.„C. f^^' tube. Inimv.ne senmi. Antigeu. complement. salmc. suspeiioiun. C.c. C.c. C.c. C.c. Co. 0.0 .3 ■ 0.2 <-J 0.2 1 0.01 25 0.1 2 0.01 0.2 0.1 0.0 S^ U.z ^^ 3 0.01 0.15 0.1 0.05 °^ 0.2 ■- 4 0.01 0.1 0.1 0.1 ^ 0.2 |t; 5 0.01 0.05 0.1 0,15 _|^ 0.2 o^ 6 0,01 0.025 0.1 0.2 i^ 0.2 7 0.0 0.4 0.1 0.0 g^ 0.2 g-M S 0.0 0.3 0.1 0.0 ^S 0.2 ^^ 9 0.0 0.2 0.1 0.05 £? 0.2 |^ 10 0.0 0.1 0.1 0.1 Ml 0.2 |« 11 0.0 0.05 0.1 0.15 g^ 0.2 g 12 0.02 0.0 0.1 0.2 a 0.2 = If an exact determination of complement value is desired the hemoly- sin titration may be followed by a complement titration as given above. The unit of complement is the smallest amovnt that completely hemolyzes 0.1 c.c. of .5 per cent, erythrocyte suspension sensitized by one unit of hemolysin. If the balance of the hemolytic system is to be obtained by varying complement instead of hemolysin, two units of hemolysin instead of one unit should be used in titrating complement. In our opinion more uniform and accurate results are secured by A-arying hemolysin than by varying complement. Antigen. — There are two requirements for a good antigen, a long range and .specificity.'^ The range of an antigen is determined by mixing varying amounts of the antigen with a constant amount of a previously tested homologous immune serum, a constant amount of complement and a constant amount of sensitized erythrocyte suspen- sion. The specificity of an antigen is determined by using heterologous in.stead of homologous immune serum in the titration. The technique employed in this laboratory in the titration of an antigen is given above. Readings are made after the erythrocytes have settled, and meantime the titration should be kept in the ice-box to prevent a con- tinuation of hemolysis. An immediate reading may be made by centrifu- galizing the tubes. If fixation is complete through 0.025 c.c. (tube 6) a 10 per cent, dilution of the antigen should be titrated in the same manner to determine the unit of antigen, which is the smallest amount that with two units of homologous immune serum (or 0.01 c.c. of a human serum which has given a + + + + reaction) gives complete fi.xation of complement. The anticomplementary dose is the samllest amount of antigen that is in itself inhibitory. The longer the range of the antigen the greater is the probability of success in diagnosis. An amount of antigen that fixes complement completely with a serum of high anti- body content may give incomplete or no fixation with a serum of low ' The range of an antigen i.9 the difference between the anticomplementary dose (the smallest amount o( antigen that is in itself inhibitory) and the minimum fixing dose the antigen unit. PLATE V a. Complete absence of hernolysis before settling of cells. b Complete absence of hemolysis after settling of cells. c. Partial hemolysis. d. N'early complete hemolysis. c. Complete hemolysis. STANDARDIZAriOA' OF REAGENTS 185 antihody content. Hence in making;' (liaii;n()stic tests, where the detec- tion of even a small anionnt of antihody is desired, it is ad\isalile to use nuicii more than one iniit of anti^'en. .I.v tlif iiiii.riniiini (iiiiimiif of antigrii Ihnt iikii/ he ii.scd with sajity in inic-foiirtli the iinticiuii jilciiiciititrii dune an (tntigcu i>f laiig range in nccennari/. If in tahle inhiliition is complete (Plate V, a and h) in the first five or six tubes, hemolysis is complete (Plate A', e) in tubes S to 12, and there is \ery slight inhibition (Plate \", (I) in tube 7 only, then (1.1 c.c. is the amount of antigen to be used in diagnostic tests. Antigen nhanld edirai/n J>e ni) diluted that 0.1 c.c. may be used. If one-fourth the anticomplementary dose gi\es complete fixation with a heterologous immune serum the antigen is non-specific and unsuit- able for tests. Oeeasionalhi an antigen in lytic for erythrocyten. In such a case, tubes containing the largest amount of antigen show more hemo- lysis than those containing less. The fixation cur\T instead of dropping (Fig. 70, a) first rises and then drops again (Fig. 70, }>). If a lytic antigen is also anticomplementary and has a long fixation range it may be used, otheru-ine it should be discarded. Co)nplete fixation Fig. 70 - per cent. valine. C.c, Senaitized erythrocyte an.spension. C.c. 1 0.1 0.1 0,1 0,0 ^a 0.2 2 U.O'I 0.1 0.1 0,01 o r- 0,2 3 O.OS 0.1 0.1 0,02 0,2 4 0.07 0.1 0.1 0,03 r^ CO 0,2 5 0.06 0.1 0.1 0.04 ^ n 0,2 r. 0.05 0.1 0.1 . 05 T3 o 0,2 7 0.04 0.1 0.1 . 00 0,2 s 0.03 0.1 0.1 . 07 0,2 O.02 0.1 0.1 0.08 0,2 lU O.Ul O.I 0.1 . 09 M ^ 0,2 11 0.0 0.2 0.1 0.0 3 Oj 0,2 12 0,2 0,0 0.1 0.0 S +^ 0,2 3 P The Wassermann Reaction. — Wassermann, Neisser, and Bruck were first to apply the Bordet-Gengou phenomenon to the diagnosis of syphilis. The antigen originally employed and still preferred by some workers consists of a saline extract of liver from a syphilitic fetus. The finely divided tissues are mixed in the proportion of one to four with normal salt solution, to which 0.5 per cent, carbolic has been added, agitated at room temperature for twenty-four hours, centrifuged, and the supernatant fluid drawn off into sterile vessels and kept in the ice- box until needed. Since the cultivation of the Treponema pallidmn has been made possible, antigens have been made from the organisms in pure culture instead of from luetic tissue; but the results have, on the whole, been less satisfactory, i. e., fewer cases of syphilis give a positive Wassermann reaction with a Treponema pallidum antigen than with a luetic tissue antigen, or even with a lipoid antigen from normal tissues. The exact nature of the antigen that produces the antibodies (called by Citron Luesreagine) taking part in the Wasser- mann reaction is unknown. It appears to be neither the pure spiro- chetes, nor a pure lipoid substance. Although a pure lipoid cannot stimulate the production of antibodies when inoculated into an experi- mental animal, it reacts in vitro with the Luesreagine in the blood of the syphilitic. The Wassermann reaction is a lipotropic or lipoidophilic reaction and not due to the interaction of specific antigen and anti- body. An extract of heart, liver, or kidney in 96 per cent, alcohol may be used. Some serologists recommend the addition of cholesterin to a crude alcoholic extract; such an antigen gives a higher percentage of positive reactions than most other antigens, but its reliability has not yet been established. In our experience a eholesterinized antigen is of a special value in determining the effectiveness of antQuetic treatment, as old cases of sj'philis that are or have recently been under treatment are more apt to react with this antigen than with a crude alcoholic antigen. In making a fresh diagnosis it is not wise to depend on a eholesterinized antigen alone, since cases of scarlet fever, leprosy, and other non-syphilitic conditions have been found to react strongly with tliis ISS PRINCIPLES OF MICROBIOLOGY antigen. These false positive reactions do not occur with a crude alcoholic antigen. A positive reaction is, however, presumptive of syphilis and a negative reaction has more value in exclufling a diagnosis of s.yphilis than has a negative reaction witli the crude alcoholic antigen. A stiff and stal)le antigen is Noguc'hi's acetone insolnhle fraction of beef heart, li\'er, or kidney, prepared by extracting macerated tissue with ten times the amount of absolute aclohol at room temperature for several days, filtering, evaporating the filtrate to dryness, taking up the residue with ether, treating the ethereal solution with five times its volume of acetone, and making a saturated solution of the precipi- tate in absolute methyl alcohol. In our laboratory the most satisfactory results have been obtained with a crude alcoholic extract of guinea-pig hearts prepared as follows: hearts (from pigs l)led to death for complement) are minced and washed in tap-water until free from lilood and macei-ated in C. P. 96 per cent, alcohol in the proportion of one gram of heart to 5 c.c. of alcohol. Extraction is allowed to take place in the ice-box for at least three months and then in the incubator for a week. The extract is filtered through paper and kept in the ice-box, where it remains stable for at least a year. The titer of different antigens varies from one in twenty to one in a hundred. In diluting the antigen the first 10 c.c. of saline are added drop by drop and the remainder slowly, the mixture being effected by gently rotating the receptacle, not by shaking. The table on page 189 gives the classical Wassermann technique. The readings are made according to Citron's standard. Our technique differs in three respects from Wassermann's: in the size of the test, in the amount of antigen used, and in the method of fixation. In our experience the results obtained through using all the reagents in one-tenth the amounts used by Wassermann are absolutely reliable, provided the test is accurately performed, and the sa^'ing in material is considerable. Another advantage in the small size is that a very small amount (about 0.2 c.c.) of the patient's serum is sufficient for a test. Concerning the amount of antigen to be used in the test, we have found the use of a constant amount to give the same results as the use of a varying amount. For fixation of complement a four-hour period in the ice- box is allowed instead of incubation, as it has been found that about 10 per cent, more positive reactions in cases of syphilis are obtained by this method, and without any apparent danger of obtaining false positive reactions. Though biologically non-specific the Wassermann reaction is clinically specific except, perhaps, in cases of leprosy, yaws, sleeping sickness, and scarlet fever.' With these exceptions a positive reaction indicates the presence of luetic infection, either active or latent. A positive reaction may be given in any stage of syphilis but is most apt to occur in the secondary stage. A negative reaction at any stage of the disease does not exclude the possibility of syphilis. Antiluetic treatment, especially mercury, frequently results in the reaction becoming negative, though the disease may still be active. ' With the use of the crude alcoholic antigens, in one-quarter the anticomplementary (lose, wc have never obtained false reactions in such cases even by ice-box fixation. Luetic Number of tube. liver extract diluted. C.c. Patient's serum. C.c. 10 per cent, com- plement. C.c. 0.85 per cent. saline. C.c. Sensitized er,\throc,\'te suspension. C.c. 1 1.0 0.2 1.0 O.S Z g; 2,0 o 3 0.5 2.0 0.1 0.2 1.0 1.0 1.4 1.8 c3 a .■ |4| 2.0 2.0 i 2.0 0.0 1.0 0.0 Q a. J= 2.0 TEST FUR GONOCOCCUS INFECTION 189 Classical Wassekmann Test ^ If the Wassermann reaction is persistently negative for two years after cessation of treatment, tests being made at intervals of three months, it is fairly certain that a cure has been effected. The inges- tion of alcohol or the administration of an anesthetic within twenty- four hours of collection of the blood-specimen interferes with the accuracy of the test, alcohol weakening the reaction and an anesthetic giving rise to false positive reactions. The Complement-fixation Test for Gonococcus Infection. — This method was first applied to the study of gonococcus infection by Miiller and Oppenheim (190G). The technique developed by Schwartz and McNeil has been widely followed and the value of the test in the diagnosis of secondary gonococcus infections, especially in conditions in which a bacteriologic diagnosis is difficult, as in arthritis, has been thoroughly established. A polyvalent antigen, one made from as many different strains of the gonococcus as possible, is essential. ^ A twenty- four-hour growth on salt-free veaP agar neutral to phenolphthalein is washed off with sterile neutral distilled water, autolyzed in a 56° C. water bath for one hour, and in an 80° C. water bath for one hour (to destroy a ferment that would render the antigen unstable), filtered through paper pulp and a Berkefeld filter,^ and heated on three successive days at 56° C. for one-half hour for sterilization. This antigen is stable for at least six months and is highly specific.^ Immediately before use the antigen must be made isotonic by the addition of one part of 9 per cent, saline solution to nine parts of the antigen. The test is performed in the usual way. The optimum period for fixation has been found to be six hours in the ice-box. Readings must be made with great care. A positive diagnosis should not be made unless 0.02 c.c. of serum gives complete inhibition of hemolysis, ' In this lalDOratory ten strains of gonococci isolated by Torrey are used in the prepa- ration of the antigen. - Medium from bob-veal is most desirable. ' New Berkefeld filters are very alkaUne, and before use for the filtration of bacterial antigens tliey should be boiled in distilled water at least three times for five minutes each time, and scrubbed thoroughly with a small iirush in fresh water, after each boiling. After the filter is set up, hot, neutral, distilled water should stand in it for about five minutes. Then hot, neutral distilled water .should run through under gentle pressure until tlie fluid is clear and neutral to phenolphthalein, when the filter is ready for use. Under high pressun.^ a filter still alkahne might test neutral. After use, the filter should lie boiled in distilled water, scrubbed, and dried in the air. A filter used for gonococcus antigen should never be used for any other bacterial antigen, unless it is first boiled in I per cent, .sodium hydroxide solution and reneutralized. ' The specificity' of a gonococcus antigen may be best determined by an antigen titra- tion with an antimcningococcus serum of high antibody content. 6 190 PRINCIPLES OF MICROBIOLOGY as serum containing heterologous amboceptors, for example, strepto- coccus, may give a + reaction with gonococcus antigen. Only gonococcus amboceptors give a + + , + + + , or + + + + reaction. Cases of anterior gonorrheal urethritis and acute vulvovaginitis rarely give a positive complement-fixation test. A positive reaction is indicative of the presence or recent activity in the body of a focus of living gonococci. A positive reaction may persist for from six to eight weeks after a cure has been effected. Persistently negative results obtained through a considerable period of time indicate the probability of a cure. The Complement-fixation Test for Glanders. — Complement fixation has proved to be a valuable aid in the diagnosis of glanders. It is generally considered specific and more reliable than the agglutination test. In this laboratory the antigen is prepared from a twenty-four- hour growth of B. mallei on salt-free veal-agar 1.6 per cent. acid. The growth is washed off with sterile distilled water, autolyzed for six to eight hours at 80° C, filtered through paper pulp and a Berkefeld, and the filtrate sterilized at 56° C. on three successive days for a half-hour. This antigen, like the gonococcus and all other aqueous extracts, must be made isotonic before use. The test is performed like the others, but the optimum period for fixation has been found to he 6 to 18 hours in the ice-box. The New York Health Department condemns all horses that give a -|- + + + complement-fixation reaction when it is confirmed by the eye mallein test, though + + and + + + are con- sidered certain indications of glanders infection.^ Streptococcus Infections. — The value of complement fixation in the diagnosis of streptococcus infections is still uncertain. In this labora- tory fairly satisfactory results are obtained with a saline antigen pre- pared as follows: A twenty-four-hour growth on salt veal-agar neutral to phenolphthalein is washed off with 0.85 per cent, saline solution. The emulsion is heated at 60° C. one hour, left in the ice-box twenty-four hours, and centrifugalized. The supernatant fluid is used for tests, either water bath or incubator being used for fixation. The test seems to be specific, but is far from perfected. Pertussis.— Complement fixation has been used for the determina- tion of the etiological cause of whooping cough and for the diagnosis of the disease. The results of many investigators, including ourselves confirm the findings of Bordet, that the Bordet-Gengou bacillus is the etiologic cause of whooping cough. As to the diagnostic value of the test, reports vary. The figures given by the workers who use active serum are undoubtedly too high, as investigations in this laboratory have proved that active serum may give non-specific fixation with even a highly specific antigen. We have found the most satisfactory antigen, that is, the strongest and most specific, to be obtained by shaking and then autolyzing at 56° C. A twenty-four-hour culture on the Bordet- ' Macroscopic lesions are always shown by horses niving a + + + + coniplement- hxatiou test, rarely by those giving a -|--|-ora-|--|--|- reaction. COMPLEMENT-FIXATION TEST FOR TUBERCULOSIS 191 (leiigou potato-blood-agar medium is scraped off with a platinum spud and put in sterile distilled water. The emulsion is shaken for about two hours, left in a thermostat at 56° C. for about eighteen hours, and centrifugalized. The supernatant fluid is used for tests, being made isotonic immediately before using. Water bath or incubator is used for fixation; room temperature, at which some workers allow fixation to take place, gives such variable results as to be unsuitable for routine work. We obtain a positive reaction in about 50 per cent, of whooping-cough cases in the paroxysmal stage. The ad- ministration of whooping-cough vaccine may increase the strength of the complement-fixation reaction, but experiments with normal individuals have proved that vaccine in itself, in the absence of whoop- ing cough, does not bring about a positive reaction. Immune serum of high antibody content may be produced by the intraperitoneal inoculation of rabbits once a week with a live culture of the Bordet- Gengou bacillus, beginning with a dose of one-eighth of a slant and increasing to three slants, the dosage depending on the condition of the rabbit. The height of immunity is usually reached through five or six inoculations ; if other inoculations are given, the antibody content decreases. The rabbits are bled nine days after the last inoculation. Complement-fixation Test for Tuberculosis. — The complement- fixation test for tuberculosis is of uncertain value. Koch's old and new tuberculins have been used as antigens, but may give non-specific fixation. Besredka's antigen, a thirty-day culture from egg-bouillon medium sterilized at 115° C., is said to give a high percentage (over 90 per cent.) of positive reactions in cases of active tuberculosis and to be of value in the diagnosis of the disease. It has been reported that syphilitics may give a positive tuberculosis reaction and that the test is therefore not specific. As an individual may suffer from both diseases at the same time the occurrence of both reactions is not a proof of non- specificity. The subsequent development of tuberculosis in cases that at the time of the complement-fixation test showed no clinical symp- toms suggests that the statistics of non-specificity that have been published may be inaccurate. Bronfenbrenner's work^ has been par- ticularly careful and thorough and maintains the specificity of the tuberculosis reaction with the use of the Besredka antigen. Our own findings confirm his insofar as specificity is concerned. Our percentage of positive reactions among cases of active tuberculosis is much lower than Bronfenbrenner's. Meningitis. — The complement-fixation method has been successfully applied in epidemic meningitis by Briick, but the diagnosis can more readily be made by the bacteriologic examination of cerobrospinal fluid. Complement fixation is a valuable means of differentiating strains of meningococci. The antigen used here for this purpose is prepared in the same manner as gonococcus antigen. Water-bath fixation is used for all titrations. ' Personal communication of preliminary work. 192 PlilNCIPLES OF MICKUBIOLOGY Typhoid. — Complement fixation is a valuable method of corroborating the Widal. Its exact clinical value and specificity have not yet been established. The use of a highly polyvalent antigen is essential. Gar- bat's method of preparing the antigen is to wash off with sterile distilled water a twenty-four-hour growth on agar. Heat the emulsion at 60° to 70° C. for twenty-four hours, shake with glass beads for twenty- four hours, and centrifugalize until the supernatant fluid is absolutely clear. A positive reaction usually appears only after bacteria have disappeared from the circulation; it becomes stronger during con- valescence and persists for several months afterward. CHAPTER XI. THE NATURE OF THE SUBSTANCES CONCERNED IN AGGLUTINATION. Fig. 71 By the phenomenon of agglutination is meant the aggregation into clumps of uniformly disposed microorganisms in a fluid because of the action of a hemologous immune serum. If the organisms are motile they become immobile. This phenomenon had probably been noted by several observers (Charrin and Roger in 1889) but it was first extensively studied by Gruber and Durham in 1896, who determined that the serum of those passing through many infections contained a specific substance (agglu- tinin) which caused the infecting organisms to clump. Several months later Widal reported that in typhoid fever the development of agglutinins could be used for diagnostic purposes. It was thus demonstrated by these studies and those of Griinbaum, Bordet and others that through agglutinins a new means was available for the identification of bacteria. As to the nature of these phenomena a number of theories have been advanced. As in the case of the immune body, there is positive proof that the agglutinin com- bines directly with agglutinable sub- stances in the bacterial body, the two bodies affecting a loose combination. But since a certain amount of salt is necessary (Bordet) for the reaction it must be classed as a physico-chemical reaction. Bordet believes that all the antibodies act very much alike in their method of union and that the effects produced vary with the antigen in c|uestion and the characteristics which, on account of its own nature, it can produce as soon as it unites with the appropriate antibody. He does not believe in diff- erent families of antibodies, but in an infinite variety of antigens. Ehrlich, on the other hand, considers that the agglutinin consists of a haptophore or combining atom group which is stabile anrl of a ferment grouj) which is labile (receptors of the second order). The latter causes the phenomenon of agglutination. In some types of infection there is a great accumulation of agglu- tinins in the blood. Thus in typhoid patients and convalescents distinct 13 Reccptor>< of the second order are pictured in c. Here e represents the haptophore group, and d the zymophore group of the receptor, / being the food molecule with which this receptor combines. Such receptors are possessed by agglutinins and precipitins. It is to be noted that the zymophore group is an integral part of the receptor. 194 PRINCIPLES OF MICROBIOLOGY agglutination has been observed in dilutions of 1 to 5000, and this action persisted for months, though not, of course, in the same degree. Even normal blood-serum, when undiluted, often produces agglutination through group agglutinins. But the specific agglutinins, which are formed only in consequence of an infection, are characterized by this, that they produce agglutination even when the serum is highly diluted, and, furthermore, that after this dilution the action is generally specific — i. e., the high dilutions of cholera-immune serum agglutinate only cholera bacilli, of typhoid-immune serum only typhoid bacilli, etc. This specificity, however, for some bacteria such as the colon and dysentery group, is not always absolute, as the group agglutinins previously present may be in unusual amount. It was formerly assumed that agglutination was a prerequisite for haderiolysw. This, however, is not so, for both in cholera and in typhoid immunity bacteriolytic substances have been observed with- out agglutinins, and agglutinating substances without bacteriolysins. Characteristics of Agglutinins. — ^The union of agglutinin with re- ceptors in bacteria is a physicochemical reaction, and is quantitative. By chemical means it is possible again to separate a portion of the agglutinin from bacteria saturated with it and use it to agglutinate bacteria anew. Before agglutination occurs sodium chloride or a similarly acting compound must be present. The amount of bacteria in the emulsion used to test the amount of agglutinin must, there- fore, be known. An emulsion one hundred times as dense as another would require one hundred times as much agglutinin to give an equally complete reaction. Agglutinin acts upon dead bacteria. Heated at .55° C. agglutinins are not destroyed although bactericidal action is annulled. Agglutinins changed by heat, acids, and other influences become "agglutinoids," which are comparable to toxoids, complementoids, etc., ('. (J., agglutinating sera heated to a certain temperature loses its power to agglutinate but act upon bacteria so that they are unable to be agglutinated by active serum Heat diminishes the agglutinability of bacteria when above 60° C. Dreyer found that if a twenty-four-hour bouillon culture of BaciUvs coli required 1 part of agglutinin to agglutinate it, then if heated to 60° C. it required 2.3 parts; if to 80° C, 18 parts; if to 100° C, 24.6 parts. He found the surprising fact that long heating of the culture restored to some extent its ability to be agglutinated by smaller amounts of agglutinins. Heated thirteen hours to 100° C, the culture was agglutinated by four parts. Heating the serum above 60° C. injures the agglutinin sligiitly, above 70° C. greatly, and above 75° C. destroys it. Weak and strong acids agglutinate bacteria, while medium acidity docs jiot. Alkalies inhibit agglutination. Agglutinin does not dialyze through animal membranes. In diluted solution agglutinin slowly deteriorated. Dried, it lasts longer. It is precipitated with the globulins by ammonium sulphate. When a GROUP AGGLUTINATION 195 solution containing- agglutinin is passed through a stone filter the first few cubic centimetres contain no agglutinin. The next contain a moderate amount and the remainder the same as the solution. It is important to remember that in low dilutions of a serum agglu- tination may fail, while iu higher dilutions agglutination may take place readily. The Development of Agglutinin. — Experimental or natural infection of animals and men is followed in seven to ten days by an appreci- able de^'elopment of agglutinin. This development is much greater for some bacteria than for others. Group Agglutination. — Many varieties of organisms have, among the different protein substances composing their bodies, some that are common to other microbes which are more or less allied to them (Fig. 72). If these substances are of the tj'pe that excite agglutinins, we have from an animal immunized by any one of them a serum acting on other organisms somewhat in proportion to the amount of agglutinin producing protoplasm which they have in common with the infecting organism. These agglutinins, acting on common substances in other microorganisms which are generally allied varieties are called, there- fore, group agglutinins. Thus, in a case, the infecting paratyphoid bacilli type B were agglutinated 1 to 5700; typhoid bacilli, however, only 1 to 120, while paratyphoid bacilli type A were agglutinated only 1 to 10. In a case of typhoid fever an agglutination of paratyphoid type B occurred with a dilution 1 to 40, while typhoid bacilli were agglutinated with 1 to 300. Fig. 72 B C B E F D G J Typhoid Bacillus Colon Bacillus Dysentery BacillU'3 Specific and common agglutinins producing protoplasm. The bacteria which are agglutinated bj^ one and the same serum need not at all be related in their morphologic or other biologic charac- teristics, as at first assumed. Conversely, microorganisms which, because of the characteristics mentioned, are regarded as entirely identical are sometimes sharply differentiated by means of their ag- glutination. In other words, the "groups" arrived at by means of a common agglutination have no necessary relation to species as the term is usually employed, but only of chemical similarity. This is indicated by the diagrams in Fig. 72. The letters indicate chemical substances capable of stimulating the production of agglutinin and of combining with it when made. Thus both the typhoid and colon will l!J(i PRINCIPLES OF MICROBIOLOGY stimulate and react to B agglutinins, but the typhoid bacilli will only react to the type A and the colon to the D type. Because of this lack of absolute specificity of the agglutinated reaction the diagnosis of the type of injection or' the absolute identification of bacteria through the agglutination or bacteriolytic tests can only be determined in those cases where the group agglutinins are not abundantly present. This suffices for some infections such as those caused b\' the typhoid bacillus and the cholera spirillum, but not for others such as those due to the colon group of bacilli. The Relative Development of Specific and Group Agglutinins.— The study of a large number of series of agglutination tests obtained from young goats and rabbits injected chiefly with typoid, dysentery, paradysentery, paracolon, colon, and hog-cholera cultures has shown that there is considerable uniformity in the development of the specific and group agglutinins. The specific agglutinins develop a larger amount of their total in the early days, being in the second week usually fnnn five to one hundred times as abundant as the group agglutinins. Later the total amount of the group agglutinins tends to approach more nearly to that of the specific, and may reach as high as 20 to 50 per cent. In a number of tests carried out by us we found that many group agglutinins supplement specific ones in their action, causing by their addition an increased agglutinating strength. In our experience the variety of microorganism used for inoculation is, if equally sensitive, agglutinated in a higher dilution by the combined specific and group ag- glutinins produced through its stimulus than any microorganism affected merely by the group agglutinins. It is true that bacteria not injected were at times agglutinated in higher dilutions than the variety injected; this, if not due to greater sensitiveness, was caused by normal group agglutinins present in the animal before immunization. For this reason untreated horse serum is a ^ery dangerous substance to use in differentiating the intestinal bacteria. The great height to which the group agglutinins may rise is seen in the following table: AfTfilutiiiiu in the Srruni uf ;i Horse Injected with Paradysentery Baeillus, Culture Type Manila. Culture. After 18 injections. After 21 injections. 1:3000 1;.5000 1:10,000 1:3000 1 :,^3000 1:10,000 Paradysentery type Manila ++ — — ++ ++ + + •^'"•""B. X ++ ++ _ ++ ++ + + The great amount of agglutinins acting upon the colon bacillus X. is remarkable. A serum is here seen to be acting in dilutions as high as 1 to 10,1)1)0 iijion a culture ])ossessing different chiiracteristics from the one iiseil in the injections. .Vlthoiigli a considerable proportion of the group agglutinins act- ing on cohm bacillus X. was undouljtedly due to the stimulus of the injections of the paradysentery culture, still a portion of them was THE USE OF ABSORPTION METHODS 197 pnilial)ly due to tlic ng-glutiiiins tU'vcloix-d l)y tlie stiiiiiihis of tlic al)s()rl)e(l intestinal l)acteria. In the table given ))el()\v is seen tlie marked aeeuinulation of ajijilutinins wliicii may occur in a normal horse before injections are begun: A ,\'ouug horse before inociilHtion. ■ 1:100 1 :500 Dysentery B., Japan + — Paradysentery, Mt. Desert . + — Paradysente^^^ Manila . + + + + Colon B. X " + + + 1:1000 1:5000 The Relative Accumulation of the Group and Specific Agglutinins for the Organism Injected and for Allied Varieties. — A test was carried out with different types of dysentery l)aeilli. _For the Manila culture of Flexner, which is nearest to the colon in its characteristics, the specific agglutinins were in the serum of an animal which had recei\'ed injec- tions of the Manila cultures at the end of the fourth month five times as abundant as the group agglutinin acting on the Mt. Desert culture of Park, which represents a type lying between the Flexner and Shiga cultures. For the dysentery bacillus (Shiga) the development of agglu- tinins was the least (Fig. 73). Another point of interest is that the proportional amounts of agglu- tinins from the different cultures varied at different times. If on tests made of a single bleeding we had attempted to draw conclusions as to the relative development of specific and group agglutinins between the cultures, we would have had an imperfect view. Many conflicting statements in literature are undoubtedly due to this lack of appreciation of the variability in the relative amount of these two types of agglutinins during a long process of immunization (Fig. 74). The Use of Absorption Methods for Differentiation between Specific and Group Agglutinins due to Mixed Infection and to a Single Infection. — It is now well established that if an infection is due to one microorganism there will be specific agglutinins for that organ- ism and group agglutinins for that and other more or less allied organ- isms. If infection is due to two or more varieties of bacteria, there will be specific agglutinins for each of the microorganisms and group agglutinins produced because of each of them. The following experiments will illustrate these points: A rabbit immunized to B. typhi agglutinated B. typhi 1 to 5000, B. coli (31) 1 to 600. After saturation with B. typhi all agglutinins were removed for both microorganisms. A rabbit immunized to both B. typhi a,nd B. coli (31) agglu- tinated B. typhi 1 to 4000, B. coli (31) 1 to 1000.' (After saturation with B. typhi the serum did not agglutinate -S. typhi, but agglutinated B. coli (31) 1 to 900.) After saturation with B. coli (31) it failed to agglutinate B. coli (31), but still agglutinated B. typhi 1 to 3500. Some other strains of B. coli still agglu- tinated in 1 to 20 or more because many strains included in this groiiji act as differently toward each other in respect to agglutinins as they do to the typhoid baccili. 198 PRINCIPLES OF MICROBIOLOGY Fig. 73 2d 3d 4th 5th th* 7th 500 ^ — ^ 400 / N 1. 300 / \ 2 00 / y- V 100 y y \ V 00 •r::: — — ,^ \ The rise and fall of common and specific agglutinins during seven months in a ralibit injected with the Manila culture. Colon bacillus X. Paradysentery type (Mt. Desert). Paradysentery type (Manila). Dysentery type (Japan). • Test dates for all four sera * Injections stopped. 1st 2d Fig. 74 3d 4th 6th 6th 7th l:5000 1:4500 / 1 :4000 1:3500 1:3000 / / .-i. 1:2 500 .^ f I.-2000 ^ S' |:| 500 i\o' >y nf/ 1:1000 9>-^:^ .\'&. '/ I: 500 ^ ^/ l: 00 — *' Similar conditions to those noted in previous chart, except that a young goat has been used for the injections of the colon bacillus X. The great accumulation of common agghi- tinins for the paradysentery bacillus in the third month of the injections of the bacillus X is very striking. • Tests made. The following tables give the outcome of several experiments: Absorption by the Tyjjhoid Bacillus of Group Agglutinins Acting upon a Number of Varieties of B. coli which were Produced by Another Variety of B. eoli. Agglutination by Serum of Rabbit Immunized to Colon Bacillus X. After attempt at absorption with typhoid bacilU at 22° C. 5000 20 30 30 10 less than 10 less than 10 li'ss than 10 Before addition of typhoid bacilli. Colon bacillus X 6000 Colon bacillus 1 500 Colon bacillus 2 500 Colon bacillus 3 250 Colon liacillus 4 250 Colon bacillus 5 10 Colon bacillus 6- IS less than 10 T\phoid bacillus less than 10 LOSS OF CAPACITY IN BACTERIA 199 The absorption tests were recent agar cultures to a 10 pc t'our-hour bouillon culture, twenty-four hours at about 2 in a simple dilution of serum Thus, in an extreme instance a with bouillon or salt solution hours, lost 30 to 40 per cent. 15 to 20 per cent. Left for per cent. carried out by adding' the ))acil!i from r cent. st)lution of the serum in a twcnty- The mixture was allowed to stand for 2° C. It was found tliat the agglutinin when left at 37° C rapidly deteriorated, scrum positive at 1 to 1500, when diluted 1:25 and left at 37° C. for twenty-four of its strength; at 22° C. it lost at times three hours, the loss only was 5 to^lO Fig. 75 ^^°0°H^fe *J 1 :i 800 E 3 o a 5 c i- o o-- u V, '^ l:i600 oi 01 ■t i X |:I400 a. ^ 1 H - 0; > Manila ^ l:i200 O < 5 Man la V I:IOOO HtOfsert. Coney 1- (V QJ C 800 1 'f Japan 1 Normal c 1 1: 600 i: ■D I: 400 ' 1 : ' 1 = 1 1 \ ^ a VI 1 a I: 200 j j ! 1 i 3^« ■ l 1 ^ ' l: 100 «: 1 S 1 ^1 o O L O o I: 00 hi T 1 1 ! i 1 ^ 1 : III! 7 1 : II 1 1 Showing the effect of satiirating with bacilli of types of Shiga, Manila, and Mt. Desert, a serum from a horse which had recei'\'ed combined injections of dysentery bacilli of the three types. Note that the Manila type removed almost all the specific and group agglutinins acting upon its own type and the group agglutinins acting upon the Coney Island and normal types, leaving the specific agglutinins for tj^pes Shiga and Mt. Desert. The same is true for types Shiga and Mt. Desert when thej' were used. Manila paradysentery. — Japan dj'sentery. Mt. Desert paradysentery. and Atypical paradysentery. The great number of varieties of the colon group of bacilli that are in the normal intestine and which are al^sorbed slightly in health and more markedly in intes- tinal diseases make the use of absorption test for diagnostic purposes too com- plicated except for peculiarly important cases. Loss of Capacity in Bacteria to be Agglutinated or to Absorb Agglutinins Because of Growth in Immune Sera. — The loss of these characteristics by growth in sera has been demonstrated by Marshall and Knox. The experiments of Collins and ourselves are recorded because they were undertaken in a slightly different way and also because a certain number of confirmatory observations are of value. The maltose fermenting paradysentery bacillus of Flexner was grown on each of eleven consecutive days in fresh bouillon solutions of the serum froin a horse immunized through oft-repeated injections of the bacillus. The solutions used were 1.5, 4, and 15 per cent. The serum agglutinated the culture before its 200 PRINCIPLES OF MICROBIOLOGY treatincut in ililuluiiis uj) to 1 to 800, and was strongly liactcricidal in annnals. After the elc\-en Iransfors the culture gi-own in the 15 per cent, solution ceased to he aKglutinated hy the serum and ceased to alisorh its specific agglutinins. The cultures grown in the 1.5 and 4 per cent, solutions agglutinated well m dilutions up to 1 to (iO and 1 to 100 and continued to absorb agglutninis. The recovery of the cajMcity to l)c agglutinated was very slow when the culture was transi)lantcd from time to time on nutrient agar. After growth for sixteen weeks, during which it was transplanted forty-three times, it agglutinated in dilu- tions of 1 to 200. The culture grown in 4 per cent, agglutinated 1 to 500, and the one in 1.5 per cent. 1 to SOO. This diminution and final cessation of development of agglutinable substance in bacteria grown in a serum rich in agglutinin and immune bodies is interesting both as showing the variation of the bacteria and as one means of adapting themselves to resist destruction, since the bacteria which ceased to produce agglutinable substance probably also produced less substance with affinity for other antibodies. This inhibi- tion of the production of agglutinable substance was also very note- worthy in the case of pneumococci grown in serum media. Non-agglutinable Strains. — Sometimes strains of microorganisms are obtained which possess all the other characteristics of a type and yet do not agglutinate. When grown on artificial media for some time they may gain the property. Organisms freshly obtained from the blood and tissues frequently agglutinate to a much less degree than after several transfers on media. Fig. 76 1 and 2' 3 and 4 5 and 6 7 and S 8 and 9 9 and 10 11 and 12 1,3 and 14 15 and 16 R(4:ifioii of agglutinative to bactericidal powei-. Horse injected with culture of dysentery bacilli over a period of sixteen month.s. Agglutination index. Bacterieidal index. • Test dates. Relation between Agglutinating and Bactericidal Power. — In spite of proof to the contrary good observers hold to the belief that there is some relation between the agglutinating and the bactericidal strength of a serum. The tests we carried out on the serum" of a number of horses showed no such relation. In Fig. 7() are recorded a number 1 Months. ^[ACRnSCOPTC method of KSTI mating AaaiA'TINlNH 201 of C(mi])arativc tests during a pcrioil of sixteen months. While such an ex}H'riiiient sliows no definite relation hetween ai;i;lnl inin and immune bodies it must In- renieniliered that these antihodies are the products of processes whicii are ndxcrncd liy similar laws, and so they ha\e many points in common. Variation in the Agglutinating Strength of a Serum. — There is usually a continued increase in the amount of agglutinin in the blood of an infected person from its first appearance at the end of from four to twenty-one days until convalescence and then a decrease. At times, however, there is a marked variation from day to day, so that it may be abundantly present one day and very slight in amount the next. Mode of Obtaining Serum from Blood or Blisters for Examination. — Fluid blood-serum can easily be obtained in two ways: First, the serum may be obtained directly from the blood, thus: The tip of the finger or ear is pricked with a lancet-shaped needle, and the blood as it issues is allowed to fill by gravity a capillary tube having a central bulb. The ends of the tube are than sealed by heat or with melted wax, or candle-grease, and as the blood clots a few drops of serum separate. To obtain larger amounts of serum for a microscopic examination the blood is milked out from the puncture into a small homeopathic \'ial or test-tube. One cubic centimetre of l)lood can easily be collected in this way. The vial is then corked and placed on the ice to allow the serum to separate. As a ride, one or two drops of serum are obtainable at the end of three or four hours. Second, the .serum may be obtained from blisters. This gives more serum, but causes more or less delay. The method is as follows: A section of cantharides plaster, the size of a 5-cent piece, is appplied to the skin at some spot on the chest or abdomen. A blister forms in from six to eighteen hours. This should be protected from injury by a vaccine shield or bunion plaster. The serum from the blister is collected in a capillary tube, the ends of which are then sealed. Several drops of the serum can easily be obtained from a blister so small that it is prac- tically painless and harmless. The serum obtained is clear and admirably suited for the test. A piece of blotting paper soaked in strong ammonia when placed on the skin and covered by a watch- glass or strips of adhesive plaster will quickly raise a blister. A little vaselin should be smeared on the skin surrounding the blotting paper. The whole blood may be used. It is dropped on glass or stiff paper, dried, and when it reaches the laboratory it is brought into solution. The Reaction. -The reaction can be obser^'ed either macroscopically by sedimentation of the agglutinated clumps in a test-tube or micro- scopically in a hanging drop. The Macroscopic Method of Estimating the Amount of Agglutinins. — The tests are carried out with sets of test-tubes in racks as in the complement-fixation tests (see Chapter X). Some prefer tapering tubes so that the sediment can be more easily estimated. As in complement- fixation tests, great care must be taken to ha\'e the test-tubes clean and 202 PRINCIPLES OF MICROBIOLOGY free from chemicals. The bacterial suspension used, as in the micro- scopic test, must be standardized. The addition of the bacilli on a 24 hours' slanted agar tube to 150 c.c. of normal saline solution gives a good suspension. There must be a uniform cloudiness, not too heavy, with no signs of antiagglutination. A series of dilutions "are made in which the suspensions of bacteria are of similar strength. Salt Suspension of Final ibc. Serum. solution. bacteria. solution. C.c. C.c. C.c. C.c. 1 0.4 1..5 0.1 1 to 5 2 0.2 1.7 0.1 1 to 10 3 0.1 1.8 0.1 1 to 20 4 0.05 1.S5 0.1 1 to 40 5 0.0 1.9 0.1 control After thorough mixing, the tubes are allowed to stand in the incu- bator for one hour. They are then observed and the amount of flocculent precipitate noted. They are then placed in the ice-box at 10° or under, examined in twelve to twenty-four hours, and readings again made. The tubes holding the greatest dilution of serum in which the fluid has cleared by the complete precipitation of all the bacteria shows the measure of the agglutinins. The control tube must be closely examined to note any sedimentation. Comparison of Microscopic and Macroscopic Methods. — The reaction is the same in both and one is as reliable as the other. The ice-box readings are apt to be higher than those obtained by micro- scopic examination. Dead cultures are more frequently used in the macroscopic method because the motility is of no importance. The growth of bacteria in fresh blood containing agglutinins inhibits the development of agglutinable substance in bacteria or causes them to produce substances which prevent the union of agglutinin with them. Bacteria should therefore not be grown on serum media when they are to be used in agglutination tests. Even the addition of ascitic fluid to broth has some effect. Microscopic Reaction. — If the reaction is observed through the micro- scope in a hanging drop, a formation of clumps is seen which, if it takes place rapidlj', reveals the reaction almost completed at the first glance, that is, most of the bacilli are in loose clumps and nearly or altogether motionless (Figs. 77 and 80). Between the clumps are clear spaces con- taining few or no isolated bacilli. If the reaction is a little less complete a few bacilli may be found moving slowly between the clumps in an aim- less way, while others attached to the clumps by one end are apparently trying to pull away, much as a fly caught in fly-paper struggles for free- dom. If the agglutinating substances are present, but still less abundant, the reaction may be watched through the whole course of its develop- ment. Immediately after mixing the blood and the culture together it will be noticed that the bacifli move more slowly than before the addition of serum. Some of these soon cease all progressive movement. PSEUDOB.E ACT IONS 203 and it will be seen that they are gathering together in small groups of two or more, the individual bacilli being still somewhat sej);! rated from each other. Gradually they close up the spaces l)etweeii them, and clumps are formed. According to the completeness of the reaction, either all of the bacilli may finally become clumped and immobilized or only a small portion of them, the rest remaining freely motile, and those clumped may appear to be struggling for freedom. With blood containing a large amount of agglutinating substances all the grada- tions in the intensity of the reaction may be observed, from those shown in a marked and immediate reaction to those appearing in a late and indefinite one, by simply varying the proportion of blood added to the culture fluid. The reaction takes place more quickly at a higher temperature (36° C). Fig. 77 Gruber-Widal reaction. Bacilli gathered into one large and two small clumps, the few isolated bacteria being motionless or almost so. Pseudoreactions. — If too concentrated a solution of dry blood from a healthy person is employed a picture is often obtained which may be mistaken for a reaction. Dissolved blood always shows a vary- ing amount of detritus, partly in the form of fibrinous clumps, and prolonged microscopic examination of the mixture of dissolved blood with a culture fluid shows that the bacilli, inhibited by sub- stances in the blood, often become more or less entangled in these clumps, and in the course of one-half to one hour very few isolated motile bacteria are seen. The fibrinous clumps alone, especially if examined with a poor light by a beginner, may be easily mistaken for clumps of bacilli. Again, the bacilli may become fixed after remaining for one-half to two hours, by slight drying of the drop or the effect of substances on the cover-glass. The reaction in disease is chiefly due to specific substances, but clumping and inhi- bition of movement similar in character may be caused by group agglutinins such as exist in normal horse and other serums. This is a very important fact to keep in mind. 204 PRINCIPLES OF MICROBIOLOGY In urder to lielp tlie student thoroughly to understancl wliat com- prises a reaction, Wilson ])repared a set of drawings, \\liic'h are here reproduced. The culture to be tested should be about twenty Fig. 79 ^i /\' \> Kh — ''"^^'^W, ~M Microscopic field, showing the top of a han drop in a normal tj'phoid culture. ging [icroscopic field, showing a cross- section of the drop in Fig. 7S. hours' growth, either in bouillon or on agar. If on the latter a sus- pension is made in broth or normal salt solution. A loopful of the fluid containing the bacteria is placed on the cover-glass, and to it an equal cjuantity of the desired serum dilution is added. Fig. so FiQ. 81 Microscopic field, showing the top f)f a droi) with the typhoid reaction. Microscopic field, showing a cross-section of the drop in Fig. SO. In making the hanging drop to be examined it is necessary to have it of such a depth that it will show at least three focal planes, other- wise the examination will be incomplete and imsatisfactory. The l\SEUDORE ACT JONS 205 moist chamber must be well sealed by vaselin so as to prevent dryiiiK, and kept at a temperature of at least 20° and not over 35° (\ Fig. 78 shows a microseopie field of the toj) of a hanginr;; drop of a normal liouillon culture of typhoid l:)acilli. The culture is twenty hours old and the organisms are freely motile. This represents the control drop used for camparison with the drop of the same culture to which has been added a little of the blood of a person suspected to have typhoid. Note these points in Fig. 7S; the organisms are evenly distributed throughout the field, except at the edge of the drop, where they are gathered in great numbers; they show great activity here, seemingly trying to crowd to the very edge. This attraction is probably due to the action exerted on the organisms by the oxygen in the air, which naturally exerts positive chemotaxis on all aerobic organisms. Fig. 79 shows a cross-section of the drop represented in Fig. 7S, and it will be noticed that the bacilli are evenly distributed throughout the drop, except at one place in the focal plane a, and again in the focal plane c. It sometimes happens that there is a substance adhering to a sup- posedly clean cover-glass which attracts the bacilli to that point, where they appear as fairly well defined clumps, more or less like the true clumps due to the agglutinating substance in typhoid blood. The increase in organisms at the bottom of the drop in the focal plane c is easily accounted for by the fact that gravity naturally carries the dead and non-motile organisms to the bottom, these frequently assum- ing the character of clumps. If a field can be found in any focal plane of the hanging drop free from clumps, one can be quite sure that any clumping present is not due to any agglutinating substance which necessarily will affect organ- isms in every focal plane. Fig. 80 shows the microscopic appearance of the top of a drop where the reaction is present. Notice first that the organisms have been drawn together in groups and that the individuals of each group appear to be loosely held to gether. Vie\ved under the microscope these clumps are practically quiescent, there being very little move- ment either of the individual organisms or of the clump as a whole. The edge of the drop is practically free from organisms, showing that the air no longer exerts any influence on them. Fig. 81 shows a cross-section of the hanging drop shown in Fig. 80. The clumps are evenly distributed throughout the drop, with perhaps some increase in the numbers and compactness of the clumps at the bottom. Fig. 82 shows the microscopic appearance of the foj> of a hanging drop of a bouillon culture to which has been added some blood of a patient suffering from a febrile condition not caused by typhoid infection, but which exerts a slight non-specific influence on the ty])hoid organisms. It will l)e seen that the reaction is incomplete and that there are many organisms at the edge of the drop. The air exerts the same influence on the bacilli that it did before the addition 206 PRINCIPLES OF MICROBIOLOGY of the blood. Note the character of the clumps, generally small and compact at the centre, with the bacilli at the edge of the clump, usually attached by one end only. Very frequently these clumps have the appearance of being built up around a piece of detritus present in the clump. All the organisms comprising the clump seem to have retained part, at least, of their motility, those on the edges being particularly motile, so far as their free ends are concerned. When motility is very much inhibited these clumps have a peculiar trembling movement, which is like the molecular movement described by Brown. Fig. S3 shows a cross-section of the drop represented in Fig. 82. Note the same character of the clumps in every focal plane: the large number of motile bacilli and the number attracted at the edge of the drop by the air. Fig. 82 Fiu. 8.3 Microscopic field, showing the top of a drop of culture with reaction not due to typhoid. Microscopic field, showing a cross- section of Fig. 82. Precipitin. — A substance similar to and possibly identical with agglutinin is precipitin. This substance was discovered by Kraus in 1897. He found that when a little immune serum was added to the bacteria-free filtrate of a culture of the organism used to produce the immunization there occurred a precipitate. This same reaction took place between the serum of an animal and various proteid sub- stances, such as white of egg, blood-serum, or milk, with which the animal had been injected. That part of the proteid-producing pre- cipitin is called precipitinogen (antigen). Precipitins in their develop- ment, their resistance to heat and chemicals, and in their specific and non-specific forms are similar to agglutinins. The specificity of pre- cipitins is, like that of the agglutinins, not absolute. Grou]) precipitins act upon similar chemical substances derived from cells having very PRODUCTION OF PRECIl'ITATING SERA 207 different eharacteristies. The precipitin test is mostly employed in blood-identification and in testing sera and tissne extracts rather than bacterial filtrates. As the reaction depends on the formation of a precipitate it is important that the solution of serum and antigen be absolutely clear before being placed together. The test is carried out by placing a constant amount of the precipitinogen in a row of test-tubes and to each of these adding different amounts of immune serum (precipitin). A series of control tubes of each serum above should be observed. The tubes are placed in the incubator for twenty-four hours. To prevent contaminating growths . 5 per cent, carbolic acid is first added to each tube. As the action of bacterial precipitins seems to be parallel with the action of the agglutinins, it is possible that where tube reactions are depended upon, some confusion may occur as to which substance is really affected by certain processes or agents, especially those having a solvent action upon the bodies of the bacteria. Production of Precipitating Sera. — Animals, usually rabbits, are in- jected with broth cultures or with emulsions of agar culture, or with a foreign proteid, just as in the production of agglutinins. CHAPTER XII. EXTRACT OF LEUKOCYTES. .OPSONINS. Thk original theory of Metclinikoff, that the leukocytes were the oiil\- actual protective bodies which warded oft' disease, and that they did this 1)3' attacking the bacteria, was founded on the fact that certain of the white cells possess the power of taking up into themselves patho- genic bacteria, which are there destroyed. It was later observed that these cells have the property of taking from the blood many lifeless foreign elements. The question thereby arose as to whether these cells engulfed and then killed the bacteria, or whether perhaps other substances previously prepared them before the cells took them up. The latter supposition was found to be the fact. Neufeld called these substances bacterio- tropins and Wright opsonins. The leukocytes and the chemical substances of the blood thus both play an important part. The death of the bacteria also liberates positive chemotactic substances, and the disintegration of the white-blood cells gives rise to bactericidal bodies. We find that phagocytosis is most marked when the disease is on the decline or the infection mild, but is usually absent in rapidly increasing infection. This would seem to indicate that the course of the infection is often already determined before the leukocytes become massed at the point of its entrance. The first determining influence is given by the condition of the tissues and the amount of bactericidal substances contained in them, and then, later, in cases where the bacteria have been checked, comes the additional help of the leukocytes. If the tissues are wholly free of bactericidal and sensitizing substances, neither they nor the leukocytes, nor both combined, can pre\'ent the bacterial increase. The simple engulfing by the cells of bacteria is not neces- sarily a destructive process. Metchnikoft' belie^•es that the polymor- phonuclear leukocytes are especially antibacterial in relation to acute infections. The large phagocytes are conceived to deal chiefly with the resorption of animal cells and with immunity to certain chronic diseases, such as tul)crculosis. The ])resent great interest in the sul)ject of the o]jsoniiis is largely due to the in\'estigations and influence of Wright.' We should, how- r\(T, recognize the important earlier work of others. Denys and Lcck'f had pre^'ifiusly shown that in the case of rabbits immunized against strc|)tococi'i, the increased ])liagocytosis was due to an altera- ^ Greek "upsoiio" — I cuter for. OPSONINS 209 tion in the serum and not to eliang-es induced in the k'ukoeytes. They demonstrated that the leukocytes of tlie immunized animal when placed in normal serum showed no greater phagocytic activity than normal leukocytes did, and that therefore the substances in the serum favoring phagocytosis united with the bacteria. Wright and Douglass showed definitely that phagocytosis of bacteria in normal serum depends upon a special substance in the serum which becomes fixed to the bac- teria and prepares them for phagocytes. They called this substance opsonin. Neufeld and Rimpau discovered the same point independ- ently. Wright dealt mostly with normal serum, while Neufeld used serum from immunized animals. Wright originated the idea of estimating the changes in the opsonic power of the blood for the purpose of guiding the use of vaccines in the treatment of bacterial infections. Thus he states: Fig. 84 1 fi j 6 / S / \ 1 3 / \ R 1 2 / \ / \ > 1 [ / \ / \, 9 A / \ ^ 1^ >--. ~~ll .8- \ ^ / ' / ^ / '' / / V 1 DATE sl l + l 5 |g| 7 |y 1 !) |l()| 11 \V2\ 13 U jlo Ootoh"!-. 1918 Opsonic curve showing the shght immediate rise and the later negative and positive phases follomng inoculation. The changes here are more regular than generally occurs. "I have found that there exists in the serum of the successfully inoculated patient an increase of opsonin. This is a substance which lends itself to very accurate measurement by a modification of Leishman's method. By the aid of this method the patient's progress or regress can be very accurately followed." Where vaccines are injected', Wright states, there "supervenes a negative phase where there is a diminished content in protective substances. This is succeeded bv a positive phase. This inflowing wave of protective substances rapidly flows out again, but leaves behind in the blood a more or less perma- nentlv increased content of protective substances. When a small dose of vaccine is given the negative phase may hardly appear, but the positive phase may be correspondingly diminished. Where an unduly large dose of vaccine is inoculated the negative phase is prolonged and much attenuated. The posi- tive phase may in such a case make default. "It will be obvious that, if we, in the case of a patient who is already the subject of a bacterial invasion, produce by the injection of an excessive dose of vaccine a prolonged and well-marked negative phase, we may, instead of benefiting the patient, bring about conditions which will enable the bacteria to run riot in his system. 14 210 PRINCIPLES OF MICROBIOLOGY "Now, consideration will show that we may obtain, according as w'e choose our time and our dose wisel)^ or unwisely, either a cumulative effect m the Fig. 8,5 Opsonic outfit. THE OPSONIC INDEX 211 direction of a positive phase or a cumulative effect in the direction of a nega- tive phase, "\^'c may, in other cases, b>' the agency of two or more successive inoculations, raise the patient by successive steps to a higher level of immu- nity, or, as the case may be, bring down by successive steps to a lower level. We can select the appropriate time and dose with certainty only by examining the blood and measuring its content in protective substances in each case before reinoculating." An immense amount of investigation lias revealed the fact that the index cannot be obtained accurately enough hi single tests to be a safe guide to be used in diagnosis or treatment unless the variation from the normal is exceptionally great and that the opsonic content is not a safe guide for the measure of the total antibodies in the blood. THE OPSONIC INDEX. Technique. — Wright's techniciue of measuring the opsonic power is a slight modification of the Leishmani method and is as follows: An emulsion of fresh human leukoc.ytes is made by dropjjing twenty- drops of blood from a finger prick into 20 c.c. normal salt solution containing 1 per cent, sodium citrate. The mixture is centrifuged, the super- natant clear fluid removed, and the upper layers of the sedimented blood cells transferred by means of a fine pipette to 10 c.c. normal salt solution. After, centrifuging this second mixture the supernatant fluid is pipetted oft' and the remaining suspension used for the opsonic tests. Such a "leukocyte emulsion," of course, contains a mixture of leukocytes and of red-blood cells; the proportion of leukocytes, how- ever,. is much greater than in the original blood. The bacterial emul- sion is prepared by gently rubbing a little of the culture to be tested in salt solution (0.85 to 1.2 per cent.). When thoroughly mixed the fluid is centrifuged for a few minutes so as to remove any clumps. The emulsion should be so thick that in a trial test the leukocytes take up about five apiece on the average. One volume of the leukocytes is mixed with one volume of the bac- terial suspension to be tested and with one volume of the serum. This is best accomplished by means of a pipette whose end has been drawn out into a capillary tube several inches in length. With a mark made about three-quarters of an inch from the end it is easy to suck up one such volume of each of the fluids, allowing a small air-bubble to inter- vene between each volume. All three are now expelled on a slide and thoroughly mixed by drawing back and forth into the pipette. Then the mixture is sucked into the pipette, the end sealed, and the whole put into the incubator at 37° C. The identical test is made using a nor- mal serum in place of the serum to be tested. Both tubes are allowed to incubate fifteen minutes and then the end of the tube is l)roken off, a large drop mounted on a clean slide the surface of which was previously roughened by emery-paper, and a spread made with a second slide as in ordinary blood-work, only a little thicker and using no force whatever. ' Lcishmun, British Medical .Journal, January, iyU2. 212 PRINCIPLES OF MICROBIOLOGY After drying in the air the smears are stained without previous fixation either with a 1 per cent, aqueous solution of methylene blue or some other suitable stain. The degree of phagocytosis is then determined in each by counting a consecutive series of fifty or one hundred leukocytes and finding the average number of bacteria ingested per leukocyte. This number for the serum to be tested is divided by the number ob- tained with the normal serum and the result regarded as the opsonic, index of the serum in question. Thus, if the tubercle bacilli, sensitized by a patient's blood, are taken up by the leukocytes to the average number of three per leukocytes, and bacilli from the same emulsion sensitized by normal blood are taken up by leukocytes to the average of five, then the index will be three-fifths of one, or 0.6. In this case the index would indicate a deficiency in opsonins. The presence of a high opsonic index Wright regards as indicative of increased resist- ance. He further states that the fluctuation of the opsonic index in normal healthy individuals is not more than from 0.8 to 1.2, and that an index below 0.8 is, therefore, almost diagnostic of the presence of an infection with the organism tested. A number of workers use the washed whole blood. This saves in original labor, but makes the search for a suitable number of leukocytes more difficult. Simon's Method. — Simon has suggested a modification of Wright's method. He estimates the percentage of phagocyting cells in the mixture containing the serum to be tested and compares this with the mixtures containing normal serum. He also suggests that dilu- tions of blood be tested. The Dilution or Extinction Method recommended by Dean and by Klein. The degree of dilution of the serum necessary for the extinc- tion of its opsonic index is determined; that is, the serum to be tested is diluted until a dilution is found which shows the same small amount of phagocytosis shown in preparations in which no serum is used, namely, an index below 0.5. Klein claims that results by this method are more accurate than by the method of Wright. The method is too tedious for practical use in routine work. Most workers are now agreed that the use of the opsonic index is limited to experimental investigations. The Accuracy with Which the Opsonic Power of the Blood Can Be Determined by Wright's Method. — An examination of any slide will show that the different leukocytes vary in their size and in their content of bacteria. This is due partly to variation in phagocytic activity, and partly to the interference of the red-blood cells, which are present in great numbers in the emulsion and separate the bacteria in different degrees from the white cells. These and other reasons bring it about that the different leukocytes vary greatly in the number of bacteria they take up and in their distribution on the slide. Partly to overcome this, large numbers of leukocytes are counted. Beyond one hundred, or at most one hundred and fifty, the increase of accuracy hardly compensates for the extra labor. THE OPftONIC TNDEX 213 The following table shows the ditTerence between connting larger or smaller nnmbers of cells in five opsonic tests as (leterniiued by eonntlng ditt'erent numbers of cells in one specimen: Ol'SONlC InUKX lOsTIMATLIlNS IN FlVK UluoD-SI'BC'I MKNS. Ce Us couutod. 50 1.18 101) 1.22 150 1.18 200 1.18 (iOO 1.28 1,200 1.34 Averaj^c number of bacteria in each Ieul1, / \ \ / \ i^'" A ~ ^■'i / \,W---.-t--- / \- - \ i /-. -/■- ,-+-■ \ 1 \ 7^--^- f / ■~> ~J \l \ f \ ^ 'A s 1 \ ^ V \ S 1 " A \/ \ r" < s / \ , \ - s/ N / s / '^-? - — 1 X = injection of vaccine. 3 types of lines = 3 cases. Fig. 87 aiai-cU 1!>U7 ■J a 4 . « , >i '-i 10 11 VI i:! TBC. i.r l.G 1.5 1.4 \ \ — \ \ \- \ TBC. \ \ A / TBC. GREENWALD- CALLAKAN- TDC. DOLDVAN- i.y l.'Z ""^X / ' — ^ h- -^-\V / \, /^ \^ / \ \ 1 ^^= \~j^' - ""^. \j/ t / 1 1.1 =;'^ H\ ' r \ ^\~ \ ' ^^ 1.0 .0 =X-x.\^ v- \ K K n^K ><' >^ KN ■,/- ^^J \ ~=5i?vN^ 1 jf y\ \ K - .8 [ : :■ ■'■---«ei^ \ '/;t- -^ V- — — r ^t^i^i / Ssy^y V-^i 1 "■"■-■^ ■^--JT i \ 1 _ 1- Dotted and crossed lines, normal persons. Continuous lines, tuberculosis. The Variation in the Amount of Opsonins in Supposedly Healthy Persons.— It has already been noted that in getting our measure we test a number of persons and exclude the blood of those which varies greatly from the a\'erage. We are so in the habit of 21(1 PRINCIPLES OF MICROBIOLOGY seeiiifi; the index of normal blood placed at unity because it is each day the measure of com])aris(m that even investigators are apt to think of the indices of normal ])ersons as being unchanged from day to day. This is not the fact. A glance at the next chart (P'ig. 87), in which four cases of tuberculosis are charted together with two normal persons, shows that the variation is only slightly greater in infected than in normal cases. If one normal person is charted against another for several weeks, marked differences will usually appear. The indices of the twenty-one normal cases tested against staphylococci (page 214) illustrate this variation in the amount of opsonins in normal blood. The Opsonic Index Cannot be Known at the Time the Treat- ment IS Given. — Most of those who have not carried out inoculations under the guide of the opsonic tests think that the vaccinator is guided at the moment of injection by his knowledge of the opsonic power of the blood at the time. A moment's thought reveals that this is an absolute impossibility. In fact, except under very unusual conditions, it is impossible to have the test of the opsonic power reported within twenty-four hours, and in the treatment of the poor in out-patient practice longer intervals usually elapse, so that the treatment is given on a test made either the day before or, more often, on from three to seven days before. As can be seen b}' the three curves in Fig. 86, which are quite as uniform as the average, it is impossible to judge what the index is at any moment by looking at the indices of blood taken from one to seven days previously. Other methods have been devised to get more accurate information upon the opsonic contents of the blood. The dilution method and that combined with determining the percentage of phagocytes ab- sorbing bacteria are the most valuable^ (p. 212). For experimental work they have advantages, but for practical use in governing the dos- age of vaccines they have most of the drawbacks of Wright's method. The Diagnostic Value of Opsonins. — The presence of a great excess or deficiency of opsonins for a microorganism, or of marked variation in the index after massage or exercise, has been thought by some to indicate the type of infection. Extreme caution should be used in making such an application of the index determinations. The Nature of Opsonins. — Wright and Neufeld, in their orig- inal experiments, differed as to the effect of heat on opsonins. Further investigation has shown that opsonins in those not immunized are largely thermo-labile, while opsonins developed after immunization are resistant. Muir and Martin believe from their experiments that the thermo-labile opsonin of normal serum and the thermo-stable opsonin are two entirely distinct classes of substances. The thermo- stable substance is of the nature of a true antibody and possesses the comparatively specific qualities of antibodies in general. Powerful complement absorbers have no effect on the thermo-stable opsonin, • Simon, .Tour. Exp. Merlicino, vol. ix, No. .5, 1907, p. 4,87. LFAJKOCYTE EXTRACT IN INFECTIONS 217 hut do remove ulmost completely the thermo-hihile opsonin. Neiifeld agrees witli them. Emulsions of other than thi' orf^anisms used in inununizatioii do uot ahsorl) a large pereeiitai;e oF tlie imunnie opsiiniu, hut do of the eom{)leuH'nt opsonin. We lune i-irried out ahsorption exi)erimeuts with staph\ loeoeei, colon, and tubercle bacilli. Our results were similar to those of Muir and Martiu. Opsonin Deficient in Cerebrospinal Fluid and in Exudates. — Opie^ has showu that exudates produced by injecting microorgan- isms usually have little or no opsonin for the \-ariety injected or for other varieties. Hektoeu has showed that opsonins, like other anti- bodies, are almost absent in the spinal fluid. McKenzie and Martin- show^ed that in a case of cerebrospinal meningitis the spinal fluid showed no immune bodies, while the blood contained them in abun- dance. Comparison between Opsonins and Bactericidal Substance IN THE Serum. — We ha\-e made comparative tests between the op- sonic and bactericidal power of the cell-free serum in typhoid infection and found that they did not run parallel. The frequent rapid increase in opsonic power within twelve hours of an injection of bacteria is striking and very different from the development of bactericidal strength. LEUKOCYTE EXTRACT IN INFECTIONS. Hiss^ recommends the treatment of certain diseases with a leukocyte extract. He makes this extract as follows: The leukocytes are obtained by double pleural inoculations with aleuronat into the animal (rabbit, dog). The amount of leukocyte- filled fluid obtained after twentj'-four hours from rabbits has usually been from 30 to 60 c.c. This is immediately centrifugalized, the serum poured ofT, and the extracting fluid (distilled water) added in amounts about equal to the fluid poured oft'. The cells are then thoroughly emulsified in the distilled water, allowed to stand for eight hours at 37.5° C, and then at ice-box temperature until used. Varying amounts of the entire fiuid (after shaking) are inoculated. Hiss's animal ex- periments were made on rabbits and guinea-pigs infected with staphyl- ococcus, streptococcus, pneumococcus, typhoid bacillus, or menin- gococcus. Hiss stated that animals suffering from severe septicemias and poisonings following intravenous injection of any one of the above organisms have shown the beneficial effect of treatments with extracts of leukocytes, and have, in many instances, survived infections fatal to the control animals in thirty-six hours, even when treatment has been ■ Opie, Jour. Exp. Med., vol. ix. No. 5, p. 515. 2 Jour, of Path, and Bact., vol. xii, p. 539. ' Jour. Med. Research, xiv, No. 3. 21S PRINCIPLE,^ OF MICROBIOLOGY delayed as late as twenty-four hours. Zinsser' has carefully studied the nature of the substances extracted from leukocytes. He finds they contain no complement, are not destroyed by heating to 56° C, and are no more abundant in cells derived from immunized than from normal rabbits. The Hiss leukocytic extract has now been used by a number of ob- servers for some six years. Cases of pneumonia, erysipelas, septicemia, and some other infections have been treated. It is difficult to deter- mine just what the value of the treatment is. No harmful results have been noticed. In a certain number of cases the temperature and symptoms have bettered, in a way which seemed clearly to indicate that the extract had done good. In other cases no results whatever were apparent. In our personal experience we have not seen definite results. It is usually given subcutaneously in 10-c.c. doses everj' four to six hours. As high as 500 c.c. have been given in some cases. ' .Jour, Med. Research, xxii, No. .3. CHAPTER XIII. ACTIVE DIMUNIZATION. VACCINES. The success of vaccine virus in immunization against smallpox led Pasteur to his successful attempts to immunize sheep against anthrax by the use of attenuated living cultures of the anthrax bacillus, and to inoculate human beings against rabies by the inoculation of attenuated rabies virus. Later experimenters learned, as already stated, that the parenteral injection of the whole bodies of microorganisms or of extracts of them produced more or less immunity. This could be determined both by the resistance of the animals to infection or by the estimation of the various antibodies in the serum. This success led to the use of injection of measured doses of living or attenuated organisms or their extracts into persons already diseased, with the hope that healthy tissues at a distance from the diseased area might make antibodies to add to the insufficient amount produced in the infected area. Experi- mental investigations showed that the cells constantly poisoned were not in as good condition as normal cells to make antibodies and that the bacterial substances entering the blood were neutralized by anti- bodies in the serum as soon as they had accumidated so that the antigen acted less strongly to produce additional antibodies. Koch attempted this in tuberculosis by means of graded doses of the various tuberculins and Wright and others in various infections. A general consideration of the subject only is given here as the use of vaccines which have been successful will be considered under the individual pathogenic microorganisms. There are two points to be kept in constant view, namely, that the giving of bacterial vaccines during infection is adding more or less poisonous proteins and that unless the body tissues respond to produce antibodies, harm rather than good is obtained. Experience has taught us that those who become infected are less able to produce antibodies. In such diseases as pneumonia, typhoid fever, septicemia, and other general infections due to different microorganisms, the treatment by bacterial vaccines has not met with sufficient success to be recognized by most careful observers. Here, an overdosage of the foreign more or less toxic protein should be carefully avoided. The vaccine treatment of these diseases must still be considered in the experimental stage and the exaggerated claims of certain manufacturers as wholly unwarranted. Localized Infections. — The results here in a considerable variety of in- fections have been good. The clinical observations are also borne out by the increase of the various immune bodies in the cases treated. The Use of Single and Multiple Varieties of Bacteria. — The classification of bacteria upon any grounds other than their production of antibodies 220 PRINCIPLES OF MICROBIOLOGY is useless in vaccine therapy. Two otherwise characteristic strains of colon bacilli may be as widely sei)arated from the antibody standpoint as a colon and a typhoid bacillus. This adds very much to the difficulty of treating many infectious and leads frequently to the necessity of using cultures made from the infected persons (autogenous cultures) to prepare the vaccines. In mixed infections the patient should theoretically be immunized against each of the varieties causing the different infections. The organism of the patient may not find the task of responding to a series of different vaccines (always supposing that each of these is adminis- tered in appropriate and properly interspaced doses) much more difficult than the task of responding to one variety of vaccine only. General Opinion of Results. — Although during the past few years many thousands of cases of infection have been treated by vaccines, there is at present considerable difference of opinion as to their value. The majority of observers agree that it is in subacute and chronic infections that vaccines give the best results. Thus a case of acute streptococcus septicemia, which after a week or ten days shows a tendency to abate with localization in a joint or in a valve of the het^rt, offers a much better chance for vaccine treatment than such a case during the early more acute stage. Pneumonia, which persists after partial recovery, a gonorrheal joint, a persistent pus sinus, a localized inflammation due to colon bacilli, are all considered suitable for treatment. The use of vaccines in cases of acute inflammation of the mucous membranes of the intestines, bladder, throat, etc., have in most hands given rather negative results. Most believe that the inflammation is lessened, but that, as a rule, the bacterial infection, though it becomes latent, still remains and that a relapse may occur later. The different bacteria seem to differ in the successes with which they are used as vaccines. Nearly all observers report the greatest success with staphylococcus infections. These not only when they are severe as an osteomyelitis or an extensive carbuncle, but also when they are mild, such as ordinary furuncles or acne, seem to be aided by the vaccine treatment. Gonorrheal joints respond well, while acute gonorrhea of the mucous membranes responds little if at all. The streptococcus infections seem on the whole to be more resistant, but considerable success has been met with in treating subacute and chronic joint affections following local points of infection, as in a tonsil or tooth. The pneumococcus also seems resistant, perhaps even more so than the streptococcus. Numerous cases of pneumonia have been treated with somewhat doubtful results. Some observers treat these pneumococcus and streptococcus inflammations with daily doses of 10,000,000 instead of the large semiweekly inoculations. Various sinus infections of the head have been treated, but with doubtful results. Streptococcus and other inflammations of the gums and teeth have yielded apparently good results. The treatment of tuberculosis with the different tuberculins has given perhaps the most extended use of vaccines. The majority of observers are convinced that in cases of PREl'ARATJON OF BACTERIAL VACCINES 221 tuberculosis, in which the symptoms h;i\^e become quiescent, whether they are incipient or fairly advanced, tubercuhn injections tlo good. Some beheve in giving very minute doses, and only slightly increas- ing their size. Others believe that different cases will receive with benefit different amounts, and so try to detect which will stand larger amounts. Nearly all physicians try to prevent definite reactions. The treatment of tuberculosis is considered in detail under Tuberculosis. The results of vaccine treatment, so far as we have observed them, have not come very quickly or been striking. There may be a slight betterment after the second or third injection, but at other times this improvement is delayed until ten or twelve have been given. We are always skeptical of the influence of vaccines when sudden improvement ensues. We remember, several years ago, two cases of septicemia, one of which received vaccine and the other did not. Both made a striking recovery. If circumstances had not prevented giving the vacccine to the second case, it would have seemed as almost certain that the vaccine had produced the cures. We believe that a final decision as to the value of vaccines would l)e arrived at more quickly if it were possible frequently to control treated cases with similar ones not treated, so that one might obtain more readily a correct impression of the effect of the injections. Even then it will undoubtedly be a number of years before we will clearly appreciate the class of cases which vaccines will benefit and those which they will not. Probably only the observations made by especially trained observers will ever give us this correct impression. Preparation of Bacterial Vaccines. — Cultures are preferably grown on agar, although broth may be used. The growth after twenty-four to forty-eight hours, depending on the rapidity of growth of the organism, is washed off in a small amount of saline solution. This suspension should be well shaken to give an even distribution of bacteria and is then standardized. The Wright method is most commonly used. A capillary pipette (see p. 210) is marked about one inch from the tip. The finger is pricked and blood drawn up to this mark; a bubble of air is then allowed to enter the tube and the bacterial supension then drawn up to the mark. The contents are then mixed on a slide and thin smears (as for Ijlood) made. On the under lens of the eye-piece a one-quarter inch square is marked out with blue pencil, and using an oil-immersion objective the number of bacteria and red cells appearing in this square are counted. About fift>' sciuarcs are counted and the average per square obtained. The number of red cells per c.c. being known, the number of bacteria are obtained according to the following proportion : Number of red cells ■ Number of bacteria • • -^^q (mUhon) : X (millions) per square " per square Other methods are employed as direct counting either by the Prescott method, in a counting chamber as used tor red cells, centrifuging and deter- mining the column of bacteria, or by determining the weight of the bacteria after evaporation and drying. Methods of Killing the Bacteria. — Heated above 58° the bacteria show marked changes in their chemical composition, and do not yield 222 PRINCIPLES OF MICROBIOLOGY the same amount of antibodies as those not so heated. Even lower degrees, such as 56°, are preferable to 58°. It has lately been suggested that instead of killing by heat the vaccines be killed by adding, when made, 0.5 per cent, carbolic acid, or 0.25 per cent, lysol. Vaccines can be kept for a period of three months with only slight loss of activity. The vaccines may be diluted to the required dosage when used. Amount of Vaccine Injected. — The intervals and size of dosage are still under active investigation. Some advise daily small doses, others larger doses every 2 to 7 days. We prefer giving small injections every other day. Certainly the interval should not be longer than 4 days. The dose should be gradually increased at each injection. Differ- ent observers advise that for each organism the following number of millions be given : Minimum. Maximum. Average. Staphylococcus . 50.0 ni. 2000.0 m. 250.0 m. Streptococcus 5.0 ni. 500.0 m. 25.0 m. Pnoumococcus . 5.0 m. 500 . m. 25.0 m. Gonococcus . 5.0 m. 500.0 m. .30 . m. B. coli group 25.0 m. 1000.0 m. 100.0 m. B. pyocyaneus . 25.0 m. ■ 1000.0 m. 100.0 m. Sensitized Vaccines. — Besredka^ has suggested a plan of injecting virus which has been immersed in specific serum; in other words, of using virus which has been sensitized. Heated vaccines alone some- times give severe symptoms when inoculated, e. g., typhoid, cholera; but after exposure to serum, inoculations of the sensitized vaccines are found to gi\''e only slight local and general reactions. Immunity, moreover, is produced rapidly by this procedure. Such sensitized vaccines can be given in doses thirtyfold those of pure virus. Marie^ has used the same method in rabies treatment. The method is as follows: For bacteria: A twenty-four-hour agar culture of bacteria is mixed with specific antiserum at room temperature and allowed to stand for three hours. The bacteria are then washed free from unattached serum by repeated centrifugalizations with sterile normal salt solution. Typhoid and cholera bacteria are killed at this stage of the procedure by heating at 5G° C. for one hour. Plague bacilli are killed before the addition of the serum. By this method the endotoxins are neutralized and the symptoms of intoxication are less severe. Besredka and those working with him have used sensitized living typhoid bacilli for treatment and immunization. AVe have had no experience with these. It probably is incorrect to consider that many of the sensitized bacilli are alive after a few days' storage. Autogenous and Stock Vaccines. — The usual jiractice is to use a stock vaccine for the first iuoculation and autogenous vaccines later. Some- times it is impossil)!e to obtain a culture from the patient. The staphy- ' Review of previous work. Bull. Inst. Pasteur, 1910, viii, 241. = Sec Raljies, Sec. II. VACCINES AS IMMUNIZING AGENTS 223 lococci, tubercle, and typhoid hacilli from diilVrent cases seem much alike, so that it is less important with these organisms to get autogenous vaccines than when streptococci, pneiunococci, or colon Imcilli must be used. A stock vaccine of certain organisms like the gonococcus require a stock vaccine composed of the ditt'erent varieties in the group. Vaccines as Immunizing Agents. — The injection of vaccines in healthy subjects for the prevention of disease has been made so extensively that no one doubts the advisability of their use. Typhoid vaccines are used extensively in the army and among persons going into special danger. Cholera vaccines and \'accines against bubonic plague also have been widely used. (See Individual Organisms, Part II.) CHAPTER XIV. PROTEIN HYPERSENSITIVENESS OR ANAPHYLAXIS. Perhaps the most important recent addition to our scientific knowledge has been the development of our conception of the fact and meaning of protein hypersensitiveness. Magendie is probably the first to record oliservations on this subject. He noted that rabbits which were injected intravenously for the first time with egg albumen showed no untoward effects, but that if after a lapse of time a second injection was given, serious and perhaps fatal results followed. Richet, in 1902, noted that when the blood of rabbits, which had received a previous protein injection was transferred to other rabbits, it rendered them sensitive to a second injection. The fact that this transfer of serum rendered them susceptible to protein poisons instead of protecting them caused Richet to give the term anaphylaxis to this condition. Arthus showed that in animals sensitized to horse serum a later injection given subcutaneouslj' produced striking cutaneous reactions ranging from infiltration to gangrene. Theobald Smith studied and reported the reaction in guinea-pigs and Rosenau and Anderson noted the fact that the yomig of anaphylactic mothers inherited the same protein sensitiveness. Vaughan and others explain the poisoning of the second injection as follows: The introduction of a foreign protein into the tissues or blood of an animal develops in that animal a proteolytic ferment which is specific for the protein injected or for one very closely allied to it. The specific ferment remains as a zymogen in the cells of the animal and in its blood and tissue fluids, and is activated when the same protein is again injected. All protein poisons contain a toxic and a non-toxic portion ; the first is not specific, while the second is. The first injection of the protein ])roduces no symptoms because there is no proteolytic ferment present. The second injection, if suflficient time has elapsed for the formation of the ferment, causes symptoms because, the specific ferment having been produced, the protein when reinjected is split up into its poisonous and non-poisonous portions, and the former causes symptoms of in- toxication or anaphylaxis. Those changes occur not only in the plasma, but also in the cells, and the symptoms depend on whether the second injection was made locally or into the general blood current. The second injection causes no symptoms unless, as in all other immunity reactions time enough is allowed to elapse for the cells to assimilate the proteins and for specific ferments to be produced. Gay and Southard applied the term anaphylactin to this ferment. Friedberger calls the poisonous part of the split protein anaphylatoxin. EFFECT 225 Amount Rcquircdio Sensitize: Very small amounts such as 0.0001 c.c. of horse serum sensitizes an animal by whatever way inoculated. Incubation. — This varies in different animals. In man it is usually seven to tweh-e days. It gradually reaches its maximum and lasts for four months to a year or more. Where the reaction is present after much longer periods there has probably intervened a later sensitization, just as must have happened where an anaphylactic shock occurs in persons after a primary serum injection. Quantity of Foreign Protein Required to Produce SymjAovhi. — This is much larger than the amount which sensitizes. Much smaller amounts produce symptoms when given intravenouslv- than when given sub- cutaneously. Thus, in a guinea-pig, 0.01 c.c. of serum intraperitoneally has as much effect as 0.5 c.c. subcutaneously. The following proteins and others produce the antiferments : 1. Animal proteins in sohition: Foreign serum, hemoglobin, milk albumin, egg albumen, etc. 2. Cellular animal proteins: Red-blood cells, leukocytes, cells of organs and tumors. 3. Vegetable proteins : Extracts of bacteria, seeds, etc. 4. Cellular vegetable proteins: Living or dead bacteria and yeasts. Specificity. — \Yhen an animal is sensitized with one protein it reacts most strongly to one of the same chemical structure, but also slightly to others nearly allied; thus a guinea-pig sensitized to egg albumen of the hen reacts slightly to egg albumen of the duck. This is similar to the group reaction to agglutinins, precipitins, etc. Antiana'phyla.vi.?. — According to Besredka and Steinhardt animals recovering from a second injection of serum are immune for a variable time. The effect of protein sensitization may be manifest in many ways Thus von Pirquet and Schick in 1905 described the symptoms which frequently follow in man after an injection of horse serum. These symptoms follow almost as frequently primary as secondary injections, but the time of their appearance is late with the primary injections and early with the secondary ones. The probable explanation is that after the injection of the first serum the cells gradually assimilate it and in a few days develop the specific proteolytic ferment. This acts upon the portion of the serum not yet assimilated and splitting it into the poisonous and non-poisonous portions, the toxic symptoms of fever, rash, etc., develop. The development of the symptoms in infectious diseases is attributed by Vaughan and others to this same fact of protein cleavage, as in the case of serum injections. Danger. — x\bout 1 in 20,000 primary injections of diphtheria anti- toxic sera causes the immediate appearance of anaphylactic shock, in which symptoms of respiratory embarrassment and convulsions develop. About 1 in 50,000 of these cases ends fatally. Friedberger and Hartock have shown that with the occurrence of anaphylactic symptoms there is a diminution of complement. The best studied instance of anaphylaxis is that produced in the 15 22G PRINCIPLES OF MICROBIOLOGY guniea-pig by the injection of a foreign proteid, such as horse serum, egg white, milk, etc. For example, if a guinea-pig is injected with a small quantity (about 0.01 c.c.) of horse serum, and, after an interval of ten to twelve days, is again injected with horse serum, but with a comparatively large amount (3-5 c.c. subcutaneously; 0.25 c.c intravenously), it will probably die in a short time. In a very sen- sitive animal symptoms appear within ten minutes and death occurs within an hour. The chief symptoms are respiratory failure, clonic spasms, and paralysis. If a smaller dose of serum be given to the sensitized animal it may show only slight symptoms and recoverwith the production of immunity. This phenomenon was noticed simul- taneously in several laboratories but was first definitely described by Theobald Smith, and is frequently called the Smith phenomenon. Tuberculin and mallein reactions are well-known instances of ana- phylactic manifestations (see special subjects). Doerr, of Vienna, ^ closes a most excellent review of recent work on anaphylaxis or protein sensitization with the following paragraph: "While it must be admitted that the action of those infective bac- teria, which are not known to produce specific toxins, remains with- out explanation, and while the theories which have been developed by Von Pirquet, Friedberger, Vaughan, Schittenhelm, Weicherdt, and others have opened up a new way to the understanding of incubation, fever, and crises, still it must be borne in mind that the premises of these theories do not possess the force of chemical facts. It has not been positively shown that the symptoms of anaphylaxis are due to the parenteral cleavage of proteins, that the true anaphylactic poison is identical with that produced in vitro, and that both come from the antigen. Even if we agree with Dold, Sachs, and Ritz, that so far as the role of anaphylaxis in the infectious diseases is concerned, it is irrelevant from what matrix and by what processes the hypothetical anaphylactic poison is produced, even then all the difficulties are not removed. Numerous infecting agents are not anaphylactogens; they do not differ in their effects upon sensitized and non-sensitized animals; and even when there are differences, they are slight compared with those seen when the protein antigens derived from the higher plants are employed. The relatively simple structure of the bacterial proteins is the cause of this. Therefore, it is questionable whether one has the right to explain the phenomena of the infectious diseases with serum sensitization as a starting point. Moreover, the infections are not so monomorphic as some suppose from a superficial consideration." Serum Sickness. — Besides these rare accidents there are the disa- greeable after-effects which wc group under the name serum sickness. Under this name we now include the various clinical manifestations following the injection of horse, serum into man. The principal symptoms of this disease are a period of incuI)ation varying from eight to thirteen days, fever, skin eru])tions, swelling of the lymph ' Handbuch fl. path. MikroorKauisrncu, Zweite Auflago. KoUe and Wassermanu. SERUM SICKNESS 227 glands, leukemia, joint symptoms, edema, and allniminuria. The term' "serum sickness" was first used by von Pirquet and Schick, ^ from whose excellent monograph the following data are chiefly taken. In 1874 Dallera reported that urticarial eruptions may follow the transfusion of blood. In the year 1894 the use of diphtheria antitoxin introduced the widespread practice of injecting horse serum. In the same year several cases were reported in which these injections were followed by various skin manifestations, mostly of an urticarial char- acter. Following these came a great mass of evidence which made it clear that following the injection of antidiphtheric serum these sequelae were usually comparatively harmless. Due to Serum as Such. — Heubner in 1894 and von Bokay some- what later expressed the opinion that these manifestations were due to other properties than the antitoxin in the serum, and this has proved to be the case. It has also been shown that the skin eruptions and other symptoms follow in a considerable degree according to the amount of serum injected, and this has led to attempts to eliminate the non-antitoxic portion of the serum as much as possible.- The serum reaction has been studied by many investigators, but is not yet fully understood. Von Pirquet and Schick believe that the phenomena classed under serum sickness should be classed with those of anaphylaxis. It is probable that man cannot be sensitized in the same way as the guinea-pig, the most susceptible of the laboratory animals. Children have, in numerous instances, been injected with antidiphtheric horse serum at short and long intervals without, so far as we are aware, causing severe symptoms. Certain serums — for example, the anti- tubercle serum of Maragliano — are habitually used by giving injections at intervals of days or weeks. The rare fatal cases so far reported have all followed primary injections. While it ma}' be true that the sensitizing of guinea-pigs by a previous injection of serum is analogous to the condition present in man which gives rise to the sudden symptoms following an injection of antitoxic serum, there is, in our experience, no reason to avoid a second im- munizing injection of serum when it is really indicated. A subcu- taneous injection in man comparable to the amount required to produce sickness in a guinea-pig would be over 200 c.c. We should hesitate, however, to give a large intravenous injection in a sensitized child. Banzhaf and Famuleuer have recently shown that chloral in large doses will prevent sickness in sensitized guinea-pigs. ' V. Pirquet and Schick, Die Serum Krankheit, Wien, 190.5. 2 See Banzhaf, The Preparation of Antitoxin, Johns Hopkins Hospital Bulletin, 1911, xxii. No. 241. PART II. PATHOGENIC MICE00E(4ANISMS INDIVIDU- ALLY CONSIDERED. CHAPTER XV. THE PATHOGENIC MOLDS (HYPHOMYCETES, EUMY- CETES) AND YEASTS (BLASTOMYCETES). THE HYPHOMYCETES. The majority of the molds are not pathogenic for human beings, and interest us more as organisms which are apt to infect foodstuffs and media. Some are, however, true parasites, and produce a number of rather common diseases; for example, ringworm, favus, thrush, and pityriasis versicolor. Certain of the commoner molds (mucor, aspergillus) have also been reported from time to time as present in pathologic conditions in man as well as in the lower animals. Many varieties have been found in plant diseases, and others may be a source of danger to man indirectly. Indeed, when they form poisonous substances, as in the infection of grain by claviceps jmrpitrea (ergot poisoning), they are distinctly dangerous. Paltauf reported the case of a man who died after enteritis with secondary peritonitis. The autopsy showed multiple abscesses in brain and lungs, besides the lesions in the intestines and peritoneum, in all of which a species of mucor was found. Two other cases of primary mucor infection in humans were reported by Furbringer. A number of species of mucor have been found in ear and eye infec- tions; for example, Mucor corymbifer (Fig. 89) has been found in ophthalmia. A number of varieties are pathogenic for lower animals. Aspergillus is found thus very frequently in lower animals, especially in birds, where a kind of pseudotuberculosis is often produced. Quite a number of similar cases have been reported in man, and it is supposed that the infection may be carried from birds to man. Aspergillus fumigatus (Fig. 90), is the most frequent variety found. Penicillium minimum (similar to glaucum. Fig. 88) has been found by Liebermann in inflammation of the ear. The more common patho- genic forms for man are those producing the various hair and skin 230 ■PA THOGENIC MICROORGANISMS lesions mentioned above. These will now be described (see p. 27 for general characteristics for molds; see also Phite I). Fill. NS Penirillium glauouni. Gelatin culture. Spread stained mth gentian violet. .500 : 1. (From Itzerott and Niemann.) Fig. 89 Mucor corymbifer, Coh. Mycelium with underlying branched carriers, sporangia at have burst. '}>. (After Lichtheim.) Trichophyton (Tinea, Ringworm Fungus).— Ringworm of the body or hairless parts of the skin, Tinea circinata, and ringworm of the hairy parts. Tinea tonsurans and Tinea barbae or Tinea sycosis, are due to fungoid organisms placed in the imperfecti group (p. 2S). THE HYPIfOMyrETES 231 Methods of Examination.— Lii'tftf; SpeciineHs.^Phi.co scales or hairs in hot liq. jKitassa' for a few seconds. Examine nnder CDver-glass. I'cnnaiiviit .•specimens. — Ileniovo fat with chloroform, then jilace material in formic acid and heat nntil boil (2 to 3 minutes). I^emove acid by washing in distilled water, stain witli Lietfler's methylene blue. \\'asli, deliydrate in absolute alcohol, clear in xylol and mount in balsam. Sabouraud's media are best for gi-owth of the organism. According to Sabouraiul, whose conclusions are based on an exten- sive series of microscopic examinations of cases of tinea in man and animals, of cultivation in artificial media, and of inoculation on man and animals, there are two distinct types of the fungi causing ringworm in man — one with small spores {2fx to 3^) which are known as micro- spora, and one with large spores (7^ to 8/x) which are called viegalo- spora. They differ in their mode of growth on artificial media and in Fig. 90 Aspergillus fumigatus. Gelatin culture. Spread stained with gentian-violet. .500 : 1. (From Itzerott and Niemann.) their pathological effects on the human skin and its appendages. The small spored type is the common fungus of Tinea tonsuram' of children, especially of those cases which are rebellious to treatment, and its special seat of growth is in the substance of the hair. T. megaJosporon (Fig. 91) is essentially the fungus of ringworm of the beard and of the smooth part of the skin; the prognosis as regards treatment is good. One-third of the cases of T. tonsurans of children are due to megalospora. The spores of T. microsporon are contained in a mycelium; but this is not visible, the spores appearing irregularly piled up like zoogiea masses; and, growing outside, they form a dense sheath around the hair. The spores of T. megahsporon are always contained in distinct mycelium filaments, which may either be resistant when the hair is broken up or fragile and easily breaking up into spores. The two types show distinct and contsant characters when grown in artificial cultures. The cultures 232 PA THOGENIC MICROORGA NISMS of T. microsporon show a downy surface and white color; those of T. inegaJosporon a powdery surface, with arborescent peripheral rays, and often a yellowish color. Although the morphological appearances Vu:,. yi Hair riddled with ringworm fungus. Megalosporon variety. Fig. 92 ^■■^- ../ ■'>''^S , h'i-ly ■ :■ -'MWL «' half of the cases treated show no appreciable decrease in size; the majority of the others which did show marked improvement at first, after decreasing in size for a few weeks, again began to increase and were no longer influenced by the treatment. 256 PATHOGENIC MICROORGANISMS "I ha\'e now a number of cases of spindle- celled sarcoma which have remained well beyond three years; one case of mixed (round and spindle) celled, after remaining well three years, had a return in the abdomen, and died about eight months later. The result here certainly establishes the correctness of the early diagnosis. "It should be distinctly stated that all of the tumors under con- sideration were inoperable, as I have never advised treatment except in such cases." Some surgeons have not had nearly as favorable results as Coley. There seems to be no question about good results having been obtained in a small percentage of cases. The injections cause fever and loss of flesh and strength. Production of Toxic Substances. — There is no doubt that the strepto- coccus causes fever, general symptoms of intoxication, and death by means of toxic substances which it forms in its growth; but we know very little about these substances or how they are producefl. The cell substance of streptococci possesses only shght toxicity. Ruediger' has shown that a specific streptolysin is formed which produces a true antibody. The poisons while partly extracellular are mostly contained in the cell substance. Heat destroys a portion of them. They appear to attack especially the red-blood cells, and this hemolytic action seems to be to some degree in proportion to the virulence of the organism. Susceptibility to Streptococcus Infection. — As with the ever-present stajjhylococci, whose virulence, as we have seen, is usually slight, the streptococci are more likely to in\'ade the tissues, forming abscesses or erysipelatous and phlegmonous inflammation in man when the standard of health is reduced from any cause, and especially when by absorption or retention \-arious toxic organic products are present in the body in excess. It is thus that the liability to these local infec- tions, as complications of operations or sequela? of various specific infectious diseases, in the victims of chronic alcoholism, and consti- tutional affections, etc., are to be explained. It seems established that the absorption of toxic products formed in the alimentary canal as a result of the ingestion of improper food, or in consequence of abnormal fermentative changes in the contents of the intestine, or from constipation predispose to infection. Immunity. — In none of the streptococcus inflammations do we notice much apparent tendency to the production of immunizing and curati\'e substances in the blood by a single infection. Se\'eral general infections usually progress to a fatal termination after a few days, weeks, or months. It is true, however, that cases of erysii)elas, cellulitis, and abscess, after periods varying from a few days to months, tend to reco\'er, and to a certain extent, therefore, we may assume that ])rotective agents have been jiroduced. In these cases, however, we know from ex|)ericnce that faulty treatment, by lessening the local or general resistance, would, as a rule, cause the 1 Jimr. Anier. Med. Assoc, 1UU3, xli, ij(j2. . THE STREPTOCOCCI 257 subsiding infection again to progress perhaps even to a more serious extent tlian the original attack. Koch and Petruschky tried a most interesting experiment. Tlie>' inocuhxted cutaneously a man suffering from a malignant tumor with a streptococcus obtained from erysipelas. He developed a moderately severe attack, which lasted about ten days. On its subsidence they reinoculated him; a new attack developed, which ran the same course and o\er the same area. This was repeated ten times with the same results. This experiment provetl that in this case, at least, the immunizing substances produced by repeated attacks of erysipelas were insufficient to make the tissues and lymph sufficiently bactericidal to prevent infection. The severe forms of infection, such as septicemia following injuries, operations, and puerperal infections, show little tendency to be arrested after being well established. Having, then, in remembrance, the above facts, let us consider the results already obtained in the experimental immunization and treatment of animals and men suffering from or in danger of infection with streptococci. Knorr succeeded in producing a moderate immunity in rabbits against an intensely virulent strepto- coccus by injections of \'ery slightly virulent cultures. Marmorek was the first to attempt the production of a curative serum on a large scale. Influence of Serum from Immunized Animals upon Streptococcus Infections in Other Animals. — In the table are given the results fol- lowing the injection of small amounts of a serum which represents in immunizing \-alue what about one-third of the horses are able to produce when given in gradually increasing doses the living, virulent streptococcus. In the following experiments the serum and culture were injected subcutaneously in rabbits at the same time, but in oppo- site side of the bodv : Showing Strength or Average Grade of Antistreptococcic Serum given BY Selected Horses after Six Months or Injection of Suitable Amounts of Living Streptococci. Weight of rabbit. 1. Inoculated together . . 1430 2. Inoculated together . . . 13.50 3. On opposite sides . . . 1770 4. On opposite sides . . . 1630 Controls: 1. Rabbits injected with culture 17.50 only. 2. Rabbits injected with culture 1S70 only. Amounts inoculated. 0.25 c.c. 0.01c. c. 0.125 c.c. 0.01 c.c. 0,1 c.c. 0.01 c.c. 0.1 c.c. 0.01 c.c. 0.001 c.c. 0.001 c.c. Results. Lived Lived Lived Lived Died in 4 days Died in 24 hrs. .'Vutopsy. Streptococc infection. Streptococcic infection. The above results have been repeatedly obtained, and are abso- lutely conclusive that the serum of properly .selected animals, which have been repeatedly injected with living streptococci in suitable doses possesses bactericidal properties upon the same streptococcus when it comes in contact with it within the bodies of animals. 17 258 PATHOGENIC MICROORGANISMS Definite protection from the serum has been obtained by many rehalile observers since Marmorek's first reports. Polyvalent Serum. — Results of investigators show that the majority but not ah streptococci met with in celkihtis, erysipelas, and abscess will be influenced by the same serum. Those obtained from cases of pneumonia and endocarditis and other exceptional infections are apt to have individual characteristics. In order that the serum may have specific antibodies for the variety of streptococci causing each separate infection, each horse is now injected with a large number of different varieties of streptococci. This serum will not be quite as efficient as if made b.y the streptococcus infecting the treated case, but will be fairly efficient for all cases. Dosage. — It is found that the immune bodies to be effective must be in sufficient concentration. The intravenous injections are usually 100 c.c. for an adult. Preparation of the Serum. — Antistreptococcus serum is obtained from the horse after treatment by repeated injections of living or dead streptococcus cultures derived from human sources. As a rule, a number of varieties are given at the same time so that the serum will be active against any variety causing the infection. If the serum is to be used in scarlet fever, the streptococci used should be from cases of scarlet fever. The procuring of a serum of the highest potency requires a considerable number of animals, for some produce with the same treatment a more protective serum than others. The serum must be sterile from streptococcus as well as from other contaminations. Stability of the Serum. — It is fairly stable but, after several months, the serum loses some of its protective value. It should be kept in a cool and dark place. Standardization of the Value of the Serum. — There is at present no satisfactory way. The value of the serum is sometimes measured by the amount required to protect against a multiple of a fatal dose of a Yury virulent streptococcus of the same type as the one used to inject the horses. The dose is usually a thousand times the average fatal amount of a very virulent streptococcus. Other methods of standarization, such as the estimation of the amount of opsonins or agglutinins present, are also used. Therapeutic Results.— To estimate the exact present and future value of antistreptococcus serum is a matter of the utmost difliculty. Many of the cases reported are of little or no help, because, no cultures having been made, we are in doubt as to the nature of the bacterial infection. Ill the cases of puerperal fever, erysipelas, and wound infection that we have seen, the apparent results under the treatment have not been uniform. We have frequently obser^'ed favorable results which appeared to be due to the serum when doses of 50 to 100 c.c. were given intravenously. In a, numlier of cases of septicemia where for days chills had occurred daily they ceased absolutely or lessened under daily doses of 20 to THE STREPTOCOCCI 259 50 c.c. The temperature, though ceasmg to rise to such heights, did not average more than one or two degrees lower than before the injec- tions. In some cases the serum treatment was kept up for four weeks. Some cases convalesced; others after a week or more grew worse and died. In some cases the temperature fell immediately upon giving the first injection of serum, and after subsequent injections remained normal, and the cases seemed greatly benefited. As a rule, in these cases no streptococci or any other organisms were obtained from the blood. In bronchopneumonia, laryngeal diphtheria, tonsillitis, smallpox, and phthisis, we have seen little effect. The results obtained here in New York by both physicians and sur- geons have not, on the whole, been very encouraging. In some of the cases where apparently favorable results were obtained other bacteria than streptococci were found to be the cause of the disease. We believe that the following conclusions will be found fairly accurate : The serum will in animals limit an infection already started if it has not progressed too far. The apparent therapeutic results in cases of human streptococcus infection are variable. In some cases the disease has undoubtedly advanced in spite of large injections, and here it has not seemed to have had any effect. In other cases good observers rightly or wrongly believe they have noticed great improvement from it. Except rashes, few have noticed deleterious results, although very large doses have been followed in several instances, for a short time, by albuminous urine. In suitable cases we are warranted, we believe, in trying it, but we should not expect very striking results. For our own satisfaction, and to increase our knowledge, we should always have satisfactory cultures made when possible, and the strep- tococci, if obtained, tested with the serum used in the treatment. In the cases where we want most to use the serum, such as puerperal fever, septicemia, ulcerative endocarditis, etc., we find that it is very difficult to make a bacteriological diagnosis from the symptoms, and in over one-half of the cases even the bacteriological examination carried out in the most thorough way will fail to detect the special variety of bacteria causing the infection. This is often a great hindrance to the proper use of curative antistreptococcic serum, for it, of course, has no specific effect upon the course of any infection except that due to the strepto- coccus and the full effect only on its own type. Care should be taken to get the most reliable serum; much on the market is worthless, and as it is weak, and the testing for strength is difficult or impossible, full doses (50 to 100 c.c.) of serum should be given if the case is at all serious, for the dose is limited only by the amount of liorse senun which we feel it safe to give, not because we have given sufficient protective substance. Intravenous injections give better results than those given subcutaneously. Studdiford has obtained good results by adding to the intravenous injection the packing of the septic uterus with gauze impregnated with the serum. 200 PATHOGENIC MICROORGANISMS Scarlet Fever. — In Vienna for some years the serum of horses treated at each injection with a number of strains of streptococci derived from scarlet fever cases has been used in this disease. The serum given in large doses of 100 to 200 c.c. has apparently given good results in about half of those treated. It is only used in severe cases. Moser has chiefly advocated its use. One of us had the opportunity to look over the histories of his cases. Although left in doubt as to its value, it appears to us as worth a trial. Our own results and Nicoll's, in cases occurring in the Health Department hospitals, have been rather favorable. Streptococcus Vaccine. — The use of streptococcus vaccines as a pre\'entive of scarlet fever was originated by Gabritchewsky and has been practised on an extensive scale largely in Russia with apparently favorable results. Jesjakoff in a study of 15,000 vaccinated cases con- cludes that three inoculations extending over a period of two weeks protect against the disease. In this country and elsewhere small series of cases reported from time to time tend to confirm his observations and the continuance of the procedure with the hope that it may be of value in lessening the susceptibility to scarlet fever of persons constantly exposed to it. Complement Fixation. — The method and results of this test have been considered in Part I, p. 190. Bacteriological Diagnosis. — Streptococci, using the name in a broad sense, can often be demonstrated microscopically by simply making a smear preparation of the suspected material and staining with methy- lene-blue solution or diluted Ziehl's fluid. In order to demonstrate them microscopically in the tissues, the sections are best stained by Kiihne's methylene-blue method. In all cases, even when the micro- scopic examination fails, the cocci may be found by the use of culture media, such as broth or plated agar at .37° C. To obtain them from a case of erysipelas it is best to excise a small piece of skin from the margin of the erysipelatous area in which the cocci are most numerous; this is crushed up and part of it transferred to ascitic or serum bouillon, and part is streaked across freshly solidified agar in a Petri dish on which a drop of sterile rabbit's blood had previously been placed. Both are kept in the incubator at .37° C In septicemia the culture method is always required to demonstrate the presence of streptococci, as the microscopic examination of specimens of blood is not sufficient. For this purpose from 10 to 15 c.c. of the blood shoidfi be drawn from the \'ein of the arm aseptically by means of a hypodermic needle, and to each of three tubes containing 10 c.c. of melted nutrient agar kept at about 43° C. 1 c.c. of blood is added. After thoroughly mixing the contents are poured into Petri dishes. The remainder is added to several flasks containing 100 c.c. of nutrient broth, ill order to produce a ilevelopment of the cocci, which are found in small numbers in the blood. Petruschky is of the opinion that the. cocci can be liest shown in blood by niiimal inoculation. Having with- drawn from the ])atieiit 10 c.c. of blood by means of a hypodermic syringe, under asejjtic precautions, he injects a portion of this into the THE STREPTOCOCCI 261 al)(loiniiiiil ca\ity of a mouse, wliilc thf otluT portion is planted in bouillon. Mice thus inoculated die from se[)ticeuiia when \iruleut strepti)cocei are present in only \ery small numhers in the blood. If a successful inoculation takes place we can, tlirouj;h the absence or presence of the development of capsules, often differentiate between the pneumococcus and the streptococcus, \\hich cultures may fail to y method of Hiss. The capsule is best seen in stained preparations from the blood and exudates of fibrinous pneumonia or from the blood of an inoculated animal, especially the mouse, in which it is commonly, though not always, present. It is seldom seen in preparations from cultures (except in pneumococcus mucosus) unless special media are emplo\'ed. Flagella are not present. Staining. — ^It stains readily with ordinary aniline colors; it is not decolorized after staining by Gram's method. The capsule may be demonstrated in blood or sputum either by Gram's or Welch's (glacial acetic acid) method, or the copper sulphate method of Hiss. Biology. — It grows equally well with or without oxygen; its parastic nature is exliibited by the short range of temperature at which it usually grows — viz., from 25° to 42° C. — best at 37° C. In the cultivation of this organism neutral or slightly alkaline media should be employed. 264 PA THOOENIC MICROORGA NISMS The organism when freshly isohited grows feebly on the serum-free culture media ordinarily employed for the cultivation of bacteria — viz., on nutrient agar and gelatin, in bouillon. The best medium for its growth is a mixture of one-third human or animal blood serum or ascitic or pleuritic fluid and two-thirds bouillon, or nutrient agar streaked with human, horse, or rabbit blood. Growth on Agar. — Cultivated on plain nutrient agar, after twenty- four to forty-eight hours at 37° C, the deep colonies are hardl.y visible to the eye. Under the microscope they appear light yellow or brown in color and finely granular. The surface colonies are larger, equalling in size those of streptococci, but are usually more transparent. If blood serum or ascitic fluid be added to the agar the individual colonies are larger and closer together, and the growth is more distinct in con- sequence and of a grayish color. The surface colonies are almost circu- lar in shape under a magnification of (50 diameters, finely granular in structure, and may have a somewhat darker, more compact centre, surrounded by a paler marginal zone. With high magnification cocci in twos and short rows often distinctly separated are seen at the edges. Fic. 106 Fig. 107 Pneumoooccus from bouillon culture, resem- bling streptococcus. Pneumococci in peritoneal pus. Stained with fuchsin. X 1000 diameters. Clear spaces indicate capsules. Growth on Blood Agar. — The colonies on blood agar are greenish and no hemolysis is present. This is a useful characteristic in isolating pneumococci from sputum, although many streptococci give similar colonies. Growth on Blood Serum. — The growth on Loffler's blood-serum mixture is very similar to that on agar, but somewhat more vigorous and characteristic, appearing on the surface as a delicate layer of dew- like drops. Growth in Bouillon. — In bouillon, at the end of twch'e to twenty-four hours in the incubator, a slight cloudiness of the liquid will be found to have been THE DrpLocorcrs of pneumonia 205 produced. On miorost'opif cxaiiiiimlidn vovv'\ can be seen to he ari'anged in pair.s or longer or shorter chains. .\i'ler one or (wo transpian1alio?is (he |Mieu- niococci I'rcqnently fail to grow. Growth in Milk. -U grows icadily in milk, causing coagulalion with Ihe production of acid, Ihougli coaguhition is not conslant \\i1h some I'oinis niler- metliatc between the .streptococcus and ]ineumococcus. Growth on Gelatin. — The growth on gckitin is slow, if there is any develop- ment at all, owing to the low temperature — viz., 24° to 27° C. — above wliicli even the most heat-resistant gelatin will melt. The gelatin is not liquefied. Special Media. — When cultures are grown on sermn-free media the vitalit)' of some cultures may indeed be indefinitely prolonged; l)ut after transplanta- tion through several generations it is found that the cultures begin to lose in virulence, and that they finally become non-virulent. In order to restore this virulence, or to keep it from becoming attenuated, it is necessary to interrupt the transplantation and jiass the organism through the Ijodies of susceptible animals. The vitalitj' is prolonged and the virulence less rapidly lost if serum ascitic fluid or blood is present in the medium. Serum or ascitic broth, serum semi- solid (stab cultures) or blood-streaked agar are the most satisfactory media for the preservation of cultures. Action of Bile and Bile Salts. — If 0.1 c.c. of rabbits' bile be added to 1 or 2 c.c. of a broth culture of pneumococci the culture becomes clear and the cocci become dissoh-ed. A 10 per cent, solution of sodium taurocholate has the same action. Streptococci are not dissolved by bile. Serum and glucose interfere with the reaction. Hiss Seram Water with and without Inulin. — These are very useful. The inulin is fermented by typical pneumcjcocci with coagulation of the serum, while most streptococci fail to ferment tlie inulin. This medium is, therefore, of considerable diagnostic value. Calcium Broth with or without Dextrose. — This medium has proven of great vdhw for the propagation of cuhures where agglutination tests are to be carried out. The addition of' a small piece of marble to each' tube of broth is the most satisfactory w'ay of preparing it. ^larble broth for this purpose was suggested independently by Bolduan and Hiss. Resistance to Light, Drying, and Germicidal Agents. — On artificial culture media the pneumococci tend to die rapidly. This is partially due to the acid produced by their growth. In sputum they live much longer. Pneumonic sputum attached in masses to clothes, when dried in the air and exposed to diffuse daylight, retains its virulence, as shown by injection in rabbits, for a period of nineteen to fifty-fi^'e ciays. Exposed to direct sunlight the same material retains its ^'irulence after but a few hours' exposure. This retention of virulence for so long a time under these circumstances is accounted for by the protective influence afforded by the dried mucoid material in which the micrococci were embedded, (xuarnieri observed that the blood of inocuhited animals, when rapidly dried in a desiccator, retained its \irulence for months; and Foa found that fresh rabbit blood, after inoculation and cultivation in the incubator for tw^enty-four hours, when removed at once to a cool, dark place, retained its virulence for sixty days. There are many conditions, therefore, in which the virulence of the micrococcus is 2GG PATHOGENIC MICROORGANISMS retained for a considerable length of time. To germicidal agents pneu- mococci are very sensitive. The fine spray expelled in coughing and loud speaking that remains suspended in the air soon dries so com- pletely that probably no pneumococci survive after two hours. Attenuation of Virulence. — This niay be produced in various ways. The loss of \irulencc which occurs when the micrococcus is trans- planted through several generations in culture fluid containing no blood has already been referred to. An attenuation of \'irulence, it has been claimed, takes place also spontaneously in the course of pneumonia. This attenuation is probably onlj^ apparent. If a little sputum be taken at different periods in the disease and planted in ascitic bouillon the resultant cultures do not vary greatly in virulence. The average A'irulence for rabbits of cultures made from pneumonic sputum is greater than in those from normal sputum. Restoration and Increase of Virulence. — The simplest and perhaps the most reliable method of restoring lost virulence for any susceptible animal is by passage through the bodies of highly susceptible animals of the same species. Growth in fresh blood also increases it for the homologous animal. Maintenance of Virulence — This is best done by drying the spleen of an infected animal in a dessicator. The spleen should be removed just before or immediately after death. The virulence is preserved in this way for a month or longer. Toxin Production. — We have little exact knowledge upon the nature of the substances produced by or through the growth of the pneumo- cocci in animal tissues or artificial media. Rosenow showed that the autolysis of virulent pneumococci in NaCl solution brings into the solution a group of substances which inhibits the action of the pneumo- cocco-opsonin. Such extracts are also toxic for animals. Occurrence in Man during Health. — It is probable that in crowded communities the pneumococcus is present on the mucous membranes of most persons. We have found it generally present not only in the throats of persons living in New York City, but also in those of persons living on farms and in the Adirondack Mountains. It is commonly present only on the mucous membranes of the bronchi, trachea, pharynx, and nostrils. The healthy lung seems to be generally free from it. Judging from animal tests it is very possible that the virulence for man of the organisms present in health is much less than the virulence of those in a pneumonic lung. Pathogenicity in Man. — Characteristic or atypical pneumococci are present in full}' 95 per cent, of characteristic cases of lobar pneumonia. Usually no other bacteria are obtained from the lungs. Atypical cases usually show the same conditions, but they may be due to strepto- cocci; influenza bacilli, etc. The more recent the infection the greater is the number of bacteria found in the diseased lung area. As the dis- ease progresses these decrease in number until finally at the crisis they disappear from the tissues, though at this time and long after convales- cence thej' may be present in the sputum. In atypical forms of pneu- THE DIPLOCOCCWS OF PNEUMONIA 2()7 monia they may remain longer in the tissues, and in walking pneumonia they may be absent in the original centres of infection or present only as attenuated varieties, while the surrounding, newly formed foci may contain fully \irulent cocci. It luis been sliown by Nctter that more than one-half of tjie cases of bronchopneumonia, whether ])riniary or secondary to some other disease, as measles and diphtheria, both in children and adults, are due to the diplococcus of pneumonia. Others, such as Pearce, have foimd other microorganisms, expeciallj' the strep- tococci, in the majority of cases. These findings will be considered at the end of the chapter. The pneumococci are found partly in the alveoli and bronchioles of the inflamed lung and partly in the lymph channels and blood-capil- laries. Most of the organisms are found free, but a few are found in the leukocytes. Tlirough the lymph channels they find their way to the pleura and to adjacent lymph glands. From the capillaries they find their way to the general blood-current, and thus to distant parts of the body. In about 20 per cent, of cases the pneumococci are so abundant that they can be found in cultures made from 5 to 10 c.c. of blood. In a number of instances the fetus has been found infected. The pneumococci are also responsible for : Inflammations Complicating Pneumonia. — In every case of lobar pneumonia and in most cases of bronchopneumonia pleurisy is developed, which is excited by the same microorganism that was predominant in the pneumonia. With pneumococci the exudate is usually moderate and of a fibrinous character, but may be more abundant and of a sero- fibrinous or purulent character. When the pleurisy is marked it is more apt to continue after the cessation of the pneumonia. Pleurisy due to pneumococci is more apt to go on to spontaneous recovery than that due to streptococci or staphylococci. The most frequent pneumococcic infections next to pleurisj^, fol- lowing a pneumonia, are those of the middle ear, pericardium, endo- cardium, and meninges, and these not infrequently arise together. Pneumococcic inflammations of the heart valves are apt to be fol- lowed by extensive necrosis and growth of vegetations. In these cases pneumococci can sometimes be found in the blood for many weeks. Pericarditis due to pneumococci is a frequent complication, but is usually very slightly developed. Meningitis due to pneumococci may be either fibrinous or purulent or both and is apt to be secondary to otitis, mastoiditis, or pneumonia. Arthritis, periarthritis, and osteo- myelitis are rarer complications of a pneumococcic pneumonia. Besides moderate parenchymatous inflammation of the kidney, which occurs in most cases of pneumonia, well-marked inflammation may occur in which pneumococci exist in the kidney tissues in large numbers. How is the pneumococcus conveyed from its original seat in the lungs to distant internal organs? Chiefly by means of the blood- vessels and lymphatics, in both of which it has been found in great numbers. Their presence in the blood after death has been amply proved by numerous investigations. In many instances, they have 2riS PATHOGENIC MICROORGANISMS been recovered from t]ie blood fluriiig life. Lamltert, as a rule, found tliem in all fatal eases twenty-four to forty-eif;lit Iiours before death. The eou\'eyaiiee of the infective af;;ent by means of the blood and the lymj)]! to all parts of the body, explains the multiplicity of the affections complicating a jjneumonia, which are caused by this micrococcus; and not only the secondary, but also the primary diseases, as of the brain and meninges, may be explained in the same way. Knowing that the saliva and nasal secretions under normal conditions so frequently afford a resting place for the micro- cocci, we have only to assume the production of a suitable culture medium for these parasites in the body, brought about by an abnormal condition of the mucous membranes from exposure to cold, or a reduc- tion of the vital resisting power of the tissue cells in any of the internal organs, caused by disease, traumatism, excess of various kinds, etc., to comprehend readily how an individual may become infected with pneumococci, either primarily affecting the lungs and secondarily other organs in the body, or primarily attacking the middle ear, the peri- cardial sac, the pleura, the serous cavities of the brain, etc. Presence in Inflammatory Process Not Secondary to Pneumonia. — It is now known that the pneumococcus may infect and excite dis- eases in many tissues of the body independent of any preliminary localization in the lung. As a rule, these processes are acute and usually run a shorter and more favorable course than similar inflammations due to the streptococci. The most frequent primary lesions excited by the pneumococcus after lobar pneumonia, bronchopneumonia, and bronchitis are prob- ably meningitis, otitis media with its complicating mastoiditis, endo- carditis, pericarditis, rhinitis, tonsillitis, conjunctivitis, and keratitis; septicemia, arthritis, and osteomyelitis; inflammations of the epi- didymis, testicles, and Fallopian tubes; peritonitis, etc. Pneumococcic peritonitis and appendicitis are not so very frequent. The exudate is usually seropurulent. Conjunctivitis due to pneumococci frequently occurs in epidemic form and is frequently associated with rhinitis. From statistics coflected by Netter the following percentages of diseases were caused by the pneumococcus: Pneumonia 65.9 per cent, in adults. Bronchopneumonia 15,8 per cent, in adults. Meningitis 1.3.0 per cent, in adults. Empyema 8.5 per cent, in adults. Otitis media 2.4 per cent, in adults. Endocarditis 1 . 2 per cent, in adults. In 46 consecutive pneumococcus infections in children there were: otitis media 29 cases. Bronchopneumonia 12 eases. Meninaitis 2 cases. Pneumonia 1 case. I'l'-uri.sy 1 case. Pericardii is 1 ease. THE DIl'LOCOCCUS OF PNEUMONIA 209 The pneumococ'fus and streptococcus are the two most frequent organisms found in otitis media. The cases due to the pneumococcus are apt to run the shorter course, but have a tendency to spread to the meninges and cause a meningitis. The pneumococci may also find their way into the bUiod-current. This usually follows after sinus thrombosis. In bronchitis the pneumococcus is frequently met with alone or in combination with the streptococcus, the influenza bacillus, or other bacteria. In certain epidemics pneumococcic bronchitis and pneumonia simu- late influenza \'ery closely and cannot be dift'erentiated except by bac- teriological examinations. Primary pneumococcic pleurisy is frequent in children: it is very often purulent, but may be serous or serofibrinous. Its prognosis is better than that in cases due to other organisms. Frequently we have streptococci and staphylococci associated with the pneumo- cocci. Pathogenesis in Lower Animals. — Most strains of the Micrococcus lanceolatus are moderately pathogenic for numerous animals; mice and rabbits are the most susceptible, indeed some strains are intensely virulent for these animals, while guinea-pigs and rats are much less susceptible. Pigeons and chickens are refractory. In mice and rabbits the subcutaneous injection of small or moderate quantities of pneu- monic sputum in the early stages of the disease, or of a twenty-four- hour ascitic broth culture from such sputum, or of a pure, virulent ascitic broth culture of the micrococcus, usually results in the death of these animals in from twenty-four to forty-eight hours. The course of the disease produced and the postmortem appearances indicate that it is a form of septicemia — what is known as sputum septicemia. After injection there is loss of appetite and great debility, and the animal usually' dies some time during the second day after inoculation. The postmortem examination shows a local reaction, which may be of a serous, fibrinous, hemorrhagic, necrotic, or purulent character; or there may be combinations of all of these conditions. The blood of inoculated animals immediately after death often contains the micrococci in very large numbers. For microscopic examination they may be obtained from the blood, and usually from j)leural and peritoneal exudates when these are present. True localized pneumonia does not usually result from subcutaneous injections into susceptible animals, but injections made through the thoracic walls into the substance of the lung ma,\- induce a typical fibrinous pneumonia. This was first demonstrated by Talamon, who injected the fibrinous exudate of croupous pnemnonia, obtained after death or drawn during life from the hc]jatizcd ixirtions of the lung, into the lungs of rabbits. Wadsworth showed that by injecting virulent pneumococci into the lungs of rabbits which had been ininuniized, a typical lobar pneumonia was excited, the bactericidal property of the blood being sufficient to prevent the general invasion of the bacteria. 270 PATHOGENIC MICROORGANISMS Pneumonia may be produced in dogs, and in rabbits less easily, by intratracheal injections. Varieties of the Pneumococcus. — As among all other microorgan- isms minutely studied, different strains of pneumococci show quite a wide range of variation in morphology and virulence. Some of the \'ariations are so marked and so constant that they make it necessary to recognize several distinct varieties of the pneumococcus, and to class as pneumococci certain varieties which have before this been classed as streptococci — e. g., the so-called Streptococcus vmcosus capsulatus {Streptococcus inucosus Schottmiiller), when first isolated from pneumonic exudate or elsewhere, and planted on artificial cul- ture media containing serum, grows as a rounded coccus with a small dense distinct capsule, principallj^ in short or medium chains; it pro- duces a large amount of mucus-like zooglia, forming very large spread- ing colonies; it promptly coagulates fluid serum media containing inulin. It is also very virulent for mice, but only moderately virulent for rabbits. After a number of culture generations on ordinary nutrient agar it apparently loses most of these characteristics. It then grows in small colonies principally as naked diplococci which may be elon- gated and pointed, produces no zooglia, and loses most of its virulence for mice and rabbits. It still coagulates inulin serum media, and when transferred to serum media regains its former morphological characteristics. For these reasons we consider this organism a distinct variety of the pneumococcus. This variety of pneumococcus has been isolated by us from the lungs after death following lobar pneumonia, out of twenty consecuti\'e autopsies, as the only organism present twice, and with another variety of pneumococcus once. Together with other ^'arieties it was isolated from four out of twenty specimens of pneumonic sputum, and from sixty specimens of normal throat secre- tion five times. In 1905 Park and Williams showed that this variety should be placed with the pneumococci under the common name Pneumococcus mucosus. Agglutination Reactions. — That agglutinins are produced in animals b}^ the injection of pneumococci was shown by Neufeld, Clairmont, and others. It was first thought that this test might be used as a means of diagnosis. We found (Collins), however, that when a large number of strains are tested, the variability of the reaction is so great as to render it impractical as a means of diagnosis. In the case of the pneu- mococcus mucosus Collins found greater uniformity of reaction, all strains tested reacting alike and the agglutinins of one member of the group were absorbed by the other members. Recently Hanes has corroborated this work. Neufeld, Cole and Dochez have shown that certiiiu pneumococci may be grouped according to serum reactions. According to Cole and Dochez, the groups based upon their agglutiiux- tion reactions are as follows: Groups 1 and 2, typical pneumococci; Group .3, pneumococcus mucosus; Group 4, heterogeneous strains. The members of the first three groups agree with those in the group in their specific serum reactions. Each strain in the last group seems THE DIPLOCOCCUS OF PNEUMONIA 271 independent as far as serum reactions are concerned; it is really not a group in the sense of the first three, but rather an assembling together of isolated strains. The following table by Cole gives the types he found in pneumonia in a series of cases occurring in New York City, and the relative mor- tahty of the cases according to the infecting type : Infecting Strain iVInrtality type. ' Cases. per cent. I!)eath3. per cent. 1 34 47 8 24 2 13 18 8 61 3 10 14 (i (iO 4 15 21 1 7 Total .... 72 100 23 32 In different localities and at different times in the same locality, the type percentage will probably vary greatly. Pneumococci taken from normal throats or from slight inflammation fall usually into Group 4 among the heterogenous strains. The strains belonging to Group 1 are widely scattered, being found not only througli- out the United States but also in Europe. Inununity. — Following an attack of pheumonia some immunity is established, but this lasts but a short time. Early in the history of this organism experiments were begun for the production of immunity in animals by means of preventive inoculations. Later it was found that after successive injections of gradually increasing doses of virulent pneumococci into certain animals (horse, sheep, goat, rabbit), a serum of some protective and curative power in experimental animals was obtained. The mode of action of this serum is still the subject of study. According to Wright, Neufeld, and others, its activity is due to the presence of certain substances called opsonins (Wright), or bacterio- tropic substances (Neufeld), which act on the bacteria in such a way as to prepare them for ingestion by the phagocytes. The extent to which phagocytosis brings about the crisis and healing in pneumonia is not known. Therapeutic Experiments.' — The number of cases reported in which the blood-serum of animals artificially immunized against pneu- monic infection has been used for the treatment of the disease in human beings, although numerous, has not led to the formation of a definite opinion as to the final value of this as a therapeutic agent. In the cases we have observed there has been, in some a slight immediate lowering of the temperature, in others no apparent change. As a rule, the cases did rather better than was expected, but certainly no striking curative effects were apparent. The apparent good results in some and the absence of these in others are probaljly explained by the facts brought out by Cole and Dochez that certain strains can be influenced and others cannot. It seems probable that the serum may be able to pre^'ent > See Harvey Lecture. Cole, Arch. Int. Med., 1914, xiv, 56. 272 PATHOGENIC MICROORGANISMS a general infection from taking place from the diseased lung, even though it may fail to influence the local process. It has also been shown that these injections of antipneumococcic serum are practically harmless. In the case of infections with the pneumococcus mucosus, serum therapy seems hopeless, as passive immunity is not produced in animals. In the case of Group 4, heterogeneity of the group makes it unlikely that a serum would be of value. Infections by Groups 1 and 2 are the most hopeful types for senmi thcapy using either univalent or bivalent sera. As is shown, the preceding tables Group 2 causes a more serious disease and likewise serum treatment seems less beneficial than in the milder infections due to Type 1. To apply a univalent serum the type of infecting organism must be known or the proper serum cannot be selected. The following method is employed at the Rockefeller Institute Hospital: The sputum coughed up from the lung is injected intra- peritoneally into a mouse. After four to five hours the mouse is killed and the peritoneal cavity washed out with salt solution. The washings are then centrifuged slowly to throw down the fibrin and cells and the supernatant fluid is then mixed in equal parts with known Types 1 and 2 sera, and the presence or absence of agglutination determined after incubation for one to two hours. Type 3 can usually be diagnosed by the mucoid character of the peritoneal exudate. In pneumococcus septicemia no marked results have been seen. Large injections of 1(]0 to 150 c.c. of a. polyvalent serum are given intra^'enously. Vaccines. — The use of injections of dead pneumococci in pneu- monia and other acute pneumococcic inflammations has not been followed by appreciable beneficial results in those cases which have come under our observation. In subacute cases the results appear to be more favoraljle. With better understanding of the varied infecting types better results may become possible. CHAPTER XVIII. :\IENL\GOC'0(XT'S OR .MICROCOCCUS (INTRACELLl'LARIS) :\1ENINGITIDIS, AND THE RELATION OF IT AND OF OTHER BACTERIA TO MENINGITIS. AViiiLE, as has already been stated, the piieumocoecus is a cause of isohited cases of meningitis the meningococcus is the most frequent cause of purulent menhigitis either sporadic or endemic. In 1887 Weichselbaum^ discovered a micrococcus in the exudate of cerebro- spinal meningitis in six cases, two of which were not complicated by pneumonia. He obtained it in pure cultures, studied its characteristics, and showed that this organism was clearly distinguishable from the pneumococcus and especially by its usual presence in the interior of pus cells, on which account he called it Diplococcvs intraceUvIaris meningitidis. In 1895 Jaeger and Scheurer drew especial attention to the etiological relationship of the organism to the epidemic form of cerebrospinal meningitis. They also believed it to be very probable that in most cases of primary meningitis it is from the mucous mem- brane of the nasal ca^'ities and the sinuses opening out from them that both the diplococcus of pneumonia and the micrococcus intracellularis find their way through the blood or perhaps directly through the lymph channels to the meninges. The former we know to be almost constantly present in the nasal cavities, and the latter we have reason to believe is not infrequently there. The nasal secretion of carriers is more dan- gerous to the communit>' than that of infected persons because carriers are not recognized as such usually and mingle freely with the general public. The prevalence of epidemics in winter and spring, a time favor- able to influenza and pneumonia, also suggests the respiratory tract as the place of the infection and where an increase in virulence takes place. We do not as yet know why meningitis follows in some persons and not in others after infection of the mucous membranes. Morphology. — This organism occurs as biscuit-shaped micrococci, usually united in pairs, but also in groups of four and in small masses; sometimes solitary and small degenerated forms are found. It has no well-defined capsule. Cultures resemble strongly those of gonococci. In the sediment from spinal fluids (see Fig. 108) meningococci are both intra and extracellular. When they are intracellular the prognosis is better and frequently in cases where they are extracellular at first they later become intracellular. Staining. — They stain with all the ordinary aniline dyes and are readily decolorized by Gram's solution. A smear from a culture shows charac- 1 Wcichselbaimi, Fortschr. d. Med., 1887, p. .573, 18 274 PA THOGENIC Ml CROOHGANISMS teristic irregularity in staining, some of the cocci taking the counter stain poorly and some staining deeply. The positive cocci described by Jaeger and others were prob- FiG. los ably contaminating organisms. The cells have no definite capsule. Cultivation. — They grow be- tween 25° and .38° C, best at about .3.5° C. They are most easily isolated and kept on 2 per cent, glucose ascitic agar neutral to phenolphthalein. The reaction of the media is very important. A culture can rarely be isolated on plain nutrient agar. After several generations on glucose ascitic agar they will grow quite luxuriantly on neutral salt-free veal agar for a few generations but tend to die out. Different strains of meningococci vary in the ease with which they may be cultivated, their ability to ferment carbohydrates, virulence for animals, agglutinability, degree of digestibility in leukocytes, and power of resistance to immune serum. \ Uiploc'occus intracellularis meiiiiit^itidis in pus cells. X 1100 diameters. When grown for some time, a tolerably good growth develops at the end of forty-eight hours in the incubator on nutrient agar or glucose agar. This appears as a flat layer of colonies, about one-eightli of an inch in diameter, grayish-white in color, finely granular, rather viscid, and non-confluent unless very close together. On Loffler's blood serum the groAvth forms round, whitish, shining, viscid-looking colonies, with smooth and sharplj^-defined outlines; these may attain diameters of one-eighth to one-sixteenth of an inch in twenty four hours. The colonies tend to become confluent and do not liquefy the serum. From the spinal fluid in acute cases, where the organisms are apt to 1)6 more abundant, a great many minute colonies may develop instead of a few larger ones. On agar plates the deep-ljdng colonies are almost invisible to the naked e3'e; somew'hat magnified they appear finely granular, with a dentated Ijordcr. On the surface they are larger, appearing as pale disks, almost trans- parent at the edges, but more compact toward the centres, which are yellowish- graj' in color. On blood-agar or serum agar the growth is much more luxuriant than on jilain agar and larger than the gonococcus. Cultivated in artificial media, while it often lives for weeks, it may die within four days, and requires, therefore, to be transplanted to fresh material at short intervals. Resistance. — It is readily killed by heat, disinfectants, sunlight, and drying. A few cocci may remain ali\'e for one to three days in the dried state. To maintain cultures it is necessary to n^ake transfers every other day. Pathogenesis. — This organism has a low-grade and very variable pathogenicity for laboratory animals. We used over one hundred guinea-pigs one winter in trying to establish a fatal dose of li\'e culture AGGLUTINATION CHARACTERISTICS ' 275 and as many another winter trying to find the fatal dose of meningo- coccic extract and were never able to arrive at any definite conclusion. There seemed to be no rhyme or reason to the results. Following the intraperitoneal injection the temperature of the pig falls, the hair stands out, the abdomen becomes distended and the muscles rigid. The animal hunches up in a corner and seems very sick. Not infre- quently there is prolapse of the rectum. If the dose is fatal death usually occurs in from ten to forty-eight hours. At autopsy there is fluid exudate in the abdomen and sometimes in the pleural cavity, congestion or hemorrhage of the adrenals, hemorrhages into the mesen- tery, central tendon of the diaphragm and into the whole peri- toneum. Frequently it is possible to recover the meningococcus from the heart's blood when live culture has been used. The organisms do not have to multiply to produce death and the autolysate or killed culture is just as fatal as live culture. Death is probably due to a bac- terial poison freed by the disintegration of the meningococci. Rabbits injected either subcutaneousl.y or intravenously lose weight ^apidlJ^ Presence in the Nasal Cavity of the Sick and Those in Contact with Them. — In 1 of his 6 cases Weichselbaum succeeded in obtaining dip- lococci from the nasal secretion. In 1901 Albrecht and Ghon demon- strated them in healthy individuals. Scheurer, in his 18 cases, found the diplococci in the nasal secretions of all of them during life. In 50 healthy individuals examined they were found in the nasal secretions of only two, one being a man suffering at the time from a severe cold. This man, it is interesting to note, had been employed in a room which had just previously been occupied by a patient with cerebrospinal meningitis. Lately, there has been a tendency to throw doubt on these findings, but from our experience in the 1906 epidemic in New York, one can state that the meningococci are usually present in great numbers in the nose and nasopharynx in most cases of meningitis during the first twelve days of illness. After the fourteenth day they cannot usually be found. In one case Goodwin of our laboratory obtained them on the sixty-seventh day. She also found them in five persons out of sixty tested who had been in close contact with the sick, and in two of fifty medical students. Pathogenicity for Man. — The most marked lesions occur at the base of the brain. The cord is always affected. This is not true to the same extent in other bacterial infections. In some epidemics the course of the disease is very rapid. The mortality varies from between 50 and 80 per cent. Complicating Infections. — Occasionally we find secondary to the cere- brospinal meningitis, and due to the micrococcus, pneumonia, cystitis, conjunctivitis, inflammation of the middle ear, arthritis, endocarditis, etc. The most frequent and serious complication of meningococcic meningitis is pneumonia probably due in many cases to the pneumo- coccus. Agglutination Characteristics. — A considerable percentage of cultures of meningococci are relatively inagglutinable. Strains that are agglu- 27G PATHOGENIC MICROORGANISMS tillable respond to the agglutinins developed in an animal immunized with a true strain. Careful absorption tests are capable in almost, if not all, instances of separating true meningococci from other Gram- negati^'e organisms. This serum reaction is practically never used for diagnosis because it is so variable and unreliable. Parameningococcus. — Doptor (1909) obtained from nasal secretions se\'eral strains of cocci which, though similar to other meningococci in morphology and cultures, were different from them in some serologic properties. These cocci he called parameningococci, and he considered that in some cases they mia'ht invade the system and cause meningitis. His work has been corroborated by other French observers, and, just now, by Wollstein in this country. Wollstein, testing many strains from different parts of the world, for agglutination, opsonization, complement fixation and serum protection, comes to the conclusion that while certain strains exhibit differences enough to warrant the employment of them in the production of a polyvalent serum, they show too many variations in serum reactions to be separated into a distinct class. They should rather be considered special strains among menin- gococci. This conclusion is in general accord with those obtained through the extensive researches of Elser and Huntoon (1909). Localization of Meningococci. — In addition to the situation already noted the meningococcus is frequently found in the blood in the early days of the disease. Elser found it in blood cultures in 10 out of 40 cases. The meningococci have also been found in the herpes and in the urine, a fact to be considered in ordering tlie hygiene of the sick room. Serum Treatment. — It is difficult to apportion the credit for the pro- duction of the first protective serum. Bonhoft' and Lepriere produced in animals a serum which showed definite protection. The world-wide epidemic beginning in 1904 stimulated a number of laboratories to pro- duce sera in horses with the idea of treating human cases. Thus Kolle and Wassermann, Jochmann, Flexner, and ourselves immunized horses. The usual method was to begin with cultures recently obtained from human cases and grow them on ascitic agar or jjlain nutrient agar in tubes. The growth was scraped oft', added to physiological salt solution, and heated to 55° to 60° for one hour. Li\'ing cultures were often substituted later. The original injections were quite small, being only one or two mod- erate-sized platinum loopfuls. Each succeeding injection was doubled in size each time, until the maximum dose of the growth on two Petri dishes was given, when the size of the injection was not changed. The injections were given about every eight days. Horses give the best sennn after eight months to one year. Kolle and Wassermann injected one horse with the watery extract of recent cultures. They also used both the intravenous and subcutaneous methods. The Therapeutic Use of Serum. — In 1905 there was inaugurated in Hartford, the use of subcutaneous injections of diphtheria anti- toxic serum in meningitis. This influenced us to prepare and try the subcutaneous injection of an antimeningococcus serum. The results SERUM TREATMENT 277 reported by the physicians in some 20 eases (\u\ not seem to estahhsli that any henefieial ett'eets were olitained, so no further serum was issued. Later Kolle and Wasserniann reported somewhat t'a\-orahle results in a number of eases from the subcutaneous injection of a serum pre- pared by tliem. INIeanwliile a serum jjrepared l)y Joehmann was empU)yed by the intraspinal method in a series of eases. This method soon supplanted the subcutaneous injections. The first successful use of an immune serum in cases of human cerebrospinal meningitis, by the intraspinal method, should, therefore, so far as we know, be credited to Joehmann and the physicians who used his serum in the winter of 1905 and 1906. He reported a series of cases treated by the intraspinal method before the Congress for Internal Medicine held in jMunich in April, 1900, and published his paper on INIay 17, 1906. The serum was prepared by injecting horses with increasing doses of meningococcus killed at about 58° C. The doses were given every eight days, beginning with a loopful and increasing until the growth on the surface of ascitic agar covering two Petri dishes was used. After this dose was reached living cultures were given. Characteristics of Serum. — The serum was show'n to possess both bactericidal and opsonic power. He reported forty cases of cerebro- spinal meningitis which had been treated, but gave details concerning 17 patients which all occurred in one hospital and were treated by Kromer. Five of these patients died and twelve recovered, a mortality of 29 per cent. He directed that after lumbar puncture, 20 to 50 c.c. of fluid should be removed and then 20 c.c. of immune serum injected. These injections should be repeated once or twice if the fever did not abate or returned. He noticed in general a bettering of the headache, stiffness of neck, and mental condition. Joehmann showed that in animals colored fluids injected into the spinal canal in the lumbar region passed the full length of the canal. Although the serum prepared in different laboratories in Europe was regularly used after Jochmann's report, it did not receive much attention in this country until Flexner, through his important experi- ments on infected monkeys, which demonstrated the value of the intra- spinal injections of the serum, aroused medical interest and paved the way for him to try out the serum on a large scale. All cases treated by him were subjected to most careful bacterial tests and clinical observation. Eighteen months later, Flexner and Jobling published their report which fully corroborated the earlier results of Joehmann. The serum prepared at the Rockefeller Institute for Medical Research has beeii sent to many places, both in this country and in Europe. The results obtained have been of the utmost value in arriving at the value of the intraspinal treatment. The following is abstracted from their latest report. There were 1294 cases of the disease in which the bacteriologic diag- nosis was made and the serum treatment used. In the first table the cases are subdivided according to certain age periods, and in the second 278 PATHOGENIC MICROORGANISMS the total cases of each age period are further subdivided according as the serum was injected in the three arbitrarily chosen periods of duration of the disease. Table I. — Cases uf Epidemic C'eroljiuspiijal Meiiiiiyilis Ticatcd with the AiUinieuiiigitis Serum. Cases Analyzed According to Age Groups. Per cent. Per cent Age. No. of cases. Recovered. Died. recovered. died. Under 1 year 129 65 64 50.4 49.6 1 to 2 years 87 60 27 69.0 31.0 2 to 5 years 194 139 55 71.6 28.4 5 to 10 years 218 185 33 84.9 15.1 10 to 20 years . 300 2.54 106 70.6 29.4 Over 20 years 288 180 108 62.5 37.5 Age not given 18 11 7 61.1 38.9 Totals 1294 894 400 69.1 .30.9 The highest mortality 49.6 per cent, is shown to have occurred in the first year of life. Even so it is greatly reduced, for under the older forms of treatment it was between 90 and 100 per cent. The average mortality in all the age periods was 30.9 per cent. Table II. — Cases of Epidemic Cerebrospinal Meningitis Treated mtli the Antimeningitis Serum. Cases Analyzed According to Day of Injection. Injected Ist to 3rd days. Injected 4th to 7th day. Injected later than 7th day Age. Rec. Died. % Rec. Died. % Rec. Died. % I'nder 2 years 12 1 7.7 28 9 24.3 81 78 49.1 2 to 5 years 24 6 20.0 49 17 25.8 63 30 37.3 .5 to 10 years 49 6 10.9 61 8 11.6 61 16 20.8 10 to 20 years 58 9 13.4 73 73 31.1 115 56 32.7 Over 20 years 20 14 41.2 39 16 40.0 101 63 38.4 Totals 163 36 18.1 250 123 27.1 421 243 86.6 Table II is instructive in bringing out the importance of early injections of the serum. The results in the first two years of life are especially noteworthy. The extraordinary figures given under the first period of injection, namely, in the first three days of the disease, can hardly be maintained. But the influence of period of injection is shown by the rapid rise in mortality in the subsequent two periods. The rule of the effects of early injection is preserved in the age periods up to the period of twenty years, when it disappears. The discrepancy occurring in the highest age period cannot be wholly accounted for at present. The explanations which suggest themselves are that among older individuals there tends to be a large number of very se^'ere, rapidly fatal or fulminating cases of the disease, or that older persons are less subject to the beneficial action of the serum. As regards the actual proposition, it may be stated that adults not infrecjuently respond SERUM TREATMENT 279 promptly to the serum injections })y ahru]it termination of the disease or ameUorated symptoms and jiathologic conditions. The total figures do not, ho\ve\'er, fail to indicate tjiat the early injections are more etfecti\e than the later ones, as is shown by the percentage mortality in the first-to-third-ilay i)eriod of IS.l, in the fourth-to-seventh-day period of 27.1, and the period later than the seventh day of 3G.G. Directions for Use of Serum. — The following directions are issued by the Research Laboratory of the New York City Board of Health. The antimeningitis serum is a specific immune serum of therapeutic value only in meningococcic meningitis. It is administered subdurally, the subcutaneous route being practically worthless. Perform a lumbar puncture under aseptic precautions in the third or fourth lumbar space. A general anesthetic should ne^'er be used. In hypersensitive patients a local anesthetic may be advisable. Have the patient lying on the side with the back arched so that their will be the greatest possible distance between the spines of the vertebrae. Find the notch nearest a line connecting the crests of the ilia. Intro- duce the needle, preferably a Quncke needle, in the midline and push forward and a little upward. The distance the needle goes in depends upon the age of the patient and the muscular development. It varies from 5 to 3 inches. Allow the cerebrospinal fluid to flow out until the pressure is so reduced that only 3 or 4 drops come per minute. If the fluid is cloudy, inject the serum immediately. Where possible, further injections are made only after bacteriological examination has determined the cause to be the meningococcus. The antimeningitis serum does no harm in meningitis due to other organisms. The serum is warmed to body temperature and injected very slowly under the least possible pressure. A funnel and the tube arrangement allowing it to run in by gravity should be used. The barrel of an ordinary syringe may be used as a funnel. The rubber tubing should be I to J of an inch in diameter and long enough so that the funnel can be raised 12 to 15 inches. In general, the average dose for an adult is 20 to 40 c.c. and for infants and children 2 to 20 c.c. The amount depends as much upon the quantity of cerebrospinal fluid withdrawn as upon the age. An infant will frequently stand 10 to 15 c.c. without difficulty. The dose should usually be at least 5 to 10 c.c. less than the amount of cerebrospinal fluid withdrawn. Where serum apparently runs in freely after a dry tap, it is advisable to proceed very slowly and to watch the patient carefully for the slightest change in pulse and respiration. In cases with very thick exudate which will not flow through the needle, gentle suction with a syringe may be tried. If that fails, a little serum injected will sometimes start the flow. In very severe cases it is best to inject the -serum every twelve hours until there is improvement. In moderate and mild cases, it should be repeated each day for the first four days. Further administration depends upon the patient's general condition and the bacteriological examination of 2S0 PATHOGENIC MICROORGANISMS the fluid. Usually four to six injections are necessary, but as many as fifteen or more may have to be employed. Durinti' or immediately after the injection of serum the respiration may entirely cease or the pulse may become ver\' rapid and thready. Such an occurrence, while alarming, is not necessarily serious and is best treated by immediate withdrawal of some of the serum if the needle is still in place. If the needle has been withdrawn, or, if after some of the serum is removed the symptoms do not ameliorate, artificial respira- tion should be resorted to for the respiratory condition and adrenahn or other stimulants hypodermically for the heart. Hospitals and those having available blood-pressure apparatus will find it of service in administering the serum. The successful treatment of cases of meningitis must always depend upon experience and judgment. It cannot be reduced to a rule of thumb. In all cases with meningeal symptoms a lumbar puncture should be done. No ill effects follow, on the contrary the relief of the pressure frequently produces beneficial results. Bacteriological Diagnosis. — The fluid should be collected in a sterile container. It may be clear, cloudy, or blood}'. If it is clear it may be a normal fluid from a case of pohomyelitis or tuberculous meningitis. If it is cloudy it may be due to the meningococcus; streptococcus pneumococcus, pneumococcus mucosus, influenza bacilli, or other rarer organisms. The blood in a bloody fluid may be due to a previous hemorrhage or to the accidental piercing of a vein. The two conditions may be differentiated by centrifuging the fluid. If the supernatant fluid is yellow or reddish the hemorrhage is old. Clear fluid indicates a recent hemorrhage. It is unfortunate to have much blood in the fluid for it 'obscures the microscopic picture and then unless the culture is positive it is difiScult to make even a tentative diagnosis. Clear fluids should always be centrifuged preferably for one hour at high speed. Cloudy fluids showing no organisms should also be centrifuged. The sediment should be used to make cultures and smears. The smears are examined: (1) for pus cells and (2) for tubercle bacilli if the fluid is clear or for other organisms if it is cloudy. The sediment from clear fluids should always be stained with the tubercle stain and that from cloudy fluids with Gram's stain. Once in a long while a cloudy fluid will be found to be tuberculous or a clear fluid to be due to some pyogenic organism but these occurrences are so rare as to be practically negligible. Gram's stain diflterentiates influenza bacilli, and meningococcus from the streptococcus, pneumococcus, and pneumococcus mucosus. The finding of Gram-negative cocci either intra or extracellular is presump- tive evidence of meningogoccic meningitis, but it is always well to follow it up with a culture. In order that cultures may grow fluids should be examined at the earliest moment possible, for meningococci in fluids over tweK'e hours old are frequently autolysed so that they will not grow. OTHER ORGANISMS EXCITING MENINGITIS 2S1 The following table gi\'es the main difiVrential ])oints in making diagnosis from s])inal flnids: Pressure. .■Vmount, .\ppear- Cylnloyy. Bact. Albumin. Clolmlin. Aninial O-C. ance. Inoo. Normal Noru.al. 5-10 Clear. Very few cells. Sterile. ± — Negative. Meningismus Increased . 10-100 Clear. Very few cella. Sterile. ■ ± — ■ Negative . Infautile Increased . 20-100 Clear; Early polynu- Sterile. + -+ + +-+ + Negative paralysis some- times slight fibrin web. cleosis; later lymphocytosis up to 95 per cent.; endo- thelial cells or pneu- monia. Tuberculous Increased. 30-120 Clear ; Lymphocytosis Tubercle ++-+++ + +-++-I- Tubercu- meiiiugitia. fibrin web. up to 95 per cent. bacilli. losis in 4 weeks. Epiilemic cer- Increased . 5-120 Cloudy. Polynucleosis Meningo- + +-+ + + +++-+ + + + ebrospinal up to 98 per coccus. meningitis. cent. Meningitis Increased. 20-100 Cloudy. Polynucleosis Infecting ++-++++++-++++ due to other up to 98 per organisms. organisms. cent. Differential Diagnosis from Gonococci. — i\s a rule, the portion of the body from which the organisms are obtained reveals their source. When this is insufficient careful cultural and serological tests are required. ]\lcXeil has obtained a specific complement-fixation reaction. Other Gram-negative Cocci Resembling Meningococci. — Micro- coccus pharyngis (siccus) (von Lingelsheim) , Diplococcus mucosus, Chromo- genic Gram-negative cocci, Micrococcus catarrhalis. — These ma>' all be differentiated by cultural characteristics. Other Organisms Exciting Meningitis. — 1. The pneumococats. This diplococcus is one of the most frequent exciters of meningitis, both as a primary and a secondary infection. 2. The Streptococcus pyogenes, Pneumococcus and Staphylococcus. jNIeningitis due to these organisms is almost always secondary to some other infection, such as otitis, tonsillitis, erysipelas, endocarditis, suppurating wound of scalp and skull, etc. 3. The Bacillus influenzae. Numerous reports have been published of the presence of influenza bacilli in the meningeal exudate. Those that are reliable state in almost every instance that the meningitis is secondary to infection of the lungs, bronchi, the nasal cavities of their accessory sinuses. 4. The coloji bacillus, the typhoid bacillus, that of bubonic plague and of glanders, all may cause a complicating purulent meningitis. 5. In isolated cases of meningitis complicating otitis media and other infections, other bacteria, such as the Micrococcus tetrageniis, the Bacillus pyocyaneus, the gonococcus, etc., may be found. 6. The tubercle bacillus. This is a very frecjuent cause. It is after the meningococcus the most frequent cause. Meningitis due to other organisms than the meningococcus is almost invariabh' fatal. 282 PATHOGENIC MICROORGANISMS MICROCOCCUS CATARRHALIS (R. PFEIFFER). Micrococci somewhat resembling meningococci are found in tlie mucous membranes of the respiratory tract. At times they excite catarrhal inflamma- tion of the mucous membranes and pneumonia. These arc at present included under the designation of Micrococcus catarrhalis. Microscopic Appearance. — They usuallj^ occur in pairs, sometimes in fours; never in chains. The cocci are coffee-bean in shape and slightlj' larger than the gonoooccus, and are negative to Gram's stain. The micrococci are not motile and produce no spores. Cultivation. — They grow between 20° and 40° C, best at 37° C. and less rapidly at somewhat lower temperatures, developing on ordinary nutrient agar as grajdsh-white or yellowish-white, circular colonies of the size of men- ingococci. The borders of the colonies are irregular and abrupt as though gouged out. They have a mortar-like consistencj''. On serum-agar media the growth is more luxuriant. Gelatin is not liquefied. Bouillon is clouded, often with the development of a pellicle. Milk is not coagulated, but dextrose serum media may be. Gas is not produced. Location of Organisms. — In the secretion of normal mucous membranes they are occasionally present. In certain diseased conditions of the mucous membranes they may be al^undant. Pathogenic Effects in Animals. — For white mice, guinea-pigs, and rajjbits, some cultures are as pathogenic as meningococci, while others are less so. Differential Points Separating them from the Meningococci. — These organisms have undoubtedly been at times confused. Some assert that the meningococci grow only aljove 2.5° C. Many cord cultures of menmgococci grow below this point. Some assert that the meningococci will not grow on 5 per cent, gtycerin agar. Many undoubted cultures do. Careful agglutinin absorption tests are of great differential value, but can only be carried out safely bj^ one accustomed to them. The meningococci tested by us have removed all the agglutinins acting upon meningococci from a specific meningococcus serum while the allied organisms have removed only about 60 per cent, of them. The probability is that the organisms described by different writers as Micrococcus catarrhalis were not all the same variety, and some of them were meningococci. Vaccine Therapy. — Good reports have been made of the results of injecting the dead organisms in a number of infections due to this micrococcus. References. Eher and Huntoon. Jour. Med. Res., 1909, xx, 377. Flexner and Jobling. Jour. Exp. Med., May 1, 1913, xvii. No. .5, 260. A. Sophian, in the Journal o( the American Medical Association for March 23, 1912. Wollsliiin. Jour. Exp. Med., 1914, xx, 201. CHAPTER XIX. THE GOiNOCOCCUS OR MICROCOCCUS G0N0RRH(E.T: —THE DUCREY BACILLUS OF SOFT CHANCRE— MICROCOCCUS MELITENSIS. The period at which gonorrhea began to inflict man is unknown. The earUest records make mention of it. Wlierever civihzed man has penetrated gonorrhea is prevalent among the people. Except for a period after the fifteenth century it was generally recognized as a communicable disease and laws were made to control its spread. The differentiation between the lighter forms of gonorrhea and some other inflammations of the mucous membranes was, however, almost impos- sible until the disco\'ery of the specific microorganism by Neisser, in 1879. The organism was first observed in gonorrheal discharges, and, described by him under the name of "gonococcus;" but though several attempted to discover a medium upon which it might be cultivated, it was reserved for Bumm, in 1885, to obtain it in pure culture upon coagulated human-blood serum, and then after cultivating it for many generations to prove its infective virulence by inoculation into man. The researches of Neisser and Bumm established beyond doubt that this organism is the specific cause of gonorrhea in man. Gonorrhea is in almost all cases among adults transmitted through sexual inter- course. Gonorrheal ophthalmia is a frequent accidental infection at birth and vaginitis in the young child is frequently produced by the carelessness of the nurse or mother carrying infection. Microscopic Appearance. — Micrococci, occurring mostly in the form of diplococci. The bodies of the diplococci are elongated, and, as shown in stained preparations, have an unstained division or inter- space between two flattened surfaces facing one another, which give them their characteristic, "coffee-bean" or "kidney" shape. The older cocci lengthen, then become constricted in their middle portion, and finally divide, making new pairs (Fig. 109). The diameter of an associated pair of cells varies according to their stage of development from 0.8m to 1.6;U in the long diameter — average about 1.25m — by 0.6m to 0.8m in the cross diameter. Extracellular and Intracellular Position of Gonococci. — In gonor- rhea, during the earliest stages before the discharge becomes purulent, the gonococci are found mostly free in the serum or plastered upon the epithelium cells, but later almost entirely in small, irregular groups in or upon the pus cells and epithelial cells, and always extranuclear. With the disappearance of the pus formation more free gonococci appear. Discharge expressed from the urethra usually contains more 2S4 PATHOGENIC MICROORGANISMS free organisms tlian the natural flow. Gonoeocci are sometimes irregular in shape or granular in appearance, involution forms, found particularly in older cultures and in chronic urethritis of long standing. Single pus cells sometimes contain as many as one himdred gonoeocci and seem to be almost bursting and yet show but slight signs of injury. These diplo- cocci are also found in or upon desquamated epithelial cells. There is still discussion as to whether the gonoeocci actively invade the pus cells or only are taken up by them. There is no evidence that the gonoeocci are destro.yed by the pus cells (Fig. 110). In gonorrhea of the conjunctiva they are contained in the epithelial cells, sometimes in large numbers. They form dense groups which change as they do in older cultures, producing metachromate granules in round, swollen, pale blue bodies. These masses finally present an appearance somewhat like the cell inclusions found in "trachoma" (p. 439). Fig. no Smear from pure culture of gonocofc-us on Gonococcus in pus cells. X 100 diamc- agar. X 1100 diameters. (Heimiin.) ters. Staining.— The gonococcus stains readily with the basic aniline colors. L5fller's '.solution of methylene blue is one of the best stain- ing agents for demonstrating its presence in pus, for, while staining the gonoeocci deeply, it leaves the cell protoplasm but faintly stained. Fuchsin is apt to over,stain the cell substance. Beautiful double- stained preparations may be made from gonorrheal pus by treating cover-glass smears with methylene blue and eosin. Numerous methods for double staining have been employed, with the object of making a few gonoeocci more conspicuous. None of them ha\'e any specific characteristics such as the Gram stain. It is now established that gonoeocci from fresh cultures and from recent gonorrheal infections are, when properly treated by Gram's method, quickly and surely robbed of their color and take on the contrast stains. The removal of the stain from gonoeocci in old flakes and threads from chronic cases is not so certain. This difference is mostly due to the fact that equally uniform specimens cannot be prepared. The decolorized APPEARANCE OF COLONIES 285 Sonococci are stained by dipping the films for a few seconds into a 1 : 10 dilution of carbol-fuchsin or a solution of bisniarck brown. This stain- ing should be for as short a time as suffices to stain the decolorized organisms. This method of staining cannot be depended upon alone absolutely to distinguish the gonococcus from all other diplococci found in the urethra and \-ul\'o\aginal tract, ft)r, especially in the female, other diplococci are occasionally found which are also not stained by Gram's method. It serves, howe^'er, to distinguish this micrococcus from the common pyogenic cocci, which retain their color when treated in the same way, and in the male urethra it is practically certain, as few organisms have been found in that location which in morphology and staining are identical with the gonococcus. It is certainly the most distinctive characteristic of the staining properties of the gonococcus, and it is a test that should never be neglected in differentiating this organism from others which are morphologicalh' similar. Biology. — Grows best at blood-temperature; the limits being roughly 25° and 40° C. It is a facultative anaerobe. It is not motile and produces no spores. Culture Media. — The gonococcus requires for its best growth the addition to nutrient agar of a small precentage of blood-serum or some ecjuivalent. The following media have proven of value: 1. Human blood from the sterilized finger streaked on common nutrient agar. 2. Human ascitic, pleuritic, or cystic fluid, 1 part added to and mixed with 2 parts melted 5 per cent, glycerin nutrient, f.5 per cent, agar having a temperature of 55° to 60° C. The whole after mixing being jjoured into a Petri dish or cooled slanted in a tube. The same propor- tions of nutrient broth and ascitic fluid make a suitable fluid medium. One per cent, glucose may be added. 3. Swine serum nutrose media. Wassermann strongly recommends this mixture. In our hands it has given good results. 4. Nutrient or 5 per cent, glycerin agar. When considerable pus is streaked on simple agar media a good growth of gonococci is usually obtained. After continued cultivation gonococci cultures frec|uently grow on media containing no serum. Some strains grow on ordinary glycerin or glucose nutrient agar and even on plain nutrient agar from the start. Alkaline ^'eal agar gives good growths of started cultures. Viability. — Cultures frequently die in forty-eight to seventy-two hours when kept at room temperature. In the ice-box they may live for several weeks. They frequently live for one week in the thermostat at 36° C. on plain nutrient agar. Appearance of Colonies. — A delicate growth is characteristic. At the end of twenty-four hours there will have developed translucent, very finely granular colonies, with scalloped margin. The margin is sometimes scarcely to be dift'erentiated from the culture medium. In color they are grayish white, with a tinge of yellow. The texture is finely granular at the periphery, presenting pimctated spots of higher refraction in and around the centre of yellowish color (Fig. 111). .'S(l PATHOGENIC MICROORGANISMS Surface Streak Culture. — Translucent grayish-white growth, with rather thick edges. Resistance. — The gonococcus has but httle resistant power against outside influences. It is killed by weak disinfecting solutions and by desiccation in thin layers. In comparatively thick layers, however, as when gonorrheal pus is smeared on linen, it has lived for forty-nine days, and dried on glass for twenty-nine days (Heiman). It is killed at a temperature of 45° C. in six hours and of 60° in about thirty minutes. Occurrence of Gonococci. — Outside of the human body or material carried from it gonococci have not been found. Toxins. — In the gonococcus cells substances are present which are toxic after heating and contact with alcohol. Injected in considerable amounts into rabbits, they cause infiltration and often necrosis. Ap- plied to the urethral mucous men- brane there is produced an inflam- mation of short duration. In gon- orrhea the secretion is believed to be due to these intracellular toxins. Repeated injections give only slight immunity. The filtrate of recent gonococcus cultures contains little toxin. Pathogenesis. — Non-transmissi- ble to all animals. Both the living and dead gonococci contain toxic substances which cause death or injury when injected in large quan- tities. The etiological relation of the gonococcus to human gonorrhea has been demonstrated beyond ques- tion by the infection of a number of healthy men with the disease by the inoculation of pure cultures of the microorganism. Disease Conditions Excited by Gonococci. — Affections due to this organism are usually restricted to the mucous membranes of the urethra, prostate, neck of bladder, cervix uteri, vagina, and conjunctiva. The conjunctival, vaginal, and rectal mucous membranes are much more sensitive in early childhood than in later life. The usual course of the inflammation is as follows : The gonococci first increase upon the mucous membranes which show congestion, infiltration with serous exudate and accumulation of leukocytes. The cocci then penetrate the epithe- lial layer down to the submucous connective tissue. Recovery or a prolonged chronic inflammation may then persist. The original infec- tion of the urethra or vagina and cervix may remain localized or spread to adjacent parts or through blood and lymph l:>e carried to all parts of the bod}'. Gonococci thus cause many cases of endometritis, metritis, salpingitis, oophoritis, peritonitis, prostitis, cystitis, epididymitis, and Colonies of gonococci on pleuritic fluid agar. (Heiman.) DURATION OF INFECTIONS AND OF CONTAGIOUS PERIOD 287 arthritis. Abscesses of considerable size, periostitis, and otitis are occa- sionally due to the gonocoecus. Endocarditis and Septicemia. — Cases of gonocoecus endocarditis and septicemia are not infrequent. Gonocoecus septicemia may occur in connection with other localizations or alone. Nearl,y every year one or two of these cases are met with in every general hospital. In a considerable number of cases where gonococci are obtained from the blood the patients recover. The fever is sometimes typhoid-like in character. Complications. — General infections with gonococci are often fol- lowed or accompanied by neuralgic afi'ections, muscle atrophies, and neuritis. Urticaria occasionally occurs. Immunity. — Immunity in man after recovery from infection seems to be only slight in amount and for a short period if present at all. It is known that the urethra in man or cervix uteri in woman may contain gonococci which lie dormant and may be innocuous in that person for years, but which may at any time excite an acute gonorrhea in another individual or, under stimulating conditions, in the one carrying the infections. Animals may, however, be immunized, and their blood is both bactericidal and slightly antitoxic. Therapeutic Use of Serum and Vaccine. — The use of sera in acute gonorrheal joint inflammation has given in a considerable percentage of cases good results and seems to be worth trying. It seems to be useless in acute gonorrhea of the mucous membranes. Vaccines (heated cultures) have also been used with apparently real benefit in joint inflammations and even in very localized chronic infections of the urethra, bladder, and elsewhere. They have also been used in acute vaginitis in young children. In our cases the symptoms abated sooner than we expected, but the gonococci persisted. The dose is from twenty to a thousand millions given every three to seven days. The benefit of serum and vaccine in septicemia is doubtful. Sensitized serum seem to do good in some cases. Complement Fixation. — By the method of McNeil (p. 189) very good results have been obtained, and the method is used regularly in the New York City Health Department. Agglutination. — Torrey has shown that gonococci resemble pneu- mococci in that there are a number of different strains which have different specific and but little common agglutinins. The agglutina- tion test is of no practical value in diagnosis. Duration of Infections and of Contagious Period. — There is no limit to the time during which a man or woman may remain infected with gonococci and infect others. We have had one case under obser- vation where twenty years had elapsed since exposure to infection, and yet the gonococci were still abundant. It is now well established that most of the inflammations of the female genital tract are due to gonococci, and the majority of such infections are produced in innocent women by their husbands who are suffering from latent gonorrhea. 28S PATHOGENIC MICROORGANISMS Bacteriological Diagnosis of Gonorrhea. — In view of the fact that several non-goiiorrheal forms of urethritis occasionally exist, and also that micrococci morphologically similar to Neisser's diplococcus are at times found in the normal vulvovaginal tract of adults, it be- comes a matter of importance to be able to detect gonococci when present, and to differentiate these from the non-specific organisms. Besides this, the gonococci which occur in old cultures and in chronic urethritis of long standing sometimes take on a very diversified appear- ance. From a medicolegal and social stand-point, therefore, the differ- ential diagnosis of the gonococcus has in certain cases a very practical significance. There are three methods of differential diagnosis now a\'ailable — the microscopic, the cultural, and complement fixation. The method employed in the latter test is given on p. 189. Animal inoculations are of no value, as animals are not susceptible, and, of course, human inoculations are generally impossible. In the microscopic diagnosis it should be borne in mind that after the acute serous stage has passed, the specific gonococci in carefully made preparations are always found largely within the pus cells. Diplococci morphologically similar to gonococci occurring in other portions of the field and outside of the pus cells should not be considered specific by the test only. It should also be remembered that the gonococci are decolorized by Gram's method, while other similar micrococci which occur in the urethra are, as a rule at least, not so decolorized. Organisms having these characteristics can for all practical purposes be considered as certainly gonococci if obtained from the urethra. From the vulvovaginal tract the certainty is not so great, since other diplococci are found in gonorrheal pus from this area more frequently than from the urethra which stain as gonococci; here cultures should also be made. In chronic urethritis Heiman allows the patient to void his urine either immediately into two sterilized centrifugal tubes or first into two sterile bottles. The first tube will contain threads of the anterior urethra; the second tube will be likely to contain secretion from the posterior urethra and from the prostate gland if, while urinating, the patient's prostate be pressed upon with the finger. Tubes containing such urine are placed in the centrifuge and whirled for three minutes at twelve hundred or more, revolutions per minute, the threads are thrown down. The centrifuged sediment will be found to contan. most of the bacteria present, epithelial cells, and, at times, spermatozoia When the examinations are negative and it is important to be certain, either massage or injections of a solution of silver nitrate may be employed. The latter by causing a temporary irritation with increase of secretion will almost surely cause a discharge of gonococci if any infection was present. In acute cases where the pus is abundant the specimen for examina- tion may be collected, when the patient is before one, by passing a sterilized platinum-wire loop as far up into the urethra as possible and withdrawing some of the secretion. BACTERIA RESEMBLING GONOCOCCI 289 In vulvovaginitis the procedure should he as foUows: For obtaining the vaginal material the labia are held well apart by an attend- ant wearing sterile rubber gloves. Sterile slender cotton swab is used which passes easily into the vagina without touching any external part but entrance. (If pus from the cervix is desired a speculum should be used.) T?he swab is rubbed gently about vaginal wah, then withdrawn anil rolled (not rubbed) quickly over a shde (slide sterilized and held face down if cultiu'e is to be made) . In making this smear care is ^ised not to pass the sivab oi>er the same surface twice. In this way a beautifuUj' spread film is made. The swab is then returned to its holder (and covered and numbered if culture is to be made). The air-dried slide is rewrapped in its filter paper and numbered. The Technique for Making Culture is as FoUows: A small amount of rich, sterile ascitic fluid is added to the tubes containing the swab. (If patient is at a distance from laboratory the ascitic fluid is sent in a separate tube, and is inoculated from swab just after smear is made; then sw&h is withdrawn from ascitic fluid tube, placed in its own tube, and both tubes are sent immediately to the laboratory.) After being stirred up in this fluid the swab is withdrawn and smeared in strokes radiating from the centre over ascitic-agar (1-4) plates containing 2 per cent, glucose. Other plates of the same medium are stroked with platinum loopfuls of the ascitic fluid emulsion. From two to four plates are made and placed in thermostat at 36° C. After twentj^-four hours the plates are examined and if gonococcus-like colonies are seen they are fished. Then a smear is made from the whole of one of the most characteristic streaks, stained by Gram's method and examined for gonococcus-like organisms. Grouping Cases. — From the microscopic examination of luell-m.ade and well- stained smears (stained by Gram's method), and, where necessary, from cultures and from clinical appearance as well, the eases are divided into four groups, as follows: 1. Positive cases, i. e., those showing leukocytes filled with morphologically typical gonococci in smear or showing typical cultures, or showing l)oth. 2. Suspicious cases, i. e., those showing in smears any suspicious intracellular diplocooci and 50 per cent, or more of polymorphonuclear leukocytes. 3. Observation cases, i. e., those showing in smears 50 per cent, or over of polymorphonuclear leukocytes, but no suspicious intracellular diplococci; or those having the clinical symptoms of discharge and inflammation and showing less than 50 per cent, of polymorphonuclear leukocytes. 4. Negative cases, i. e., those showing in smears less than 50 per cent, poly- morphonuclears, and no suspicious intracellular diplococci, and no clinical evidence of the disease. 5. Isolation of Groups. — Each group is kept isolated. 6. Later Smears. — From the first three groups smears are made once a week until a negative smear is obtained. From negative groups smears are made if any suspicious symptoms appear. 7. Negative Diagnosis from Later Smears. — Three successive well-made and well-stained negative smears from first three groups, at intervals of not longer than three days, means a negative diagnosis. 8. Later Ctdtures. — From cases where morphologically typical gonococci persist in smear, cultures may be made and gonococcus-like organisms isolated and studied to find out if they are true gonococci. Bacteria Resembling Gonococci. — A few micrococci which resembled gonococci in form and staining have been descrilied. These assume importance largely because they may be confused with the gonococcus. They occur occasionally on the conjunctival and vaginal mucous mem- branes. One of these microorganisms, the Micrococcus catarrhahs (see p. 282), has an importance of its own. Others are probably unimpor- 19 2!)0 PATHOGENIC MICROORGANISMS taut. When absolute certainty is demanded cultural and serologic tests must be applied. Differential diagnosis from meningococci is given on p. 281. MALTA FEVER. The Micrococcus Melitensis. — This microorganism was first dis- covered in the spleen in a case of Malta fever by Bruce in Malta in 1887. The disease is mostly confined to the shores of the Mediter- ranean, but cases of it have been observed in Porto Rico, China, Japan, the Philippines, South America, and South Africa. This disease does not seem to be directly transmitted from person to persons. Clinical Symptoms. — Prodromal symptoms follow an incubation period of five to fourteen days. Headache, sleeplessness, loss of appetite, or vomiting accompany a high fever. The fever lasts for weeks, with intermissions and remissions. The fever period of one to three weeks may occur from time to time during a period of many months. The spleen and liver are enlarged. Neuralgic pains are severe. The fatal cases appear similar to severe cases of typhoid fever. Autopsy. — The spleen is large and very soft. The liver is also large and congested. Both organs show parenchymatous degeneration. Distribution of Micrococci. — These are abundant in the blood and all organs from the second day to the end of the disease. Morphology and Biology. — Very small rounded or slightly oval organisms, about 0..30/.1 in their greatest diameter. It is usually single or in pairs. In old cultures involution almost bacillary forms occur. They are not motile. Staining. — They stain readily with aniline dyes and are negative to Gram. Cultivation. — At 37° C. they grow rather feebly on nutrient agar and in broth. The colonies are not usually visible until the third day. They appear as small round disks, slightly raised, with a yellowish tint in the centre. The broth is slightly clouded after four to six days. The culture remains alive for several M'eeks or months. In gelatin the growth is very slow. Gelatin is not liquefied. Pathogenesis in Animals.— Monkeys are susceptible. They pass through the disease much like man. They can be infected by sub- cutaneous or mucous inoculation. Guinea-pigs and rabbits may also be infected but the disease progresses slowly in them. In Malta it has been found that about half of the goats pass the organisms in feces and so contaminate their milk. This is believed to be a source of infection. By safeguarding the milk the disease has been largely eliminated. Horses and cows are also susceptible. Therapeutic Results. — Injections of heated cultures have been thought to give good results. Methods of Diagnosis.— The diagnosis of Malta fever can frequently only be made with the help of bacteriological examination. Malaria, typhoid fever, and sepsis are the three diseases most apt to be con- founded with it, MICROCOCCUS ZYMOGENS 291 Cultures are made by spreading over the surface (if a number of agar plates freshly drawn blood. Frequently no organisms develop. The agglutination test is then required. Many bloods of persons suffering from other infections agglutinate the micrococcus of Malta fever in low dilutions so that 1 : 500 or over is required for a positive diagnosis. Animals injected with the coccus produce a serum agglutinating in high dilutions. Under suitable precautions this can be used to identify suspected cultures. Laboratory Infection. — A number of workers have infected them- selves with more or less serious results. MICROCOCCUS ZYMOGENS. MacCallum and Hastings^ observed this micrococcus in a case of acute endocarditis. It has since been found in a few other pathologic processes. It occurs in pairs and short chains. It grows well on agar, ferments lactose and glucose, and slowly liquefies gelatin. 1 Jour. Exp. Med., 1S99, iv, 521. CHAPTER XX. THE BACILLUS AND THE BACTERIOLOGY OF DIPHTHERIA. The lesions of diphtheria are caused by toxemia. The concen- trated poison at the seat of the exudate causes intense local inflam- mation, while in the more severe cases the absorbed poison diffused throughout the body causes widespread cellular injury, giving rise to definite injury of the cells of the muscle, nerve, and other tissues. Historical Notes. — This specific contagious disease can be traced back under various names to almost the Homeric period of Grecian history. From time to time during the following centuries we hear of epidemics both in Italy and in other portions of the civilized world which indicate that the disease never absolutely ceased. In I77I Bard, an xAmerican, advanced strong reasons for believing that membranous croup and pharyngeal diphtheria were different manifestations of the same disease process. In 1821 Bretonneau published his first essay on diphtheria in Paris and gave to the disease its present name. His observations were so extensive and so correct that little advance in knowledge took place until the causal relations of the diphtheria bacilli and their associated microorganisms to the disease began to be recognized. Evidence of Causal Relationship. — As early as 1840 observers began to notice microorganisms in the pseudomembranes. Gradually the observa- tions became more exact. The most importance was attributed to micrococci. In the year 1883, however, bacilli which were very peculiar and striking in appearance were shown by Klebs to be of constant occurrence in the pseudomembranes from the throats of those dying of true epidemic diphtheria. He described the peculiar staining of the organisms. One year later Loffler separated these bacilli from the other bacteria and grew them in pure culture. When he inoculated the bacilli upon the abraded mucous membrane of susceptible animals more or less characteristic pseudomembranes were produced, and fre- c^uently death or paralysis followed with characteristic lesions. These animal experiments have been fortified by a number of accidental human infections in laboratories with pure cultures of bacilli with sub- sequent development of diphtlieria. The Diphtheria Bacillus.— This bacillus is one of the most inter- esting i)f bacteria. Grown in tlie animal body or in suitable culture llnid, it produces a jjowerful toxin. Its morphology and staining are ])(.'culiar. Outside of tiie body it grows best on serum media. Morphology. — When co\'er-glass preparations made from the exu- date or from the cultures grown on blood serum are examined, the STAINING 293 diphtheria bacilU are found to possess the following morphological characteristics: The tliameter of the bacilli \'aries from U.ofi to O.Hn and the length from l/x to (>/j. They occur singly and in pairs (see Figs. 77 to 84) anil very infrt'ipiently in t'hains of three or four. The rods are straight or slightly curved, and usually are not uniformly cylindrical throughout their entire length, but are swollen at the end, or pointed at the ends and swollen in the middle portion. The average length of the bacilli in pure cultures from different sources frequently varies greatly, and even from the same culture individual bacilli differ much in their size and shape. This is especially true when the bacilli are grown in association with other bacteria. The two bacilli of a pair may lie with their long diameter in the same axis or at an obtnse or an acute angle. The bacilli possess no spores, but have in them highly refractive bodies, some of which are the starting point for new bacilli (see p. 37). There are no flagella. For mode of division, see p. 37. Fig. 112 Fig. 113 One of the very characteristic forms of diphtheria bacilli from blood-.serum cultures, showing clubbed ends and irregular stain. X 1100 diameters. Stain, methylene blue. Extremely long form of diphtheria bacillus. This culture has grown on artificial media for twenty years and pro- duces great amounts of toxin. X 1100 diameters. Staining. — The Klebs-Loffler bacilli stain readily w-ith ordinary aniline dyes, and retain fairly well their color after staining by Gram's method. With Loffler's alkaline solution of methylene blue, and to a less extent with Eoiix's and dilute Ziehl's solutions, the bacilli from blood-serum cultures especially, and from other media less constantly, stain in an irregular and extremely characteristic way. (See Fig. 112.) The bacilli do not stain uniformly. In many cultures round or oval bodies, situated at the ends or in the central portions, stain much more intensely than the rest of the bacillus, usually showing metachromatism (metachromatic granules. See p. 37 and Plate III). Sometimes these highly stained bodies are thicker than the rest of the bacillus; again, they are thinner and surrounded by a more slightly stained portion. Other bacilli have barred staining. The bacilli stain in this peculiar manner at a certain period of their growth, so that only a portion of the organisms taken from a culture at any one time will show the 294 PATHOGENIC MICROORGANISMS Fig. U4 Fig. 115 >\ >' *'^> ^ vi % Diphtheria, bacilli characteristic in shapes, but showing even staining. X 1000 dia- meters. Stain, methylene blue. Fig. 116 VJi,- r «> %. B. diphtheria agar culture. Bacilli small and uniform in shape. X 1000 diameters. Fig. 118 W - ■ VJ^l B. diphtheriue. Forty-eight hours' agar culture. Many segments; long, Indian- chibbcd ends. One year on artificial media. X 1410 diameters. Non-virulent diphtheiia bacilli, show- ing stain with Neisser's solutions. This appearance was formerly supposed to be characteristic of virulent bacilli. Bodies of liacilli in smear, yellowish-brown; points, dark blue. Fig. 117 B. diphtheriae. Forty-eight hours' agar culture. Thick, Indian-clubbed rods and moderate number of segments. One year on artifi(aal culture media. X 1410 diameters. Fig. 119 B. diphtherice. Twenty-four hours' agar culture. Coccus forms. Segmented granular forms on LofHer's serum. Only variety found; in cases of diphtheria at Children's Home. X 1410 diameters. THE MORPHOLOGY OF THE DIPHTHERIA BACILLUS 295 characteristic staining. Tlie yoiiuj;' cultures have the most rej,nilar fcimis, an eigliteen-hour growtli sliowing more chilihed forms tluin at tweh'c hours. After twenty-four hours the bacilH do not stain (juite as weH. In still older cultures it is often difficult to stain the bacilli, and the staining, when it does occur, is frequently not at all characteristic. The same round or oval bodies which take the methylene blue more intensely than the remaintler of the bacillus are brought out still more distinctly by the Neisser stain. The Neisser stain is carried out by placing the cover-slip smear of diphtheria or other baciUi in solution No. 1 for from two or three seconds, and then, after washing, in No. 2 for from three to five seconds. The bacilli will then appear either entirely browii or will show at one or Isoth ends a dark blue, round body. With characteristic diphtheria bacilli, taken from a twelve to eighteen hours' growth on serum, nearly all will show the blue bodies (Fig. 115), while with pseudo type (Fig. 121), to be described hereafter, few will be seen. The solutions are as follows : No. 1. Alcohol (96 per cent.) 20 parts. Methylene blue (Griibler) 1 part. Distilled water 950 parts. Acetic acid (glacial) 50 parts. No. 2. Bismarck brown 1 part. Boiling distilled water 500 parts. The Neisser stain has been advocated in order to separate the viru- lent from the non-virulent bacilli, without the delay of inoculating animals; but in our hands, with a very large experience, neither the Neisser stain not other stains, such as the modifications of the Roux stain, have given much more information as to the virulence of the bacilli than the usual methylene-blue solution of Loffler. A few strains of virulent bacilli fail to show a marked characteristic stain, and quite a few pseudodiphtheria bacilli show the dark bodies. There are also in many throats bacilli which seem to have all the staining and cultural characteristics of the virulent bacilli, and yet have no relation to the disease diphtheria. They are therefore non-virulent in the sense that they produce no diphtheria toxin. As will be stated more fully later, nothing but animal inoculations with the suspected bacilli together with control injections of diphtheria antitoxin will separate harmless bacilli from those capable of producing diphtheria. The Morphology of the Diphtheria Bacillus on Serum-free Media. — This varies considerably with different culture media employed. On glycerin agar or simple nutirent agar there are two distinct types. One grows as smaller and, as a rule, more regular forms than when grown on serum culture media (Fig. 116). The other type shows many thick, Indian-clubbed forms with a moderate number of segments (Figs. 117-119). Short, spindle, lancet, or club-shaped forms, staining uniformly, are aU observed. The bacilli which have developed in the pseudomembranes or exudate in cases of diphtheria resemlile in shape young iwicilli grown on ))lood-serum, but stain more evenly. 200 PATHOGENIC MICROORGANISMS Biology.—The Klebs-Loffler bacillus is non-motile and non-liquefy- ing. It is aerobic and facultative anaerobic. It grows most readily in the i)resence of oxygen. It does not form spores. It begins to develop, but grows slowly at a temperature of 20° C, or even less. It attains its maximum development at 37° C. In old cultures in fluid media, Williams has observed fusion of one bacillus with another. The fused forms live the longest. Growth on Culture Media. — Blood-serum. — Blood-serum, especially coagulated in the form of LofHer's mixture, is the most favorable medium for the growth of the diphtheria bacillus, and is used particu- larly for diagnostic purposes in examining cultures from the throats of persons suspected of having diphtheria. For its preparation, see p. .'315. If we examine the growth of diphtheria bacillus in pure culture on blood serum we shall find at the end of from eight to twelve hours small colonies of bacilli, which appear as pearl-gray, whitish-gray or, more rarely, yellowish-gray, slightly raised points. The colonies when separated from each other may increase in forty-eight hours so that the diameter may be one-eighth of an inch. The colonies lying together become confluent and fuse into one mass when the serum is moist. The diphtheria colonies after a growth of twelve hours become larger than those of the streptococci but remain smaller than those of the staphylococci. Growth on Agar. — On 1 per cent, slightly alkaline, nutrient or glycerin- agar the growth of the diphtheria bacillus is less certain and luxuriant than upon blood serum; but the appearance of the colonies when examined under a low-power lens, though very variable, is often far more characteristic. (Fig. 120). For this reason nutrient agar in Petri dishes is used to obtain diphtheria bacilli in pure culture. The diph- theria bacillus obtained from cultures which have developed for some time on culture media grows well, or fairly well, on suitable nutrient agar, but when fresh from pseudomembranes one prevalent type of bacilli grows on these media with great difficulty, and the colonies de\'elop so slowl>' as to be frequently co^'ered up by the more luxuriant growth of other bacteria when present, or they may fail to develop at all. If the colonies de\'elop deep in the substance of the agar they are usually round or oval, and, as a rule, present no extensions; but if near the surface, commonly from one, but sometimes from both sides, they spread out an apron-like extension, which exceeds in surface area the rest of the colony. When the colonies develop entirely on the surface they are more or less coarsely granular, and usually have a dark centre and vary markedly in their thickness. The colonies from some are almost translucent; from others are thick and almost as lux- uriant as the staphylococcus. The edges are sometimes jagged, and frequently shade off into a delicate lace-like fringe; at other times the margins are more even and the colonies are nearly circular. Peculiarites in the growth of the diphtheria bacillus upon agar are of prac- tical importance. If a large number of the bacilli from a recent culture are imiilanted upon a properl>' prepared agar plate a certain and fairly vigorous GROWTH IN ASCITIC OR SERUM BOUILLON 297 growth will always take place. If, however, the agar is inoculated with an exudate from the throat, which contains hut a few bacilli, no growth wliatever may occur, while the tubes of coagulated blood-serum inoculated with the same exudate contain the bacilli alumdautly. Because of the uncertainty, therefore, of obtaining a growth by the inoculation of agar with luicilli unaccustomed to this medium, agar is not a reliable inetlium for use in primary cultures for diag- nostic jjurposes. A mixture composed of two parts of a 1.5 per cent, nutrient agar and one part of sterile ascitic fluid makes a medium upon which the bacillus grows much more luxuriantly, but not so characteristically. Isolation of the Diphtheria Bacillus from Plate Cultures. — Nutrient plain or glycerin-agar should be freshly melted and poured in the Petri dish for this ]mr- pose. After it has hardenecl, the medium in a number of ])lates is streaked across with bacteria from colonies on the serum culture, which appear in size and color like the diphtheria bacilli. Other plates are made from a general Fig. 120 mixture of aU bacteria, selected, as a rule, from the drier portion of the serum. Still others are inoculated from the pellicles of ascitic broth cultures. The plates are left in the incubator for about sixteen hours at 37° C. In the examination of the plates one should first seek for typical colonies, then, if these are not found, for any that look most nearly like the characteristic picture. Diphtheria col- onies are very apt to be found at the edges of the streaks of bacterial growth. The pickings from the colonies are inoculated upon Loffler's blood-serum, or into ascitic bouillon. Growth in Bouillon. — The diphtheria bacilli from about one-half the cul- tures grow readily in broth slightly alkaline to litmus; the other cultures grow feeblj'. The characteristic growth in neutral bouillon is one showing fine grains. These deposit along the sides and bottom of the tube, leaving the broth nearly clear. A few cultures in neutral Ijouillon and many in alkaline bouillon produce for twenty-four or forty-eight hours a more or less diffuse cloudiness, and frequently a film forms over the surface of the broth. On shaking the tube this film breaks up and slowly sinks to the bottom. This film is apt to develop during the growth of cultures wliich have long been cultivated in bouil- lon, and, indeed, after a time the entire development maj' appear on the surface in the form of a friable pellicle. The diphtheria bacillus in its growth causes a fermentation of meat-sugars and glucose, and thus, if these are present, changes the reaction of the bouillon, rendering it distinctly less alkaline within forty- eight hours, and then, after a variable time, when all the fermentable sugars have been decomposed, more alkaline again through the progressing fermenta- tion of other substances. Among the products formed by its growth i's the diphtheria toxin. Growth in Ascitic or Seriim Bouillon. — All varieties of diphtheria bacilli grow well in this medium, even when first removed from the throat. They almost always form a slight pelhcle at the end of twenty- four to forty-eight hours. This culture medium is, as pointed out hy Williams, of the greatest value in attempts to get pure cultures of the diphtheria bacillus from solidified serum cultures containing Colonies of diphtheria Vjaeilh. diameters. X 200 298 PATHOGENIC MICROORGANISMS few bacilli among manj' other bacteria. Plate cultures are made from the pellicle. The fluid is prepared by adding to the nutrient bouillon 25 per cent, ascitic fluid or blood-serum. Growth on Gelatin. — Tlie growth on this nii'diuni is much slower, more scanty, and less characteristic tliau that on the other media mentioned. This largely is on account of the lower temperature at which it must be used. Growth in Milk. — The diphtheria bacillus grows readily in milk, beginning to develop at a comparatively low temperature (20° C). The milk remains unchanged in appearance as lactose is not fermented by the diphtheria bacillus. Pathogenesis. — In Lower Animals. — The diphtheria bacillus through its toxins is, when injected into their bodies, pathogenic for guinea- pigs, rabbits, chickens, pigeons, small birds, and cats; also in a lesser degree for dogs, goats, cattle, and horses, but hardly at all for rats and mice. In spite of its pathogenic qualities for these animals true diph- theria occurs in them with extreme rarity. As a rule, supposed diph- theritic inflammations in them are due to other bacteria which cannot produce disease in man. The cat is the only animal that we have known to contract true diphtheria from contact with the disease. Cobbett reports a case in a colt. At the autopsy of animals dying from the poisons produced by the bacilli, the characteristic lesions described by Loffler are found. At the seat of inoculation there is a grayish focus surrounded by an area of congestion; the subcutaneous tissues for some distance around are edematous; the adjacent lymph nodes are swollen; and the serous cavities, especially the pleura and the peri- cardium, frequently contain an excess of fluid usually clear, but at times turbid ; the lungs are generally congested, the suprarenals are markedly congested. In the organs are found numerous smaller and larger masses of necrotic cells, which are permeated by leukocytes. The heart and certain voluntary muscular fibers and nervous tissues usually show degenerative changes. Occasionally there is fatty degeneration of the liver and kidneys. The number of leukocytes in the blood is increased. From the area surrounding the point of inoculation virulent bacilli may be obtained, but in the internal organs they are only occasionally found, unless an enormous number of bacifli have been injected. Par- alysis, commencing usually in the posterior extremities and then grad- ually extending to the whole body and causing death by paralysis of the heart or respiration, is also produced in many cases in which the inoculated animals do not succumb to a too rapid intoxication. In a number of animals we have seen recovery take place three to six weeks after the onset of the paralysis. Tissue Changes in Natural (Human) Infection. — The characteristic lesions are a pseudomembranous inflammation on some of the mucous membranes or occasionally on the surface of wounds and the general hyperplasias and parenchymatous inflammations produced by the absorbed toxic substances. Pneumonia is apt to occur as a compli- cation of laryngeal diphtheria. The membrane may be simply a thin THE PRODUCTION OF TOXIN IN CULTURE MEDIA ■ 299 pellicle, which is easily removed without causing bleeding or it may he thick and firmly attached and leaving when removed a. ragged hieeding surface. The tissue beneath the pseudomemliranc is always intensely injected and often hemorrhagic. The cells show marked ilegencrative changes. Causes of Death. — These are chiefly toxemia, laryngeal obstruction and bronchopneumonia. Septicemia due to other bacteria is f'reciuently an additional factor. Diphtheria Toxin.— This poison was assumed by Loffler (f884) to be produced by the bacilli, but it was first partially isolated by Roux and Yersin, who obtained it by filtration through porous porcelain from cultures of the living bacilli. It has not yet been successfully analyzed, so that its chemical composition is unknown, but it has many of the properties of proteid substances, and can well be desig- nated by the term active proteid. It resembles in many ways the ferments. x\fter injection into the body there is a latent period before its poisonous action appears. The poison produced is probably com- posed of a mixture of several nearly related toxins. Diphtheria toxin is totally destroyed by boiling for five minutes, and loses some 95 per cent, of its strength when exposed to 75° C. for the same time; 60° C. destroys very little. Lower temperatures only alter it very gradually. Kept cool and from light and air, it deteriorates very slowly. Desiccated at low temperatures and kept dry in a vacuum it keeps unaltered for long periods. The Production of Toxin in Culture Media. — The artificial production of toxin from cultures of the diphtheria bacilkis has been found to depend upon definite conditions, which are of practical importance in obtaining toxin for the inocu- lation of horses, and also of tlieoretic interest in explaming why cases of appar- ently equal local severity have such different degrees of toxic absorption. The researches of Roux and Yersin laid the foundation of our knowledge. Their investigations have been contmued b}' Theobald Smith, Spronck, ourselves, and others. After an extensive series of investigations we (Park and Williams) came to the following conclusions: Toxin is produced by fully virulent diph- theria bacilli at all times during their life when the conditions are favorable. Under less favorable conditions some bacilli are able to produce toxin while others are not. Diphtheria baciUi may find conditions suitable for luxuriant growth, but unsuitable for the production of toxin. The requisite conditions for good development of toxin, as judged b)f the behavior of a number of cul- tures, are a temperature from about 32° to .37° C, a suitable culture medium, such as a 2 per cent, peptone nutrient bouillon made from veal, of an alkalinity which should be about 9 e.c. of normal soda solution per liter above tlie neutral point to litmus, and prepared from a suitable peptone (Witte) and meat. The culture fluid should be in comparatively thin layers and in large-necked Erlen- meyer flasks, so as to allow of a free access of air. The greatest accumulation of toxin in bouillon is after a duration of growth of the culture of from five to ten days, according to the peculiarities of the culture employed. At a too early period toxm has not sufficiently accumulated; at a too late period it has begun to degenerate. In our experience the amount of muscle-sugar present in the meat makes no appreciable difference in the toxin produced when a vigor- ously growing bacillus is used, so long as the bouillon has been made sufficiently alkaline to prevent the acid produced by the fermentation of the sugar from producing in the bouillon an acidity sufficient to inhibit the growth of tiie bacilli. 300 PATHOGENIC MICROORGANISMS With the meat as we obtain it in New York, we usually get better results with unfcrniented meat than with fermented. In Boston, with the same bacillus, Smith gets more toxin from the bouillon in whicii the sugar lias been fermented by the colon bacillus. Instead of colon bacilli, j'east may be added to the soaking meat, which is allowed to stand at about 25° C. The preliminary fer- mentation of the meat sometimes produces poisonous substances which are deleterious to the horses. We have obtained especially good results with veal broth made from calves two to four weeks old (bob veal) . When strong toxin is desirable the muscle is separated from all fat, tendon and fibrous tissue before Ijeing chopped. Under tlie best conditions we can devise, toxin begins to be produced by l)acilli fi'om some cultures when freshly sown in bouillon some time during the first twenty-four hours; from other cultures, for reasons not well understood, not for from two to four days. In neutral bouillon the culture fluid frec^uently becomes slightlj' acid and toxin production maj^ be delayed for from one to three weeks. The greatest accumulation of toxin is on the fourth daj^, on the average, after the rapid production of toxin has commenced. After that time the number of li^'ing bacilli rapidh^ diminishes in the culture, and the con- ditions for those remaining alive are not suitable for the rapid production of toxin. As the toxin is not stable at 35° C, the deterioration taking place in the toxin already produced is greater than the amount of new toxin still forming. Bacilli, when repeatedly transplanted from bouillon to bouillon, gradually come to grow on the surface only. This characteristic keeps the bacilli in contact with the oxj^gen and seems to aid in the de^'clopment of toxin. Comparative Virulence as Estimated by Toxin Production of Dif- ferent Cultures. — The ^'irule^c•e of diphtheria bacilli from different sources, as measured by their toxin production, varies considerably. Thus, as an extreme instance, 0.002 c.c. of a forty-hour bouillon culture of our most virulent bacilli will kill a guinea-pio;, which it would require 0.1 c.c. of culture of our least virulent bacillus to kill. This difference frequently depends on the unequal growth of the bacilli, one culture having fifty times as many bacilli as the other. When the different strains are grown on ascitic broth, upon which their growth is usually good, the majority of cultures are nearly equal in virulence, but some still show marked differences. Moreover, the diphtheria bacilli difi'er in the tenacity with which thej' retain their virulence when grown outside the body. The bacillus that we have used to produce toxin in the laboratory of the Board of Health has retained its virulence unaltered for twenty years in bouillon cultures. Other bacilli have apparently lessened their capacity for toxin production after being kept six months. Brown and Smith^ report on a culture in which 5 c.c. was the minimum toxic dose of the filtrate. The passage of diphtheria bacilli through the bodies of susceptible animals does not increase their toxin production to any considerable extent. Comparative Virulence of Bacilli and Severity of Case.— From the severity of an isolated case the virulence of the bacilli cannot be deter- mined. The presence of slight amounts of antitoxin in the blood of the person attacked and the association of other bacteria are at least two of the disturbing factors. The most virulent bacillus we have ever found was obtained from a mild case of diphtheria simulating tonsillitis. ' .Jour. Med. Research, vol. xxx, No. .3, p. 44.3. PERSISTENCE OF DIPHTHERIA BACILLI IN THE THROAT 301 Another case, however, infected by the bacilhis proved to l)e \'ery severe. In locahzed epidemics the average se\'erity of tlie cases prob- ably indicates rouglily tlie virulence of the bacillus causing the infection, as here the indi\idual susceptibility of the different persons infected would, in all likelihood, when taken together, be similar to that of other groups; but even in this instance special conditions of climate, food, or race may influence certain k)calities. It must be remembered that bacilli of like toxic pt)wer may differ in their liability to infect the mucous membrane. Mrulence has thus two distinct meanings when used in ■ connection with diphtheria bacilli. Virulent Bacilli in Healthy Throats.— Fully virulent bacilli have frequently been found in healthy throats of persons who have been brought in direct contact with diphtheria patients or diphtheria carriers without contracting the disease. It is, therefore, apparent that infec- tion in diphtheria, as in other infectious diseases, requires not only the presence of virulent bacilli, but also a susceptibility to the disease, which may be local or general. We now know that 50 per cent, of all persons are protected from infection because of having antitoxin present in their blood. Among the predisposing influences which con- tribute to the production of diphtheritic infection may be mentioned the breathing of foul air and living in overcrowded and ill-\'entilated rooms, impure food, certain diseases, more particularly catarrhal inflammations of the mucous membranes, and depressing conditions generally. Under these conditions an infected mucous membrane may become susceptible to disease. In connection with Beebe (1894) we made an examination of the throats of 330 healthy persons who had not come in contact, so far as known, with diphtheria, and we found virulent bacilli in S, only 2 of whom later developed the disease. In 24 of the 330 healtliy throats non-virulent bacilli similar to the viru- lent diphtheria bacillus were found. Very similar observations have since been made in Boston and by others in many widely separated countries. In 1905 Von Sholly in our laboratory examined 1000 throats of those who had not knowingly been in contact with diphtheria and found virulent diplitheria bacilli in 0.5 per cent, of the cases. We have foimd virulent bacilli in about 5 per cent, of cases of scarlet fe\'er. This indicates they are more prevalent in throats than a single culture from normal cases indicate. Persistence of Diphtheria Bacilli in the Throat. — The continued presence of \'irulent diphtheria bacilli in the throats of patients who have recovered from the disease has been demonstrated by all inves- tigators. In the investigations of 1894 we found that in 304 of 605 consecutive cases the bacilli disappeared within three days after the disappearance of the pseudomembrane; in 17(i cases they persisted for seven days, in (i4 cases for twelve days, in 3() cases for fifteen days, in 12 cases for three weeks, in 4 cases for four w(>eks, and in 2 cases for nine weeks. Since then we have met with a case in which they persisted with full virulence for eight months. It is safe to say that in o^'e^ 10 per cent, of the cases a few bacilli persist two weeks after the disappear- 302 PATHOGENIC MICROORGANISMS aiice of the exudate and in over 1 per cent, four weeks. It is extremely difficult to prove that a case is absolutely clear as the bacilli may remain hidden in the epithelial cells of some tonsillar crypt and not be detected by cultures. Diphtheria-like Bacilli Not Producing Diphtheria Toxin.— In the tests of the bacilli obtained from hundreds of cases of suspected diph- theria which have been carried out during the past twenty years in the laboratories of the Health Department of New York City, in over 95 per cent, of cases the bacilli derived from exudates or pseudomem- branes and possessing the characteristics of the Loffler bacilli have been found to be virulent, that is, producers of diphtheria toxin. But there are, however, in inflamed throats as well as in healthy throats, either alone or associated with the virulent bacilli, occasionally bacilli which, though morphologically and in their behavior on culture media identical with the Klebs-LofHer bacillus, are yet producers, at least in artificial culture media and the usual test animals, of no diphtheria toxin. Between bacilli which produce a great deal of toxin and those which produce none we find few minor grades of virulence. We believe, therefore, in accordance with Roux and Yersin these non-virulent bacilli should be considered as possibly attenuated varieties of the diphtheria bacillus which have lost their power to produce diphtheria toxin. This supposition is, however, not proven and it may be that the ancestors of these bacilli were never toxin producers. These ob- servers, and others following them, have claimed that the virulent bacilli can be artificially attenuated ; but the reverse has not been proven that bacilli which produce no specific toxin have later been found to develop it. In our experience some cultures hold their virulence even when grown at 41° C. for a number of months, while others become slightly attenuated rather quickly. We have never yet been able to change a virulent culture into an absolutely non-virulent one, and we have never accomplished the reversed process. We believe therefore that for practical purposes bacilli which produce no toxin in animals can be disregarded as a possible source of human diphtheria. It may be added here that no facts have come to light which indicate that bacilli which do not produce diphtheria toxin in animals ever produce it in man. It must also be borne in mind, however, that such proof is necessarily very difficult to obtain. Bacilli Resembling the Diphtheria Bacilli. — Diphtheria-like bacilli are also found which resemble diphtheria bacilli very closely except in toxin production, but differ in one or more particulars. Both these and the characteristic non-virulent bacilli are found occasionally upon all the mucous membranes both wlien inflamed and when apparently normal. Diphtheria-like Bacilli Pathogenic to Guinea-pigs Producing no Diphtheria Toxin. — These bacilli are obtained frequently from normal or slightly- inflamed throats or from other mucous membranes, and may be slightly pathogenic in guinea-pigs, since they may kill, as we found, in doses of 2 to .5 c.c. of broth culture subcutaneously or intraperi- HOFM ANN'S PSEUDODIPHTHERIA BACILLI 303 toneally injected. Animals are not protected by diphtheria antitoxin from the action of these bacilli showing that their poisonous action is not due to diphtheria toxin. At autopsy the bacilli are usually found more or less abundantly in the blood and internal organs. The fact that large injections of antitoxic serum hastens the death of guinea- pigs injected with these bacilli has given rise to the notion that injections of antitoxin might be dangerous in persons in whose throats these bacilli were present, either as saprophytes, ui possibly, as inciters of slight disease. It is not the antitoxin, but the serum, which in large doses injures the vitality of the guinea-pigs and so slightly hastens death. The amount of serum required to produce this effect is far in excess of that properly given in man. These bacilli were first described by Davis^ from our laboratory and later by Hamilton in 1904. The pos- sibility of their being present affords no reason to avoid giving antitoxin in suspected cases. When pathogenic in man they are usually only feebly so. Hofmann's Pseudodiphtheria Bacilli. — Besides the typical bacilli which produce no diphtheria toxin, but which, so far as we can deter- mine, are otherwise identical with the Loffler bacillus, there are other bacilli found in positions similar to those in which diphtheria bacilli abound, which, ^^°- i^i though resembling these organisms in many particulars, yet differ from them as a class in others equally important. The variety most prevalent is rather short, plump, and more uniform in size and shape than the true Loffler bacillus (Fig. 121). On blood-serum their colonj' growth is very similar to that of the diphtheria bacilli. The great majority of them in any young culture show no polar granules when stained by the Neisser method, and stain evenly p.uedodiphtheria bacilli. throughout with the alkaline methylene (b. hofmanni.) blue solution. They do not produce acid by the fermentation of glucose, as do all known virulent and many non- virulent diphtheria bacilli; therefore, there is no increase in acidity in the bouillon in which they are grown during the first twenty- four hours from the fermentation of the meat-sugar regularly present. They are found in varying abundance in different localities in New York City, in about 1 per cent, of the normal throat and nasal secretions, ancl seem to have now at least no connection with diphtheria; whether they were originally derived from diphtheria bacillus is doubtful; they certainly seem to have no connection with it now. They have been called psemlodiphtheria hacilli, and more properly, B. hofmanni} In bouillon they grow, as a rule, less luxuriantly than the diphtheria bacilli. Some of the varieties of the pseudodiphtheria bacilli are as long as the 1 Medical News, April 29, 1899. ' Clark, Jour, of Inf. Dis., 1910, vii, 335. 304 PATHOGENIC MICROORGANISMS shorter forms of the virulent bacihi. When these are found in cultures from cases of suspected diphtheria they may lead to an incorrect diag- nosis. These bacilli are found occasionally in all countries where search has lx>en made for them. There are also some varieties which resemble the short pseudobacilli in form and staining, but which produce acid in glucose bouillon. Bacillus Xerosis. — Diphtheria-like bacilli were found by Kutschert and Neisser (1SS4) in the condition known as xerosis conjunctiva?. Since then, they have been found frequently in normal conjunctivae. These bacilli went by the name of xerosis bacilli. Under this name, no doubt, different observers have placed several different varieties of bacilli morphologically somewhat similar to the diphtheria bacilli. Knapp first limited the name to bacilli that ferment saccharose and not dextrin, in contradistinction to diphtheria bacilli which ferment dextrin and not saccharose. The fermentation tests for three of the diphtheria group, according to Knapp, may be shown as follows : Species. Dextrose. Levulose. Galactose. M altcse. Saccharose. Dextrin. B. diphtheria . + + + -1- — + B. xerosis . + + + -f- + — B. hofmanni — — — — — — Note. — One per rent. sugar in Hiss scrum-water media These reactions do not separate a large number of the non-virulent bacilli morphologically like the diphtheria bacilli, which are found in throats under varying conditions. Persistence of Varieties of the Bacillus Diphtheria and of Diph- theria-like Bacilli. — The fact that there are distinct differences between strains of bacilli producing diphtheria toxins which are as great as between these and some strains of bacilli producing no diphtheria toxins has, we think, been fully established. But that such varieties are true sub-species with constant charac- teristics, one variety not changing into another of the established forms, has not been accepted by all. On the contrary, the opinion is held by some investigators that all of the \'arious forms of diphtheria-like bacilli are the result of more or less transitory variations of the same species, and hence that the virulent forms are the result of a rapid adaptation to en\'ironment and consequent pathogenesis of the non- virulent forms, both typical and atypical. This question is of great practical importance in methods of handling the persons harboring these bacilli. Wesbrook, Wilson, and McDaniel,' make a provisional classification based upon the morphology of the individual bacilli, into three groups, called granular, barred, and solid, two of the groups into seven types and the other into five, two of the types corresponding with those in the other groui)s not ha\ing been seen. They state that tliere is generally a sequence of tyj)es in the variations which appear thrf)ughout the course of the disease,- the granular types, as a rule, predominating at ' Transactions of the Association of .\n)erican Physicians, 19UU. RESISTANCE TO HEAT, DRYINit, AND CHEMICALS 3U5 the outset of the disease, and tliese gix'ing plaee wliolly or in jiart to the barred and solid types shortly before the disappearance of diplitheria- like organisms. The inference drawn from this work is that the diphtheria bacillus may be rather easily, especially in the throat, converted into non- granular, solidly staining forms of the " pseudodiphtheria" type, and that the converse may occur, and that therefore all diphtheria-like bacilli must be considered a possible source of danger. In studying the subject Williams^ (1902) came to the following con- clusions: Though some cidtures change on some of the media, each changes in its own way, and each culture still has its distinct individ- uality. After many culture generations, especially when transplanted at short intervals, the different varieties of virulent diphtheria bacilli tend to run in lines parallel with a common norm, which seems to be a medium-sized, non-segmented bacillus, producing granules in early cultures on serum and growing well on all of the ordinary culture media. The non-virulent morphologically typical bacilli must be classed with the virulent varieties as one species, though there is little doubt that more minute study would show that the former constitute a distinct group. The atypical pseudo forms, however, which show no tendency to approach the norm of the typical forms, must be classed as distinct species. All of the pseudo and the non-virulent morphologically typical varieties when inoculated into the peritoneum of guinea-pigs in immense doses cause death. In studying successive direct smears from the throats of diphtheria patients no evidence of change from one type to the other was noted. Attempts have been made to give more virulence to some of these varieties by successive peritoneal inoculations, and by growing the organisms in symbiosis with several other organisms, but in no instance has any increase of virulence or decided change in mor- phological or cultural characteristics been noted. Since there are so many different forms or varieties of diphtheria- like bacilli, it is quite possible that some of them are derived from strains of the diphtheria bacillus and that under certain conditions they readily regain its characteristics. This seems to be the only way to explain the apparent discrepancies in the results obtained by different observers. Such closely related varieties, however, do not appear to be common and we have up to the present been unable to obtain them. So we may safely say that in this region, at least, non- virulent diphtheria-like organisms retain their characteristics under \-arious artificial and natural conditions, and that they may be regarded from a public health standpoint as harmless. Resistance to Heat, Drying, and Chemicals. — The thermal death point with ten minutes' exposure is about t)()° C, with five minutes 70° C. Boiling kills almost instantly. It has about the average resistance of non-spore-bearing bacteria to disinfectants. In the dry state and exposed to diffuse light diphtheria bacilli usually die in from a few hours ' Juurnal uf Med. Re.sc;uch, .June, 1902. 20 30(5 PATHOGENIC MICROORGANISMS to a few days, but they ma\- live for months; when in tlie dark, or pro- tected by a film of mucus or albumin, they may li\-e for even longer periods. ' Thus we found scrapings from a dry bit of membrane to con- tain vigorous and virulent living bacilli for a period of four months after removal from the throat, and if the membrane had not been at that time completely used, living bacilli could probably have been obtained for a much longer period. On slate- and lead-pencils, toys, tumblers, as well as on paper money, they may live for several weeks, while on metal coins they die in tweh'C to thirty-six hours. In culture media, when kept at the blood-heat, they usually die after a few weeks; l}ut under certain conditions, as when sealed in tubes and protected from heat and light, they retain their life and virulence for years. The bacillus is not sensitive to cold, for we found about 10 per cent, of the bacilli to retain their vitality and virulence after exposure for two hours to several hundred degrees below zero. At temperatures just below freezing they may remain alive for a number of weeks. Transmission of Diphtheria. — The possibility of the transmission t)f diphtheria from animals to man cannot be disputed; we ha^-e met with two instances where cats had malignant diphtheria, and many other animals can be infected, but there are no authentic cases of such transmission on record. So-called diphtheritic disease in animals and birds is usually, if not always, due to other microorganisms than the diphtheria bacilli. The virulent bacilli have been found on soiled bedding or clothing of a diphtheria patient, or drinking-cups, candy, shoes, hair, slate- pencils, etc. These sources of infection by which the disease may be indirectly transmitted are, however, the less important ones. The usual source of the bacilli are the discharges of diphtheria patients; the secre- tions of the nose and throat of convalescent cases of diphtheria in which the virulent bacilli persist from the healthy throats of individuals who acquired the bacilli from being in contact with others having \'irulent germs. When we consider the number of healthy carriers and that it is only the severe types of diphtheria that remain isolated during their actual illness, the wonder is not that so many, but that so few, persons contract tlie disease. Susceptibility to and Immunity against Diphtheria. — An individual susceptibility, both generaj and local, to diphtheria, as in all infec- tious diseases, is necessary to contract the disease. Age has long been recognized to be an important factor in di])htheria. Children within the first six months of life are but little susceptible, but exceptionally infants of a few weeks are attacked, the greatest degree of susceptibility being between tiie second and tenth year. After that age susceptibility decreases. Young animals born of mothers imminie to diphtheria possess nearly the same degree of immunity as their mothers. The}' gradually lose this but retain traces up to six to twelve months. The human infant is now known to receive immunity from its mother. Diphtheria Antitoxin. — As the result of animal experiments, it is now known that an artificial immunity against diphtheria can be produced, by the action of diphtheria toxin on the cells causing the development USE OF ANTITOXIN IN TREATMENT AND IMMUNIZATION .307 of substances directly antidotal to the diphtheria toxin. The blood- serum of about 50 per cent, of persons who have recovered from diphtheria has been found to possess to some degree this protective property, which it acquires about a week after the beginning of the disease, and loses again before the end of a year. Those not possessing antitoxin probably have protection from bactericidal substances which have developed because of the infection. Moreover, the blood-serum of more than one-half of all individuals, usually adults, who have never, so far as known, had diphtheria, has a slight amount of antitoxin. Neutralizing Characteristics of Antitoxin. — Diphtheria antitoxin has the power of neutralizing diphtheria toxin, so that ^^'hen a certain amount is injected into an animal before or together with the toxin it overcomes its poisonous action. There is a direct action of the anti- toxin upon its corresponding toxin. The various attempts to separate the toxin and antitoxin from neutral mixtures h&xe been failures, and it is found that neutralization takes place according to the law of multiple proportions, i. e., to save an animal from 1000 fatal doses of diphtheria toxin recjuires little more than a hundred times as much antitoxin as is required for ten fatal doses, the resistance of the animal itself accounting for the differ- ence. The general characteristics of antitoxin are considered in Part I. Use of Antitoxin in Treatment and Immunization. — The antitoxin in the higher grades of globulin solution or serum is identical with that in the lower grades; there is simply more of it in each drop. In treat- ment, however, for the same amount of antitoxin we have to inject less foreign proteids with the higher grades, and, therefore, have some- what less danger of rashes and other deleterious results. The amount of antitoxin required for immunization is 300 to 500 units for an infant, 500 to 1000 for an adult, and proportionately for those between these extremes. The larger doses are advised when the danger of infection is very great. After the observation of the use of antitoxin in the immunization of several thousand cases, we have absolute belief in its power to prevent an outbreak of diphtheria when given in the amounts ad^dsed for at least two weeks, and also of its almost complete harmless- ness in the small doses required. When double the above quantities are given the immunity is prolonged on the average for abour one Week. If it is desired to prolong the immunity the antitoxin injection is repeated every ten days. For treatment, mild cases should be given 2000 units, moderate cases 2000 to 8000 units, and severe cases 10,000 to 20,000 units. One injection suffices, if the proper amount is given. A glance at the charts will make this clear. The large amount of anti- toxin given in bad cases is for the purpose of quickly reaching the cells already attacked by the toxin and not because of the amount of toxin to be neutralized. Intra^'enous injections give most rapid effect, and should be used in all malignant cases. It takes forty-eight to seventy- two hours for the absorption into the blood of the greater part of the antitoxin from the subcutaneous tissues. This in bad cases may be a fatal delay. Antitoxin is only to a very slight extent absorbed when given by the mouth, 308 PATHOGENIC MICROORGANISMS Results of the Antitoxin Treatment of Diphtheria.— The conclu- sions a^^i^'ed at by Biggs and Guerard, after a review of a large amount Fig. 122 1 2 HOURS 24 ■ r> /\ y .^ 3.0 y y y ^y y y y ' y^p ^-^ ^¥^ -^ -4^ ^ -^nO^^ ^t.0^ 7 • ^^..^^ ^ ^ y ^ ^ 1 / y ^,,„'^ 1 1 'Cy^ 1 _ ^ — ■ • H / ^} / ^.^ fl J ^ y — — ;7^ r^ ^ ' _, bO V^^ — .] / .S / • /, ^-9 ^--' \.. ■ ■ — — — in POUNDS , 1 /' " — ■ — ■ ^ ■" Amount of antitoxin in 1 c.c. of serum from persons, at different intervals of time, after a single subcutaneous injection of 10,000 units. of the statistics and opinions published since the beginning of the anti- toxin treatment in 1892, were as follows: ^ "It matters not from what point of view the subject is regarded, if the evidence now at hand is properly weighed, but one conclusion is Fir.. 123 Amou)it of antitoxin in 1 c.c. of serum from persons, at different interv.als of time, after a single inlrnvenous injection of 10 000 units. or can be reached— whether we consider the percentage of mortality from diphtheria and croup in cities as a whole, or in hospitals, or in private practice; or whether we take the absolute mortality for all the DETERMINATION OF THE PRESENCE OF ANTITOXIN 309 cities of Germany wliose population is o^■er 15,000, and all the cities of France whose population is over 20,000; or the absolute mortality for New York City, or for the jiTcat hospitals in France, (jcrmany, and Austria; or whether we consider only the most fatal cases of diph- theria, the laryngeal and operative cases; or whether we study the question with relation to the day of the disease on whicli treatment is commenced, or the age of the patient treated; it matters not how the subject is regarded or how it is turned for the purpose of com])arison with previous results, the conclusion reached is always the same, namely, there has been an average reduction of mortality from the use of antitoxin in the treatment of diphtheria of not less than 50 Fig. 124 / DAVe UNITS 1 2/3 4 5 6 7 14 CO .8- .6 .4 .2 5.0 .8 • 6 .4 .2 4.0 .8 1 — /— _.^ IS 1 — ^^ ^ i U)A- -<^?°' ^,^^^4,0 / ^ / / / / / / / •/^-— -* •"--^ _^ \ "'/ J*" V. ^^ •" 4 J^ '^ ^^ .2 3.0 / / jT^ N ■^ / y^ V ,^\ / ^ -^ ^ / ^^ ■^ .4 .2 2.0 .8 .6 .4 .2 1.0 .8 .6 .4 / / ^^'^ ■*■ L. \ A / ^ ■ yj' •--. ^\ 50 p„ // J^ - '>-i — An per c quan not i coun with exter cities per c of ai large has b De The ■So lactise Park ( lount of antitoxin in 1 c.c. of serum from persons, at difTerent intervals of time (days), after a single subcutaneous injection of 10,000 units. ent., and under the most favorable conditions a reduction to one- ter, or even less, of the previous death rate. This has occurred n one city at one particular time, but in many cities, in differet tries, at different seasons of the year, and always in conjunction the introduction of antitoxin serum and proportionate to the it of its use." The combined statistics of deaths in 19 of the chief of the world show there were in the last four years not over 30 ent. as many deaths as in the four years preceding the introduction ititoxin. Except where immunization has been practised on a scale, no marked reduction in the number of cases of diphtheria een evident. termination of the Presence of Antitoxin in the Living Body.— 'Schick. Reaction} — Recently Schick published a method by which hick (B.) Die, Diphtheritoxin-Hautreaktion des Mensohenals Vorprobeder Prophy- hen Diphtherie heilserum in,iection, Munch med. Woch., 191.3, Ix, 2608-2610. W. H.), Zingher (A.), and Serota (H. M.), Archives of Pediatries, 1914. 310 PATHOGENIC MICROORGANISMS the presence of antitoxin in the blood and tissue can be determined very easily. xA. minute quantity of toxin is injected intracutaneously, and a local reaction follows if there is less than y\j of a unit of antitoxin per c.c. of blood. T\m amount is considered sufficient to i)rotect against diplitheria. The exj)laiiation of the test is that when no antitoxin is present the toxin acts on the cells of the skin; when antit(jxin is present it neutralizes the toxin so that no poisoning occurs. A negative reaction therefore indicates the presence of antitoxin. A standard diphtheria toxin is diluted at first 1: 10 in 0.5 per cent, phenol; this dilution will keep in the ice-box with little deterioration for at least two weeks. For use further dilutions are made in normal saline, of such strength that 0.1 c.c. contains J-jj of the minimum lethal dose for the guinea-pig. This amount is injected intracutaneously on the flexor surface of the arm or forearm. If the injection has been made properly, a definite wheal appears which lasts for several minutes. A positive reaction appears in twenty-four to thirty-six hours, and is characterized by a circumscribed area of redness and slight infiltration which measures from 1 to 2 cm. in diameter. It persists for seven to ten da\'s, and on fading shows superficial scaling and a persistant brownish pigmentation. The test represents a true irritant action of non-neutralized toxin. Pseudoreactions are seen occasionally in young adults, who may have a large amount of antitoxin. These are local sensitization phenomena of a general protein character, and can be distinguished from the true reaction. They appear earlier, are more infiltrated, less sharply circumscribed and disappear in twenty-four to forty-eight hours. On fading they leave a faintly pigmented area which may show superficial scaling. They can ailso be obtained with dilutions of plain broth. By the use of this test a large numljer of indi\'iduals have been shown to be naturally immune. Negative reactions were obtained in 93 per cent, of the newborn, in 57 per cent, during the first year of life, in 37 per cent, between two and five years, and 50 per cent, between five and fifteen years. In adults the negative reactions were as high as 90 per cent. By applying the test, therefore, passive immunization with antitoxin of a large number of individuals can be omitted and the disagreeable symptoms of sensitization avoided. Of 400 cases of scarlet fever, showing a negative Schick reaction and who received no immunizing dose of anitoxin, not one developed chnical diphtheria. Of these cases 25 per cent, were carriers of virulent diphtlieria bacilli. Active Immunization with Toxin-antitoxin Mixtures. — Immunization of horses with mixtures of toxin and antitoxin has been used by us for years, but for human beings it was first recommended by Theobald Smith in 1907, and carried out by v. Behring in 1912. Mixtures of toxin and antitoxin which are neutral or slightly toxic to the guinea- pig are injected in small and repeated doses, subcutaneously or intra- cutaneously. Local signs, like redness, infiltration and tenderness and occasionally constitutional sym]jtoms, such as fever, headache, and malaise, are noted. yVn increase of antitoxin is generalh' found RESULTS FROM USE OF ANTITOXIC GLOBULIN SOLUTION 311 in individuals who have natviral iinniuiiity. TJiis increase is nnted often within six days after one injection of the vaccine. Xon-imniune patients, as determined l)y the Scliici< reaction, react less readily to active inimuni/.ation. We found that oidy 22 \k'\- cent, of snsceptihle indiviihials will produce sufficient antitoxin to surely protect them against diphtheria. Another practical objection is that the immunity in this group is slowly produced so that during the first two weeks after the injection it is practically negligible. Usually this is the period during wliich we desire the greatest immunity, because it is tlien that the danger of infection is greatest in places like tlie measles and scarlet fever wards of a contagious disease liospital. Indi\iduals who are definitely exposed to infection should therefore be passi\ely immunized, even if the toxin- antitoxin mixture has been given. The mixtures of toxin and antitoxin used for acti^•e immunization represent from 50 to 90 per cent, of tlie L+ dose of toxin to each unit of antitoxin. Mixtures containing from SO to 90 per cent, of the L+ dose of toxin will cause paralysis in guinea-pigs, but are still harmless to man. Within these limits they can be injected in doses of 0.25 to 1 c.c. and repeated at the end of seven to ten days. The injections are given subcutaneously, or combined subcutaneously and intracu- taneously in the interscapular region. The production of sufficient antitoxin in susceptible cases after active immunization can be determined clinically by the Schick test. A negati\'e reaction indicates that the jierson has become immune. According to our animal tests appreciable antitoxin will last from one to two years. In fiorses we found it disappeared in some within nine months, while in others it persisted for more than a year. The homologous antitoxin is produced in some cases in sufficient amount to be used in the passive immunization of individuals who are especially susceptible to anaphylactic symptoms like cases of asthma. Danger in Giving Subcutaneous Injections of Antitoxin. — About 1 in 10,000 persons develop, within a few minutes after an injection of serum, alarming sj'mptoms. About 1 in 50,000 of the injected die. About thirty deaths in all have been reported. In 140,000 persons injected by New York City Health Department Inspectors there have been two deaths due to serum. About the same proportion is reported from Boston. The persons suffering severe symptoms have usually been subject to asthma, while tlie fatal cases usually have the patlio- logical changes known as status lymphaticus. X few of these rare cases die almost instantly. As a rule, when death occurs it takes place within a few- minutes after the development of symptoms. Usualh' the respira- tory rather tlian the circulatory centre seems to be affected. Persons wlio have not reacted badly to a first injection do not need to fear a second. So far as known all fatal results have followed the first injection. Results from the Use of Antitoxic Globulin Solution. — The curative effect proved to be identical with that of the wdiole serum. Our tests showed clearly that not only the toxin, but also the poisons ]iroduced in the animal by injections with virulent bacilli are neutralized as com- 312 PA THOGENIC MICROORGANISMS pletely Idv the globulin solution as by the antitoxic serum from which they are separated. The injections of the globulin solution were found to be followed by decidedly less severe rashes than the whole serum, and it was especially noted that there were very few who had any constitu- tional disturbances even when tlie development of the rashes did occur. The following comparative table gives a summary of the constitu- tional and local reactions obtained in the treatment of 50 cases of diph- theria in young children, with a lot of antitoxic serum received from three horses which was found to produce an excessive amount of dis- turbance, and of an equal number of similar cases treated with a solution of the antitoxic globulins derived from a portion of the same lot of serum is as follows : Children treated with the whole serum. 28 per cent. Marked constitutional symptoms accom- panied by a severe and persistent rash Moderate constitutional symptoms accom- panied by a well-developed erythema or urticaria 18 per cent. Very slight constitutional disturbance accompanied by a more or less general urticaria or erythema 20 per cent. No appreciable constitutional disturbance but more or less general urticaria or erythema 4 per cent. No appreciable deleterious after-effects whatever 30 per cent. Children who were treated with the .intitoxic gobulins. per cent. 4 per cent. 5 per cent. .34 per cent. .54 per cent. Days. .A-ntitoxic globiUin cases Whrile serum cases DUR.tTlON OF EASHE.S. 1 2 3 . . . . 5 7 5 . . . . 1 4 10 3 10 8 Totals —23 5—36 The concentration of antitoxin made possible by the elimination of the non-antitoxic substances is not only a convenience but is of a distinct importance, as it tends to encourage large doses. Some pro- ducers supply a product which is so rich in proteid as to be almost semisolid. This is not quite so well absorbed as the less concentrated product. The total solids in the globulin solution should not be more than twice those in the serum. The antitoxic globulin solution tends to become slightly cloudy when kept at moderate or high temperatures and substances such as solutions of carbolic acid and tricresol precipitate it. Development of Agglutins for Diphtheria Bacilli. — By the in- jections of the bodies of diphtheria bacilli into animals agglutinins have been developed in sufficient amount to act in 1 : 5000 dilutions of the serum. The serum produced from diphtheria bacilli does not agglutinate pseudodiphtheria bacilli in high dilutions. The serum of patients convalescent from diphtheria has, as a rule, little agglu- tinating power. This test is not used in diagnosis. The Persistence in the Man's Blood of Injected Antitoxin Produced in the Horse. — Antitoxins and other antibodies produced in an animal PSEUDOMEMBRANOUS EXUDATIVE INFEAMM ATJONS 313 disappear more rapidly when introduced into the blood of another species than into one of the same species. In man an alien serum nnist be used in all except exceptional cases. In oiu- experiments in guinea-i)igs \vc \va\v fo\nid that the homol- ogous antitoxin was retained in apj^rcciable amounts for at least six months, while the heterologous antibodies were noticeable to the same extent for only four weeks (see Human Chart, \). 309). There is a rather rapid loss of horse produced antitoxin during the first few days and then a slow loss becoming more and more gradual until final elimination at the end of ten days to three weeks. The larger the amount of anti- bodies injected the longer will be the time before the elimination of effective amount. Mixed Infection in Diphtheria. — Virulent diphtheria bacilli are not the only bacteria present in human diphtheria. Various cocci and bacilli more particularly streptococci, staphylococci, pneumococci, and influenza bacilli, are also found actively associated with LofHer's bacilli in diphtheria, playing an important part in the disease and lead- ing often to serious complications (sepsis and bronchopneumonia). Investigations indicate that when other pathogenic bacteria are asso- ciated with the diphtheria bacilli they mutually assist one another in their attacks upon the mucous membrane, the streptococcus being particidarly active in this respect, often opening the way for the invasion of the LofHer bacillus into the deeper tissues of supplying needed con- ditions for the development of its toxin. In most fatal cases of broncho- pneumonia following laryngeal diphtheria we find not only abundant pneumococci or streptococci in the inflamed lung areas, but also in the blood and tissues of the organs. As these septic infections due to the pyogenic cocci are in no way influenced by the diphtheria antitoxin, tliey frequently are the cause of the fatal termination. Other bacteria cause putrefactive changes in the exudate, producing alterations in color and offensive odors. Pseudomembranous Exudative Inflammations Due to Bacteria other than the Diphtheria Bacilli. — The diphtheria bacillus, though the most usual, is not the only microorganism that is capable of pro- ducing pseudomembranous inflammations. There are numerous bacteria present almost constantly in the throat secretions, which, under certain conditions, can cause local lesions very similar to those in the less-marked cases of true diphtheria. The streptococcus and pneu- mococcus are the two forms most frequently found in these cases, but there are also others, such as the Vincent's bacillus, which, under suitable conditions, excite this form of inflammation, but without serious constitutional symptoms. The pseudomembranous angina accompanying scarlet fever, and to a less extent other diseases, may not show the presence of diph- theria bacilli, but only the pj^ogenic cocci, especially streptococci, or, more rarely, some varieties of little-known bacilli. The deposit cover- ing the inflamed tissues in these non-specific cases is, it is true, usually but not always, rather an exudate than a true pseudomembrane. 314 PATHOGENIC MICROORGANISMS Relation of Bacteriology to Diagnosis. — We belie\'e that all expe- rienced clinicians will agree that, when left to judge solely Ijy the api^earance and symjjtoms of a ease, there are certain mild exudative inflammations of the throat of similar appearance some of which trans- mit diphtheria while others do not. The doubtful cases that have the diphtheria bacilli in the exudate, are capable of giving true characteristic diphtheria to others, or later develop it characteristically themselves, while those in whose throats no diphtheria bacilli exist can under no condition gi\'e true charac- teristic diphtheria to others or develop it themselves. It is, indeed, true, as a rule, that cases presenting the appearance of ordinary folli- cular tonsillitis in adults are not due to the diphtheria bacillus. On the other hand, in small children mild diphtheria very frequently occurs with the semblance of rather severe ordinary follicular tonsillitis, due to the pyogenic cocci, and in large cities where diphtheria is prevalent all such cases must be watched as being more or less suspicious. Without doubt most observers agree with us in thinking that if in any case exposure to diphtheria is known to have occurred, even a slightly suspicious sore throat should be regarded as probably due to the diph- theria bacilli. If, on the other hand, no cases of diphtheria have been known to exist in the neighborhood, even cases of a more suspicious nature would probably not be regarded as diphtheria. Now that we know about 50 per cent, of children have a fairly constant supply of antitoxin in their blood, we have reason to believe that many of these doubtful cases are simply diphtheria carriers in which the bacilli are taking little or no part in making the lesions. Like any carriers they are dangerous to others. The presence of irregular-shaped patches of adherent grayish or yellowish-gray pseudomembrane on some other portions than the tonsils is, as a rule, an indication of the activity of the diphtheria bacilh. Restricted to the tonsils alone, their presence is less certain. Occasionally, in scarlatinal angina or in severe phlegmonous sore throats, patches of exudate may appear on the uvula or borders of the faucial pillars, and still the case may not be due to the diphtheria bacilli; these are, however, exceptional. Thick, grayish pseudomem- branes which cover large portions of the tonsils, soft palate, and nostrils are almost invariably the lesions produced by diphtheria bacilli. The very great majority of cases of pseudomembranes of exudative laryngitis, in the coast cities at least, whether an exudate is present in the pharynx or not, are due to the diphtheria bacilli. Nearly all membranous affections of the nose are true diphtheria. W'hen the membrane is limited to the nose the symptoms are, as a rule, very slight; but when the nasopharynx is involved tlie symptoms are usually grave. Most cases of pseudomembranes and exudates, entirely confined to portions of the tonsils in adults, are not due to the diphtheria bacilli. Cases presenting the appearances found in scarlet hvev, in which a thin, grayish membrane lines the borders of the uvula and faucial pillars, are rarely diphtheritic. As a rule, pseudomembranous inflam- TECHNIQUE OF THE BACTEmOLOaiC niAGNOFilK 315 Illations coin])licating scarlet fever, syphilis, and other infections dis- eases ari' due to the actixity of the pathoiicnic cocci and other hacteria, inchiced by the intiained conditions of tlie mucous menihranes due to the scarlatinal or other jjoison. The possihility of these persons heing carriers of dii)htheria bacilli must always he ke])t in mind. Paralysis following a pseudomembranous inflamiiiation is an almost positive indication that the case was one of diphtheria, although slight paralysis has followed in a \'ery few cases in which careful cidtures have rexealed no diphtheria bacilli. These, if not true diphtheria, must be considered \'ery exceptional cases. Bacteriologic Diagnosis. — From the abo\e it is apparent that fully developed characteristic eases of diphtheria are readily diag- nosticated, but that many of the less marked, or at an early period undeveloped, cases are difficult to differentiate the one from the other. In these cases cultures are of the utmost value, since they enable us to isolate those in which diphtheria-like bacilli are found, and to give preventive injections of antitoxin to both the sick and those in contact with them, if this has not already been done. As a rule, cultures do not give us as much information as to the grax'ity of the case as the clinical appearances, for before the lapse of the twelve hours required for the laboratory report the extent of the disease is usually possible of determination. The reported absence of bacilli in a culture must be given weight in proportion to the skill with which the culture was made, the suitableness of the media, the location of the disease, and the knowledge and experience of the one who examined it. Diphtheria does not occur without the presence of the diphtheria bacilli; but there have been many cases of diphtheria in which, for one or another reason, no bacilli were found in the cultures by the examiner. In many of these cases later cultures revealed them. The ^e^'erse is also true, the presence of diphtheria bacilli in throats without clear clinical signs of diphtheria in no sense makes it a case of diphtheria. In a convalescent ease the absence of bacilli in any one culture indicates that there are certainly not many bacilli left in the throat, but even repeated cultures cannot absolutely prove their total absence, for in some deep tonsillor crypt a few bacilli may remain in the epithelial cells. The physician must have intelligence to use advantageously laboratory findings. Technique of the Bacteriologic Diagnosis. — Collection of the Blood-serum and its Preparation for Use in Cultures. — A covered glass jar, which has been thor- oughly cleansed with hot water, is taken to the slaughter-house and filled with freshl}' shed blood from a calf or sheep. The blood is received directly in the jar as it spurts from the cut in the throat of the animal. After the edge of the jar has been wiped it is covered with the lid and set aside, where it may stand quietly until the blood has thoroughly clotted. The jar is then placed in an ice-chest. If the jar containing the blood is carried about before the latter has clotted, very imperfect separation of the serum will take place. The blood is allowed to remain twenty-four hours on the ice, and then the serum which sur- rounds the clot is sijjhoned off lay a rubber tube and mixed with one-third its quantity of nutrient beef-broth, to which 1 per cent, glucose has been added. .31fi PATHOGENIC MICROORGANISMS This constitutes the Loffler blood-serum mixture. This is poured into steriHzed tubes. Care should be taken in filling the tubes to avoid the formation of air bubbles, as they leave a permanently uneven surface when tlie serum has been coagulated by lieat. About 3 c.o. are sufficient for each tube if the small size is employed; if not, .5 o.c. are required. Tlie tulies, having been filled are placed slanted at the ]iroi)cr angle and then k(-i)t for two houi's at a temperature just below 95° C. The degree of heat must Ijc carefully watched, as otherwise the temperature may go too high, and if the scrum is actually boiled the culture medium will be spoiled. After sterilization by this process the tubes contain- ing the sterile, solidified blood-serum can be placed in covered tin boxes, or stopped with sterile paraffined corks and kept for months. The serum thus prepared is quite opaque and firm. Swab for Inoculating Culture Tubes. — The swab we prefer to use to inoculate the serum is made as follows: A stiff, thin, iron rod, six inches in length, is roughened at one end by a few blows of a hammer, and about this end a little absorbent cotton is firmly wound. Each swab is then placed in a separate glass tube, and the mouths of the tubes are plugged with cotton. The tubes and rods are then sterilized by dry heat at about 150° C. for one hour, and stored for future use. These iron rods have proved more serviceable for making inocu- lations than platinum-wire needles or wooden sticks, especiall.y in young chil- dren and in laryngeal cases. It is easier to use the cotton swalj in such cases, and it gathers up so much more material for the inoculation that it has seemed more reliable. The wood unless very carefully selected is apt to break and is too thick to use in the nose. For convenience and safet)' in transportation "culture outfits" have been devised, which consist usually of a small wooden box containing a tube of blood- serum, a tube holdmg a swab, and a record Islank. These "culture outfits" may be carried or sent by messenger or express to an}' place desired. Directions for Inoculating Culture Tubes with the Exudate. — The patient is placed in a good light, and, if a child, properly held. The swab is removed from its tube, and, while the tongue is depressed with a spoon, is passed into the pharynx (if possible, without touching the tongue or other parts of the mouth), and is rubbed gently but firmlv against any visible membrane on the tonsils or in the pharynx, and then, without being laid down, the swab is immediately inserted in the blood-serum tube, and the portion which has previously been in contact with the exudate is rubbed a number of times back and forth over the whole surface of the serum. This should be done thoroughly, but it is to be gently done, so as not to break the surface of the serum. The swab should then be placed in its tube, and l)oth tubes, thin cotton plugs having been inserted, are reserved for examination or sent to the laboratory or collecting station (as in New York City). If sent to the health department laboratories for examination the blank forms of report which usually accompnay each "outfit" should be filled out and forwarded with the tubes. Where there is no visible membrane (it may be present in the nose or larynx) the swab should be thoroughly rubbed over the mucous membrane of the pharynx and tonsils, and in the nasal cavities, and a culture made from these. In very young children care should be taken not to use the swab when the throat contains food or vomited matter, as then the bacteriological examination is rendered more difficult. Under no conditions should any attempt be made to collect the material TECHNIQUE OF THE BACTERIOLOOIC DIAGNOSIS 317 shortly after the application of strong disinfectants (especially solutions of corrosive sublimate) to the throat. Cultures from the nostrils are often more successful if the nostrils are first cleansed with a spray of sterile normal salt solution. Examination of Cvltures. — The culture tubes which have been inoculated, as described above, are kept in an incubator at 37° C. for twelve hours, and are then ready for examination. When great haste is required, even five hours will often suffice for a sufficient growth of bacteria for a skilled examiner to decide as to the presence or absence of the bacilli. On inspection it will be seen that the surface of the blood serum is dotted with numerous colonies, which are just visible. No diagnosis can be made from simple inspection; if, however, the serum is found to be liquefied or shows other evidences of contamina- tion the examination will probably be unsatisfactory. In order to make a microscopic preparation a clean platinum needle is inserted in the tube and quite a large number of colonies are swept with it from the surface of the culture medium, a ])art being selected where the most suitable colonies are found. A sufficient amount of the bacteria adherent to the needle is washed oft' in a tiny droplet of water previously placed on the cover-glass and smeared over its surface. The bacteria on the glass are then allowed to dry in the air. The co^•e^-gIass is then passed quickly through the flame of a Bunsen burner or alcohol lamp, three times in the usual way, covered with a few drops of Loffler's solution of alkaline methylene blue, and left without heating for five or ten minutes. It is then rinsed off in clear water, dried, and mounted in balsam. When other methods of staining are desired they are carried out in the proper way (see Methods of Staining). In the great majority of cases one of two pictures will be seen with the -j-j oil-immersion lens — either an enormous number of character- istic Loffier bacilli, with a moderate number of cocci, or a pure cul- ture of cocci, mostly in pairs or short chains. (See Streptococcus.) In a few cases there will be an approximately even mixture of Loffier bacifii and cocci, and in others a great excess of cocci. Besides these, there will be occasionally met preparations in which, with the cocci, there are mingled bacilli more or less resembling the Loffier bacilli. These bacilli, which are usually of the pseudodiphtheria type of bacilli (see Fig. 121), are especially frequent in cultures from the nose. In not more than one case in twenty will there be any serious diffi- culty in making the diagnosis, if the serum in the tube was moist and had been properly inoculated. In such a case another culture must be made or the bacilli plated out and tested in pure culture. Direct Microscopic Examination of the Exudate. — Kn immediate diagnosis without the use of cultures is often possible from a micro- scopic examination of the exudate. This is made by smearing a slide or cover-glass with a little of the exudate from the swab, drying, heating, staining, and examining it microscopically. This examination, howe^'er, is much more difficult, and the results are more uncertain than when the co\'ers are prepared from cultures. The bacilli from the membrane 318 PATHOGENIC MICROORGANISMS are usually less typical in appearance than those found in cultures, and they are mixed fl'ith fibrin, pus, and epithelial cells. They may also be very few in number in the parts reached by the swab, or bacilli may be met with which closely resemble the Loffler bacilli in appearance, but which differ greatly in growth and in other characteristics, and have absolutely no connection with them. When in a smear containing mostly cocci a few of these doubtful bacilli are present, it is impossible either to exclude or to make the diagnosis of diphtheria with certainty. Although in some cases this immediate examination may be of the greatest value, it is not a method suitable for general use, and should always be controlled by cultures. When carried out in the best manner an experienced bacteriologist may obtain markedly accurate results. Higley in New York in a series of consecutive throat cases made the same diagnosis from the direct examination of smears as the Health Department laboratory made from the culture. To get the exudate he used a probe armed with a loop of heavy copper wire which has been so flattened as to act as a blunt curette. He makes thus thin smears from the exudate. After drying and fixing by heat the smears are stained for five seconds in a solution made by adding five drops of Kiihne's carbolic methylene blue to 7 c.c. of tap-water. After washing and drying stain for one minute in a solution of 10 drops of carbol-fuchsin in 7 c.c. of water. The dilute solution should be freshly prepared. The diphtheria bacilli will appear as dark red or violet rods, and their contour, mode of division, and arrangement are manifest. minimal Inoculation as a Test of Virulence. — If the determination of the virulence of the bacilli found is of importance, animal inocu- lations must be made. Experiments on animals form the only method of determining with certainty- the virulence of the diphtheria bacillus. For this purpose, alkaline broth cultures of forty-eight hours' growth should be used for the subcutaneous inoculation of guinea-pigs. The amount injected should not be more than one-fifth per cent, of the body-weight of the animal inoculated, unless controls with antitoxin are made. In the large majority of cases, when the bacilli are virulent, this amount causes death within seventy-two hours. If a good growth is not obtained in nutrient bouillon, ascitic broth should be used. At the autopsy the characteristic lesions already described are found. Bacilli which in cultures and in animal experiments have shown them- selves to be characteristic may be regarded as true diphtheria bacilli, and as capable of producing diphtheria in man under favorable conditions. For an absolute test of specific virulence antitoxin must be used. A guinea-j)ig is injected with antitoxin, and then this and a control animal, with 2 c.c. of a brotli culture of the bacilli to be tested; if the guinea-pig which received the antitoxin lives, while the control dies, it was surely a diphtheria bacillus which killed by means of diphtheria toxin — or, in other words, not simply a virulent bacillus, but a virulent diphtheria bacillus. When the bacilli to be tested grow poorly in a simple VINCENT'S ANGINA 319 nutrient k)iiill()n they slunild be grown in Ijouillon to wliich one-third its quantity of ascitie fluid has been added. Quite a number of Ijaeilli ha\-e been met with which killed 250 gram, guinea-pigs in doses of 2 to 15 C.C., and yet were unaffected by antitoxin. These liacilli, though slightly virulent to guinea-pigs, produce no diphtheria toxin, anrl so cannot, to the best of our belief, produce diphtheria in man (see p. 302). Fig, 125 Vincent's bacillus with accompanj'ing spirochetes. Vincent's Angina. — The local symptoms are similar to a slight case of diphtheria. Exudate or pseudomembrane forms on the tonsils and tends to become necrotic leaving a superficial ulcer, which is slow in healing. The general disturbance, outside a little fever, is usually slight. The disease runs its course in from one to two weeks. It has been frequently noticed that the disease begins with an eruption of vesicles as in aphthous stomatitis. Paralysis never follows from this infection. The bacilli found by Vincent in the lesions are G^u to 12/i long by O.b/u to O.S/x broad. Their ends are tapering. They are fre- quently bent like the letter S and resemble spirilla^. The bacilli stain with methyl blue irregularly so that light and dark bands alternate (see Fig. 125). Stained by the method of Romanowsky there appear sharply defined chromatin bodies in the blue .stained protoplasm. The bacilli are not motile. These spindle-shaped bacilli have not been grown in pure culture, indeed there is doubt as to their nature. When direct smears are made from the exudate tiny spirochetes are usually found mixed with the bacilli. Certain necrotic conditions of the mucous membrane of the cheek and about the teeth are accompanied by microorganisms very similar to those described by Vincent. CHAPTER XXI. THE BACILLUS AND THE BACTERIOLOGY OF TETANUS. Tetanus is a disease which is characterized by a gradual onset of general spasm of the voluntary muscles, commencing in both man and the horse most often in those of the jaw and neck, and extending in severe cases to the muscles of the body. The disease is usually associted with a wound received from four to fourteen days previously. It has long afflicted man. The writings of Hippocrates clearly describe the symptoms. In 1884 Nicolaier, under Fliigge's direction, produced tetanus in mice and rabbits by the subcutaneous inoculation of particles of gar- den earth. The Italians, Carle and Rattone, had just before demon- strated that the pus of an infected wound from a person attacked with tetanus could produce the same disease in rabbits. Finally, Kitasato, in 1889, obtained the bacillus of tetanus in pure culture and described his method of obtaining it and its biological characters. Occurrence in Soil, etc. — The tetanus bacillus occurs widely through- out the world as a common inhabitant of the soil, especially in places where manure has been thrown, being abundant in many localities, not only in the superficial layers, but also at the depth of several feet. It has been found in many different substances and places — in hay-dust, in horse and cow manure (its normal habitat is the intestine of the herbivora), in the mortar of old masonry, in the dust from horses' hair, and in the dust in rooms of houses, barracks, and hospitals. The tetanus bacilli are more numerous in certain localities than in others — for example, some parts of Long Island and New Jersey have become notorious for the number of cases of tetanus caused by small, woiuids — and they are fairly common in New York City. As a rule they are more abundant in regions where the temperature is high. In some islands and countries in the tropics cases of puerperal tetanus and tetanus in the newborn are very frequent. Tetanus bacilli are found in the intestines of about 15 per cent, of horses and calves living in the vicinity of New York City. They are also present to a somewhat less extent in the intestines of other animals and of man. Morphology. — From young gelatin cultures the bacilli appear as motile, slender rods, with rounded ends, O.S^u to 0.5/x in diameter by 2id to 4/.1 in length, usually occurring singly, but, especially in old cultures, often growing in long threads. They form round or nearly round spores, thicker than the cell (from l^u to 1.5m in diameter), occupy- ing one of its extremities and giving to the rods the appearance of small pins (Fig. 12(1). RESISTANCE OF SPORES TO DELETERIOUS INFLUENCES 321 Staining. — It is stained with the ordinary anihne dyes, and is not tiecolorized by Gram's method. The spores are readily stained and may be demonstrated by double-staininj;- with Ziehl's metliod. The flageUa are fairly easily stained on freshly developed bacilli taken from cultures which have been at short intervals several times transplanted. Biology. — An anaerobic, liquefying, moderately motile bacillus. It has abundant peritrichic flagella. Forms spores, and in the spore stage it is not motile. It grows slowly at temperatures from 20° to 24° C, and best at 38° C, when, within twenty-four to thirty hours it forms spores. At temperatures of 20° to 24° C. spores form in from six to ten days. It will not in pure culture grow in the presence of oxygen, but grows well in an atmosphere of hydrogen gas. With certain other bacteria the tetanus bacillus grows luxuriantly in the presence of oxygen. Tetanus bacilli with spores in Jdis- tended ends. X 1100 diameters. Growth in Media. — The bacillus of tetanus grows in ordinary nutrient gela- tin and agar of a sliglitly all'Caline reaction. The addition to the media of 1.5 per cent, of glucose causes the development to be more rapid and abundant. It also Fig. 126 grows abundantly in alkaline bouillon in an atmosphere of hydrogen. On gelatin plates the colonies develop slowly; they resemble somewhat the colonies of the Bacillus sub- tilis, and have a dense, opaque centre sur- rounded by fine, diverging rays. Lique- faction takes place more slowl_v, however, than with BaciUus suhlilis, and the resem- l)lance to these colonies is soon lost. The colonies on agar are ciuite character- istic. To the naked eye they present the appearance of Ught, fleeoj^ clouds; under the microscope, a tangle of fine threads. The stab cultures in gelatin exhibit the appearance of a cloudy, linear mass, with l^rolongations radiating into the gelatin from all sides (arborescent growth) . Liquefaction takes place slowly, generally with the production of gas. In stab cultures in agar a growth occurs not unlike in structure that of a miniature pine tree. Alkaline bouillon is rendered somewhat turbid by the growth of the tetanus bacillus. In all cases a production of gas results, accompanied Ijy a char- acteristic and very disagreeable odor. It develops in milk witiiout causing coagulation. Resistance of Spores to Deleterious Influences. — The spores of the tetanus bacillus are very resistant to outside influences; in a desic- cated condition they may retain their vitality for several years, and are not destroyed in two and a half months when present in putrefy- ing material. They withstand an exposure of one hour to 80° C, but are usually killed by an exposure of ten minutes at 10.5° C. to live steam. They resist the action of 5 per cent, carbolic acid for ten hours. A 5 per cent, solution of carbolic acid, however, to which 0.5 per cent, of hydrochloric acid has been added, destroys them in two hours. They are killed when acted upon for three hours by bichloride of 21 322 PATHOGENIC MICROORGANISMS mercury (1 : 1000), and in thirty minutes when 0.5 per cent. HC] is added to the solution. Silver nitrate solutions destroy the spores of average resistance in one minute in 1 per cent, solution and in about five minutes in 1 : 1000 solution. With regard to the persistence of tetanus spores upon objects where they have found a resting place, Henrijean reports that by means of a splinter of wood which had once caused tetanus he was able after ele\'en years again to cause the disease by inoculating an animal with the infective material. Isolation of Pure Cultures. — The growth of the tetanus bacillus in the ani- mal body is comparatively scantj^, and is usually associated with that of other bacteria; hence, the organism is difficult to obtain in pure culture. The method of procedure which is most successful, consists in inoculating freshly boiled slightly alkaline nutrient agar or glucose bouillon with the tetanus-bear- ing material (pus or tissue from the inoculation wound), keeping the culture under anaerobic conditions for twenty-four to forty-eight hours at a temperature of 37° C, and, after the tetanus spores have formed, heating it for one-half an hour at S0° C, to destroy the associated bacteria. The spores of the tetanus bacillus are able to survive this exposure, so that when anaerobic cultures are then made in the usual way the tetanus colonies develop. When the tetanus bacilli are the onlv spore-bearing bacteria present, pure cultures are readily obtained; when other spore-bearing anaerobic bacteria are present, the isola- tion of a pure culture may be a matter of difficulty, but even then the pres- ence of tetanus toxin in the culture fluid will indicate the presence of tetanus baciUi. Pathogenesis. — In mice, guinea-pigs, rabbits, horses, cattle, goats and a number of other animals inoculations of pure cultures of the tetanus bacillus cause tetanus after an incubation of from one to four days. In the smaller animals tetanus usually develops first in the muscles nearest the point of inoculation. A mere trace of an old culture — only as much as remains clinging to a platinum needle — is often sufficient to kill very susceptible animals like mice and guinea-pigs. Other animals require a larger amount. Rats and birds are but little susceptible, and fowls scarcely at all. These never develop tetanus from natural infec- tion. It is a remarkable fact that an amount of toxin sufficient to kill a hen would suffice to kill 500 horses. It is estimated that if 1 gram of horse requires 1 part of toxin to kill, then 1 gram of guinea-pig requires (J parts, 1 of mouse 12, of goat 24, of dog 500, of rabbit 1500, of cat bOOO, of hen .360,000. Horses frequently develop tetanus after injuries or operations. Cultures from different cases vary in their toxicity. On the inoculation of less than a fatal dose in test animals a local tetanus may be produced, which lasts for days and weeks and then ends in recovery. On killing the animal there is found at autopsy, just at the jjoint of inoculation, a hemorrhagic spot, and no changes other than the.se here or in the internal organs. A few tetanus bacilli may be detected locally with great difficulty, often none at all; possibly a few may be b)und in the region of the neighboring lymphatic glands and even in the blood. hVom this scanty occurrence of bacilli the conclusion has been reached that the bacilli of tetanus, when inoculated in pure culture, do not multiply to any great extent in the living body, but TOXINS OF THE TETANUS BACILLUS 323 only produce lesions through the absorption of the poison which they develop at the point of infection. It has been found that pure cultures of tetanus, after the germs have spondated and the toxins been de- stroyed by heat, can lie injected into animals without producing tetanus. But if pathogenic streptococci or staphylococci or even non-pathogenic organisms are injected simultaneously with the spores, or if there is an etl'usion of blood at the point of injection, or if there was a previous or later bruising of the tissues, the animals surely die of tetanus. Natural Infection. — Here the infection may be considered as prob- ably produced by the bacilli in their spore state, and the conditions favoring infection are almost always present. A wound of some kind has occurred, penetrating at least through the skin, though perhaps of a most tri\ial character, such as might be caused by a dirty splinter of wood, and the bacilli or their spores are thus introduced from the soil in which they are so widely distributed. If in any given case, the tissues being healthy, the ordinary saprophytic germs are killed by proper disinfection at once, a mixed infection does not take place, and tetanus will not develop. If, however, other bacteria, especially pathogenic or putrefactive types accompany the tetanus bacilli, or if the tissues infected be bruised or lacerated, the spores may develop and produce the disease. Gelatin and catgut are occasionally found to contain tetanus spores. Tetanus in Man. — Man and almost all domestic animals are sub- ject to tetanus. It is a comparatively rare disease in the United States, during times of peace, except after the Fourth of July celebration, when a considerable number of cases develop. Injuries causing crushing of tissues with accompanying contamination are the most likely to be followed by tetanus. Until immunization became the practice more than one hundred persons yearly developed tetanus after blank cart- ridge wounds. On examination of an infected individual very little local evidence of the disease can be discovered. Generally at the point of infection, if there is an external wound, some pus is to be seen, in which, along with numerous other bacteria, tetanus bacilli or their spores may be found. Although rather deep wounds are usually the seat of infection, at times such superficial wounds as an acne pustule or a vaccination may give the occasion for infection. In rare cases tetanus has developed from the infection of necrotic mucous membranes as in diphtheria. Not only undoubted traumatic tetanus, but also all the other forms of tetanus, are now conceded to be produced by the tetanus bacillus — puerperal tetanus, tetanus neonatorum, and idio- pathic tetanus. In tetanus neonatorum infection is introduced through the navel, in puerperal tetanus through the inner surface of the uterus. It should be ))orne in mind that when there is no external and visible wound there may be an internal one. The lesions in the nervous system are still obscure. Congestion, cellular exudate into the peri\'ascular spaces, and chromatolysis of the ganglion cells are common. Toxins of the Tetanus Bacillus. — It is evident from the localization of the tetanus bacilli almost wholly at the point of inoculation and 324 PATHOGENIC MICROORGANISMS their moderate multiplication at this point that they exert their action through the production of powerful toxins. These toxins are named, according to their action, the tetanospasmin and the tetanolysin. The first only is of importance. One ten-thousandth of a cubic centimeter of the filtrate of an eight-day bouillon culture of a fully virulent bacillus is sufficient to kill a mouse. The purified and dried tetanus toxin pre- pared by Brieger and Cohn was surely fatal to a 15-gram mouse in a dose of 0.000005 gram. The toxin is precipitated by saturating the broth with ammonium sulphate and having been collected on the filter is compressed to eliminate the fluid clinging to it. The appalling strength of tetanus toxin may readily be appreciated when it is stated that it is twenty times as poisonous as dried cobra venom. The quantity of the toxin produced in nutrient media varies accord- ing to the age of the culture, the composition of the culture fluid, reaction, completeness of the exclusion of oxygen, etc. For some reason more toxin develops in broth inoculated with masses of tetanus spores in nutrient agar than with bacilli. The variation in strength is partly due to the extreme sensitiveness of the toxin, which deteriorates on keeping at blood-heat or on exposure to light. It is sensibly aff'ected by most chemical reagents and is largely destroyed by heating to 55° to 60° C. for a few minutes. It retains its strength best when protected from heat, light, oxygen, and moisture. The tetanus cultures retain their ability to produce toxins unaltered when kept under suitable conditions; but when subjected to deleterious influences they maj' entirely lose it. Production of Toxin for Immunization of Horses. — The usual medium for the development of the toxin is a slightly alkaline veal bouillon containing 1 per cent, of peptone and 0.5 per cent. salt. The younger the calves from which the bouillon is prepared the better the results. The cultures are kept at .37° for ten days and then filtered. For other methods of preparing toxin and for further characteristics of toxin, see Part I. See also later in this chapter. Action of Tetanus Toxin in the Body. — After the absorption of the poison there is a lapse of time before anj^ effects are noticed. With an enormous amount, such as 90,000 fatal doses, this is about nine hours; with 30,000, ten; with 3000, twelve; with 300, twenty hours; with ten fatal doses, thirty-six to forty-eight hours; with two fatal doses, two to three days; with one fatal dose, four to seven days. Less than a fatal dose will produce local symptoms. The parts first to be affected with tetanus are, in about one-third of the cases in man, and usually in animals, the muscles lying in the vicinity of the inoculation — for instance, the hind foot of a mouse inoculated on that leg is first affected, then the tail, the other foot, the back and chest muscles on both sides, and the forelegs, until finally there is a general tetanus of the entire body. In mild cases, or when a dose too small to be fatal has been received, the tetanic spasm may remain confined to the muscles adjacent to the point of inoculation or infection. The symptoms following a TECHNIQUE OF TESTING ANTITOXIN SERUM 025 fatal dose of toxin vary greatly with the method of uijeetion. Intra- peritoneal injection is followed by symptoms which can hardly l)e distinguished from those due to many other jwisons. Injection into the hraiu is followed by restlessness and ei)ileptiforni con\'ulsions. In man the first symptoms are usually those of a contraction of the muscles of the lower jaw and then those of the neck. Presence of Tetanus Toxin in the Blood. — The blood during the first four days of the disease usually contains toxin. After that time antitoxin usually develops and soon makes the blood antitoxic. In St. Louis some years ago the serum of a horse dying of tetanus was given by acci- dent in doses of 5 to 10 c.c. to a number of children, with the develop- ment of fatal tetanus. In this connection Bolton and Fisch showed by a series of experiments that much toxin might accumulate in the serum before symptoms became marked. Tetanus Antitoxin. — Behring and Kitasato were the first to show the protective and curative effects of the blood-serum of immunized animals. It was found that animals could be protected from tetanus infection by the previous or simultaneous injection of tetanus anti- toxin, provided that such antitoxic serum was obtained from a thor- oughly immunized animal. From this it was assumed that the same result could be produced in natural tetanus in man. Unfortunately, however, the conditions in the natural disease are very much less favorable, inasmuch as treatment is usually commenced not shortly after the infection has taken place, but some hours after the appearance of tetanic symptoms, when the poison has already attacked the cells of the central nervous system. The Production of Tetanus Antitoxin for Therapeutic Purposes. — The tetanus antitoxin is developed in the same manner as the diphtheria antitoxin — by inoculating the tetanus toxin in increasing doses into horses. 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. This overneutralized toxin stimulates the production of antitoxin. Recently we have preferred to inject the horses with 5000 units of tetanus antitoxin and then give increasing doses of straight toxin. After some months of this treatment the blood of the horse contains the antitoxin in sufficient amount for therapeutic use. Some horses have produced as high as 600 units per c.c. Antitoxin Unit and Technique of Testing Antitoxin Serum. — Tetanus antitoxin is tested exactly in the same manner as diphtheria antitoxin, except that the unit is different. In April, 1907, the pro- ducers of serum in the United States agreed to a unit of antitoxin which is approximately ten times the size of the unit of diphtheria anti- toxin. A unit is defined as the amount of antitoxin required to just neutralize 1000 minimal 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 toxin. 32(J PATHOGENIC MICROORGANISMS The amount of antitoxic serum wliieli neutralizes an amount of test toxin which would destroy 40,000,000 gi'ams of mouse contains 1 unit of antitoxin liy the Ciernian standard. In the French method the amount of antitoxin wliich is reciuirecl to protect a mouse fi'om a dose of toxin sufficient to kill in four daj's is iletermined, and the strength of the antitoxin is stated liy deter- mining the amount of serum required to jirotect 1 gram of animal. If 0.001 c.c. l^rotected a 10-gram mouse the strength of that serum would be 1 : 10,000. The toxin used for testing is preserved ])y jjrecijMtating it with saturated ammonium sulphate and drying and preserving the precipitate in sealed tuljes. As recjuired, it is dissolved in 10 per cent, salt solution as above stated. For small testing stations the best way is to obtain some freshly standardized anti- toxin and compare serums with this. Persistence of Antitoxin in the Blood. — Ransom has clearly shown that the tetanus antitoxin, whether directly injected or whether pro- duced in the body, is eliminated equally rapidly from the blood of an animal, provided that the serum was from an animal of the same species. If from a different species it is much more quickly eliminated. Tlie same author found some interesting facts in testing the anti- toxic values of the serum of an immunized mare, of its foal, and of the milk. The foal's serum was one-third the strength of the mare's and one hundred and fifty times that of the mare's milk. In two months the mare's serum lost two-thirds in antitoxic strength, the foal's five-sixths, and the milk one-half. Theories as to the Methods by which the Toxin Produces its Effects. — Gamprecht and Stintzing concluded from their experiments that the toxin from the wound passed to the central nervous system partly directly by the peri- and endoneural lymph spaces of the nerves which directly connected with subdural space and partly indirectly from the blood. The local tetanus they considered as due to the contact of the poison with the motor end plates. The experiments of Meyer and Ransom and of Marie and Morax have proved to them that the poison is transported to the central nervous system by the way of the motor nerves — and by no other channel. These authors thought that they had shown that the essential element for the absorption and transpor- tation of the toxin is not the lymph channels, but the axis-cylinder, tlie intramuscular endings of which the toxin penetrates. The poison is taken up quite rapidly. Marie and Morax were able to demonstrate the poison in the corresponding nerve trunk (sciatic) one and a half hours after the injection. Absorption, however, and conduction are dependent to a large extent on the nerves being intact. A nerve cut across takes very much longer to take up the poison (about twenty- four hours), and a degenerated nerve takes up no poison whatever. In other words, we see that section of the nerve prevents the absorption of the poison by way of the nerve channels. Similarly section of the spinal cord prevents the poistju from ascending to the brain. The poison which passed through the general lymph channels to the blood was partly returned to the tissue fluids throughout the body and taken up by nerve endings and thus produced general tetanus. According to Me\'er and ivansom, the reason wh^■ the sensory nerves UNION OF TOXIN WITH GRAY MATTER. OF THE BRAIN 327 do not play any role in the conduction of the poison lies in the pres- ence of the spinal ganglion, which places a bar to the advance of the poison. Injections of toxin into the posterior root leads to a tetanus dolorosns, which is characterized hy strictly localized scnsiti\'cness to pain. Ascending centripetally along the motor paths, the poison reaches the motor spinal ganglia on the side of inoculation; then it affects the ganglia of the opposite side, making them hypersensitive. The visi- ble result of this 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 af- fected portion is irritated. In the further course of the poisoning the toxin as it ascends continues to affect more and more motor centres, and also the neighboring sensory apparatus. This leads to spasm of all the striated muscles and general reflex tetanus. Zupink showed that local tetanus did not develop when toxin was injected where it did not come in contact with muscle. He believed, therefore, that the muscle spasm was due to direct action of the toxin on the muscle fibers. Field in our laboratory has shown that not only tetanus toxin, but diphtheria toxin and inert colloids can be demonstrated in the sciatic nerves after they have been injected subcutaneously or intramuscularly, and after varying periods may be found in the spinal cord. He believes that the toxins are absorbed by way of the lymphatics of the nerves, and not by waj' of the axis-cylinder. A later experiment of Cernovodeanu and Henni tends to confirm this contention. They ligated all the muscles and bloodvessels in a guinea-pig's leg, leaving intact only the sciatic nerve, skin and bone, and then injected a large amount of tetanus toxin below the point of ligation. The animals in which this was done never developed tetanus. In this case there was only a very slight flow of lymph into the ligated area, and so there could be only a slight flow up the nerve. If the toxin gets into the blood the only path of absorption to the central nervous system is stfll by way of the motor-nerve tracts. There seems to be no other direct path, as, for example, by means of the bloodvessels supplying the central nervous system. Even after intro- ducing the poison into the subarachnoid space, owing to the passage of the poison into the blood, there is a general poisoning and not a cerebral tetanus. This at least is the case if care has been taken dur- ing the operation to avoid injuring the brain mechanically. A very much smaller amount of toxin is required to produce fatal tetanus if it is injected into a nerve than if it is injected into the blood. The Union of Toxin with the Gray Matter of the Brain and Spinal Cord. — This union is a loose one and the toxin can be partially freed from its union by action of proteolytic ferments. A number of different elements of the cell substances seem to have this power of binding the toxins. Heating to 65° C. for ten minutes destroys the capacity to fix toxin. These brain substances which unite with toxin are certainly not of the nature of antitoxin and the brain cells if they produce anti- :]2S PATHOGENIC MICROORGANISMS toxin at all certainly share the power with other cells that have no power to hind toxin. Marie, in a recent commiuiication , notes that adrenalin neutralizes tetanus toxin and that lecithin compounds are undoubtedly concerned in the mechanism of tetanus toxin on nerve cells. Rapidity of Absorption and Loss of Tetanus Antitoxin from Tissues. — The absorption of antitoxin administered subcutaneously takes place rather slowly. In his animal experiments Knorr found the maximum quantity in the blood only after twenty-four to forty-eight hours. From that time on the amount again steadily decreased, so that by the sixth day only one-third the optimum quantity was present. By the twelfth day only one-fiftieth and at the end of three weeks no antitoxin what- ever could be demonstrated. We injected one of our laboratory assis- tants with 10,000 units of tetanus antitoxin subcutaneously. The blood antitoxic strength was found to be as follows: 18 hours, 0.6 units; 24 hours, 0.8; 48 hours, 1; 72 hours, 1; 148 hours, 0.7. The two important facts to be noted are the slow absorption of antitoxin from the subcutaneous tissues and its long retention in the blood. The two charts showing the absorption and disappearance of diphtheria anti- toxin apply equally to tetanus antitoxin (see pages 308 and 309). When injected intravenously the antitoxin very quickly passes into the lymph. Ransom, in 1901, was able to demonstrate it in the thoracic duct of a dog fifteen minutes after intravenous injection. Only after very massive intravenous doses and a considerable interval of time are small traces found in the cerebrospinal fluid. This is the reason that passively and actively immunized animals become tetanic if the poison is injected directly into the central nervous system or into a peripheral nerve. Antitoxin injected subdurally passes almost entirely over into the blood within twenty-four hours. So long as the toxin circulates in the blood it is neutralized by anti- toxin in about the same proportion as in test-tube experiments. By means of intravenous injections of antitoxin Ransom was able to render the lymph free from toxin in a very few minutes. According to Marie and Morax, toxin injected into the muscles is already demon- strable in the nerve tissue at the end of one and a half hours — i. e., it has already entered the channel, where it is reached with difficulty by the antitoxin. Donitz injected various rabbits intravenously, each with 1 c.c. of a toxin solution containing twelve fatal doses. There- upon he determined the dose of antitoxin which, when intravenously given, would neutralize this poison after various intervals of time. The antitoxin was of such a strength that in test-tube experiments 1 c.c. of a 1 : 2000 solution just neutralized the amount of toxin em- ployed. He found that at the end of two minutes double the dose required in vitro would still neutralize the poison; at the end of four minutes about four times the dose was required, and at the end of eight minutes ten times. When one hour had been allowed to elapse forty times the original dose just sufficed to protect the animal from death, but not from sickness. From the foregoing it is not difficult to formulate the conditions INTRASPINAL ADMINISTRAriON OF TETANUS ANTITOXIN 320 under which an antitoxin introduced into the organism can exert its neutrahziug power on the toxin. We see that the poison deposited at any given phice takes either of two paths to tlie central nervons system, one a direct path by way of tlie local perii)hcral nerves and the other an indirect path tlirougli the lymph channels and blood to the end plates of all other motor nerves. Only that portion can be surely neutralized which (a) still lies unabsorbed at the site of inocula- tion, or (6) whicli, though it has passed into the blood and Ij'mph, has not yet combined with the motor-nerve cells. A curative effect can therefore result from antitoxin introduced intraspinally or intravenously- only so long as a fatal dose of poison has not been taken up by the nerves, nerve cells, and possibly by the nerve lymphatics or fibers. The ana- tomical relations of tlie spinal cord and brain are such that fluids injected subdurally gain access to the surface of the brain and the spinal roots. This has led to experiments to discover whether the cells endangered by the tetanus toxin would be better protected by subdural or intravenous injections. Experiments on the Curative Value of the Intraspinal Administra- tion of Tetanus Antitoxin. — While tetanus antitoxin has proved most efficacious in the prevention of tetanus, its employment as a curative agent has been much less successful. This failure to produce more uniformly good results is chiefly due to its too late administration. Insufficient dosage and the use of the subcutaneous method have also been important factors. AVhile the subcutaneous use of antitoxin, which at first was the usual method employed, may prove of value in large doses given within the first few hours after the onset of symptoms, nevertheless it frequently fails because of slow absorption and the time required to reach the tissues of the central nervous sj-stem. In the light of our present knowledge, this method can be justified only by an inability of the physician, for one reason or another, to give an intraspinal or an intravenous injection. The latter method is being more and more recommended, and when employed before the disease has made too much headway has given much better results than the subcutaneous. From time to time since 1903, single cases or small series of cases have been reported in which the antitoxin has been given intraspinally. Thus Neugebauer' in 1905 reported forty-three cases from Continental and American sources with a mortality of twenty-two, or 51 per cent., and also three patients treated by him of whom two recovered. Many of these patients received also intravenous or subcutaneous injections or both. The results seemed to some observers to be better than M'hen other methods were used without intraspinal dosage, but on the whole, this method of treatment has not made much headway. Experimental proof of the greater value of the intraspinal method has not until very recently been at all convincing. Permin of Copenhagen, working on rabbits and dogs, showed that local tetanus could be prevented by the 1 Wiener klin. Woeh., 1905, xviii, 450. 3.30 PATHOGENIC MICROORGANISMS Himultaneous injection of tetanus toxin intramuscularly and a dose of antitoxin given in the spine, whereas with the same dose of antitoxin given intravenously, local tetanus occurred. Four hours after the giving of the toxin neither method of preventing the occurrence of local tetanus was efficacious, and when nine hours were allowed to elapse between the giving of toxin and antitoxin, the animals could not be saved. The following series of experiments was undertaken with the object of determining to what extent tetanus antitoxin given in the spine has greater curative power, when the disease is actually established, than when given in the circulation. The comparative ineffectiveness of subcutaneous injections of tetanus antitoxin in developed cases is inci- dentally brought out. The use of guinea-pigs for this purpose has not been recorded so far as we know. They possess, however, a marked susceptibility to tetanus together with comparative freedom from intercurrent diseases. Administration and Result of Antitoxin in Experiment IV. Weight. Condition Amount No. Gm. of leg. Method. in units. Result. 116 290 Fairly stiff Control D 3 days 42 310 Fairly stiff Control D 3 days 296 2.50 Slightly stiff H. 100 D 8 days 227 27.5 Fairly stifT H. 100 D 4 days .399 300 Fairly stiff H. 100 D 5 days 216 255 Slightly stiff N. 200 D 4 days 287 255 Fairly stiff N. 200 D 3 days 289 280 Fairly stiff N. 200 D 3 days 306 285 Slightly stiff N. 200 D 3 days 59 25.5 Stiff Sp. 10 Discharged April 23; normal .304 275 Fairly stiff Sp. 10 Discharged April 23 drags leg .321 320 Fairly stiff Sp. 10 Discharged April 23 drags log 102 300 Stiff 10 32.5 Stiff 272 350 Stiff 263 285 Stiff 123 325 Stiff 294 350 Stiff Six Given Antitoxin Twenty-two and a Half to Twenty-three Hours after Inoculation. H. 100 D 5 days H. 200 D 4 days H. 200 D 4 days Sp. 50 Discharged April 23; drags leg Sp. 50 D 5 days Sp. 50 Discharged April 23: drags leg This would seem absolutely conclusive of the superiority of the intra- spinal method of giving antitoxin over the intravenous method. It will be noted that only those animals receiving the antitoxin in the spine were able to survive. This result is the more striking since the amount of antitoxin given them was only a fractional part of that given in the circulation. An attempt was made to give the antitoxin to four guinea-pigs intraneurally. lender an anesthetic the sciatic nerve of the RESULTS OF USE OF ANTITOXIN FOR IMMUNIZATION 331 affected limb was cut down on and freed from the surrounding tissues, as much antitoxin as could be introduced into the nerve sheatli injected, and the remainder intranniscuUirly dircctlx' along the course of the nerve. Owing to the small caliber of the nerve, most of the antitoxin passed to the deep intramuscular tissues. The animals lived no longer than those receiving subcutaneous injections. In all of these experiments no guinea-pigs were discharged from observation until there was absolutely satisfactory evidence that the disease had long ceased to make any progress. While a number of the animals, perhaps most, on being discharged exhibited more or less stiffness in the inoculated leg lasting for weeks or even months, they were nevertheless in the best of health in other respects. This condi- tion appears to be identical with that seen in human tetanus, in which the stiffness in different groups of muscles is often very prolonged even when the patients are perfectly convalescent. Since beginning this work, we have obtained records of four consecu- tive clinical cases of tetanus in which an intraspinal injection of anti- toxin was given and all the patients recovered. Two of these occurred in private practice and two in hospitals. In all of them, in addition to the antitoxin used intraspinally, very much larger amounts were also given by other methods. On experimental and clinical grounds the following recommendation for the treatment of tetanus with antitoxin would seem to be amplj^ justified : In every case strongly suspected of being tetanus, from three to five thousand units of tetanus antitoxin should be given at the first possible moment intraspinally, slowly by gravity, and always, if pos- sible, under an anesthetic. In order to insure its thorough dissemina- tion throughout the spinal meninges the antitoxin should be diluted if necessary, to a volume of from 3 to 10 c.c. or more, according to the patient's age. When fluid is drawn off previously to the giving of the antitoxin, an amount of the latter somewhat less than that of the fluid withdrawn should be given. A number of cases of "dry tap " have been observed in the disease by those so expert in spinal puncture as to leave no room for doubt that the canal was properly entered. In such cases only a small amount of tetanus antitoxin should be injected (from 3 to 5 c.c). In brief, tetanus antitoxin should be used in precisely the same way as antimeningitis serum. Results of the Use of Antitoxin for Immunization. — The striking results which have been obtained, both in human and in veterinary practice, with the prophylactic injection of tetanus antitoxin, would seem to warrant the treating of patients with immunizing doses of serum — at least, in neighborhoods where tetanus is not uncommon — when the lacerated and dirty condition of their wounds may indicate the possibility of a tetanus infection. Splended results have followed this practice in many places. It is the custom at many dispensaries in New York City and elsewhere to immunize all Fourth of July wounds by injecting 1000 units. None of these have ever developed tetanus. Even the few cases of human 332 PATHOGENIC MICROORGANISMS tetanus rejwrted as occurring after single injections of antitoxin prove the \'alue of immunizing injections, for the mortality was low. They teacJi, however, that where tetanus infection is suspected the antitoxic serum should lie gi\'en a second and even a third time at intervals of seven days. Results of Antitoxin in Treatment. — In cooperation with Dr. (Jjrus W. Field, one of us (Park) tried a number of experiments upon guinea- pigs to test the importance of intravenous and of intraneural injections of antitoxin in animals in which tetanus had already developed. Forty guinea-pigs have been experimented upon. These were injected in the lower part of the hind leg with ten to twenty times the fatal dose of a mixture of tetanus toxin and bacilli. Within from one to two hours after the development of the first definite symptoms of tetanus the animals were operated upon and given antitoxin. The experiments show clearly that moderate doses of antitoxin given after the develop- ment of tetanus did not save the animals from death or even prolong life, while very large doses usually did both. Seventy-five per cent, of those receiving 500 units recovered. The surprising result developed that amputation of the infected leg at the hip-joint hastened the death of the animals in every case. Control animals which had not been infected stood the amputation perfectly well, and made good recoveries. Without antitoxin, excision of a piece of the nerve did not materially prolong life, nor did ligation of the nerve. In the guinea-pigs receiving antitoxin the ligation of the nerve seemed to be of benefit. The results of the experiments showed that large doses of antitoxin given shortly after the de^'elopment of tetanus usually saved the animals, and that most of the toxin was absorbed by the blood and not by the nerves of the infected part. Every minute of delay after the appearance of tetanus was of importance. With Dr. Matthias Nicol, Jr., I have recently compared intravenous with intraspinal injections. The results with intraspinal injections were a little better than with intravenous. Repeated injections did not give any better results than a single large injection. In actual cases in which the treatment was given within six hours of the development of symptoms the results have been sur- prisingly good. The recoveries have been over 70 per cent. In some cases no beneficial results appeared. We have seen numerous cases of generalized tetanus that after a large intravenous injection have markedly improved and finally recovered, and these cases have certainly done better on the average than apparent!}' similar ones receiving palliative treatment alone. Lambert, who some years ago made an exhaustive study of tetanus, states that in a total of 114 cases of this disease treated with antitoxin, according to published and unpublished reports, there was a mortality of 40.35 per cent. Of these, 47 were acute cases — that is, cases with an incubation period of eight days or less and with rapid onset, or cases with a longer period of incubation, but in- tensely rapid onset of symptoms; of these the mortality was 74.46 per cent. Of the chronic type — those with an incubation period of nine days or more, or those with shorter incubation with slow onset — there METHODS OF EXAMINATION TN A CASE OF TETANUS 333 were 61 cases, with a mortality of 16.39 per cent. Witli a still larger number of cases the results indicate that with tetanus antitoxin about 20 per cent, better results are obtained than without. Differential Diagnosis between Tetanus and Tetanus-like Bacilli. — The differential diagnosis of the bacillus of tetanus is, generally speaking, not difficult, inasmuch as animal inoculation affords a sure test of the specific organism. No other microorganism known produces similar effects to the tetanus bacillus, nor is any other neutralized by tetanus antitoxin. The other characteristics also of this bacillus are usually distinctive, though microscopic examination alone cannot be depended on to make a differential diagnosis. Difficulty arises when other anae- robic, or aerobic bacilli, almost morphologically identical with the tetanus bacillus, are encountered which are non-pathogenic, such as the Bacillus 'pseudoteianicus anaerobius, already mentioned, and the Bacillus pseudotetanicus aerobius. It is possible, however, that both these bacilli, when characteristic in cultures, are only varieties of the tetanus bacillus, which, under unfavorable conditions of growth, have lost their virulence. These non-virulent types do not, as a rule, have spores absolutely at their ends, and the spores themselves are usually more ovoid than those in the true tetanus bacilli. Methods of Examination in a Case of Tetanus. — (a) Microscopic. — From every wound or point of suppuration film preparations should be made and stained with the usual dyes. The typical spore-bearing forms are looked for, but are usually not found. At the same time other bacteria are noted if present. (6) Cultures. — Bits of tissue, pus, cartridge wads, etc., are collected and dropped into glucose bouillon contained in small flasks or tubes. (c) Inoculation. — A salt solution emulsion of material from the wound is inoculated into mice or guinea-pigs subcutaneously. Successful results are usually obtained, if the bacilli or their spores are present. CHAPTER XXII. INTESTINAL BACTERIA. Significance of Bacteria in Intestines. — The constant presence of great numbers of Imcteria in the intestinal tract has been the subject of much investigation which has given somewhat conflicting results. On the one hand, certain experiments seem to show that the presence of intestinal bacteria is not essential to life. For example, Nutall and Thierfelder experimenting with guinea-pigs succeeded in keeping the intestines free from bacteria for a limited time, during which the young pigs remained well. Furthermore, Levin makes the interesting statement that the intestinal tract of polar animals is for the most part sterile. On the other hand, the supporters of the opposite theory, namely, that certain intestinal bacteria are necessary for perfect physiologic action, state that in their experiments on feeding animals with sterile food they found that development was retarded; thus Schottelius claims this for chickens, and Mme. MetchnikofF obtained similar results with young frogs. However, whether or not the presence of bacteria in the intestinal canal is essential to tlie animal economy, it is, never- theless, evident that microorganisms play a certain role in aiding or inhibiting some of the alimentary processes dependent upon biological activity. Recently new interest has been added to the subject by the work of IMetchnikolf who claims that old age is hastened by the in- creased growth and action of certain putrefactive bacteria normally found in small numbers in the intestines; and he, Herter, and others consider that the development of these harmful varieties may be checked by the growth of the obligate intestinal bacteria or by some substituted variety which has no harmful action upon the host. Conditions Influencing Development of Bacteria. — The intestinal canal presents such varying conditions dependent upon so many dif- ferent factors that of necessity its flora will reflect great diversity. As the organisms gain access to the tract chiefly through the air, food, and drink ingested, the character of these will influence the nature of the flora. The condition of the oral cavity and that of the respiratory passages on account of swallowing Iiacteria will also have an influence on the kind of bacteria found. Some few microorganisms, such as the colon group and the obligate anaerobes, have become established as regular inhabitants of the intestines and find in the difl'erent locali- ties of the canal their liest enviroinnent. Together with these may be found those bacteria which having been ingested with various sub- stances have survi\'ed the action of the gastric and intestinal fluids. REGIONAL DISTRIBUTION OF BACTERIA IN DIGESTIVE TRACT 335 The length of time that the intestin,al contents are retained at any one point of the tract will cavise an increase or decrease of certain types, as well as the total number, since all portions of the canal arc not equally adapted to the development of any one species nor to bacteria as a class. Under absolutely normal conditions organisms, which are not de- stroyed, pass through the intestinal tract without entering the body of the host, but if injury occurs to the intestinal wall or the normal resistance of the liody's tissues is lowered for any reason they may pass into the circulation. Maklezow claims that after twenty-two hours of fecal impaction, intestinal organisms were found in the circulation. In chronic constipation intestinal bacteria are found in the urine. Escherich found that in idem and immediately after birth the meconium is sterile, unless, in exceptional cases where the mother has suffered from a severe bacterial infection and the invading organ- isms are found in the fetus. From three to seven hours after birth a few bacilli, cocci, and yeasts may be found, having presumably entered by the anus; after eighteen hours the number and kinds of bacteria increase, being taken in by the food or the swallowing of saliva. The stools of artificially-fed infants show a greater variety of organisms than those of the breast-fed child. Anaerobic Conditions in the Intestines. — Virchow first questioned the presence of free oxygen in the entire intestinal canal and con- cluded it was essentially anaerobic, the oxygen which is taken in being quickly absorbed or combined with hydrogen. The character of the flora also indicates an anaerobic condition of the small intestines with more or less aerobic conditions in the lower part of the colon and rectum. The anaerobes play the chief part in intestinal putrefaction, and certain varieties are thought to be at least the predisposing if not the chief cause of many cases of appendicitis. Regional Distribution of Bacteria in Digestive Tract. — Many different organisms may be found at times in all parts of the tract, but each species finds its best envorinment in some one location and is here found with greater frequency. In the stomach, very few bacteria develop, the sarcina^, B. gcustricus, and cloaca? group are rather constant, the larger number and variety taken in being destroyed to a great extent by the gastric juice. The fact that great numbers of bacteria are destroyed by the diges- tive juices, together with a rapid passage of the partly digested food and the strict anaerobic condition, accounts for the very few bacteria that are usually found in the upper part of the small intestines. It is in this location that the obligate anaerobes, which are usually spore bearers an(l often Gram-positive organisms, such as the U. putrificua of Bienstock, B. capsidatus aerogenes, and B. hifidus are usually found. The chief bacteria of the lower part of the small and the upper part of the large intestines are members of the B. coll group which reach their highest development in the cecum and upper colon. Here, too, other 33G , PATHOGENIC MICROORGANISMS organisms which have been held in check by the above chemical and mechanical causes, finding a more suitable soil, develop, and a marked increase is found in many Gram-positive bacilli and cocci of various types. In the lower colon and rectum with the accumulation of specific antagonistic substances formed from the abundant growth higher up, many forms, especially of B. coli group, are more or less destroyed. The flora can be materially changed in dogs by the diet, as has been shown by Herter. The .range of variation of the bacteria that appear normally from time to time in the intestinal tract is so very great that no one grouping, except in the most general sense, such as fermenters or non-fermenters of glucose, anaerobes, or aerobes, seems to apply to all cases. Ford isolated 50 distinct species from the human feces. Methods Used in Examination of Normal Feces. — The material should be taken from a perfectly fresh stool, preferably after a dose of castor oil has been given. This induces a quick and thorough emptying of the intestinal tract, with the least alteration of the chemical nature of the feces. The use of bhnd tuljes or flushing are apt to give only the contents of the lower part of the colon and rectum and are useful only when the examination is to be hmited to this area. To 1 gram of the material 100 c.c. of normal salt solution is gradually added, first rubbing up the feces in a small quantity of the diluent and then shaking thoroughly as more is added. Definite amounts of this emulsion can be used for plating. Two per cent, glucose agar with defibrinated blood added to it is very satisfactory for the isolation of anaerobes, while beerwort agar (10 per cent, sterile beerwort added to the usual stock agar) is used for the aoidophile group. A very convenient method, according to Zinsser, of growing anaerobic cultures is to take crystallizing dishes of different sizes so that one dish fits within the other, leaving a space of three-fourths of an inch all around. The larger dish is placed over the smaller dish as a cover, they are then wrapjied in filter paper and in this way can be easily sterilized. When ready for plating 1 c.c. of defibrinated lilood is placed in the bottom of the smaller dish and into this is poured 10 c.c. of glucose agar which has been inoculated with 1-10 c.c. of the above emulsion. By gentljf tipping the dish back and forth the blood and agar become very well mixed. This is covered with a petri dish or the com- panion crj'stallizing dish and allowed to stand until it is perfectly cold. In the larger dish are placed two pieces, aljout I5 inches to 2 inches long, of caustic soda and the dish is filled to about j its capacity with pyrogallic acid. The smaller dish is carefully inverted over this and sufficient sterile water poured into the larger dish to cover the acid. The whole is then sealed with paraffin or oil poured over the water that is collected outside the smaller dish; this pre- vents continual absorption of the oxygen from the air. Plates are made from })eerwort agar and grown both anaerobically and aerobically. Fishmgs are made from these plates upon corresponding tube media and the cultures are further testerl on such media as maj' be best adapted to each special organism. Substitution of One Variety of Bacteria for Others. — It is possible for the usual flora of the intestines to be almost entirely replaced temporarily by an invading organism. This occurs in disease when . the microorganism producing a specific disease of the intestines is found in almost pure cultures, as in dysentery or cholera. As already stated, Metclmikoff claims that this possibility of substitu- tion can be used in cases where intestinal putrefaction is excessive and thus check the process by the introduction of a lactic acid bacterium. LACTIC ACID MILKS 337 Tliis work of Metchnikoff and his followers has led to an extensive study of organisms causing lactic acid fermentation. Fig. 127 B. bulgaiicus; seventh day (44°) colony. Whey agar plate. X 50 diameters. (White and Avery.) Lactic-acid Milks. — For many j'ears the people of western Asia and eastern Europe have looked upon sour milk as an essential part of daily diet. In western Europe and America buttermilk has been a favorite drink with many, but it never assumed as much importance as in the east. The term sour milk covers all milks or parts of milks in which lactic acid fermentation is pronounced. The ordinary buttermilk sours because of the growth of lactic acid bacteria in the raw milk which have been derived from the local surroundings. Sour milk from the dealers may be such milk, but it is more usually heated milk to which some special culture of bacteria (starter) has been added. Sour milk is usually nearly fat-free, but more or less of the cream may remain in it. Hansen, of Copenhagen, has for some time supplied a lactic acid bacillus which has been much used. Another starter now popular is one suppled by Metchnikoff, which he obtained from the east. There are a number of preparations of sour milk used at present, among these are : Kumyss, in which the fermentation is due to lactic acid bacteria and yeasts, and thus contains not only lactic acid, but carbon dioxide and about 1 per cent, of alcohol. 22 338 PATHOGENIC MICROORGANISMS Maadzoun and Yoghvri, the common sour milk of southeastern Europe, containing chiefly the B. hulgaricvs and streptococci and diplococci, all producing lactic acid. Zoohk (matzoon) made by adding to heated milk the same bacteria as occur in maadzoun. Yohourd, blabberade, and other sour milks are made by the use of much the same organisms. Fig. 128 B. bulgaiicus. X 1000 diains. (Piffard.) The bacilli at present of most interest are those resembling the B. hulgaricvs (B. of Massol) which are present in the eastern milks and are now through the advocacy of Metchnikoft' used alone or in con- nection with a lactic acid streptococcus to ])roduce much of the souring of milk of Europe and America. In 1906, Cohendy studied the action of this bacillus and found that it produced a large amount of lactic acid, 3.23 per cent, being found after ten days at 36° C. From other preparations slightly different bacilli were found which produce a firm clot, while the B. hxlgariciis produces a soft curd. vSome bacilli which resemble the B. bylgarlni.s in many respects produce gas as well as acid. The bacilli in all strains of B. biilgaricKS- show wide \'ariations in length from 2/.i to 50;U. Chains of bacilli occur in some strains to a more marked degree than in others. The bacilli are non-motile, non- sporulating, (iram-positive, except when in inx'olution forms, when they are said to be (iram-negative. Difficult to cultivate in most media. When freshly isolated, growth obtained only on media con- taining whey or malt or milk. Grow equally well in aerobic and anae- robic conditions. Ojjtimum temperature for growth is 44°, fair growth at 30°, slight at 2ri°, none at 2(1°. (Jelatin is not liquefied. (\)lonies LACTIC ACID MILKS 339 on whey agar are round, grayish white, and measure 0.5 to 1.3 mm. Periphery of colonies mostly filamentous. The growth in whey jjro- FiG. 129 'Lactic acid" milk containing B. bulgaricus antl a lactose fenneuting streptococcus. Fig. 130 Yeast cells and lactose fermenting bacilli in "fermented milk." X 1000 diams. duces clouding, but this disappears in 5 to 14 days, leaving a sediment. Coagulates milk in S to IS hours at 44°, and after longer time at lower 340 PA THOGENIC MICROORGANISMS temperatures. The lactic acid formed is either inactive or levoro- tatory. A small quantity of volatile acid is also produced. No appreciable peptonization of the curd. The bacilli are non-pathogenic. These bacilli are probably widely distributed in nature, being frequently present in the intestines of man and animals. White and Avery, who have made an exhaustive study of this group of bacilli, consider that they all belong to one group which is identical with the group Bacterium caucasicum (Kern). There are apparently at least two distinct types which differ in the amount and kind of lactic acid ft)rmed. Baldwin showed that when milk plus a lactic acid bacillus was added to a mixed diet, the ethereal sulphates in the urine may be increased. Fig. 131 B. Ijul^arifiis from normal imHith; furty-i'ight hour eoloiiic8 on aficl nhey afiar at 42° C. Small foloiiy X 40 diameters. LarKC eolony X 220 diameters. (Heinemann and Hofferan). Means of Supplying Lactic Acid Bacteria for Therapeutic Uses. — These are supjjlied in fermented milks, fluid cultures, and in tablets. Bendick has shown in work done in the research laboratory that most of the tablets are sterile and that the fluid cultures contain few living bacilli after storage for a few weeks. Prevalent Intestinal Bacteria.^B. Bifidus. — Tissier found the B. hifidiiH in tlic stcKils nf breast-fed infants wiiicli at times forms nearly the entire llnra. lie found it, though less fretiuently, in arti- ficially fed infants, lie als(.) isolated it in the superficial ducts of the mammary gland of the mother. It is ii strict anaerobe. In the feces and fresh cultures it j^resents the form of a slender bacillus with one end PREVALENT INTESTINAL BACTERIA 341 tapering and the other chih-shaped. It 3/i and even i/i. varies in leiii;th from 2/j to It occurs mostly ;i« a diiilohacillus (sec Ki;;. 132) witii the pointed ends adja- cent and the swollen entls free, but at times this order is reversed and a fusi- form appearance results. As the line of separation is often ol)scure and as the two organisms come together at different angles these various arrangements give the impression of many different forms. The bacilli lie sometimes in parallel groups, but are seklom entangled. In old cultures, the swollen ends seen in the young cultures become bifurcated, others take the stain irregularly and Tissier designates this the vesicular form. In some instances se\'eral Ijacilli become grouped together at different angles, giving the appearance of multiple branching forms. As the medium becomes more acid, the bifurcated forms become more numerous. Vesicular forms bear a relation to ^-itality and bifucated forms a relation to media. It is non- motile, stains by Gram's method, old cultures staining irregularly. Does not seem to possess spores. Killed at 60° for 15 minutes. Does not die out quickly. Can be transplanted after three weeks. Grows best at 37° C, but also grows at 20° C. Fig. 132 X B. bifidus, representing the variou.s forms described; the irregularly stained or \'e.sieLihir forms being from old cultures. X al^out ISOO diameters. On glucose agar, after three days, fine regular colonies, oval in shape, appear. It is innocuous to guinea-pigs. It can be cultivated on beerwort agar and on glucose agar. In stab cultures made from the feces, in either medium the bacilli may be found in almost pure cultures at the bottom of the stalj after two to fifteen days; the other organisms djdng out unless the enterococcus which is a facultative anaerobe, is present, then B. bifidus, being less adaptable, is overgrown. Fermentation tubes of glucose broth inoculated with fecal material will show an abundant growth of B. bifidus in the sediment. Conradi and Kurpjuweit claim that in infants the B. hifidiis plays the same role in inhibiting the growth of the more harmful organisms as the B. coli does in the adults. Cahn states that the B. bifidus is not found so constantly in artificially fed infants, but that the B. acidophilus of Mora takes its place. :142 PATHOGENIC MICROORGANISMS B. Acidophilus. — This organism lielongs to the acidophile group and difi'ers from the bifidus in several respects. It never shows the bifid forms. It is only found in the artificially fed infants and in milk from cows. Colonies are irregular and send out filaments. It is a facultative anaerobe. Some consider it to be the same as the B. bifidus. Enterococcus. — Thiercelin in 1903 described the Enterococcus profei- formis (Fig. 133) as occurring as a coccus, diplococcus, streptococcus, staphylococcus, tetrad filaments and rods. It has a capsule barely visible, sometimes forming a halo. Fig. 1.3.3 Reprosents tho gradation of tho Enterococcus (Thiercelin) from the apparent bacillary forms to the coccus without a capsuk^. X 1000 diameters. The arrangement depends upon the mode of divisions. When the organism assumes the form of a bacillus the division takes place in line of the short axis, the capsule being tough does not rupture but encloses 2 or 4 more organisms. This form is observed in culture media con- taining alcohol, quinine, chromic acid, permanganate, and especially bichromate of potash. This bacillary form, before division takes place, is confusing as it is difficult to tell whether or not the culture is pure until transferred to a medium in which alcohol is present when the forms all become coccal. In strongly alkaline broth it grows in tetrads, on agar it resembles the staphylococcus, on gelatin the same. In broth containing a little methylene blue, picric or acetic acid and in hay infusions it is a distinct streptococcus. It is present in normal stools but may become pathogenic. It is found in the upper respiratory tract, skin, vagina, and is obtained in pure cultures from purulent flischarges, being easily isolated by ordinary methods. It is .sensitive to heat and direct sunlight, rather resistant to dis- infectants, does not grow in distilled water and sparingly in broth containing 2 per cent, sodium carbonate or nitrate. Grows in sterilized tap-water, tloes not ferment sugars, does not constantly coagulate milk, does not liciuefy gelatin, grows well in the digestive fluids. It produces no gas, indol, or odor in the jjresence of sugar. B. AEROGENES CAPSULArUS 343 It produces a toxin that kills mice in 24 to 4S hours. Tlie orjjanisms are found in all the organs after death. B. Putrificus. — Bienstock found in putrid mixtures an anaerobic, spore-bearing bacillus resembling tetanus in its morphology, which is capable of decomposing fibrin in the absence of oxygen, in this case the end products of putrefaction, such as indol, are not formed. When, hoAvever, B. putrificus is associated with some aerobes it acts upon fibrin in the presence of oxygen, forming the characteristic pntrefacti\e products, which are further split up l)y the aerol)es, forming indol. This action is not observed with all aerobes, for ex- ample, with B. coli and B. huiis aerogenes inoculated on fibrin with B. jjuirificvs. B. putrificus is found commonly in the small intestines where it enters through the respiratory and digestive tract; that putrefaction does not occur, normally is supposed to be due to the presence of in- hibiting bacteria. It is isolated with difficulty from the feces. Is an obligate anaerobe with drum-stick spores. It is a slender rod with blunt ends, and sometimes forms threads, especially on liquid gelatin, is actively motile with flagella arranged on either side. Liquefies blood- serum with the production of a foul odor. Is Gram-positive. Is not pathogenic for animals. B. Aerogenes Capsulatus {B. welchii, B. perfringens). — Found usually in small numbers in healthy adults. Increased in old age. It is considered by Herter to be the chief cause of intestinal putre- faction. For a full description of its pathogenicity and other char- acteristics, see p. 353. Klein found in the feces of patients during an outbreak of diarrhea at St. Bartholomew's Hospital, London, an organism which he named the B. aerogenes sporogenes, which may be a variety of B. welchii. BiBLIOUHAPHY. Bienstock. Archiv. f. Hygiene. 1899, xxxvi. E^chcrich. Darnibakterien des Siiuglings und ihre Beziehiingeii zur Physiologic der Vcrdauung, Stuttgart, 1SS6. Herter. Bacterial Infections of the Intestinal Tract, New York, 1907. Klein. Ueber einen pathogenen anaerol)en Darmbazillen B. enteritidis sporogenes, Centralbl. f. Bakt., 1895, Bd. xviii. MacNeal, Lalzer, and A'err. Fecal Bacteria of Healthy Men, .Tour. Infect. Dis., 1909, vi, 123 and .571. Meichnikoff, E. Sur les microbes de la putrification iiitestinale, C. R. Acad. Sci., 1908, cxlvii, 579. Etude sur la flore intestinale, Ana. Inst. Past., 1908, xxii. Thiercelin. Formes d'involution dc enterocoque enterobacteria, Coniptes-rendus de la Society de Biologic, 1902-190.3. Tissicr. Recherches sur La Flora Intestinale, Normale et Pathologique du Nourrison, 1900. CHAPTER XXIII. THE COLON-TYPHOID GROUP OF BACILLI. THE COLON GROUP. Theee are a number of varieties of bacilli normally occupying the intestines of man and animals which, because thej' have similar charac- teristics and live in the colon, are generally grouped together as colon bacilli. Many of the varieties occurring in animals seem identical with those found in man. These bacilli are only pathogenic under unusual conditions. The specific pathogenic typhoid, paratyphoid, including the types responsible for meat poisoning, dysentery, and paradysentery bacilli, also have among themselves and between them and the colon bacilli resemblances and are often classed together in the group of the colon-typhoid bacilli. The chief common characteristics of this whole group are: (1) a similar morphology, i. e., short, rather plump rods with a tendency to thread formation; (2) a Gram-negative staining reaction; (3) similar growths on agar and gelatin; (4) non-liquefaction of gelatin (a few closely related organisms, such as B. cloaca, liquefy gelatin very slowly). In order to see more clearly the main points of difference between the subdivisions of this great group the following tabulations may be studied. Group of Colon-typhoid Bacilli. Colon group. Paratyphoid group (intermediate group) B. (coli) communis B. (coli) comn:iunior B. (acidi) lactici B, aerogenes types B. para typhosus, A B. paratyphosus, B B. paratyphosus, C B. entcritidis (Similar types, intermediates between colon and typhoid have been isolated from water and other sources, probably non-pathogenic.) Normal inhabitants of the intes- tines, under certain conditions, become pathogenic. Usually pathogenic for man or ani- mals in varying degree. B. typhosus Pathogenic for Dysentery group. B. dysenteriae (mannite not fermented) B. paradysenterice (man- nite fermented). Three varieties. Pathogenic for man, paradysen- teria? usually less so than dysen- tery type. Alkaiigenes group. B. alkaligenes. Occasionally pathogen MORPHOLOGY 345 Chief Diffkrential Points uetween Members of the Typhoid-Colon Group. 1 i Milk. Colonies. RuBsell medium. Ort!;anisui. -D a ■4 Q o 1 3 Q "3 ~6 < d Sl.lrit. Butt. B. (noli) communis + + ++ ++ * + * + + ♦ —. Red Red Red Red, gas B. (coli) communior ++ ++ ++ ++ * + * + + * — Red Red Red Red, gas B. (acidi) laetici . ++ ++ « + * + + * — Red Red Red Red, gas B. at'rogenes . + + ++ ++ — * + * + + * — Red Red Red Red, gas B. paratyphoid, A ++ + + + + d= Slight Color- Blue Blue Red, gas B. paratyphoid, B ++ - - ++ ++ - zt - Strong less Color- less Blue Blue Red, gas B. enteritidis ++ — — ++ ++ — — Strong Color- less Blue Blue Red, gas B. typhosus . + — — — + — =t — Slight Color- Blue Blue Red B. dysenteric + — - — — — zt — Slight less Color- less Color- Blue Blue Red B. paradysentcrije + — . — + + * Slight Blue Blue Red less B. alkahgenes — — — — — — — Strong Color- Blue Blue Blue less ' Variable, used for further subdivision. -}- acid positive; gas negative. -t- * Usual, exceptions found. H — h acid positive; gas postive. icid negative; gas negative, slight reaction. The Colon Group. — The first description of an organism of the colon type was by Emmerich (1885), who obtained it from the intes- tinal discharges of cholera patients. A similar organism was found by Escherich (1886) in the feces of healthy infants. He gave it the name of Bacterium coli commune. It has since been demonstrated that closely allied tj'pes of bacilli are normal inhabitants of the intes- tines of most of the lower animals. They are transferred through the feces as manure and sewage to cultivated land, surface waters, etc. During warm weather they may multiply outside of the animal body. Those strains having the chief cultural characteristics of the original strain are classed as colon bacilli, while those differing con- siderably from it are, while considered in the general group, given different names, such as paracolon, "atypical coli," etc. These types are especially common in water and are often due to the suppression of certain characteristics due to unfavorable environment. The colon group is divided into four main subgroups, B. communis, B. communior, B. aerogenes, and B. acidi laetici. These subgroups have numerous varieties. The colon group has interest not only because it excites disease at times in man and animals, but also because it is an index of fecal pollu- tion from man or animals. If from man it indicates the possibility of infection with the typhoid or dysentery bacilli. For the significance of the colon group in water see Bacteriology of Water. B. (Coli) Communis. — This type is described as an example of the group. Morphology. — B. communis varies in morphology. The typical form (Fig. 134) is that of short rods with rounded ends, from 0.4;u to 0.7/i in diameter by 1/x to 3yu in length; sometimes, especially where the culture media are not suitable for their growth and in tissues, 346 PA THOGENIC MICROORGA NISMS the rods are so short as to be almost sphericah resembling micrococci in appearance, and, again, they are somewhat oval in form or are seen as threads of 6^ or more in length. The various forms may often be associated in the same culture. The bacilli occur as single cells or as pairs joined enfl-to-end, rarely as short chains. There is nothing in the morphology of this bacillus sufficiently characteristic for its identification. Flagella. — l^pon some varieties seven or eight peritrichic flagella have been demonstrated, the non-motile types show none. The flagella are shorter and more delicate than those characteristic of the typhoid bacilli. Staining. — The B. communis stains readily with the orrlinary aniline colors; it is always decolorized by Gram's method. Under certain conditions the stained bacilli exhibit bipolar granules. Fig. 134 Colon liarilli. Twenty-four-hovir agar culture. X 1100 diameters. Biology. — It is an aerobic, facultative anaerobic, non-liquefying bacillus. It develops best at 37° C, but grows well at 20° C, and slowly at fO° C. It is usually motile, but the movements in some of the cultures are so sluggish that a positive opinion is often difficult. In fresh cultures, frequently, only one or two individuals out of many show motility. The B. coli does not form spores. Cultivation. — The B. communis develops well on all the usual culture media. Its growth on them is usually more abundant than that of the typhoid bacillus or the d.ysentery bacillus, but the difference is not sufficient for a differential diagnosis. Gelatin. — In gelatin plates, colonies are developed in from eighteen to thirty -six hours. They resemble greatly the colonies of the typhoid bacillus, except that many of them are somewhat larger ancl more opaque. (See Figs. 43-45, page 110.) When located in the depths GAS PRODUCTION 347 of the gelatin aiul examined by a low-power lens they are at first seen to be finely grannlar, almost homogeneous, and of a j)ale yellowish to brownish color; later they become larger, denser, darker, and more coarsely granular. In shape they may be round, oval, or whetstone- like. When the gelatin is not firm the margins of many colonies are broken by outgrowths, which are rather characteristic of colon bacilli. In stab cultures on gelatin the growth usually takes the form of a nail with a flattened head, the surface extension generally reaching out rapidly to the sides of the tube. Nutrient Agar. — In plate cultures: Surface colonies mostly circular, finely granular, and rather opacjue. The deep colonies are apt to have protuberances. In streak cultures an abundant, soft, white layer is quickly developed, but the growth is not characteristic. Bouillon. — In bouillon the B. communis produces diffuse clouding with sedimentation; in some cultures a tendency to pellicle formation on the surface is occasionally seen. Potato. — On potato the growth is rapid and abundant, appearing after twenty-four to thirty-six hours in the incubator as a yellowish- brown to dark cream-colored deposit covering the greater part of the surface. But there are considerable variations from the typical growth ; there may be no visible growth at all, or it may be scanty and of a white color. These variations are often due to variations in the potato. Milk. — IMilk is usually coagulated in from one to four days at 37° C. Coagulation is due principally to the production of lactic and acetic acid. Some strains produce acid but coagulation does not occur. It is possible that a lab ferment is partly responsible for the coagulation. Chemical Activities. — Behavior Toward Carbohydrates. — In cultures of 7)'. communis many carbohydrates, especially sugars, become fermented with production of acid and gas. The important fermentation products, both cjualitatively and quan- titati\'ely, are produced from grape-sugar, probably, according to the following reaction : 2CGH,=0t + HsO = 2CiH,0, + CHjCOOH + C2H5COOH + 200. + 2H5 Grape-sugar. Water. Lactie acid. Acetic acid- Ethyl alcohol. Carbonic Hj'drogen. acid. There are reasons to think that lactic acid is first produced and that from this other acids and products develop. Under aerobic conditions lactic is produced in excess of acetic acid, while in the absence of oxygen the reverse is apt to be true. Gas Production. — When B. communis is grown in a solution of glucose (dextrose), CO2 and H2 are produced, in the proportion of ICO2 to iHo up to lC02to3H2. Anaerobic conditions aid gas formation. Very slight traces of gases other than H and CO2 are produced. The amount of gas varies in different varieties; the closed arm of the tube half-filled, and the H and CO2 in the proportion 2 to 1, is the characteristic type. The fermentation is not a simple hydrolytic action, but one in which combinations between the C and O atoms are sundered and formed. 348 PATHOGENIC MICROORGANISMS This is not an oxidation process, but a change through breaking down — that is, a true decomposition. What oxidation takes place is chiefly due to the oxygen Uberated from sphtting the sugar molecules. For the carbohydrates fermented, see table, p. 345. Effect of B. Communis in Nitrogenous Compounds. — Indol Formation. — B. communis does not liquefy gelatin nor peptonize any albumins. They do, however, break down some of the higher nitrogenous compounds into smaller atom groups. The first noted of these compounds was indol, C^Y{i{ ^^t CH. This is one of the most important products of -L rl/ colon acti\'ity, although some varieties lack the ability to produce it. The maximum amount of indol is present about the tenth day. In the intestinal canal in health very little indol appears to be produced by the bacilli. Sulphurated hydrogen is liberated from sugar-free proteid substances. Reduction Processes. — Nitrates are reduced to nitrites and from them ammonia and free nitrogen. Litmus and other dyes are also reduced. Toxins. — The bodies of dead bacilli contain pyogenic and other substances, which, injected into the circulation, produce paralysis of the striped muscle fibers, convulsions, coma, and death. Extracts from some cultures produce irritation of the mucous membranes of the large intestines with d,ysenteric symptoms. Growth with Other Bacteria. — The B. communis as well as other members of the colon group act antagonistically to many of the pro- teolytic bacteria in the intestinal tract, and so inhibit alkaline putre- faction otherwise caused bj' the latter. In milk the same antagonism exists, probabh' because of the acidity caused by the colon growth. Reaction to High and Low Temperatures. — B. communis is killed at 60° C. in from five to fifteen minutes. Frozen in ice a large propor- tion die, but some resist for six months. Frozen in liquid air 95 per cent, are killed in two hours. Resistance to Drying and Antiseptics. — Simple drying destroys the majority of organisms dried at any one time, but some bacilli of the number dried may remain alive, especially when held in the texture of threads, for five or six months, or all may die in forty-eight hours. To most antiseptics they are moderately resistant. They are killed in five to fifteen minutes in a 1 per cent, solution of carbolic acid. Effect of Acids. — B. communis grow in a wider range of acids and alkalies than most other bacteria. They develop in from 0.2 to 0.4 per cent, of mineral acids, in from 0.3 to 0.45 per cent, of vegetable acids, and in from 0.1 to 0.2 per cent, of alkalies. Effect of Intestinal Juices. — Gastric juice kills unprotected B. communis ))acilli unless it is too greatly diluted by food. All the members of the typhoid-colon group are more resistant to the gastric juices than most non-spore-bearing bacteria. With the food they readily pass from the stomach into the intestines. They grow in bile and in the intestinal juices. PATHOGENESIS IN MAN 349 B. (Coli) Communior, this species ditt'ers from B. cnunnuniH only in its sugar fermentations, fermenting saccharose witli tlie production of gas. B. (Acidi) Lactici, this type differs from botli of tiic preceding in faihng to ferment dulcit. Most of the differential work has been done with strains from feces, similar work Avith strains from pathological lesions of various parts of the body has been, so far as we know, only limited in amoiuit. For this reason the collective term B. coli is used in the following descrip- tions. Pathogenesis. — In Lower Animals. — Intraperitoneal and intravenous inoculation of guinea-pigs and rabbits ma\' produce death, which, when it follows, usually takes place within the first forty-eight hours, accompanied by a decided fall of temperature, the symptoms of enter- itis, diarrhea, etc., and finally fibrinopurulent peritonitis. Subcutaneous inoculation in rabbits is followed usually by abscess formation at tlie point of inoculation. Dogs and cats are similarly ali'ected. Cystitis and pyelonephritis may be produced by direct injections into the bladder and ureters, if the urine is artificially suppressed. Angiocholitis and al)scess are produced by direct injections into the li\'cr. Osteomyelitis may follow the intravenous injections of cultures into young rabbits. From experiments on animals it would appear that the explanation of the pathogenesis of tlie colon bacillus is undoubtedly to be found in the toxic effects of the chemical substance and products of the cells. In Man. — In normal intestines with intact mucous membranes the toxic products formed by B. culi are absorbed but little or not at all, and the bacilli themselves are prevented from invading the tissues by the epithelial layer and the bactericidal projierties of the body fluids. Possil)ly there is an acciuired immunity to the colon \'arieties which have long inhabited the intestines. B. coli was at first regarded purely as a saprophyte. Later, because of the postmortem invasion and the great ease of growth of the colon bacillus on ordinary media, the other extreme was taken of attributing too much to it. The bacilli previously present in the intestines can, either by an increase in virulence or by a lowered resistence in the person, invade the tissues. Thus in the case of ulceration in typhoid fever B. coli may enter the blood, or in perforation produce peritonitis. In dying condi- tions they at times pass through the intact mucous lining. In the gall- bladder or urinary tract the spread of bacilli from the intestines may cause disease. The specific serum reaction in the body is a sign of infec- tion, but great care has to be observed in deciding that it is present, as group agglutinins also occur. Up to the present time it is very difh- cult to state in any colon infection whether the bacilli were previously present in the intestines or were derived from outside sources through water, food, or direct contact with other cases. 35(1 PATHOGENIC MICROORGANISMS ' Intestinal Lesions. — The lesions present in intestinal infection attri- buted to B. coli are those of enteritis; the duodenum and jejunum are found to contain fluid, the spleen is somewhat enlarged, and there are marked hyperemia and ecchymosis of the small intestines, together with swelling of Peyer's patches. Virulence of B. Coli from Normal and Diseased Intestines. — The virulence varies with the culture and the time since its recovery from the intestines. Other things being equal, it is usually more virulent from an intestinal inflammation. From severe diarrhea the colon bacilli in 0.25 c.c. bouillon culture may kill guinea-pigs if given intraperitoneally, while from the healthy bowel 2.5 c.c are usually required. B. Coli in Sepsis. — When lesions of the intestinal mucous mem- branes exist, or in colon cystitis, pyelitis, or cholecystitis, there is fre- quently' just before death a terminal dissemination of the bacilli and consequent septicemia. Here special symptoms of intoxication may occur, such as diarrhea, changes in temperature, heart weakness, and hemorrhages. In most of these cases infection proceeds from the intes- tines, but in not a few from the wounded urethra or bladder. The colon septicemia is detected by blood-cultures. At times very few bacilli are foimd, and then the blood-infection may be less important than the local one. Cases occurring in typhoid and cholera have been observed, especially in relapses in typhoid. In very young infants a malignant septicemia with tendency to hemorrhages is due to B. coli. In a few cases in which B. coh but no typhoid bacilli were present the course of the disease has been similar to typhoid fever. An epidemic due to colon infection of water has been noted. Infection through food and water are usually brought about by other closely allied bacilli not belonging to the colon group. B. Coli in Diarrhea. — In diarrhea we find increased peristalsis, less absorption of foodstuff, increased and changed intestinal secretions. Tissier observed that under treatment with cathartics the colon ^•arieties increased, while the anaerobic forms are inhibited. In diarrhea, there- fore, we should expect favorable conditions for multiplication and inhil)iting causes to be lessened. This makes one question the signi- ficance of numerous epidemics which have been reported of acute diarrhea in children from one to five years of age in which almost pure cultures of colon bacilli have been found. The .symptoms in such epi- demics begin with high fever which often rapidly- falls, and frequent stools, only watery or containing mucus and streaks of blood. These symptoms may quickly abate or go on to a toxic state characterized by heart weakness and drowsiness. This may lead to lung complica- tions or death. B. Coli in Peritonitis. — Here tlie lesions must be considered as being due to mixed infection. Not only perforation of the intestines in man, but injury to the intestinal walls, allows colon infection of the peri- toneum to take jjlace. At first most of these cases were believed to be a pure colon infection, but now it is known that this idea came largely from tlie overgrowth of colon bacilli in the cultures. More careful INFLAMMATION OF THE URINARY TRACT 351 investigations, thronsh cultures and smears, lia\'e demonstrated the fact that streptococci, and less frequently staphylococci aufl pneu- mococci, are also usually present in peritonitis arising from intestinal sources. The colon bacilli found even in the same case commonly comprise many varieties. B. Coli in Inflammation of the Bile Tract. — The normal healthy gall-bladder is usually sterile. This is true in spite of the fact that bile is apparently a good culture medium for the colon group. Simple tying of the neck of the gall-bladder usually causes a colon infection to take place within twenty-four hours. Obstruction of the bile-duct through various causes is fairly common in man. The gall-bladder then becomes infected, and following the inflammation of the mucous mem- branes there is often the formation of gall-stones. Some cases of jaun- dice are believed to be due to colon inflammation of the gall-ducts. Atypical varieties of B. coli are frequently isolated from gall-bladder infections. Inflammation of the Pancreas. — Welch was the first to record a case of j)ancreatitis with multiple fat necroses due to B. coli, infection, A few more cases have since been reported due to members of the colon group, either alone or in conjunction with the pyogenic cocci. Inflammation of the Urinary Tract. — As far back as 1879 Bouchard noted cystitis due to bacilli of the colon group. When cystitis is estab- lished the bacterial infection frecjuently spreads to the pelvis of the kidneys, causing a pyelitis or suppurative nephritis. The same process often occurs in man. In most ca,ses of chronic cystitis the ureters and pelves of the kidneys become involved; any malformation of the ureters aids the process. From the pelvis the bacteria push up into the urinary tubules and excite inflammation and multiple abscesses. Colon infection of the different parts of the urinary tract may occur at any age, from infancy upward. Instead of starting in the bladder it may begin in the kidney itself, the colon bacilli coming from the blood or peritoneum. In many of these cases the bacilli isolated from the urine are clumped in high dilutions of the blood from the patient. Although other bacteria — the pyogenic cocci, the proteus, the typhoid bacillus, etc. — may excite cystitis, still in 90 per cent, of all cases some of the colon group are found, and this percentage is even higher in young children. The clinical picture of colon infection is ver>' variable. The lightest cases progress under the guise of a bacteriuria. The urine is passed a little more frequently and shows a fine granular cloudiness. The reaction is acid. The cell elements are but little increased. There is an excess of mucus. Albumin is absent or present in only a trace. The condition may last for weeks or months and then spontaneously disappear or grow worse. With a somewhat more se^'ere infection there is painful urination, perhaps tenesmus, increase of pus cells and slight fever. In a conical glass a sediment of pus cells forms at the bottom, and clear urine remains above. If the infection passes to the kidney colicky pain and tenderness over the region of the kidneys is usually present. The most important syni])tom of pyelitis is an irregular inter- 352 ' PATHOGENIC MICROORGANISMS mitteiiit fever resembling malaria. The albumin is increased in the urine and red-blood cells may be seen. If a general nephritis arises the symp- toms are all intensified and an anemic condition may develop. Septi- cemia may finally result. In most of these cases the microscopic examination is sufficient to make a probable diagnosis, since the bacteria- are so abundant. The variety of colon bacillus present can, of course, only be told by cultures and other means. In the urine they appear as diplobacilli, or partly in short, almost coccus, forms, partly in long threads. As a rule, motility is absent. Not infrequently the cultures appear to be identical with those of the Bacillus aerogenes. The characteristics of the urine itself have much to do with the probability of infection ; the more acid urines being less likely to afford a proper soil for growth. Some uriries are bactericidal even when they are neutral. The substances producing this condition are not known. The colon bacilli in the urine produce no appreciable effect on the reac- tion, but give up some of their toxins, which upon absorption cause the deleterious local and general effects. The serum of the patient usually agglutinates the cultures from the urine in 1 : 20 or 1 : 50 dilutions, but this property is sometimes absent, especially in light cases. In all cases in addition to the introduction of the colon bacillus a preflis])osing condition must be present, such as more or less marked retention of urine by an enlarged prostate or stricture, any unhealthy state of the mucous membrane or general depression of vitality. B. Coli as Pus Formers. — Members of this group are frequently the cause of abscesses in the region of the rectum, urethra, and kidney. They rarely produce pus in other locations. B. Coli in Inflammation Not Previously Mentioned. — Broncho- pneumonia, lobar pneumonia, and pleurisy have occasionally been caused by colon bacilli, probably from blood-sources. Not a few cases of meningitis and spinal meningitis in infants, follow localized B. coli infections. The symptoms are not well developed, as a rule. Some cases of endocarditis have also been noted. Immunity. — Natural infection in man, or inoculation into animals, is followed by the production of antibodies; agglutinins, precipitins, bactericidal substances, and opsonins being produced. The attempt to separate the members of this group by agglutination have shown the great dissimilarity of the different members in their immune reactions although group reactions are marked. Natural agglutinins for this grou]) are commonly present in the serum of man and animals. Curative Vaccine Treatment. — Localized inflammations due to the B. coli have been treated quite successfully by injections of dead organisms. An injection of 25 millions can be made daily or 300 millions every few days. The dose should be increased at each injection depend- ing on tlic reaction, lOOO million being the usual maximum dose. Autogenous vaccines should always be prepared if possible. Methods of Isolation. — They may be isolated from lesions on ordi- nary media or bile media or Endo- or Conradi plates may be used. The B. AEROGICNES 353 latter are used in isolation frona feces or other mixed material. Blood cultures may be made in bile or broth. For the examination of water for members of the colon group, see Bacteriology of Water. Bacillus Aerogenes and Allied Encapsulated Bacilli. — The members of this group differ from the members of the colon group, already de- scribed, in producing a viscid or mucoid growth, and in smears a capsule is commonly demonstrable. For this reason the group is spoken of by some as the capsulatus group, and the aerogenes types described as allied varieties. There are wide variations in the group, varying from forms almost identical with the colon types already described, to forms in which the capsule production is marked, and the ability to ferment lactose is slight or absent. In the absence of biologic differentiation of types, the varieties are best considered according to their source. For this reason the term B. aerogenes is collectively used for the types normally found in feces, milk, etc. B. Aerogenes. — Normally present in feces and also found in sewage and water where it has the same significance as other types of the colon group. It is constantly present in milk and is one of the chief causes of the souring of milk and cream. Morphologically it is variable in length and capsule production. In cultures the growth is abundant and viscid or mucoid in variable degree. It usually ferments the various carbohydrates more vigorously than other colon types, the closed arm of the fermentation tube being usually full or nearly full of gas. Different strains differ in the carbyhodrates attacked. Indol production is variable. B. aerogenes is much more resistant to acids and other deleterious substances than the other colon types. Pathogenesis. — Probably slight, although it has been isolated from infections of the urinary tract, peritoneum and liver and gall-bladder. In some of the cases reported there is a possibility that the infection was really due to other of the colon types. In the absence of reliable methods of differentiation there is always a question whether the more serious infections are not due to the more virulent bacillus of Friedlander. Bacillus Pneumoniae, Friedlander (B. Mucosus Capsulatus). — This bacillus was first described by Friedlander in 1882. He confused this bacillus with the pneumococcus, then undescribed, and regarded it as the causative agent in lobar pneumonia. Morphology. — It varies from coccoid forms to longer bacilli; they may be single, in pairs, or in short chains. They are surrounded by a wide capsule which is easily demonstrable not only in the material directly from the lesions but also in smears from cultures. It is Gram- negative and non-motile. Biology. — It grows freely on ordinary media. On agar the colonies are characteristically mucoid, with a tendency to confluence. Indol is not produced. The fermentative properies of different strains is very varied not only in the sugars fermented, but also in the amount of acid or gas or both produced. Milk-sugar is usually fermented, but milk is not coagulated. 23 354 PATHOGENIC MICROORGANISMS Pathogenesis. — It is pathogenic for mice and guinea-pigs in variable degree. Injection intraperitoneally or into the huig is followed by peri- tonitis or local hepatization with septicema. Rabbits are killed by some strains after intraperitoneal or intravenous injection; subcutaneous injection, however, commonly causes only a local lesion. In man it causes both broncho- and lobar pneumonia. This tj'pe of pneumonia is relatively infrequent and is characterized by its very high mortality. The bacilli are present in the sputum as a rule in almost pure culture. It is commonly present in the upper respiratory tract and the accessory sinuses, and may cause inflammation. By extension infection of the ear occurs, which may be complicated by meningitis. The pleura or the pericardium may be infected as a comphcation of pneumonia. A septicemia may develop as a complication of pneumonia or infection elsewhere. Inflammations of the eye are occasionally due to this organism. B. Ozsena. — This organism is culturally indistinguishable from the preceding. It receives its name from its constant presence in ozena or fetid rhinitis. Abel and others consider it the causative agent in this disease, basing their opinion on its constant presence. Bacillus of Rhinoscleroma. — This bacillus differs from the Bacillus pneuvioniw only in its weaker fermentative properies. Its name comes from its frequent presence in rhinoscleroma. Its etiological relationship to this disease is not established. Immunity and Serum Reactions of the Capsulatus Types. — Active im- munity cannot as a rule be produced in animals by the injection of killed cultures or the products. Careful injections of animals, however, lead to the production of agglutinins, precipitins, and complement fixing antibodies. The attempt to differentiate the various types by means of serum reactions has as yet led to no uniform results. In common with other encapsulated bacteria, they are not easily affected by sera. Forges found that the absence of agglutination could be attributed to the presence of the capsule and devised a method of destroying the capsule by first treating the bacterial suspension with acids and heat and then neutralizing with alkali. Vaccine Therapy. — Only a limited number of cases have been treated, some with reported improvement, others not. Other Bacilli Allied to the Colon Group. — There are various types of which B. cloacw is an example, whicli resemble the colon bacillus in many respects but liquefy gelatin in varying degree, some ^'ery slightly and only after prolonged growth. CHAPTER XXIV. THE TYPHOID BACILLUS. Tins organism was first observed by Eberth, and independently by Koch, in 1880, in the spleen and diseased areas of the intestine in typhoid cadavers, but was not obtained in pure culture or its prin- cipal biologic features described until the researches of Gaffky in 1884. The methods of identification employed by Gaffky were found insuffi- cient to separate the t>'phoid bacillus from other bacilli of the colon- typhoid group. The absolute identification of the bacillus only became possible with the increase of our knowledge concerning the specific immune substances developed in the bodies of immunized animals. Its etiologic relationship to typhoid fever has been particularly difficult of demonstration, for although pathogenic for many animals when subcutaneously or intravenously inoculated, it has been impossible to produce infection in the natural way or produce gross lesions corre- sponding closely to those occurring generally in man. Nevertheless the specific reactions of the blood-serum of typhoid patients, the constant presence of the Bacillus typhosus in the intestines and some of the organs of the typhoid cadavers, the very frequent isolation of this bacillus from the roseola, spleen, blood, and excretions of the sick during life, the absence of the bacilli in healthy persons, unless they have at some time been directly exposed to, or are convalescent from, typhoid infection, all these have demonstrated scientifically that this bacillus is the chief etiologic factor in the production of the great majority of cases desig- nated as typhoid fever. Morphology and Staining. — Typhoid bacilli are short, rather plump rods of about 1^ to 8// in length by 0.5/x to O.S^t in diameter, having rounded ends, and often growing into long threads. They are longer and somewhat more slender in form than most of the members of the colon group of bacilli (Figs. 13.5 and 13G). The typhoid bacilli sUdn with the ordinary aniline colors, but a little less intensely than do most other bacteria. Like the bacilli of the colon and paratyphoid groups, they are decolorized by Gram's method. Bipolar staining is sometimes marked. Biology. — The typhoid bacillus is a motile, aerobic, facultative, anaerobic, non-licpiefving bacillus. It develops best at 37° C; above 40° and below 30° growth is retarded; at 20° it is still moderate; below 10° it almost ceases. It grows slightly more abundantly in the presence of oxygen. It does not form spores. Resistance. — When a number of typhoid bacilli are dried most of them die within a few hours and a few frequently- remain alive for 356 PA THOGENIC MICROORGA NISMS months, but sometimes all the bacilh die very quickly. In their resistance to heat and cold they behave like the average non-spore- bearing bacilli. With rare exceptions they are killed by heating to 60° C. for one minute. Fig. 13.5 Fig. 136 Typhoid bacilli from nutrient agar. X 1100 diameters. Typhoid barilli from nutrient gelatin. X 1100 diameters. Motility. — Typhoid bacilli, when living under favorable conditions, are very actively motile, the smaller ones having often an undulat- ing motion, while the larger rods move about rapidly. In different cultures, howe^^er, the degree of motility varies. Fig. 138 #. FlaKclla. heavily stained, attached to l.aeilli. Typhoid bacillus with faintly stained flagella. (Van Ermengen's method.) (Lofflcr's method.) Flagella. — These are often numerous and spring from the sides as well as the ends of the bacilli, but many short rods have but a single terminal flagellum (Figs. lo7 and 138; see also Plate III). Cultivation. — Its growth on most sugar-free 'culture media is quite similar to tliat of the BacUlm mli, but it is somewhat slower and not quite so luxuriant. TNDOL REACTION 357 Growth on Gelatin Plates (Fig. 139). — The colonics growing deep down in this phite medium have nothing in their appearance to distin- guish them from submerged colonies of the colon gronp; they appear as finely granular round points with a sharj) margin and a, >'ello\vish- browu color. The superficial colonies, however, ])articularly when young, are often cpiite characteristic; they are transparent, bluish- white in color, with an irregular outline, not unlike a grape-leaf in shape. Slightly magnified they appear homogeneous in structure, but marked by a delicate network of furrows. Surface colonies from some varieties of colon bacilli give a similar picture. In stick cultures in gelatin the growth is mostly on the surface, appear- ing as a thin, scalloped extension, which gradually reaches out to the sides of the tube. In the track of the needle there is but a limited ^^'^- 1^9 growth, which may be granular -'' or uniform in structure, and of a - . yellowish-brown color. There is no liquefaction. Growth in Bouillon. — This me- dium is uniformly clouded by the typhoid bacillus, but the clouding is not so intense as by the colon bacillus. When the bou- illon is somewhat alkaline a deli- cate pellicle is sometimes formed on the surface after eighteen to "^ j twenty-four hours' growth. -v -^'"-J", jf Growth on Agar. — ^The streak ^x^ f'^* , - ' cultures on agar are not dis- V ' - ' tinctive; a transparent, filiform, gravish streak is formed. , c ■, , mx i i i ,',\ ° " A superficial colony (1) and a deep colony (2) Growth on Potato. — Ine growth of typhoid bacilli in gelatin. X 20 diameters. on this medium was formerly of great importance in identification, but now other media, giving more specific characteristics, have been discovered. When characteristic, the growth is almost invisible but luxuriant, usually covering the surface of the medium, and when scraped with the needle offers a certain resistance. In some cases, however, the growth is restricted to the immediate vicinity of the point of inoculation. Again, the grow^th may be quite heavy and colored yellowish brown, and wdth a greenish halo, when it is very similar to the growth of the colon bacillus. These differences of growth on potato appear to be chiefly due to variations in the substance of the potato, especially in its re- action. For the characteristic growth the potato should be slightly acid. A new lot of potato should always be tested with a typical typhoid bacillus as a control. Indol Reaction. — It does not produce even a trace of indol in peptone- water solution. This test was proposed by Kitasato for differentiating 358 PATHOGENIC MICROORGANISMS the typlioid ])ucil]us from other similar bacilli such as those of the colon grou]), which, as a rule, give the indol reaction. The typhoid bacillus, like the colon l>acillus, produces alkaline substances from peptone. Neutral Red. — In stick cultures in glucose agar the typhoid bacillus produces no change, while the colon bacillus decolorizes the medium and produces gas. Effect of Inhibiting Substances in Culture Fluids. — The typhoid bacil- lus is inhibited by weaker solutions of formaldehyde, carbolic acid, and other disinfectants than is the colon bacillus. Most typhoid-like bacilli resemble the typhoid bacillus in this respect. Some sub- stances, such as brilliant green, inhibit the colon bacillus more. Action on Different Sugars. — The determination of the action upon sugars of any bacillus belonging to the typhoid or colon group is one of the most important of all the cultural differential tests. It has been given in detail in the table, p. 345. While the typhoid bacillus does not produce gas from dextrose galactose, and levulose, it does produce acid from these substances. Milk. — The typhoid bacillus does not cause coagulation when grown in milk. A very slight acidity is evidenced in litmus milk from the action of the tv'phoid on traces of fermentable substances other than lactose. Production of Disease in Animals. — It is impossible experimentally to produce the characteristic lesions usually met with in human typhoid fever in animals. Sickness or fatal results without the appear- ance of the typical pathologic changes may be produced by animal inoculations, but in most cases they could easily be traced to the toxemia produced by the substances in the bodies of the bacilli injected. Typhoid bacilli, freshly obtained from typhoid cases and introduced subcutaneously in animals, rapidly die. In the peritoneal cavity they may increase, causing a fatal peritonitis with toxic poison- ing. By accustoming bacilli to the animal body a certain degree of increased virulence for the animal can be obtained, so that smaller amounts of culture may prove fatal. Among the most successful efforts in this direction are the experiments of Cygnaeus and Seitz, who, by the inoculation of typhoid bacilli into dogs, rabbits, and mice, produced in the small intestines conditions that were histologically, and to the naked eye, analogous to those found in the human subject. Their results, however, were not constant. Very similar results followed inoculation of virulent strains of colon bacilli. In rabbits a septicemia can be produced by intravenous inoculation. Localization in the gall- bladder follows and a "carrier" state similar to that found in man results. Distribution of Bacilli in the Human Subject. Toxic Effects.— Typhoid fever during its early stages, at least, is accompanied by a bacteremia. The bacilli thus pass to all parts of the body and become localized in certain tissues, such as the bone marrow, lymphatic tissues and s]jleen, liver and kidneys. Wherever found in the tissues the ELIMINATION OF TYPHOID BACILLI FROM BODY P.fjf) tyi)lu)i(l bacilli are usually observed to l)e arraiif^cd iu i;roui)s or Foci; ouly occasionally arc they found sinj^'ly. These foci are formed during;' life, as is proved by the dei;■enerati^'e changes often seen about them; but it is possible that the bacilli may also multiply somewhat after death. Important Primary Characteristic Lesions in Man.— The lesions of the intestines which are most prt)nouncetl in the lower part of tlic ileum consist of an inflammatory enlargement of the solitary and agmiiiated lymph nodules. Necrosis with ulceration frequently follows the hyperplasia ui the more severe cases. In the se\'erest eases the ulceration and sloughing may involve tlie muscular and peritoneal coats and perforation may occur. Peritonitis and death usually follow. In rare cases the perforation is closed by adhesions. The minute changes are an hj^serplasia of normal elements of the lym- phatic tissue, namely, the lymph cells and the endothelium of the trabeculae and sinuses. In severer forms necrotic changes are apt to intervene. These changes are attributed to the toxic substances formed by the typhoid bacilli, but may be directly brought al)out by the occlusion of the nutritive bloodvessels, as pointed out by Mallory. The mesenteric lymph nodes undergo changes similar to those in the ileum. The spleen is enlarged because of congestion and hyperplasia. The liver and, to a less extent, the kidnej^s are apt to show foci of cell proliferation. In typhoid fever, as in other infectious diseases, toxic poisoning may be mani- fested by disturbances in the circulatory, respiratory, and heat-regulating mechanism as well as by manifest lesions. In a few cases the intestinal lesions are absent. Some of the inflammatory complications which occur in typhoid fever are due to the growth of the bacillus in excessive numbers in unusual places in the body; but many of them are due to a secondary infection with other bacteria, expecially the pyogenic cocci and bacilli of the colon group. Unusual Location of Typhoid Lesions Occurring as Complications of Typhoid Fever. — Cases of sacculated and general peritonitis, ab- scess of the liver and spleen, subphrenic abscess, osteomyelitis, peri- ostitis, and inflammatory processes of other kinds have been reported as being due to the typhoid bacillus. In certain cases of typhoid pneumonia, serous pleurisy, empyema, and inflammations of the f^rain and spinal cord or their membranes, typhoid bacilli exclusively have occurred. The inflammation produced may or may not be accom- panied by the formation of pus. There are indeed a number of cases now on record in which the typhoid bacillus has played the part of ini-s prodvcer. The Importance of Mixed Infection. — Frequently when complications occur in typhoid fever they are due to secondary or mixed infection with the staphjdococcus, pneumococcus, streptococcus, pyocyaneus, and colon bacillus. Frequently these bacteria are found side by side with typhoid bacilli; in such cases it is difficult to say which was the primary and which was the secondary infection. Elimination of Typhoid Bacilli from Body. — Not infrequently typhoid bacilli are found in the secretions. They are present in the urine in about 25 to 50 per cent, of the cases during some part of the disease. Slight pathological lesions in the kidneys almost always occur in typhoid fever, but severe lesions also sometimes occurs. In some cases the urine is crowded with typhoid bacilli. 360 PATHOGENIC MICROORGANISMS In cases of pneumonia due to the tyijhoid bacillus it is abundantly present in the sputa, and care should be taken to disinfect the expec- toration of typhoid patients. In typhoid fever the bacilli are almost always present in the gall-bladder. The bacilli are usually eliminated by the feces, being derived from the ulcerated portions of the intestines; their growth within the intestinal contents is, with few exceptions, not extensive. They may be excreted, however, very early in the disease, and have been found in the stools even during the incubation period. Not only do the very great majority of cases examined bacterio- logically and pathologically, but the epidemiological history of the disease, proves that the chief mode of invasion of the typhoid bacillus is by way of the mouth and stomach. The infective material is dis- charged principally by means of the excretions and secretions of the sick — namely, by the feces, the urine, and occasionally by the sputum. Occurrence in Healthy Persons. — The bacilli usually disappear from the body in the fourth or fifth week, but may remain for months or exceptionally years in the urine and throughout life in the gall- bladder. They have been found in deep abscesses one year after recovery from typhoid fever. Typhoid Carriers. — Examinations of convalescent typhoid cases show that about 1 to 5 per cent, continue to pass typhoid bacilli for years, perhaps for life. Petruschky in 1898 reported that typhoid bacilli sometimes remained in the urine of typhoid convalescents for months. Gushing soon after observed a case who had had ty- phoid fever five years before. In 1902 Frosch, and a little later Conradi and Drigalski, reported persons who passed typhoid-infected feces months after recovery from typhoid fever. Some bacilli carriers did not know either that they had had typhoid fever or been in contact with it, and others knew only that they had been in contact with it. In Washington, typhoid bacilli were isolated 0.3 per cent, of persons with no known exposure, but who did not or had not had typhoid. These cases may be called "healthy or normal carriers" in contra- distinction to "convalescent carriers." Lentz in 1905 found out of a large number of examinations that about 4 per cent, of persons convales- cent from typhoid fever were typhoid carriers. In our laboratory we have found six in one hundred and forty institution convalescents. The focus of infection is either the gall-bladder or gall-ducts. The majority are women. A remarkable case of a cook was under our care for three years. A visitor of the family in which this woman was cook developed typhoid fever some ten days after entering the household. This was in 1901. The cook had been with the family three years and it is difficult to judge which infected the other. The cook went to another family. One month later the laundress in this family was taken ill. In 1902 the cook obtained a new place. Two weeks after arrival the laundress was taken ill with typhoid fever; in a week a second case dc\'eloped and soon seven members of the household were sick. COMMUNICABILITY 3G1 In 1904 the cook went to a lioine in I>oiig Island. There were 4 in the family as well as 7 ser^'ants. Witliin ;*> weeks iifter arrival, 4 servants were attacked. In !!)()() the (xH)k went to another family. Between Angust 27 and September 3, (> ont of its 11 inmates were attacked with typhoid. At this time the cook was first suspected. She entered another family on September 21. On October 5, the laundress developed typhoid fever. In 1907 she entered a family in New York City, and two months after her arrival two cases developed, one of which proved fatal. Altogether during fi-\-e years this cook is known to have been the cause of 2G cases of typhoid fever. The cook was removed to the hospital March 19, 1907. Cultures taken every few days showed bacilli off and on for three years. Some- times the stools contained enormous numbers of typhoid bacilli and again for days none would be found. We recently traced some hundreds of cases of typhoid fever to a milk supply produced at a farm, looked after by a typhoid carrier who had typhoid fever forty-seven years ago. Treatment of Typhoid Carriers. — Medicinal treatment or surgery seems so far to have yielded only slight results. Urotropin in very large amounts is reported to have cured one case, in which operation alone had failed. Removal of the gall-bladder cannot be relied upon, as the bacilli may be present in the gall-ducts. Meader reports some success with long-continued treatment with killed cultures. Duration of Life Outside of the Body. — It is of importance to know for what length of time the typhoid bacillus is capable of liv- ing outside of the body; but, unfortunately, owing to the great diffi- culties in proving the presence of this organism in natural conditions, our knowledge on this point is still incomplete. In feces the length of life of the typhoid bacilli is very variable, depending on the composi- tion of the feces and on the varieties of bacteria present; sometimes they live but a few hours, usually a day, exceptionally for very long periods. Thus, according to Levy and Kayser, in winter typhoid bacilli may remain alive in feces for five months. Foote says that they can be found in living oysters for a month at a time, but in nu- merous experiments we have not been able to find them after five days. Their life in privies and in water, however, is usually very much shorter. As a rule, they can be detected in river water no longer than seven days after introduction, and often not after forty-eight hours. The less the general contamination of the water, the longer the bacilli are apt to live. The life of the typhoid bacillus varies according to the abundance and varieties of the bacteria associated with it, and accord- ing to the presence or absence of such injurious influences as deleterious chemicals, high temperature, light, desiccation, etc., to which it is known to be sensitive. In ice typhoid bacilli rapidly die, none prob- ably ever live as long as six months (see p. 372). Communicability. — The bacilli maj^ reach the mouth by means of infected fingers or articles of various kinds, or by the ingestion of infected food, milk, water, etc., or by more obscure wa>'s, such as the 3(12 PATHOGENIC MICROORGANISMS eating of raw oysters and clams or the contamination of food by files. Of tlie greatest importance, however, is the production of infection by contaminated drinking-water or milk. In a very hirge numl;er of cases indirect proof of this mode of infection has been afforded by find- ing that the water had been contaminated with urine or feces from a case of typhoid. In a few instances the proof has been direct — namely, by finding typhoid bacilli in the water. Examples of in- fection from water and milk have frequently come under our direct observation. The following instances may be cited: A large force of workmen obtained their drinking-water from a well near where they were working. Typhoid fever broke out and continued to spread until the well was filled up. InA'estigation showed that some of the sick, in the early stages of their disease, repeatedly infected the soil surrounding the well with their urine and feces. Another example occurred in which typhoid fever broke out along the course of a creek after a spring freshet. It was found that, far up near the source of the creek, typhoid feces had been thrown on one of its banks and had then been washed into the stream. In the late epidemic at Ithaca some 1500 cases developed among those using the infected water supply of the town. The students and townspeople not drinking the infected supply escaped. The epi- demic at Scranton, Pa., during the winter of 1907 was most interesting. A little over 1 per cent, of the inhabitants were attacked. No pollu- tion of the water with typhoid infected feces or urine could be discov- ered, although typhoid bacilli were isolated from the water of a small intercepting reservoir by Dr. Fox. This was only accomplished by using large quantities of water. The bacillus isolated was identical by all known tests with the typhoid cultures from cases of typhoid fever. An instance of milk infection secondary to water infection was in the case of a milk dealer whose son came home suffering from ty- phoid fever. The feces were thrown into a small stream which ran into a pond in which the milk cans were washed. A very alarming epidemic of typhoid developed, which was confined to the houses and asylums supplied with this milk. During the Spanish-American war not only water infection, but food infection was noticed, as in the case of a regiment where certain companies were badly infected, while others nearly escaped. Each company had its separate kitchen and food supply, and much of the infection could be traced to the food, the contamination coming partly through the flies. Several epidemics have been traced to oysters. Contact infection is probably responsible for as many cases as are due to the causes, already gi^-en. Even during outbreaks due to these causes, contact infection plays a large role in the development of secondary cases. These cases develop either by contact with known typhoid cases or through convalescent or healthy carriers in the households. The early undiagnosed cases may have typhoid bacilli in their stools and act as a source of contagion; like- wise short, mild cases develop which are never surely diagnosed. VACClNAriON AGAINST TYPIIOfD FEVER 3fi3 For these reasons "typlioid preciiutions" sliould not await positive cliaj^'nosis, hnt shonid he instituted on tiie sligiitest snspieion. Individual Susceptibility. — In this, as in all infeetious diseases, utdividiKil .s- II, SVC pi ill i I it 1/ plays an important role in the produetion of infeetion. Without a suitable soil upon wliieh to fi-row, the seed cannot thrive. There must in many be some disturbanee of the di- gestion, excesses in drinking, etc., or a general weakening of the power of resistance of the individual, caused by bad food, exposure to heat, overexertion, etc., as occurs with soldiers and prisoners, for example, to bring about the conditions suitable for the production of typhoid fever. Immunization. — After recovery from typhoid fever a considerable immunity is present which lasts for years. This is not absolute, as about 2 per cent, of those having typhoid fever have a second attack, which is usually a mild one. Specific immunization against experi- mental typhoid infection has been produced in animals by the usual method of injecting at first small quantities of the living or dead typhoid bacilli and gradually increasing the dose. The blood-serum of animals thus immunized has been found to be highly bactericidal and to possess some protective power against toxic extracts of the bacilli. It is also rich in agglutinins, precipitins, and opsonins. These characteristics have also been observed in the blood-serum of persons who are con- valescent from typhoid fever. The attempt has been made to employ the typhoid serum for the cure of typhoid fever in man, but, although a number of indi\'idual observers have reported good results with one or another of the sera, most consider that little or no good is derived from serum. Vaccination During Typhoid Fever. — The value of vaccines in the treatment of typhoid fever has not as yet been definitely determined, although used by Richardson and later by Watters and Eaton' and Callison.^ The use of vaccines early in the disease in doses of 500,000,000 every second or third day would seem to shorten the course of the disease in a certain number of cases and to produce a somewhat lower death rate. Very recently Boinet^ has reported favorable results in twenty-five cases treated with sensitized vaccines prepared according to the method of Besredka. Vaccination Against Typhoid Fever. — The vaccination against typhoid fever is now a procedure of established worth. Its use began in 1896, when Pfeiffer and Kolle and, independently, Wright, demon- strated that persons injected with killed typhoid bacilli developed the same antibodies in their blood as are found in recovered cases of typhoid fever. Wright then introduced its use in the English army, and the results led to its introduction in other countries. Vaccination was started in the American army in 1909 under the supervision of Russell. The rate per 1000 in the whole American army has dropped from 3.2 in 1908 to 0.03 in 1913. During the recent maneuvers in Texas 1 Med. Rec, 1911, Ixxix, 797. = Ibid., 1911, Ixxix, 1129, ' Ann. dc I'lnst. Pasteur, 1914, xxviii, .540. 304 PATHOGENIC MICROORGANISMS there were 20,000 men in the encampments with only two cases of typhoid during a period of four months. This remarkable improve- ment over the rate in encampments of previous years is ]jrol)al)ly due, in large part, to improvements in camp sanitation, but not altogether. The soldiers visited ate and drank at Galveston and San Antonio, and in the former city there were 192 cases and in the latter 49 cases of typhoid fever during the same period of time. The civil population in this way acts as a control Similar results have been obtained in the English army. To cite one instance only. Firth reports that in India for 1911 the rates per thousand were: vaccinated 1.7, deaths 0.17; unvaccinated 6.7; deaths 1.15. These figures are interesting not only because of the reduction in cases, but it gives a fair estimate of the reduction in the death rate of typhoid among those vaccinated. Similar results have followed its use in civil life. It has given good results even in the presence of an epidemic. CuUinan, for instance, vaccinated 500 persons in an asylum during an epidemic. Of the vac- cinated only 1.36 per cent, contracted typhoid, and these were nearly all in the incubation period of the disease; of the unvaccinated, 114 nurses, 14.9 per cent., contracted typhoid. The use of vaccine during exposure has not led to any results, therefore that would warrant the fear of the production of a "negative phase," that is, reduction of resistance to infection. Since January, 1913, the Department of Health of New York City has detailed inspectors to vaccinate on request. The members of families where typhoid exists are especially urged to be vaccinated. The inspectors have vaccinated 2794 persons, of which about 80 per cent, have received full doses. Of these, 1014 were not directly exposed to infection and none have developed tj'phoid. The exposed persons numbered 1780, among these 31 cases of typhoid developed, the great majority recei%dng only one injection, and were already in the incuba- tion period of the disease. Two cases receiving three doses developed typhoid fever within ten days of the last dose. Of the remaining cases two nurses exposed to infection developed typhoid, one four months, the other eight months after injection. One died of hemorrhage. Another case died six months after vaccination of typhoid complicated by pregnancy with miscarriage. Her mother was a typhoid carrier, and four of her children had typhoid about the same time. Of about 8000 persons exposed to typhoid during the above period of time there have been 190 cases of typhoid. During the same time vaccine for over 2000 persons has been given to private physicians and institutions. No cases of typhoid have been reported among the persons injected. We know of two other cases of typhoid after vaccination, one a nurse who died of hemmorrhage, the other a physician who recovered. These cases show that the protection conferred is only relative, and with an undue amount of exposure infection may result. The duration of the immunity is shown liy the experience of Firth in the Indian army. He found that the immunitv diminished after USE OF DRIED BLOOD 365 two and one-half years, but even after four to five years some im- munity persists, the rate among the A'accinated being only one-fourth of that among the unvaccinated troops. Three doses of vaccine are administered, the site of injection being the subcutaneous tissues over the insertion of the deltoid. The first dose is 500,000,000, the second and third 1,000,0()(),0()0 typhoid bacilli. The period between injections is seven to ten days, the injection being given in the late afternoon, so that if a reaction occur the person will be in bed. The reaction usually consists of a reddening and swelling at the point of injection, which is tender. In some instances this area is large and the axillary lymph nodes become swollen and tender. A general reaction is usually absent, or if present consists of slight malaise. In a few cases, less than 1 per cent., a more marked reaction occurs with prostration and considerable rise of temperature. The reaction is of no importance further than the discomfort caused. The vaccine usually employed is a suspension of the bacilli grown on agar, standardized by the method of Wright and killed by heating to 56° C. Sensitized vaccines of either killed or live bacilli have also been used, but their use has been too limited to warrant their general adoption as superior to the vaccine in general use. Diagnosis by Means of the Widal or Agglutination Reaction, — The chief practical application of our knowledge of the specific substances developed in the blood of persons sick with typhoid fever has been as an aid to diagnosis. Gruber-Widal Test. — The first application of the use of serum for the early diagnosis of typhoid fever on an extensive scale was made by Widal, and reported with great fulness and detail in a communication published in June, 1896. For history and theory of test, see p. 201. Since then the serum test for the diagnosis of typhoid fever has come into general use in bacteriologic laboratories in all parts of the world, and though the extravagant expectations raised at the time when Widal first announced his method of applying this test have not been entirely fulfilled, it has, nevertheless, proved to be of great assistance in the diagnosis of obscure cases of the disease, and is now one of the recognized tests for the dift'erentiation of the typhoid bacillus. It should also be mentioned that to Wyatt Johnson, of Montreal, belongs the credit of introducing its use into municipal laboratories, suggesting that dried blood should be employed in place of blood-serum, and in October, 1896, the serum test was regularly introduced in the New York Department of Health Laboratory for the routine examina- tion of the blood-serum of suspected cases of typhoid fever. Since then all health departments have followed the example set by those of Montreal and New York. Use of Dried Blood. — Directions for Preparing Specimens of Blood. — The skin covering the tip of the finger or the ear is thoroughly cleansed, and is then pricked with a needle deeply enough to cause several drops of blood to exude. Two fair-sized drops are then placed 3(36 PATHOGENIC MICROORGANISMS on a glass slide, one near either end, and allowed to dry. Glazed paper may also be employed, but it is not as good, for the blood soaks more or less into it, and later, when it is dissolved, some of the paper fibre is apt to be rubbed off with it. The slide is placed in a box for protection. Preparation of Specimen of Blood for Examination. — In preparing the specimens for examination the dried blood, if accuracy is desired, is first weighed and then brought into solution bj' adding to it the quantity of normal salt solution to make the desired dilution, remembering of course to allow for the loss in water through drying. A drop of each dilution is placed on individual cover-slips and to each is added a drop of an eighteen-hour broth culture of typhoid. These are then mounted on hollow shdes in the ordinary way. (See p. 72.) The drops, after being mixed, have in a 1 : 10 dilution a distinct reddish color and in 1 ; 20 a faint pink tinge. The cover-glass with the mixture on the surface is inverted over a hollow slide (the edges about the concavity having been carefully smeared with vaselin, so as to make a closed chamber). Ordinarily the dried blood is not weighed, but the measure of dilution is estimated by the color of the drop. To judge this the beginner must carefully make dilutions of fluid blood and notice the depth of color in 1 : 10 and 1 : 20 dilutions. Besides the faulty judg- ment of the dilution color by the examiner, the variation in depth of color of different specimens of blood makes the estimation of dilu- tions more or less inaccurate, but fortunately this does not greatly interfere with the value of the test. Use of serum or fluid blood. (See Agglutination for methods.) Advantages and Disadvantages of Serum, Dried Blood, and Fluid Blood for the Serum Test. — The dried blood is easily and quickly obtained, and does not deteriorate or become contaminated by bacterial growth. It is readily transported, and seems to be of nearly equal strength with the serum in its agglutinating properties. It must in use, however, be dilutefl with at least five times its bulk of water, otherwise it is too viscid to be properly employed. The amount of dilution can only be determined roughly by the color of the resulting mixture, for it is impossible to estimate accurately the amount of dried blood from the size of the drop, and it is generally considered too much trouble to weigh it accurately. Serum, on the other hand, can be used in any dilution desired, from a mixture which contains equal parts of serum and broth culture to any dilution desired; and this can be exactly measured by a graduated pipette, or, roughly, by a measured platinum loop. The disarlvantages in the use of serum are entirely due to the slight difficulty in collecting and transporting it. If the serum is obtained from blood after clotting has occurred a greater quantity of blood nnist l)e drawn than is necessary when the dried-blood method is used. For scientific investigations and for accurate results, par- ticularly ill ol)scure cases, the use of serum is to be preferred to dried blood. Practically, however, the results are nearly as good for diag- nostic purjtoses from the dried blood as from the serum. DILUTION OF THE BLOOD-SERUM 'M')7 The Culture to be Employed. — It is important that the culture em- ployed for serum tests should l)e a suitable one, for although all cul- tures show the reaction, yet some respond much better and in higher dilutions than others. Cultures freshly obtained from typhoid cases are not as sensitive as those grown for some time on nutrient media. Those kept for a long time on artificial media sometimes show a decided tendency to spontaneous agglutination. Artificial cultivation is usually accompanied by increase of capacity for agglutination. At present a strain known as Mt. Sinai is used at the laboratory. A broth culture of the typhoid bacillus developed at 25° to 35° C, not over twenty- four hours old, in which the bacilli are isolated and actively motile, has been found to give us the most satisfactory results. If the broth culture is heavy it should be diluted. Cultures grown at temperatures over 38° C. are not apt to agglutinate so well as those grown at lower temperatures. Stock cultures of typhoid bacilli can be preserved on nutrient agar in sealed tubes and kept in the ice-box. These remain alive for months. From time to time one of these is taken out and used to inoculate the broth. Dilution of the Blood-serum to be Empolyed and Time Required for the Development of Reaction. — The serum test, as has been pointed out, is quantitative and not qualitative, that is, the diagnostic reaction must be in dilutions higher than occur because of the presence of normal or group agglutinins. (See p. 195.) It is most important to remember that it is purely a matter of experience to determine in any type of infection what agglutinating strength of a serum is of diagnostic value. The results obtained in the Health Department Laboratories, as well as elsewhere, have shown that in a certain proportion of cases not tj'phoid fever a slow reaction occurs in a 1 : 10 dilution of serum or blood ; but very rarely does a complete reaction occur in this dilution within fifteen minutes. When dried blood is used the slight tendency of non-typhoid blood in 1-: 10 dilution to produce agglutination is increased by the presence of the fibrinous clumps, and perhaps by other substances derived from the disintegrated blood cells. From many eases examined it has been found that in dilutions of 1:20 a quick reaction is almost never produced in any febrile dis- ease other than due to typhoid or paratyphoid bacillus infection, while in typhoid fever such a distinct reaction often occurs with dilutions of 1 : 100 or more. It is possible that some cases of paratyphoid infection give a prompt reaction in 1 : 20 dilutions, but if this is so, it is not a serious drawback. The rare cases of persons who, though never having had typhoid fever, yet are typhoid-bacillus carriers usually have specific agglutinins in their blood. The mode of procedure as now employed is as follows: With serimi, one part of a 1: 10 dilution is adderl to one of the bouillon culture. With dried blofid, a solution of the blood is first made, and the dilution guessed from the color. To obtain an idea of the dilution by the color, known amounts of blood are dried and then mixed with definite amounts .3GS PATHOGENIC MICROORGANISMS of water ; the colors resulting are fixed in the memory as guides for future tests. If there is no reaction — that is to say, if within five minutes no marked change is noted in the motility of the bacilli, and no clumping occurs — nothing more is needed; the result is negative. If marked clumping and immobilization of the bacilli immediately begin and become complete within five minutes, this is termed a marked immediate typhoid reaction, and no further test is considered necessary, though it is always advisable to confirm the reaction with higher dilutions up to 1 : 50 or more, so as to measure the exact strength of the reaction. If in the 1 : 20 dilution a complete reaction takes place within thirty minutes, the blood is considered to have come from a case of typhoid infection, while if a less complete reaction occurs it is considered that only a probability of typhoid infection has been established. By many the time allowed for the development of the reaction with the high dilutions is from one to two hours, but to us twentj' minutes with the comparatively low dilution of 1 : 20 seems safer and more convenient. Positive results obtained in this way may be considered conclusive, unless there be grounds for suspecting that the reaction may be due to a previous fairly recent attack or to recent vaccination. The failure of the reaction in one examination by no means excludes the presence of typhoid infection. If the case clinicall\' remains doubtful, the examination should be repeated every few days. Use of Dead Cultures. — Properly killed typhoid bacilli respond well to the agglutination test. For the physician at his office the dead bacilli offer many advantages. The reaction is slower than with the living cultures and is observed either macroscopically or microscopically. A number of firms now supply outfits for the serum test. These outfits consist of a number of small tubes containing an emulsion of dead typhoid bacilli. Directions accompany the outfit. Proportion of Cases of Typhoid Fever in which a Defimte Reaction Occurs, and the Time of its Appearance. — As the result of a large num- ber of cases examined in the Health Department Laboratories, it was found that about 20 per cent, give positive results in the first week, about 60 per cent, in the second week, about SO 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 98 per cent, of the cases in which repeated examinations were made (hospital cases) a definite typhoid reaction was present at some time during the illness. Persistence of the Reaction. — A definite typhoid reaction has been observed from three months to a year after convalescence, and a slight reaction, though much less than sufficient to establish a diagnosis of typhoid infection, from one to fifteen years after the disease. In persons in whom the typhoid bacilli persist tine serum reaction may last as lung as tlie bacilli remain in the body. Typhoid BaciUi in the Blood. — A blood-culture is usually positive during the lirst week of tyi^hoid fever, and is the best method for early diag- nosis. They appear in the blood even in the first few days of the disease. In the first week, nearly 100 per cent, positive results are obtained. TYPHOID BACILLI IN FECES ;]()9 in the second week 50 per cent, and then progressively less till the end of the disease. In relapses they reappear. Method. — The blood is drawn from the median basilic vein by syringe and is inoculated into either broth or bile medium. If broth is used, several flasks are used with sufficient broth to dilute the blood 50 times or more, so that if there are any bactericidal substances in the blood they will be too diluted to act Bile media (see p. 105) are more convenient, as the bulk need not be so large. The bile inhibits coagulation. Three parts of bile medium are used to one part of blood. The bile is placed on Endo, or Conradi, after eighteen hours' incubation. It should also be examined after two to three days, as growth is sometimes delayed. Another method, not as satisfactory as the preceding, but valuable in emergencies, is to inoculate the blood into ten times its volume of distilled water. Hemolysis results and the proteins supply the nutrient substances for growth. Typhoid Bacilli in Feces. — The following table by Schroder gives the number and time of positive results: No. examined. Positive. Per cent. First week .... . . 115 11 9.6 Second week .... . , 160 45 28.1 Third week .... . . 71 16 22.5 Fourth week .... . . 41 9 21.9 Fifth week .... . . 26 4 15.3 Sixth week . . . . . . 12 2 16.6 Convalescent . . . . . . 190 10 7.9 These results were obtained, using several media in combination. The more promptly the stools are plated after passage, the better is the chance of obtaining typhoid bacilli. Hiss, examining the stools very quickly after passage, obtained a very high percentage of positive results, in 43 consecutive cases, 37 of which were in the febrile stage and 6 convalescent. In a number of instances only one stool was examined, but even under these adverse conditions the average of positive results in the febrile stage was 66.6 per cent. Out of 26 cases of typhoid fever examined in hospitals, 21 were in the febrile stage and 5 convalescent. In the febrile ca.ses in 17 the presence of typhoid bacilli, often in great numbers, was demonstrated. Thus in these care- fully followed cases the statistics show over 80 per cent, of the febrile cases positive. The bacilli were isolated from these cases as early as the sixth day, and as late as the thirtieth day, and in a case of relapse on the forty-seventh day of the disease. These observations, with regard to the appearance of the bacilli in the stools during the febrile stage and their usually quick disappearance, except in the permanent typhoid carriers, after the defervescence, have been confirmed by others. We have, however, a carrier under observation who was discharged from the hospital after three negative examinations. The bacilli are isolated in some cases in which no Widal reaction was demonstrated, 24 370 PATHOGENIC MICROORGANISMS or before its appearances. Between the seventh and twenty-first day of the disease, experience seems to indicate that the bacilh may be obtained from about 25 per cent, of all cases on the first examination and from about 75 per cent, after repeated examinations. In some samples of feces typhoid bacilli die out or are overgrown within twenty- four hours; in others they remain alive for days or even weeks. This seems to depend on the bacteria present in the feces and upon its chemical character. Probably the presence of typhoid bacilli in some stools and their absence in others must be explained largely by the character- istics of the intestinal contents. The short life of the typhoid bacillus in many specimens of feces suggests that stools be examined as quickly as possible. In fact, unless the physician wishes to take the trouble to have the sample of feces sent immediately to the laboratory, it is hardly worth while for the bacteriologist to take the trouble to make the test. Method. — The feces, if solid, are rubbed up with broth, otherwise they can be used without further preparation. The fluidified feces should not consist of merely a loopful of the outer portion, but a mixture of a generous amount. The density of the suspension to employ is a matter of experience. Plates are then streaked (see p. 108) and the colonies of typhoid can then be agglutinated directly either microscopically or by using the slide method. In the latter a drop of saline for control and a drop of high-titre serum are placed on a slide and the colony rubbed up in the saline and then in the serum. With appropriate dilutions of the serum agglutination will take place almost immediately. For fishing, Russell's medium is used, which gives a tentative indica- tion of the nature of the organism, which can be confirmed b}' agglu- tination and further cultural tests. Typhoid Bacilli in the Urine. —Of great interest is the frequent occurrence of typhoid bacilli in large numbers in the urine. The results of the examinations of others as well as our own indicate that the typhoid bacilli are not apt to be found in the urine until the end of the second week of the fever, and may not appear until much later. From this on to convalescence they appear in about 25 to 50 per cent. of the cases, usually in pure culture and in enormous numbers, even as high as 100 million per c.c. They are found until several weeks or months after convalescence; in exceptional cases they persist for years. When we think of the chances such cases have to spread infection as they pass from place to place, we begin to realize how epidemics can _ start without apparent cause. The more we investigate the persistence of bacteria in convalescent cases of disease, the more diffi- cult the prevention of their dissemination is seen to be. The disinfection of the urine should always be looked after in typhoid fever, and con\'alescents should not be allowed to go to places where contamina- tion of the water supply is possible, without at least warning them of the necessity of great care in disinfecting their urine and feces for some weeks. IMPORTANCE OF ICE IN PRODUCTION OF TYPHOID FEVER 371 Method. — If the bacilli are numerous, as evidenced by examination in the hanging drop, they are easily obtained by direct plating, other- wise the urine should be centrit'uged and the sediment plated, and larger amounts inoculated into bile medium for enrichment. Typhoid Bacilli in Rose Spots and Spleen. — Although the bacilli have been frecpiently isolated from rose spots, it is a less convenient method than blood-cultures. Spleen puncture has been employed and although cultures are frequently positive the operation is dangerous, and has been abandoned. Detection of Typhoid Bacilli in Water. — There is absolutely no dou1)t that the contamination of streams and reservoirs is a frequent cause of the outbreak of epidemics of typhoid fever, but the actual finding and isolation of the bacilli is a very rare occurrence. This is often due to the fact that the contamination has passed away before the bacteriologie examination is undertaken, and also to the great difficidties met with in detecting a few typhoid bacilli when they are associated with large numbers of other bacteria. The greater the amount of contamination entering the water, and the shorter the time which elapses between this and the drinking of the water, the greater is the danger. A recent isolation of the typhoid bacillus is that from the small storage reservoir supplying Scranton. The bacillus was isolated by Fox from about 1 liter of water. Tested alongside of a culture from one of the Scranton cases it seemed identical The Importance of Ice in the Production of Typhoid Fever. — The total number of instances of typhoid fever which have been directly traced to ice infection are remarkably few. One was in France, where a group of officers placed ice made from water polluted by a sewer in their wine anfl afterward a large percentage developed typhoid fever, while those of the same companv' not using ice escaped. A second case was a small epidemic which occurred in those w'ho used ice from a pond. It was found that water directly infected with typhoid feces had flowed over its frozen surface and been congealed there. The fact that freezing kills a large percentage of typhoid bacilli makes it indeed possible to conceive that ice from moderately infected water contains so few li\'ing typhoid bacilli that only the exceptional person here and there becomes infected, and so the source of the infection remains undetected. If this be true and scattered cases occur, there should be at least some increase on some or every year in March, April, and May in such a city as New York, where four-fifths of all the ice consumed is from the Hudson River, which is known to be contaminated with typhoid bacilli. When we examine the records for the past ten years we find no in- crease of typhoid fever in Greater New York during those months, with the one exception of 1907, when we had in the borough of Man- hattan a sharp outbreak lasting four weeks. This outbreak did not occur at all in Brookljai. As the people of Brooklyn drank different water, but received ice from the same places of the Hudson River as 372 PATHOGENIC MICROORGANISMS those of Manhattan, this directed attention to the water or milk rather than the ice. Examination of the Croton watershed at the time showed that a small epidemic of typhoid existed there and that pollution of the water was probable. This suggested still more strongly that the water and not the ice was the cause of the typhoid infection. It happened that most of the cases occurred in those living in the section of the upper West Side, where only well-to-do people live. An investigation showed that the majority of the infected had used only artificial ice and several had used no ice in their water at all. Life of the Typhoid Bacillus in Ice in Laboratory Experiments. — The first important investigation was that of Prudden, who showed that typhoid bacilli might live for three months or longer in ice. This experi- ment is frequently wrongly interpreted, as when a recent writer states: " It has been amply demonstrated that the germs of typhoid fever are not killed by freezing and that they have been known to live in ice for long periods of time." It is true that in Prudden's experiment a few typhoid bacilli re- mained alive for three months, when the experiment was terminated, but those were but a small fraction of 1 per cent, of the original num- ber. Following Prudden's experiment Sedgwick and Winslow in Boston and Park in New York Tity carried on independently a series of experi- ments. These led to the same conclusions. A table summarizing a final experiment of ours in which twenty-one different strains, mostly of recent isolation, were subjected to the test is given below: Life of Twenty-one Stbain.s of Typhoid Bacilli in Ice. Before freezing Frozen three days Frozen seven days Frozen fourteen days Frozen twenty-one days .... Frozen fi\'e weeks P^rozen nine weeks Frozen sixteen weeks Frozen twenty-two weeks .... In these experiments twent\'-one different flasks of Croton water were inoculated each with a different strain of typhoid bacilli. In one a little of the feces rich in typhoid was directly added. The in- fected water in each flask was then pipetted into thirty tubes. These tubes were placed in a cold-storage room in which the temperature varied from 20 to 28° F. At first tubes were removed and tested twice a week, later once a week. The object of using so many different strains was because it has become evident that some cultures live longer than others. At the end of five weeks the water infected with six cultures was sterile, at the end of sixteen weeks only four strains remained alive. Interesting investigations of Hudson River ice were carried out in 1907 by North. Average number Percentage typhoid of bacilli in ice. bacilli living. 2,560,410 100.0 1,089,470 42.0 :J61,136 14,0 203,.300 8.0 10,280 0.4 2,950 0.1 127 0.005 107 0.004 DIFFERENTIAL DIAGNOSIS 373 There was noticed a considerable diflVrenee between tlie nnmber of bacteria in tlie toj), middle, and bottom layers of ice. This is natural, since while water in freezin<>' from above downward markedly purihes itself, 7") per cent, of the solids and a fair proportion of bacteria beinj;' eliminated, yet this cannot happen in the case of the snow blanket which becomes flooded by rain or by cutting holes through the ice. Here all impurities, such as dust and leaves which have fallen on the surface and dirt which may come from the water, remain with the bacteria which they carry, since all are retained in the porous snow. The bacteria in freshly cut bottom ice generally show the least destruc- tion by freezing. The river water in the forty specimens averaged ISOO bacteria per c.c, the top ice 306, the bottom ice 3(i, and the middle ice 14. Only four specimens of top ice had over 500 bacteria per c.c. ; none of the specimens of middle or bottom ice. The great destruction by freezing is noticeable in these figures. Even the top ice soiled by the horses and men gathering it contained but 16 per cent, as many bacteria as the water from which it was obtained. The bottom ice, the last to be frozen, had but 2 per cent, of those in the water. Conclusions in Regard to Ice Pollution. — The danger from the use of ice produced from polluted water is always much less than the use of the water itself. Every week that the ice is stored the danger be- comes less, so that at four weeks it has become as much purified from typhoid bacilli as if subjected to sand filtration. At the end of four months the danger becomes almost negligible, and at the end of six months quite so. Diiferential Diagnosis. — The typhoid bacillus is easily separated from the other members of the group, and for practical purposes it is sufficient identification if the colony on the special plating media and the growth on Russel's medium is characteristic and is agglutinated in relatively high dilutions of serum. If a strain does not agglutinate freely it should be transferred daily on plain agar and again tested. The range of group agglutinins in the serum used should be known or error will result. In important examinations or strains from unusual sources as water, etc., a more extensive cultural study should be done. Likewise, absorp- tion of agglutinins and possibly the production of agglutinins for a known strain should be tested. B. (fecaUs) AUcaUgenes. — This group resembles the typhoid bacillus but produces acid from no sugars. It is frequently present in the intestines. It is usually only a saprophyte, but has been found in a few cases of disease in man. Other typhoid-like bacilli have been isolated from cholera infected swine, cow's feces, and water. CHAPTER XXV. PARATYPHOID GROUP. Gartner, in ISSS, found the Bacillus cnteriiidis in association with a meat-poisoning epidemic. A cow sick for two days with profuse diarrhea had been shxuglitered and the meat sold for food. Of the persons eating the meat fifty-seven became ill and one died. Similar bacilli were isolated by Smith from swine suffering from hog cholera, from mice by Loffler, and the bacilli became known as the hog cholera group. Similar bacilli were isolated by others from cases resembling typhoid fever. Schottmiiller reported in 1900 that he was able to differentiate the strains from 5 cases into two groups, one resembling typhoid very closel}', the other corresponding to the enteritidis types. The work of Durham and Buxton gave the basis for classification into subgroups. Numerous types have been isolated from infections in man and animals, and most of the terms in use refer to their source; but many cannot be differentiated one from the other, so fall into distinct groups according to their bacteriological and immvme reactions. Paratyphoid A. — Disease Produced. — First isolated by Gwyn, later by Schottmiiller,^ Brown, and Kayser, and others. It produces a typhoid- like disease in man, but is of relati\'ely infrecjuent occurrence. The bacillus may be present in the feces, urine, blood, and bile. The post- mortem findings have been variable. In some the typical intestinal lesions of typhoid infections were present, in others they were absent. A diffuse inflammation of the intestines may be found. Morphology and Biology. — Similar to typhoid in many respects, but resembles Paratyphoid B. in its sugar fermentations (see table, p. 345). Communicability. — Communicability is tlie same as for typhoid. It has been isolated in a few instances from animals, and perhaps in water and foodstuff's, but its distribution can in no way be compared with that of the other members of this group. Diagnosis. — See below. Paratyphoid B. — This group includes many types, isolated not only in disease of man but also in the disease of many animals. Disease Produced in Man. — This varies and may be subdivided into several clinical types, depending on whether the disease is primarily an infection or an intoxication. Typhoid-like Type. — A variable jjcriod of incubation is usually present. The onset is commonly more acute than in typhoid fever, and is usually, accompanied by chills. As a rule it runs a shorter and milder course. ^ MiiHer strain. COMMUNICABILITY 375 In some epidemics, aborti\'c attacks occur with no symptoms except fever for a short time. Tlie hacili may be present in the feces, blood, urine, and gall-bladder; in tlie first two, early in the disease and com- monly during' the whole ct)nrse. ("om|)li("itions similar to those in typhoid may occur. (t astro-enteric Type. — The onset is acute and the symptoms come on promptly after eating the infected food or after twenty-four to forty- eight hours. The symptoms are those of an acute enteritis ushered in liy chills and rise of temperature. Nervous symptoms are common anil may be se^•e^e. The bacillus can be isolated from the blood as early as the first day of the disease. In the mild cases cultures are nega- tive. They are present in the stools during the acute stage but quickly disappear in the acute cases. Cholera-hlie Type. — This differs from the preceding only in the degree of the toxic and intestinal symptoms. The bacillus may be present in the blood. Localized infections occur as complications, occasionally as primary infections without any other symptoms of paratyphoid infection elsewhere, such as pyelitis (sometimes associated with septicemia), cystitis, arthritis, periostitis, phlebitis, cholecystitis, etc. It occasion- ally occurs as a complicating infection of other diseases. Occurrence in Healthy Persons; Bacillus Carriers. — The bacillus may be found in the urine after convalescence for shorter or longer periods of time. Its localization in the gall-bladder and its excretion in the feces by chronic carriers is fairly frecjuent. As a rule the acute cases do not develop into carriers. Careful bacteriologic examination has revealed bacilli of the paratyphoid type in the feces of a considerable per- centage of healthy persons. Morphology and Biology. — They closely resembe the other members of this group. For differential characteristics, see table, p. 345. Frequency of the Disease. — In this country the disease is relatively infrequent, though statistics are not available. It is frequent in Europe, especially in certain districts. Communicability. — In coiltrast to typhoid the individual case and the carrier either through contact or by the contamination of waterand milk, etc., are not the only sources of infection. Another important source is the wide distribution of the bacillus in domestic animals. Food infection, how^ever contaminated, plays the important role. Infection has followed the consumption of the following foods: milk and milk products; fish; potato salad; variously prepared string beans. In water it is more capable of a saprophytic existence than the typhoid bacillus, and has been isolated more often. Meats or edible organs from cattle, swine, horse, sheep, geese, as well as various products containing these, are the main sources of infection. Although contamination of meat may occur in other ways the important source in epidemics is meat from infected animals. Such meat may by contact infect meat from healthy animals. Pickled, salted, and smoked meat may be a source of infection. Cooking cannot be relied upon to 376 PATHOGENIC MICROORGANISMS kill tlif bacilli hecause of the slow penetration of the heat. It is occa- sionally found in the feces, meat, and organs of some of the domestic animals, although a,pi)arently healthy. It has also been isolated from meat products and market milk. B. Enteritidis. — In human pathology this bacillus plays the same role as the B. jMratyphosus B. Meat is the usual source of infection, although other foodstuffs have been the cause. The disease is almost always of the acute type (see above). It is found in the feces, urine, and blood. Chronic carriers have not been found, although the bacilli have been found in gall-stones, and probably do occur. They may be present in the stools of healthy persons exposed to infection. Contact infection occurs, but infrequently. The pathological lesions are the same as those caused by the B. Paratyphosus B. Infection of domestic animals occurs and the dissemination of the bacillus is the same as for paratyphoid B. It has not been found in water, probably not in milk, and infrequently in meat products. In cultures it is identical with B. paratyphosus B. For methods of diagnosis see below. Virulence. — Although varying in degree, the various members of the group are highly virulent for white mice, guinea-pigs, rabbits, and rats. Death usually results from a septicemia. Toxic substances are freely produced in fluid media, which are heat resistant. The pxtratyphoid A. is probably the least virulent of the group. Immunity. — In experimental animals immunity is produced by feed- ing and by injection. Immunity is produced not only against the homo- logous strain but, as a rule, also against related strains. It has been suggested that because of the cross-protection of the members of these groups and the production of group antibodies active against typhoid or paratyphoid when one or the other is injected that a poly^'alent vaccine of the typhoid-paratyphoid group be used. Differential Diagnosis of Members of the Groups. — The cultural differentiation onlj' separates the A type from the B and enteritidis type. The members of the latter two groups, whether from man or animals, show no constant differences. Immunity reactions, such as agglutination, fails to separate the members of the different groups, although the different groups are easily separated. As with other allied bacteria, however, group agglu- tinins are present. Other immune reactions give the same results. Variation in Types. — The related enteritidis-paratyphoid strains show a wide ability for variation, not only culturally but in their patho- genicity and virulence. The above-described differences hold good for strains isolated from natural infections. There is every reason to believe that a strain from man can adapt itself and develop a full degree of A'irulence for an animal host, and ince versa. Even its original agglutination reactions may vary in degree, so that there is an ajjparent shifting from the paratyphoid to the enteritidis type; the antigenic properties do not change, however, in a parallel manner. Because of adaptability of the members of the group the pres- ence of any organism belonging to this group should be looked upon as a potential source of infection for man. ENTERITIDIS TYPES 377 Diagnostic Methods.-- The diagnostic iiietliods are the Wiihil reac- tion, hlood-culture, isolation from the stool or nrine or other sites of infection. The use of the Widal reaction for a dill'ercntial (lia,nnosis, from typhoid or between infection with dill'erent nienihers of the j^rou]), may occasionally lead to errors because of the i)resence of group agglu- tinins. For surety of diagnosis the other methods should be employed. Their application is the same as in typhoid fex'er. For final diagnosis immune sera against the various types is necessary. The range of group agglutination of the sera used must be known l)efore they are used for identification. Paratyphoid-like Bacilli. — B. Paratyphosus C. — Bacilli having all char- acteristics of the paratyphoid group, but not agglutinated by paratyphoid or enteritidis serum, have been isolated from swine suffering from hog cholera, feces of man, meat, etc. Other similar types have been isolated, showing cidtural ^•ariation from the paratyphoid group, as indol pro- duction, absence of gas in glucose media, etc. Bacilli of these types have been found in infections in man. Types of the Paratyphoid-Enteritidis Group found in Animal Dis- eases. — B. Paratyphosus B. Types. — B. sinyestifer, hog-cholera bacillus, commonly present as a secondary in\'ader. The disease is caused by a filtrable virus. B. typhi murium, mouse typhoid bacillus, used for the destruction of mice, successfully when the technical details for the maintenance of virulence are carefully carried out. Instances of infection in man have been reported, and in Prussia its use is restricted by law. B. psittacosis, the cause of enteritis in parrots, infectious for man, the disease being spoken of as psittacosis. Pseudotube rculosis of guinea-pigs. A paratyphoid bacilli is found in the pseudotubercular lesions in the spleen and liver and lymph nodes. Sporadic cases or epidemics are common. Recently the condition developed in a series of guinea-pigs which we had injected with milk to test for the presence of tubercle bacilli. Diarrhea of calves, an acute contagious disease of the first few days of life, characterized by diarrhea and the development of a septicemia, or by the development of a septic pneumonia or of septicemia alone. Colon-like bacilli as well as paratyphoid and enteritidis types have been isolated. Enteritidis Types. — Rat Viruses. — Danyz isolated an organism from an epidemic of mice w^hich had a high degree of virulence for rats and mice. It has been widely used for the extermination of rodents. Similar strains have been isolated by others. They are obtainable in the market as Ratin, Danyz-Virus, Liverpool Virus, etc. Recently infection in man has been reported. Domestic animals, meat and meat products, should be protected from contact with the virus. B. Paratyphosus C. — Infectious Abortion in Mares. — Various inves- tigators have isolated paratyphoid types from infected mares. The recent studies of Meyer and Boerner revealed an organism belonging to the above group. It is possible that other members of the group mav cause this disease. CHAPTER XXVI. DYSENTERY GROUP. Dysentery may be divided into acute and chronic. Amebiie appear to be tlie cliief exciting factor in most cases of chronic dysentery, tliough bacilli of the colon group also play a part. In temperate climates acute dysentery is but very rarely due to amebie, but usually to the bacilli identified by Shiga or to allied strains identified by Kruse, Flexner, and Park. The usual summer diarrheas are not excited by the dysentery bacilli. Historical Note. — In 1897 Shiga found in the stools of cases of dysentery a bacillus which had not been before identified. In 1900 Flexner and Strong isolated bacilli which they at that time considered the same as those isolated by Shiga. In the same year Kruse in Germany isolated bacilli from cases of asylum dysentery which differed, however, in their agglutinative properties. Park and Dunham, 1902, isolated a bacillus from a severe case of dysentery during an epidemic at Seal Harbor, Mt. Desert, Maine, which they showed differed from the Shiga dysentery bacillus in that it produced indol and differed in agglu- tinating characteristics. At first they considered it the same as the Flexner strain, but it was shown later by Park to be a distinct variety, and later found by him in widely separated epidemics. ^lartini and Lentz in December, 1902, found that the Shiga type was present in separate epidemics in Europe, but also that other types similar to those isolated by Flexner, Park, Kruse, and others were found. These types differed from the Shiga type in fermenting mannite and in agglutination. In January, 1903, Hiss and Russell showed that a strain isolated by them differed from the Shiga type in same characteristics. German observers at first were inclined to consider the Shiga type as the only one producing dysentery, wlaile the American obser^'ers con- sidered both types of equal importance. Park investigated se\'eral epidemics and isolated not only the Shiga type but two mannite fer- menting types, thus definitely proving the importance of the mannite fermenting types. The results obtained by others were the same, so that no doubt exists that all of the types produce true dysentery. Morphological and Cultural Characteristics of Dysentery Bacilli. — Microscopic. — Similar to bacilli of the colon group. Staining. — Similar to bacilli of the colon group. Motility. — No definite motility has been observed. The molecular movement is very active. Flagella are absent. Appearance of Cultures. — On cjelatin the colonies appear more like the t\i)lioid than the colon bacilli. Gelatin is not liquefied. On agar PATHOGENESIS 379 growth is somewhat more dehcate than that of the average colon eiiltures. Ou Potato. — A (leHcate growth just visible or distinctly lirownish. In Bunillon. — Ditt'use cloudiness with slight deposit and sometimes a pellicle. See table (p. 34")) for comparison with other members of colon- typhoid group. The fermentation reactions vary, and on this bases the dysentery group is divided into subgroups and types. Glu- Man- M.al- Saccha- cose. iiite. tose. rose. Indol B. dysentcriie .... . . + — — — — B. pariidysenteri^: Type 1 (Mt. Dcsort, Y) . . + + — — + Type 2 (Flexner) . . . . . + + + — + Type 3 (Strong) . . . . . + + — + + In differential tests 2 per cent, mannite and 2.5 per cent, maltose give surer and more prompt reactions than the usual 1 per cent, Fig. 140 Fio. 141 ? » « S } V 1 » f % % /., t ^ y* •> f" * .» '/ / % # 1 '- / t • » ; • ^ * • # ■ 1 V/- Dj senteri, bacilli. X 1000 diam eters Colony of dysentry bacilli in gelatin. X 40 diameters. Pathogensis. — Animal Tests. — No characteristic lesions with one ex- ception have followed the feeding of large ciuantities of bacilli. Dogs at times have had diarrhea with slimy stools, but section showed merely a hyperemia of the small intestine. The disease can be produced and occurs spontaneously in monkeys. Many animals are very sensitive to bacilli injected into vein or peritoneum; 0.1 mg. of agar culture injected intravenously produced diarrhea, paralysis, and death; 0.2 mg. under the skin have killed, and the same amount in the peritoneum has caused bloody peritonitis, with lowered temperature and diarrhea. Both small and large animals are very sensitive to killed cultures. The autopsy of animals dying quickly from injection into the peri- 3Sn PATHOOENIC MICROORGANISMS toneum of living or dead bacilli shows the j^eritoneum to be hypcr- emic, the cavity more or less filled with serous or bloody serous exudate. The liver is frequently covered with fibrinous masses. The spleen is moderately or not at all swollen. The small intestine is hlled with fluid, the large intestine is usually empty. The mucous membrane of both is hyperemic and sometimes contains hemorrhages. Conradi found ulcer formation in one case. ' Subcutaneous injections of dead or living cultures are followed by infiltration of tissues and frequently by abscess formation. The dysen- tery bacilli are not found in the blood or organs of animals. Toxins. — A highly poisonous endotoxin is found in autolysates and in filtered broth cultures. When injected into animals death results and on autopsy the same lesions are found as following the injection of live or dead bacilli. This endotoxin can be neutralized by large doses of im- mune serum. Kraus and others claim to have demonstrated the presence of an extracellular toxin against which an antitoxin can be produced. The Shiga type is the more toxic, the others less so or irregularly so. In Man. — The etiological significance of the dysentery bacillus in man is not only shown by its constant presence and by the presence of immune bodies in the serum of infected persons, but also by experi- mental infection of man. Thus Strong infected two condemned criminals with pure cultures. Jehle infected himself. Kruse reported two acci- dental laboratory infections, and others have occurred. As a general rule infection with the Shiga type is more severe and the mortality higher than infection from other types. But the severity of indi\'idual cases vary widely during an epidemic. Prevalence of the Disease. — The disease is distributed over the whole world. The Shiga type and Type I of the paradysenterj- varieties are most commonly found. Character of Disease in Man. — In the onset, acute dysentery is sudden and ushered in by cramps, diarrhea, and tenesmus. The stools, at first feculent, then seromucous, become bloody or composed of coffee- ground .sediment. At the height of the disease there are ten to fifty stools in the twenty-four hours. After two to seven days the blood usually disappears. In temperate climates the mortality varies from 5 to 20 per cent. Bacillary dysentery is a disease especially of the mucous membrane of the large intestines. The epithelium is chiefly involved. In the lightest cases a catarrhal inflammation is alone present, in the more severe the lymph follicles are swollen and some necrosis of epithe- lium takes place. In severe cases in adults the lesions are of a diphtheritic character and may be very marked. The entire lumen of the intestines may be filled with a fibrinous mass of pseudomembrane. In young children, even in fatal cases, the lesions may be more superficial. The following macroscopic and microscopic report of the intestinal findings on a fatal case of bacillary dysentery in an infant is a typical picture: Small Intestines. — Slightly distended. Mesenteric glands large and red. Peyer's patches are swollen slightly without ulceration. PROTECTIVE INOCULATION 381 Large Intedines. — Outer surface vessels cong'ested and prominent, on section, covered with a yellowish mucus. Mucous mcml:)raiie seems to be aljscnt in places. Solitary follicles are elevated and enlarged, esiiecially in the region of sigmoid flexure. In some instances the centres of the follicles are depressed and appear to be necrotic. Large Intestines. — Mucous glantls are for the most part normal, but over tlie solitary follicles they have broken down somewhat and contain pol3aiuclear leukocytes. The interglandular stroma in these places has undergone necrosis. The necrotic area extends down into the -submucosa in the region of the solitary follicles. The capillaries of the solitary follicles are much dilated and congested. The submucosa is thickened and slightly edematous. The connective-tissue cells appear to have undergone a slight hyaline degeneration. The musculature is not affected, neither is the peritoneal coat. Small Intestines. — Normal. Distribution of the Bacilli. — The bacilli are only found in the mtes- tines. They do not in\'ade the rest of the body. The feces, therefore, are the only excretions containing them. Duration of Life Outside of the Body. — The resistance to the ad\'erse conditions outside of the body is relatively smalh The Shiga type is the least resistant. They die in stools in one to two days. In water they die out in several days to a week, exceptionally after a longer time. In general it resembles the typhoid bacillus in its resistance. Communicabihty. — Contact infection is mainly responsible for its spread. Especially dangerous are the mild or subacute cases, or carriers. Such infection may be direct or indirectly by contamination of food, dishes, linen, and clothes; water may also be infected and be the cause of epidemics. The bacillus has been isolated from water in such epidemics. Bacillus Carriers. — Both healthy and convalescent carriers are found. The former may be equal to one-fourth or even one-half of the number of cases. The duration of the carrier state is usually short, some become chronic carriers. The Widal reaction is present in many of these carriers. Convalescents commonly excrete the bacilli for weeks and some become chronic carriers. The excretion is irregular, and slight relapses occur, when the bacilli are most numerous. The importance of finding and isolating the carriers during an epidemic is obvious. Susceptibility. — The frequent occurrence of healthy carriers and mild cases along with severe and fatal cases shows the varying resistance to infection. Disturbances of digestion, alcohol, or other conditions lower- ing the general resistance are factors in infection. Immunity. — Immune bodies appear in the blood some time after infection. In animals immune bodies are also produced by injection. Agglutinins, bactericidal substances, precipitins, and opsonins are found. Neutralization of the toxic products is also possible with immune sera. True antitoxin is also probably produced. Active immunity in animals is produced with difficulty against the Shiga type because of its high toxicity. Against the other types, however, immunity is easily produced. Protective Inoculation. — Some encouraging reports have ai)peared. With the Shiga type, however, the reactions are very severe. For this 382 PATHOGENIC MICROORGANISMS reason Shiga has attempted the simultaneous injection of immune serum and vaccines. Oral immunization has also been attempted. Serum Therapy. — The use of immune sera for treatment have been very good. Employed early in the disease in large doses, it not only limits the se\'erity and duration of the disease, but also reduces the mortality very decidedly. Where various types of bacilli are prevalent ' a polyvalent serum is necessary. Diagnosis. — The use of the Widal reaction is limited, because the reaction does not appear during the acute stage, but later it may be used. Group or normal agglutinins are commonly present and interfere greatly with the value of the reaction in the paradysentery types. The action on the infecting subgroup is usually sharp, but infection by the types of the paradysentery group can frequently not be differentiated. A Widal reaction is commonly present in carriers. For diagnosis the serum should agglutinate in 1 : 50 in infections with B. dysenteriw and in 1 : 100 in other types. Isolation of the bacillus is the only method of diagnosis applicable (luring the acute stage. The mucus flakes in the stool should be selected for plating. The methods of isolation and identification are the same as for typhoid or paratyphoid, except that the crystal ^'iolet should be omitted from the Conradi medium. The baciUi are so abundant in the mucus of most cases that they can be readily isolated from nutrient agar plates. If Endo is used the plates should be fresh. Wlien the color returns the growth of the Shiga types is commonly inhibited. Differential Diagnosis of Type. — The cultural difi'erences have been given. The actual nature of the bacillus should be A'erified by agglu- tination, although the cidtural characters will suffice for a tentati\'e diagnosis from a case clinically typical. Agglutination. — Sera from rabbits or goats are used, as horses develop normal and group agglutinins to a high degree. For practical purposes two sera, one for B. dysenterice and a poly- valent serum for the paradysentery types may be used, further difl'er- entiation being determined culturally. In the use of sera for differen- tiation of the latter groups univalent sera for each of the types must be on hand and all the precautions against error from group agglutinins should be observed. Variations in agglutinability of freshly isolated strains adds to the difficulty. Variability. — Cultures, after prolonged artificial cultivation, may show variations in their sugar fermentations. By passage through animals (monkey, etc.) the original type is returned. CHAPTER XXVII. BACILLUS PYOCYANEUS (Bx\CILLUS OF GREEN AND OF BLUE PUS). BACILLUS PROTEUS (VULGARIS). BACILLUS PYOCYANEUS. The blue and green coloration which is occasionally found to ac- company the purulent discharges from open wounds is usually due to the action of the Bacillus yyocyaneus. According to recent in- vestigations, this bacillus appears to be very widely distributed and not infrequently the cause of infection. It was first obtained in pure culture and its significance noted by Gessard. Morphology. — Slender rods from 0.3,u to 1^ broad and from 2/x to 6^ long; frecjuently united in pairs or in chains of four to six ele- ments; occasionally growing out into long filaments and twisted spirals. The bacillus is actively motile, a single flagellum being at- tached to one end. Does not form spores. Stains with the ordinary yig. 142 aniline colors; does not stain with ., Gram's solution. . ". _ * ",- Biology. — Aerobic, liciuefying, / . \-» ' '^ / ' ' '*\ motile bacillus. Capable also of ,' - ' " ., *, ^^-• an anaerobic existence, but then _. ^ - ' ■ ' "-^ ■ . *'^ -^ produces no pigment. Grows ,, / " * ^'."''V- \ readily on all artificial culture » . -•» -^ " .;" > ^ '- j media at the room temperature, i ^- • ' ' j! ' '—"'•" -j though best at 37° C, and gives ' ,\ • • . y^'*\ ' j to some of them a bright green , ^ • , f ~'"/^ , color in the presence of oxygen. \-;,'** . %" ' > ^^^ In gelatin-plate cultures the col- ' » , ' « ;„' -^^ ♦ onies are rapidly developed, im- ' % » • , , *■ parting to the medium a fluores- cent green color; liciuefaction begins Bacillus pyoeyancus. (From Kolle and at the end of two or three days, Wassemmnn.) and by the fifth day the gelatin is usually all liquefied. The deep colonies, before liquefaction sets in, appear as round, granular masses with scalloped margins, having a yellowish-green color; the surface colonies have a darker green centre, surrounded by a delicate, radiating zone. In stick cultures in gelatiii liquefaction occurs at first near the surface, in the form of a small funnel and gradually extends downward ; later the liquefied gelatin is separated from the solid part of the medium by a horizontal plane, a greenish- 384 PATHOGENIC MICROORGANISMS yellow color being imparted to that portion which is in contact with the air. On agar a wrinkled, moist, greenish-white layer is developed, while the surrounding medium is bright green; this subsequently becomes darker in color, changing to blue-green or almost black. In bouillon the green color is produced, and the growth appears as a delicate, flocculent sediment. Milk is coagulated and assumes a yellowish-green color. Pigment. — Two pigments are produced by this bacillu.s — one of a fluorescent green which is common to many bacteria. This is soluble in water but not in chloroform. The other (pyocyanin) of a blue color is soluble in chloroform, and may be obtained from pure solution in long, blue needles. This pigment distinguishes the Bacillus pyocyaneiis from other fluorescing bacteria. Ferment, — Besides the ferment causing liquefaction of gelatin there is one which acts on albumin. It resists heat. This ferment called pyocyanase has the power to dissolve bacteria, and it has been stated to have some protective power when injected into animals. It has been used locally in diphtheria in a number of cases. We do not think it has any advantage over the cleansing preparations. Distribution. — This bacillus is very widely distributed in nature; it is found on the healthy skin of man, in the feces of many animals, in water contaminated by animal or human material, in purulent discharges, and in serous wound secretions. Pathogensis. — Its pathogenic efi'ects on animals have been care- fully studied. It is pathogenic for guinea-pigs and rabbits. Sub- cutaneous or intraperitoneal injections of 1 c.c. or more of a bouillon culture usually cause the death of the animal in from twenty-four to thirty-six hours. Subcutaneous inoculations produce an extensive inflammatory edema and purulent infiltration of the tissues; a sero- fibrinous or purulent peritonitis is induced by the introduction of the bacillus into the peritoneal cavity. The bacilli multiply in the body and may be found in the serous or purulent fluid in the subcutaneous tissues or abdominal cavity, as well as in the blood and various organs. When smaller quantities are injected subcutaneously the animal usually' recovers, only a local inflammatory reaction being set up (abscess), and it is subsequently immune against a second inoculation with doses which would prove fatal to an unprotected animal. It is interesting to note that Bouchard, Charrin, and Guignard have shown that in rabbits, which have been inoculated with a culture of the bacillus anthracis a fatal result may be prevented by inoculating the same animal soon after with a pure culture of the bacillus pyo- cyaneus. Loew and Emmerich have shown that the enzymes produced in the pyocyaneus cultures are capable of destroying many forms of bacteria in the test-tube, and have a slight protecting value in the body. The p.yocyancus bacillus jiroduccs these effects not only through ferments, but by intracellular toxins. Our knowledge of the pathogenic importance of the Bacillus pyo- cyaneus in human diseases has been much increased by recent inves- BACILLUS PROTEUS ■ii<5 tig-ations. Its presence in wounils greatly delays the jjrocess of re- pair, and may give rise to a general depression of the vital powers from the absorption of its toxic products. This bacillus has been obtained in pure culture from pus derived from the tympanic cavity in disease of the middle ear, from cases of ophthalmia, and broncho- pneumonia. Kruse and Pasquale have found the organism in three cases of idiopathic abscess of the liver, in two of them in immense numbers and in pure culture. Ernst and Schiirmayer report the presence of the bacillus pyocyaneous in serous inflammations of the pericardial sac and of the knee-joint. Ehlers gives the history of a disease in two sisters who were attacked simultaneously with fever, albuminuria, and paralysis. It was thought that they would prove to be typhoid fe\'er or meningitis, but on the twelfth day there was an eruption of blisters, from the contents of which the bacillus pyo- cyaneus was isolated. Krambals refers to seven cases in which a general pyocyaneus infection occurred, and adds an eighth from his own experience. In this the bacillus pyocyaneus was obtained post- mortem from green pus in the pleural cavity, from serum in the pericardial sac, and from the spleen in pure culture. Schimmel- busch states that a physician injected 0.5 c.c. of sterilized (by heat) culture into his forearm. As a result of this injection, after a few hours he had a slight chill, followed by fever, which at the end of twelve hours reached 38.8° C; an erysipelatous-like swelling of the forearm occurred, and the glands in the axilla were swollen and pain- ful. Wassermann reports an epidemic of septic infection of the new- born, starting in the umbilicus. In all there were ele^'en deaths. Lartigau found it in well-water, and in great abundance in the intesti- nal discharges of a number of cases made ill by drinking the water. It has also been found in a certain number of cases of gastro-enteritis where no special cause of infection could be noted. We may therefore conclude from these facts that the Bacilhis pyo- cyaneus, although ordinarily but slightly pathogenic for man, may under certain conditions, as in general debility, become a dangerous source of infection. Children would seem to be particularly susceptible. The differential diagnosis of the pyocyaneus from other fluorescing bacteria is easy enough as long as it retains its pigment-producing property. When an agar culture is agitated with chloroform a blue coloration demonstrates the presence of this bacillus. When the pyocyanin is no longer formed, however, the diagnosis is by no means easy, particularly when the pathogenic properties are also gone. Immunity. — Animal infection is followed by the production of anti- toxic and bactericidal substances. No practical use has been made of this knowledge. BACILLUS PROTEUS (VULGARIS). This bacillus, which is one of the most common and widely dis- tributed putrefactive bacteria, was discovered by Hauser (1885) along with other species of proteus in putrefying substances. These 25 3S(j PATHOGENIC MICROORGANISMS bacteria were formerly included under the name " Baderivm tervto" by previous observers, who applied this name to any minute motile bacilli found in putrefying infusions. Morphology. — Bacilli varying greatly in size; most commonly occur- ring 0.6/i broad and l.'I/ji long, but shorter and longer forms may also be seen, even growing out into flexible filaments which are sometimes more or less wavy or twisted like braids of hair. The bacillus does not form spores, and stains readily with fuchsin or gentian violet. Biology. — An aerobic, facultative anaerobic, liquefying, motile bacillus. Grows rapidly in the usual culture media at the room temperature. Growth on Gelatin. — The growth upon gelatin plates containing 5 per cent, of gelatin is very characteristic. At the end of ten or twelve hours at room temperature small, round depressions in the gelatin are observed, which contain liquefied gelatin and a whitish mass con- sisting of bacilli in the centre. Under a low-power lens these depres- sions are seen to be surrounded by a radiating zone composed of two or more layers, outside of which is a zone of a single layer, from which ameba-like processes extend upon the surface of the gelatin. These processes are constantly undergoing changes in their form and posi- tion. The young colonies deep down in the gelatin are somewhat more compact, and rounded or humpbacked; later they are covered with soft down; then they form irregular, radiating masses, and simu- late the superficial colonies. But it is difficult to describe all the forms which the proteus vulgaris takes on in all the stages of its growth on gelatin plates. When the consistency of the medium is more solid, as in 10 per cent, gelatin the liquefaction and migration of surface colonies are more or less retarded. In gelatin-stick cultures the growth is less characteristic — liquefaction takes place rapidly along the line of puncture, and soon the entire contents of the tube are liquefied. Upon Nutrient agar a rapidly spreading, moist, thin, grayish-white layer appears, and migration of the colonies also occurs. Milk is coagulated, with the production of acid. Cultures in media containing albumin or gelatin have a disagree- able, putrefactive odor, and become alkaline in reaction. Growth is most luxuriant at a temperature of 24° C., but is plentiful also at .37° C. It is a facultative anaerobe and grows also in the absence of oxygen, but the proteus then loses its power of liquefying gelatin. It produces indol and phenol from peptone solutions. The proteus develops fairly well in urine, and decomposes urea into carbonate of ammonia. Pathogensis. — This bacillus is pathogenic for rabbits and guinea- pigs when injected in large quantities into the circulation, the ab- dominal cavity, or subcutaneously, producing death of the animals with symptoms of poisoning. Hauser has obtained the Bacillus proteus {imlgaris) from a case of purulent peritonitis, from purulent puerperal endometritis, and from a phlegmonous inflammation of the BACILLUS PUnrEUS 387 hand. Brunner also reports similar infections in wliich tliis organism was found associated with pus cocci, and Charrin describes a case of pleuritis during pregnancy, in wliich the proteus was present and a foul-smelling secretion was produced. Death in this case, which ensued without further complication, is said to have been due j)robably to the poisonous products of the proteus. An interesting example of pure toxemia resulting from the toxin of the proteus is reported by Levy : While conducting some experi- ments on this organism he had an ojjportunity of making a bacterio- logical examination in the case of a man who died after a short attack of cholera morbus. From the vomited material and the stools he obtained a pure culture of the proteus; but the blood, collected at the autopsy, was sterile. In the meantime seventeen other persons who had eaten at the same restaurant were taken sick in the same way. Upon examination at the restaurant it was found that the bottom of the ice-chest in which the meat was kept was covered with a slimy, brown layer, which gave off a disagreeable odor. Cultures from this ga^•e the proteus as the principal organism present. In- jections into animals of the pure cultures produced similar symp- toms as occurred in the human subjects. Levy concludes that in so-called "flesh poisoning" bacteria of this group are commonly concerned, and the pathogenic effects are due to toxic products e^•olved during their development. Next to the para- typhoid-enteriditis types they are probably the most frecjuent cause of meat poisoning. Booker, from his extended researches into this subject, concludes that the proteus plays an important part in the production of the morbid symptoms which characterize cholera infantum. Proteus vulgaris was found in the alvine discharge in a large proportion of the cases examined by him, but was not found in the feces of healthy infants. "The prominent symptoms in the cases of cholera infantum in which the proteus bacteria were found were drowsiness, stupor, and great reduction in flesh, more or less collapse, frecjuent vomiting and purging, with watery and generall.y offensive stools." Next to the Bacillus coll covimvnis the Profev.i vulgaris appears to be the microorganism most frequently concerned in the etiology of pyelonephritis. In cases of cystitis and of pyelonephritis this bacillus is often found in pure cultures or associated with other bac- teria. It probably gets into the l)ladder chiefly through catheteri- zation. From the animal experiments of the authors above men- tioned, simple injection of pure cultures of proteus into the bladder, without artificial suppression of urine, invariably produces severe cystitis. The fact that this organism grows in urine is sufficient to account for the extension of the purulent process finally to the kidneys. The Proteus ruh/aris is usually a, harmless parasite when located ill the mucous membrane of the nasal cavities. Here it only decom- poses the secretions, with the production of a putrefacti\'e odor. It is found occasionally in the discharge from cases of otitis media in con- nection with other bacteria. CHAPTER XXVIII. THE BACILLUS AND THE BACTERIOLOGY OF TUBER- CULOSIS. A KNOWLEDGE of phtliisis was certainly present among men at the time from which our earhest medical descriptions come. For over two thousand years many of the clearest-thinking physicians have considered it a communicable disease; but it is only within compara- tively recent times that the infectiousness of tuberculosis has become an established fact in scientific medicine. Villemin, in 1865, by infecting a series of animals through inoculations with tuberculous tissue, showed that tuberculosis might be induced, and that such tissue carried the exciting agent of the disease. He also noticed the difference in virulence between tuberculous material of human and bovine sources, and sa.ys that not one of the rabbits inoculated with human material showed such a rapidly progressive and widespread generalization as those receiving material from the cow. Baumgarten demonstrated early in 1882 bacilli in tissue sections which are now known to have been tubercle bacilli. But these investigations and those of others at the same time, though paving the way to a better knowledge of the disease, proved to be unsatisfactory and incom- plete. The announcement of the discovery of the tubercle bacillus was made by Koch in March, 1882. Along with the announcement satisfactory experimental evidence was presented as to its etiological relation to tuberculosis in man and in susceptible animals, and its principal biological characters were given. He submitted his full re- port in 1884. Innumerable investigators now followed Koch into this field, but their observations served only to confirm his discovery. Distribution of Bacilli. — They are found in the sputum of per- sons and animals suffering from pulmonary or laryngeal tubercu- losis, either free or in the interior of pus cells; in miliary tubercles and fresh caseous masses in the lungs and elsewhere; in recent tuber- culous cavities in the lungs; in tuberculous glands, joints, bones, serous effusions, mucous membranes, and skin affections. They are also found in the feces of those suffering from tuberculous disease of the intestines or of those swallowing tuberculous sputum. They are frequently present in the blood in very small numbers, in large number only ill acute miliar\- tuberculosis. Morphology. —The tubercle bacilli ai-c slender, non-motile rods of about 0..')M in diameter by 1.5^ to 4;u in length. (Plate VI, Figs. 1 and 2.) The morphology is extremely variable, especially on culture media, and varies with the type of medium used. Commonly they occur singly or PLATE VI Tubercle bacilli in red. Streptobacilli in blue. Tubercle bacilli in red. Tissue in blue. X lOOO diameters. X llOO diameters. FIG. 8 FIG. 4 1 Leprosy bacilli in nasal secre- tion of person suffering from nasal lesions. (Hansen.) Short smegma bacilli in red, rest of material in blue. X SCO diameters. X llOO diameters. RESISTANCE 389 in pairs, and are then usually slightly curved; frequently tliey are ob- served in smaller or larger hunches. Under excejjtioiial conditions branching and club-shaped forms are ol)servcd. The tubercle bacillus, therefore, is closely allied to nocardia. In stained pre])arations there are often seen unstained portions. In old cultures irregular forms maj- develop, the rods being occasionally swollen at one end or presenting lateral projections. Here also spherical granules appear which stain \\ith more difficulty than the rest of the bacillus and also retain the stain with greater tenacity. The bacilli, however, containing these bodies are not appreciably more resistant than those not having them; therefore they cannot be considered true spores. The bacilli have a thin capsule, shown in one way by the fact that they appear thicker when stained with fuchsin than with methylene blue. Chemical Constituents of the Tubercle Bacilli. — Water 86 per cent., dry substance 14 per cent., 25 per cent, of which is soluble in alcohol and ether, consisting of free fatty acids and fatty acids combined with the higher alcohol "mykol" to form a wax; lecithin proteins, largely nucleo-albumins, nuclein, protamin, nucleic acid and inorganic bases constitute the remainder. Staining Peculiarities. — These are very important, for bj' them its recognition in microscopic preparations of sputum, etc., is rendered possible. Owing to content of waxy substance it does not readily take up the ordinary aniline colors, but when once stained it is very difficult to decolorize, e\'en by the use of strong acids. The more recently formed bacilli are much more easily stained and decolorized than the older forms. Methods of Staining. — Carbol-fuchsin. — Steam three minutes, decolorize with 3 per cent, hydrochloric acid in alcohol, counter-stain witli methylene blue. Herman Stain. — A, crystal violet, 3 per cent, solution in alcohol; B, ammonium carbonate, 1 per cent, solution in water; mix one part of A witli three parts of B just before using; steam as above, decolorize with 10 per cent, nitric acid, wash in alcohol, and eounter-stain in Bismarck brown. Biology. — The bacillus of tuberculosis is a imrasitic, aerobic, non- moiile bacillus, and grows only at a temperature of about 37° C, limits 30° to 42° C. It does not form true spores. Resistance. — The bacilli, because of the protection given by their w'axy substances, it has been assumed, have a somewhat greater resisting power than most other pathogenic bacteria. Frequently a few out of a great number of bacilli resist desiccation at ordinary temperatures for months; most bacilli die, however, soon after drying. There is a greater resistance shown by the tubercle bacillus than by most other non- sporebearing bacilli to the action of the products of bacterial growth as in souring milk, in water, and in sewage. In water and sewage they may remain viable for weeks. They frequently retain their vitality for several weeks, or even months, in putrefying material, such as sputum. Upon cultures the bacilli do not live longer than three months, unless the media be favorable, such as egg or serum; transplants after this time may fail to grow. A few bacilli, sufficient to infect guinea-pigs. 390 PA TIIOGENIC MICROORGA NISMS may persist much longer. Cold has little effect upon tliem. A¥lien dry, some of the organisms stand dry heat at 100° C. for twenty minutes hut are dead in forty-hve minutes; hut when in fluids and separated as in milk, they are quickl\' killed— viz., at 00° (.'. in twenty minutes, at 05° (\ in fifteen minutes, at 70° (". the gre;it majority in one minute, all in five minutes, at 80° C the great majority in one-half minute, all in one minute, and at 95° C. in one-half minute. In some experiments they appear to withstand a higher temperature. As pointed out by Theobald Smith, when milk is heated in a test-tube in the usual wa.y the cream which rises on heating is exposed on its surface to a lower temperature than the rest of the milk, and as this contains the greatest percentage of the bacteria some of them are exjaosed to less heat than those in the rest of the fluid receive. Rosenau points out another source of error : If a moderate number of Fkj- 1-13 killed bacilli are injected, limited lesions will arise and caseation may follow. On killing and autopsying the animals, tubercle bacilli can then be demonstrated in smears from the lesions, and the inocula- tion is considered positive. If, however, this material is reinjected into a second pig, the latter will show nothing on autopsy. This capacity of dead bacilli to cause macroscopic lesions has long been shown by Prudden and Hodenpyl. Its importance, however, is not sufficiently considered. The resisting power of this bacil- lus to chemical disinfectants, dry- ing, and light is considerable, but not as great as it is apt to appear, for, as in sputum, the bacillus is usually protected by mucus or cell protoplasm from penetration b.y the germicidal agent. It is not al- ways destroyed by the gastric juice in the stomach, as is shown by successful infection experiments in susceptible animals by feeding them with tubercle bacilli. iThey are destroyed in sputum in six hours or less by the addition of an equal cjuantity of a 5 per cent, so- lution of carbolic acid.\ Bichloride of mercury is less suitable for the disinfection of sputum as it combines with the mucus and forms a more or less protecting envelope. Iodoform has no effect upon cul- tures until 5 per cent, is added. The fumes from four pounds of burning sulphur to each 1000 cubic feet of air space will kill tubercle bacilli in eight hours when fully exposed to the action of the gas, providing they are moist, or abundant moisture is present in tlie air. Formaldehyde gas is quicker in its action, but not much more effi- cient. Ten ounces of formalin should be employed for each 1000 cubic feet of air space. The tubercle bacillus resists the action of alkaline Tul^erflc barilli. Impression prepara- tion from small colony on coaeulatod Ijlood- scrum. X 1000. CULTIVATION OF THE TUBERCLE BACILLUS m hypochlorite sohition ("antiformiii") in dihitioiis wliieh quickly dis- solve nou-acid fast hacteri.i. The tubercle bacillus in sputum when exposed to direct sunlight is killed in from a few minutes to scA'eral hours, according to the thick- ness of the layer and the season of the year; it is also usually destroyed by diffuse daylight in from five to ten days when placed near a window in fine powder. Protected in cloth the bacilli survive exposure to light for longer periods. Tuberculous sputum expectorated upon sidewalks, etc., when left undisturbed in the shade may be infectious for weeks, but when exposed to the action of direct sunlight, will in many cases, especially in summer, be disinfected by the time it is in condition to be carried into the air as dust, but not before children and flies get into it. This action of sunlight and other more impor- tant hygienic reasons, suggest that the consumptive patients should occnpj' light, sunny rooms. Fig. 144 Fig. 145 Tubercle bacilli, bovine. X 1000 diameters. Tubercle bacilli, human, diameters. X 1000 Dried sputum in rooms protected from abundant light has occa- sionally been found to contain virulent tubercle bacilli for as long as ten months. For a year at least it should be considered dangerous. The Rontgen rays have a deleterious effect on tubercle bacilli in cul- tures, but practically none upon those in tissues. Multiplication of Tubercle Bacilli in Nature Takes Place Only in the Living Animal. — The tubercle bacillus is a strict parasite — that is to say, its biological characters are such that it could scarcely find natural conditions outside of the bodies of living animals favorable for its multiplication. Under exceptional conditions, such as in freshly expectorated sputum, tubercle bacilli may increase for a limited time. Cultivation of the Tubercle Bacillus. — On account of their slow groAvth and the special conditions which they require, tubercle ba- cilli cannot be grown in pure culture by the usual plate method on ordinary culture media. Koch first succeeded in cultivating and isolating this bacillus on coagulated beef serum, which he inoculated by carefully rubbing the surface with sections of tuberculous tissue and 3f)2 PATHOGENIC MICROORGANISMS then leaving tlie eulture, protected from evaporation, for several weeks in the incubator. Cultures are more readily obtained of human or avian than of bovine bacilli. Growth on Coagulated Dog or Bovine Serum or on Egg. — On these, one of which is generally used to obtain the first culture, the growth is usually visible at the end of ten days at 37° C, and at the end of three or four weeks a dis- tinct and characteristic development has occurred. On serum small, grajush- white points and scales first appear on the surface of the medium. As develoji- mcnt progresses there is formed an irregular, membranous-looking layer. On egg the growth is in the form of more or less elevated colonies which may become confluent. Growth on Nutrient 3-5 Per Cent. Glycerin Agar. — Owing to the greater facility of preparing and sterilizing glycerin agar, it is now usually emploj^ed in preference to blood-serum for continuing to produce later cultures. When numerous bacilli have been distributed over the surface of the culture medium, a rather unLform, thick, white layer, which subsequently acquires a slight yellowish tint, is developed; when the bacilh sown are few in number, or are associated in scattered groups, separate colonies are developed, which acquire considerable thickness and have more or less irregular outhnes. The growth appears similar to that shown upon bouillon as seen in Fig. 146. Fig. 146 Growth of tubercle baeilli upon glycerin bouillon. (Kolle and Wassermann.) Growth on Nutrient Veal or Beef Broth Containing 5 per cent, of Glycerin. — This is of importance, because in this way tuberculin is produced. On these media the tubercle bacillus also grows readily if a very fresh thin film of growth from the glycerin agar is floated on the surface. Glycerin broth is used for the development of tuberculin and must be neutral to litmus, viz., between 1.5 per cent, to 2 per cent, acid to phenolphthalein. The small piece of pellicle removed from the previous culture continues to enlarge while it floats on the surface of the liquid, and in the course of three to six weeks covers it wholly as a single film, which on agitation is easily broken up and then settles to the bottom of the flask, where it ceases to develop further. The liquid remains clear. A practical point of importance, if a quick growth is desired, is to remove for the new cultures a portion of the pellicle of a growing bouillon culture, which is very thin and actively increasing. PURE CULTURES OF TUBERCLE BACILLUS FROM SPUTUM 393 Growth on Potato. — A good growth from culturcw ;iiul Hoinetimcs even from tissue lakes place on potato, and this forms tlie most uniform niedimn for stock cultui'es. * Obtaining of Pure Cultures of the Tubercle Bacillus from Sputum, Infected Tissue, and other Materials. — On account of the time re- quired and the difhculties to be overcome, this is never desirable ex- cept when careful investigations of importance are to be luidertaken. Pure cultures can be obtained directly from tuberculous material if the tubercle bacilli are present in sufficient number and mixed infection is not present using the proper blood-serum or egg culture medium; but it is difficult to get material free from other bacteria which grow much more rapidly and take possession of the medium before the tubercle bacillus has had time to form visible colonies. It is usually necessary first to inoculate guinea-pigs, subcutaneously or intramus- cularly, preferably in the thigh, and then obtain cultures from the animals as soon as the tuberculous infection has developed. In this way, due to the susceptibility of the guinea-pig to tuberculosis, cultures may be obtained from material containing very few tubercle bacilli, although contaminating bacteria may be very numerous. Animals inoculated usually die at the end of three weeks to four months. It is better, however, not to wait until the death of the ani- mals, but at the end of four to six weeks to kill a guinea-pig without vio- lence, using illuminating gas, chloroform, or ether in a closed tin or jar. (Animals which develop tuberculosis acutely are apt to have abundant tubercle bacilli and give successful cultures, while the chronic cases usually have few bacilli and may give unsuccessful cultures.) The animal after being killed is tied out in trays, and after washing with a 5 per cent, solution of carbolic acid, immediately autopsied. The skin over the anterior portion of the body having been carefidly turned back, the inguinal nodes are removed with fresh instruments. The nodes on the side of injection are especially favorable for cultures. The abdomen is then opened and the spleen and retroperitoneal nodes removed. As the organs are removed they should be placed in Petri dishes and thoroughly minced with knife and forceps. Fresh instru- ments should be used for each operation. The sternal nodes may be used for cultures, but the lungs are almost useless as the majority of cultures will be contaminated. The minced tissue is then placed on the surface of the culture media, both egg and glycerin egg being used, and evenly and thoroughly smeared over its surface, then the cotton plug is dipped in hot paraffin and the tube corked with a tightly fitting charred cork, to keep the media from drying. The tubes are incubated in the inclined position. On egg, growth is visible in from seven to ten days, and well marked at the end of three weeks. Many tubes should be inoculated as it is only with the dexterity acquired by practice that contaminations are avoided. As will be noted further on, the growth of the bovine type will be very sparse and on glycerin egg probably negative. .394 PATHOGENIC MICROORGANISMS Pathogenesis. — The tubercle bacilhis is pathogenic not only for man, hut for a large number of animals, such as the cow, monkey, pig, cat, etc. Young guinea-pigs arc very susceptible, and are used for the detection of tubercle bacilli in suspected material. When inocu- lated with the minutest dose of the living bacilli they usually succumb to the disease. Infection is most rapidly produced by intraperitoneal injection. If a large dose is given, death follows in from ten to twenty days. The omentum is found to be clumped together in sausage-like masses and converted into hard knots, which contain many bacilli. There is no serous fluid in the peritoneal cavity, but generally in both pleural sacs. The spleen is enlarged, and it, as well as the liver and peritoneum, contains large numbers of tubercle bacilli. If smaller doses are given the disease is prolonged. The peritoneum and inte- rior organs — spleen, liver, etc., and often the lungs — are then filled with tubercles. On subcutaneous injection, for instance, into the thigh, there is a thickening of the tissues about the point of inocu- lation, which may break down in one to three weeks and leave a sluggish ulcer covered with cheesy material. The neighboring lymph nodes are swollen, and at the end of two or three weeks may attain the size of hazel-nuts. Soon an irregular fever is set up, and the animal becomes emaciated, usually dying within four to eight weeks. If the injected material contained only a small number of bacilli the wound at the point of inoculation may heal up and death be postponed for a long time. On autopsy the lymphatic nodes are found to have undergone cheesy degeneration; the spleen may be very much enlarged, and throughout its substance, which is colored dark red, are distributed masses of nodules. The liver is also commonly increased in size, streaked brown and yellow, and the lungs are filled with grayish-white tubercles; but, as a rule, the kidneys contain no nodules. Occasionally the lesions are limited to the inguinal and retroperitoneal nodes. Tubercle bacilli are found in the affected tissues, but the more chronic the process the fewer the bacilli present. Injection into the thigh is to be preferred for diagnostic purposes, the swelling of the local lymph nodes being then palpable. As soon as this is appreciable the node may be removed with or without killing the pig, the presence or absence of tuberculous lesions noted, and smears made for the detection of tubercle bacilli, thus saving time. It must be remembered that the pig may not show the usual picture of gener- alized tuberculosis, but only a swelling of the local lymph nodes. Fortunately tubercle bacilli are usually easily demonstrable in smears made from the crushed nodes. If there is any doubt the remaining tissue should be emulsihed and reinjected into a second set of pigs. Another point to be considered is that other organisms may, rarely, give a picture diflScult to distinguish macroscopically from tuberculosis, as, for instance, streptothrix. To safeguard against error smears should be stained and tubercle bacilli demonstrated. Chronic guinea- pig septicemia may be accompanied by lesions in the spleen which might be taken for tuberculous lesions. POINT OF ENTRANCE OF INFECTION 395 Rabbits are very susceptible to tiibereulosis of tlie Iiovine type, less so to that of the Imniaii tyjM'. This will be gi\'eii more in detail under the ditferenees between human and l)o\iue tuberculosis. Monkeys are very susceijtible to infection with both tyjx's of bacilli. Cats, dogs, rats, and mice are susceptil)le, the last two usually show no tuberculous lesions, but there is great multiplication of the bacilli in the tissues. Tubercle Toxins. — The tubercle bacillus produces no true toxins. The bodies of the bacteria contains substances which cause necrosis of tissue with subsequent caseation or abscess. In broth cultures, after filtration, are present substances which produce fever and inflam- matory reactions of tissues. These substances as well as extracts from tubercle bacilli are highly toxic for tuberculous animals, little if at all for normal animals, and cause fever only in the former and the tissue inflammation spoken of is marked about tuberculous lesions. Tliese poisons will be considered in detail later in connection with tuberculins. Action upon the Tissues of the Poisons Produced by the Tubercle Bacillus. — Soon after the introduction into the tissues of tubercle bacilli, either living or dead, the cells surrounding them begin to show that some irritant is acting upon them. The connective-tissue cells be- come swollen and undergo mitotic division, the resultant cells being distinguisheci by their large size and pale nuclei. A small focus of proliferated epithelioid cells is thus formed about the bacilli, and according to the intensity of the inflammation these cells are sur- rounded by a larger or smaller number of the lymphoid cells. When living bacilli are present and multiplying the lesions progress, the central cells degenerate and die, and a cheesy mass results, which later may lead to the formation of cavities. Dead bacilli, on the other hand, unless bunched together, give off sufficient poison to cause the less marked changes only (Prudden and Hodenpyl). Of the gross pathological lesions produced in man by the tubercle bacilli the most characteristic are small nodules, called miliary tubercles. When young, and before they have undergone degeneration, these tubercles are gray and translucent in color, somewhat smaller than a millet seed in size, and hard in consistence. But miliary tubercles are not the sole tuberculous products. The tubercle bacilli may cause diffuse growth of tissue identical in structure with that of miliary tubercles, that is, composed of a basement substance, containing epithelioid, giant, and lymphoid cells. This diffuse tuberculous tissue also tends to undergo chees.y degeneration. Point of Entrance of Infection. — Infection by the tubercle bacillus takes place usually through the respiratory tract or the digestive tract, including the pharynx and tonsils, more rarely through w'ounds of the skin. Tuberculosis may be considered to be caused chiefly by the direct transmission of tubercle bacilli to the mouth through soiled hands, lips, handkerchiefs, milk, etc., or by the inhalation of fine particles of 39fi PATHOGENIC MICROORGANISMS mucus thrown off f)y coughing or loud speaking, or of tuberculous dust contaminated by S])utum or feces. Tuberculosis of Skin and Mucous Membranes. — When the skin or mucous membranes are superficially infected through wounds there may develop lupus, ulceration, or a nodular growth. The latter two forms of infection are apt after an interval to cause the involvement of the nearest lymphatic nodes. Tuberculosis of Respiratory Tract. — The lungs are the most frequent location of clinically recognizable tuberculous inflammation. On account of their location they are greatly protected from external infection. Most of the bacilli are caught upon the nasal or pharyngeal mucous membranes. Only a small percentage can find their way to the larynx and trachea, and still less to the smaller bronchioles. From the examination of the lungs of miners as well as from experimental tests there is no doubt but that some of the bacilli may find their way into the deeper bronchi. The deeper the bacilli penetrate the more unlikely that they can be cast out. On the other hand the lungs are the most likely point of localization of tubercle bacilli which find their way into the blood-stream. It is in this way that the lungs frequently become infected. It is now well established that infection taking place through the intestine may find its way by the blood to the hmgs and excite there the most extensive lesions with or without leaving any trace of its point of entrance. Even if infection of the lung is slight or even absent the tubercle bacilli then find their way to the bronchial nodes where lesions may develop. Lesions either active or latent, any where in the body may be the source of a subsequent pulmonary infec- tion. Should bacilli find their way into the blood infection of the lung tissue will result if the resistance is lowered for any reason. The nasal cavities are rarely affected with tuberculosis, but more often the retropharyngeal tissue. Tuberculosis of this tissue as well as that of the tonsils is apt to give rise to infection of the lymph nodes of the neck. It is believed that just as bacilli may pass through the intestinal walls to infect the mesenteric nodes, so bacilli may, without leaving any trace, pass through the tonsils to the nodes of the neck. Primary infection of the larynx is rare. Secondary infection is fairly common. The region of the vocal cords and the interarytenoid space are the special sites attacked. Infection by Inhalation of Dried and Moist Bacilli. — A common mode of infection is by means of tuberculous sputum, which, being coughed up by consumptives, is either disseminated as a fine spray and so inhaled, or, carelessly expectorated, dries and, broken up by tramping over it, sweeping, etc., distributes numerous virulent bacilli in the dust. As long as the sputum remains moist there is no danger of dust infection, but only of direct contact; it is when it becomes dry, as on handkerchiefs, bedclothes, and the floor, etc., that the dust is a source of danger. A great number of the expectorated and dried tubercle bacilli undoubtedly die, especially when exposed to the action of direct sun- INFECTION BY INHALATION OF DRIED AND MOIST BACILLI 397 light; but when it is considered that as many as five bilHon virulent tubercle bacilli may be expectorated by a single tuberculous individual in twenty-four hours, it is evident that even a much smaller pro- portion than are known to stay alive will suffice in the immediate vicinity of consumptives to produce infection unless precautions are taken to prevent it. The danger of infection is greatest, of course, in the close neighborhood of tuberculous patients who expectorate profusely and indiscriminately, that is, without taking the necessary means for preventing infection. We found that of 100 tuberculous men admitted to one of the consumption hospitals, only 20 claimed to have taken any care to prevent the contamination of their surround- ings by their sputum. There is much less danger of infection at a distance, as in the streets for instance, where the tubercle bacilli have become so diluted that they are less to be feared. In rooms the sputum is not only protected from the direct sunlight, but it is constantly broken up and blown about by the walking, closing of doors, etc. In crowded streets on windy days infected dust must sometimes be in the air unless the expectoration of consumptives is controlled. Exhaustive experiments made by many observers have shown that particles of dust collected from the immediate neighborhood of con- sumptives, when inoculated into guinea-pigs, produce tuberculosis in a considerable percentage of them; whereas, the dust from rooms inhabited by healthy persons or dust of the streets does so only in an extremely small percentage. Fliigge is probably right in think- ing that the dust which is fine enough to remain for a long time in suspension in the air is usually free from living bacilli. It is in the coarser though still minute particles, those in which the bacilli are protected by an envelope of mucus, that the germs resist drying for considerable periods. These are carried only short distances by air currents. Such results as those obtained by Straus, who, on examining the nasal secretions of twenty-nine healthy persons living in a hospital with consumptive patients, found tubercle bacilli in nine of them, must be accepted with some reserve, since we know that in the air there are bacilli which look and stain like tubercle bacilli and yet are totally different. It may be said that the danger of in- fection from slight contact with the tuberculous is not so great as it is considered by many, but that on this account it is all the more to be guarded against in the immediate neighborhood of consumptives. Those who are most liable to infection from this source are especially young children; the adult members of the family, the nurses, the fellow- workmen, and fellow-prisoners of persons suft'ering from the disease are relatively very less likely to become infected. In this connection, also, attention may be drawn to the fact that rooms which ha^■e been recently occupied by consumptives are not infrequently the means of producing infection (as has been clinically and experimentally demon- strated) from the deposition of tuberculous dust on furniture, walls, floors, etc. The danger is not apt to last beyond three months. Fliigge has drawn attention to the fact that in coughing, sneezing, etc., very 398 PATHOGENIC MICROORGANISMS fine particles of throat secretion containing bacilli are thrown out and carried by air currents many feet from the patient and remain suspended in the air for a considerable time. To encourage us we now have a mass of facts which go to show that when the sputum is care- fully looked after there is very little danger of infecting others except by close personal contact. Tuberculosis of Digestive Tract. — Tuberculosis of the gums, cheeks, and tongue is rare. The tonsils and pharynx are somewhat more often involved. The stomach and esophagus are almost never at- tacked. The small intestines are rather frequently the seat of infection from bacilli swallowed with the food or dust-infected mucus. In a striking case four previously healthy children died within a short period of one another. Their nurse was found to have tuberculosis of the antrum of Highmore, with a fistulous .opening into the mouth. She had the habit of putting the spoon with which she fed the children into her mouth so as to taste the food before it was given to them. As already noted, the bacilli frequently pass through the mucous membrane to the lymph glands without leaving any lesions. Infection by Ingestion of Milk and Milk Products. — Milk serves as a conveyor of infection, whether it be the milk of nursing mothers or the milk of tuberculous cows. In this case evidence of infection is usually shown in the mesenteric and cervical lymph nodes or general- ized tuberculosis maj^ be ■ caused, while the intestinal walls are fre- quently not affected. Bacilli accompanied by fat pass much more readily through the intestinal mucous membrane or that of the tonsils and pharynx. The transmission of tubercle bacilli by the milk of tuberculous cows has been abundantly proved. Formerly it was thought that in order to produce infection by milk there must be a local tuberculous affection of the udder; but it is now known that tubercle bacilli may be found in the milk in small numbers, when adjacent tissue is infected and when careful search fails to detect any udder disease. Schroder has shown that the feces are a very dangerous factor in the dissemination of tubercle bacilli. He compares feces in cattle to sputum in man, since the tubercle bacilli are swal- lowed by cattle and are to a great extent passed through the intestinal tract without destruction. He found that when milk from phthisical cows having healthy udders was obtained so as not to become infected by feces it was free from fjacilli, but when obtained without special precautions it was frequently infected. The milk of every cow which has any well-developed internal tuberculous infection must there- for be considered as possibly containing tubercle bacilli. Rabino- witsch, Kempner, anfl Mohler also proved beyond question that not only the milk of tuberculous cattle, which showed no apprecial)le udder disease, but also tliose in which tuberculosis was only detected through tulicrrnliii, frerinently contained tubercle bacilli. |)ifl'erent observers have found tubercle bacilli in 10 to .30 per cent, of the samples of un- heated city milk. Butter may contain tubercle bacilli in higher per- centages of samples examined. When we consider the prevalence of BOVINE INFECTION IN MAN 399 tii})erciilosis anions' t"ittle we can readily realize that, even if the bovine bacillus infects hnman beings with difficulty, there is danger to children when they are exposed to this source of infection. The milk from cattle suffering from udder tuberculosis usually' contains a few hundred bacilli per c.c, but may contain many millions. It is also important to mention the fact that mixed milk from a herd, though tending to dilute the milk of cows excreting tubercle bacilli, may be badly infected from one cow, especially if this cow has udder disease. Taking the abattoir statistics of various countries, we find that about 10 per cent, of the cattle slaughtered were tuberculous. A less probable source of infection by way of the intestines is the flesh of tuberculous cattle. Here the danger is considerably less, from the fact that meat is usually cooked, and also because the muscular tissues are seldom attacked. In view of the finding of the bovine type of bacilli in a considerable percentage of the cases of tuberculous children tested, the legislative control and inspection of cattle and milk is an absolute necessity. iVs a practical and simple method of preventing infection from suspected milk, sufficient heating of the milk used as food must commend itself to all. Human tubercle bacilli may be found in milk as instanced by one sample of city milk examined in the Research Laboratory by Hess. Method of Examining Milk for Tubercle Bacilli. — Thirty c.c. of milk are centrifuged at high speed and 10 c.c. of the lower milk and sedi- ment collected. Four cubic centimeters of the cream is thinned with a little sterile water and injected into two guinea-pigs. The sediment is injected in amounts of 3 to 5 c.c. into other pigs. Larger amounts than this are apt to kill too many pigs from the associated bacteria. Subcutaneous injection is to be preferred. There are certain precau- tions that must be taking in drawing conclusions as the different types of acid-fast "butter bacilli" may cause lesions, and their presence will be noted in smears made from these lesions. To avoid this source of error, two methods are resorted to. If cultures are made from the sus- pected lesions on glycerin agar, these bacilli develop in a few days, whereas tubercle bacilli would not. When one is ready to kill the pigs, 2 c.c. of old tuberculin should be injected into each pig late in the day. The following morning the tuberculous pigs will be dead or dying." Autopsies should be done on all to confirm the test. The milk should be as fresh as possible to prevent the growth of other bacteria. Bovine Infection in Man. — Numerous investigations have been made on this point. To Ravenel probably belongs the credit of isolating the first bovine bacillus from a child. The following tables summa- rizing the results of a large series of cases give a fair idea of inci- dence of such infection. As will be seen, children are especially the ones infected, and usually the point of entry is clearly alimentary as shown by the lesions. Cervical adenitis and abdominal tuberculosis are the most frequent types of infection. Generalized tuberculosis due to bovine infection is less frequent. Bone and joint tuberculosis is most commonly of the human type. The meninges are less 400 PA T HOG EN I C MI CROORGA NISMS Table I. — Tabulation of cases reported.' Adults sixteen Children five to Children under years and over. sixteen years. five J ears. Diagnosis. Human. Bovine. Human. 497 Bovine. Human. Bovine. Pulmonary tuberculosis 3 6 28 1 Tuberculous adenitis axil- lary 2 — — — 2 — Tuberculous adenitis cer- vical 27 1 17 14 9 11 Abdominal tuberculosis 15 4 7 8 9 11 Generalized tuberculosis, alimentary origin 6 1 3 4 16 13 Generalized tuberculosis . 27 — 4 1 56 3 Generalized tuberculosis, including meninges, ali- mentary origin . — — 1 — 5 -10 Generalized tuberculosis. including meninges . 4 — 10 — 51 — Tubercular meningitis — 1 — 2 2 Tuberculo.sis of bones and .joints 31 1 31 3 20 — Genito-urinarytuljcrculosis 16 — 1 — — — Tuberculosis of skin . 9 3 4 6 2 — Miscellaneous cases; Tuberculosis of tonsils — — — 1 — — Tuberculosis of mouth and cervical nodes — 1 — — — — Tuberculous sinus or ab- scess 1 — — — — — Sepsis, latent bacilli — — — — 1 — Totals .... 635 14 85 37 201 51 Mixed or double infections: 10 Pulmonary tuberculosis. 20 27 Abdominal tuberculosiw. 70 Generalized tuberculosis 5!,' (Alimentary origin). cases. years, years. years. years. 18 years. Generalized tuberculosis. Generalized tuberculosis, includiiig meninges (alimentary origin). Generalized tuberculosis, including meninges. 30 9 4 years. months. years. Sputum, human and l^ovine types. Sputum, human and bovine types. Mesenteric nodes, human type. Retroperitoneal, human and bovine type. Spleen, human type. Mesenteric node, bovine type. Lung, culture not obtained. Mesenteric nodes, human and bovine type Bronchial node, human type. Mesenteric nodes, human and bovine type Bronchial nodes, bovine type. Mesenteric nodes, human type. Meninges, human type. Bronchial nodes, human type. Mesenteric nodes, bovine type. Bronchial nodes ) injected together. Spleen, /bovine and human types. Meninges, human type. Lung, human type. Mesenteric nodes, human type. [ Bronchial nodes, human and l:)ovinc types. Total Cases. 1033. 'Summary of cases reported up to July, 1912, exclusive of cases examined at the Research Laboratory (See Table 11), In contrast to the next table, the above con- lains a largo percentage of selected cases of alimentary types of tuberculosis, many of which showed only slight lesions. 4/^- years. j BOVINE INFECTION IN MAN 401 Tablk II. — The Relative Proportion of Human and Bovine Tubercle HacMlli Infections in a Large Series of Unselected Cases^ Examined at the Research Laboratory. Diagnosis of eases ex- amined. Puhuonary tuberculosis. Tiilior-ruloiis adonitis in- tiuinal and axillarj' Tuhcrculous adenitis cer- vical . Ahdnniinal tuberculosis Generalized tuberculosis alimentary oi'it^in . Generalized tuberculosis Generalized tuberculosis including meninges Tubercular meningitis. Tuberculosis of bones and joints. Gcnilo-urinary tuberculosis Tubercidosis of skin Tuberculous abscess Adults sixteen Children five to years and over. sixteen ,\'ears. Children under five years. Human. Bovine. Human. Bovine. Human. Bovine. 281 13 20 Totals 91 Notes. Clinical diagnosis only known and therefore no positive details as to the extent of lesions else- where. See next. In two cases cultures were from axillary nodes but the primary focus was cervical. Another case died shortly afterward with pulmonary tuber- culosis. Milk supply of one child subsequently examined. Tubercle bacilli isolated. Only three cases given under this heading. Many of the cases in the following subdivisions showed marked intes- tinal lesions and some possibly were of ahment- ary origin. One bovine case had tuber- culous osteomyelitis of the metatarsal bone. No autopsy. Extent of lesions elsewhere un- known. The adult b' of the two bacilli, calf experiments were resorted to. This was necessary as the supposed bovine cultures from children would ha\'e to be virulent for calves to the same extent as cultures from bovine material. The commonly used method was the subcutaneous inoculation in the side of the neck with 50 mg. of culture. The human type of bacillus caused only a local lesion or at most a spreading to the nearest lymph node. The bovine bacillus, on the other hand, caused a generalized tuberculosis which was or was not fatal. Sufficient data has been accumulated to make this test practically unnecessary for the determination of type. Differences in Morphology. — The bovine bacillus tends to be shorter, thicker, and solidly stained; the human type tends to be longer, slimmer, usually bent, and shows beading and irregularities in staining. AVe have found this dift'erence most marked on glycerin egg, slight or impercep- tible on other media. Besides the above differences Theobald Smith made the interesting discovery that the production of acid differed with the two types when grown on glycerin broth. The bovine type renders the bouillon less and less acid; this may even progress until the medium becomes slightly alkaline to phenolphthalein. The human type causes a preliminary fall in the acidity; as growth progresses the acidity is then gradually increased, and may exceed the original acidity of the broth used. This difference is e\'ident in tuberculin made from the two types of bacilli. The bovine tuberculin is alkaline or very slightly acid while human tuberculin is markedly acid. The change is only noticed when glycerin is used in the media. Whether this difference is specific is doubtful. The work of more recent investigators would seem to show that this difference, like all differences between the types, is purely ciuantitative, and that different strains vary in their reactions and give intermediate reactions between these two extremes. Bird (Avian) Tuberculosis. — Tuberculosis is \'ery common and infec- tious among fowl. The bacillus grows easily and freely on glycerin media. It tends to form a moist or even slimy growth, and commonly produces an orange pigment. It is able to grow at a higher tem- perature than mammalian tubercle bacilli, the latter failing to grow abo\e 41 ° C\ ; the former growing at even higher temperatures. Guinea- jiigs are less susceptible to inoculation with a\ian tubercle bacilli, and tiie \-irulcnce for these animals is usually quickly lost. Uabbits are much more susceptilile. Rats and mice are spontaneously infected with avian tubercle bacilli and are supposed to be an imi)ortant factor in spreading METHODS OF EXAMINATION FOR TUBERCLE BACILLI 415 the disease. Birds are refractory, with few exceptions, to infection witii the mammaUan tubercle bacillus. Parrots, however, are susceptible to infection with all three types and commonly have spontaneous tuberculosis caused by the human t.\])e of bacillus. Stability of the Different Types of Bacilli. — The fact that the agglu- tination reactions and the tuberculin reactions of the different types is similar shows their close relationship. This has led to the endeavor to change one type into the other. This is usually done by passage through animals. The results have been peculiar. Some cultures ha^'e been passed through a series of calves without any change ex- cept for a moderate increase in virulence. Other cultures seem to have completely changed their type. We believe that this is not a change of type, but an additional bovine infection. vStrong negati\'e evidence is the fact that the bovine bacillus when infecting man loses none of its characteristics, though present in the human body for years. Tuberculosis of Cold-blooded Animals. — The bacilli of this group are of interest mainly because of the recent claims of Friedmann that immunity against the tubercle bacillus is produced by their injection, and the publicity given to his claims that vaccines of these organisms have a curative influence in tuberculosis. The results of the use of the so-called Friedmann vaccine have, however, been unsatisfactory. A similar attempt to employ the cold-blooded types for immunization is the use in cattle as advised by Klimmer. These types have been isolated from spontaneous, tuberculous-like lesions of frogs, lizards, turtles, fishes, etc. They have little resemblance to the mammalian types of tubercle bacilli other than their acid-fastness. They grow rapidly and luxuriantly on ordinary media and their optimum of growth is at 20° to 30° C, higher temperatures inhibiting their growth. They are not pathogenic for warm-blooded animals, although toxic symptoms or limited lesions may be produced by the injection of large doses as with most other non-pathogenic acid-fasts. Tuberculins prepared from them are not toxic for tuberculous mammalia except in large doses, so that a specific reaction can be excluded. It is possible but very improbable that they produce some antibodies which would protect against mammalian bacilli to a limited extent. Methods of Examination for Tubercle Bacilli and Other Associated Bacteria. — One of the most important results of the discovery of the tubercle bacillus relates to the practical diagnosis of tuberculosis. The staining peculiarities of this bacillus renders it possible by the bacteriological examination of microscopic preparations to make an almost absolutely positive diagnosis in the majority of cases. A still more certain test in doubtful cases is the subcutaneous or intraperi- toneal injection of guinea-pigs, which permits of the determination of the presence of numbers of bacilli, so small as to escape detection b.y microscopic examination. For the animal test, however, time is recjuired — at least three weeks, and, if the bacilli present are very few in number, at least six weeks — before any positive conclusion can be reached, for when only a few bacilli are present tuberculosis develops 416 PATHOGENIC MICROORGANISMS slowly in animals. In disinfection experiments where many dead bacilli are injected, care must be taken to exclude the local effect of dead bacilli. In doubtful cases a second guinea-pig should be injected from the first. Microscopic Examination of Sputum for the Presence of Tubercle Bacilli.— 1. Collection of Material. — The sputum should be collected in a clean bottle (two-ounce) with a wide mouth and a water-tight stopper, and the bottle labelled with the name of the patient or with some other distinguishing mark. The expectoration discharged in the morning is to be preferred, especially in recent cases, and the material should be coughed up from the lungs. Care should be taken that the contents of the stomach, nasopharyngeal mucus, etc., are not discharged during the act of expectoration and collected instead of pulmonary sputum. If the expectoration be scanty the entire amount discharged in twenty-four hours should be collected. In pulmonary tuberculosis the purulent, cheesy, and mucopurulent spu- tum usually contains bacilli; while pure mucus, blood, and saliva, as a rule, do not. When hemorrhage has occurred, if possible, some purulent, cheesy, or mucopurulent sputum should be collected for examination. The sputum should not be kept any longer than neces- sary before examination, for, though a slight delay or even until putre- faction begins, does not vitiate the results so far as the examination for tubercle bacilli is concerned, it almost destroys any proper inves- tigation of the mixed infection present; it is best, therefore, to ex- amine it in as fresh a condition as possible, and it should be kept on ice until examined if cultures are to be made. 2. Methods of Examination. — Examinaiion for Tuherde Bacilli. — Pour the specimen into a clean, shallow vessel, having a blackened bottom — a Petri dish placed upon a sheet of dull black paper answers the purpose — and select from the sputum some of the true expectora- tion, containing, if possible, one of the small white or yellowish-white cheesy-looking masses or "balls." From this make rather thick cover- glass or slide "smears" in the usual way. In doubtful cases a number of these coarse or fine particles should be placed on the slide. The material being thick should be evenly spread and very thoroughly dried in the air before heating. (For methods of staining see p. 3S9.) Occasionally one is able to demonstrate the presence of tubercle bacilli with the Hermann stain where Ziehl's carbol fuchsin gives nega- tive results. At least two smears should be made and examined, if possible, before a negative report is given. Many of the incipient cases will recjuire several examinations before bacilli are found. Some will remain consistently negative. It must be remembered that lesions may exist and that without ulceration the bacilli do not find their way into the sputum. Methods for Concentrating the Bacilli.— Uhlenhuth advises the use of antiformin. This is a patented preparation consisting of a mixture of sodium hydroxide and sodium hypochlorite solution. If this is mixed with sputum so that the total strength is about 15 per cent. METHODS FOR CONCENTRATING THE BACILLI 417 of antiformin, the sputum quickly becomes fluid. This siiould he thinned with water or alcohol to help reduce the specific gravity of the mixture and centrifup;ed. The sediment is then mixed with water and recentrifuged, and the washed sediment used for smears. Besides the dissolving action, antiformin kills most of the bacteria in the sputum, but not the tubercle bacilli, though they are slowly affected, so that sediment may be used for cultural purposes or injection into guinea-pigs. A comparison of the above methods made by us gave the follow- ing results: Of twenty-eight sputa negative with carbol-fuchsin, two showed bacilli after a few minutes search with the crystal violet stain. On restaining with carbol-fuchsin and giving only a light counterstain with methylene blue the negative slides were also positive. Of the remaining twenty-six, four (15 per cent.) were quickly positive in the antiformin sediment when stained with crystal violet, whereas only three were positive with carbol-fuchsin and only after restaining as above. It is advisable, therefore, in using carbol-fuchsin to have only a light counterstain to make the method most efficient, and control the results with crystal violet if negative. In place of sedimenting the bacilli, the dissolved sputum may be shaken up with a hydrocarbon. When the hydrocarbon separates out from the sputum the waxy tubercle bacilli adhere to it and are collected in a layer between the dissolved sputum and the hydrocarbon. If the hydrocarbon is heav}-, chloroform, they are carried down, if light, ligroin, they are carried up. Kinyoun has modified the original ligroin method as follows: We have had very satisfactory results with its use. As a routine method it saves time and gives a high percentage of positive results. Bottles of about 15 c.c. capacity containing about 2 c.c. of a 1 per cent, solution of cresol are used for collection. The cresol is added to limit decomposition of the sputum if its transit to the laboratory is delayed. When received 1 c.c. of ligroin (specific gravity, not less than 0.715 or more than 0.72) is added and the bottle filled with an alkaline solution of hypochloride of lime. If the bottle is full, about one-third must be poured out to allow for the addition of the solution. The hypochlorite solution is prepared as follows: Three packages of chlori- nated lime are weighed, and for each 90 grams, 65 grams of sodium car- bonate are taken. The lime is mixed with 500 to 600 c.c. of water and the carbonate is dissolved in 1500 c.c. of water by boiling. The carbonate is then added to the lime and thoroughly mixed. After standing twelve to twenty-four hours the solution is filtered oft'. The amount of avail- able chlorine is estimated and the solution diluted so that the chlorine is 5.6 per cent. Then 7.5 grams of caustic soda are added to each 100 c.c. of the filtrate. The solution should be kept cool and in the dark. Fresh lots should be prepared about every three months. After the addition of the ligroin and the solution of chlorine the bottles of sputum are placed in a shaking machine and thoroughly shaken for five to ten minutes. The bottles can then be allowed to stand until 27 418 PATHOGENIC MICROORGANISMS the ligroin rises, which talies several hours, or this can be hastened, placing the bottles in a centrifuge, with special cups to accommodate the bottles, and run for about ten minutes at moderate speed. When the ligroin rises to the top a soapy layer develops at_ the point of contact with the fluidified sputum and the tubercle bacilli are collected in this layer. The soapy layer is taken up with a platinum loop and smears made on glass slides fixed by heat and stained. Indi- vidual slides must be used and the slides must be stained separately or error will result, as the bacilli are not firmly fixed to the slides. For this reason, staining in the cold is more convenient, and the fol- lowing formulfe is especially suited for this purpose: Carbolic acid (crystals) 8 grams. Alcohol, 9.5 per cent 20 c.c. Water 100 c.c. The staining requires three minutes. The slides are then decolorized with acid alcohol and counterstained in the usual wa,y. A counterstain is desirable to aid in focussing on the small amount of detritus present. Care must be exercised so that the bacilli are not carried from one slide to another. All the antiformin methods must be used with caution, as it is easy to see how error can creep in from contamination with other acid-fast bacilli. Detection of Tubercle Bacilli in Urine and Feces, Etc. — The cath- eterized urine is centrifuged. If little sediment appears, the upper portion of the fluid is removed and more urine added and again centrifuged. If the urine is rich in salts of uric acid, the same may be diminished by carefully warming the urine before treating it. If too alkaline add a little acetic acid. A possible source of error is the presence of smegma bacilli. The feces are examined for any purulent or mucous particles. If none are found, larger masses of feces are removed and then the rest diluted and centrifugalized. The antiformin methods are a great aid in the examination of feces. The examiner must remember that bacilli swallowed with the sputum may appear in the feces. In examin- ing cerebrospinal fluid for tubercle bacilli it must be remembered that the majority of the bacilli are entangled in the delicate clot that forms. Wherever possible the fluid after withdrawal should be allowed to stand until this filmy clot develops, which is then fished out and examined. If this is impossible the fluid should be centrifuged and the sediment stained. This is also the case in other serous fluids, but in ascitic or pleuritic fluid they are usually very few in number. Examination for Other Bacteria (Mixed Infection). — With regard to the huderlologicdl dlngnosis of pulmonary phthisis, many consider that it is not enough to show only the presence of tubercle bacilli; it is held to be of importance, both for purposes of prognosis and treatment, that the presence of other microorganisms which may be associated with the tubercle bacillus should also be determined.' It CULTIVATION 419 is now usual to distinguish pure tuberculosis of the lungs from a mixed infection. Phthisis due to the tubercle bacillus alone, which constitutes but a small percentage of all cases, may occur almost without febrile reaction; or when fever occurs the prognosis is un- favorable, thus indicating that the disease is already advanced. It is in the uncomplicated forms of phthisis, moreover, where one must expect if an\'T\'here the best results from treatment. The majority of cases, however, of pulmonary tuberculosis show a mixed infection, especially with varieties of the streptococcus, pneumococcus, or in- fluenza bacilli. These cases may be active, with fever, or passive, without fever, according, perhaps, as the parenchyma of the lung is invaded by the bacteria; or they are only superficially located in cavities, bronchi, etc. Mixed infection with the staphylococcus, other micrococci, and with the influenza bacilli have also been frequently met with by us. The tetragenus has not been often detected by us in thorougly washed fresh sputum. At present the facts seem to prove that the tubercle bacilli have in the great majority of cases, at least shortly before death, a much more important role than the associated bacteria. Sputum Washing. — Some of the associated bacteria found in the expectora- tion come from the diseased areas of the lungs, while others are merely added to the sputa as it passes tlirough the mouth or are developed after gathering. To endeavor to separate the one from the other we wash the sputa. The first essential is tliat the material is to be washed within a few minutes, and certainly within an hour after being expectorated. If a longer time is allowed to inter- vene, the bacteria from the mouth will penetrate into tlie interior of the mucus, and thus appear as if they came from the lungs. Sputum treated twenty-four hours after its expectoration is useless for examining for anything except the tubercle bacillus. A rough method is to pour some of the specimen of sputum to be examined into a convenient receptacle containing sterile water, and with- draw, by means of a sterilized platinum wire, one of the cheesy masses or thick "balls" of mucus. Pass this loop five times through sterile water in a dish; repeat the operation in fresh water in a second and third dish. Spread what remains of the mass on cover-glasses and make smear preparation; stain and examme. With another mass inoculate ascitic bouillon in tubes and agar in plates. When we wish thoroughly to exclude mouth bacteria, a lump of the sputum raised by a natural cough is seized by the forceps and transferred to a bottle of sterOe water and thoroughlj^ shaken; it is then removed to a second bottle of bouillon and agam thorougly shaken. From this it is passed in the same way through four other bottles of bouillon. A portion of the mass is now smeared over cover-glasses, and the rest inoculated in suitable media, such as agar in Petri dishes, and ascitic fluid bouillon in tubes. If desired, the bacteria washed off in the different washings are allowed to develop. Inoculation of Animals. — The inoculation of suspected material into guinea-pigs produces tuberculosis; even if the number of bacilli is very small. When no bacilli can be detected by microscopic examination this can be done for diagnostic purposes. The material should be injected subcutaneously as already described. Cultivation. — This requires so much time that it is not used except in important investigations upon the nature of the tubercle bacilli. The special methods have already been given. CHAPTER XXTX. OTHER ACH)-FAST BACILLI: BACILLUS OF LEPROSY, BACILLUS OF RAT LEPROSY, BACILLUS OF JOHNE'S DISEASE IN CATTLE, AND THE GROUP OF NON- PATHOGENIC ACID-FAST BACILLI. LEPROSY BACILLUS B. LEPRiE. The bacillus of leprosy was discovered by Hansen and Neisser (1879) in the leprous tubercles of persons afflicted with the disease. This discovery was confirmed by many subsequent observers. Morphology (in Tissues). — Small, slender rods resembhng the tubercle bacilli in form, but somewhat shorter and not so frequently curved. The rods have pointed ends, and in stained preparations unstained spaces, similar to those observed in the tubercle bacillus, are seen. They stain readily with the aniline colors and also bj^ Gram's method. Although differing slightly from the tubercle bacillus in the ease with which they take up the ordinary aniline dyes, they behave like tubercle bacilli in retaining their color when subsequently treated with strong solutions of the mineral acids and alcohol. The slight difference in staining characteristics is too little to be relied upon for diagnostic purposes (see Plate VI). Bacilli Isolated from Leprous Lesions. — Clegg reported in 1908 that he had been able to cultivate an acid-fast bacillus by growing it in symbiosis with ameba and cholera. Since then Duval, Kedrowski, Twort, and many others have reported their results. Various organisms have been isolated which may be grouped as follows:' Bacilli of the Diphtheroid Type. — In serum media the colonies are yellowish white and develop best at 37° C, although a slight growth occurs at room temperature. When growth occurs on broth, the medium remains clear, and a pellicle is produced. The morphology is variable. They are either solidly staining or irregularly stained like other types of diphtheroids. They are gram-positive and may show some resist- ance to decolorization after staining with carbol-fuchsin, especially the metachromatic granules. Pdt.hngFriirlt.j/ none or questionable. Acid-fast Chromogenic Bacilli.— This ty])e of bacillus is difficult to isolate but after isolation grows freel>' at lioth .37° and 20° ('. on most of the ordinary media. The growth is luxuriant, moist, and a 1 The results of all the investigation.s cannot be given. For a fuller discussion and bibliography see the excellent resume of Wolbach and Honeij, Journ. Med. Research, 1914, xxix, 367. LEPROSY BACILLUS 421 yellow to deep orange color develops. They vary in morphology from coceoid to filamentous bacilli, some showing metachromatic graimles, others showing clear areas. They are acid-fast hut less so tluui the tubercle bacillus. I'athugcuictty. — Lesions similar to leprosy produced in Japanese dancing mice and in monkeys. Anaerobic Bacilli. — In this place it is sufficient to state that such organisms have been isolated. Acid-fast Non-chromogenic Bacilli. — These types are characterized liy their feeble, slow growth on artificial media, and growth only takes place at 37° C, and then only on special media. Morphologically they vnvy from plump to long, slender bacilli, often beaded or bipolar in appearance. Piithogenicity, none. Fig. 147 Leprosy bacilli in nodule. (KoUe and Wassermann.) What conclusion is to be drawn from such variable results is difficult to say. Has the diphtheriod bacillus any relationship to the acid-fast types? This is a possibility when one considers that under certain cir- cumstances it shows some resistance to decolorization. Then, too, the leprosy bacilli in tissue may decolorize easily although they are abundant, as shown by staining with polychrome methylene blue. On the other hand, the repeated isolation of diphtheroids from the lymph nodes in Hodgkin's disease and even from the lymph nodes in other conditions raises a strong element of doubt as to the etiologic significance of this organism. A careful comparison of the diphtheroids from various conditions, especially their resistance to decolorization, might give us some help. As to the chromogenic acid-fasts, the character of their growth, viz., the luxuriant growth upon ordinary media at low temperature is not that of a* highly specialized parasite. As to the apparently successful animal inoculations it must be remembered that lesions have been produced by acid-fast bacilli, known not to be pathogenic. Why such 422 PATHOGENIC MICROORGANISMS bacilli should be frequently insolated from leprous lesions is still to be explained. The non-pigmented types are more consistent with our idea of what the bacillus sh(juld be like, but whether it is actually the etiologic organism remains to be seen. The serum reactions, such as agglutination and complement-fixation, have added no evidence as to the etiologic significance of any one of the bacilli isolated. Each of the bacilli mentioned have been agglu- tinated by sera of lepers. The complement-fixation reaction awaits a successful specific test for the individual acid-fast organisms. Pathogenesis. — Numerous inoculation experiments have been made on animals with portions of leprous tubercles, but there is no con- clusive evidence that leprosy can be transmitted to the lower animals by inoculation. The inference that this bacillus bears an etiologic relation to the disease with which it is associated is based chiefly upon the demonstration of its constant presence in leprous tissues (Fig. 147). The bacilli are found in all the diseased parts, and usually in large numbers, especially in tubercles on the skin, in the conjunctiva and cornea, the mucous membranes of the mouth, gums, and larynx, and in the interstitial processes of the nerves, testicles, spleen, liver, and kidneys. The rods lie almost exclusively within the peculiar round or oval cells of the granulation tissue which composes- the lep- rous tubercles, either irregularly scattered or arranged parallel to one another. In old centres of infection the leprosy cells containing the bacilli are larger and often polynuclear. Giant cells, such as are found in tuberculosis, are claimed to have been observed by a few investigators (Boinet and Borrel). In the interior of the skin tuber- cles, the hair follicles, sebaceous and sweat-glands are often attacked, and bacilli have sometimes been found in these (Unna, etc.). Quite young eruptions often contain a few bacilli. A true caseation of the tubercles does not occur, but ulceration results. During acute exacer- bations with development of new lesions bacilli have been observed in the blood. In the anesthetic forms of leprosy the bacilli are found most com- monly in the nerves and less frequently in the skin. They have been demonstrated in the sympathetic nervous system, in the spinal cord, and in the brain. The Bacillus lepra occurs also in the blood, partly free and partly within the leukocytes, especially during the febrile stage which precedes the breaking out of fresh tubercles (Walters and Doutrelepont). The bacilli have also been found in the intestines, in the lungs, and in the sputum, but not in the urine. With regard to the question of the direct inheritance of the disease from the mother to the unborn child there is considerable difference of opinion. Some cases have been reported, however,, in which a direct transmission of the bacillus during intra-uterine life seems to be the only or most plausible explanation of the infection. At the same time, we have no positive experimental evidence to prove that such an infection does take place. Although many attempts have LEPROSY BACILLUS 423 been made to iul'ect lioalthy individuals with material eoiitainirig the l)aeiih of leprosy, the results are not eonclusive. h]vv]\ the experi- ments made by Arning, who sueeessfuUy infected a eoiidenmed crim- inal in the Sandwich Islands with fresh leprous tubercles, and which have been regarded as positive evidence of the transmissibility of the disease in this way, are by no means conclusive; for, according to vSwift, the man had other opportunities for becoming infected. These negative results, together with the fact that infection does not more frequently occur in persons exposed to the disease, may possibly be explained by the assumption that the bacilli contained in the tuberculous tissue are mostly dead, or much more probably that an individual susceptibility to the disease is recjuisite for its pro- ductions. The widespread idea, before the discovery of the leprosy bacillus, that the disease was associated with the constant eating of dried fish or a certain kind of food, has now been entirely abandoned. The relation of leprosy to tuberculosis is sufficiently evident from their great similarity in many respects. This is rendered still more remarkable by the fact that leprosy reacts, both locally and generally, to an injection of tuberculin in the same manner as tuberculosis, but to a somewhat less extent. Rat Leprosy. — The interest in this disease lies in the fact that diph- theroid and a chromogenic acid-fast bacilli similar to those described above, have been isolated from leprous rats. Bacillus of Johne's Disease, Chronic Enteritis or Paratubercular Dysentery of Cattle. — This disease is comparatively common in this country and is characterized by chronic diarrhea and emaciation, commonly leading to death. The intestinal mucosa is thickened, and the lesions are not limited. Tubercle formation and necrosis are absent. The bacilli are present in the lesions in enormous numbers. Twort succeeded in cultivating the organism and his work was verified by Holth and Meyer. A tuberculin made from this organism will probably be of diagnostic value. Animals suffering with disease do not react to the ordinary tuberculin test but do react to large doses of tuberculin made from the avian type of tubercle bacillus. The bacillus is not pathogenic for guinea-pigs and rabbits, although local abscesses may be produced. Non-pathogenic Acid-fast Bacilli. — These have no importance further than historical interest and the fact that they may be present in materials suspected of containing tubercle bacilli and thus lead to error. They vary widely in their acid-fastness, especially when artifi- cially cultivated. Differential staining methods have been divised to separate them from the tubercle bacillus and although in a general way the decolorization by prolonged action of acid and alcohol is presumptive evidence against suspected bacilli being tubercle bacilli, it is an unsafe procedure. Tubercle bacilli vary in their acid-fastness but the non- pathogenic types vary even more widely, some being extremely resist- ant to decolorization. Many of the non-pathogenic types grow rapidly 424 PATHOGENIC MICROORGANISMS at low temperatures and in cultures can thus be quickly difFerentiated from tubercle bacilli. They can be separated from tubercle bacilli })y inoculating ani- mals in which no progressive lesions will develop, although limited lesions may he produced if they are injected in large numbers. The guinea-pig may be injected with 2 c.c. of tuberculin and if infected with tuberculosis will die, but if by other acid-fast bacilli, will show little or no reaction. If a second group of guinea-pigs are inoculated with a small amount of the infected tissue from the inoculated pigs there will develop progressive tuberculosis, if the doubtful bacilli were tubercle bacilli, and practically no lesions if they were grass bacilli. Cultures from the lesions may also be an aid in differentiation. Bacillus of Lustgarten. — This bacillus was found by Lustgarten in 1884 in syphilitic lesions or ulcers. It is undoubtedly a saprophyte. It is very similar morphologically to the smegma bacillus and may be identical with it. It is of historical interest only. Smegma Bacillus. — This bacillus is present in smegma from the prepuce or vulva. Its only interest is the danger of mistaking it for tubercle bacilli in the examination of urine, especially if the latter be carelessly collected. Timothy and Other Grass Bacillus. — On various grass, in cow manure, in butter and in milk there have been found bacilli with varying degrees of acid-fastness. Similar bacilli have also been demonstrated in water. They make the direct microscopic examination of such material for tubercle bacilli of little value and the nature of any acid-fast organisms so found must be determined by animal inoculations. CHAPTER XXX. GLANDERS BACILLUS (BACILLUS MALLEI). This bacillus was discovered and pro\'ed to lie the cause of glanders, by isolation in pure culture and inoculation into animals, by several bacteriologists almost at the same time (1882). The bacilli were first obtained in impure cultures by Bouchard, Capitan, and Charrin, and first accurately studied in pure culture by Lofiler and Schiitz. They are present in the recent nodules in animals aft'ected with glanders, and in the discharge from the nostrils, pus from the specific ulcers, etc., and occasionally in the blood. Morphology. — Small bacilli with rounded or pointed ends, from nutrient agar cultures, 0.25;u to 0.5^ broad and from L5jU to 5^ long; usualh' single, but sometimes -, J • ■ ■ ;. Fig. 148 united in pairs, or growing out to long filaments, especially in potato cultures. The bacilli frequently break up into short almost coccus-like elements (Fig. 148). Staining. — The bacillus mallei staina with difficulty with the ani- line colors, best when the aqueous solutions of these dyes are made feebly alkaline; it is decolorized by Gram's method. This bacillus presents the peculiarity of losing very quickly in decolorizing solu- tions the color imparted to it by the aniline staining solution. For this reason it is difficult to stain in sections. Loffier recommends his alkaline methylene-blue solution for staining sections, and for decolorizing, a mixture containing 10 c.c. of distilled water, 2 drops of strong sulphuric acid, and 1 drop of a 5 per cent, solution of oxalic acid; thin sections to be left in this acid solution for five seconds. Biology. — An aerobic, non-motile bacillus, whose molecular move- ments are so active that they have often been taken for motility. It grows on various culture media at 37° C. Development takes place slowly at 22° C. and ceases at 43° C. The bacillus does not form spores. Exposure for ten minutes to a temperature of 55° C, or for five minutes to a 3 to 5 per cent, solution of carbolic acid, or for two Glanders bacilli. Agar culture. X 1100 diameters. 426 PATHOGENIC MICROORGANISMS minutes to a 1 : 5000 solution of mercuric chloride, destroys its \itality. As a rule, the bacilli do not grow after luuing been preserved in a desic- cated condition for a week or two; in distilled water they may li^-e 25 days. It is doubtful whether the glanders bacillus finds conditions in nature fa\'orable to a saprojJiytic existence. A solution of chlorinated lime, containing 1 part of free chlorine per 1000, is useful as a disinfectant of stables and utensils; it kills the bacillus in from one to two minutes. Cultivation. — (For obtaining pure cultures see page 428.) — It grows well in the incubating o^'en on glycerin agar. Upon this medium, at the end of twenty-four to forty-eight hours, whitish, transparent colonies are developed, which in six or seven days may attain a diameter of 7 or 8 mm. On hlood-serum a moist, opaque, slimy layer develops, which is of a yellowish-brown tinge. The growth on cooked potato is especially characteristic. At the end of twenty-four to thirty-six hours at 37° C. a moist, yellow, transparent layer develops; this later liecomes deeper in color, and finally takes on a reddish-brown color, while the potato about it acquires a greenish-yellow tint. In bouillon the bacillus causes diffuse clouding, ultimately with the formation of a more or less ropy, tenacious sediment. It grows on media about 1 .5 per cent, acid to phenolphthalein, and both with and without oxygen. Milk is coagulated with the production of acid. Pathogenicity. — The bacillus of glanders is pathogenic for a num- ber of animals. Among those which are most susceptible are horses, asses, guinea-pigs, cats, dogs, ferrets, moles, and field mice; sheep, goats, swine, rabbits, white mice, and house mice are much less sus- ceptible; cattle are immune. Man is susceptible, and infection not infrequently terminates fatally. When pure cultures of Bacillus mallei are injected into horses or other susceptible animals true glanders is produced. The disease is characterized in the horse by the formation of ulcers upon the nasal mucous membrane, which ha\'e irregular, thickened margins, and secrete a thin, virulent mucus; the submaxillary lymphatic glands become enlarged and form a tumor which is often lobulated; other lymphatic glands become inflamed, and some of them suppurate and open externally, leaving deep, open ulcers; the lungs are also involved, and the breathing becomes rapid and irregular. In farcy, which is a more chronic form of the disease, circumscribed swellings, varying in size from a pea to a hazel-rmt, appear on different parts of the body, especially where the skin is thinnest; these suppurate and leave angry- looking ulcers with ragged edges, from which there is an abundant purulent discharge. The bacillus of glanders can easily be obtained in pure cultures from the interior of suppurating nodules and glands which have not yet opened to the surface, and the same material will give successful results when inoculated into susceptible animals. The discharge from the nostrils or from an open ulcer may contain com- ])aratively few bacilli, and these being associated with other bacteria which grow more readily on the culture media than the Bacillus mallei, GLANDERS BACILLUS 427 make it difficult to obtain pure cultures from sucli material by the plate method. In tliat case, however, j;iiiiica,-pi,t;' inocuhi.tioiis are useful. Oi test animals j;-uiiica-pii;s an- formed the Prowazek inclu- sions found in trachoma, and when we found that these bacilli and the inclusions were found coincidently and repeatedly in so many cases diagnosed as acute papillary trachoma, the possibility became a probability and we proceeded to study the morphology of the cultures more minutely. The morphology varies somewhat with the age of the inoculated culture, the date from isolation, the kind of medium, and the strain. In 48 hours the forms become somewhat more irregular. Then in 3 days most of the bacilli have become extremely minute, many showing only as reddish granules (the "elementary bodies of Prowazek"), while scattered through the culture are swollen spheroidal bodies taking a fainter clear blue stain (the larger "initial bodies of Prowazek"), in some of which are minute reddish granules (more of the "elemen- tary bodies of Prowazek"). A number of irregular light blue bodies are also scattered through the culture. Where the bacteria are densely grouped more red granides may appear in the centre of the group than at the periphery and more blue bodies at the periphery than in the centre. In short, all of the changes described by Prowazek and others as characteristic of trachoma inclusions are seen in the growing cultures of these hemoglobinophilic bacilli. Similar day-to-day studies were unrlertaken with the other types of bacteria found most frequently in the eyes diagnosed as trachoma, e. g., streptococci, staphylococci, gonococci, a minute Gram-negative, non-hemoglobinophilic bacillus not before described, found in a few cases of papillary conjunctivitis, xerosis bacillus, and Micrococcus tetrac/aui.i, but in none of these varieties except the gonococcus were similar changes found in the same marked degree. The fact that the gonococcus cultures showed such definite appear- ances similar to the trachoma inclusions led us to make a special study of a series of ophthalmia neonatorum cases. In all of the inclusion cases where gonococci are found, apparent transition forms between gonococcus and inc'hision are very evident, and we find from a further microscopic study of these slides that the inclusions on the whole present certain characteristics different from those found in our series produced, according to our hypothesis, by nests of growing hemogk)binophilic bacilli. THE HORDKT-dKNdOU HACILLVS 441 From this coniparatix'c study of "inclusions" and culturfs \vc liave reached the following conchision: In many cases of "papillary conjunctixitis" and a certain number of cases of ophthalmia neonatorum, as well as in a certain number of cases of inflammation of the mucous niemliranes of other parts of the body {e. g., vagina, urethra), the trachoma inclusions found are due to one or more ^•arieties of hemogk)binophilic bacilli; in a certain number of cases of gonorrheal ophthalmia as well as in gonorrheal inflammation of the mucous membranes of other parts of the body, the trachoma inclusions are due to the gonococcus. According to one of the later reports of Leber and Prowazek and the recent report of Noguchi and Cohen, certain inclusion conjunctivitis cases may be caused by micro- organisms other than the two mentioned above. THE BOEDET-GENGOU BACILLUS (B. PERTUSSIS). In 1906 Bordet and Gengou announced that they had discovered the etiologic factor of whooping cough to be a small bacillus found in pre- dominating numbers in whooping-cough sputum. To this organism they gave the name Bacillus pertussis. Their claim that this bacillus is the real cause of whooping cough they based upon their results with the complement-fixation test which they had been first to describe. Morphology. — The pertussis bacillus is a short, oval rod, varying in size from about 0.2/x to 0.3|U in diameter and from 0.5m to 2p. in length. It occurs singly, sometimes in twos joined at the ends, and very exceptionally in short chains. Motility. — It is non-motile. Staining. — The pertussis bacillus is decolorized by Gram's method. It is stained faintly by the ordinary aniline dyes. Bi-polar staining is demonstrated very well by Gram's method and by Toluidine's methylene blue. Cultivation. — The pertussis bacillus grows best at 3.5° C. to .37° C. It grows slowly at room temperature. It is aerobic, facultative anaerobic. When first isolated in pure culture it grows only upon the glycerin-potato-blood-agar, recom- mended by Bordet and Gengou. In later generations it grows upon the ordinary culture media. Isolation. — The pertussis bacillus is isolated from sputum with difficulty, owing to the fact that in cultures it is frequently overgrown by other organisms found in the respiratory- tract. The sputum should be collected from the patient during the early stage of the disease, not over one week. The thick grayish portion of the sputum should be selected for the culture. This material is streaked over the surface of a plate of Bordet-Gengou medium. The plates are incubated at 35° C. to 37° C. In forty-eight hours to three days very minute, dis- crete, elevated colonies appear. When these colonies occur in abun- dance, an area of hemolyzed blood may be seen at their periphery. In pure cultures the hemolysis is marked. 442 PA THOGENIC MICROORGA NISMS Identification.— (a) Differential Diagnosis by Culture.— Occurring in whooping-coTigh sputum are other bacilli so closelj' resembhng the pertussis bacillus in morphologic and staining characteristics that they cannot be distinguished in smears. These bacilli, however, ^caii be difl'ereiitiated by their growth upon \'arious culture media. The most important of these organisms is the influenza ))acillus, which will never grow without the presence of hemoglobin in the culture medium. There is frequently found a Gram-negative bacillus which makes a pro- fuse growth upon all media from the first generation in pure culture. The following table gives the chief points of differentiation: (i. pertussis R. infliienzce Growth on j Bordet- Coagulated i Glycerin- i Bordet-Gengou Gengou horse-blood ascites-agar I Plain- plates. ' slants. slants. slants. agar slants. Hemoiyzes or , lightens the 1 medium. First generation in pure culture ; abundant ! moist growth in twenty-four hours. ( Darkens the First generation:! medium. delicate growth.! Intermediate group of Hemoiyzes Gram-negative bacilli, medium. the First generation: abundant moist spread in twenty-four hours. After several generations abundant tenacious growth in ■ fortj'-eight hours. First gene- ' ration: abundant moist spread' in twenty- *, four hours. First genera- tion: profuse moist spread in twenty- four hours. After several generations j abundant ' tenacious growth in j forty-eight hours. Never grows.' First genera-' tion: profuse ! moist spread in ■ twenty- four hours. After several gene rat ion.-* tenacious growth OCCIU'S slow I, \'. Never grow?. First gene- ration : profuse moist spread in twenty- four hours (b) Complement Fixation. — Bordet and Gengou regarded the com- plement fixation as the main support for their claim of the specificity of their organism. They report positive complement fixation in the majority of human cases tested by them. (Dther investigators, how- ever, have reported negative tests of complement fixation. The irregular reports given of the complement-fixation test in human beings may be accounted for by difterent methods used without making comparative studies and suflBcient controls, and, more impor- tant still, by the occurrence of distinct varieties in the species Bacillus ■pertussis. The question of the best method of making the complement- fixation test is still under experiment. Difl'erent observers have used difierent methods of preparing both antigen and serum and have em- ployed different hemolytic systems. In experimental animals, specific antibodies are produced which give a positive complement-fixation test. Pathogenicity.— The pathogenicity of the pertussis bacillus for man still lacks positive proof. Several investigators have reported its patho- genicity for monkeys, dogs, rabbits, and guinea-pigs. Mallory claims that a certain number of bacilli between the cilia of the epithelial cells in the trachea and bronchi constitute the specific lesion. He claims that he has fulfilled Koch's laws by the findinos of these bacilli in experimental animals in the same situation and by the THE BACILLUS OF SOFT CHANCRE 443 recovery of the culture from these animals. lie acknowledges, however, that his results in animals are complicated hy the fact that these animals arc frequently infected by the Bacillu.'! hri)i)clii.seiitirus (accepted as tiie cause of distemper in dogs) which is morphologically similar to the pertussis bacillus and that it apparentlj' has the same power (as Theo- bold Smith and others have pointed out) to cling to the cilia of the epithelial cells in the respiratory tract. In human beings Mallory states, judging from the examination of hundreds of controls, only pertussis cases showed this lesion. THE BACILLUS OF SOFT CHANCRE. This bacillus was first specifically described and ol)tained in pure culture by Ducrey in 1889. An experimental inoculation is followed in one to two days by a small pustule. This soon ruptures and a small round depressed ulcer is left. About this other pustules and ulcers develop which tend to become confluent. The base of the ulcer is covered with a gray exudate and its edges are undermined. There is no induration such as in the syphilitic chancre. The secretion is seropurulent and very infectious. The process usuahy extends to the neighboring lymphatics, which become swollen and may result in abscesses. These are known as "buboes." Morphology. — About l.S/x long and 0.4/i thick, growing often in chains and in cultures, sometimes twisted together in dense masses. It stains best with carbol-fuchsin, and shows polar staining. It is Gram-negative. Cultural Characteristics. — The following method of cultivation has given the best results: Two parts of liciuefied agar at 50° C. are mixed with one part human, dog, or rabbit blood. The blood from the cut carotid of a rabbit may be allowed to run directly into the agar tube, to which the pus from the ulcerated bubo is then added in proper proportion, and the whole placed in the incubator at 35° C. The pus may be obtained by puncture and aspiration from the unbroken ulcer, or if the ulcer is already open it is first painted with tincture of iodine and covered with collodion or sterile gauze. After twenty-four to forty-eight hours, some pus having collected under the bandage, inoculations are made from it. The bacillus grows well also in uncoagulated rabbit-blood serum or in condensation water of blood agar. In twenty-four to forty-eight hours, on the surface of the media, well-developed, shining, grayish colonies, about 1 mm. in diameter may be observed. The colonies remain separate, but only become numerous after further transplantation. The best results are obtained when the pus is taken close to the walls of the abscess. Glass smears show isolated bacilli or short parallel chains with distinct polar staining. After the eleventh generation of the culture, and from all old cul- tures, on inoculation the characteristic soft chancre is produced in 444 PATHOGENIC MICROORGANISMS man. Animals in general cannot be infeeted, hut i)ositi\-e results have been obtained with monkeys and eats. The organisms are especially' charafteristie in tlie water of con- densation from blood-agar, the bacilli being thinner and shorter, with rounded ends; sometimes long, wavy chains are found. In rabbit- blood-serum at 87° C. a slight clouding of the medium is produced and small flakes are formed, consisting of short bacilli or moderately long, curved chains, showing polar staining. The bacillus lives several weeks on blood-agar at 'M" C, but it soon dies in cultures on coagulated serum. All other ordinary culture media so far tried have given negative results, and even with the media described development is difficult and often fails entirely. The chancre bacillus possesses but little resistance to deleterious outside influences. Hence, the ^'arious antiseptic bandages, etc., used in treatment of the affection soon bring about recovery by preventing the spread of inoculation chancre. CHAPTER XXXII. MICROORGANISMS BELONGING TO THE HEMORRHAGIC SEPTICEMIA GROUP. A NUMBER of bacilli of similar characteristics have been described as causing certain infectious diseases of lower animals, marked by the appearance of hemorrhagic areas throughout the body (hemorrhagic septicemia of Hueppe). The bacilli are short, bipolar-staining, non- motile, non-sporebearing organisms. They are Gram-negative and do not liquefy gelatin. They are found in rabbit septicemia, fowl cholera, swine plague,' and a similar disease in cattle. The bacillus of bubonic plague seems to be closely related to the bacteria of this group. BACILLUS OF CHICKEN CHOLERA. ( Bacilluti Avi^eptlcui!.) In ISSO Pasteur carried on some fundamental studies on the bacillus of chicken cholera. The bacillus was isolated from a widely disseminated acute disease of fowls and smaller birds. Characteristics. — It is a short (0.5-1 /x long), non-motile bacillus with marked polar staining. In general, its characteristics are similar to those of other numbers of this group. Pathogenicity. — Pure cultures are very pathogenic for chickens and rabbits, they are less so for sheep, pigs, and horses. Chickens are infected even by feeding minute amounts. A septicemia is produced which is rapidly fatal. It is accompanied by diarrhea, depression, and finally coma. The lesions are hemorrhagic inflammation of the mucous membrane of the intestines and sometimes of lungs, with enlargement of the spleen and liver. Immunity. — Pasteur immunized chickens with attenuated cultures. The method, however, has not been found practicable. Ivitt and Mayer state the serum of horses immunized with the bacillus of chicken cholera shows some protection against the bacillus of swine plague. BACILLUS OF SWINE PLAGUE (SCHWEINESEUCHE). ( Bacillus Seuiscpficiin.) This organism is morphologically and culturally similar to the B. avisepticux. It difters in pathogenesis in that it is naturally a disease of swine, characterized by a more or less chronic bronchopneumonia ' German, Schweiiiseuche. 44G PATHOGENIC MICROORGANISMS V followed by septicemia. The gastro-intestinal tract is not markedl atfected. The disease is generally fatal in young pigs. A polyvalent serum has been produced which has given some practical results. The "bacillus of hog cholera" (schweinepest) may often be found as a mixed infection with the B. seuise'pticus . BACILLUS OF BUBONIC PLAGUE (BACILLUS PESTIS). Historically we can trace the bul)onic plague back to the third century. In Justinian's reign a great epidemic spread over the Roman Fro. 1.52 Fig. 1.5.3 'f'-'^'X^ Btifillus pcstis from agar pulture. Bacillus pestis from bouillon culture. X 1100 diam. X llOOdiam. empire and before it terminated destroj^ed in many portions of the country nearly .50 per cent, of the people. The fourteenth century saw the whole of Europe stricken with this "black death." Europe and America have of late been practically free, but in India the disease still breaks out in all its horrors so that at the present time over 500,0(10 jKTsons die annually from it. Among the most fatal forms of infection is that of the lungs. Pneumonic cases are not alone very serious, but they readily spread the infection. The bacillus exciting the disease was discovered simultaneously by Kitasato and Yersin (1894) during an e])idemic of the bubonic plague in China. It is found in large numbers in the scropurulent fluid from the recent buboes characteristic of this disease and in the lymphatic glands; more rarely in the internal organs excejrt in pneumonic cases when the lungs and sputum contain im- mense numbers, it occurs in the blood in acute hemorrhagic cases and shortly before death. It also occurs in malignant cases in the feces of men and animals. The bacillus, as we have stated, is closely allied t(j the hemorrhagic septicemia group. BACILLUS OF BUBONIC PLAGUE 447 Invdliition forms on salt agar. Wassermaiin.) (Kolle and Morphology.— The bacilli in smears from acute abscesses nr infecteil tissues are, as a rule, short, thick rods with rounded ends. The central portion of the bacillus is slightly convex. When lightly stained the two ends are more colored than the middle portion. The bacilli are mostly single or in pairs. Bacilli in short chains occur at times. Ft- 154 The length of the bacilli varies, but on the average is about 1.6m (1.5m to 1.7^), breadth 0.5m to 0.7m. Besides the usual oval form, the plague bacillus has many exceptional ^'ariations which are character- istic of it. In smears, especi- ally from old buboes, one looks for long bacilli with clubbed ends (similar to involution forms (Fig. 154), yeast-like forms, and bladder shapes. Some of these stain with dif- ficulty. When obtained from cultures the baciUi present not only the forms already men- tioned, but also long chains. Staining. — They stam readily with the ordinary aniline dyes, and especially well with methylene blue, the ends being usually more deeply colored than the central portion; they do not stain by Gram's method. Biology. — An aerobic, non-motile bacillus. Grows best at 30° to •55° C. Does not form spores. Grows on the usual culture media, which should have a slightly alkaline reaction. Does not liquefy gelatin. Grows well on blood-serum media. It grows rapidly on glycerin agar, forming a grayish-white surface growth. The bacilli appear, as a rule, as short, plump, oval bacilli, but a few present elongated thread forms which are very characteristic. In bouillon which is kept still a very characteristic appearance is produced, the culture medium remaining clear while a pellicle forms on the sur- face from which projections sprout downward (stalactite formation) toward a granular or grumous deposit which forms on the walls and on the bottom of the tube. In bouillon and most fluid media the growth is in the form of short or medium chains of very short, oval bacilli, which look almost like streptococci. It does not coagulate milk, but produces a slight acidity. It produces no indol in peptone media. Pathogenicity. — This bacillus is pathogenic for rats, mice, squirrels, guinea-pigs, monkeys, and rabbits. Fleas, flies, and other insects may also become infected. The chief lower animal infected is the rat, and the chief carrier of like disease 448 PATHOGENIC MICROORGANISMS is the flea [Xcnopayda cheopis), which by its bites usually die within two or three days after inoculation. Then at the point of inocula- tion is found a somewhat hemorrhagic infiltration and edema, with enlargements of the neighboring lymph glands, hemorrhages into the peritoneal cavity, and parenchymatous congestion of the organs. The " spleen sometimes shows minute nodules resembling miliary tubercles. Microscopically the bacilli are found in all the organs and in the blood. The disease is rapidly communicated. During epidemics, rats, mice, and flies, in large numbers, become infected and die, and the disease is frequently transmitted through them to man. Very small wounds are sufficient to allow entrance of the germs. Mere rubbing of a culture on the skin of rats and guinea-pigs may produce the disease. The organism is found at times in the feces of sick animals, in the dust of infected houses, and in infected soil. Fig. 15.5 Bafilli ill smear from acutely inflamed gland. Ground sciuirrels in California have been shown to be susceptible to infection and they are supposed to help spread the disease. The virulence of the bacilli in cultures and in nature seems to vary considerably-. It may rapidly diminish when grown on artificial media. The growth in cultures becomes more abundant after frequent trans- plantation. The virulence of the organism is increased by successive inoculation in certain animal species, and then its pathogenic prop- erties for other species are less marked. In man the ijneumonic form of the disease is most dangerous as a means of direct infection to man. Immunity.— Like typhoid infection, a single attack of the plague bacillus protects, with rare exception, from a second infection. Yersin, Calniette, and Borrel have succeeded in immunizing animals against the bacillus of bubonic plague by the intravenous or intraperitoneal injection of dead cultures, or by repeated subcutaneous inoculation. They als(j succeeded in immunizing rabbits and horses, so that the BACILLUS OF BUBONIC I'LACUE 449 serum afforded protection to small animals, after suheutaneous injection, of \'irulent cultures, and even cured those wliicii had been inoculated, if administered within twelve hours after injection. The serum has considerable antitoxic as well as bactericidal properties. It also contains specific agg'lutinins which may be made use of in diagnosis. ^Nlore recently this serum has been applied to the treatment of bubonic plague in man, with ])romising results. Experience has shown that the treat- ment is more efficacious the earlier the stage of the disease. When treatment is begun in the first day of the attack, fever and all alarming symptoms sometimes disappear with astonishing rapidity. In cases treated at a later stage larger doses of the serum are required, and even in the favorable cases sujipuration of the buboes is not always prevented. In some of the early cases and in many of the rather late ones the serum fails. When the disease is far advanced the serum is powerless. For immunizing purposes the senun should be valuable, and a single injection would probably give protection for several weeks. Vaccines. — Haffkine, in India, has applied his method of preventive inoculation to the bubonic plague, as he previously did with cholera, and apparently with equally good results. This method consists in an inoculation of dead cultures, and is essentially a protective rather than a curative treatment. It gives after six to ten days a consider- able immunity, lasting a month or more. Duration of Life Outside of the Body. — In cultures protected from the air and light the plague bacilli may live ten years (Wilson) or more. In the bodies of dead rats they may live for two months. In sputum from pneumonic cases the bacilli lived ten days. ITpon sugar sacks, food, etc., they may live six to fifteen days. Resistance to Deleterious Influences. — The bacilli resemble the colon bacilli in their reaction to heat and disinfectants. Boiling for one to two minutes kills them. Carbolic acid, 5 per cent, solution, kills cultures in one minute. [Mercuric chloride 1 to lOOO kills in ten minutes. Bacteriologic Diagnosis. — When the lymph glands are acutely inflamed but not yet suppurated, cut down on one and make cultures on nutrient agar slanted in tubes. If pus has formed withdraw a little by means of the hypodermic needle. There should also be made smears from the suspected bubo, or in case of pneumonia from the sputum. If the patient is dead, cultures from the spleen and heart's blood are also taken when possible. Suspected animals, such as rats and mice, when freshly killed, and after being immersed in an antiseptic solution to kill fleas, etc., are examined as in man; when decomposed, rats aufl guinea-pigs should l)e inoculated. Plague-like Bacilli in Rodents. — McCoy and Chapin (l'.)12) found an organism { ISiiri/lii.s- hilnrcii.'ie) in a disease of Calituriiian ground squirrels, wliicli show lesions similar to those of plague. The bacilli have been cultivated by McCoy and Chapin on an egg-yolk medium. They are very minute (0.3^ to O.lfi long) and ma>- show a capsule. They are most abundant in the spleen of victims of the disease. 29 CHAPTER XXXIII. THE ANTHRAX BACILLUS AND THE PATHOGENIC ANAEROBES. BACILLUS ANTHRACIS. An'I'HKAX is an acute infectious disease which is very jirevalent among animals, particularly sheep and cattle. Geographically and zoologically it is the most widespread of all infectious disorders. It is much more common in Europe and in Asia than in America. The ravages among herds of cattle in Russia and Siberia and among sheep in certain parts of France, Hungary, Germany, Persia, and India are not equalled by any other animal plague. Local epidemics have (occasionally occurred in England, where it is known as splenic fever. In this country the disease is rare. In infected districts the greatest losses are incurred during the hot months of summer. The disease also occurs in man as the result of infection, either through the skin, the intestines, or in rare instances through the lungs. It is found in persons whose occupations bring them into contact with animals or animal products, as stablemen, shepherds, tanners, butchers, and those who work in wool and hair. Two forms of the disease have been described — the external anthrax, or malignant pustules, and the internal anthrax, of which there are intestinal and pulmonary forms, the latter being known as "wool-sorters' disease." Owing to the fact that anthrax was the first infectious disease which was shown to be caused by a specific microorganism, and to the close study which it received in consequence, this disease has probably contributed more to our general knowledge of bacteriology than any other infectious maladjr. Pollender in 1849 observed that the blood of animals suffering from splenic fever always contained minute rod-shaped bacteria. Davaine in 1863 announced to the French Academy of Sciences the results of his inoculation experiments, and asserted the etiologic relations of the microorganism to the disease, with which his investigation showed it to be constantly associated. For a long time this conclusion was energetically opposed until, in 1877, Koch, Pasteur, and others estab- lished its truth by obtaining the liacillus in pure cultures, and showing that the inoculation of these cultures produced anthrax in susceptible animals as certainly as did the bloofl of an animal recently dead from the disease. Morphology. — Slender, cylindric, non-motile rods, having a breadth (jf l/x to 1.25m, and ranging from 2^ or .3^ to 20^ or 2.5^ in length. Some- BACILLUS ANTHRACIS 451 times sliort, isolated rods are seen, and, again, shorter or longer chains or threads made np of several rods joined end-to-end. In snitid)le cultnre media very long, flexible filaments may be observed, which are frequently united in twisted or plaited cord-like bundles. (See Figs. 15(3 and 157.) These filaments in hanging-drop cultures;^ before the de^'elopment of spores, appear to be homogeneous or nearly so; but in stained preparations they are seen to be composed of a series of rectangular, deeply stained segments. When obtained directly from the blood of an infected animal the free ends of the rods are slightly rounded, but those coming in contact with one another are quite square. In cultures the ends are seen to be a trifle thicker than the bodj' of the cell and somewhat conca^'e, giving the appearance of joints of bamboo. At one time much stress was laid upon these peculiarities as distinguished Fui. 1.56 Fig. 157 Anthrax Imrilhis. X 900 di;imutCT!i Agar culture. Spores heax'ib' ytaiued (iu wpcciineu rod). Bodies of disintegratiuji bacilli faintly stained (in specimen Ijluc). X 1000 diameters. marks of the anthrax bacillus; but it has been found that they are the eff'ects of artificial cultivation and not necessarily characteristic of the organism under all conditions. The bacillus is inclosed in a transparent envelope or capsule, which in stained preparations (from albuminous material) may be distinguished by its taking on a lighter stain than the deeply stained rods which it surrounds. Under favorable conditions in cultures spores are developed in the bacilli. These .spores are elliptic in shape and about one and a half times longer than broad. They first appear as small, refractive grannies distributed at regular intervals, one in each rod. As the spore develops the mother-cell becomes less and less distinct, until it disappears alto- gether, the complete oval spore being set free by its dissolution. (See Fig. 157 and Plate III, Fig. 22). Irregular sporulation sometimes takes 452 PATHOGENIC MICROORGANISMS place, and occasionally there is no spore formation, as in varieties of non-spore-bearing anthrax. Staining. — The anthrax bacillus stains readily with all the aniline colors, and also by Gram's method, when not left too long in the de- colorizing solution. In sections good results may be obtained by the employment of Gram's solution in combination with carmine, but when only a few bacilli are present this method is not always reliable, as some of the bacilli are generally decolorized. McFadyean-Heine Methylene-blue Reaction. — In imperfectly fixed film preparations (pass through flame three times in a second with film side up) the capsule disintegrates. When a solid film is stained for a few seconds in an old solution of methylene blue, washed in water and dried with filter paper, the bacteria are surrounded by a varying amount of a reddish-purple amorphous or fixed granular deposit. McFadyean says this does not occur with other morphologically similar bacteria. Biology. — The anthrax bacillus grows easily in a \'ariety of nutrient media at a temperature from 18° to 43° C., 37° C. being the most favorable temperature. Under 12° C. no development takes place, as a rule, though by gradually accustoming the bacillus to a lower tempera- ture it may be induced to grow under these conditions. Under 14° C. and above 43° C. spore formation ceases. The lower limit of growth and of sporulation is of practical significance in determining the question whether development can occur in the bodies of animals dead from anthrax when buried at certain depths in the earth. Kitasato has shown that at a depth 1.5 meters the earth in July has a temperature of 15° C. at most, and that under these conditions a scanty sporulation of anthrax bacilli is possible, but that at a depth of 2 meters sporulation no longer occurs. The anthrax bacillus is aerobic — that is, its growth is considerably enhanced by the presence of oxygen — but it grows also under anaerobic conditions, as is shown by its growth at the bottom of the line of puncture in stick cultures in solid media; but under- these conditions it no longer produces the peptonizing ferment which it does with free access of air. Furthermore, the presence of oxygen is absolutely necessary for the formation of spores, while carbonic acid gas retards sporulation. This explains, perhaps, why sporulation does not take place within the animal body either before or after death. It is also capable of leading a saprophytic existence. The bacillus is non-motile. Growth in Gelatin. — In gelatin-plate cultures, at the end of twenty- four to thirty-six hours at 24° C, small, white, opaque colonels are de^'eloped, which, under a low-jKiwer lens, are seen to be dark gray in the centre and surrounded l.)y a, greenish, irregular border, made up of wa\'\' filaments. As the colony develops on the surface of the gelatin these wavy filaments spread out, until finally the entire colony consists of a light gray, tangled mass, which has been likened to a Medusa head (Fig. 15S). At the same time the gelatin begins to liquefy, and the colony is BACILLUS ANTIIRACIS 453 soon surrounded by the liquefied niecHuni, upon the surfaee of wliieh it tloats as an irreg'ular, wliite pellicle. In ijcldfiii-.'itivk ciilhirc.s at first de\elo])ineiit occurs alont; the line of pnncture as a delicate white thread, from which irrei;uiar, hair-like prdjectidns soon extend j)er- pendicularly into the culture niediiun, the {growth being' most luxuriant near the surface, but continuing also below. At the end of two or three days liquefaction of the medium commences at the surface and gradually progresses downward. Growth on Agar. — The growth on agar-plutc cultures in the incubator at 37° C. is similar to that on gelatin, and is still more characteristic and beautiful in appearance. A grayish-white layer is formed on the surface within twenty-four hours, which spreads rapidly and is seen to be made up of interlaced threads. Growth in Bouillon. — The growth is characterized by the formation of flaky masses, which sink as a sediment to the bottom of the tube, leaving the supernatant liquid clear. Fig. 158 Colonics of Biioillus .inthracis upon gelatin plates: n, at the end of twenty-four hours; h. at the end of forty-eight hours. X SO. (F. Fliigge.) Spore formation, as alreadj' noted, only takes place in the presence of oxygen, and at a temperature of 15° to 43° C. There is no develop- ment of spores at a greater depth than 1.5 meters in the earth, or in the bodies of living or dead animals; but spores may be found in the fluids containing the bacilli when these come in contact with the air, as in bloody discharges from the nostrils or from the bowels of the dead animal. There are certain non-spore-bearing species of anthrax. Sporeless varieties have also been produced artificially by cultivating the typical anthrax baciflus under certain conditions, among which may be mentioned the addition of antiseptics, as carbolic acid, and of continued 454 PATHOGENIC MICROORGANISMS Fi(i. 159 high temperature (43° C). Varieties differing in their pathogenic power may also be produced artificially. Pasteur produced an " attenu- ated virus" hy keeping his cultures for a considerable time before replanting them upon fresh soil. Anthrax cultures containing spores retain their vitality for years; in the absence of spores the vitality is much more rapitlly lost. When grown in liquids rich in albumin the bacilli attain a considerable degree of resistance; thus dried anthrax blood has been found to retain its virulence for skty days, while dried bouillon cultures only did so for twenty-one days. Dried anthrax spores may be preserved for many years without losing their vitality or virulence. They also resist a comparatively high temperature. Exposed in dry air they require a temperature of 140° C. main- tained for three hours to de- stroy them; but suspended in a liquid they are destroyed in four minutes by a temperature of 100° C. Pathogenesis. — The anthrax bacillus is pathogenic for cattle, sheep (except the Algerian race), horses, swine, mice, guinea-pigs, and rabbits. Rats, cats, dogs, chickens, owls, pig- eons, and frogs are but little susceptible to infection. Small birds — the sparrow particularly — are somewhat susceptible. Man, though subject to local infection and occasionally to internal forms of the disease, is not as susceptible as some of the lower animals. In susceptible animals the anthrax bacillus produces a true septicemia. Among test animals mice are the most susceptible, succumbing to very minute injections of a slightly virulent virus; next guinea-pigs, and lastly rabbits, both of these animals dying after inoculation with virulent bacilH. Infection is most promptly produced by introduction of the bacilli into the circulation or the tissues, but inoculation by contact with wounds on the skin also causes infection. It is difficult to produce infection by the ingestion even of spores; but it may readily be caused hy inhalation, particularly of spores. Subcutaneous injections of these susceptible animals results in death in from one to three days. Comparatively little local reaction occurs immediately at the point of inoculation, but beyond this there is an extensive edema of the tissues. Very few bacilli are found in the blood in the larger vessels, but in the internal organs, and especially in the capillaries of the liver, the kidneys, and the lungs, they are present Srrtion of li\'er from moiiso dead of anthrax sfpfic.eniia. X lOUO tliameters. (From Itzci-ott and Niemann). BACILLUS ANTHRACLS 455 in great numbers. In some places, as in tlie glomeruli of tiic kidneys, the eapillaries will he seen to he stutt'ed full of hacilli, and hemorrhages, prohably due to rupture of capillaries by the mechanical pressure of the bacilli which are developing within them, may occur. The patho- logic lesions in animals infected by anthrax are not marked except in the spleen, which, as in other forms of septicemia, is greatly enlarged. Occurrence in Cattle and Sheep. — Cattle and sheep are affected chiefly with the intestinal form of anthrax, infection in these animals commonly resulting from the ingestion of food containing spores. The bacillus itself, in the absence of spores, is quickly destroyed by the gastric juice. The disease usually takes a rapid course, and the mortality is high — 70 to 80 per cent. The pathologic lesions consist of numerous ecchymoses, enlargement of the lymphatic glands, serous, fatty, and hemorrhagic infiltration of the mediastinum and mesentery, of the mucous membranes of the pharynx and larynx, and particularly of the duodenum, great enlargement of the spleen, and parenchymatous changes in the lymphatic organs. The blood is very dark and tar-like. Bacilli are present, especially in the lymph spaces, in enormous masses. Sheep are also subject to external anthrax, infection taking place by way of the skin; cattle are seldom infected in this way. At the point of inoculation there develops a hard, circumscribed boil — the so-called anthrax carbuncle; or there may be ditTuse edema with great swelling of the parts. When death occurs the appearances are similar to those in intestinal anthrax, except that the duodenum is usually less affected; but in all cases metastasis occurs in various parts of the body, brought about, no doubt, by previous hemorrhages. Occurrence in Man. — The disease does not occur spontaneously in man, but always results from infection, either through the skin, the intestines, or occasionally by inhalation through the lungs. It is usually produced by cutaneous infection through inoculation of exposed surfaces — the hands, arms, or face. Infection of the face or neck would seem to be the most dangerous, the mortality in such cases being 2G per cent., while infection of the extremities is rarely fatal. External anthrax in man is similar to this form of the disease in animals. There are two forms: malignant pustule or carbuncle, and, less commonly, malignant anthrax edema. Ill malignant pustule, at the site of inoculations, a small papule develops, which becomes vesicular. Inflammatory induration extends around this, and within thirty-six hours there is a dark, brownish eschar in the centre, at a little distance from which there may be a series of small vesicles. The brawny induration may be extreme. There may also be considerable edema of the parts. In most cases there is no fever; or the temperature at first rises rapidly and the febrile phenomena are marked. Death may take place in from three to five days. In cases which recover the symptoms are slighter. In the mildest form there may be only slight swelling. Malignant anthrax edema occurs in the eyelids, and also in the head and neck, sometimes the hand and arm. It is characterized by the 45() PATHOGENIC MICROORGANISMS absence of the papule and vesicle forms, and by the most extensive edema. The edema may become so intense that gangrene results; such cases usually prove fatal. The l)acilli are found on microscopic examination of the fluid from the pustiile shortly after infection; later the typical anthrax bacilli are often replaced by involution forms. In this case resort may be had to cultures, animal inoculation, or examination of sections of the extirpated tumor. The bacilli are not present in the blood until just before death. Along with the anthrax bacilli pus cocci are often found in the pustule penetrating into the dead tissue. Internal anthrax is much less common in man; it does, however, occur now and then. There are two forms of this: the intestinal form, or mycosis intestinahs, and the pulmonic form, or wool-sorters' disease. Intestinal anthrax is caused by infection through the stomach and intestines, and results probably from the eating of raw flesh or unboiled milk of diseased animals. That the eating of flesh from infected animals is comparatively harmless is shown by Gerlief, who states that of 400 persons who are known to have eaten such meat not one was affected with anthrax. On the other hand, an epidemic of anthrax was produced among wild animals, according to Jansen, by feeding them on infected horse-flesh. It is evident, therefore, that there is a possibility of infection being caused in this way. The recorded cases of intestinal anthrax in man have occurred in persons who were in the habit of handling hides, hair, etc., which were contaminated with spores; in those who were conducting laboratory experiments, and rarely it has been produced by the ingestion of food, such as raw ham and milk. The symptoms produced in this disease are those of intense poisoning: chill, followed by vomiting, diarrhea, moderate fever, and pains in the legs and back. The pathological lesions are similar to those described in animals. Wool-sorters' disease, or pulmonic anthrax, is found in large establish- ments in which wool and hair are sorted and cleansed, and caused by the inhalation of dust contaminated with anthrax spores. The attack comes on with chills, prostration, then fever. The breathing is rapid, and the patient complains of pain in the chest. There may be a cough and signs of bronchitis. The bronchial symptoms in some instances are pronounced. Death may occur in from two to seven days. The pathological changes produced are swelling of the glands of the neck, the formation of foci of necrosis in the air passages, edema of the lungs, pleurisy, bronchitis, enlargement of the spleen, and parenchymatous degenerations. Prophylaxis against Anthrax Infection.— Numerous investigations have been undertaken with the object of preventing infection from anthrax. The efforts of Pasteur to effect immunity in animals by preventive inoculations of "attenuated virus" of the anthrax bacillus opened a new field of productive original research. Following in his wake many others have devised methods of immunization against anthrax infection; but the one adopted by Pasteur, Chamberland, and Roux has alone been practically employed on a large scale. According BACILLUS AN'rilRACIS SYMPTOArATICI 457 to these authors, two aiitlirax cultures of ditt'ereiit deiirees of virulence attenuated by cultivatiou at 42° to 4;!° C, are used for iuoculation. \'aecuie No. I kills mice, hut not ,i>-uinea-i)i.i!,'s; \'accine No. 2 kills guinea-pigs, hut not rahhits. 'Plie animals to he inocnlated--Ai/,,. sheep and cattle — are first gi\en a suhcutancous injection of one to several tenths of a cubic centimeter of a four-(la,\-old Ixniillou culture of vaccine No. 1 ; after ten to tweh'c days they receive a similar dose of vaccine No. 2. Prophylactic inoculations given in this way have been widely employed with apparently good results. Seriun Treatment. — The serum of immunized animals has been used in Italy with apparently some good results. ^Ye have had no personal experience. Bacterial Cultures for Diagnosis. — The detection of the anthrax bacillus is ordinarily not difficult, as this organism presents morpho- logic, biologic, and pathogenic characteristics which distinguish it from all other bacteria. In the later stages of the disease, liowever, the bacilli may be absent or difficult to find, and cultivation on artificial media and experimental inoculation in animals are not always followed by positive results. E^'en in sections taken from the extirpated pustule it is sometimes difficult to detect the bacilli. In such cases only a probable diagnosis of anthrax can be made. It should be remembered that the bacilli are not found in the blood until shortly before death, and then only in varying quantity; thus blood examinations often give negative results, though the bacilli may be present in large numbers in the spleen, kidneys, and other organs of the body. The suspected material should be streaked over nutrient agar in Petri plates and inoculated in mice. Differential Diagnosis. — Among other bacteria which may possibly be mistaken for anthrax bacilli are Bacillus .nihtilis and the bacillus of malignant edema. The former is distinguished by its motility, by various cultural peculiarities, and by being non-pathogenic. The latter dift'ers from the anthrax bacillus in form and motilit}', in being decolorized by Gram's solution, in being a strict anaerobe, and in various pathogenic properties. The diagnosis of internal anthrax in man is b}' no means eas\', unless the history points definitely to infection in the occupation of the individual. In eases of doubt cultures should be made and inoculations performed in animals. BACILLUS ANTHRACIS SYMPTOMATICI (BACILLUS OF SYMP- TOMATIC ANTHRAX). Like the bacilli of anthrax and of malignant edema, both of which it resembles in other respects also, the bacillus of symptomatic anthrax is an inhabitant of the soil. It is found as the chief cause of the disease in animals — principally cattle and sheep — known as "black leg," "quarter evil," or symptomatic anthrax (rauschl)rand, German; charbon symptomatique, French), a disease which is characterized by 458 PATHOGENIC MICROORGANISMS a pecailiar emphysematous swelling of the subcutaneous tissues and muscles, especially over the quarters. Clinically it is sometimes con- fused with anthrax. Morphology. — Bacilli ha\'ing rounded ends, from (l.r)/x to ().(i^ broad and from 3^i to ^p. long; mostly isolated; also occurring in pairs, joined end-to-end, but never growing out into long filaments, as the anthrax bacilli in culture and the bacilli of malignant edema in the bodies of animals are frequently seen to do. In the hanging drop the bacilli are observed to be actively motile, and in stained prepara- tions flagella may be demonstrated surrounding the peripherj^ The spores are elliptic in shape, usually thicker than the bacilli, lying near the middle of the rods, but rather toward one extremity. This gives to the bacilli containing spores a somewhat spindle shape. Stains with the ordinary aniline dyes, but not with Gram's method or only with difficulty and after long treatment or intense colors. Biology. — Like the bacillus of malignant edema, this is a strict anaerobe, and cannot be cultivated in an atmosphere in which oxygen is present. It grows best under hy- Fio. 160 drogen, and does not grow under _.--_^ carbonic acid. This bacillus de- y^ A f t velops at the room temperature in / ' \ . the usual culture media, in the ab- / »■ ' sence of oxygen, but it grows best ' ^ VI in those to which 1.5 to 2 per cent. ( / V , I \ °^ glucose or 5 per cent, of glycerin I ' 1 I has been added. i~ * ''\ M 1 Growth on Agar. — The colonies on \- < I agar are somewhat more compact * . 1 than those of malignant edema, but \ , * / I they also send out projections very -, /, y often. In agar-stick cultures, in the *^^ • 'l incubator, growth occurs after a day or two also some distance be- Batiiiii of symptomatic anthrax, showing low the surfacc, and is accompanied spore«. (After zettnow.) ^y the production of gas and a peculiar disagreeable acid odor. Pathogenicity.— The bacillus of symptomatic anthrax is pathogenic for cattle (which are immune against malignant edema), sheep, goats, guinea-pigs, and mice; horses, asses, and white rats, when inoculated with a culture of this bacillus, present only a limited reaction; and rabbits, swine, dogs, cats, chickens, ducks, and pigeons are, as a rule, naturally immune to the disease. The guinea-pig is the most susceptible of test animals. When susceptible animals are inoculated subcutan- eously with pure cultures of this organism, or with spores attached to a silk thread, or with bits of tissue from the affected parts of another animal dead of the disease, death ensues in from twenty-four to thirty- six hours. At the autopsy a bloody serum is found in the subcutaneous tissues, extending from the point of inoculation over the entire surface BACILLUS ANTIIRACIS SYMPTOM ATICI 459 of the abdomen, and the muscles present a dark red or black appearance, even more intense in color than in malignant edema, and there is a considerable development of gas. The lymphatic glands are markedly hyperemic. The disease occurs chicHy in cattle, more rarely iu sheep and goats; horses are not attacked spontaneously — /. c, by accidental infection. In man infection has never been produced, though ample opportunity by infection through wounds in slaughter-houses and by ingestion of infected meat has been given. The usual mode of natural infection by symptomatic anthrax is through wounds which penetrate not only the skin, but the deep, intercellular tissues; some cases of infection by ingestion have been observed. The pathologic findings present the conditions above described as occurring in the experimental infection. Distribution Outside of the Body. — Symptomatic anthrax, like anthrax and malignant edema, is a disease of the soil, but it shows a more limited endemic distribution than the former, and is differently dis- tributed over the earth's surface than the second of these diseases, being confined especially to places over which infected herds of cattle have been pastured. It is doubtful whether the bacilli are capable of development outside of the body like anthrax. In the form of spores, however, reproduction may take place; by contamination with these, through deep wounds acquired by animals in infected pastures, the disease is spread. Toxins. — Under favorable conditions extracellular toxins are formed so that the filtrate of cultures is very poisonous. Injections of the toxin into animals excite the production of antitoxins. Differential Diagnosis. — The principal points of differentiating this bacillus from the bacillus of malignant edema, which it closely resembles, are: it is smaller; it does not develop into long threads in the tissues; it is more actively motile, and forms spores more readily in the animal body than does the bacillus of malignant edema. It is pathogenic for cattle, while malignant edema is not; and swine, dogs, rabbits, chickens, and pigeons, which are readily infected with malignant edema, are not, as a rule, susceptible to symptomatic anthrax. Preventive Inoculations. — It is well known to veterinarians that recovery from one attack of symptomatic anthrax protects an animal against a second infection. Artificial immunitj' to infection can also be produced in various ways: by inoculations with cultures which have been kept for a few days at a temperature of 42° to 43° C. and have thus lost their original virulence, or by inoculations of filtered cultures, or of cultures sterilized by heat. P'or the production of im- munity in cattle it is advised to use a dried powder of the muscles of animals which have succumbed to the disease, and which have been subjected to a suitable temperature to insure attenuation of the virulence of the spores contained therein. Two vaccines are prepared, as in anthrax — a stronger vaccine by exposing a portion of the powder to a temperature of 85° to 90° C. for six hours, and a weaker vaccine by exposing it for the same time to a temperature of 100° to 104° C. In- 4()0 PA THOOENIC MICROORGANISMS ociilations are made with this attenuated virus into the end of the tail — first the weaker and later the stronger. These give rise to a local reaction of moderate intensity, and the animal is suhsequeiitly immune from the elfects of the most virulent material and from the disease. Fourteen days are allowed to elapse l)etween the two inoculations. The results obtained from this method of preventive inoculation seem to have been very satisfactory. According to the statistics, including many thousand cattle treated, the mortality, which among 22,300 non-inoculated cattle was 2.20 per cent., has been reduced to O.IG per cent, in 14,700 animals inoculated. When danger of immediate infection exists, it is advisable to inject some antitoxin with the vaccine. This lessens the reaction and gives immediate immunity. If an antitoxic serum is at hand it should be given in cases seen early in the disease. THE GROUP OF MALIGNANT EDEMA BACILLI. This group is widely distributed, being found in the superficial layers of the soil, in putrefying substances, in foul water, and by in- vasion from the intestine, in the blood of animals which have been suffocated. One such organism was discovered (1877) by Pasteur in animals after infection with putrid flesh, and named by him " vibrion septique." He recognized its anaerobic nature, but did not obtain it in pure culture. Koch and Gaffky (1881) carefully studied this micro- organism, described it in detail, and gave it the name " Bacillus edematii maligni" (Fig. 161). Fig. 161 2 3 4 Bacilli of malignant edema. 1, bacilli; 2, with spores; 3 and 4, deep colonies in dextrose nutrient agar. (Kolle and Wassermann.) In earlier times infection of man was quite often, but now with the surgical precaution used it rarel}- happens. This bacillus belongs to a group which ha\'e lateral flagelhe, produce oval spores, and grow only anaerobically. Morphology.— The edema bacillus is a rod of from 0.8m to V in width, and of very varying length, from 2^ to lO/i or more, according to the conditions of its cultivation and growth. It is usually found in THE CROUP OF MALICNANr EDEMA BACILLI 4G1 pairs, joined end-to-eiul, but may occur in chains or lon^ filaments. It forms spores, and tliese are situated in or near tlie middle of the body of the rods. Exceptionally the spores are near the ends. The spores y-ATY in length and are oval in form, being often of greater diameter than the bacilli, to which they give a more or less oval shape. The bacilli fitain readily by the usual aniline colors employed, but are usually decolorized by Gram's method. Freutag found that very young cultures were stained, while older ones were decolorized. Biology. — A strictly anaerobic, liquefying, motile bacillus. Forms spores which are very resistant. It grows in all the usual culture media in the absence of oxygen. Development takes place at 20° C, but more rapidly and abundantly at 37° C. Growth in Gelatin. — This bacijlus may be cultivated in ordinary nutrient gelatin, but the growth is more abundant in glucose gelatin containing 1 or 2 per cent, of glucose. After two or three days small, almost transparent, circular colonies appear | to 1 mm. in diameter. Later, as liquefaction increases, the colonies become grayish and then confluent. Gas bubbles are formed and the gelatin liquefies. Growth on Agar. — On agar plates the colonies appear as dull, whitish points, irregular in outline, and when examined under a low-power lens are seen to be composed of a dense network of interlacing threads, radiating irregularly from the centre toward the periphery. Blood-serum is rapidly liquefied, with the production of gas. Cul- tures of the malignant edema bacillus give oft' gas with a peculiar, disagreeable odor. Resistance. — The spores are very resistant and because of this the soil remains infected. Pathogenicity. — The bacillus of malignant edema is especially patho- genic for mice, guinea-pigs, and rabbits, although man, horses, dogs, goats, sheep, calves, pigs, chickens, and pigeons are also susceptible. A small quantity of a pure culture injected beneath the skin of a sus- ceptible animal gives rise to an extensi\'e hemorrhagic edema of the subcutaneous connective tissue, which extends over the entire surface of the abdomen and thorax, causing hyperemia and redness of the superficial muscles. No odor is developed, and there is little, if any, production of gas. In infection with garden earth, owing to the presence of associated bacilli, the eft'used serum is frothy from the de^'elopment of gas and possesses a putrefactive odor. The disease, in natural infection caused by the contamination of wounds with earth or feces, runs the course above described. Simple abrasion of the skin is not sufficient to produce infection; owing to the bacillus being capable only of an anaerobic existence, the poison nuist penetrate deep into the tissues. Malignant edema is confined mostly to the domestic animals, the horse, sheep, cattle, and swine, but cases luivc also been reported in man. Animals which recover from malignant edema are sul)sequently im- mune. Artificial immunity may be induced in guinea-pigs by injecting filtered cultures of the malignant edema bacillus in harmless quantities. 4G2 PATHOGENIC MICROORGANISMS In man the chief symptom is the sudden appearance of subcutaneous edematous swelling accompanied by high fever. In light cases this remains circumscribed; in severe cases it spreads widely and the case ends fatally. Appreciable quantities of gas usually fail. Autopsy shows a serous or hemorrhagic infiltration of the subcutaneous tissue and intramuscular connective tissue. In the inflamed tissue the bacilli with and without spores are found. Prevention. — Most cases are produced by injecting subcutaneously albuminous fluids infected by the bacilli. Care should be taken that fluids to be injected do not become infected by dust or dirt. BACILLUS AEROGENES CAPSULATUS (BACILLUS WELCHH). This bacillus was found by Welch in the bloodvessels of a patient suffering with aortic aneurysm; on autopsy, made in cool weather, eight hours after death, the vessels were observed to be full of gas bubbles. Since then it has been found in a number of cases in which gas has developed from within sixty hours of death until some hours after death. External cutting operations on the urethra and operations upon the uterus have been followed in a nvimber of cases by infection. It has been found in ovarian abscesses and in infections of the genito- urinary tract. These cases are, as a rule, marked by delirium, rapid pulse, high temperature, and the development of emphysema and dis- FiG. 162 A <^, - -'*'-?"--&»., , ^-o,'^. "*^ X -^ if . "ij ^ Bacillus aerogenes capsulatus. 1, bacilli; 2, spores; 3, culture in dextrose nutrient agar. coloration of the diseased area or of marked abdominal distention when the peritoneal cavity is involved. This bacillus is present, as a rule, in the intestinal canal of man and animals and is apt to be fotmd in the dust of hospitals and elsewhere. Herteri has shown that it is present in excessive numbers in certain diseases of the digestive tract. These cases are ajrt to develop anemia. Morphology.— Straight or slightly curved rods, with rounded or sometimes square-cut ends; somewhat thicker than the anthrax bacilli ' .Journal of Biolog. Cheni., 1906, ii, p. 1. BACILLUS AEROGENES CAPSULATUS 463 and varying- in length; occasionally long threads and chains are seen. The bacilli in the animal body, and sometimes in cnltures, are enclosed in a transparent capsule. Spores are usually absent in the tissues and often in cultures. Dunham showed that the culture isolated by Welch formed spores when grown on blood-serum. Some strains since isolated make spores readily. It is possible that these differences may be due to the fact of there being several strains. Biology. — An anaerobic, non-motile, non-liquefying bacillus. Dif- ferent strains of this bacillus vary in their tendency to make spores. It is stained by Gram, but is more easily decolorized than many bacteria. Growth is rapid at 37° C, in the usual culture media in the absence of oxygen, and is accompanied by the production of gas. Nutrient gelatin is not liquefied by the growth of this bacillus, but it is gradually peptonized. If agar colonies are developed which are from 1 to 2 mm. or more in diameter, grayish-white in color, and in the form of flattened spheres, ovals, or irregular masses, beset with hair-like projections. Bouillon is diffusely clouded, and a white sediment is formed. Alillc becomes acidified and coagulated, then partially digested, giving a worm-eaten appearance to the clot. Pathogenicity. — Usually non-pathogenic in healthy animals, although Dunham found that the bacillus taken freshly from human infection is sometimes very virulent. When quantities up to 2.5 c.c. of fresh bouillon cultures are injected into the circulation of rabbits and the animals killed shortly after the injection, the bacilli develop rapidly, with an abundant formation of gas in the bloodvessels and organs, especially the liver. This procedure is one of the best methods of obtaining the bacilli: The material suspected to contain the bacillus alone or associated with other bacteria is injected intravenously into rabbits, which are killed five minutes later and kept at 37° C. for sixteen hours, and cultures made from the liver and heart's blood. It is suggested by Welch that in some of the cases in which death has been attributed to the entrance of air into the \'eins the gas found at the atuopsy may not have been atmospheric air, but may have been produced by this or some similar microorganism entering the circulation and developing shortly before and after death. The same may be true for gas in the uterine cavity. Bacillus Enteritidis Sporogenes. — This resembles B. Welchii. It produces spores readily. Klein considers that when taken in milk it may produce diarrhea. This is disputed by others. It is also considered to be an e\-idence of sewage pollution, but this is not at all certain since it occurs in cultivated soils (Jordan). CHAPTER XXXTV. THE CHOLERA SPHHLLUM (CHOLERA VIBRIO) AND SIMILAR VARIETIES. In 18S3 Koch separated a characteristically curved organism from the dejecta and intestines of cholera patients — the so-called "comma hacUlus." It was absent from the blood and viscera, and was found only in the intestines ; and the greater the number, it was said, the more acute the attack. Koch also demonstrated an invasion of the mucosa and its glands. The organisms were found in the stools on staining the mucus flakes or the fiuid with methylene blue or fushsin, and sometimes alone; by means of cultivation on gelatin they were readily separated from the stools. Numerous control observations made upon other diarrhoeic dejecta and upon normal stools were negative; the comma bacillus was found in choleraic material only, or occasionally in small numbers in the stools of healthy persons who came in contact with cholera. Soon, however, other observers described comma-shaped organisms of non-choleraic origin. Finkler and Prior, for instance, found them in the diarrheal stools of cholera nostras, Deneke in cheese, Lewis and Miller in saliva. All of these organisms, however, differed in some respects from Koch's comma bacillus, and it has since been proved that none of them is affected by the specific serum of animals immunized to cholera. After a time, therefore, the exclusive association of Koch's vibrio with cholera or those in contact with it became almost generally ackowledged, until now it is regarded by bacteriologists everywhere to be the specific cause of Asiatic cholera. Certain sporadic cases of cholera-like disease, however, are undoubtedly due to other organisms. Morphology. — Curved rods with rounded ends which do not lie in the same plane, of an average of l\p. in length and about OAfx in breadth. The curvature of the rods may be very slight, like that of a comma, or distinctly marked, particularly in fresh unstained prepara- tions where the adhesion of two individuals presents the appearance of a half-circle. By the inverse junction of two vibrios S-shaped forms are produced. Longer forms are rarely seen in the intestinal discharges or from the cultures grown on solid media, but in fluids, especially when grown under unFa\'orable conditions, long, spiral filaments may develop. 'J'he spiral forms are best stndied in the hanging drop, for ill the dried and stained pre])aTations the spiral character of the long lilaments is often obliterated. In film preparations from the intestinal contents in typical eases it will be f(jund that the organisms are present in enormous innn})ers, and often in almost pure culture. In old cultures lilOLOCY 4(J5 irregularly clubhed and thickened involution forms are freciuent, and tlie presence iu the organisms of small, rounded, highly refractile bodies is often noted. Staining.— The cholera spirillum stain.'! with the aniline colors usually employed, but not as readily as many other bacteria; a diluted aqueous solution of carbol fuchsin (1-10) is recommended as the most reliable staining agent. It is decolorized by dram's method. The organisms _ exhibit one long, fine flagellum attached to one end (Cholera-like spirilla often have two or more end flagella.) In sections the vibrios are stained best by alkaline methylene-blue solution and \\-aslK\l in water slightly acidulated with acetic acid. Fiu. lOo ViG. 104 C'ontucl smear of c-oIokn' of cholera spirilla from afj;ar. X 7()U diameters. (Dunham.) C'holera spirilla preparation from gela- tin-plate culture of eholera. X 800 diam- eters. Biology. — The cholera spirillum is aerobic, liquifying, and extremely motile. It grows readily on ordinary culture media, best at 37° C, but also at room temperature, 22° C. In gelatin plate cultures, characteristic colonies are produced. After twenty-four hours' growth, there is a uniform granular appearance of the surface, which because of the high degree of refraction gives the appearance of being powdered with broken glass. Color is practically absent or there ma\- be a slight yellowish tint. The al)sence of color aids one in separating the cholera colony from those developing from coli in the .stool. If growth is continued, liquefaction appears about the colony and the appearance gradually changes. The characteristic colony in gelatin has been of the greatest practical importance. With the introduction of special selective media, however, the use of gelatin has been practically discontinued. In gelatin stab-culture, a small funnel of liquifaction api)ears after twenty-four hours. This deepens and broadens, until at the end of a week liquifaction may be complete. On agar a moist shiny grayish-yellow layer develops. On the surface of agar-plates the indi^■idual colonies are characteristic. They are 30 4(iH PATHOGENIC MICROORGANISMS round, transparent and have a rather distinctive opalescent sheen. This characteristic appearance is used practically for isolation. On Diendonne alkaline blood-agar the colonies are large and circular. On transmitted light there is a glassy transparency, on reflected light they are grayish. On alkaline egg-agar the colonies, when examined by "transmitted light, appear to be deep in the agar and have a dis- tinctive hazy appearance due to the development of a_ halo about the colony. A zone of clearing may develop where growth is continued. As some "fecal bacteria other than vibrios may develop on these media, the development of a typical colony is a great aid in isolation. Fiu. 105 ('(ilri'a colonii'.s in gelatin; twenty-four to thirty-six liours' growtli. X about 20 diameters. Loffler's blood-serum is rapidly liquified at 37° C. Milk is not coagu- lated. Glucose and saccharose are fermented with great rapidity, acid only being formed. Growth in fluid media is abundant and usually characteristic, most so in peptone-water. Peptone-water is diffusely cloudy to a moderate degree, but at the surface the cloud is much denser. Due to the greater supply of oxygen, the vibrios seek the surface and multiply here more freely. Gholera strains «'hich have been in culti^'ation for some time show changes in morphology and in the appearance of colonies. They grow less typically on selective media and fluid cultures may develop a wrinkled pellicle. Their digestive [powers are also lessened, as gelatin-liquefaction or the liquefaction of Loffler's serum medium. Variations can also be induced by special conditions. Reaction of Media. — Cholera grows best on media that are sharply alkaline to litmus. They can grow in an excess of alkali sufficient to inhibit the growth of many of the bacteria found in feces. This is most evident where an alkali-albumin mixture is used to increase the alkalinity. (See Special JNIedia.) Cholera-red Reaction. — All cholera strains give this reaction. This is important and many non-cholera spirillie do not give this reaction. Hemolytic Production. — Six strains were isolated by Gottshlich at El Tor from Pilgrims to Mecca, who died with diarrheal symptoms but ' had shown no clinical evidences of cholera. These strains are called RESISTANCE AND VITALITY 4B7 "El Tor" strains. They give tlie serum reactions of cholera vibrios but produce a strong hemolysin. Xraus and Ruffer believed them not to be true cholera, as hemolysin production was considered by the former as an attribute possessed only by non-cholera types. Further investigations have shown that cholera vibrios may develop, lose, or show wide ^•ariations in their ability to produce hemolysins. It is apparently a potential power of all vibrios and of no value in differen- tiation. For this reason the El Tor strains must be considered as cholera vibrios. Resistance and Vitality. Development Outside of Body. — If a culture be spread on a cover-glass and exposed to the action of the air at room temperature the bacilli will be dead at the end of two or three hours, unless, the layer of culture is very thick, in which case it may take twenty-four hours or more to kill all the bacilli. This indicates that infection is rarely if ever produced by means of dust or other dried objects contaminated with cholera bacilli. The transmission of these organisms through the air, therefore, can only take place for short distances, as by the spray from infectious liquids. The cholera bacillus is also injuriously affected by the abundant growth of saprophytic bacteria. It is true that when associated with other bacteria, if present in large numbers, and if the conditions for their development are particularly favorable, the cholera bacillus may at first gain the upper hand, as in the moist linen of cholera patients, or in soil impregnated with cholera dejecta; but later, after two or three days, even in such cases, the bacilli die off and other bacteria gradually take, their place. Thus, Koch found that the fluid contents of pri\'ies twenty-four hours after the introduction of comma bacilli no longer contained the living organisms; in impure river- \\-ater they were not demonstrable for more than six to seven days, as a rule. In the dejecta of cholera patients thej' were found usually only for a few days (one to tliree days), though rarely they have been observed for twenty to thirty days, and on one occasion for one hundred and twent}- days. In imsterilized water they may also retain their ^'itality for a relatively long time; thus, in stagnant well-water they have been found for eighteen days, and in an aquariiun containing j)lants and fishes, the water of which was inoculated with cholera germs, they were isolated several months later from the mud at the bottom. Koch found them in the foul water of a tank in India, used by the nati\'es for drinking purposes. In running ri\er-water, however, they have not been observed for over six to eight daj's. In milk they are finally destroyed by acidity due to the growth of the milk bacteria, in sterilized milk they may survive eight to ten days. For the cholera organisms the conditions faA'orable to growth are a warm temperature, moisture, a good supply of oxygen, and a considerable proportion of organic material. These conditions are fully met with outside the body in but \'ery few localities. The common bacillus has the a^'e^ag■e resistance of spore-free bacteria, and is killed by exposure to moist heat at 56° C. in half an hour, at 4GS PATHOGENIC MICROORGANISMS 8(J° for fi^'e minutes, at 95° tt. 100° C. in one minute. The bacilli have been found alive kept for a few days in ice, but ice which has been preserved for several weeks does not contain living bacilli. Chemical disinfectants readily destroy the vitality of cholera vibrios. For disinfection on a small scale, as for washing the hands when con- taminated with cholera infection, a 0.1 per cent, solution of bichloride of mercury, or a 2 to 3 per cent, solution of carbolic acid or, better, lysol may be used. For disinfection on a large scale, as for the disin- fection of cholera stools, strongly alkaline milk of lime is an excellent agent. The wash of cholera patients, contaminated furniture, floors, etc., may be disinfected by a solution of 5 per cent, carbolic acid and soap water. For the disinfection of drinking water, chlorinated lime, 1 to 2 parts per million of free chlorine may be used for fairly pure water. Fi^'e i)arts per million would probably he effecti\'e e\'en in polluted water. Pathogenesis. — Not one of the lower animals is naturally subject to cholera. It has been shown that the comma bacillus is extremely sensitive to the action of acids, and is quickly destroyed by the acid secretions of the stomach of man or the k)wer animals, when these secretions are normally produced. Koch sought to produce infection in guinea-pigs per view naturales by first neutralizing the contents of the stomach with a solution of carbonate of soda and inhibiting peri- stalsis by the use of opium. Animals thus treated in six to eighteen hours after infection show an indisposition to eat and other signs of weakness, its posterior extremities become weak and apparently para- lyzed, and, as a rule, death occurs within forty-eight hours with the symptoms of collapse and fall of temperature. At the autopsy the small intestine is found to be congested and filled with a watery fluid, con- taining the spirillum in great numbers. These results, however, are somewhat weakened by the fact that experiments made with some other bacteria morphologically similar to the comma bacillus of Koch but specifically dift'erent, occasionally produced death when introduced in the same wa>' into the small intestines of guinea-pigs. Later, several investigators have succeeded in producing in animals a disease similar to cholera. Thomas injected a dilute suspension of cholera spirilla into the ear vein of young rabbits, and thus caused symptoms and lesions of the intestinal mucous membrane resembling those of cholera in man. The spirillum was reco^'ered from the feces. Metchnikoft' was similarly successful with suckling rabbits by rubbing a small amount of a culture on the teats of a mother ralibit. Intraperitoneal injections with cholera spirilla kill guinea-pigs acutely, but intestinal lesions are rarely found. There are several cases on record which furnish the most satisfactory evidence that the cholera s])irillum is able to produce the disease in man. In 1S84 a student in Koch's laboratory in Berlin, who was taking a course on cholera, i)ecame ill with a severe attack of cholera. At that time there was no cholera in Germany, and the infection could not ha^'e been produced in any other way than through the cholera COAfMUNIC ABILITY 469 ciiltuR's wliieh were heing used for the iiistruetidii uf students. In 1892 IVttenkdFer and Emmerich e\])erinu'nted (in themselves by s\valh)\vini;' small quantities of fresh chdlera cultures (ihtained from Ilamliurg. Pettenkofer was atfected with a mild attack of cholerine or se\ere diarrhea, from which he recovered in a few days without any serious effects, hut Emmerich became very ill. On the ni Peptone-w;iter • Poptone-water. fDircct I Sclfftivp Modia fSmcar, if positivp, hanging drop -( Mirrcisfopif )■ [ '. with and without serinn. Platr i, Examination J / .Agglutination of \ j for pure fulturcs for vorifica- \ susi)icious colony, j [ tion, etc. J Direct Microscopic Examination. — This is of great value in suspected cases but of no use in the examination of carriers. The presence of many typical, extremely motile vibrios warrants a tentative diagnosis of cholera. In exceptional cases, not cholera, a great abundance of \'ibrios may be found. This led us in one instance to unnecessarily isolate a nurse who, while caring for a cholera patient, developed nervous diarrhea. ISOLATION OF CHOLERA VIHRU) FROM STOOLS 478 Peptone-water. — Inoculate' with feces and incuhate six to twelve liours. Hxaniine smears from surface .urowtli. If tlie xihrios are numerous, ])i-e- pare liansing' drojis with and without iinnuine serum. If \-ihrios are not found in the snu'ar, or if too few in numher for haiii;iii,n' dru]) oiisei'\a- tion, suhinocuhite into pei)tone-water, or selecti\e nie(ha, or hoth. Subculture Peptone-water. — This second enriclunent is prohahly never required except in tlie examination of sus])ected carriers. In four instances we have found cholera vibrios in subculture where they were not e\ident in smears from the first peptone culture. This second enrichment helps also to exclude some of the vibrios other than cholera, some will have died out, some will not have enriched beyond the amount present in the first peptone culture; these are not cholera. Smears from the surface growth are examined and if positive, examination of hanging drops carried on Where haste is not a factor as in carrier examinations the examination of the first ])e])tone culture may be omitted. In making smears and hanging drops from peptone-water especial care should be taken not to shake the tubes and that the loop is just touched to the surface. In the examination of smears little time need be spent; if \ery few vibrios are present, further enrichment is necessary. If they are present in sufficient numbers for agglutination, they are found at once. The use of the surface growth for agglutination is open to certain criticisms, but in practical work it has given us accurate results. Where cholera or cholera-like vibrios, having the same abilit\' to enrich as true cholera, are present, the surface growth is nearly a pure culture. The reliability of the peptone method, as outlined, was best shown in a series of examinations where we found two carriers. In this series 50 per cent, of the stools contained vibrios. The results were checked by the isolation of pure cultures. W^ith some experience a great number of examinations can be carried through in this way, using peptone- water only, with a minimum of preparation and equijjment. Selective Media. — Inoculation may be done directly from the feces or after enrichment in peptone-water. Tlie ad\'aiitages of such media is that they may be heavily inoculated. The colonies which develop are used for agglutination. A^arious modifications of Dieudonne's have been suggested. The alkaline-egg medium has the advantage that a distinctive vibrio colony develops allowing quick selection for agglutination should non-vibrios develop. A secondary plating on plain agar is necessary, if pure cultures are to be obtained from selective media. The other bacteria, which are only restrained, may be trans- ferred in fishing a colony and thus yield mixed cultures. Alkaline agar may be employed for plating either tlirectly from the feces or from peptone enrichments. As stated, the colony is distinctive and no difficulty will be encountered if the vibrios are fairly numerous.' ' The direct plating of stooLs on agar or gelatin is sometimes of practical importance. In no disease but cholera do vibrio colonies approach or exceed in number those develop- ing from the ordinary fecal bacteria. Even without identification by agglutination, such a condition gives us a practically certain diagnosis of cholera. 474 PATHOGENIC MICROORGANISMS Saccharose Peptone-water. — This medium has been suggested by Bendick to avoid the time-consuming microscopic examination of peptone tubes. The stool is first inoculated in peptone-water and the surface growth then inoculated into this medium. Because of the ability of the cholera vibrio to rapidly split saccharose, decolorization occurs in five to eight hours. The tubes which do not decolorize in this time can be discarded, the decolorized are examined for vibrios, which, if present, are isolated by plating. Because of the presence of a fermentable sugar, the growth is diffused and the surface is not satisfactory for agglutination. To avoid this difficult}^ duplicate peptone tubes could be planted, however, and used for agglutination where the saccharose peptone tubes were decolorized. This method promises to be of the greatest value where many specimens must be examined in the search for carriers. Examination of Suspected Carriers. — The simplest procedures possible must be employed where the daily examinations may run into many hundreds. The peptone method outlined reduces the bacteriological work ^•ery much, especially if the first peptone tubes are not examined. The collection of stools under these circumstances is impracticable. Ordinary swabs moistened in peptone-water may be inserted into the rectum to obtain fecal material. Individual glass specula may be employed to aid in the introduction of the swabs. Where feasible much time is saved by giving an identification card a number and dropping the swab at the time of taking into a similarly niunbered tube of peptone- water. The sterilization of the peptone tubes, supported in blocks of wood or racks with a cover instead of using cotton plugs and the use of individual wire loops (sterilized in bundles) for transfers or smears saves a great deal of time. The removal and replacing of cotton plugs and the burning of the platinum loop usually employed is thus avoided. In transferring the wire is dropped into the second tube. The exposure of the tubes when the cover is removed for inoculation or transfer does not lead to interference by contamination.^ Isolation from Water. — As the cholera vibrios are few in number in water a large volume should be used. About a liter of water is taken and 100 c.c. amounts placed in Ehrlenmeyer flasks and to each is added 10 c.c. of a tenfold strength peptone-water. These are shaken and incubated for eighteen hours. The surface growth is then subinoculated in peptone-water for further enrichment if necessary and plates made for isolation. Especial care must be taken in fishing as vibrios other than cholera may be present in the water, and there is some evidence that cholera vibrios lose their agglutinability to some extent after living in water. These difficulties may be surmounted by many fishings and the use of a high titer serum in low dilutions for selecting cultures for final identification. Spirilla (Vibrios) More or Less Allied to the Cholera Spirillum. — Various types of spirilla may be isolated from stools, water, and other sources. 1 Creel, Anier. Journ. Public Health, December, 1911. ISOLATION OF CHOLERA VIBRIO FROM STOOLS 475 Some are practically identical, niorijhologically and culturally, witli true cholera. It woidd be well to limit the term "cholera-like vibrios" to this gToiip. Another group of viljrios, similar to cholera in some respects but differing in others might be termed "non-cholera vibrios. Much of the practical interest in these types was lost when the sero- logical methods for identification were introduced. In emergency work, where immune serum is not at hand, they are of extreme practical importance. A few of the types are of interest because of their patho- genicity for animals. Some of these vibrios enrich in mixed cultures in peptone-water like true cholera, others enrich to a limited extent or die out. The majority of the vibrios found in stools during routine examinations for cholera spirilla can be excluded culturally if serum be not available. Of 50 \'ibrios thus isolated we found that 43 did not give the cholera-red reaction. Of the 7 that did, 2 produced no acid from glucose and 4 produced acid and gas. One produced acid only, but could be excluded, as it formed a tenacious pellicle on peptone-water. The following is a short list of vibrios of interest historically or because of their pathogenicity for animals: Vibrio Metschnikovii; source, epizootic gastro-enteritis of fowls, present in in- testinal contents and in blood. Typical vibrio with one flagellum, liquifies gelatin and gives cholera-red reaction. A minute amount of culture inoculated into a cutaneous wound causes a fatal vibriosepticemia in pigeons and guinea- Vibrio ]Massa\'ah; source stools, considered cholera vibrio when first isolated, four flagellce, pathogenicitj' like vibrio Metschnikovii. Mbrio septicus; source, stools, case of cholerine, cholera-like culturally and morphologically, minute amounts cause a rapidlj^ fatal septicemia in guinea-pigs. Vibrio Nasik, produces a very strong toxin, 0.5 to 1 c.c. of a four-day broth culture intravenously kills rabbits in 10 to 15 minutes, a strong hemolytic toxin is produced. Sph'iUuni Finkler-Prior ; sources feces, cholera nostras, does not give cholera- red reaction. Vibrio Ivanoff and vibrio Berlionensis; source, former, artificially inoculated stools for disinfection tests, latter, water artificiaUy inocidated to determine viability of cholera vibrio in water. Both are probably variants produced by artificial conditions, they give the immune reactions of cholera spiriUa. CHAPTER XXXV. PATHOGENIC IMICROClRGANISlMS BELONGING TO THE IIKiHER BACTERIA (TRICHO^IYCETES). Observers are still of difi'erent opinions in reo;ai'(] to the classifi- cation of this irronp of organisms (see Chapter II). Foulei'ton and his associates have made an extensive study of tliis group, lioth sa]jrophytic and parasitic varieties, and they agree with some others in cal- hng attention to the acid-fast character of some of the varieties and to the appar- ent relationship of the group to B. tuberculosis, B. mallei, and B. diphtheriw. To us, however, the relationship does not seem to be close enough to place all of these organisms in one group. We have shown (Chapter II) that the apparent branchmg in B. diphtherice is not a true branching. If not classed with the true liacteria, they should either be put in a group I;)y themselves or be classed with tlie cladothrix group since their apparent l^ranching takes place in a manner similar to that described as occurring in the latter grou)5. Foulerton considers all organisms in the group classed as higher bacteria as belonging to a single genus, streptothrix, which he places with the h>'phomy- cetes, or mold fungi, because of their growth in branching threads from spore- like bodies. He saj^s that streptothrix and actinomyces are absolutely synony- mous terms, and that the majority of pathologists consider them so. Wright and others do not agree with this view (see below). It seems to us tliat more mumte work, Ijoth clinical and experimental, should be done on this group of infections before a classification can be accepted. Leptothrix Infections. — Leptothrix forms are frequently found in the human mouth (Leptothrix huccalis) , and one or two writers have chiimed that under certahi conditions these may become pathogenic, but since no eorroborati\'e work has been done, and very little is known aljout tlie grouj), no opinion can be formed of the worth of these ob- servations. Cladothrix Infections. — The organisms found in the comparatively few cases \vhich ha\-e been considered by their obse^^'ers to be due to cladothrix have not been minutely enough studied to decide definitely as to their true or false branching, tlie characteristic chosen to separate them from the nocardia; hence it is difficult to separate the two groups, but an attem]:)t should be made, since the difference said to exist between them is an important one from a morphologic stand-point. Clinically, however, according to the reports, the cases cited are very similar to those said to be due to nocardia (streptothrix) and to actino- myces. Gasten found in a case f)f clinically typical actinomycosis, in which abscess cavities were found along the spinal column, not the usual actinomyces in the yellow, granular pus, but a fine mass of filaments. THE ACTINOMYCES 477 Cultures gTt'W on all the ordinary media, best at iiicuhator tempera- ture, but also at lower temperature on <;x'';itiii. The gt'latin stiek eulture, whieh was espeeially charaeteristie, formed on the surface a whitish button; delicate threads stretched out in all directious from the point of inoculation. On agar and potato rumpled, folded films with white deposit forms on the surface, which contained spores. Animal inocidation ga\e positi\'e results oidy in a few cases of intra- peritoneal injection of rabbits and guinea-pigs. Purulent nodules were found in the peritoneum. Gasten called the organism Cladotlirix li(iuefaci('rif<. Eppiiigcr found in ])ostmortem examination of a case of chronic cerebral abscess, which was the result of purulent meningitis, in the pus and abscess walls, etc., a delicate fungoid growth which he suc- ceeded in cultivating on various media. On sugar agar it formed yellow, rumpled colonies which finally develojied into a skin. On potato it grew rapidly, but the colonies remained small, at first a white granular deposit, which afterward turned red, and on the twentieth day resembled a crystallized almond. It did not grow' well on gelatin. In bouillon it formed on the surface a small white granule, which became deeper in the centre as it grew and sank to the bottom as a white deposit. The bouillon remained clear. Microscopically, the fungus consisted of fine threads without branches which exhibited distinct motility. No fiagella were observed. It was judged to be a cladothrix, to which the name "asteroides" was given by the author. It proved to be quite pathogenic for rabbits and guinea-pigs, and produced an infection called pseudotuberculosis. Mice were not affected by inoculation. THE ACTINOMYCES. The little clumps produced by this group of parasites were first seen by Von Langenbeek in 1845 and the organisms were later discovered by Bollinger (1877) in the ox. They were given the name of actinomyces, or ray fungus, by the botonist Harz. They were reported in human beings by Israel in 1878. The characteristics of the microorganisms, first described minutely by Bostrom (1890) and by Wolf and Israel (1891), differed greatly and have led to confusion. Bostrom's organism grew best areobically and developed well at room temperature. He noted the intimate rela- tion of the organism with fragments of grain, and this led to the finding of similar microorganisms in the outer world on grains, grasses, etc. There is no doubt that some suppurative processes have been due to organisms of these characteristics, but they do not seem to excite true actinomycosis. Wolf and Israel described a microorganism from two human cases, which differs from that described by Bostrom, but agrees with the microorganisms obtained by most of the more recent investigators from typical actinomycosis. It grew best imder anaerobic conditions 47S PATHOGENIC MICROORGANISMS and did not grow at room temperature. Its growth was much less luxuriant than Bostrom's microorganism. On the surface of anaerobic agar slant cultures on the third, fourth, and fifth day numerous minute isolated dew-drop-like colonies appeared, the largest pinhead in size. These gradually became larger and formed ball-like, irregularlj' rounded, elevated nodules varying in size up to that of a millet-seed, exceptionally attaining the size of a lentil or larger. As a rule, the colonies did not become confluent, and an apparent!}' homogeneous layer of growth was seen to be made up of separate nodules if examined with a lens. In some instances the colonies presented a prominent centre with a lobulated margin and appeared as rosettes. A characteristic of the colonies was that the}' sent into the agar root-like projections. In aerobic agar slant cultures no growth or a slow and yevy feeble growth was obtained. In stab cultures the growth was sometimes limited to Fiii. 100 Smear from IjouiUou culture ot ac-tinomyces. X 1.500 diameters. (From Wright.) the lower portion of the line of inoculation or was more vigorous there. In bouillon, after three to five days, growth appeared as small white flakes, partly floating and partly collected at the bottom of the tube. Growth occurred in bouillon under aerobic conditions, but was better under anaerobic conditions. The organisms here grow in branching and interlacing filaments, which later tend to break into segments (see Fig. 16(1). The microorganism in smear preparations from agar cultures appeared chiefly as short homogeneous, usually straight, but also comma-like or bowed rods, whose length and breadth varied. In many cultures short plump rods predominated, and in others longer, thicker, or thinner individuals were more numerous. The ends of the rods often showed oval or ball-like swellings. Swollen clubs were formed irregularly in the presence of blood or serous fluids. On intraperitoneal inoculation guinea-pigs and rabbits after four to seventeen weeks showed, after jjeing killed, tumor growths mostly THE ACTINOMYCES 479 in tlic ])eritiineal ca\'ity and in one instance in the s])leen. Microscopic examination of tlie tumors showed in all cases but one the presence of typical actinoniyces colonies, in most cases with typical "clubs." The general histological appearance of tiie tumors was like that of actino- myotic tisue. Wolf in a later paper reports that an animal inoculated in the peri- toneal ca\'ity with a culture of the same organism had lived a year and a half. At the autopsy several tumors were found in the peritoneal cavity, and in the liver a large typical tumor in which were many colonies which by microscopic examination were shown to be typical club-bearing actinomyces colonies. Wright in 1905 made an extensive study of actinomycosis and added greatly to our knowledge of it. Naked-eye Appearance of Colonies of Parasite in Tissues. — In both man and animals they can be readily seen in the pus from the affected regions as small, white, yellowish or greenish granules of pin- head size (from 0.5 to 2 mm. in diameter). When pus has not formed they lie embedded in the granulation tissue. Fni. 1G7 .A f\-|ji(.';il "clulj "-bearing colony of actinomyces. X 325 diameters. (From Wright.) Microscopic Appearance. — Microscopically, these bodies are seen to be made up of threads, which radiate from a centre and present bulbous, club-like terminations (Hg. 167). These club-like termi- nations are characteristic of the actinomyces. They are generally arranged in pairs, closely crowded together, and are very gKstening in appearance. They are more common in bovine than in human lesions. They have been thought to be reproductive elements, but they are probably simply a reacti(jn of the filament end to the host tissue. The threads which compose the central mass of the granules are from 0.3/j to 0.5^ in diameter. The threads show true branching and in the older colonies show a segmentation which gives them the apisearance of chains of cocci. Sometimes the whole centre of the 4.S0 PATHOGENIC MICROORGANISMS colonies seems to be a mass of coccus-like bodies most of which are considered spores or conidia ; the clubs are from 6m to S/j. in diameter. The threads and spores are stained with the ordinary aniline colors, also by Gram's solution ; when stained with gentian violet and by Gram's method the threads appear more distinct than when stained with methylene blue. The cluljs usually lose their stain by Gram's method and take the contrast strain. Isolation of Actinomyces. — There are two cultural \'arieties, one grows aerobically and the other anaerobically. The anaerobic variety is grown with difficulty. A large number of solidified blood-serum or serum agar tubes are inoculatetl with the hope that one or two will develop a growth. The culture appears much like one of tubercle bacilli. It grows, however, into the medium, and takes on a yellowish hue. Wright recommends that granules, preferably obtained from closed lesions, are first thoroughly washed in sterile water or bouillon and then crushed between two sterile glass slides. In bovine cases make sure the granule has filamentous masses, for, if not, no culture will grow. The crushed granule is transferred to a tube of melted 1 per cent, glucose agar at 40° ('. The material is thoroughly distributed by shaking and the tube placed in the incubator. A number of granules after washing should be placed on the inside of a sterile test-tube and allowed to dry. In this Avay, should the material be contaminated, the drying of the granules for se^'eral weeks may kill off the other organisms. The tube should lie examined dail}'. If a number of living filaments were added to the agar a large number of colonies will develop. These will be most numerous in the depth in a zone five to twelve millimeters below the surface (aerophiles). The cultures are cpiite resistant to outside influences; dried, they may be kept for a year or more; they are killed by an exposure of five minutes to a temperature of 7.5° (\ Experimental Inoculation in Animals. — True progressi\-e infection is rarely or never obtained by the injection of pure cultures in rabbits, guinea-pigs, or other small animals. In the bo\'ines, however, the disease has been pr(jduccd from cultures. The cultures form the char- acteristic "club"-bearing colonies in the tissues of the experimental animals. These colonies are either enclosed in small nodules of connec- tive tissue or are contained in suppurative foci within nodular tumors made up of connective tissues in varying stages of development. Wright does not accept the prevalent belief, based on the work of Bostrom, Gasperini, and others, that the specific infectious agent of actinomycosis is to l:)e found among certain branching microorganisms, widely disseminated in the outer world. He thinks that these forms belong to a separate genus, Nncardin, and that those cases of undoubted infection by them should l)e called nocardiosis and not acthiomycosis. The term actinomycosis should be used only for those inflammatory [)rocesscs tlic lesions of which contain the characteristic granules or "driisen." That Nocardia ever forms these characteristic structures in lesions produced Iiy them has not been con\'incingly shown. THE ACTINOMYCES 481 Occurrence. — Actinomycosis is ((uitc ]jrc\alciit a.moiiK' cattle, in which it occurs endeniically; it is more rare among swine and horses. jNIany cases ha\'e in recent years been reported in man. The disease is rarely communicated from one animal to another and no case is known where a direct history of human contagion has been obtained. The cereal grains, which from their nature are capable of penetrating the tissues, have been found in centres of actinomycotic infection in the lower animals. The microorganism may also be introduced by means of carious teeth. Cutaneous infection has been produced by wood splinters, and infection of the lungs by aspiration of fragments of teeth containing the fimgus. The presence of the microorganism in cereal grains, which was formerly accepted, is denied l)y Wright and therefore certainly placed in doubt. The further distribution of the fungus after it is introduced into the tissues is effected partly by its growth and partly by conveyance by means of the lymphatics and leukocytes. Not infrequently a mixed infection with the pyogenic cocci occurs in actinomycosis. Characteristics of Disease in Man and Animals. — In the earliest stages of its growth the parasite gives rise to a small granulation tumor, not unlike that produced by the tubercle bacillus, which contains, in addition to small round cells, epithelial elements and giant cells. After it reaches a certain size there is great proliferation of the surrounding connective tissue, and the growth may, particularly in the jaw, look like, and was long mistaken for, osteosarcoma. Finally, suppuration occurs, which, according to Israel, may be produced directly by the fungus itself. The course of the disease is very chronic. Usually the first sign is a point of infiltration about the lower jaw or lower on the neck. This almost painless swelling increases and finally softens in its centre. The necrotic tissue finally forces a passage externally or, passing downward, infects the pleura, lungs, mediastinum, or ribs. As a rule, the disease is not accompanied by fever. In cattle the disease is usually situated in some portion of the head, especially in the jaw, tongue, or tonsils, hence called lumpy jaw, wooden tongue, etc. Primary lung, intestinal, and skin lesions are not infrequent. These local lesions sometimes scatter and produce a general infection and the udder may be involved. Treatment. — In 1892 Nocard showed that cases in animals might be cured by iodide of potassium, calling attention to the fact that Thomassen had recommended this treatment in 188.5. It is given in doses of Ij to 2^ drams once a day. Salmon and vSmith (U. S. Bureau of Animal Industry, Circular No. 9(1) give directions as to its use. Mycetoma (Madura Foot). — This is a puinilent inflammation of the foot occurring jjrimarily in warm dimates. The inflammation is accomijanied by much irregular enlargement of the foot. Three varieties of this condition have been described based upon the color of the granules found in the diseased area: (1) white, (2) black, and (3) red. Tiie white ^'ariety has been studied by Mu.sgrave and Clegg 31 482 PATHOGENIC MICROORGANISMS (1907), who have isolated an organism resembUng somewliat aetino- myces and somewhat the organism isolated by Wright (1898) from a black variety of the disease which is probably a true mold. NOCARDIA (STREPTOTHRIX) INFECTIONS. The most familiar name of this group of microorgansims is strepto- thrLx, but this name had already been used for another genus; there- fore, according to the rules of nomenclature, nocardia, which name was proposed by Trevisan in 1889 for the organism discovered by Nocard in farcin des beitfs of cattle, should be employed. Wright calls attention to the misuse of the term streptothrix, and gives the reasons for the employment of the term nocardia in its place. From widely scattered localities and at long intervals of time reports have been published describing unique cases of disease produced by varieties of microorganisms belonging to the genus nocardia. In some of these cases points of similarity can be recognized in the clinical symptoms and the gross pathologic lesions, while others differ widely in both respects. They have been found in brain abscess, cerebrospinal meningitis, pneumonic areas, and in other pathologic conditions. Eppinger injected cultures into guinea-pigs and rabbits, and observed that they caused typical pseudotuberculosis. Consolidation of portions of both lungs, thickening of the peritoneum, and scattered nodules resembling tubercles were noted by Flexner in a case of human infection as due to nocardia in which the pathologic picture of the disease re- sembled so nearly that of tuberculosis in human beings that the two diseases could be separated only by finding the causative microorganism in each case. But in no two cases reported up to the present time have the descriptions of the microorganisms found agreed in all par- ticulars. In some cases no attempt at cultivation was made. In other cases numerous and careful plants on various culture media failed to develop the specific organism. As late as the year 1904 Tuttle was able to find the reports of only twelve cases in which nocardia -was found in sufficient abundance to have been an important, if not the principal, factor in producing disease. These cases were all fatal, and only once was the character of the disease recognized during life. As the clinical symptoms and the lesions in the human subject as well as in the animals experimentally inoculated with nocardia often resemble those of miliary tuberculosis, the question is naturally suggested whether cases of nocardia tuberculosis are not more numerous than the few reported cases would indicate. The almost universal prevalence of genuine tuberculosis and the extreme gravity of the disease have so long occupied the attention and study of the medical profession that much is taken for granted, and in cases in which the symptoms and lesions resemble with some closeness those characteristic of the well- known disease they may easily be set down without question to the account of the tubercle bacillus. The cases of nocardiosis reported NOCARDIA INFECTIONS 483 which simulated tuberculosis have beeu fatal, and the lesions for the most part have been widely distributed, but in a number of cases old lesions have been found which sug'gest that the disease may have been localized for a longer or shorter time, and then, by some accident, may have become rapidly general. In this resjject also these cases may resemble tuberculosis. Whether all cases of nocardiosis in the human subject are general and fatal or, as in tuberculosis and actinomycosis, whether there may be cases of localized disease which recover, are questions which have not yet been decided. The methods employed to demonstrate the presence of tubercle bacilli render nocardia more or less in\-isible. Again, unless the observer keeps in mind the possi- bility of nocardia infection, he may not appreciate the importance of finding slender threads with or without branches, and may consider them accidental bacilli, or varieties of leptothrix or non-pathogenic fungi. As the lungs ha-\-e appeared to be the seat of the primary infection in most of the cases of human nocardiosis it is very desirable that all cases presenting the physical signs of tuberculosis, in which repeated examinations fail to discover the tubercle bacillus, should be system- atically examined for threads. In this way alone can the frequency of the disease be determined. Gram's method of staining or the Ziehl- Neelsen solution decolorized with aniline oil seem to be the most reliable agents for demonstrating these organisms. Varieties of nocardia are widely distributed and are not very infrequently met with, but as yet, with the exceptions mentioned above, very little is known about them. Tuttle's report of a case of general nocardia infection at the Presbyterian Hospital gives such a good clinical, bacteriologic, and pathologic picture of a case of this infection that a considerable portion of it is repeated here: Six days before her admission to the hospital her ilhiess began -with a severe chill and fever and pain in her left side and back. The following day the pain in the side was worse and breathing was difficult. She began to cough and had some expectoration, but no blood was noticed in the sputa. At irregular inter- vals she had alternating hot and cliilly sensations. On admission, the patient complained of pain in the left side of the chest, cough, fever, wealoiess, and prostration. Her temperature was 103° and her pulse and respirations were rapid. The history of the disease and the pliysical signs indicated an attack of acute lobar pneumonia, the area of consolidation being small and situated in the lower part of the left upper lobe in front. Frequent and violent coughing, with ahnost no expectoration, pain in the affected side and in the lumbar region, restlessness and sleeplessness, and involuntary urination were the symptoms noted during the first four days in the hospital. The pneumonic area increased somewhat and extended iiackward to the posteitor axillary hne, and the temperature was continuous at 103° to 103 . 5°. On the fifth day the temperature fell two degrees and signs of resolution appeared in the consohdated area. The apparent improve- ment, however, was of short duration. On the sixth daj^ the temperature rose to 104.. 5°, and continued to rise each day, reaching 107.. 5° shortly before death, which occurred on the ninth day in the hospital and the fifteenth day of the disease. There were repeated attacks of profuse s^-eating. On the da}' before her death three indurated swellings beneath the skin were noticed. One. on the 484 PATHOGENIC MICROORGANISMS left forearm, about the size of a walnut, apparent! j' contained pus. Two, of smaller size, were situated in the right groin. Blood-cultures from a vein in the arm, taken on the sixth day, remamed sterile. The leukocjrte count on the seventh day was 36,000. Autopsy.— On the right arm, the left forearm, the abdominal wall, and on both thighs there are eight or ten sUghtly projecting, rounded, fluctuating, sub- cutaneous swellings from one-half inch to one inch in diameter. The skin oyer most of these nodules is unaltered, but over the larger ones there is a slight bluish Fig. 168 Fig. 169 Streptothrix from bouillon culture. Tuttle.) (From Young"'streptothrix threads showing ter- minal buds. (From Tuttle.) Fig. 170 Portion (jf kidney showing minute and large areas of infection. discoloration. The nodules arc eomjaosed of bluish-gray, thick, mucilaginous matter, which is very tenacious and can he drawn out into long threads. The lower Inlie is thickly studded with miliary tubercles, and scattered through the entire lung are supiiurating foci. I.iver and spleen normal. Kidneys:" The description of one applies to both. The surface is everywhere and evenly dotted with miiuite white spots, which suggest septic emlwli rather than tubercles A few prominent white nodules from one-quarter inch to one-half inch in diameter contain thick, tenacious matter (Fig. 170). Section shows that the entire substance of the kidney is densely studded with these minute white granules NOCARDIA INFECTIONS 485 The gross inilliological conditions were iiitef])rete(l before noeiu'diu was found us follows: All old tuliereulous uodulo in fhc right lung; ueute niijiary tuljer- eulosis in the right lung and periloiicuiii; acule loliai- iineumoiiia., affei'ting the left lung; septic infarctions and pyemic abscesses of both lungs, heart muscle, both kidneys, pancreas, mesenteric lyni()]i nodes, and subcutaneous connective tissue. The miliary tubercles of the right lung and peritoneum presentetl the characteristic appearance of genuine tuberculosis. They were minute, hard, gray, almost translucent nodules, while the granules in tlu; kidneys were of an opacjue white or yellowish-white color. Microscopic Examination. — Smears from the abscess beneath tlie skin and on the surface of the kidneys were stained with methyl blue, carbol-fuchsin, and by Gram's method. The smears resemble those made of tenacious sputum. There is a large amount of mucoid material containing a considerable number of leukocytes. Occasionally irregu- larly curved, thread-shaped microorganisms are found. They A'ary considerably in length and thickness, and broken and apparently degenerating fragments are seen. The more slender threads are e\\enly stained, but some fragmentation or beading of the protoplasm can generally be observed. The thicker threads and broken fragments show deeply stained globides and irregular bodies in a faintly visible rod or thread-shaped covering. Some branching threads are obser^'ed, but more commonly they are not branching. No other microorganisms are found in the smears. Sections from the lower lobe of the right lung, stained with hematoxylin and eosin, show in certain places the identical microscopic appearances which are considered characteristic of tuberculosis. Stained by Gram's method, with care not to decolorize too completely, threads like those described in the abscesses are found in great abundance, but rather faintly stained. No threads can be found within the typical tubercles witli giant cells, but in the zones of small cells around them thej^ are seen in great numbers, winding about among the cells and forming a sort of network. In the minute foci of small cells one or two fragments of threads are generally seen, and a moderate number in the small abscesses. In the areas of more diffuse infiltration these threads are abundant. No other microorgan- isms can be found except in the pneumonic area of the left lung, where some groups of cocci are seen. Culture Experiments. — Six tubes of LofHer blood-serum were in- oculated from the kidneys and kept at 37° C. On the third day minute white colonies appeared in some of the tubes, and on the fifth day all the tubes showed from three to ten or twelve similar colonies in each. The colonies increased in size until some of them reached a diameter of one-eighth of an inch. The color, at first white, changed to yellowish- white and then to a decided pale yellow. The well-de\'eloped colonies cling firmly to the surface of the medium and were not easily detached or broken up. The growths in all of the tubes were absolutely pure, and consisted of branching threads like those found in the sections. LofHer's blood-serum seems to be the most suitable medium for cultures. The growth on this medium is more rapid and abundant than on any of the other media tried. 4S6 PATHOGENIC MICROORGANISMS On plain agar and glycerin agar tlie growtli is the same as on blood serum, bu.t is less rapidly developed. In bouillon the growth is slow. If the tube is not disturbed or jarred, minute white tufts are seen clinging to the surface of the glass. But if the tube is shaken e\'en slightly the\' sink slowlj' to the bottom, forming a white, fluffy layer. These growths when undisturbed resemble minute balls of thistle-do\ra. The yellow color is not apparent even in the mass at the bottom of the tube. It is strictly aerobic. Morphology (Figs. 168 and 169).— On blood-serum the threads are comparatively thick and coarse, but those growing in bouillon are very slender and delicate. The main trunk also is often thicker than the branches. When unstained they are homogeneous gray threads, without any appearance of a central canal or double-contoured wall. There is ixeyer any segmentation of the threads. When properly stained there is always a distinct beading or fragmentation of the protoplasm, but oA'erstaining with fuchsin produces rather coarse, evenly red rods. The branching is irregular and without symmetry, and the branches are placed at a wide angle, very nearly, and some- times quite, at right angles. This is best seen in specimens taken from liquid media. The irregularly^ stellate arrangement of the branches, which was observed by Eppinger in his original specimen, is often seen in young organisms floated out from a liquid medium. Spore Formation. — On examining the deep orange or red-colored growth upon potato, one is surprised to find that the threads have entirely disappeared and that the specimen consists of moderately large coccoid forms. These represent the spore form of the organism, and when planted upon blood-serum the branching threads again appear. The spores stain readily with carbol-fuchsin and are not easily decolorized. They are spherical, or nearly so, but often appear somewhat elongated, apparently from beginning germination. They are killed by exposure to moist heat of 65° to 70° C. for an hour, but are more resistant to dry heat. Drying destroys the threads after a comparatively short time, but the spores retain their vitality for an indefinite period. A dried-up potato culture retains its vitality at the end of almost four years. The identity of Tuttle's microorganism is not fully established. It is undoubtedly a nocardia, but it does not agree in all particulars with any of the varieties described. Animal Inoculations. — A number of rabbits and guinea-pigs were inoculated subcutaneously upon the abdomen and in the neighborhood of the cervical, axillary, and inguinal lymph nodes with colonies broken up in salt solution. Indurated swellings were produced at the point of inoculation and a number of abscesses resulted. The abscesses developed rapidly and some of them opened spontaneously, while others were incised. The material evacuated did not resemble ordinary pus, but was thick and mucilaginous and exceedingly tenacious, like that from the subcutaneous abscesses of the patient described above. NOCARDIA INFECTIONS 487 The microscopic appearance was tlie same, and tlic nocardia threads were found in considerable numbers. Se\'eral rabbits and guinea-pigs and two cats receix'ed ])critoncal inoculatit)ns, but none of them showed any sign of infecticMi. When rabbits were inoculated intravenously, a rapidly fatal general infection was produced, and the lesions were similar in kind and distribution to those described in the human subject. Other Cases Reported. — Ferre and Faguet found in Bordeaux, in a cerebral abscess in the centrum ovale, a branching fungus, colored by Gram, which corresponded to nocardia. It grew on agar in round, ochre-colored colonies; on potato there was little growtli visible; slimy, tough colonies, which became gray and remained free from white dusting on the surface. Inoculations in rabbits and guinea-pigs were negative. Varieties of nocardia have been found in the human vagina. We ha^'e found a variety of nocardia in several cases of stillbirth with infection of the placenta with the same organism. Nocardia in Cases Simulating Actinomycosis or Tuberculosis. — Sabraces and Riviere found, in a case of cerebral abscess and a case of chronic lung disease with occurrence of subacute abscesses, fungi which differed from actinomyces. The organisms were contained in the lungs and pus, in the latter in pure culture. They grew best at 37° C. in the presence of oxygen. On agar plates round, wart-like colonies were found with yellowish under and whitish upper surface. Grew particularly well on fat and glycerin media; in milk a flesh- colored rim was developed; in gelatin agar a rough, brownish deposit, becoming black with age. Gelatin was liquefied. The culture had a strong odor of old mold. A yellowish pigment was usually produced which dissolved in ether; in an atmosphere of pure oxygen a brown pigment. Animal experiments gave positive results only when to a fourteen-day-old bouillon culture lactic acid was added; then pseudo- tuberculosis was produced. Numerous cases have since been observed in which nocardia proved to be the cause of chronic lung diseases, clinically suspected to be tuberculosis. Treatment. — Recently autogenous vaccines have been tried in certain cases, but it is yet too soon to determine with what result. References. Bostroem. Beitr. Path. Anat., etc., 1890, ix. Foulerlon. The Strepotrichoses and Tuberculoses, Lancet, 1911), clxxviii, 551, f)2(j, and 769 Musgrave and C'legg. Phila. Jour. Sci., 1907, iii, 2, 477. Nocard. Ann. de I'lnst. Past., 1888. Tuttle. Med. and Surg. Rep., Presbyterian Hosp., New York, 1904. vi, 147. Wright. Jour. Exp. Med., 1898, iii, 421, and Jour. Med. Res., 1905, viii, .349, and Osier's Modern Medicine, 1907, i, 327. CHAPTER XXXV F FIT;rR ABLE VI RI ^SES. 1 )ISEASES OF UXKXOWX ETIOLO(iY. FILTRABLE VIRUSES. There exists a class of infectious diseases from whicli it has been quite impossible up to the present time to demonstrate visibly any individual microSrganisms, although infective material from such diseases may, -with certain precautions, be passed through stone filters of varying degrees of porosity (for types of filters, see p. 92) ; the filtrates will contain the virus and be capable of reproducing the disease with all its characteristics when inoculated into a susceptible animal. Examined microscopically, even with the highest powers, the filtrate is limpid, and, except in a few diseases which will be described in detail later on, not a sign of characteristic particulate matter can be seen. Such a filtrate, therefore, contains either ultramicroscopic organisms or small organisms with refraction and staining powers so faint that they cannot be demonstrated by our present methods. Certahi precautions must be observed in such filtrations. In the first place the filter must be shown by actual test to be free from infec- tion. Any and all of the test organisms must be absolutely retained and none pass into the filtrate. A few minute species of germs have been found to pass certain grades of filters, especially under pressure. Thus- the bacillus of guinea-pig pneumonia, which is O.b/x x O.ljx, passes Berkefeld Y (Wherry) ; a spirillum isolated by von Esmarch passes, according to him the Berkefelds, Chamberland F, and other filters; a minute water flagellate was found by Borrel to pass through the coarser filters. Recently, Wolbach and Binger have shown that two spirochetes (new species) found in pond water may pass through Berke- felds V, N, and ^N, by pressure and by suction. It is seen, therefore, that certain known organisms within the limits of visibility may pass even some of the finer filters. The filtration must be completed within a moderate time, because even bacteria as large as the typhoid bacillus may, in media containing a certain amount of albuminous material, grow in time through the filter. The material to be filtered should, furthermore, be greatly diluted and first filtered through filter paper in order to avoid the clogging action of extraneous material. If after all the proper precautions have been taken the filtrate is pathogenic it must be shown that the symptoms are due to a micro- organism and not to a toxin. This may be decided ))y inoculating a series of animals successively with the filtrate obtained from a pre- \'ioush' so inoculated animal. DISEASES PRODUCED BY FILTRABl.E AGENTS 489 From our present knowledge, filtrable agents may he divided into two groups: (1) tiiose which have not yet been niorpiiologically demon- strated (idtramierosei)pie'!'); (2) tliose wiiieii are shown to be within the limits of \isil)ility. A third group may be made of the diseases produced b.y viruses of c(uestionable liltral)ility. GROUP I.— DISEASES PRODUCED BY FILTRABLE AGENTS OF UNKNOWN MORPHOLOGY. Foot and Mouth Disease. — This is probably the first disease shown to be produced by a filtrable agent. It is a highly infectious disease of cattle. Other domestic animals, as well as man, may also be attacked. In man it usually occurs among people handling infected cattle and by drinking the milk of infected animals. The disease is characterized by the appearance of vesicles in the mouth and around the coronet of the foot as well as between the toes. Loffler and Frosch, in 1898, discovered that, after diluting the contents of an unbroken \'esicle with twenty to forty times its volume of water and passing the resulting fluid through a Berkefeld filter, the filtrate contains the virus. This virus remains infectious for some time. One attack of the disease usually produces immunity. Loffler claims to have produced a serum which has immuniz- ing properties. Mosaic Disease of Tobacco. — This is the second disease in which a filtrable virus was demonstrated. The disease causes the young tobacco leaves to become de^'oid of chlorophyll in spots which enlarge, turn brown, and the underlying tissue becomes necrotic. Beijerinck in 1899 showed that the filtrate from a porcelain filter promjjtly rejjro- duced the disease on tobacco leaves. Cattle Plague (Rinderpest). — This fatal Furopean and African disease of cattle is characterized by inflammation of the intestinal mucous membrane. The blood is infectious and filtrates of it through Berkefelds and Chamberland F (Nicolle and Adil-Bey, 1902) produce the disease. No organism can be seen. Immunitj- follows one attack. It can also l)e produced by a subcutaneous inoculation of bile from an infected animal. Yellow Fever. — The undiluted serum from cases of this disease has been shown by the American commission (1901) and others (see p. 590) to pass the Berkefelds and the Chamberland B filters as clear filtrates, and in this form to be infectious; therefore some forms at least of the specific organism are probably ultramicroscopic. Rabies. — Xo bacteria have been discovered that are considered as factors. The probability of the Negri bodies being protozoa and the exciting factor is considered in Chapter XLV. The virus of rabies has been shown to be filtrable (Remlinger, 190.3). Poor and Steinhardt (1913) showed that gland virus is more filtrable than that from the brain. It may pass through the coarser Chamberland filters. Hog Cholera. — De Schweinitz, Dorset, Bolton, and INIcBrjde demon- strated in 1905 that the blood of hogs suft'ering from hog cholera con- 490 PATHOGENIC MICROORGANISMS tains a filtrable virus which is capable of producing the disease on inoculation into healthy hogs. This virus passes Chamherland B and F filters. It lea\-es the body in the urine and probably enteres another animal through contaminated food. King, Baeslack, and Hoffman found in 19i;3 a short motile spirochete {Hpirocheta suis) in the blood in a series of cases of hog cholera. They think this organism may be a stage of the specific organism which may produce filtrable granules. This disease is a very fatal and contagious disease of swine char- acterized by fever and ulcerative enteritis. Immunity follows one attack. An immune serum is produced. Animals are also immunized by a mixture of infected blood and immune serum (sensitized virus). The hog cholera bacillus was earlier supposed to be the cause of this disease. South African Horse Sickness. — This is a warm-weather disease,. more common in animals that do not pass the night under cover. The horses are uneasy, have difficulty in breathing, and a reddish froth exudes from their mouths. The temperature rises in the day time, but has a tendency to drop at night. In severe cases an edematous swelling of the head and neck may appear. MacFadyen succeeded in passing blood serum of a diseased horse (diluted) through the Berkefelds and a Chamberland F, not through a Chamberland B filter. Chicken Sarcoma. — In 1910 Rous discovered a spindle-celled tumor in chickens, which he has reproduced in other chickens by transplanta- tion, by inoculation of dried tumor tissue, and by the inoculation of Berkefeld filtrates from emulsions of tumor cells. Novy's Rat Virus. — Novy has found an extremely virulent filtrable virus from a disease of rats, which passes all filters. He has not yet published full reports. GROUP II. DISEASES PRODUCED BY FILTRABLE AGENTS SHOWN TO BE VISIBLE. Contagious Pleuropneumonia of Cattle. — This malady affects bovines, but not other species. Typically, there is an inflammation of the lungs and the pleura which is invasive and causes necrosis of the diseased parts. Nocard and Roux succeeded in cultivating the organism in collodion sacs placed in the peritoneal cavity of rabbits, using a mixture of serum and bouillon. After two weeks a very faint turbidity appeared in the sacs; coincidently the fluid became infective. Nocard' and Roux described the organisms as minute spheres and spirals just within the limits of visibility. They showed (1899) that the organisms passed the Berkefelds and a Chamberland F, but not a Chamberland B filter. Immunity is produced by a single attack. It has also been produced artificially by the inoculation of cultures or virulent exudates. Epidemic Poliomyelitis.— This is a disease which affects chiefly the central_ nervous .system. It occurs usually in children, and appears sporadically and in epidemics in many countries. In New York, in 1907, there was an epidemic of over 2000 cases and in Texas, in 1912, DISEASES PRODUCED BY FfLTRABLE AGENTS 491 there was a large oiitln-eak. In nature, the virus enters probably through the upper air passages. Tiie ehief symptoms of tlie disease are fever, with or without sore throat, followed in a few days by paresis and paralysis. The niortalit}- is Iom'. There is usually permanent injury to parts of the motor areas of the nervous system, witli resulting deformity. The prineipal mieroseopic ehanges are a marked exudation of polynuclear leukocytes into the lymph spaces and the cerebrospinal fluid. The changes are usually specially marked in the anterior com- missure and the cornea of the cervical and lumbar regions of the cord, but the whole nervous system is more or less affected. The bloodvessels may become thrombosed and ruptured. The disease seems to be closely related to, if not identical, with certain diseases manifested by acute encephalitis and bulbar paralysis, e. g., Landry's paralysis. That the disease also bears a marked resemblance to rabies is quite apparent. The finding of the virus in the salivary glands and the demonstration of its filtrability give added evidence of its similarity to rabies. Until recently, nothing was definitely known of the etiology. Now we have a series of studies which seem to have practically cleared up the subject. Landsteiner and Popper (1909) reported the transmission of acute poliomyelitis to apes. They inoculated the spinal cord intraperi- toneally and produced typical symptoms and lesions, but did not succeed in transmitting from ape to ape, proliably because they used a mild case. Flexner (1909) and Lewis transmitted the disease from monkey to monkey by means of intracerebral inoculations. Landsteiner and Levaditi (1909) also transmitted it from monkey to monkey. They found that the virus lives some time outside of the body; that the degenerated ner\'e cells are taken up by phagocytes, and there is an analogy between the lesions of poliomyelitis and those produced by rabies. They demonstrated that the virus is filtrable. Leiner and Wiesner transmitted the disease from monkey to monkey, found young animals more susceptible than older ones, and spinal fluids, blood, and spleen negative. Flexner transmitted the disease by inoculating into the blood or peritoneal cavity, also by the subcutaneous method, and independently found the virus to be filtrable. Landsteiner and Leva- diti found the virus in the salivary glands and suggested the saliva, moist or dry, as a source of contagion. Soon after this (1911) Noguchi and Flexner announced that they had obtained cultures in media similar to that used by Noguchi in cultivating spirochetes (see p. 103). Li such media, in about five days the pieces of tissue employed become surrounded by an opalescent haze which increases for about five days more, and a sediment gradually forms. Giemsa's stain shows the presence of minute globoid bodies (0.15 to 0.3 diam.) in pairs, short chains, and masses. Cultures were also obtained from the filtered virus. Monkeys inoculated with these cultures for a variable number of culture generations may die with typical lesions of the disease. The authors consider these bodies the cause of the disease. They further report that the cultures are fil- trable. This work awaits corroboration. Levaditi states that he cannot 492 PATHOGENIC MICROORGANISMS obtain the results of Noguchi, Ijut that he obtains e^'idenc■e of growth l)y the li\ing tissue method. Jval)bits have also been^ found to be somewhat susceptible to the ^•i^us (Kraus and Meuieke, Marks). lAIarks states that the disease in rabbits does not resemble that in man. Immunity.— One attack seems to gi\-e certain immunity. Flexner and Amoss state that, experimentaliy, the virus inoculated into the blood is capable of being neutralized by intraspinous injections of immune serum. Relapsing Fever.— Todd and Wolbach have found (1914) that Spirocheta duttuni may be forced through e\'en the finer grades of Berkefeld filters by a pressure of from fifty to ninety pounds to the sciuare inch. GROUP m.— DISEASES PRODUCED BY VIRUSES OF QUESTIONABLE FILTRABIUTY. Smallpox and Related Diseases. — The details of the disease smallpox are considered in a later chapter. In 19(18 Casagrandi reported that the ^■irus was filtrable under pressure through coarser Berkefeld filters. Vaccine virus had already been reported as filtrable by some investi- gators, and as non-filtrable by others. Our results agree with those of the latter ol)servers. The opinion as to the filtrability of the other "pox" diseases is not unanimous. Dengue. — Ashburn and Craig claim to have reproduced dengue in susceptible individuals by a similar procedure to that employed in 3'ellow fever. The ^•irus passes a Berkefeld filter. The intermediary host ill natural infection is claimed by them to be Cidex jatigans. Measles. — This very definite exanthematous disease still remains among those of unknown etiolog}-. Both cell inclusions (Field, Ewing, and others) and bacilli have been reported as having an etiologic relation- ship. The reports ha\'e not been corroborated. In 1905, Hektoen produced measles in two human cases by the inoculation of blood drawn from an infected case at an early stage of the disease. Anderson and Goldberger in 1911 inoculated monkeys with measles blood and demonstrated the virus in the blood of the inoculated animals and in the secretion of the upper air passages. They also demonstrated the period of infectivity of the blood. They reported positive results with Berkefeld filtrates. Scarlet Fever. — Scarlet fever is an acute febrile, highly infectious dis- ease, characterized by a diffuse punctuate erythematous skin eruption, accompanied by catarrhal, croupous, or gangrenous inflammation of the upper respiratory tract and by manifestations of general systematic infection. The disease was probal)ly known long before the Christian era, but the present name does not appear until the time of Sydenham (1()85), who dift'erentiated the disease from measles. It is \'ery generally disseminated, but is much more common in temperate climates than in the tropics. The specific exciting factor is thought by many to be a streptococcus, of the Streptococcus pyogenes type, but the CA-idence in fa\-or of this view is very slight (see Streptococcus pyogenes). DISEASES PRODUCED BY VIRUSES 493 Mallory (1914) found certain liodies which lie (h:'scrihcs as follows: "They occur in and between the epitiiehal cells of the ci)idermis and free in the superficial lymph vessels and spaces of the corinni. The great majority of the bodies \-ary from LV to 7^ in diameter, and stain delicately but sharply with methylene blue. They form a series of bodies, including the formation of definite rosettes with numerous segments, wdiich are closely analogous to the series seen in the asexual development (schizogony) of the malarial parasites, but in addition there are certain coarsely reticulated forms which may represent stages in sporogony or be due to degeneration of the other forms." He gave the name Ci/da^terion .scarlafinale to these bodies in consequence of the frequent wheel and star shapes of the rosette. Field and others have not been able to corroborate these findings. The reports that a filtrable virus is found lack corroboration. Trachoma. — This condition has already been considered in a previous chapter. Bertarelli reported that he was able to produce a specific filtrate. Nicolle also reported positive results with filtrates. The descriptions of the disease, however, are too vague to allow an opinion as to the truth of these reports. Tjrphus Fever. — The occurrence of this infectious disease in epidemic form has disappeared from civilized lands. The fact that the body louse may transmit the disease helps explain why it has always been classed as a filth disease. It still occurs endemically in parts of Europe and North and South America. In Mexico it occurs in epidemics where it is known as Tabardillo. In New York, it occurs occasionally in mild form under the name of Brill's disease. Brill thought it was a new disease, but Anderson and Goldberger have shown that typhus fever and Brill's disease are the same. Nicoll and Krumwiede observed four cases which clinically verified these findings. The disease is characterized by high temperature and a petechial rash. Nicolle (1909) show-ed that the old world typhus can be transmitted to the chimpanzee and from this to the macacus with typical eruption in each case. He also showed that the disease is transmitted by the ordinary body louse (Pediculus ve.siimenti). Anderson and Goldberger (1909) were the first to transmit typhus fever of Mexico (tabardillo) to monkeys. Thej' were able to transmit directly from human beings to the macacus and capuchin and from monkey to monkey. Ricketts and Walker (1910) also found that the macacus was directly susceptible to the disease. They based their diagnosis chiefly upon a rather indefinite fever and in most cases somewhat distinct symptoms of illness. They also found that the monkey may pass through an attack of typhus so mild that it cannot be recognized clinically, but it results in immunity. The immunity test is a reliable ])roof of the previous occurrence or non-occurrence of tyi)h\is at least within n ])eriod of one month. They found that t>pluis was transmittt'd to the monkey Ijy the bite of the louse. They further state that in stained ])reparations of blood of patients taken from the se\enth to the twelfth days of th(' disease the^' in\'ariabl\' found a few short bacilli similar to those which 494 PATHOGENIC MICROORGANISMS belonged to the hemorrhagic septicemia group. In moist preparations they saw similar forms in all cases. No motility was observed. No cultures could be obtained. They examined the dejecta of many lice and found similar bodies in large numbers in infected lice and occasion- ally in non-infected lice. Pediculus capitus may also transmit the disease. Recently, Plotz reported the isolation in pure cultures of a gram positive pleomorphic anaerobic bacillus from the blood of six typhus cases and six cases of Brill's disease. He states that he obtained com- plement fixation with the blood of typhoid patients. Attempts at filtration by Ricketts and Wilder and Anderson and Goldberger were at first negative. But Nicolle, Connor and Wilder showed that filter-ed blood inoculated into monkeys made them immune. The corroboration of these findings by the others makes greater the possibility of the virus having a filtrable stage. Several other diseases of less importance have been listed as belonging to those produced by a filtrable agent. A short review of the subject up to 1912 is given by Wolbach. OTHER DISEASES OF UNKNOWN ETIOLOGY. Rocky Mountain Spotted Fever. — This is an acute infectious disease characterized by fever and a more or less hemorrhagic eruption. Some years ago, Wilson and Chowning thought they found a protozoan in the blood similar to Babesia, a Texas fever. Their findings have not been corroborated. Their investigations proved, however, that rabbits are susceptible to the disease and that a tick of the genus Dermacentor probably carries the infection. Then Ricketts and Gomez made some very interesting studies on the disease. They found that guinea-pigs and monkeys are susceptible as well as rabbits, and they further found that in guinea-pigs and monkeys an attack of spotted fever produces a strong active inherited immunity characterized by a serum with high protective but low curative power, and that the production of the serum in the horse with the use of serovaccination in man may give practical results. They found a moderate number of diplococcoid bodies in the blood of infected guinea-pigs and monkeys, and fewer in man. They found that the virus is transmitted by the infected female tick to her young through the eggs. If the larva? from these eggs are allowed to feed upon normal guinea-pigs, these animals come down with the disease. Immense numbers of these apparent organisms are found in affected eggs and none were found at first in normal eggs. x\fterwards Ricketts found a few, but he tliought these might be an avirulent species of the same organism. The salivary glands, alimentary sac and ovaries of infected female ticks are swarming with these bodies, while normal ticks seem to have none. Lasth', Ricketts found that these ))odies agglutinate with sijecific serum, 1 to iSdO dilution. Mumps.— Comparatively few studies have been made of this infec- tious disease, probably because of its low mortality. By puncturing OTHER DISEASES OF UNKNOWN ETIOLOGY 495 the parotid gland, Laveran and Catrin ol)tained a diplococcus which stains easily, is (Tram-negative, and grows on ordinary culture media. No satisfactory specific studies have been made of this organism. Pellagra. — This disease has been much studied recently. The theory that it is due to the ingestion of damaged corn has received a check from the work of Siler, Garrison and MacNeal (1913) who state that they were unable to get evidence to support this theory. They consider the disease a specific infection caused by unknown means. References. Anderson and Goldberger, Public Health Report, 1910, 1912 and 191.3. Jour. Anier. Med. Assoc, 1911 and 1912. Ashium and Craig. Jour. Int. Dis., 1907, iv, 440. Ewing. Jour. Inf. Dis., 1909, vi, 1. FieU. Jour. Exp. iSIed., 1903, vii, .34.3. Flexner and Lewis. Jour. Amer. Med. Assoc., 1909, liii; .and Jour. Exp. Med., 1909, xii. Flexner. Huxley Lecture, Lancet, 1912, ii, 1271. Flexner and Noguchi. Jour. exp. Med., 1913, xviii. Flexner and Co-workers. Jour. Exp. Med., 1913 and 1914. Hektoen. Jour. Ind. Dis., 1903, ii. King, Baeslack and Hoffman. Jour. Int. Dis., 1913, xii, 39 and 206. Kraus u. Meinicke. Deut. Med. Woch., 1909, xxxv. Loffler and Frosch. Cent. f. Bakt., 1898. Landsteiner and Levaditi. Compt. Reudus Soc. de Biol., 1909. Mallory. Jour. Med. Res., 1904, x, 483. Marks. Jour. Exp. Med., 1911. Nicolle. Compt. rend. Acad. Sci., 1909 and 1911; Assoc, de I'Inst. Past., 1910, 1911, and 1912. Nicoll, Kruinwiede, Pratt and BuUovm. Jour. Amer. Med. Assoc, 1912, lix, .521. Plotz. Jour. Amer. Med. Assoc, 1914, Ixii. Poor and Steinhardt. Jour. Inf. Dis., 1913, xii, 202. Read, Carrol, Agranionie and Lazear. Philadelphia Med. Jour., 1900. Remlinger. Ann. de I'lnst. Pasteur, 1903, 834. Ricketts and Wilder. Jour. Amer. Med. Assoc, 1910, Ix, 309. Rous and Murphy. Jour. Exp. Med., 1911, xiii. Wilder. Jour. Inf. Dis., 1911, 9. Wolbach. Jour. Med. Res., 1912, xxviii, 1. CHAPTER XXXVII. FLAGELLATA. The flagellates that are pathogenic to man belong chiefly to the genera Trypanosoma and Leishmania, but certain other genera are formed occasionally in man which may be mentioned because of the possibility of their' becoming pathogenic. According to the classifica- tion of Calkins (190S) the flageUates parasitic in man are from three orders, the Monadida, the Heteromastigida, and the Polymastigida; according to that of Doflein and Koehler (1913), they are from two, Protomonadina (one to three flagella), and Polymmtigina (four to eight flagella). Six families, according to the morphology of the flagella, are distinguished, three of which contain forms parasitic for man, namely, Cercoinonadidw (one flagellura and no blepharoplast), Trypanosomed/B (one flagellum, a blepharoplast, and an undulating membrane), and BodonidtB (two flagella). Under the Trypanosomedw, which include most of the pathogenic forms, are placed the genera Leptomunas, Herpetomonas, Leishmania, Trypanosoma, and Schizoiypamim and Endotrypanum. Under the Hodonidw. are placed the genera Bodo and Prowazckia, and in the order Polymatigina the genera Trichomonas and Lamhlia have been found in human beings. Hartmann puts the Trypanosomata, with other blood-parasites, in an order, the Binudeata, and makes the Spirocheta an appendix of this order. According to this arrangement the Hemosporidia are taken from the Sporozoa and placed with the Trypcmosomata in this order, the malarial organisms supposedly lose through tlieir parasitism many of the characteristics ascribed to this order. Material and Methods for Study. — A number of flagellates (Bodo, for in- stance, sec p. 528) are found in the large intestine of the lower animals. The toad, the grass lizard, and the guinea-pig may contain some interesting forms. As these forms are easily obtained and remain alive a long time outside of the l^ody, they are well fitted for class study. The feces are obtained by pressing lightly over the anus of the animal, or if the whole intestinal tract is to be examined, by sacrificing the animal and dissecting out the parts wanted. The material is placed in a clean watch- glass and thinned if necessary with physiologic salt solution. Hanging drops may be made in physiologic salt solution or in such a solution made a little thick by the addition of gelatin in order to retard the motion of the flagellates somewhat so they may be better studied. Permanent preparations may be made according to directions given on p. 7:i. As most of the pathogenic menilx^rs of ihis group may be difficult to obtain in the living conililion at any stated time, they uuist be studied by students i)rhicipally in stained smears and sections. If one can obtain rats infected with Tr. lewisi, others with fjno or more path- ogenic forms; still others with Spirocheta ohermeim, the infecting organisms can be kept alive ljy frecjuent reinoculation of the heart's blood, subcutaneously LEPTOMONAS, HERI'ErOMONAS, AND CRITHIDIA 497 or iiitraperitoueally iiito the fresh Muiinal, or cultures may be carried on (sec lielow) . But this is an expensive and tiresome worlv in those laboratories where such work is not being carried on, and generally one must rely on the permanent preparation. In the de\'elopment in the second host one must also study the stained specimens in the great majority of instances. The fresh specimens of blood are obtained from the tail tip of the rat, or the ear of the dog; they may he examined after dilution with physiologic salt solution in the hanging ilrop, or in a drop spread under a cover-glass and ringed with -^-aseliu. For permanent preparations films of the blood are spread, fixed, and stained in the usual way; Giemsa's method of staining (p. 81) is very satisfactory. For section work of the \'arious oi'gans the fixatives and methods given on page 84 may he used. Special methods are given under each organism. Artificial Cultures of Blood-flagellates (see p. ]0(j). — These, according to Novy and ^lacNeal, may be made on a culture medium consisting of a mixture of ordinary nutrient agar with variable amounts of fresh defibrinated rabbit or rat blood. The best all around results are ob- tained with ecjual parts of blood and agar. The agar is melted and cooled to 50° C, then the blood is added and thoroughly mixed. The tubes are inclined until the medium stiffens, when they should be inoculated af once with blood or other infected material containing living try- panosomes. The surface of the medium should be very moist, so water of condensation may form. Generally evidence of growth may be observed in three or four days. CERCOMONAS. The members of this genus are round or oval flagellates with a long anterior flagellum and a more or less pointed posterior extremity which is sometimes ameboid. The vesicular nucleus is situated anteriorly, and passing through the organisms from flagellum to posterior extremity is an axial elastic fibril (Fig. 171). Division into two daughter forms has been observed. A number of cercomonada, none of them well studied, have been observed in different animals as well as in man. They are very numerous in stagnant water. Cercomonas homiuis (Davaine, 1854) was observed in the dejections of a cholera patient by Davaine. The body is 10^ to 12;^ long and pear-shaped, pointed posteriorly. The flagellum is twice as long as the body. Davaine also reported a smaller form in the stools of a typhoid patient. Other observers have noticed similar forms in human stools, some associated with ''Amoeba coli." Similar forms have been seen also in an echinococcus cyst of the liver, in the sputum from a case of lung gangrene, in the exudate of a hydropneumothorax, and a few times in the urine. They are all probably harmless invaders. LEPTOMONAS, HERPETOMONAS, AND CRITHIDIA. Certain flagellates found in the digestive tract of mosquitoes, flies, and other insects are very similar to trypanosomes. Among them 32 498 PArilOGENIC MICROORGANISMS several species have been recognized, but they need to be more fuhy studied iu order to determine their definite relationship to each other and to the genus trypanosoma. Leptomonas is described as having a single flagellum directed forward and arising near a blepharoplast situated in the anterior part of the cell. Herpetomonas is distinguished from leptomonas by a flagellum containing two filaments and by a delicate filament extending from the blepharoplast toward the posterior end. Crithidia has a rudimentary undulating membrane. The dis- tinctions between these three genera and the genus Trypanosoma which have been recognized are; (1) the former contain no undulating mem- brane or only a rudimentary one, and (2) their centrosome or blepharo- plast usually lies at the side of, or anterior to, the nucleus instead of posterior to it, as in Trypanosoma (Fig. 171). Fig. 171 a O c d e .Schematic drawings of flagellates l^eloiigiriK to the trypanosomidae, showing; differential points: a, cercomonas; h, leptomonas and Leishmanial c, herpetomonas; d, crithidia; e, trypanosoma. These distinctions, Novy claims, may disappear in the cultural forms of the three genera, when trypanosomes may show a rudimentary undulating membrane and an anterior blepharoplast. His caution in regard to confusing these insect flagellates with developmental stages of \'ertebrate blood parasites should be remembered. The Leishman-Donovan bodies found in kala-azar are closely related to the genus Leptomonas. They are considered a separate genus, Leishmania. LEISHMANIA (LEISHMAN-DONOVAN BODIES AND ALLIES). r.ertain fevers of severe malarial-like types known in dift'erent sec- tions of the tropics by dift'erent names (dum-dum fever, cachexial malaria, Ivala-azar) have been shown to have a causal relationship by the finding of similar protozoon-like bodies in the lesions. These bodies were first minutely described by Leishman in 1903 as beino- LEISII MANIA 499 present in certain eells in the spleen of eases ocenrrin^- in Iiidiii, called hy him duin-duni fever. He considered them as possibl,y trypaao- somes, but did not name them. Later in the same year Donovan de- scribed similar bodies in cases of what he called malarial cachexia. The bodies were first called the Leishman-Donovan bodies; then Laveran and Rlesnil who examined Dono\'an's preparations and considered the organisms similar to those causing Texas fever in cattle, called them Piroplasma donomni. Ross, however, thought they constituted a dis- tinct genus which he called Leishmania. This genus is now accepted, hence they are known as Leifihmania chmovani. Rogers and Patton Fi.i. 172 .% Leisbuiaiua tropica in a caye of tropical ulcer. Smear preparation from the lesion stained with Wright's Romanowsky blood-staining fluid. The ring-like bodies with white central i;)ortions and containing a larger and a smaller dark mass are the inicroorganisms. The dark masses in the bodies are stained a magenta, while the peripheral portions of the bodies Iti typical instances are stained a pale I'obin's egg blue. The very large dark masses are nuclei of cells of the lesion. X 1.500 approximately. (After Wright.) place them with the genus Herpetomonas, but until we know more of the limits of ^•ariation of all these forms it seems best to make them a separate genus. They IvAxe since been found in different parts of India, in China, Tunis, Algiers, Arabia, Egypt, South Africa, Italy, Greece, and Portugal. Wright in this country has reported in a case of tropical ulcer, or Delhi boil, from an Armenian immigrant, bodies which, accord- ing to his excellent photographs (Fig. 172) and description, must l)e identical with, or very closely related to, Leishman's bodies. On account of the different pathologic conditions in which they are found, however, they are classed as a ditt'erent species, Lei.s-hmania tropica Wright. The form found in infantile splenomegaly is considered another oon PA THOGENIC MICROORGA NISMS species, with the name Leishmania infantmn Nieolle. DarHng described an organism resembling that of Kala-azar found in a fatal disease of tropical America. Though the organism, he says, resembles Donomni, he thinks it has enough points of difference to be placed in a different Fi(i. \-f?, m f'ultur:,! Inr.ns of L. iuUuUnm, showins Uu- flagoll.to typo. (After Nir„lle.) genus; thereh.re he gives it the name UiMoylcuwrn mpsnhdum, and calls the fhsease histoplasmosis. He says it differs from Donovani in the form and arrangement of its chromatin nucleus and in not possessing a chromatin rod. It has a refractilc acliromatic capsule LEI SUM AN I A 501 Morphology. — The bodies as seen in the eells of tlie host are eireiilar to ellii)tieal in sha])e, from IV to 4^ in diameter, and contain two miek-i, a hu'.nx' o\'al one at one i)art of the peri])hery and a, small eirenlar or rod-sha]>ed one (hlepharojilast) near or at the o|iposite part of the ])eri- l.)hery. This smaller body stains more intensely than thi' larger one, while the cytoplasm of the parasite stains very dimly, sometimes show- ing only a faint peripheral rim. Near the rod-shaped nucleus may sometimes he seen a minute granule or rod which is the rudiment of the tlagellum. Any nuclear and cytoplasmic staining methods will bring out these points in Zenker-fixed material. Smears stain well by Wright or the Nocht-Romanowsky methods. It was not until these organisms were cultivated outside of the body that their relationship to the flagellates was established (Fig. 173 and Plate IV, i, Fig. 1, b). Site in Body. — The bodies have been found in large endothelioid cells in the spleen, liver, bone-marrow, lymph nodes, kidney, lungs, testes, skin, muscles, intestinal ulcers and in the leukocytes in the peripheral blood. In this last situation they are onlj' found in appreciable numbers in ad\'anced cases. The large cells containing the ])arasites are suppo.sed by Christophers to be the endothelial cells from the finest capillaries. Donovan states that he found small forms in the polynuclear leukocytes of the peripheral circulation when the temperature was abo^■e 103°. Marshall found them chiefly in the mononuclears. Rogers was able to demonstrate them more easily in the peripheral circulation by centrifuging the blood and examining the leukocytic layer. Nicolle and Conte and others obtained them in the mononuclears found in the fluid of blisters pro- duced experimentally on the skin. Cultivation. — Rogers has grown abundant pure cultures of the bodies in a slightl,y acid citrated blood-medium at 20° to 22° C. Nicolle and later Novy have shown that L. injantum is pathogenic for dogs and that cidtures may be obtained with comparati\'e ease from the infected animals (Fig. 173). Nicolle has also cultivated L. tropicum. In these cultures the rounded organism elongates, the fiagellum develops, and the Leishman-Dono^"an body becomes a flagellate, like the Leptomonas (Fig. 171). Insect Carriers. — Rogers and Patton have shown that the bed-bug may transmit the disease, and Patton has demonstrated the develop- ment of the organism up to the fully flagellated stage in the gut of this insect. The Sargents have shown that L. infantum may be transmitted by the dog flea. Effect on Human Host. — The pathologic changes are those following the degen- erations, subsequent to the growth of the organisms in the large mononuclear cells. The symptoms, in the cases of general infection are: (1) very much enlarged spleen and less enlarged liver; (2) progressive anemia with peculiar dark, earthy pallor of skin (Kala-azar), progressive emaciation, and muscular atrophy; (3) long-continued, uregularly remittent, and intermittent fever (97° to 104°); (4) hemorrhages, such as epistaxis, bleeding from gmiis into subcutaneous tissue, producing purpuric eruption; (5) transitory edemas of various regions. There are often complications, such as congestion of lungs, 502 PATHOGENIC MICROORGANISMS. dysentery, and cancruni oris. The blood shows practically no loss of red-blood cells, but a diminution of heniogloblin; there is a decrease in the leukocytes, pi'incipally ]ioh'iuiclears, giving a relative increase of mononuclears. Negative ponits which helji in the diagnosis are: absence of malaria, no typhoid or Malta fever reaction, resistance to medication, quinine, as a rule having no effect, though in early cases, and with large continued doses a few good results have been reported. Puncture of spleen or liver with the finding of Leishman-Donovan bodies makes the diagnosis certain. Sometimes the bodies may be found in the peripheral blood. The duration of the disease is from a few months to several years. ^ The percentage of deaths in systemic infections is great; in some forms of the disease, at the height of an epidemic, it may reach 98 per cent. Strangers from non- tropical countries are specially susceptible. The infection in children known as splenomegaljr is similar to that in the adult. The local disease known as Delhi or Aleppo boil or tropical ulcer is a compara- tively non-dangerous circumscribed chronic ulcer in which the endothelial cells contain the organisms in large numbers (Fig. 172). Recovery is foUowed by marked immunity. Complement-fixation. — Makkas and Pappassoterion state that a specific antigen gives positive results with both Kala-azar and sj'philis, but a syphiUtic or a non-specific antigen gives positive results only with syjihilis, therefore both should be used. Prophylaxis. — Segregation and perfect cleanliness, especially in regard to bedbugs and fleas, are recommended as the best means of eradicat- ing the disease. Referen(;:e,s. .S. T. Darling. The Morphology of the Parasite (Hi,stopla,sma napsulatnni), ete., .Tour. Exper. Med., 1909, xi, ,51.5. Dofiein u. Koehler. M. Kolle and Wassermann, 1913. Sec. Ed., Jena. M. Mayer. Leishmania. M. Kolle and Wassermann, 1913. Sec. Ed., Jena. Sergent. Ed. and et. Bull. Soc. Path. Exp., 1912, v, 595. Makkas and Pappassoterion. Arch. d. Med., 1911. Nicolle. Le Kala-azar infantile. Ann. Inst. Pasteur, 1900, xxiii, 301 nnfl 441. Navy, MacNeal, and Torry. Jour. Inf. Dis., 1907, iv, 223. W. S. Pation. Scientific Memoirs by Officers of Medical and Sanitary Departments of Government of India, new series. No. 31. Wenyon. .Jour. Trop. Med., London, 1912, iii, 13. Wright. Jour. Med. Research, 190.3, x, 472. CHAPTER XX XVI II. TRYPANOSOMA. Pathogenic Forms. — ^'ery many species of trypannsoma liave heen (Icserihed, ami the mimher reported as distinctly pathogenic is increas- ing. Two of the hitter are known to be pathogenic for man; a ch)sely related form that was described by Chagas in 1909 is made a new genus. The accompanying table gi\'es a list of the better-known path(j- genic forms for mammals with their chief differential characteristics. They are divided by Laveran into three groups according to the different characters of the flagellum. Their general characteristics and broader classifications are given in Part I. (See also Plate IV, i, Fig. 2). Historical Note. — The first species of trypanosome studied with any degree of fulness is the ooniparati\'ely non-virulent T. leivisi. It was probably first seen in the blood of the rat in 1845, but was not well described until 1879, when Lewds studied it more fully. Since then it has been studied by many observers. It is found in tlie Ijlood of from 2 to 3 per cent, of wild rats through- out the world. The first of the more pathogenic trypanosomes was discovered by Evans in the blood of East Indian liorses suffering from surra, but it was not well studied until 1893, when Lingard's important work on surra led, in a way, to all the subsequent work on diseases caused by trypanosomes. The next year a trypanosome was discovered bj^ Bruce in the blood of liorses and cattle suffering from nagana in Zululand and other parts of Africa. Bruce further demonstrated the important fact that the disease was transmitted by the bites of flies, the tsetse flies (Glossina). Announcements of other pathogenic trypanosomes in different parts of the tropics quickly followed. In 1896 Rouget found that dourine, a disease of equines in Algiers and South Africa, was caused by a trypanosome (T". equiperdum) . Then the South African disease of horses, called mal de Caderas, was shown by Voges to be due to a similar flagellate, while in 1902, Theiler found a variety of trypanosome in the blood of cattle in the Transvaal suffermg from the disease called galziekte, or gall-sickness. The number of trypanosomes found in the tropics is constantly increasing — both pathogenic and non-pathogenic forms. Man was thought to be comparatively immune to trypanosomes until the important discovery was made that trypanosomes are the specific cause of a definite disease known as sleeping sickness, which occurs chiefly in the African negro. In 1898 Nepvieu reported having found trypanosomes in the blood of 6 out of more than 200 cases of human beings examined for malarial organisms and in a seventh case which was apparently m good health. The eighth case is reported by Dutton in 1901. The tenth and eleventh cases were published bj^ Manson in 1902. Broden published two more cases, and Baker three. In 1904 Castellani stated that the cause of sleeping sickness of the negro is a trjrpanosome. He found trypanosomes in the centrifugalized cerebrospinal fluid of 20 out of 34 cases of this disease. His work has been fully corroborated. 504 3 C' J m ;?; ■< « o Q s o a s -p pSSd i? Rat shght. , Rat fleas (Ctenophthal- mus) and lice (Hsmatopi- nus). Acute. Stomoxyx (fly)- Acute. Glossina. Acute. Acute. ' 1 Transmitted by coitus. Acute. Hydrocherus Capybara. Glossina. Glossina. 1 1 Slight. 1 Conorhinus. p 1 1 II i.i 2-a Trypano- somiasis . Surra. Nagana. Trypano- somiasis. Dourine. Mai de cadcras. Trypano- somiasis. Trypano- somiasis. , Trypano- 1 somiasis. Sleeping- sickness. Trypano- somiasis. 1 Vertebrate host. Rats. Equida; cattle camels, etc. Equidte cattle. Equidie. Equidje. Equidae cattle. EquidiP cattle. Equids cattle. Bovidse slight. Equidie cattle. Man monkey. .^ characteristics. Very motile, nu- cleus at anterior and middle third, blepharoplast rod-shaped. Post, end usually bluntly rounded. Kinetonucleus very minute. Very motile, club shaped. Heavier build than vivax. Blepharoplast near nucleus. Very large. Dividing forms not seen in blood, blepharoplast large i a g 1 ■s s lo 1/3 lo "O lO r^ »'l O O lO OOOOC^jOOO CO cvi oi coro-M'co-^soiO d O OO OO COlDiMcOCOCOn^ CO c^ CO c^coc^coco^o^~-t; ^ p bI Throughout world. India. Zululand. Central America. North Africa. South America. Cameroon. German East Africa. Transvaal. German East Africa. Brazil. Date dis- covered. 1881 1885 1899 1910 1901 1901 1905 1910 1903 1902 1909 1 V2 T. lewisi Kent. T. evansi Steele T. brucei Plimmer and Bradford. T. hippicum Darling. T. equiperdum Dofiein. T. equinum Voges. T. vivax Ziemann. T. caprae Kleine. T. transvaaliense Laveran. T. theileri Laveran. T. (Schizotrypanum) cruzi Chagas. 02 O r-< C^ CO ■^ lO 1> GQ ■5 P •s 3 J?? ti b -^ "5 ■< ■< E -S-^^ S ^ ^^ bS S.2J d 2i C d cd &^ a « a- III i- 0.. £' 3 £■ 1 a'aa-s g a s|-is !Jj ai|.§sai a c M 3 ^ £3 1" ai a'2'a 3 c d d ■g (^ .4, (I, CO OS d *3 . t- M 03 c4 a « :h g ;2 d "0 -^ d a d ^-a ^ 3 * . q nj ^ a Is n &"- -K 1^ |l__ 1° in CO -• ^ CO tL i " " bl oT a> aT lU ;- fcc > bc > s < ((]<< C4 < 00 (M CO -^ ■V rH '"' «o CO Ci m Ci CO CO CO CO CO CO ~_^ ^ (S - s s 3"a P=1.S ■r: 1 -a Hi 1 13 fall ^1 5J TD i, Oq- >> oj op^ r^ z; ^z (^ Z p ^ 3 >o C: -M C-l r- CO CO -H C"^ CI Oi en 01 ""■ ■"■ '"' ""* '-' ID a 3 a P a ra S •3 a ■3 % i. ■1 a r5 d "O d — us J b^ H H H £-; H ^^ h" H CO OS -H M asjj "lou sauiipraos [iiib j3Aon nin]{.52i'e[j; ■]]; osjj sauii^aoios nmipSEi^ ■III rm PATHOGENIC MICROORGANISMS The trvpanosomes found in these cases resemble each other; they are, therefore, included under the same name, Trypanosoma gambiense Button. A smiilar form found liy Stephens and Fantham in 1911 in cases of sleepmg sickness m Rhodesia has been given another specific name, T. rhodesiense, chiefly because of the characteristic action in experimental animals (see table). Cliagas, in 1909, states that a trypanosome which he had discovered iii a small monkey {Callethrix hapalepenecellatu,) is the cause of human infection m liio de Janeiro. Because of its ability to grow in the tissues of infected animal.s it is classified by some as a new genus— schizotrypanum. It is carried ])y a hemiptera, genus Conorhinus. The flagellate is small with a large blephar- oplast (kinetonucleus). It grows on blood-agar readily and infects laboratory animals easily. Chagas reports developmental forms in the monkey's lung and in the gut of the Qy. Fig. 174 Agglutination of Tri/pa}tn.^o)na Icwisi. (Levaran and Mesnil.) Comparative Characteristics of the Different Species. — The form changes of the same species in the same host are so varied that few have been found absolutely characteristic of a single species, and, as physiologic properties alone are not considered final in species classi- fication, we cannot be sure that all of the organisms in this group des- cribed as separate species are so until more of the complete life histories are known. Until then each new form found witli distinct pliysiologic jjroperties, though apparently morphological!}' similar to others, may expediently be considered a new species. As an example of the difficulty of deciding whether or not a trypanosome found is a new species, the history of T. rhodesiense may be taken. All of the points brought forward by Stephens and Fantham, the discoverers, as evidence of a new species have been combatted by others; and its relation to T. gambiense on one side and T. hrucei on the other is an open question. At present the points are in favor of its being a distinct species. MOTILITY 507 Morphology. — Size. — The x'uriations rccdnlod in tXwdiiiicnslons of tlie species we are considering- may he seen hy glancing' at tlie aho^'e tahle. Tiie tryj)anosonies ])atiiogenic for man ( 7'. ijiinihicii.sc and T. rhodcKii'ii.'^i') have the smahest aA'erage size of the gronp. \Vith tiie excei)tion of T. iheilen and T. ti-(ni{tiHiaIicits(\ w hieli are mni'h liirger than any otlier of these ft)rms, the A'ariations in si/e of the different sjjeeies are not so marked as they are in the same species under different conditions. Shape. — In shape, though all follow the type, each species varies greatly according to conditions of growth and multiplication. At times they may be slender and worm-like, at others they may be so short and thick as to be almost rountl. T. lewh'i has the posterior (afiagellar) end often thinner and more pointed than the other species. T. evansi is generally a little longer and thinner than T. lewisi, while T. bnicei has a more rounded afiagellar end than either, and is generally broader, more club-shaped (Plate IV, Fig. i, 2). The cytoplasm differs slightly in the different forms. T. lewisi is relatively free from chromatoid granules, while T. hrucei has usually many. Myoneme fibrils ha^'e been demonstrated in some species and probably all contain them. An oval vacuole has been seen in some species. The nuclear apparatus is essentially similar in all forms. The two nuclei (tropho- and kinetonucleus) vary somewhat in position and size in the different species and at different stages in the same species. In T. theileri and in young forms of T. lewisi, both nuclei lie close together near the centre of the organism. In T. lewisi the tropho- nucleus is situated more anteriorly than in the other species. In T. rhodesiense the nucleus is often posterior to the centre, especially' in experimental animals. Many variations from tlie type forms are seen. Some are no doubt degen- eration and involution forms. Three forms, however, which are more or less constantly seen in all the species have Ijeen interpreted as definite phases in the life cycle. These forms were first described by Schaudinn in T. noctuce, and were interpreted by him and since then l^y others as male, female, and indifferent form. The male cells are smaller, more hyaline, and more free from granules than the female. The nucleus of each sexual cell rids itself of mafe and female chromatin, respectivel3^ The indifferent cell, on the contrary, has a com- plete nucleus. Opinions differ as to Schaudinn's interpretation bemg the correct one. More research is needed before we can arrive at a definite conclusion. Motility. — The first thing noticed on examining a fresh hanging drop of blood at a magnification of 100 to 300 diameters is active movements of the red-blood corpuscles in certain areas, and, on care- fully focusing over one of these areas, the rapidly wriggling worm-like organism may be seen. As the movements become slower, the flagellum may be seen swaying from side to side and the wave-like movements of the undulating membrane are quite discernible. Movement is twofold: (1) progression with an auger-like motion effected by the undulating membrane assisted by the flagellum; (2) contractions of the body assisted no doubt by myoneme-like structures. Relatively, r,08 PATHOGENIC MICROORGANISMS T. lewisi is most active and T. briicei least. Motility soon ceases outside of the body, continuing longer if the organism has been kept in the ice-box than at higher temperatures. Aflagellar forms, sometimes ameboid, ha\-e been frequently described in the blood of mammals. Reproduction.— The usual method of multiplication is l)inary longi- tudinal fission. In se^'eral species a rosette-like segmentation has also been observed. Longitudinal fission begins usually with di^■ision of the kinetonucleus, then of the trophonucleus and cytoplasm; but this order of division seems to be quite variable. The flagellum often appears to be di\'iding first, and probably division always starts with the centrosome-like basal granule of the ifagellum. In many cases a new flagellum seems to be formed instead of division of the old one. The details of division have not been frequently studied, but it is probable that both nuclei divide by a primitive mitosis. During di^'ision the kinetonucleus generally moves near the trophonucleus. Generally the fission is equal, but occasionally the daughter trypanosomes may be quite unequal in size. This is notably the case in division of T. lewisi where the cytoplasm may divide so unequally that the process may be compared to budding. The resulting small parasites have at first no undulating membrane, hence they resemble somewhat Lepto- monas. These young forms may divide several times in succession, producing smaller and smaller fusiform parasites. As a result, some forms are so small that they can only be seen when agglomerated or in motion (Schaudinn). The question as to whether trypanosomes undergo phases of development in their invertebrate hosts has been widely studied. Prowazek has described sexual forms of T. lewisi in the body of the louse. The subject, however, requires further research for convincing evidence. Insect Carriers. — The trypanosomiasis of vertebrates are trans- mitted bj' blood-sucking insects (dourine is possibly an exception). Bruce (1894) first showed that T. brucei was conveyed by the fly Glossina morsitans. Since then other varieties of flies also have been shown to spread the disease (table, pp. 504 and 505). Among them Glossina jxilpalis (Fig. 175) is supposed to be the chief agent in transmitting human trypanosomiasis. These flies bite by day and in full moonlight. The infective period of the insects after they have bitten a sick animal is variable. Bruce found living trypanosomes in the proboscides of the flies up to forty-eight hours. Up to one hundred and eighteen hours he found them in the flies' stomachs, after one hun- dred and forty hours he found the stomachs empty, and what appeared to be dead parasites in the excreta. Klein and others have since found that after a certain number of days (usually from tweh'e to twenty-five days) a small percentage of the biting flies, which were fed suflSciently on an infected animal become infective again. This is due to the fact that the trypanosomes have developed in the gut and have passed to the salivary glands of the fly. They remain in the glands probably during the life of the fly, and continue to be infective (Bruce, Hammer- ton, Bateman, Mackie, and others.) EFFECT ON VERTEBRATE HOST 509 Dourine in liorses is usually communicated durin};- the act of coitus. Surra may be transmitted to dogs in eating- infected animals. The rat trypanosome is conveyed by contaminative infection through the dejecta of the fleas or lice when biting a new host. Cultivation. — Novy and INIacNeal were the first (1903) to cultivate trypanosomes in the test-tube. They have grown T. leiri.ii through many culture generations extending over several years. At the last tests made the parasites were as virulent as at the beginning. The culture medium used in their work was the condensation fluid from slant tubes of ordinary nutrient agar containing variable amounts oi fresh defibrinated or laked rabbit or rat blood. The best results were obtained with a mixture of equal parts of blood and agar. At room temperature the growth is slower but surer than in the thermostat. A culture at room temperature retains its vitality for months; thus in one case the trypanosomes were alive after three hundred and six days. Novy and ^lacNeal also cultivated in mtro T . brucei, T. evansi, and various liird trypanosomes. The latter they found especiafly easy to culti\ate, while the former are much more exacting in their require- ments than is the 2\ lewisi. They recjuire two parts of blood to one of agar, and growth is best at 28° C. The primary cultures are not transferred until three weeks. These are not virulent for animals, but subcultures regain their virulence. Mice and rats die in six to four- teen days after inoculation. Guinea-pigs have a relapse in two to ten weeks. The great majority of trypanosomes experimented with have been found by various workers to be cultivatable, with more or less ease. Trypanosoma gambiense, however, seems to be more difficult to cultivate (Thompson and Sinton). Effect on Vertebrate Host (Pathogenesis). — Lower Animals. — Many of the lower vetebrates have become, through mutual toleration, natural hosts of the trypanosome. It is probable that each pathogenic trypanosome has an indigenous wild animal as natural host and that in this way the supply to strange mammals coming into the ^'icinity is kept up. These strange animals, being unaccustomed to the native trypanosomes, often succumb to the infection. Symptoms. — In general the descriptions given of the symptomatology of trypanosomiasis in various animals show a great similarit3', though there is mucli variation in individual cases. The average clinical picture, according to Mus- grave and Clegg, is as follows: After an incubation period which varies in tiie same class of animals and in those of different species, as well as with the con- ditions of infection, and during which the animal remains perfectly well, the first sjmiptom to be noticed is a rise of temperature. For some days a remit- tent or intermittent fever may be the only evidence of illness. Later on the animal becomes somewhat stupid; watery, catarrhal discharges from the nose antl eyes ajipear; the hair becomes roughened and falls out in places and the peripheral lymph nodes are enlarged. Finally the catarrhal discharges become more i)rofuse and the secretions more tenacious and even purulent; marked emaciation develops; edema of the genitals and dependent parts ap]5ears; a staggering gait, particularly of the hind parts, comes on, in some forms passing on to paralysis. This is followed by death. There may be various )10 PA T HOG EN I C MICROORGANISMS CH'cli.ymoscs and skin eruptions. Parasites are found in the blood more or less regularly after the appearance of the fever. Thej^ are often more numerous iu the enlarged lymph nodes and in the bloody edematous areas than in the general circulation. The autopsy shows general anemia, an enlarged spleen with hypertro- phied follicles, more or less gelatinous material in the adipose tissue, the li\'er slightly enlarged, a small amount of serous exudate in serous cavities, edem- atous condition, and small hemorrhages in various tissues. There is a relative increase of the mononuclears in the blood. The duration varies from a few days to many months. The prognosis seems to be influenced to a certain extent hy the species of host. It is probably always fatal in horses. Some cattle recover. The cause of death is pcssibly a toxic substance, though no definite toxin has been isolated. Mechanical disturbances (emboli, etc.) also proliably play a part in producing death. Man. — Sleeping sickness, or human trypanosomiasis, is an endemic disease in certain regions of equatorial Africa. Neither age nor sex are predisposing factors, l)ut occupation and social position seem to have a marlved influence, tlie great majority of cases occurring among very poor field worl^ers. As these workers are all negroes, the question of the relati\-e influence of race cannot be determined. The white race, however, is not innnune, as has been frequently shown. Tir,. 17.T (iliiwsina palinilis, cniTicr of ih ■ liuniaii tr>-i>arios(niii:iyis c (Kdllr and Was.sc'riiiaiin.) i,\" 'I'. L'anil)ioiisp. X 4. Symptoms.— The course of the disease is very insidious, as the trypano- soiiies may exist m the blood for a long time before entering and growing in the cerebrospinal fluid and causing the characteristic symptoms. Therefore, the symptoms may be divided into two stages. In the first stage there is only an irregular fever with enlargement of the perijiheral lymiih nodes. In the second stage the fever becomes hectic, the pulse is constantly increased; there are neu- METHODS OF EXAMINATION 511 ralgic pains, partial edemas and eiythemas, trembling of the muscles, gradually increasing weakness, emaciation, and lethargy. The sonmolence increases until a comatose condition is developed and death occui's. In the second stage try- panosomes are always found in the cerebrospinal fluid. Throughout the disease they are usually found in small numbers in the blood. Duration. — The first stage maj' last for several years; the second, from four to eight months. The percentage of deaths in cases reaching the second stage is 100. Whether some in the first stage recover is not yet certain. T. gambiense, the trjrpanosome first shown to be pathogenic lor human beings, is irregularly pathogenic for some monkeys {Macants rhestcs and others), for dogs, cats, and rats. It is less pathogenic for mice, guinea-pigs, rabbits, horses, baboons, cattle, and swine. In fact it has a ^'cry wide range of patho- genicity. Flies (Gl. jxilpalia) have been found to be infected with this trypanosome in areas which ha\'e had no human population for several years, a longer time than the life of a fly. And since trypanosomes are not known to be hereditarily transmitted, it is concluded that certain mammals are harboring the trypano- somes, thus acting as reservoirs. T. rhodesiense is more pathogenic for experimental animals than is T. gambiense. Pathological Changes. — Congestion of the meninges; increased quantity of cerebrospinal fluid; hypertrophj' of spleen, liver, and lymphatic ganglia; diminished hemoglobm and number of red cells; number of leukocytes about normal, but a relative increase of eosinophiles, mast cells, and lymphocytes. Enlargement of the superficial lymph nodes has been noted as an early symptom and has thus been made use of in diagnosis. Button and Todd found that 91 per cent, of natives in the Congo Free State, who had posterior cervical glands enlarged, showed trypanosomes in the punctured gland juice. Diagnosis of Trypanosomiasis in General. — This should be made as early as possible in order to prevent the spread of the disease. An early positi^'e diagnosis can only be made by the determination of the peripheral infection. This is done in two ways: first, by micro- scopic examination of freshly drawn blood, cerebrospinal fluid, or tissue from enlarged peripheral lymph nodes; second, by animal inoculation of the blood or other tissue. In the microscopic examination it may be necessary to examine the blood of the suspected animal for sev- eral days in succession. The parasites are rarely absent in the early stages in domestic animals for more than a few days at a time, while in man the time may be much longer. Methods of Examination. — Blood. — If the direct examination of the blood is negative, 10 c.c. should be withdrawn from the vein, and after adding a tenth of its volume of citrate of sodium it should be centrifuged for ten minutes, and the sediment examined in hanging drop and in smear. The great majority of the parasites will be found collected with the white cells in the thin white laj'ers which may easily be removed with a fine pipette. The parasites are readily detected with the low pow'er of the microscope (X 100) by areas of irregularly moving cells. If only a .small amount of blood can be obtained, the tiny tubes recommended by Wright in his opsonin work (p. 210) may be used. Cerebrospinal Fluid. — Ten c.c. of the fluid withdrawn by lumbar jKuicture should he centrifuged for fifteen minutes and the deposit should be examined under 150 to 200 diameter magnification. 512 PATHOGENIC MICROORGANISMS Cultures sometimes give positive results, especially from infections in lower animals and in infections with T. rhodesieme. The Inoculation Test. — If the trypanosomes cannot be found by the above methods, animal inoculation should always be made. Monkeys, if possible, should be used, or if monkeys cannot be ob- tained, dogs or rats may be used. A few drops to 1 c.c. of the blood or other tissue from the suspected animal should be inoculated intra- peritonealh' or subcutaneous!}'. Smears may be stained by any modification of the liomanowsky method. Giemsa's method (p. 81) gives good results (Plate IV, Fig. i, 2). Prophylaxis. — The disease is readily controlled by preventive meas- ures. There should be strict quarantine regulations governing the imjjortation of animals. When the . All means possible should be used to exterminate the reservoirs. Treatment. — The whole question of treatment is still in the experi- mental stage. The chronic course of the disease with relapses often after long intervals makes it impossible, especially in cases of human trypanosomiasis, to come quickly to a conchision in regard to the effi- ciency of any drug. Many drugs ha^-e been found to possess trypano- cidal properties. Atoxyl (p-amino-phenyl-arsenic acid, /OH V /\AS =/ O I. J \. ^^ NH2 X1N.2 / introduced by Thomas (1905) and used first l)y Thomas and Breinl in treatment of experimental trypanosomiasis, has proved to be the drug above all others to have a beneficial effect in the difi'erent forms of this disease, ^luch experimental work has been done on the dif- ferent i)hases of treatment by atoxyl and its allies, in the course of which some yevy interesting facts relating to chemotherapeutics have been demonstrated. I^hrlich has added to his "side-chain theory," while (others have advanced quite different views in regard to the action of this group. The chief facts are the following: 1. Atoxyl does not act ni vifro unless mixed with an oxidizing sub- stance, hi viix) atoxyl combines with a blood-constituent to act upon the tr_ypanosomes. 2. After the first few treatments with any of these drugs trypano- somes ma>' become resistant to the drug. This resistance is more or less specific for all members of the group to which the drug used belongs. COMPLEMENT-FIXATION ^ 513 There are a few exceptions, e.g., an atoxyl-resistins strain may still be influenced by arseno-phenyl-glycine or by orsndan. This acquired resistance lasts for some time in the species of animal used, l)ut may be quickly lost if the resisting trypanosomes are inocu- lated into another species. Passage througli an insect carrier may cause a clrug-je.'^t trypanosome to become again susceptible to tlie drug. o. The time of the reappearance of parasites after a discontinuation of treatment is more or less regular. Witli T. gambiense, in rats and monkeys, the period is generally 50 to GO days. With T. brvcei in rats, guinea-pigs, and dogs, the time is only 11 to 25 days. 4. In order to pronounce an animal cured a long period must elapse, since relapses may occur at a very late date (226 days in rats infected with T. hriicci and treated with atoxyl). In human trypanosomiasis favorable reports from atoxyl treatment still continue to come, though the percentage of cures claimed by Koch is probably not reached. Just now good reports are being received from the use of another arsenic compound, introduced by Ehrlich, namely, arseno-])henyl-glycine. Serum Therapy. — Various normal sera from different animals have been tried with practically no success. A few have prolonged life. Thus Laveran and Mesnil state that human serum injected in sufficient quantities shows manifest action on the disease, and that sometimes cure results in mice and rats. Further, b,y alternating human serum with arsenic they obtained better results still. Kanthack, Durham, and Blandford showed that animals recovering from trypanosoma infection were immune to further infection. RabinoM'itsch and Kempner haA'c made a very careful study of immune serum produced by T. lewisi. They have shown that an animal may be hyperimmunized and that then its serum, in comparatively large doses, inoculated into mice at the same time as the trypanosomes, or twent.^'-four hours before or after, allows no de\'elopment of the organisms. Laveran and Mesnil state that the serum causes the rapid destruction of the organisms by the leukocytes, though MacNeal, on the other hand, states that the trypanosomes are destroyed by a cytolytic action of the serum. This immune serum also has a similar action on the trypanosoma of dourine. The serum of animals hyperimmunized against other varieties of trypanosoma is not as active as that obtained by the inoculation of T. lewisi. Koch suggested that immunity might be produced by the inoculation of attenuated parasites, and Novy and MacNeal have succeeded in attenuating cultures of T. hriicei, and have obtained some success in protecting experimental animals against virulent cultures. Complement-fixation. — The earlier tests did not promise ]>ractical results. l{ecently, howe\'er, se\'eral in\'estigators have given more favorable rei)orts. In this country Mohler claims that the test is of great worth in diagnosing dourine, many cases of which have occurred in some of our western states (notably Iowa and Montana). INIohler, Eichhorn, Buck, and Traum state that they use a fresh antigen pre- pared from the spleens of rats dead after infection with surra (used 33 514 PATHOGENIC MICROORGANISMS Ijecause it is easier to transmit than is dourine). The antigen is simpl_y a filtered emnlsion in salt solution of the infected spleen. The emulsion from each spleen is made up to 40 c.c. by addition of salt sohition. A fresh antigen must be prepared and titered (see Part I) each day. The results have been controlled b,y autopsies of animals giving a positive reaction. References. Breinl and Nierenstein. Ann. of Trop Med. and Parasit., 1909, iii, .39.5. Brace. " Trypanosomiasi.s " in Osier's Modern Medicine, 1907, i, 460. Bruce, Hammerton, Bateman and MacKie. Proc. Roy. Soc., 1911. Ehrlich. Ueber Partial funktionen der Zelle., Miinch. nied. AVoch., 1909, v, 217. Hitidle. Obser^■ations on the life historj' of T. lewisi in the rat louse. Assoc, of Trop. Med. and Parasit. Lai'cran ct Mesnil. Trj'panosomes and Trypanosomiases, Trans. lj\' Nabarro, London, 1907. MacNeal. Jour. Inf. Dis., 1904, i, 537. MacNeal and Novy. Contrib. to Med. Research, Vaughan Anniv., 1903, p. 645. MacNeal and Nrmj- Trypanosomes of Mosquitoes, Jour, of Infect. Diseases, 1907, iv, 223. Mayer, M. Tryjianosonicn als Krankheitserreger. In KoUe und Wasscrniann's Handbuch d. Path. Mikrcbrganismcn, 1913, 2d ed., Jena. Mrsnil et Kerandel. Sur Taction preventive et curati-i-e de I'arsennphenj'lslycitc dans les trypanosomiases expcrimentales et en particuUcr dans les infections El T. gambienxe. Bull. d. 1. .Soc. d. path, exot., 1909, ii, 402. Mohler, Eichhorn, and Buck. Jour. Agrie. Res., 1913, i, 99. Musgrave and Clegg- Tryjjanosoma and Trypanosomiasis, etc., Manila, Bureau of Public Printing, 1903. Nary and MocNenl. Trypanosomes of liirds, etc., Jour. Infect. Di,seases, 1904, i- 1905, li, 2.56. Noi>!/, Perkins, and Chambers. Jour. Inf. Dis., 1912, xi, 411. Nultall. Parasitology, 1913, v, 275. Slephans and Fantham. Proc. Roy. Soc., 1910, Ixxxviii, 28. Thompson and Sinton. Amer. Trop. Med., 1912, vi, 351. Vianna. M6m. d. I'lnst. CJswald. Orig., 1911, iii, 276. ^Yoodcnck■. The Hemoflagellates and Allied Forms in Lankester's ".A Treatise on Zo61o£;y," London, 1909. Part I, first fascicle, p. 193. Ynrke, W., and Blacklock, R. The Differentiation of the more important mammalian (rj panosomcs. .imer. Trop. Med. and Parasit., 1914, viii, 1. CHAPTER XXXI X. SPIROCHETA AND ALLIES. The genus Spirocheta -was introduced by Elirenl:)erg in 1838, who differentiated it from spirillum by its flexibility. Schaudinn in 1905 thought he saw an undulating membrane in Spirocheta refringens, so he added this characteristic to the genus and considers that thus its relationship to the flagellated protozoa, genus Tryponosoma, is indicated. Since the appearance of the work of Schaudinn and Hoffmann (1905) showing the etiologic relationship of a spirochete to syphilis, the Spiro- chetce have been brought into great prominence. Numerous spirochetes and spiral organisms haA'e been described, some associated with Treponema (spironema) paUichini in syphilis, some in other lesions or in the normal secretions of both man and the lower animals; and still the ciuestion as to their classification is unsettled. The majority of observers, however, are willing to admit that the structure of many of the varieties classed with this group is more complicated than that of bacteria and that hence the group may be an intermediate one between protozoa and bacteria. The chief reasons given for considering spirochetes protozoa are: (1) the flexibility and the indications in many of longitudinal division and of undulat- ing membrane; (2) the demonstration of forms intermediate between the trypanosomes and the spirochetes {Sp. balbianii); (3) the spirochetal forms of certain trypanosomes (Tr. noctiwe); (4) stages of development in the louse and tick. In favor of the bacterial nature of spirochetes are: (1) the rigiditj' of some forms, the lack of undulating membrane in most and of definite nuclear appara- tus in all, and the evidence of transverse division in all and of flagella arising from the periplast in some; (2) the cultivation of certam forms (e. g., Sp. refrin- ge)is, by Levaditi; Sp. obermeieri, by Novy; several spirochetes by Noguchi) for many generations without development of tr^'panosome forms. So far the studies on this group show that the spirochetes and allies prob- ably occupy a position intermediate between the protozoa and the bacteria. We study them here because of the claims that they are closely related to the trypanosomes. It may be well to note briefly the chief characteristics of the more familiar non-pathogenic ones in order better to understand the relation- ships between them and the Trrpnnevia paUiditm and other pathogenic forms. Material and Methods for Study. — The large Spirochela balbianii is found in the stomach of oysters fresh from salt water. Smaller spirochetes are fre- quently found in human mouths. When fresh syphilitic or relapsing fever material can be obtained this should be examined. The Treponema pallidum (the spiral organism of s.yphilis), because of its low refractive index, is seen 51fi PATHOGENIC MICROORGANISMS when alive with difficulty by the ordinary microscope, but with the dark- stage illumination, especially if a drop of distilled water is added to the serum containing the organisms, it is seen distinctly and its motion and structure may be more easily studied. The fluid containing the organisms should be dropped on an ordinary glass slide, covered with a thin cover-glass, and well sealed with vaselin as most spirochetes are anaerobic. Material may be ob- tained from syphilitic lesions as follows: The lesion is first thoroughly washed and dried witii distilled water and sterile gauze. Part of the base and margin is then scraped with a curette until the superficial tissue is removed and blood appears. The blood is wiped away with sterile gauze untU clear serum begins to ooze. A drop of this serum is used for examination. Smears should be made as thin as possible and may be stained (1) by Giemsa according to the method on page 81 [Tr. pallidum stains reddish). A modi- fication of Giemsa, used by Schereschewsky (see References) has been highly recommended by various workers; (2) by Goldhokn's method (p. 82). Cultures. — Pure cultures have been obtained of the Spirocheta dentium in the following manner: Poured serum-agar plates are made of various dilu- tions of material from the mouth containing these spirochetes. _ After Ijeing kept in the thermostat at 37° C. under anaerobic conditions for nine to twelve days the spirochetal colonies are fished and planted in agar tulses as stick cultures. Pure cultures of Spirocheta obermeieri l^y Novi and of Spirocheta refringeyis by Levaditi have been obtained Isj^ gro-ning in collodion sacs. (For other culture experiments see lielow.) Spirocheta Balbianii (Certes). — This immense form, next largest known to the Spirocheta plicatilis Ehrenberg, may be found in tlie stomach of the oyster. It is important for study because it is appar- ently a transitional form. In fact, it is considered a trypanosome by Perrin and others. [Nliihlens gi^•es its characteristics as follows: Length 26/.1 to 120^, in width \ii to ?>ii. The body is flattened and pos- sesses an undulating membrane whicli is visible during life on some individuals. It has 4 to 8 flat, wide spiral coils. Its movements are lively, similar to those of trypanosomes, but more corkscrew-like. During motion its form is apparently easily changed. The rim of the undulating membrane does not end in a free flagellum, but one end of it seems to be attached to a triangular mass of chromatin (basal granule, blepharoplast?) which is a part of the central chromatin material. The nuclear material is arranged in a more or less spiral band along the entire centre of the organism. Before division this nuclear band, after passing through chroma- some-like changes, breaks up into pairs, and division takes place longitudinally between them. Di\'ision is often incomplete for a time, the two ends remaining attached. Spirocheta balanitidis.— This is a spirochete found by Simon in Balanitis rirciiiafa and regarded by some as the specific cause of this disease. Hoffmann and Prowazek describe it as a rather strongly re- fractive, acti\-ely motile, band-shaped organism, shorter and thicker than Spirocheta pallida, with G to II) coils, staining bluisli-red with (iiemsa's method and exhibiting an undulating membrane and at either enil a i)erii)lastic cilium. Miihlens thinks this may be identical with Spirocheta refringens. SPIliOCIIF/rES IN TUMORti 517 Le\'a(liti has recently rei)ort.ecl cultnating' it (see below under Trcpo- nenia pallidum). The Mouth Spirochetes. Three ^•al■ieties nf iioii-pathdt^eiiie hiriiis are couimonly found in normal mouths. 1. Spirocheta Buccalis (Cohn). — Length, 10^ to 'Mp.; thickness, \p. to f/j. It has i! to 10 irregular Hat coils. No true cilia have been ilemonstrated, but Schaudion, Hotfman, and Prowazek say it has an undulating membrane. It stains \'iolet with Giemsa. 2. Spirocheta Dentium (Koch). — This is much smaller than the previous form. It is as thin as the pallidmn and is somewhat similar to it in refraction, staining cjualities, and in the fixity of its coils during motion. It is somewhat smaller and stains a little more easily with LofHer's tlagella stain, and ilagella have been demonstrated. Neither definite undulating membrane nor nuclear material has been seen. It is 4// to 12^ long, and has 4 to 20 regular spirals of about the same appearance as those of the paUidiiiii. Pure cultures have been made from this spirochete as described above. 8. A middle form between these tw^o has been fovuid in the mouth. This also is somewhat similar to the pallidum, but it is larger and has less regular spirals; moreover, it stains more intensely with the blue of Giemsa, only in poorly prepared specimens does it appear red. Spirocheta refringens is also found in the mouth, but it is espe- cially interesting from the fact that it is so often found associated with the Treponema palliduin in tlie various lesions of syphilis. It is not in such large numbers as the pallidum and probalily bears the relation of a restricted secondary invader. It is generally longer than the pallidum (lOji to '?,(^^x) and much thicker (i^u to f^). In life it is much more refractive. It has 3 to 15 irregular wide, flat spirals which change their shape during motion. Its mo\-ements are much more lively than those of pallid init. With Giemsa it stains quickly and easily, a blue to a blue-violet tone, according to the length of staining. Schaudinn states that it possesses an undulating mem- brane. Levaditi claims to have demonstrated terminal cilia for this organism and to have culti^"ated it in collodion sacs in the rabl)it's peritoneum. Spirocheta Vincenti. — Accompanying the fusiform bacilli in Vincent's angina (see p. 319) are many spirochetes similar to the "middle form" found in the mouth. Whether they are identical with these spirochetes or whether they are a special variety (or, as some think, a second form of the fusiform bacillus) still remains to be determined. Their relation- ship to the disease is also uncertain. Spirochetes in Tumors. — Lowenthal, Borrel, and others found spirochetes in small numbers in certain mouse tumors. Ewing and Beebe found a few in some dog tumors and others have reported their occasional presence in both ulcerating and non-ulcerating human tumors, but apparently never in sufficient numbers to account for the tissue reaction. Gaylord, however, found that in repeated transplants of a mouse tumor, as the inoculated material became more virulent the 5 IS I'ATIIOGENIC MICROORGANISMS num))er of spirochetes greatly increased. Calkins stndied the mor- phology of Gaylord's spirochete and decided it to be a distinct species, lie has also fonnd this species in primary as well as in transplanted tumors. It is much shorter and thicker than tlie imllidviii , and has blunt ends. It closely resembles the spirochetes found comi)aratively fre- quently Jjy Tizzer and others in apparently normal mice, though the possibility of infection in these cases was not ruled out. Miscellaneous Spirochetae.— Besides the spirochetes found in syphilis, in frambesia, and the spiral organisms causing African and European relapsing fevers, all of which will be described below, spirochetes have been found (1) in the normal intestinal tract of mosquitoes and human ))eings as well as in the diarrheal stools of the latter; (2) in the blood of mice, fowls {Sp. gallinarum causing relapsing fe\'er in_ fowls and Sp. amerina found in a similar condition of geese) ; (3) in various ulcera- tive and gangrenous process of man. The fowl spirochetes have been most studied. Marchoux and Sal- imbeni were the first to show that the tick, Argas persiais, is a carrier of these spirochetes. The mechanism whereby the tick infects the fowl has been minutely worked out by Nuttall and his associates. Noguchi has cultivated several species of spirochetes by the same method he used for growing Tr. paUidurn (see below). Four of these, he stated, are new species, Sp. macrodentiuvi and Sp. inicrodentium from the mouth, Sp. pluigedensw from human genitals, and Sp. calligyrum from condylomata. Noguchi also reports the cultivation in successive transplants of Sp. recurrentis and Sp. dtdtoni. Treponema (Spirunema) Pallidum (Spirocheta pallida). — This organism is found in large numl)ers in syphilis, an infectious disease of human beings, characterized by its long course and by the definite stages of its clinical manifestations. Historical Note. — Notwithstanding the fact that syphilis is one of the oldest diseases known and studied, only recently has definite light been thrown upon its cause in the discovery of the Treponema pallidum (Schaudinn and Hoffmann). Before this it was thought that the bacillus described bj' Lustgarten (1884) and others as occurring in small numbers in the lesions of sjq^hihs bore an etiologic relationship to the disease, but there were no evidences to support this view. Many other bacteria have been erroneouslj' regarded as the prol.iable cause of syphihs. From time to time various observers have described protozoan-like bodies in syphilitic lesions, but their observations have not been confirmed. Schaudinn announced early in 190.5 that working with Hoffmann he found in the fresh exudates of chancre a spiral organism possessing characteristics similar to those of the spirochetes and he named it Spirocheta pallida. Later he concluded that this organism was individual enough (that is, it showed no undulating membrane, but possessed fiagella) to be placed in a separate genus, so he called it Treponema pallidum. He thought that the organism was the cause of the disease. Since then there have l^een extensive studies on hmnan syphilis and on experimental syphihs in lower anunals with the result that the work of Schaudinn and Hoffmann has been abundantly corroborated and many new facts liave been brought out. The Organism (Fig. 176. — The Treponema pallidwn is a very delicate structure closely resemlding in moridn)logy and staining reactions CULTIVATION 519 the Spii-oeluia dentiiitii. It is somewhat longer, 4^ to l-'O^i long (average lO/i), and thinner, \n to \fx in ilianieter. It has three to twenty sliarp, deep spirals. The relationship between tlu' length and the de|)th of the sjiirais is ditl'erent in the two speeies; in 'Tri'jiiiju'tiia pdllidiiiii length is to depth as 1 is to 1-1. T) (\^x long and I// to 1 .."i/a dee])), w hile in t^piro- cheta dentiitin the a\'erage relationship is 1 : 0.5 the sjnrals heing more shallow. The angle of the spiral tnrn is very sharp in both forms (more than 90°). Fig. 17G Fig. 177 "^ y ) The two spirochetes in the centre are Tr. pallidum; the three others, Sp. refringens. (Schaudinn and Hoffmann.) Treponema pallidum appearing as bright refrac- tive body on a dark field as shown by India ink or ultramicrosrope. Flagella-like anterior and posterior prolongations are often seen in the pallidum. The donble flagella occurring rarely at one end are interpreted by Schaudinn as beginning longitudinal division. Schau- dinn states that the division occurs very quickly (hence the reason why so few dividing forms are seen in stained preparations) and that it may be followed only by the most experienced observers during life. In the living condition the organism is not very refractive, so it is seen at first with difficulty. Its characteristic movements are rotation on its long axis, quivering moA'ements up and down the spiral which is com- parati\'ely rigid, slight forward and backward motion and bending of the entire body. By the use of the ultramicroscope the motility of the organism is clearly seen (Fig. 177). Cultivation. — In 1909 Levaditi and Mcintosh obtained impure cul- tures of spirochetes in collodion sacs containing human serum and syphilitic material and placed in the peritoneal cavity of a monkey {Macacvs cynomolyvs). Schereschewsky reported that he had ob- 520 PATHOGENIC MICROORGANISMS tained impure cultures of a spirochete from sypliilitic lesions and blood in the following culture medium : horse serum sterilized b.y heat (58° to 60° C.) until it is o( jelly-like consistency, and afterward autolysed at 37° for three days. A ])iece of tissue excised from the lesion (c. g., base of a papule or part of a lymph node) is inoculated into this medium, and grown at 37° C. The culture Iiegins in three daj's, but the optimum is reached in 5 to 12 days. Miihlens (1909) and later Hoffmann (1911), reported that they had also obtained cultures of a pallidum-like spirochete from syphilitic lymph nodes, grown at first in Schereschewsky's medium and afterward transplanted to broth and grown anaerobically. Animal experiments are negative. Levaditi and Stanesco about the same time reported growing two species of spirochetes from a case of balanitis. One, a new one, which they found very like pallidum, but non-pathogenic for monkeys, and which they named Sp. gracilis; the other Sp. halanitidis. They em- ployed as media (1) collodion sacs in tubes of fluid horse serum; (2) horse or human serum heated to 75° C. These spirochetes were never obtained in ]3ure culture. Noguchi (1911) obtained pure cultures from syphilitic lesions in the rabbit, and later from human beings. His culture medium contained in deep tubes was a mixture of 1 part of ascitic or hydrocele fluid and 2 parts of 2 per cent, agar, in which was placed a small portion of sterile rabbit's kidney or other organ. The medium was covered by a deep layer of albolene. The spirochetes inoculated along a central stick grow out into the medium as a diffuse layer, while most contaminating bacteria remain in the line of inoculation. Subcultures made away from the contaminating growths, finally become pure. Noguchi states that he has obtained syphilitic papules by scarifying monkeys {cer- copithecus and macacufs), and that such monkeys' blood gives a positive Wassermann reaction. Pathogenesis. — So far as is known, syphilis in nature appears only in man. Since 1879, when Klebs stated that he had produced syphihs in monkeys by the inoculation of human virus, various experimenters have reported its transmissibility to these animals by direct inoculation. Most of the earlier reports did not state the exact identity of the animals employed nor did they give details of methods and results. MetchnJkoff and Roux in 1903 produced a tj^pical chancre on the genital mucosa of the young chimpanzee twentj'-six days after inoculation. The essential lesion was followed hy inguinal adenitis, and thirty A&js later by a generalized papular eruption. The virus was transferred in this case to lower monkeys. Most monkeys developed a primary lesion only, but some had alsimdant secondaries. Since the discovery of the Tr. ■pallidum, experiments on monkeys have been more numerous and liave been followed by more helpful results. More has been learned about the course of the infection in man, the evidence in favor of the Tr. pallidum being the cause of the disease has been strengthened, and many interesting investigations in regard to immunity have been made. Usu- ally inoculations made by scarification on eyebrows or genitals are successful. The primary lesions are seen generally about thirty days after infection. In PATHOGENESIS 521 somewhat more than half the cases, after a slightly longer period, secondary sjauptoms (squamous papules on skm, and mucous patches in mouth) show them- selves. No tertiary symptoms ha\'e been ol)served. Ra.hhits were fii'st suc- cessfully inoculated by Mansell, then by Bertarelli. The eye and the testes are favorable localities, (ieneralized syphilis has been produced in young rabbits by intracardiac inoculation (Uhlcuhuth and Mulzor). Important features in reganl to course of the infection have been sum- marized by Ewing as follows: "If the virus is applied to the broken epithe- lium, a chancre develops, but if similar virus is inoculated into tlie subcutaneous tissue an initial lesion does not follow, innnunitjr tloes not develop, and the animals remain susceptible to subseciuent inoculation of the epithelium. Yet in several instances Neisser was unable to produce chancres in monkeys which had previously received subcutaneous mjections of s^Tihilitic material, indicat- ing that inmiunity may sometimes appear after such subcutaneous injections. Possibly the leukocj^tes of the subcutaneous tissue destroy the virus before it can begin to multiply. Hence, small superficial wounds may be more dangerous in man than deep ones. Nevertheless, it is recorded hj Jullien that two French siu'- geons, accidentally inoculated by deep needle punctures, developed pronounced signs of constitutional syphUis, as attested liy Fournier, but failed at an5r time to show signs of a chancre at the point of inoculation. It remains to be seen whether the observations of the clinicians or those of the experimental pathol- ogists represent the true laws of irfection in syphilis. "In monkej's the virus exhibits a certain choice of epithelium for its entry. The abdominal skin resists the entrjr, the eyebrows and genitals are most readily inoculable in apes, and the palpebral borders in catharinians. The period of incubation varies from thirty days, on the average, in the chim- panzee, to twenty-three daj^s in lower monkeys, but the shorter the incuba- tion, the shorter and less severe the subsequent disease. "That the ^-irus circulates in the blood in certain stages of syphihs has Ijeen clearly shown experimentally. Although Neisser inoculated human subjects with the blood of florid syphilis without effect, a result which is now explicable, Hoffmann, in two of four experiments, produced syphilis in monkey (Macacus rhesus) by inoculating the skin with human blood drawn forty days and six months after the appearance of the chancre. The resulting primary lesions were typical, appearing after the usual incubation and showing a characteristic histologic structure and the presence of Tr. pallidum. " Syphilographers are agreed that tertiary lesions are not contagious. Ex- perimental studies have shown , hov.'ever, that some tertiary lesions are capable of transmitting the disease. Sahncn had negative results with an ulcerated gumma in the eighth j^ear of the disease. Yet Neisser produced chancres and secondaries in a gibbon and in a macacus with the material from a non-ulcerated gumma (duration unknown), but the periods of incubation were very long, fifty-one and sixty-eight days. All tertiarjr lesions do not seem to contain the virus, as Neisser found the material from tubercserpiginous lesions non-infec- tious. It appears also that secondary infection and ulceration of tertiary lesions reduce their infectivity. None of these observations invalidates the clinical experience that tertiary lesions are practically harmless for the patient's neigh- bors, but they suggest greater caution in dealing with tertiary lesions. "According to Colles' law, a mother who gives birth to a syphilitic infant may not herself contract the disease, but thereafter remains immune to inocu- lation. This law may be explained by the infection by the embryo or ovum, and the transference of immunity to the mother by the blood or by some other method. The probable mode of origin of the maternal immunity is suggested by an observation of Buschke and Fischer who found spirochetes in the inguinal lymph nodes of such a case which remained entirely free from the sjaiiptoms of the disease. The observation, taken with the failure of subcutaneous and intraperitoneal inoculation to infect monkeys, may explaui the workings of Colles' law. Levaditi and Sauvage claim to have shown that Tr. pidliduni is 522 PATHOGENIC MICROORGANISMS capable of invading the ovum. Finger and Landsteiuer found the semen in one case of seeondarv lues infectious for apes, but in other eases tlien- results were negati\'e. It is, thcref(jre, only necessary to suppose an occasional escape from the genital tra<'t in order to complete the necessary conditions for the infection of the emliryo with immunity in the mother. "Neisser 'endeavored to determine the degree and duration of the mfec- tivity of the organs of monkeys and found that the virus persists especially in the blood-forming organs, spleen, lymph nodes, and marrow, while in the testicle also the virus is long preserved in active form. The other organs gave entirely negative results." The virus is not filtralile. It is readily destroyed by heat (52° C). Luetin.—N oguchi found that an extract from his cultures, which he calls luetin, gives a characteristic cutaneous reaction in syphilic infection. In normal persons there is a slight local erythema, with possibly a small papule on tire second day. In syphilitics there are three types of po.sitive reaction ; 1. Papular type. Large indurated red j^apulc which increases for about four days. 2. Pustular type. Tlie papule changes first into a vesicle and then into a pustule. :i. Torpid type. After a ten-day latent period, a small papule forms which changes into a pustule. These oliservations of Noguchi have been corroliorated by Cohan and Roliinson. Syphilis in Man. — The course of the disease is divitled into three stages: primary, secondary, and tertiary. The general character of the lesions in these stages is a more or less circmiiscribed formation of new tissue which is largely made up of small spheroidal cells alone or accompanied by fewer polyhedral cells, and occasional giant cells. The initial or primary lesion occurs in the form of a papule which develops into the so-called chancre, an ulcer with hardened base. Following this there is hyperplasia of the nearest lymph nodes. These lesions subside and six or seven weeks later the secondary lesions appear in various general eruptions on skin and mucous membranes and in other constitutional disturbances. The tertiary lesions which consist principally of the masses of new tissue called gummata are found throughout the viscera and in the periosteum. Schaudinn's spirochetes have been demonstrated in practically all the lesions of syphilis (they are most easily demonstrated in the pri- mary and secondary lesions), including the congenital types. Immunity. — Natural immunity in syphilis is A'ery peculiar. After the development of the primary lesions, man is usually insusceptible to reinoculation during the active stage of the disease, but during all stages both man and monkey can, in some cases, be reinoculated. Reinoculation in the tertiary stage gi\'es precocious lesions of the tertiary type, gunmiata, and tubercles. Neisser found reinoculation from twenty-four to one hundred and four da,ys after primary in- oculation in monkeys sometimes effective, more often negative. During the stage when the skin is refractory to inoculation secon- daries develop, showing that there is no complete immunity of the skin to the virus, since the Treponema is abundantly present in the lesions. Neisser suggests 'that cutaneous secondaries develop at ])eriods of relative deficiency of immunity. He has shown that failure SPIROCHETES IN FR.\^rBESI.\ TROI'ICA r,2:\ of reinoculation is not due to iiiiimniit>' to forei};ii infection and sus- ceptibility to auto-infection, since the patient's own \irus in holli man and monkey is inefl'eeti\'e. Passive Immunization.— Injection of larjic (piantities of sennn of sypiiilitics into eliinipanzees lias failed to ])ro(luce definite innnunity, although some of Neisser's animals after such treatment failed to take syphilis. The serum of a monkey cured of syphilis and subsequentl.\' injected over a period of fifteen months with the blood of a syphilitic subject in roseolar stage was without therapeutic effect. I-Iowe\er, this serum dried and powdered pre\-ented tlie chancre when placed on the site of inoculation one hour after the \irus. The Wassermann Reaction. — Wassermann, Xeisser, and Brnck were the first to apply the Bordet-Gengou phenomenon (see Fart I) to the diagnosis of syphilis. According to most workers, enough work has been done since then to establish its ^'alue as a diagnostic test. Some interesting points have been brought out in connection with this study, and many different methods have been recommended. This test has been very widely used and positive results have been obtained in an immense number of cases (o\er 90 j)er cent, of cases with active general infection). In general paralysis and in the majority of cases of tabes, a positi\'e reaction occurs which may be given also by the cerebrospinal fluid. On account of the difficult technique involved it can only be of use in the hands of experienced workers. Positive results have been reported in a number of other diseases as well as syphilis, but in many cases these results ha\'e not been generally accepted, and in other cases the diseases showing positive reaction as yaws, leprosy, dourine, etc., ha^-e been as a rule confined to the tropical countries, or else the positive reaction has been found only during a limited stage of the disease, as in scarlet fever, or the differ- ential diagnosis is otherwise marked, as with tuberculosis. The general opinion at the present time is that the test is of great practical value in the majority of cases of syphilis. It is used as a routine public health measure in New York City and several other centres. The reaction is practically always present during the secondary stage of the disease. The reaction gradually disappears when the disease becomes inactive or is cured. It may disappear before a cure is established, to reappear if an active process starts. Mercury treatment diminishes or annuls the reaction; while treatment with salvarsan may at first intensify it. The nature of the reaction and the technique of the test are considered in Part I. Spirochetes in Frambesia Tropica (Yaws). — Castellani in 1906 announced that he had found in yaws a spiral organism which he called Spirocheta jjertenvis. He determined that monkeys (macacxis, senuiopithecus) are susceptible to inoculations with material from yaws patients apparently containing only this spirochete. Such material filtered is inert. He states that monkeys successfully inoculated with yaws do not become immune for syphilis, neither do those haA'ing had syphilis become immune for yaws. Castellani further states that specific 524 PATHOGENIC MICROORGANISMS characteristics betAveen the two diseases are also brought out by means of the Bordet-Gengou reaction. His work has been corroborated by several obser\'ers. Frambesia lesions similar to those produced by syphilis in the testicles of rabbits have been obtained by Nichols. Le\'aditi and Nallan-Larrier state that monkeys infected with syphilis are refractory to yaws, while those infected with yaws are susceptible to syphilis; therefore, they conclude that yaws is a mild form of syphilis. Spirocheta Obermeieri (Sp. recurrentis) and Allies.— These organisms are classed with the spirochetes as protozoa by Schaudinn, Hartmann, Miihlens, and others, but by Norris, Novy, and others they are still placed with the bacteria. Novy and Knapp have made extensive studies of Sj}. obermeieri (the cause of relapsing fever in Europe) as well as of 8}}. diittoni (the cause of tick fever), spirochetes from American relapsing fever, and Sp. gaUinarum (fowl spirochete) and consider that they have demonstrated their bacterial nature and that many, if not all, spirochetes should be placed in this group. Fig. 17S Fig. 179 Photograph of Sp. ohermcicri showing Spirochi'ta ohcnntieri blood-smear, terminal flagellum. X 3000. (After Fuchsin. X 1000 diam. (From No"\'y.) Itzerott and Niemann.) Spirocheta (spirillum) obermeieri was first observed by Obermeier in 1873 in the blood of persons suffering from relapsing fever. It was found in large numbers during the height of the fever, it disappeared about the time of the crisis, and reappeared during the relapses. It was not found in other diseases. Obermeier considered it the cause of the disease, and his views were shown to be correct by the production of the disease in man and ape through experimental inoculation. Morphology. — The organisms are long, slender, flexible, spiral or wavy filaments, with pointed ends, from 1(3m to 40^ in length and from one-quarter to one-third the thickness of the cholera spirillum {'^p. to f;u). They stain somewhat faintly with watery solutions of the basic aniline dyes, better with LofBer's or Kiihne's methylene-blue solu- BIOLOGIC CHARACTERS 525 tions, or with carbol-fuehsin ; best with the Romanowsky inrtliod or its mollifications. They are negative to Gram. Novy has demonstrated a terminal tlagelhim (Fig. 17S). There are three to twelve wide, more or less irregular spirals. Biologic Characters. — In fresh preparations from the blood the spiro- chetes exhibit active progressive movements, accompanied by very rai)id rotation in the long axis of the spiral filaments or by undulat- ing rao^•ements. They are found only in the blood or blood-organs, never in the secretions, and only during the fever, not in the intermis- sions, or at most singh' at the beginning of, or for a short time after, an attack. When kept in blood-serum, or a 0.6 per cent, solution of sodium chloride, they continue to exhibit active movements for a considerable time. They may be preserved alive and active for many days in sealed tubes. They are killed quickly at 60° C, but they remain alive for some time at 0° C. Unsuccessful efforts to cultivate them in artificial culture media have been made from time to time. Koch has observed an increase in the length of the spirilla and the formation of a tangled mass of filaments. Novy finally succeeded in cultivating them in celloidin capsules placed in the peritoneum of rats. His culture remained virulent (with a slight loss) for many generations. Pathogenesis. — In man, whether the disease is acquired naturally or by artificial inoculation, the organism causes the following symptoms: After a short jjcriod of incubation the temperature rises rapidly, re- mains high for five to seven days, and then returns to normal by crisis. About seven days later there is another sudden rise of temperature, but this time the crisis occurs sooner. A second or third relapse may occur. The organisms increase in numbers rapidly in the l)lood from the beginning of the fever, large numbers often being found in every microscopic field. They began to disappear a short time before the crisis, and immediately after the crisis it is practically impossible to find them in the circulating blood. The mortality varies in different epidemics from 2 to 10 per cent. When monkeys are inoculated with human blood containing the spirilla, they become sick about three anfl a half days later, but show only the initial febrile attack or, at the most, an occasional short relapse. The organisms are found to have the same relation to the pyrexial periods as in man. Blood from one animal taken during the fever induces a similar febrile paroxysm when inoc- ulated into another animal. Metchnikoff showed that during the intermissions when the spirilla disappeared froin the circulating blood they accumulated in the spleen and were ingested in large lumibers by certain jjhagocytes and finally were destroyed. According to Lamb, a certain amount of immunity is conferred upon monkeys (Macacus radiatus) soon after an attack, Init it disappears quickly. If the serum is removed during this time it is found to ha^'c some protective action when mixed with the blood-containing spirilla, and also to cause agglutination of the organisms. Xo^'y (1906) showed 52(1 PATHOGENIC MICROORGANISMS that a powerful specific germicidal body exists in the blood of rats during- and after recovery, notably in the blood of hyperimmunized rats. An immunizing body probably distinct from this is also present. He also showed that passive immunity can be imparted by injections of recovered or hyperimmunized blood, that both active and passive immunity may last for months, and that the serum has both a prevent- ive and a curative action. Agglutinins are also present in such a serum. Breinl has shown that the immunity produced by Sp. ohermeieri does not protect against Sp. duitoni, and vice versa. Neither does it protect against the Asiatic or American variety. The strain found in Bombay seems to be more virulent than that in Europe. Spirocheta Duttoni. — The organism shown by Button (1905) to be the cause of African tick fever is very similar morphologically to Sp. ohermeieri, but Novy, Frankel, and others have shown slight differ- ences which make them belie^'e that it is another variety, if not another species of this group.. Button demonstrated that this organism can be transferred to monkeys by the bites of young ticks {Ornithoclonis mouhata) at their first feed after hatching from infected parents. He accidentally demonstrated the fact that the disease can be inoculated into human beings through a cut surface, for after a M'Oimd received at autopsy he developefl the disease which eventually caused his death. Koch corroborated the author's results. Leishman demonstrated that the second generation of ticks may also infect. He was unable to find spirochetes in tissues of ticks later than ten days after feeding, though young ticks from eggs hatched after this time were infective. He found, however, granules which he considers changed spirochetes. Hindle agrees with him. The fact that ticks may be infective long after the disappearance of typical spirochetes, if they are placed at 30° to 3.5° C. is e\'idence of the infectious nature of these granules. Moller found that a tick may remain infective for a >ear and a half or more after its initial feed from an infected host. Moller also showed that the third generation of ticks might inherit infection. Sargeant and Foley and later Balfour have observed another type of relapsing fever in Africa, which they consider due to a different variety of spirochete (iS'/J. berbera). The filtrability of Sp. duttoni is mentioned on page 492. Spirocheta Carteri. — This spirochete was described by Carter in 1S77 as causing relapsing fever in Bombay. Monkeys were inoculated by Carter successfully with the hiunan blood containing this spirochete. Spirochetes from Relapsing Fever in America. — Recently Barling has reported a study of the relapsing fever of Panama. He isolated the organisms in two cases and studied their characteristics. He finds they agree with those reported by Carlisle, Norris, and Novy for the organisms isolated by Norris, but they can only be dift'er- entiated from the other relapsing fever spirochetes by animal inocu- lations and by the disease in humans. Moreover, he finds that in all ])robability a polyvalent serum ma\' be necessary for cure, since the serum from one strain did not protect against the other strain. INSECT CARRIERS 527 Insect Carriers. — Tho spirochetes are comeyed hy tlic bites of artliro- pods. Many studies have been made on tliis sul)jeet since the worlv of Marchoux and Sahmbeni on fowl spirochetes, and of Dutton and Todd on African tick fe\er. Nuttall and his associates have added much to our l^nowledge of ticks and other artliropods as vectors. Tliat the bed-bug may occasionally be a conveyer was shown by Mackie and corroborated by Nuttall. Mackie later obtained stronger evidence that the body louse is the chief carrier. His observations have been corroborated by a nmiiber of observers. Nicolle and his colleagues found after twenty-four-hour spirochetes disappear from gut of the louse (7-". i^estiDiente and P. capitis) to reappear in from eight to twelve days and continue for eleven days and possibly longer. INIonkeys inoculated with the contents of lice fifteen days after feeding, develop relapsing fever. Reference.s. BertarelH. Ccntralbl. t. Bakt., 1906-1907, xli, 320, (i:«); xliii, 167, 2.38. Calkins. Jour. Infect. Diseases, 1907, iv. Castellani. .Tour. Hygiene, 1907, "S'ii, .558. Darling. Arch, of Int. Med., 1909, iv, 150. Ewuig. New York State .Jour. Med., 1907, vii, 177. (With good Inljliography.) Flexner. Medical News, 1905, Ixxxvii, 1105. Hoffmann. Ztschr. f. Hygiene, 1911, Ixviii, 27. Levadiii and Mclnlosh, Ann. Inst. Past., 1907, p. 785. Levadili and Naltan-Larrier. Ann. Inst. Past., 1908, xxii, 260. Hindle. Parasitology, 1911, iv, 133 and 183. Leishman. Jour. R. O. M. C, 1909, xii, 123, and Lancet, 1910, i. 1. Koch. Deut. med. Woch., 1905, and Berl. kUn. Woch., 1906. Muhlens. Zeitschr. f. Hygiene, etc., 1907, vii, 405; Klin. Yahrb., 1910, 339; Kolle und W^assermann, 1913, Sec. Ed., Jena. Nichols. Jour. Exp, Med., 1910, xii, 616 and 1911, xiv, 196. Nichols and Fordyce. Jour. Amer. Med. Assoc, 1910, Iv, 1171. Noguchi. Jour. Exp. Med., 1909, xi, 84 and 392; Ibid., 1911 and 1912. Norris, Pappenheimer and Flournoy. Jour. Infect. Diseases, 1906, p. 527. Novy and Knapp. Jour. Infect. Diseases, 1906, iii, 291 Nuttall. Parasit., 1913, v, 262. Perrin. Arch. f. Protist., 1906, vii, 131. Schaudinn u. Hoffman, Arbeit a. d. Kaiserl. Gesundh., 1905, xxii. Schercschewsky. Ccntralbl. f. Bakt., e e. Orig. Abt. I, 1908, xlv, 91. Sobernheim. In KoUe u. Wassermann, 1913, See. Ed., Jeua. Swifl. The .Jour, of Cutaneous Dis., 1909, July, and the Arch, of In(. Med., 1909, iv, 376 and 494. Uhlenhuth and Muhcr. Berliner klin. Woch., 1910-1911. CHAPTER XL. BODO. POLYMASTIGIDA. BODO LACERT^ffi (GRASSI). Bodo lacertse is frequently found in the intestinal contents of most of the higher animals, hence it is easily obtained for class study. A species of the Bodo has been observed in human urine (Bodo urin- arius), but it is probably a harmless invader. Bodo lacertce is wedge-shaped, the posterior part of the body being turned a half to a whole spiral on itself. It possesses two character- istic flagella, equal in thickness but unequal in length. In motion the longer one is directed forward, while the shorter is carried backward, functioning as a rudder, or a towing flagellum. Both flagella spring from basal granules which are well demonstrated by the iron hema- toxylin stain. They are situated in the extreme anterior part of the body and are attached to the nucleus by a delicate fibril (Fig. 180, 6, c, and Plate IV, Fig. i, 3). The movement of the organism is character istic, it consists in a rapid irregular swimming in various directions with the anterior flagellum moving from side to side. The body itself shows a slightly sinuous motion. There are two types of nuclei seen. First, the typical ^'esicula^ nucleus most frequently seen among the flagellates. This is round anfl has a definite membrane about which chromatin is arranged in irregular masses. In the centre, or eccentrically placed, is a compact karyosome. Iron hematoxylin preparations bring out an achromatic network between the chromatin masses and the karyosome. In the living condition the nucleus appears as a greenish glistening refractive vesicle. The second type of nuclear apparatus is seen in smaller organisms. This is a similar nucleus except that it is smaller and more compact; posterior to this is another nuclear-like body, varying much in shape and arrangement of chromatin (Fig. ISO, c). This is the sexual chromidia. The cytoplasm appears in iron hematoxylin stained specimens as finely reticular. It contains many deeply stained granules. There is no mouth opening. Food is taken in by osmosis. In propagation, the two types just described develop dift'erently. 'i'he first or orilinary type forms round di\'ision cysts. The flagella (hsappear and a delicate cyst membrane is formed. The increase in the size of the luicleus and the subsequent division may be followed in fife. It lasts about twenty minutes. After a single or, more seldom, BODO LACEHT.K 529 a double (Hvision oF the cell, the daughter cells, while still within the eyst, form their Hagella, become very motile, hiuilly break the cyst w<\\\ and swim out. The second type ^increases, in the free living condition by longi- tudmal division. The basal granules (li\i(le, the principal nucleus divides by mitotic dix'ision, the chromidia by aniitosis. This all can be seen_ in hematoxylin preparations. Sexual di\-ision in this species occurs in cysts by autogamy. It is not easily followed in life be- cause of the high refraction of the cyst. The changes must therefore be studied in specimens stained with iron hematoxylin. Fig. ISO Bndo UfccrUe (Grass!) : a Viyiuii,; b and c stained; 6 type without, c type with chromidium (1000 : 1). (After v. Prowazek, from Kisskalt and Hartniann.) They are shortly as follows: The nucleus becomes larger and about its membrane appear small spheres of chromatin which finally leave the nucleus and gather together, forming the so-called chromidial or sexual nucleus, while the original or somatic nucleus gradually degene- rates. The new nucleus divides amitotically into two daughter nuclei; from these two smaller jjarts are then separated, as reduction nuclei, which also degenerate. The remaining parts of the two nuclei increase in size and then fuse to form a new nucleus. The organism may then leave the cyst or the cyst may become a lasting cyst and serve to infect a, new host. Besides this method of fructification by autogamy in a cyst, is seen, 34 530 PATHOGENIC MICROORGANISMS though seldom, a copulation between two individuals of different sizes ■ which afterward become encysted and divide into two to sixteen daughter flagellates. POLYMASTIGIDA. The order polymastigida consists of flagellates having several flagefla projecting from different parts of the body. The majority of the forms known are parasitic in certain fish. Trichomonas Vaginalis.— Donne in 1837 described a form which he found in the human vagina, and which he therefore called Tricho- monas vaginalis. It has been found by other observers to be a frequent habitant of the vagina at all ages. It is also found occa- sionally in the acid urine of both sexes, the mode of infection of the female is unkno^vn. The body of the parasite at rest is pear-shaped, but during action its ameboid movements cause it to assume various shapes. The size varies from 12m to 25m long and 8m to 15m wide. Fig. 181 Fig. 182 Tricho)ito7ias vaghialis. {Blochniami.} Lainblia uitestinalis. (Schewiakoff.) The protoplasm is finely granular, excepting for two rows of larger granules which begin on either side of the nucleus and converge pos- teriorly. From the anterior part project three to four flagella, which seem to begin at a basal thickening near to, or connected with, the more or less oval, indistinctly ^'esicular nucleus. From the origin of the flagella an undulating membrane extends backward. The body also seems to possess a certain linear structure connected with the membrane. Contractile vacuoles have not been seen. Trichomonas hominis Davaine. — This form, found frequently in the human alimentary canal, is very similar to the Trichomonas vaginalis, but it is smaller and more pear-shaped. It has been found often in acute diarrheas, but no causal relation between it and the pathologic process has been shown. A similar form has been seen a few times in lung gangrene, aspira- tion pneumonia, and bronchiectasis. Lamblia intestinalis (Lambl, ]S59), a flagellate belonging to this group, parasitic in the small intestines of mice, rats, rabbits, dogs, cats, and sheep, has also been found occasionally in the human in- LAMBLIA INTESTINALIS 531 testines. It is beet-shaped, bilaterally symmetrical, ](V to 'Zip. long and 5m to 12/i wide, possessing fiagella 9/x to 14^ long. Anteriorly, this species has a characteristic concavity, the rim of which seems to be contractile, forming a sucking apparatus. The eight flagella of the organism are arranged in pairs: one anteriorly, two laterally, and one posteriorly. The nucleus is situated anteriorly and has a central constriction. The protoplasm of the body is thick and hya- line. Contractile \'acuoles ha\-e not been seen. Schaudinn (1900) observed encystment, copulation, and complicated nuclear changes in this organism. Infection follows the ingestion of the cj^sts with unclean food. The parasites fasten themseh'cs to the free surfaces of the ejjithelial cells by their sucking apparatus, but seem to exert no harmful nflu- ence on their hosts. They have been found most frecjuently in poor children who play often in dirt containing the cysts. Repeated small doses of calomel will cause their disappearance from the feces. CHAPTER XLI. AMEBA. Several authors have reported the finding of ameba? in man, especially in so-called tropical, ulcerative, or amebic dysentery, but as the first descriptions were incomplete and the laws of nomenclature were not strictly foHowed there resulted many synonyms for the same species and many species bearing the same name. At present the four that were considered as distinct species have now become three according to some authors, and only two of those de- scribed, from a practical stand-point, need be considered. These are Entameba histolytica, the form described b\' Schaudinn from tropical dysentery and considered by him the cause of that disease, and Entameba coli, the kind found in normal human intestines by Schaudinn and thought by him to be harmless. The discussion in regard to the "parasitic" and the "non-parasitic" forms is still going on. Some investigators say that certain ameba" are strict parasites of the intestinal tract of mammals; that is, that these ameba? cannot be culti- vated on artificial culture media, and that they are so different from the "sapro- phitic" forms, i.e., those that are cultivated (sic), that they must constitute a different genus. Williams, Calkins, and others, on the contrary, believe that not enough stud}^ on the "cultural forms" has been done to draw definite conclusions as to their relationship to those seen in preparations from amebic cb^sentery, Historical Note. — Stiles has given a detailed history of the generic name Ameba and of the specific one Ameba proteus, and, finall)^, of the naming of the intestinal amebae. He shows why the name Entameba should be given to the genus described by Lambl and Losch. This article illustrates very forcibly the afisurdity of liringing forth new names for organisms only half studied and of claiming that such organisms belong to new genera. The first report on intestinal ameba? of man was made by Lambl in 1860 who announced the presence of ameboid forms in the intestinal mucus of a child who had died from enteritis. Supposedly the same forms were more fully described by Losch in 1875 under the name Ameba coli; Losch found his organ- isms in stools of a patient suffering from chronic dysentery and he succeeded bj' rectal injections in producing superficial ulceration in the large intestines of dogs. He therefore claimed that this organism is the cause of dysentery. His work was corroborated by many observers. In the meantime, ameba? were found in diseases other than dysentery, and Grassi in 187!) reported them in healthy intestines. The work of Kartulis (1886), however, helped largely to establish the fact that amelw' ]ilay an important part in the etiology of dysentery in Egypt. He was the first to find the organism in abscess of the liver in tropical dysentery. In our own country among the most important workers in this field are Councilman and Lafleur (1891). They conclude that amebic dysentery should be regarded etiologically, clinically, and anatomically as a distinct disease. They disappi'(i\(', however, of the name Ameba coli aiid propose the name Ameba dy. tenter iie for the pathogenic form; but as they do not show in any way Ihan by its pathogenesis that the species they describe is a new one, their name. .S/r7?,S OF AMF.B.'E JN THE HVMAN BODY 533 acconliiii!; to tlie rules of zo()l(i)j;ical noiueiiclalurc, cniiiHil lir acccplcd. Ilmris' work, loo, is imporlant in slunving an ctiologic ri'lalionsliii) liclwccn aniiiia' and a C'crlain form of (lysentcry, Imt nintli(>r did he describe (lie nior]>liolo,u;y of his organism minutely enough to identily it with Hehaudinn's histolvtiea which is described below. C'asagrandi and Barbagallo in 1S!»7 were the Ilrst to claim that the amcba^ so far described in man show differences enough from the fresh-water ameba^ to belong to a new genus. They therefore created the genus Entameha and ga^'e the specific name Eiitanielxi hominis to ameba; of the Amcha coli type. Schaudinn and Stiles agree with them as to the generic name, Ijut consider that the correct sjxx'ific name is complicated by the fact that there are different sjjccies in this group. Many observers (Kartulis, Councilman and Lafleur, Quincke and Roos, Kruse and Pasquale), have con- sidered that there are different varieties in the human intestines, but they ha-^-e giA'en no morphologic differences ilistinct enough to classify such varieties. Schaudinn is the first who bases upon a definite moi-phology his claim (1903) that at least one species among them is pathogenic and one non-pathogenic. The latter, which he found in normal human intestines, he says resembles those already described as Arncba coli; therefore he gives it the name Entameha coli; while the former, which he found exclusively in ulcerative tropical dysentery, he calls Entameha hixtolytica. The different \'iews upon the relationship to disease of ameba? found in the human intestines may be summarized as follows: 1 . That the amebs in man have no ]iathogenic properties, hence are not the cause of amebic d^'sentery. (Cunningham, Grassi, Celli and Fiocca, Casa- grandi and Barfiagallo, and others.) 2. That any intestinal ameba may become pathogenic and cause the specific malady known as amebic dysentery. (Musgrave, Clegg, and others.) 3. That amebiB are able to keep up a preexisting inflammation. This was the original view advanced hy Losch when he described the most commonly cited form, Ameha coli, and several authors have followed Losch in this opinion. 4. That more than one species of amebce are found in man, at least one pathogenic, and one non-pathogenic. (Kartulis, Councilman and Lafleur, Quincke and Roos, Strong, Schaudinn, Craig, and others.) The study of bacillary dysentery bj' Shiga, Kruse, Flexiicr, and others (see under bacillary d^'sentery) has demonstrated that there are at least two forms of dysentery, one produced bj^ amebte and the other Ijy bacilli, and from the work on the former it now seems certain that it is produced bj' a specific form of amebae. Amebae have been reported in teeth cement and in carious teeth as well as in aljscesses of the jaw. Flexner in 1892 described an amebic organism in the latter condition, and considered it identical with the organism described Ijy Losch, Councilman and Lafleur as Ameha coli. In the same year Kartulis described similar organisms found in similar lesions. Gross and Sternberg found them in tartar of teeth. Smith has recently found amebte in practically all of a series of cases of Rigg's disease. Prowazek (1904) described a mouth ameba as a distinct morphologic species under the name of Entameha huccalis. Successful cultures have been made of amebae obtained from the intestines of man and other animals, as well as from certain fruits and vegetables. Musgrave and Clegg (1904) studied amebae in the Philippines by the cul- ture method and came to the conclusion that forms obtained from various sources were probablj^ all a single species. Williams (1911) obtained pure cultures on sterile "tissue media'" of amebaj isolated from the intestines of mammals, including one from a case of amebic dJ^senterJ^ Sites of Amebae in the Human Body. — Intestines and neighborino; tissues; abdominal cavity; abscess of liver, lung, pleura, brain, and mouth; necrosis of jaw-bone; urine; tartar of teeth. 534 PATHOGENIC MICROORGANISMS Materials and Methods for Study.— Human cases of dysentery showing amehfe in stools is so seldom on hand in the northern part of this country that they cannot be counted on. The nou-pathogenic form in human intestmes nught be obtained after administration of a saline cathartic, but generally one must depend upon saprophytic forms fo)' work with students, or upon cultures ob- tained from cases of amebic dysentery. Material rich in saprophytic forms may be obtained from an infusion in water of lettuce, cabbage, potato skms, or other vegetable material. Such an infusion should be made a week or two before it is needed, when it will be found that the pellicle which forms contains many varieties of protozoa and bacteria, among which are generally large numbers of ameboid forms. Often one may get good material from the feces of many of the lower animals, such as the lizard, toad, or guinea-pig. If one has material containing human intestinal amebfp, kittens or puppies may be fed with the cysts in order to obtain a new supply. The amebaj should be examined in both the fresh and fixed condition. Cultures may also be made as described below. Examination of the Fresh Material.— The study of the living amebaj is ex- tremely important. This may be done by making a hanging drop or hanging mass (p. 72) from fluid containing ameba?. The size, kind of motion, frequency of pulsation of contractile vacuole, and as much of the cell contents as possible should be noted. Tlie stools should be examined on the warm stage as soon as possible after their passage (not later than two hours), and should be kept at blood heat until examined. A platinum loopful of material should be taken from the shmy masses in the thinner part of the feces, diluted with physiologic salt solution, covered with a cover-glass, and examined under moderate magnification. Harris found that a drop of a watery solution of toluidine blue added to a small particle of the feces stains the entoplasm of the amebffi at once and the ectoplasm a few minutes later. The amebse seem to be ciuickly killed and often when natural forms are beautifully preserved the coverslips, after being washed in water and mounted in Tarrant's medium, may be preserved for months, but after a time the stain completelj' fades. Permanent Preparations. — Thin films are made on glass slides or cover- glasses, and immediately, before they are allowed to dry, they are placed in the fixing solution. Cover-glasses may float, film dowai, on the surface of the fixative. Among the liest fixatives are: Hot subhmate alcohol (50° C), Zenker's fluid, hot Hermann's fluid, or methyl alcohol (p. 84). Stains. — Among the many good stainmg methods the following may be mentioned : 1 . Thin Delafield's hematoxjdin, from one-half hour to several hours, then washed in water. (If overstained, the preparation may be differentiated in acid alcohol, controlling under the microscope, then washed in water.) The film or section is then passed successively through 70 to 95 per cent, and 100 per cent, alcohol, absolute alcohol -|- xjdol, xylol, cedar oil, or Canada balsam. 2. Heidcnhain's iron hematoxylin (see p. 84). The smear is put from dis- tilled water'into the iron-alum mordant for 4 to 12 hours, or overnight; well washed in distilled water; in stain from 2 to 24 hours, excess washed out in the iron mordant, controlled under the microscope (as decolorization occurs very ciuickly) until the nucleus is sharply differentiated; the chromatin of the nucleus must be a deep blue-black, and the cytoplasm a light gray; then a thorough washing in tap water and passage through the alcohols and xylol, and in Canada balsam, or cedar oil for mounting. .3. After fixation in methyl alcohol one may use Gicmsa's staining method (see p. 81), or a modification of the method suggested Ijy Van Gieson for staining the Negri bodies in smears (see p. 83). Masses containing amebas, as mucous flakes or portions of the intestinal or liver abscess wall in amebic dysentery, or pieces of decaying vegetables may be fixed in Mo in hot sublimate alcohol for one-half hour, washed in iodin alcohol for 24 hours, passed through the different strength alcohols and em- bedded in paraffin (see p. 85) for section-cutting if desired. hfORPHOLOGY 535 iM:illoiy and Wriglit rcetiiiimciul (lie foUowiiis iiielJiiKl for Iksucw; 1. Harden in alcohol. 2. Stain soclions in a .saiuraU^d aqueous solution of t liionin three to five minutes. :■! . nilferentiate in a 2 ])er cent. a(iU('ons solution of oxahc acid for one-half to one minute. 4. Wash in water. 5. Dehydrate in alcohol. 5. Clear in oleum origani cretici. 7. \\'ash off with xylol. S. Xjdol balsam. JNfallory's eoshi methylenc-blue method is also very good for sections (p. 83). Cultuies of Amebae. — I'urc mixed cultures may be made in the following way: From the material containing amebw a small loojjful is removed with a platinum wire and isolated spots are touched over tlie surface of the follomng media poured in sterile Petri dishes: Agar f .0, tap water 90.0, ordinary nutrient broth 10.0, mixed and sterilized in the regular way. It should Ije slightly allvaUne to phen- olphthalein (f per cent.). If necessary, feces contents may be thinned with phy- siologic salt solution Ijefore planting. In one to several days at 25° C. the ameba^ with the accompanying bactei'ia may o^•ergrow the entire "plate. We have found that amebip will grow as well upon nutrient agar — better with certain bacteria — as on the special media just mentioned. Klatsch preparations may be made of these cultures, or small pieces of agar and culture may be embedded entire. From such a culture the "pure mixed" cultures of Frosch may be made as follows: The ameba? which have crept out to the periphery of the growth are taken out with their accompanying bacteria and transplanted. UsuaUy one or two organisms favorable for the growth of the amebiie accompany them and in this way one may finally get the amebae growing with one definite bacterium. We have isolated from a culture a single ameba unaccompanied by bacteria by the fcllowing simple method: Under the low-power lens with a fine platinum loop an isolated ameba is draw-n to the edge of the agar plate. When it is well sepa- rated a disk of agar containing it is cut out following the margin of the objective and is transferred to a fresh agar plate. A very small quantity of a desired bact- erum is now added to the disk near the ameba, and a "pure mixed" culture results. Pure Cultures. — Certain varieties of amebse from the intestines of mammals grow without other organisms, i. e., grow in pure cultures, when inoculated on a piece of fresh sterile brain, kidney or liver placed upon Musgrav'e and Clegg's agar or on nutrient agar. They grow abun- dantly on such media at temperatures varying from 22° C. to 38° C. (Williams.) Morphology. — The morphologic characteristics of the ameboid stage, as described by various observers, seem not to have been minutely enough studied to be depended upon in differentiating the species. Moreover, descriptions have differed markedly. While Schaudinn and others, especially Craig of the more recent writers, say that it is easy to differentiate between the ameboid forms of histolytica and coli, Musgrave and Clegg, Strong, Williams and others say the points of difference are not marked The observations of Schaudinn and others may be summarized as follows: (1) Ent. coli is, on the whole, smaller than Ent. histolytica; (2) its ectoplasm is so small in amount and so slightly differentiated that it is only seen when the organism puts forth pseudopods, while the cortical zone of the Ent. histolytica is wider and is distinctly differ- entiated from the entoplasm; (3) the pseudopods of the former are small rounded delicate, and not highly refractive, those of the latter are larger, finger-shaped, firmer, and more highly refractive, thus indicating 5.3fi PATHOGENIC MICROORGANISMS the ])()\ver of the (ir- Schaudinn that Ent. histolytica during the vegetative .stage may multiply by budding as well as by binary fission, is now considered incorrect. Unequal division, however, probably frequently occurs. It is now thought by Walker, Hartmann, Craig, and others that Ent. PATHOGENICITY 537 tetra(jtii(i. (IcscrilK'd liy Morcck, Ilartmaini, and ('rai,^■ as a, distinct species, is merely a phase of Knt. /li.sidlj/tini. though Ilartiiiaim calls the species tctrdgcnd and drops the name lii.sli)h/tic(i. The observations of Schaudiini on the sexual phenomena of I'hita- meb(F, though earlier corrohorated hy Ilartmann, Craig, and others, are now considered incorrect hy these same obser\'ers. 'I'he subject awaits further in\-estigation. Viability. — The i)athog-enic amebte are apt to lose their motility very quickly alxn-e or below body heat, while the saprophytic forms remain motile at higher or lower degrees. Though the former lose their motility, they are not all killed by cold. They may still be infec- ti\-e after freezing. Musgrave kept an encysted culture from a dysen- teric stool at —12° C. for 45 days and found it still \-iable at the end of that time. A temperature of 60° C. for one hour usually kills encysted cul- tures of amebie, according to Strong, Init considerable variation has been noted in the degree of temperature necessary to destroy differ- ent strains.' Enemata of quinine sulphate and saturated solution of boric acid do not affect ameba^ in the intestinal canal, though a^L^ quinine sulphate added to the stools invariably kills them in ten minutes. They are also destroyed in stools by weak solution of hydrogen dioxide, potassium permanganate, toluidine blue, and dilute acids. Luttle found that TTyu^inr hydrochloric acid and yTj-fy- silver nitrate check motility, but do not destroy parasites except after prolonged contact. ^lusgrave and Clegg found that in cultures treated with 1 : 2500 solution of quinine hydrochlorate the parasite quickly encysts, and in from fi^'e to eight minutes may break up and disappear; ten minutes later cultures made produced no growth of amelia?, while the bacteria grew well. Pathogenicity. — Lower Animals. — Just how pathogenic Ent. coli is for lower animals cannot be determined, as we have before stated, until a more minute study of the intestinal amebfe is made. In regard to amebse from tropical dysentery (presumably Ent. histo- lytica), they have been shown to be pathogenic to young cats, dogs, and monkeys. The infection may take place in two ways: (1) By feeding material containing the cysts; (2) by rectal inoculations of the vege- tative forms. The best work done on dogs is by Harris in 1901, who found that puppies were particularly susceptible after rectal injections of fresh material from human dysentery cases. Morphine was adminis- tered before the injection in order to retard peristalsis. The disease de\'eloped in two or three days and lasted from four to sixteen days. The chief symptoms were a bloody diarrhea and progressive emaciation. The lesions observed in the intestines on postmortem examinations were a swollen and congested mucosa, over whicli were scattered numerous small ulcers. In two cases there were liver abscesses. ' An air-dried agar plate culture of " Amcba coli" given us by Dr. Calkins who obtained it from the Philippines n'as viable after three years at room temperature. 538 PATHOGEN I C MICROORGANISMS Microscopically, tlie mucosa first showed slight exudative and productive iutlammation, followed by necrosis and desquamation of the epithelial cells and their basement membrane. At the same time tlie interglandular tissues beneatli Isecame swollen and small hemorrlragcs occurred. Great mmibcrs of macrophages collected. Ulceration proceeded from above downward. Many amebffi were first seen in and between the epithelial cells, then in the connective tissue at the base or sides of the ulcers. Necrotic and suppurative processes producing varying degrees of suppurative inflammation may compHcate the lesions. The abscesses which form in the liver contain degenerated liver cells, poly- nuclear leukocytes, red-blood cells, and groups of small amebse. As controls Harris tried rectal injections of various bacteria, in- cluding the Shiga bacillus. All gave negative results, however, and he considered that the amebae showed their specific action very plainly. Though he did not describe the morphology of the organism from his cases with enough minuteness to identify it with Schaudinn's histolytica, he gave enough points to make the inference strong that it is the same species. Whether Entameba coli would produce similar dysentery in young dogs is yet to be proved. As stated above, Schau- dinn found that he could produce the typical disease by feeding young cats with cysts of Ent. histolytica, but could not get the same results by feeding the vegetative forms. Musgrave and Clegg injected "pure mixed cultures" of material from cases of clinical amebic dysentery as well as similar cultures of amebae from various sources into monkeys and produced dysen- tery. Musgrave fed monkeys with encysted amebte in bacterial cul- tures and obtained, in a small percentage of the cases, dysenteric stools and ulcerations in which amebse were found without their accompanying bacteria. Kartulis, Kruse and Pasquale, and Strong injected into the rectum the contents of liver abscesses containing apparently only the amebae and produced typical dj'sentery, with lesions similar to those seen in man. Strong states that the lower monkeys and the orang-outang in the Philippines contract the disease naturally. In Man. — According to Craig, about 50 per cent, of human beings harbor harmless amebffi in their intestines. Schaudinn states that he found this form of ameba in one-half the cases examined in East Prussia, one-fifth of those in Berlin, and 25G times in 385 examinations in Austria. Recently Walker and Sellards have carried on an extensive study of the pathogenicity of the different forms in human beings— sixty Philippine prisoners — which they divided into three groups : 1 . Twenty men fed with cultures of amebae isolated from stools or Manilla water. The same ameba was recovered in cultures from several of the cases. No lasting parasitism occurred, neither did any case of dysentery result. 2. Twenty men were fed stools containing Ent. coli. No cultures were recovered; no cases of dysentery occurred, but 17 cases were parasitized. 3 . Twenty men were fed stools containing Ent. histolytica. No cultures were recovered; four cases of dysentery occurred, and 17 cases were parasitized. TISSUE CHANGES 539 They coiichulo from these studies that they have funiished Uu-. proof of tlio pathogeiucity of one form (Eiit. histolytica), the iioii-i>athogeiiic hut parasitic iiattire of the otlier form {Ent. coli), and tlie saprophitic nature of cuhuraf forms. Tliese exiicrmients are open to a number of criticisms, ciiief of wliicli are: Probable immunity of the men, the use of only Musgrave and Clcgg's culture medium for their cultures, and their cultivation only at room temperature. The disease produced liy patliogenic amehie in man is known as amebic dt/scntcri/ (amebic colitis, amebic enteritis, amebiasis entam- ebiasis). Incidence. — The disease occurs endemically in tropical countries. It is particularly prevalent in Egypt, India, and the Philippine Islands. It occurs frequently in parts of South America and southern United States. In northern United States few cases are reported, though Patterson, who in 1909 described three cases (without a description of the ameba? present), and who calls attention to fifteen cases reported as endemic in New York City, since 1S93, states that this disease is probably more widespread than is generally thought, and that if it were searched for more carefully more cases would be recognized. Patterson adds to his report a bibliography of cases reported as orig- inating in North America. Sporadic cases are found in Russia, Ger- many, Austria, Italy, and Greece. An occasional small epidemic may occur in the milder climates. Where it is endemic, the largest number of cases occur after the heavy rains have begun in early summer. Males are more frequently attacked, because more exposes o a ; -g "Eli o S R M-: 1 O 03 m a a 9 ! s S °* ( o O 3 ^ 2 o) 0) O C 3 1 ?'SS -S SfS-g t. .2^- • Regul shap 6 to age pher latio f= -r- 3 hl\ £ 3 S S a ^ -S a "6 =* ■?9 . nj Within hours. Collec zone phery Coarse' - +i «- n S S.£ .23+^0 m >S. 5 C3 o 4,53 '''■rt .1-, ■!> 2 g ^ ftr. ^ O « ft O g 3 E iZ " t^ 'fl CM tc'rt ^ " Small othe very -} dia ^^0 -autum- 1 (perni- :)us) fever pe. Kp H ■» t- I 552 PATHOCIENIC MICROORGANISMS as the achromatic zone, and of a basic (blue) periphery, the body, including a metachromatically stained, rounded, compact (red) chromatin mass, the nucleus, which tends to give the parasite the form of a .signet ring. Later stages up to a certain number of hours show simply changes in size and outline of the body. The nucleus then divides by simple mitosis. Later it breaks up by amitotic division into an increasing number of small masses. By the time the chromatin division is completed the chromatin masses will have assumed a rounded form, and will be seen to exhibit ultimately the same strong affinity for certain dyes which is seen in the compact chromatin body of the young ring- like form. At this stage the heretofore scattered pigment appears in one clump. Good technique will always show a corpuscular remnant even at this time. The achromatic zone mentioned will be seen to develop with the chromatin, and when the next step, namely, the division of the body of the parasite, is seen to be completed, there will be as many achromatic bodies as there are chromatin bodies, each division having a share of the basic mother-body, each representing the young parasite (merozoite). A certain number of the full-grown parasites do not segment and these are the forms which commence the life cycle in the mosquito. These forms grow to produce the sexual forms, the macrogametocj'te, or female organism, and the microgametocyte, or male organism. When mature these forms are generally larger than the mature schizont of the same species, the female organism being usually larger than the male and containing more food granules and a smaller nucleus. Li the estivo-autumnal forms they are crescentic in shape, while in the other species they are spherical. In the circulating blood of human beings they show no further changes except to become freed from the corpuscle; but when the blood containing them is withdrawn and exposed for a short time to the air, an interesting series of changes in the microgametocyte is observed. The crescentic bodies are transformed into spherical bodies; the pigment of the microgameto- cj'tes becomes actively motile, due to internal agitation of the chro- matin fibrils, which presently emerge as flagella-like appendages. Their movements are very rapid, causing corpuscles to be knocked about, and finally they become detached as the microgametes, or male elements, and go in search of the female element. Li birds, one may actually observe the process of conjugation in slide preparations even without the aid of a moist chamber and heat. This transformation of male bodies never occurs in the human blood. It Mall be seen that it belongs to the sexual cycle which occurs in the stomach of the mosquito. The Sexual Cycle (Sporogony) Developing in the Mosquito.— The connnon mosquito, often daj'-flying, belongs to the genus Cvlcx; it cannot carry human malaria. It is easily distinguished from its night-flying or dusk-flying relatives. Anopheles (the malarial carrying mosquitoes comprise about eight genera of the subfamily anophelinie). THE SEXUAL CYCLE DEVELOP/NO fN THE MOSQUITO 553 l)y its assuming a ditt'orent posture on the ])erpen(licular wall. While the Ciller holds the boily more or less j)arallel with the surface, the body of the AiiDphelcs stands otf at a marked angle. Other diH'erentia! pt)ints are the following (see Figs. 188-197): Wings of Culex are unspotted; those of Aiioplirlcs are spotted (except in one rare species). The proboscis of ^Inophelcs points toward the resting surface, while that of Culex does not do so. Anopheles species bite usually in the early evening, while those of Culex bite almost at any hour of the day. The male moscjuito is readily told from the female by its plumed antenna;, those of the female being inconspicuous. The eggs and the larvis of the two genera are quite distinct as may be readily seen by glancing at Figs. 188 to 197. The anopheles mos- quitoes breed in practically any kind of a collection of water, though some species prefer slow running water to quiet pools. The best known domestic carriers are usually found in barrels and cisterns. If an ordinary mosquito (Culex) is allowed to imbibe the blood of a malarial patient whose blood shows gametocytes there will be simply a digestion of such blood in the mosquito, and no development of the malarial organisms results. If, however, certain species of Anopheles ingest such blood, immediate changes follow. It should be remem- bered that only female mosquitoes are blood sucking; hence, they alone can be responsible for the spreading of the disease. It should also be remembered that if the blood imbibed by the anopheles does not contain gametocytes, though it may contain earlier stages of the malarial organisms, no amount of such blood can cause general in- fection of the mosquito. The sexual cycle is similar in all species of the parasite. The flagellation of the male parasite described above will promptly take place in the stomach of the anopheles, 4 to 8 microgametes being formed; these conjugate with the female element (Plate VII) in a manner comparable to the impregnation of the ovum of higher animals by spermatozoids. The macrogametocyte becomes a macrogamete by the formation of a reduction nucleus which is thrown out of the organism. The product of conjugation, the ookinet (zygote), remains for a number of hours in the juices of the chyme stomach, changing gradually from a spherical, immobile body into an elongated motile wormlet (Plate VII, Sexual Forms, 1 h) . This penetrates the epithelial hning of the stomach and rests in the tunica elastico-muscularis (Plate VII, Sexual Forms, Fig. 2); here it changes into an o^'al then into a round body, which grows in the course of the next few days enor- mously, forming a cyst which projects into the body cavit}'. Meanwhile the chromatin will have become very active. It will have divided into numerous nuclei, which become arranged around inactive portions, and filamentous sporozoites develop from this chromatin and surrounding protoplasm (Plate VII, Sexual Forms, Fig. 2). These sporozoites ulti- . ±\j , X oo ±' Jlj. -103 I'^i.;. 190 Fig. 191 Fig. 192 Fig. 19.3 Fig. 194 Fig. 195 Chief comparative characteristics of Culex and Anopheles. (From KoUe and Hetsoh.) Egg of Culex, Fig. 188, laid together in "small boat," those of Anopheles, Fig. 189, separate and rounded. Larva of C, Fig. 190, hangs nearly at right angles to water surface, those of A., Fig. 191, are parallel to surface. Body of C, Fig. 192, when resting is held parallel to wall in a curved position, that of A., Fig. 193, stands at an angle of about 45° and is straight; wings of C, Fig. 194, are generally not spotted, those of A., Fig. 195, are spotted; in C. the paloae. Fie. 190. of the female are verv short nf tbp mnlo -iro ir„i,T,.^ +T,o.i tl,o TOXIN PRODUCTION 555 niately fill the cysts, wliicli rupture, setting tlicin free into tlie cavity of the mosquito's body; they then are carried by the lymph to all parts of the body of the mosquito antl tluis reach a glandular strTicture in the thoracic caA'ity of the insect, the so-called salivary gland (poison gland), in which they accumulate in large numbers. This gland is in immediate connection with the biting and sucking apparatus. If, now, such an infected mosquito "bites" a human being, the lubricating fluid of the puncturing apparatus will carry sporozoites into the latter's blood and the human cycle begins. The stages of development in the mosquito require from seven to ten daj's, but only when the temperature is favorable. Cultivation. — Bass and Johns announced in 1911 that they had succeeded, by a rather complicated method, in obtaining a certain amount of development of II. Vivax in the test-tube (see p. 10(1 for details of method). Essentially, defibrinated malarial blood is used, to which is added | per cent, dextrose. Bass and Johns state that the layer of serum about the sedimented red-blood cells must not be too deep (5 inch) and that the leukocytes must be removed if more than one generation of the parasite is wished. The Thompsons (1913) state that they have gotten several generations by less attention to these details. They draw the malarial blood into the sterile test-tube (10 c.c.) in which there is a thick wire. One-tenth c.c. of a 50 per cent, aqueous solution of glucose is added before adding blood. The blood is defibrinated bj^ gently stirring the wire for five minutes. The vdie and clot are removed, ancl the blood is poured into smaller tubes, 1 inch in each. Rubber caps are placed over cotton plugs. These tubes are kept at .37 ° to 44° C. The corpuscles settle slowlj^ leaving about one-half inch clear serum. They have found it unnecessary to remove the leuko- cytes by centrifuging. They state that the malarial organisms are not destroyed by the leukocytes in the tube but by the changes in the serum. Their organisms grow through the whole depth of the laj'er of red-blood cells. Effect on Man (Pathogenesis). — As the organism grows at the expense of the red-blood ceUs the principal change is in the blood. Melanemia, or the formation of pigment granules from the destroyed red-blood cells, is one of the most characteristic features of malaria. As the disease progresses the red corpuscles show varjdng changes in form and hemoglobin content, not only the infected corpuscles, but others as well, thus showing that the organism produces either primarily or secondarily some toxic substances. The pig- ment occurs in two forms, melanin and hemosiderm. The second onljr gives the reaction for iron and is found in the internal organs, while the first is found everywhere in the circulating blood. The pigment is taken up by the leuko- cytes. Thei'e is usually a definite reduction of both red- and white-blood cor- puscles, which is more marked in tertian and cjuartan malaria than in estivo- autumnal. There is a relative increase in the number of mononuclear leuko- cytes. The spleen shows marked hyperplastic inflammation and pigmentation. In intense estivo-autumnal cases the capiUaries of brain and other organs may be filled with the parasites. We have observed parasites also in the large nerve cells of the brain. Toxin Production. — The relationship between segmentation and paroxysm is always noted in tertian cases, and it is reasonable to sup- pose that the occurrence of the paroxysm is referable entirely t(j the Description of Plate VIII. 1. Typical young tertian form; the corpuscle shows incipient degeneration; cor- puscle to left above shows a blood-platelet. 2. Abnormal young form, showing small accessory chromatin bodj'. 3. Two parasties; one a normal young form; the second a large form in crenated corpuscle is an unusual abnormal form with very large achromatic area. 4. 5, 6. Estivo-autumnal parasites; single, double, and triple infection; central elongated chromatin bodies. These forms are about the largest usually seen in the peripheral blood; no degeneration of corpuscle. 7. Tertian parasite, about ten hoirrs old; marked degeneration of corpuscle. 8. Double infection of a corpuscle in tertian fever; marked degeneration of corpuscle. 9. 10, 11. Large tertian parasites showing division of chromatin previous to seg- mentation. 12 and 14. Complete segmentation of tertian parasite. 13. Double infection of corpuscle, one parasite reaching maturity, but showing unusually small segments; the second one atrophied. 15. Tertian parasite, old case; while the parasite is only half-grown, the chroma- tin has split into several compact masses. Degeneration of infected corpuscle. 16. Dwarfed tertian parasite, smaller than a red corpuscle, but showing five compact chromatin bodies; resemblance to quartan rosette. 17. Microgametooyte of tertian malaria; prominence of blackish pigment sur- rounding a large achromatic zone in which the microgaraetes lie coiled up. 18. Tertian macrogametocyte. 19 to 23. Crescentic bodies of estivo-autumnal malaria. 19. Typical gametocyte; pigment surrounding achromatic area; no chromatin shown; the "bib" is present. (Male?) 20. Semiovoid gametocyte. (Female?) 21. Pigment removed. Elliptical achromatic area in which the microgametes are seen. 22 and 23. Pigment removed; chromatin more compact; possibly female elements. 24. From a case of pernicious malaria with rich infection; only liyaline forms in peripheral blood. Below, a large blood-platelet. Note. — As the amphlication is not uniform, a comparison of the jiarasites with Ihe blood-corpuscles shown should be made in order to have a correct conception of their size. PLATE VIII _-p- •> c ^^i ^■J*^- a^: /3 /7 !\, ^*i 2/ /(? V / ■%*► /2 m /6 ^ J: 19 20 23 i«4 Photographs of Tertian and Estivo-autumnal Malarial Parasites in Different Stages of Developi-nent. (Goldhorn.) POINTS OF DIAGNOSIS 557 liberation of toxic sulistances resulting from metaliolic activity of the parasitewithin the corpuscle. That there should be a toxic "product seems_ highly probable, and its amoiuit must be considered in heavy infections. Cases showing an infection of 1 to 5 per cent, of all cor- puscles are not infrequent; the destruction of from 50,000 to 200,000 or more corpuscles per cubic millimeter of blood leads to the rapid deglobularization of the blood; hence the deficiency in numbers; add to this the effects of the metabolic products, and little is left to the imagination to explain the pronounced anemia. Immunity from malaria appears to exist as natural and acquired immunity. Prophylaxis.— The fact that, with the extermination of the malarial carrying mosquitoes, malarial fevers in man would be made impossible, remains established; the parasite must have its chance of rejuven- esence in the mosquito's stomach. The various methods of extermination are fully described in books which go minutely into the subject. The method of giving small doses of quinine to human beings exposed to Anopheles, and of thus getting rid of the organism itself within man, should be considered. In hot climates especially, where it is practically impossible totally to destroy the breeding places of the mosquitoes by drainage or oiling, this method is especially serviceable. In these countries, too, the use of adequate screening is of marked value. Points of Diagnosis. — By a study of the circulating parasite the examiner should be able to tell not only the species present, but also the progress the disease is making. Malarial parasites can always readily be found in recent primary infections, and it is usually only in old cases that the searcli becomes difficult; one is, however, generally rewarded by finding them if one looks long enough for them. A helpful sign is the finding of pigment in mononuclear leuko- cytes, which are seen about the time of a chill or of the period symp- tomaticall_y corresponding to it. Free pigment cannot be used as a means of diagnosis, as it may be impossible to tell it from dirt or dust. In a primary infection of lotig standing, the gametocj'tes may be found, and in relapses and in those cases treated by quinine, many atypical forms appear. A small dose of quinine may drive all parasites except the sexual forms out of the peripheral circulation; at all events, the finding of them becomes, in the absence of gametocytes, a matter of time and experience, especially also as they may be much altered in appearance. The part most and first affected is the blue staining body; later follow eccentricities of the chromatin, such as multiple bodies, and dwarfing, just sucli changes as might have occurred in time, if the body had been allowed to combat the parasite without the aid of drugs. In both cases the fe\'er curve becomes atjpical. It should be remembered that there is no quotidian form originating in this country. (Quotidian paroxysms occurring here are cither a double, tertian, or a triple c^uartan infection. The notion tliat the parasites can be found only at the time of the paroxysm is still in the minds of 558 PATHOGENIC MICROORGANISMS many; it is erroneous. The gametocytes are quite resistant to qui- nine and other drugs, and it appears as if cases in which these forms are seen are much more prone to relapse than promptly treated recent primary infections. The macrogametocytes may remain quiescent for years in the blood, and then under certain conditions, probably through parthenogenesis, may again begin to develop and multiply, thus bringing about relapses. In the estivo-autumnal forms the crescentic gametocytes are gener- ally few, but at times large numbers of them develop. Of course, they are absolutely characteristic. The young parasites are more or less characteristic in stained preparations (Plate VII). There may be as many as seven parasites in one corpuscle. Later the few heavy pig- ment granules are characteristic. In fatal cases the formation of crescents may not take place; the blood infection with young parasites is then enormous, every field of the microscope showing numbers of them. In the study of estivo-autumnal fever, as well as in that of the other forms, it is to be remembered that crescents when found indicate that the disease is of some standing, for such sexual forms are not formed until the asexual propagation is waning. The recognition of these ovoidal and crescentic bodies is easy. But as there are no readily discoverable pigmented forms in the peripheral blood in the early stages, it is necessary to be thoroughly familiar with the young estivo- autumnal forms. Polychrome staining for them cannot be too much recommended, as there is little that is characteristic about them when they have been stained with methylene blue alone. Many a serious error has been made by adhering to the antiquated idea that parasites should be looked for in the fresh blood, as these young, non-pigmented, so-called hyaline forms cannot be readily recognized by the inex- perienced, while it is an easy matter to know and classify them when properly stained. The recognition of the quartan parasite in its early stages in the fresh blood is not as difficult as that of the tertian form, because the outline is more distinct; but in stained preparations it is often indis- tinguishable from the latter. The living ameboid young form or schizont is more refractive than the young living tertian schizont, more like the estivo-autumnal form, and it is just as sluggish in its movements. Then, too, the corpuscle is often shrunken and looks as if it contained more hemoglobin than in the case of infection with the tertian parasite. The growing parasite rapidly becomes pigmented, but it shows fewer, larger, less motile pigment granules than the corresponding tertian one; moreover, the ])igment is arranged around the periphery of tlie orgaiiisjn, while in tiic tertian form it is distributed through- out the protoplasm. The quartan parasite is apt to form a band across the infected corpuscle. Segments are few in niunber, as a rule, and the parasite remains dwarfed while the infected red-blood cells are normal BLACKWATER FEVER 55!) The segments are generally arranged symnietrieally around the central piginent, gi\ing the so-ealled daisy or marguerite appear- ance to the parasite at this stage (Plate VII). In tertian fe^'er, the granular degeneration which the infected cor- puscles early undergo is diagnostic. In the first few hours it reseml)les the ordinary granular stroma degeneration with basic affinity, while it is later seen that the affinity of the then more numerous granules is more acid, or, at least, the staining is no longer orthochromatic, the blue being superimposed by a red; in other words, these granules stain later metachromatically. The greater the loss or transformation of the hemoglobin the greater the number of granules. This holds good only for tertian parasites, the estivo-autumnal variety causing practically no appreciable change though the same technique be used. Malarial-like Parasites in Other Animals. — Two genera of protozoa closely related to the malarial organisms have been found in birds: (1) the proteosoma or hemoproteus; (2) the halteridium; both found in owls (Hcevioproteiis noduw Celli and Sanfelice). Points in their life history ha-\'e been brought out by various observers, especially by Ross and by INIacCallum. The complete life cycle of both forms, as worked out by Schaudinn, is considered by him and his followers to be of fundamental importance to the understanding of the rela- tionship of blood parasites. Schaudinn states that these organisms pass through a flagellate stage in the intestinal tract of the common mosquito {Culcx pijnens) which has previously fed on owls infected with the intracellular organisms (halteridium and hemoproteus). Novy considers that this mosquito flagellate stage of Schaudinn is simply a growth of trypanosomes in the mosquito's intestinal tract which are normally found there, and that Scliaudinn did not sufficiently control his work to warrant his conclusions. Malarial-like organisms have been found also in monkeys, cattle, dogs, and frogs, but they have been little studied. An interesting article by Bernberg-Gossler on malarial organisms in monkeys was published in 1909. In it the author describes a binucleate phase of these plasmodia and agrees with Hartmann in his recent classification of these organisms. Blackwater Fever. — This is a condition whicii occurs frequently, especiafly in Europeans in tropical countries. Its etiology has been the subject of much discussion. The chief symptoms are fever, hemoglo- binuria, delirium, and collapse. It frequently ends in coma and death. A few consider it a disease entity, but the majority of observers are inclined to believe it the terminal stage of a severe malarial infection. It is frequently associated with the demonstration of the malarial organism, but they are not always foinid. It may be that the invasion of the ganglion cells of the brain by the malarial organisms are the chief cause of the s,ymptoms, aided in certain cases by the lysemia noted by Christophers and Bentley. 500 PATHOGENIC MICROORGANISMS GENUS BABESIA (PIROPLASMA). It was not until 1888 that there was a hint as to the real nature of the actual cause of "Texas fever" (bovine malaria, tick fever, hemo- globinuria) and allied diseases which attack field cattle in many parts of the world. Then Babes described inclusions in red-blood cells in Roumanian cattle sick with the disease, though he did not decide upon the nature of the organism. No new studies were reported until 1893, when Theobald Smith and Kilborne gave such a complete description of this disease and its cause as occurring in Texas cattle that little concerning it has since been discovered. These authors describe as the cause of Texas fever, pigment-free ameboid parasites appearing in various forms within the red-blood cells of infected animals. The organisms may be irregularly round and lie singly or they may be in pear-shaped twos, united by a fine line of protoplasm. Because of these double pear-shaped forms Smith and Kilborne named the organism Pyrosoma bigeminvm^ and they placed it pro- visionally among the hemosporidia. These authors also showed that the contagion '\^'as carried by a tick (see p. 560). Their work has been corroborated by many investigators in different parts of the world. Hartmann places this genus in his new order Binucleata, and he considers it an important form for showing the relationship of the endocellular blood-parasites to the flagellates. Schaudinn, in 1904, was the first to call attention to the occurrence of nuclear dimorphism in B. canis and bovis, and Luhe, Nuttall and Graham-Smith, Breinl and H indie, and others have confirmed this observation. The second nuclear mass is generally in the form of a small granule similar to the blepharoplast of undoubted flagellates. Morphology of the Parasite. — In the examination under 1000 diameters of fresh blood of sick cattle, according to Smith and Kilborne, are seen, in the red blood cejls, double pear-shaped forms and single rounded or more or less irregular forms. The size \'aries, though generally it is the same among the bodies in the same red-blood cell. The average size is 2/j, to 4/x long and l^iU to 2/j wide. The pointed ends of the double form are in apposition and generally touch, though in unstained specimens a connection between them cannot be seen. The axis forms either a straight line or an angle. The protoplasm has a pale, non-granular appearance, and is sharply separated from the protoplasm of the including red-blood cell. The small forms are generally fully homogenous, whereas the larger ones often contain in the rounded ends a large rounded body, O.V to 0.2;u in size, which is very glistening and takes a. darker stain. Within the largest forms in the centre of the ' Tin' generic iiMiiu; Pyrua utu, already in use for a well-known Ascictian genus, was altered to Piroplasnia by Patton in 1895. In the meantime Starcovici (1893) had given I he name Babesia bovis to tlic form described by Baizes; and as this form seems to be iden- tical witli that described by Smith and Kilborne the correct name of the genus should be Babesia, while the species parasitic in cattle should be called Babesia bigcmina. GENUS BABESIA ^Cil thick end is a lar.y,e round or oval body, 0.5^ to V, wliicli sometimes shows amelioid motions. Piana and Galli-Valerio (1895 and 189G) and other observers have since described definite amel)oid motion of the whole parasite. The motion of the whole parasite on the warm stage is not produced by the formation of distinct pseudopodia, but by a constant change of the boundary. The changes can succeed each other so quickly that it is scarcely possil)le to follow them with the eye. The motion may persist for hours. The single ones show motion, while the double ones remain unchanged. The parasites take most basic aniline stains well. The Romanow^sky method or its modifications gives the best results (Plate IV, Fig. iii, b). Stained by this method, the smallest forms appear as tiny rings, about one-sixth the diameter of the red-blood cell. A part of the rim takes the red nuclear stain, the rest is blue. In the large mature pear-shaj^ed organisms a loose mass of chromatin is at the rounded end and a dense, compact mass is situated nearer the pointed end. These mature, pear-shaped forms, Nuttall states, are the mark of distinction between Piroplasma (Babesia) and other intracorpuscular blood-para- sites. These pyriform bodies are generally present in pairs, and occasionally, in the acute form of the disease, sixteen pairs may be seen in a single blood cell. The number of red cells infected is about 1 per cent, of the whole. If the number increases to 5 per cent, or 10 per cent., it generally means the death of the animal. The parasites quickly disappear from the blood after the disappearance of the fever. In fatal cases many parasites are found in the red-blood cells of the internal organs. They vary in number according to the stage at wdiich death occurs, are most abundant in the kidneys (50 to 80 per cent, of all red corpuscles infected), and are foimd in fewer numbers in the li\'er, spleen, and other internal organs. R. Koch has described a bacillar form which he found in large numbers in red-blood cells of acute fatal cases in East Africa. Be- tween these and the pear-shaped forms he found all grades. This variety is probably a distinct species. Flagella-like appendages in Babesia have been described by se^'eral observers as occurring in the blood in mammals. More frequently they have been seen in the tick and in attempted cultures. Some of them lia\'e been interpreted as possible microgametes (Hartmann, Calkins), others as true flagella (Breinl and Hindle), still others as fine pseudojDodia (most observers). Smith and Kilborne showed that the infection is caused by a species of tick, Margaropus annulatus Say (Boophilus bovis) (Fig. 198), and Kossel gives Ixodes redivius as the tick causing transmission of the germ in the hemoglobinuria of Finland cattle. The ticks feeding upon the blood of cattle and other mammals become sexually mature at their last moult. They then pair, and the fertilized females, after gorging themseh'es with the blood of their host, drop to the ground. Each female then lays about 2000 eggs, 36 562 PA THOGENIC MICROORGANISMS and within the shell of each egg a large quantity of blood is deposited to serve as food for the developing embryo. The female then shrivels up, becoming a lifeless skin. The newdy hatched larva; containing in their abdomens some of the mother-blood, crawl about until they either die from sta^^'ation or have the opportunity of passing to the skin of a fresh host. If the mother-tick has drawn its supply of blood from cattle infected with piroplasma, her larva; are born infected with the parasite and become the means of disseminating the disease further. This mode of dissemination explains the long incubation period of the disease (forty-five to sixty days— thirty days for the development of the lar^-a; and the remainder for the development of the parasite within the host). It is possible that the tick embryo acquires the infection secondarily from the blood it absorbs in the egg, and that the parasites do not pass through the o^^im itself as in Nosema bombycis. This species of tick M. annulaius has been found also on sheep and ponies. Fig. 198 No. 1. Texas fever tick, Margaropus annulaliis (Boophilus bovis). No. 2. Natural size. (Mohler.) X 15. So far, it has not been possible experimentally to inoculate animals other than cattle with these parasites. Calves M'ithstand the infec- tion better than older animals and a certain degree of immunity is reached in some of the older cattle in infected districts. The piro- plasmata taken in by such animals may remain as harmless parasites for some time. If, howe\'er, such cattle are weakened from any cause, their resistance to the organism may be lowered and they may there- fore pass through a more or less severe attack of the disease. Symptoms of the Disease. — Fever (40° to 42° C), anorexia, weakness, increased pulse and respiration, decreased secretion of milk, hemoglobinuria at the height of the fever, causing the urine to appear dark red like port wuie or darker. The urine may contain albumin even if the hemoglobinuria is absent, but there are no red-blood cells present, the color being due to the coloring matter of the blood only. There is icterus of the mucous membrane if much Idood is destroyed. GENUS BABESIA 503 The prognosis ^•al■ies in different epidemics from 20 to 00 per eeiil,. Death may occur in three to five days after first symptoms appear, lleco^'ei'y is indicated by a gradual fall of the fever. Treatment. — Quinine in large doses seems to have lieljjed in some epidemics. Nuttall, Graham-Smith, and Had wen have reported curative effects from trypanblau in both canine and bovine babesiosis (Piroplasmosis). Prophylaxis.— Stalled cattle are not infected, but it is impracticable to keep large herds of cattle stalled. If the cattle are kept from infected fields for one or two years and either animals (horses and mules) are allowed to feed there the ticks may disappear. The burning of the field for one season may have a good effect. If animals cannot be taken from infected fiekls such fields should be enclosed. Ticks on animals may be killed by allowing the cattle to pass through an oil bath (paraffin, cottonseed oil, etc.), whereupon the ticks die from suffocation. The bath shoidd be repeated after a week in order to kill any lavvx which may ha\'e developed. All animals sent from infected regions should recei^'e this treatment. Animals apparently healthy before the treatment, after the disturbing influence of the bath often develop the disease in an acute form and die. Certain birds in Australia seem to feed on the ticks, therefore such birds might be proj^agated. Various attempts have been made to gi\'e protection by the inocu- lation of fresh (not older than two or three days) blood from slightly infected animals. Some partial results have been reporter!, especially when the inoculations were made during the cold months. In Aus- tralia, the inoculation of defibrinated blood from animals which have just recovered from the infection, but whose blood still contains some parasites, has been tried. vSo far no absolute protection has been pro- duced, neither does the jjarasite-free serum of animals which have entirely recovered from the disease seem to contain protective qualities. Blood-organisms. — Blood-organisms similar to those described in the hemoglobinuria of cattle have been found in cases of red water fever of cattle in England. They also occur in monkeys, dogs, sheep, horses, and pigeons. Xocard and Motas, who have made an extensive study of these parasites in the malignant jaundice (hemoglobinuria, malaria, or biliary fever) of dogs, state that though the parasites are morphologically similar to those infecting cattle, yet it is impossible to infect cattle or any other animal tried with them. They must therefore be considered a jdiysiologic variety. Cultivation. — Thompson and Fantham ha\'e reported successful development in the test-tube after the method of Bass and Johns for malaria. Nuttall and Graham-Smith report a si iidy of canine piroplasmosis, and have drawn a cycle sliow'ing tlie usual mode of multiplication in the circulating blood. They consider B. cmiis a species distinct from B. boris and B. pilheci (found b}^ Ross, in 1905, in blood of a species of cercopithecus) though no moiphologic differences are given. Christophers has descrilied proluible se.xual stages of de^•elopmcnt in the tick R. sanguineus, so that he has drawn a complete life cycle of the organism. 5GJ: PATHOGENIC MICROORGANISMS Refehences. Bass and Johns. Jour. Exp. Med., 1912, xvi, 567. Bcre?ibreo-Gosslcr. Beitriigc zur Naturgcschichte der Malariaplasmodien, Arcliiv. fiii- Protistenkunde, 1909, xvi, 245. Christophers. Jour. Trop. Med., 1907, x, 323. Craig. The Malarial Fevers, in Osier's Modern Medicine. Pliiladelphia, 1907, vol. i; also The Malarial Fevers, eto., 1909, Wm. Wood & Co., New York, first edition. Howard. Mosquitoes, in Osier's Modern Medicine, Philadelphia, 1907, vol. i. Kinoshita. Arch. f. Protistenk., 1907, viii, 294. Koch. Zeitschr. f. Hygiene, 1901, xlv, 1. Marchiafava and Bignami. Malaria, in Twentieth Century Practice, New York, 1900. Miyajami. Philip. Jour. Science, 1907, ii, 83. NiUtall and Graham- Smith. Jour. Hj'giene, 1905-1900-1907. Also in Parasitology, 1909, ii, 215, 229, 23C. Rowley. .Tour. Exp. Med., 1914, xix, 450. Ruge. KoUe and Wasscrmann's Handbuch der Pathogenen Mikroorganisnien, 1913, 2d ed., Jena. T. H. Smitli and Kilborne. United States Department of Agriculture, 1893, Bulletin No. 1. Thayer and Hcwctson. The Malarial Fevers of Baltimore, Johns Hopkins Hospital Rep., 1895, vol. v. Thompson and TJiompson. Ann. Par. and Trop. Med., 1913, vii, 509. Thompson and Fantham. Ann. Par. and Trop. Med., 1913, vii, 621. CHAPTER XLIV. SMALLPOX (VARIOLA) AND ALLIED DISEASES. Introduction. — The diseases smallpox, cow-pox, vaccinia, liorse-pox, sheep-pox, if not identical, are closely allied. Indeed, the following facts seem to prove that at least cow-pox and variola are very closely related, if not essentially the same disease: First, smallpox virus in- oculated into calves produces, after passage through several animals, an affection exactly similar to cow-pox. The successful inoculation of the first series of cattle from smallpox is a matter of great difficulty, but so many experimenters have asserted that this has been done that there seems to be no doubt as to its truth. In our laboratory not one of many attempts to accomplish it has been successful. Second, both when occurring in nature and when produced by experiment the lesions of the two diseases are similar. Third, monkeys have been successfully protected against either disease by previous inoculation of the other; also, observations go to show that human beings inoculated with cow-pox vaccine are not susceptible to inoculation with smallpox virus, and that those who have within a varied time passed through an attack of smallpox cannot be inoculated successfully with cow-pox vaccine. These facts seem positively to prove that the two diseases are produced by organisms originallj- identical, one being modified b}^ its transmission through cattle, the other through human beings. Variola is perhaps the most regularly characteristic of the diseases of man. It is highly infectious and is controlled only by vaccination. Notwithstanding the fact that we know definitely the exact site of the infective agent in this disease and that certain experimental animals are susceptible to inoculation of the material containing the infective agent, most investigators are still undecided in regard to the nature of the chief exciting factor. A few, however, claim that certain bodies found chiefly in the epithelial cells of the skin and mucous membranes in the specific lesions are protozoa causing the disease. Definition. — Smallpox (Synonyms: Variola, la variola, Blattern, Pocken, Vajuola) is an acute infectious disease characterized by an epidermic eruption of vesicles and pustules which, upon healing, produce cicatrices of varying extent and depth. Historical Note. — The first undoubted description of the disease was given by Rhazes in the tentli century, but it is evident that he did not consider it a new disease. To trace its original home seems to be impossible. It may have developed first in certain regions m Asia and Central Africa where it is at present endemic and is said to be uncontrolled bj' vaccination. Many outbreaks of the disease in the United States can be traced directly to the importation of African negroes. Tlie ilisease, carried by the interconmiunication, principally of war and com- 56G - PATHOGENIC MICROORGANISMS mercc, was widespread when Edward .Tenner showed couclusivelj' in 1798 that vaccination with cow-pox afforded protection. Now the few cases of variola that occur are seen in those who, through neglect or ignorance (sometimes willful), have not been vaccinated. Etiology of Variola and Cow-pox. — It has been repeatedly shown that no bacteria similar- to any of the known forms have a causal relation to these diseases. In our own laboratory we are able, by the inoculating of rabbits' skins, to produce extremely active vaccine virus in large quantities, absolutely free from microorganisms which grow' under the conditions of our present methods of bacterial culti- vation. Such pure active vaccine, when emulsified in equal parts of glycerin and water and filtered through tw^o or three thicknesses of the finest filter paper, gives a slightly opalescent filtrate, which in the hanging drop under high magnification shows many very tiny granules with an occasional larger one, and in smears shows no formed elements giving characteristic stains. This filtrate, from which no growth was obtained on artificial culture media, when rubbed over a freshly shaved rabbit's skin after the method of Calmette and Guerin, or when used to vaccinate human beings, gives an abundant typical reaction. These facts show that some, at least, of the infective forms cannot as yet be made to grow outside of the body, that such forms are very minute, and that they do not stain characteristically with our usual methods of staining. In a few experiments we were unable to filter the virus through a Berkefeld filter under forty pounds' pressure, but this may have been due to the fact that we did not dilute our virus sufficiently. Since then Bertarelli and a few others have reported that the virus is slightly filtrable under pressure. Park and Wilson, however, on later experiments (1913) still obtain only negative results. Steinhardt, Israeli, and Lambert (1913) report that evidence of multiplication of germs may be obtained by growing the virus on living tissue in vitro. Since Guarnieri in 1892 claimed that certain inclusions present in the epithelial cells of the lesions of smallpox in a rabbit's cornea (Fig. 199) were parasites, much attention has been given to the study of these bodies, commonly known as "vaccine bodies," yet opinions still differ as to their nature. Among the more important studies of these bodies are those, on the one hand, by Councilman and his asso- ciates, who believe them to be protozoa, and, on the other, by Ewing, who believes that all of the forms so far described are degeneration products, some specific, others not. Calkins, working with Councilman, thinks that his original tentative cycle is too elaborate. He still firmly believes that the bodies are protozoa, but that they belong among the rhizopoda and not among the microsporidia where he first placed them. Prowazek and others believe that the organisms of this group of diseases, as well as of rabies, scarlet fever, trachoma, and a few others, are all minute coccus- hke forms which have the power of producing an envelope from the host cell SMALLPOX AND ALLIED DISEASES 507 sulistaiice, such envelope with its contained orsauisui constitutins the specific Ixidy whicli others have called a jji-otozoon. Prowazek calls the group Chlii- mydozoa and says they proliably stand between the bacteria and the protozoa in systematic classification. From our studies on this whole group of diseases we ha\'e come to the conclusion that thei-e is uo close relationship between the trachoma bodies and the intracellular bodies of rabies, smallpox and scarlet fever (see pp. 439 and 571). Fic. 1U9 Epithelial cells of a ral:)1;)it'.s cornea, containing many "vaccine bodies." Tissue fixed three days after inoculation \^'ith sinallricix virus, a and c/, vaccine bodies; h and c, nuclei. X 1500 diameters. In our own work on sections, which has extended irregularlj' over a period of several years, we have gotten results which are somewhat confusing, princi- pally so because of the non-uniformity of the appearances of these bodies, Iwth by different methods and bj' the same methods at different times. There is no doubt that, whatever the nature of the bodies, they are easily affected by methods used for fixing, hardening, and staining them. This accounts in part for the varied results reported. However, in the most perfectly prepared specimens, judged according to the appearance of the red-blood cells, leukocj'tes, and tissue cells at a distance from the lesions, we have found that the vaccine bodies, especially in corneal infection, show a more or less constant series of changes, somewhat similar to those described by Calkins in his "gemmule formation" and by Tyzzer in his development of the vaccine bodies. Our best results on corneas have been obtained with the following tech- nicjue: Fix in Zenker's fluid for from four to eight hours; wash in running water overnight; place in 95 per cent, alcohol (changing in two hours to fresh) for twenty-four hours, then in absolute alcohol for twenty-four hours. Imbed in paraffin. The cuts should be from S^ to 5m thick. Stain with (1) eosin and methylene blue (MaUory) — eosin haU' an hour, methylene blue two minutes; (2) Heidenhain's iron hematoxylin; (3) Borrel modified by Calkins. The vaccine bodies may be studied for a short time in the Uving cornea by rapidly excising an inoculated cornea, spreading it on a shallow agar plate and dropping a thin cover-glass over it. The structured bodies are very clearly differentiated from the rest of the cell contents, and interesting changes have been observed in them. Too little work has been done, however, by this method, to draw any further conclusions in regard to their nature. Councilman and 568 PATHOGENIC MICROORGANISMS Tyzzer photographed these hving cornea bodies with the ultraviolet light, and the structure came out as tlie chromatin structures of known living cells. Pathogenesis. — For Lower Animals. — Various aniuuils sccni to contiaut tlic disease, or a modification of it, in natm-e. Horse-pox, slieep-pox, and cow-pox, all show similar pathologic changes. Experimentally, probably all mam- mals are susceptible though in varying degrees. Most of them are rnore sen- sitive to vaccinia than to variola. The epidermis of rabbits, for instance, shows a beautifully typical eruption after inoculation with vaccine virus, while material from smallpox eruptions produces only diffused redness. The corneal "take," however, in both instances, is similar in intensity. Monkeys are eciually susceptible to both forms of the disease. For Man. — Without vaccination human lieings seem to be equally suscep- tible to infection with variola, whatever their race or their condition in life or in whatever part of the world they live. The immunity caused by successful vaccination is not permanent^ and varies in its duration in different individuals. Although it usually gives protection for several years and may give it for ten or fifteen years, it is not well to count on immunity for more than one year, and whene\-er one is liable to exposure it is well to be vaccinated. If this vaccination were unnecessary it will not be successful, while if it is successful we have reason to believe the individual was open at least to a mild smallpox infection. Protective Substances Present in the Serum of Animals after Suc- cessful Vaccination. — It has been frequently shown that the blood- serum of a calf some days after an extensive vaccination possesses feeble protective properties, so that the injection of one or two liters of it into a susceptible calf would prevent a successful vaccination. A further and more convincing fact has been demonstrated by Huddle- ston and others, namely, that when active vaccine is mixed in certain proportions wdth serum from an animal which had just recovered from a successful vaccination, and the mixture is inoculated into a susceptible animal, there is no reaction. The Preparation of Vaccine. — For the following points we are indebted to Dr. Fielder who is now in charge of this work in New York Health Department. Seed Virus. — This may be prepared by one of the following methods: 1. Glycerinized hovine vims which has been ripened for two or three months. Such virus becomes attenuated after a varying number of calf-passages. 2. Rabbit Virus. — Rabbits vaccinated with stock bovine virus. This may also result in deterioration. 3. Human Virus. — This consists of the serum collected on sterilized bone slips from the vaccine vesicles of pre^'iously unvaccinated children. This produces good vaccine on calves; it is manifestly difficult to secure. 4. Glycerinized vaccine from calves which have been vaccinated with an emulsion of human vaccine crust. This also gives good virus, but it necessitates the constant collection of human crusts. SMALLPOX AND ALLIED DL'^EASES 569 5. Ilumun-Calf- Rabbit Sce4. — This has been found to be the most economic, efficient, ami rehable seed yet found by us. It is produced as follows: (^rusts are collected from healthy children aliout nineteen days after successful vaccination. These crusts are cut u[) and ejnnlsified with boiled water to a nuicila.ginous paste. This humanized seed is inoculated into an area about 6 inches square ui)on the abdomen of a calf, the remainder of the calf being vaccinated in the ordinary way. The pulp from this special area is separately collected and glyeerin- ized in the usual way. It is then tested bacteriologically and clinically. This bovine virus from human seed is now used in a dilution of 1 part to 121 parts of normal salt solution to vaccinate rabbits. The seed is rubbed sufficiently upon the freshly shaven skin of the back. Five days after vaccination the pulp is removeil with a curette, weighed and emulsified in a mortar with the following solution: glycerin 50 per cent., sterile water 49.5 per cent., and carbolic acid 0.5 per cent., in the proportion of 1 part of pulp to 8 parts of the solution. Four rabbits should yield from 15 to 20 c.c. of this emulsion, an amount sufficient to vaccinate one calf. The regular supply of vaccine is pro- duced by vaccinating calves with this rabbit seed in the manner to be described. Animals. — The preferable animals are female cahes, from two to four months of age, in good condition and free from anj^ skin disease. These can easily be vaccinated on the posterior abdomen and insides of the thighs by placing them on an appropriate table. It is possible that, on account of the character of the available supply, older animals mav" be desirable, but the calves take more typically and are more easily handled. When an animal is too old to be thrown and held with- out difficulty it may be vaccinated on the rump each side of the spine; but the skin there is tougher than on the posterior abdomen and insides of the thighs, and the resulting virus, though efficient, is not so easily emulsified. Vaccination. — The hair should be clipped from the entire body when the animal is first brought into the stable and the calf should be cleaned thoroughly, including the feet and the tail. Just before vaccination the posterior abdomen and insides of the thighs are shaved and the skin beneath washed in succession with soap and water, sterilized water and alcohol, and then dried with a sterile towel. On this area there are now made superficial linear incisions with a sharp knife, about a fourth of an inch apart. After they have been made they should be dried with a sterile towel or with sterile cotton and rubbed with the charged slips. One to two slips, depending on the amount of virus each slip holds, should be sufficient for vaccinating each vesicle. Collection. — On the fifth or sixth daj', depending upon the rate of development of the vaccine vesicles, they should be ready for collection. The entire shaved area is washed with sterile water and sterile cotton, and the crusts are picked off. The soft, pulpy mass remaining is then curetted off with an ordinary steel curette and the pulp placed in a sterilized vessel. The pulp should be mixed with four times its weight 570 PATHOGENIC MICROORGANISMS of glj-cerin and water (50 per cent, glycerin, 49 per cent, water, 1 per cent, carbolic acid). This may be done by passing the mixture through a special ^'acci^e mill. The more watery the pulp, especially if it is not to be used inmied lately, the smaller should be the proportion of glycerin. The emulsion so produced can then be put up for issue in vials. Capillary tubes require especial means of filling, and .small vials filled and corked answer the purpose admirably. Care of the Calves. — The calves are inspected by a Department Veterinarian at the time of purchase and during the period of detention previous to vaccination. After the vaccine has been collected the calves are immediately killed and their organs examined by the Veterinarian. If, at autopsy, an animal be found tuberculous or otherwise diseased, the vaccine is discarded. The vaccine stable at Otisville, N. Y., is a new building with screened windows and concrete floors and stalls, which are kept flushed with water to wash away the dejecta of the animals. The calves stand upon raised racks of galvanized iron. They are fed on milk, no hay or straw being used for any purpose. The calves are vaccinated and the vaccine pulp collected under careful aseptic precautions in a separate operating room, which has tiled walls and a concrete floor. The vaccine pulp when collected is placed in sterile glass containers, sealed, packed in ice and shipped at once by express to the vaccine laboratory. Preparation and Testing of Finished Product. — As soon as received, the vaccine pulp is ground up and emulsified in a solution of glycerin, 50 per cent.; sterile water, 49 per cent.; carbolic acid, 1 per cent.; in the proportion of 1 gram of pulp to 4 c.c. of diluent. Lahoratori/ Teds for Purity. — The following tests for purity are now made: 1. Plating upon agar and counting colonies of organism. This is done weekljf for five weeks. After one to three weeks the plates usually show no growth, the carbolic acid and glycerin having killed off the extraneous organisms. 2. Inoculation of glucose broth in fermentation tubes for evidence of gas-forming organisms. 3. Tests for Tetanus. — Two tests are carried out at the same time, using two different anaerobic methods. After the cultures have incu- bated six daj's, they are filtered and guinea-pigs are inoculated with the filtrate, and are watched for ten days for evidences of tetanus. 4. Test for Streptococcus. — A guinea-pig is inoculated subcutaneously with the freshly prepared vaccine and watched for ten days for evidences of streptococcus infection. Clinical Tests for Efficiency. — After all the laboratory tests for purity ha\'e been made and found satisfactory, and not until then, the vaccine is ready for use upon human beings, provided it be found to be efficient. To determine this point, fifteen inoculations of the vaccine are made upon previously unvaccinated children. These must all show a perfect SMALLPOX AND ALLIED DISEASES . 571 take in order to pass tlie \accine as up to standard. A clinical test of such A'accine is made e\ery two weeks thereafter so long as the \aecine is on the market. If one of these tests fails before the end of the period of guarantee, the \acc'ine is called in. Keeping of Vaccine.- Bulk vaccine is kept in cold storage at a temperature of 4° to 8° F. below zero. Vaccine which has been put up in packages ready for issue is kept in an ice-box at a temperature of 33° to 40° V. References. Councilman and Co-workers. Jour. Med. Research, 1904, xii, 1. Osier's Moilern Medicine, Philadelphia, 1907, vol. ii. Ewing. Jour. Med. Research, 1904, xii, 509. Prowazek and Halberstaedter. Zur Aetiologie des Trachoms, Deutsche med. Woch., 1907, vol. xxxiii. n'ilUams and Flournoij. Studies from the Rockefeller Institute for Medical Reseaich, 190,5, vol. iii. Steinhardt, Israeli, and Lambert. Jour. Inf. Dis., 1913, xiii, 294, and 1914, xiv, S7. Williams. Rabies, in Forsoheimer's System of Med., 1914, vol. v. CHAPTER XLV. RABIES. YELLOW FEVER. RABIES. Introduction. — Rabies (synonyms: Hydrophobia, Lyssa, Hundswuth, Rage) is an acute infectious disease of mammals, dependent upon a specific virus, and communicated to susceptible animals by the saliva of an infected animal coming in contact with a broken surface, usually through a bite. The name rabies is given to the disease because of its most frequent and characteristic symptom — furor or madness. Hydrophobia (Greek, fear of -vvater) is another name com- monly used, which is also given because of a frequent symptom of the disease, the apparent fear of water. Lyssa is a Greek word meaning hydrophobia. Within the gray nervous tissue of rabid animals are peculiar protozoon-like structures known as "Negri bodies" which are diagnostic of rabies. The nature of these bodies is still a question of dispute (see below). Historical Note. — Rabies is probably one of the oldest diseases in exist- ence, but because of the occurrence of so few human cases, and because the disease develops so long after the bite, its source was for a long time not known nor was it recognized as a separate disease. Hippocrates does not mention it in his writing, but Aristotle about .50 years later (about 300 b. c.) speaks of its being purely an animal disease and being carried by the bite of one animal to another. Celsus in the fii'st century was the first to give in writing a detailed description of human rabies. He speaks of it being produced by the Ijite of rabid animals and states that the wound must be thoroughlj^ bathed and then burned with a hot iron in order to prevent the development of the disease, for after symptoms appear death always follows. As Celsus was not a physician he must ha^'e gotten his knowledge from writings which have since been lost. Other writers soon after gave very true descriptions of the symptoms and handling of the disease. Many hundred years passed after this without adding anything to our knowl- edge of the disease, though authors on the subject were numerous. Van Sweiten in 1770 observed the paralytic form of rabies in human beings. At this time several authors, among them Morgagni and Zwinger, believed that the bite of a dog which was not suffering from rabies might produce the disease in man. In 1802 Bosquillon brought forth the original idea that belief in the existence of infectious material in rabies was a chimera and that hydrophobia was simply due to fright. This false idea had adherants for a long time; even now, by a few people, it is thought to be a true one. Among the host of good observers who studied the disease during the latter part of the nineteenth century, Pasteur stands out as the discoverer, in 1880, of the fact that the disease may be prevented by inoculating gradually increas- ing doses of the virus into the person or animal Ijittcn. This treatment with some modifications, the details of which will be given later, is still used, though many efforts have been made to develop an efficient serum treatment. Pasteur, PLATE IX v»yp W^.^'!^l:- ,.-BS vC~^fe :^'»-V^^i»*^'v-v . WILLIAMS, DEL Nerve Cells in Spreads froni Aninion's Horn. Magnification 1200 diameters. Figs. 1 and 2 from clog "street rabies" show "Negri bodies" (NB) ; Fig. 3 from non-rabic eat, and Fig. -4 from dog di stem, per show indefinite inclusion (I) that might be nnistaken for Negri bodies by tlie inexperienced. Negri bodies are structured, more intensely staiiiing, and more refractive. Figs. 1, 3, and 4 are stained by fuehsin-methylene blue mixture, which stains Negri bodies (NB) red with blue granules, nucleus (NUC) of nerve cells blue, and red-blood cells yello"vv (RB). Fig. 2 is stanied by Gienisa's mixture, which stains Negri bodies a robin's egg blue with red granules, nuclet,is of nerve cells red, and red-blood cells salmon pink. RABIES 573 as well as numerous other iuvestigators, tried to diseover the specific cause of rabies, but all of the results were negative. The importance of making a quick diagnosis had become so e\'ident that the efforts of many workers were directed toward this entl alone. Pasteur and his inunediatc followers relied for their diagnosis entirely upon rabbit inoculations, and this meant a fifteen to twenty days' wait before the patient knew whether or not the treatment he was receiving was necessary. In 1S9S this time was shortened to about nine days in our laboratory by Wilson, who found that guinea-pigs came down with the disease much more quickly than rabbits. From time to time it has been thought that certain histologic findings were diagnostic; for instance, the "rabic tubercles" of Babes, and the areas of "round and oval-celled accumulation in the cerebrospinal and sympathetic ganglia" of Van Gehuchten and Nelis, were said to be specific, but further study has shown that they are not absolutely specific for rabies. In many cases the whole picture of the grosser histologic changes is sufficiently characteristic to warrant the diagnosis of rabies, but often it is not so. 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 which seemed absolutely specific for hydrophobia. Negri claims that these bodies are not only specific for rabies, but that they are probably animal parasites and the cause of the disease. Negi'i's later studies confirm his previous work and add some new facts m regard to the structure of the larger bodies. His work, especiallj' as far as the diagnostic value of these bodies is con- cerned, has been corroborated by investigators in almost all parts of the scientific world, among them workers in our own laboratory who not only determined their worth in diagnosis, but investigated their nature. In our work emphasis was placed upon the fact that the demonstration of the "Negri bodies" by our "smear method" (see p. 572) wonderfully sim- plified the process of diagnosis. As a result of our studies we concluded that the Negri bodies are not only specific for rabies, but that they are living organ- isms, belonging to the protozoa, and are the cause of the disease; giving as our reasons the following facts: (a) Thej' have a definite, characteristic mor- phology; (&) This morphologjr is constantly cyclic, that is, a definite series of forms indicating growth and multiplication can l^e demonstrated; (c) The structure and staining qualities, as shown especially by the smear method of examination, resemble those of protozoa. Since this report was published many more cases of rabies have been more or less studied by us and our former conclusions have been more firmly established. Indeed, the evidence as to animal nature of these cell inclusions seemed so convincing that Williams in 1906 gave them the name Neurorydes hydro-phobice.^ Calkins has since studied these bodies and agrees with Williams as to their nature. He called attention to the similarity between their structure and that of the rhizopoda. A number of observers, however, still believe that the Negri body as a whole is principally the result of cell degeneration and that the specific organism may be contained within it. Prowazek includes rabies with his " chlamjdozoan diseases" (see p. 565). To anyone who has made a long and minute study of the two diseases, however, there can be no cpiestion in regard to the essential difference between the "trachoma bodies" and the "Negri bodies." 1 Proceedings of tlie N. Y. Pathological Society, 1906, vi, 77. 574 PATHOGENIC MICROORGANISMS Material and Methods for Study. — In New York one may still frequently oLitain fresh brains of rabid animals, from veterinarj^ hospitals or from the laboratories handling this material. Two methods have been used in helping to study the principal site of infection. (1) Animal moculations. (2) Sections and smears. The first method is used as a decisive test in diagnosis when results from the second method are doubtful. The technique of the smear method used at present iu the Research Labora- tory of the New York City Health Department is as follows; 1 . Glass slides and cover-glasses are washed thoroughly with soap and water, then heated in the flame to get rid of oily substances. 2. A small bit of the gray substance of brain chosen for examination is cut out with a small sharp pair of scissors and placed about one inch from the end of the slide, so as to leave enough room for a label. The cut in the brain should l^e made at right angles to its surface and a thin slice taken, avoid- ing the white matter as much as possible. 3. A cover-slip placed over the piece of tissue is pressed upon it until it is spread out in a moderately thin layer; then the cover-slip is moved slowly and evenly over the slide to the end opposite the label. Onl)^ slight pressure should be used in making the smear, but slightly more should be e.xerted on the cover-glass toward the label side of the slide, thus allowing more of the nerve tissue to be carried farther down the smear and producing more well-spread nerve cells. If any thick places are left at the edge of the smear, one or two of them may be spread out toward the side of the slide with the edge of the cover-glass. 4. For diagnosis work such a smear should be made from at least three different parts of gray matter of the central nervous svstem; first, from the cortex in the region of the fissure of Rolando or in the region corresponding to it in lower animals (in the dog, the convolutioir around the crucial sulcus); second, from Ammon's horn, and, third, from the gray matter of the cerebellum. 5. The smears are partially dried in air and fixed for about ten seconds in acid-free methyl alcohol to which 0.1 per cent, picric acid is added. 6. The excess of alcohol is removed by pressing fine filter paper gently over the smear. 7. The methylene-blue-fuchsin staining mixture recommended by Van Gieson is poured over the slide, warmed until it steams, poured off, and the smear is washed in running tap-water, and allowed to drj^, the excess of water being removed with fine filter paper. The staining mixture reeonmiended by Van Gieson is made by us at present in the following proportions: 30 c.c. distilled HoO; 10 e.c. sat. ale. sol. meth. blue; 0..5 c.c. sat. ale. sol. basic fuchsin. The mixture in room temperature in diffuse daylight will keep for a day, and possibly two. In the dark at room temperature it retains its staining powers a little longer. At ice-box temperature it lasts a much longer time, probably indefinitely. With this method the Negri bodies stain magenta, their contained granules blue, the nerve cells blue, and the red-blood cells yeUow (Plate IX, Fig. 1). (Jfher methods we have found useful for staining smears are; (1) Giemsa's (p. .SI), by which generally the "bodies" are a blue and the contained granules are azAire. The cytoplasm of the nerve cells stains blue also, but with a success- fully made smear the cytoplasm is so spread out that the outline and struc- ture of most of the "bodies" are seen distinctly within it. The nuclei of the nerve cells are stained red with the azur, the nucleoli a dull blue, the red-blood cells a i)inlv-yellow, more pink if tlie decolorization is used (Plate IX, Fig. 2). The "bodies" have an appearance of depth, due to theu- refractive qualities. (2) The eosin-methylene-blue method of Mallory (p. 83). With this method of staining, the cytoplasm is a magenta, light in the small bodies, darker in the larger; the central bodies and chromatoid granules are a very dark blue, the nerve- cell cytoplasm a light blue, the nucleus a darker blue, and the blood cells a RABIES 575 l)rilliaut eosin-pink. With, more dccolorization in the alcohol the "bodies" are not such a deep magenta, and the difference in color between them and the red- blood cells is not so marked. In the technique of the section work (p. 84) the most important point is the time the material is allowed to remain in Zenker. According to our experience two hours' fixation is not enough, three to eight hours is very good, and with every hour after eight hours the results become less satisfactory. Left in Zenker o-s-ernight the tissue is granular and takes the eosin stain more or less deeply, both of which results interfere with the appearance of the tiniest "bodies," especially of the very delicate, minute forms found by us in sections from fixed virus. In regard to the rest of the technique, it is sufficient to say that the changes to the difl'ercnt fluids are made with great regularity, and the final differentia- tion in alcohol of the stained sections is done most carefully. The sections may be stained by the eosin-methylene-blue method of Mallory (p. S3). In the sections made in this way we have been able to demonstrate elearljr very minute forms, as well as good structures in the larger forms. Giemsa's method for sections may also be used. Mann's method, recom- mended by others, has not given us such good results. Harris has published a staining method for both sections and smears (see References), which brings the larger bodies out clearly, but which does not seem to give enough differentiation between the smaller bodies and the nucleoli of the nerve cells. Fiu. 200 Ncgii IjikIv showing central chromatin with ring of small granules. X 2000. Morphology of the Negri Bodies. — The largest forms measured are about ISm and the smallest about 0.5/^. They are round, oval, oblong, triangular, or ameboid. The latter are more numerous in the fixed virus of rabl)its and guinea-pigs. Their structure is show-n especially well in smears. Whatever the variety or species of animal infected, the bodies present the same general characteristic structure; i. e., a hyaline-like cytoplasm with an entire nuirgin, containing one or more chromatin bodies having a more or less complicated and regular arrange- ment. Their structure varies to a certain extent with their size. In fixed \iru8, with an occasional exception, only tiny forms are found. These 570 PATHOGENIC MICROORGANISMS are rounded or sometimes wavy in outline, as if possessing slight ameboid motion, sometimes elongated, extending along the rim of the host-cell nucleus, or along one of the nerve fibrils, as if moving there; with eosin and methylene blue they take a delicate light magenta stain, very similar to that taken by the small serum globules in the bloodvessels. Many of the organisms, howe\'er, show a small chromatin granule, situated more or less eccentrically, sometimes on the very rim of the body. In the larger forms the granule is large, in the smaller it cannot always be seen; some of the larger forms show from two to several granules and occasionally' there is a body with the definite central body and the small granules about it. Detailed Characteristics of Structure in the Large Forms (Fig. 200). — In smears, as well as in sections, the cytoplasm appears quite homo- geneous; there is no evidence of a reticulum or of a granular structure outside of the definite chromatoid granules. The smears, however, have brought out one important point in regard to the cytoplasm more clearly than the sections, and that is that it is more basophilic than acidophilic in staining qualities. With the Giemsa stain, as we have already seen, it takes the methylene-blue stain more than the eosin- red, and even with the simple eosin-methylene-blue stain the protoplasm appears as a deep magenta unless much decolorized. In studying the central bodies of these organisms, as they appear in the smears, one of the first things noticeable is that they are not surrounded by a clear space — that there is no sign of a vacuolar ap- pearance in the body as there usually is in the sections. We notice next that in the great majority of the organisms the central body stands out clearly, as decidedly different in structure, and slightly so in staining qualities, from the chromatoid granules which surround it. The general type of the structure of the central body is that of many well-known protozoan nuclei; that is, the chromatin is arranged in a more or less granular ring around the periphery of the central body or nucleus, leaving an achromatic or more acid-staining centre in which is situated, generally eccentrically a \'arying-size(l karyosome. There are a number of variations from this principal tvpe, according to stage of development (PlatelX, Figs, land 2). Fragmented particles seem to be leaving the nucleus in certain forms, and in this way presvmiably the chromatoid granules are pro- duced, thus forming chromidia. The chromatoid granules are most frequently arranged in a more or less complete circle about the nucleus. They are somewhat ir- regular in outline and size, being occasionally ring-shaped, some- times elongated, often in twos, due probably to active changes of growth and division. They take generally a more mixed chromatin stain than the chromatin of the nucleus. Evidences of Division. — All stages in transverse division are seen. Many evidences of Inidding are also seen. The chromatoid granules seem to divide and pass out with part of the cytoplasm as a bud. This budding or unequal di\dsion appears to take place very early in the RABIES 577 j^Towth of the organism and to continue throughout growth until the jxirent body forms a mass of small organisms which may then break apart at the same time. The budding accounts for the number of small and large forms in a single cell (Plate IX, Fig. 1). Number. — The\- vary in number according t(j the stage of the disease and to the infecti\'ity of the part. Site. — They are situated chiefly in the cytoplasm and along the fibers in the branches of the large ner\'e cells of the central ner- vous system. In parts of smears which are more broken up the bodies may appear as if lying free, and it is these bodies, if the pressure be not too great in smearing, that show the structure best. In some cases the bodies are distinctly localized in small scattered areas of the central ner\-ous system. We have always foimd })odies in the spinal cord in abundance, but here they are especially prone to be localized in discrete groups of cells. Manouelian has found them in the ganglion cells of the sali\'ary gland. That the organisms are present in various glands of the body (sali\'ary thyroid, suprarenal capsule, etc.) is shown by the virulence of emul- sions from these organs. Cows' milk (Westbrook, McDaniel) and blood (Marie) ha-\'e also been shown to be slightly virulent. Cultivation of the Rabies Organism. — Many attempts have been made to produce artificial cultures, but no reports have been so far corroborated. Noguchi, the latest claimant, states that he has obtained virulent cultures on -the twenty-first transplant. Williams, Krauss, Barbara, and Volpino have not been able to get the same results. Diagnosis of Rabies. — In our laboratory, for the past ten years, or since we have used the smear method in routine diagnosis, there have been about 4500 cases in all examined, including suspected rabies and controls. These are divided into two groups, the first comprising the cases sent in from outside, for diagnosis only, and the second, the experi- mental cases. Since the publication of our work in Ma}', 1906, in our routine work we have considered the presence of the Negri bodies in smears as diagnostic of rabies and have made no further tests except in those cases which we have used in our experimental work. Through this experimental work, however, we ha\'e added many hundred cases to the list of those which had the comparative tests, and our former conclusions have been more firmly established. In all of our work controlled by careful animal inoculations we have never yet failed to have typical rabies develop in animals inoculated with material showing definitely structured Negri bodies. Negative results after inocvilatiou with such material must be interpreted at ])resent as due to some error in technique, such as regurgitation, or hemorrhage at the time of inoculation, emulsion improperly made, not enough of the virulent material taken l)ecause of localization of the organisms, etc. 37 57S PATHOGENIC MICROORGANISMS Possibly individual resistance of the animal inoculated might pla.y a part. We have used principally guinea-pigs, and some of them have shown enough irregularity in regard to the time in which they have come down with the disease to suggest a varied individual susceptibility, if other factors can be ruled out. On the other hand, material in which we have failed to demon- strate typically structured bodies has produced rabies. All of this material, however, since we have improved our technique, has shown suspicious small forms similar to those found in rabbit-fixed virus. But any decomposing brain may also show in smears bodies very similar to these tiny forms, therefore it is difficult to rule out rabies in such cases. Of course the animal test will probably always have to be used with brains that are too decomposed to show any formed elements except bacteria, unless a reliable chemical test can be dis- covered. Brains from animals dying of distemper may show small non-structured forms, somewhat like "fixed virus" forms (Plate IX, Figs. 3 and 4). So far we have not had rabies produced by fresh brains showing no Negri bodies and no suspicious forms, but a few observers have claimed that such material has produced the disease. Therefore, until we can standardize our technique, we must in all such cases use animal inocu- lations. We may, however, be reasonably certain that a case showing such negative material was not a case of rabies. We may summarize our knowledge in regard to the worth of the smear method in diagnosis as follows : 1 . Negi'i bodies demonstrated, diagnosis rallies. 2. Negri bodies not demonstrated in fresh brains, probably not rabies (except 4). .3 . Negri bodies not demonstrated in decomposing brains, uncertain. 4. Suspicious bodies in fresh brains, probably rabies. The localization of the Negri bodies is an important point in mak- ing diagnoses. We have found well-developed bodies distinctly local- ized in different parts of the brain in several instances. In horses there may be small, widely scattered areas of well-structured forms throughout the cerebellum, while tiny, indefinite forms are scattered through the rest of the brain examined. In human brains well-developed forms are found in the corpus striatum and not in the rest of the brain. In several dogs the localization has also been marked. The Complement Binding Test in Rabies.— This test has been tried by Heller (1907), Friedberger (1907), and Baroni (1908), with negative results. Berry (1910), in our research laboratory went over this work thoroughly and obtained similar negative results. Effect of Chemical and Physical Agents on Rabic Virus.— Rabic virus appears to become attenuated under certain conditions of tempera- ture; indeed, if it be subjected for about an hour to 50° C. or for half an hour to 60° C"., its activity is completely destroyed. A 5 per cent, solution of carbolic acid, acting for the same period, exerts a RABIES 579 similar effect, as do likewise 1 : 1000 solutions of bichloride of mercury, acetic acid, or potassium permanganate. Cumming has shown that formalin is especially deleterious to the virus. The virus also rapidly loses its strength by exposure to air, especially in sunlight; when, however, protected from heat, light, and air it retains its virulence for a long period. The virus is readily filtered through all grades of Berkefeld filters, and from the glands through the coarser Chamberland. Poor and Stein- hardt have shown that the filtered gland and brain virus seem similar. Pathogenesis. — Natural Infection. — The disease occurs in nature among the following animals given in order of their frequency: dogs, cats, wolves, horses, cows, pigs, skunks, deer, and man; in fact, as all warm-blooded animals are more or less susceptible to inoculations, all may presumably contract the disease when an open wound is brought in contact with infectious material of a rabid animal. Rabies occurs in almost all parts of the world. It is most common in Russia, France, Belgium, and Italy; it is not infrequent in Austria and in those parts of Germany bordering on Russia. In this hemisphere it is infrequent in Canada, but in the United States the cases are in- creasing in numbers. In Mexico, and South America it occurs occa- sionally; while in England, North Germany, Switzerland, Holland, and Denmark, because of the enforced quarantine laws, and of the wise provision that all dogs shall be muzzled, it is extremely rare. In Australia it is unknown, probably because the law that every dog imported into the island must first undergo a six months' quarantine has always been enforced. In the vicinity of New York the disease seems to be on the increase. In this connection the question as to how long the sputum of a rabid dog may remain virulent after it drops from the animal is an interesting one. A case came under our observation in 1906 which illustrates this point. A child of six j^ears came c1ot\ti with typical rabies in a neighljorhood where there had recently been several cases of canine rabies, but no history of a bite could be obtained. The parents were sure she had not been bitten. Six weeks before, however, the child had fallen in the street and cut her cheek severly on a jagged stone. The wound was cauterized and healed without further trouble. A mad dog had been on that street just before this occurred. It is reasonable to su]i- pose that the stone had on it some of the sputum from that dog, and so the child was infected. Such a case would not occur very often, but the possibility should be considered. In regard to the question as to whether the bite of apparently healthy animals may give the disease, it may be said that, judging from labora- tory experiments, some animals may have a light attack of the disease and recover spontaneously; though such cases, if they occur, are prob- ably extremely rare. That the bite of an infected animal may give the disease before that animal shows symptoms has been proved. Fifteen days is the longest time reported between a bite and the appearance of symptoms in the dog. Therefore, if an animal is kept under observation three weeks after biting another, without developing symptoms, he nuiy be pronounced free from suspicion. 580 PATHOGENIC MICROORGANISMS Occupation seems to have an effect upon the number of cases among humans in one waJ^ Those people who are much in the country or on the streets— in other words, those who might come most frequently in contact with rabid animals— most frequently contract the disease; otherwise neither age, sex, nor occupation has any effect. The time of the year seems to have little effect, though most cases are said to occur during the summer months. The numbers vary with different years. In 1907 for instance we had as many cases in January as in August and in September and more in June than in any other month. In another year February gave the largest number of cases . The certainty with which the disease may be produced after a bite and the rapidity of its development have been found to be governed by three factors: (1) the quantity of the rabic virus introduced; (2) the point of inoculation; (3) the strength of the virus as deter- mined by the kind of animal which affords the cultivation ground for the growth of the organism. It is a matter of common observation in man that slight wounds of the skin of the limbs and of the back or wherever the skin is thick and the nerves few either produce no results, especially when bites are made through clothes, or are followed by the- disease after an extremely long period of incubation; while in lacerated wounds of the tip of the fingers where small nerves are numerous or where the muscles and nerve trunks are reached, or in lacerated wounds of the face where there is also an abundance of nerves the period of incubation is usually much shorter and the disease generally more rabid. These facts explain whj- only about 16 per cent, of human beings bitten by rabid animals and untreated appear to contract hydrophobia. Since the establishment of the Pasteur treatment for the disease, the percentage of developed cases after bites is very much less, a fraction of 1 per cent. Symptoms. — There is always a decided incubation period after the bite which varies within quite wide limits, but in the great majority of cases it is from t went J' to sixty daj's. Any period after six months is an exception; the shortest we liave on record is fourteen days and tlie longest authentic period is seven months. A very few apparently authentic cases have been reported as developing in about one year, but reports of any time beyond this must be received with doubt. After treatment, however, a few^ cases have been reported as occurring later than this, Ijut even here the question of reinfection is not absoluteljr ruled out. The wound heals as other wounds and sometimes show's no further sjanp- toms. Occasionally, however, redness and swelling of the scar have been reported; oftener there are pains extending from the scar along the nerve paths of the brain. The syniptoms may l)e divided into three stages. First, the prodromal or melancholic stage; second, the excited or convulsive stage; and, third, the paralytic stage. When the second stage is the most pronounced the disease is called furious or convulsive raljies; when this stage is very short or practically lacking and paralysis liegins early, the disease is called dumb or paralytic rabies. In the dog raljies appears in the two typical forms, tlie furious and the paralytic. The principal symptoms of each form may be suimiiarized as fol- RABIES 581 lows: (a) Furious rabies: change of behavior, biting (especially at those to wlioiu the auuual has been alTeetionate before), increased aggressiveness, ehavacl eristic restlessness, loss of appetite for ordinary food, with desire to eat nnusual things, internuttent disturbance of consciousness, ixuMxysnis of fury, peculiar howling bark, rtqnd enuiciation, paralysis, beginning in the hind limbs, death in great niajorit}' of cases in three to six days (excepticjnally slightly longer) after the liegimiing of symptoms. (6) Paruhjlic rabies: short period of excitation, paralysis of the lower jaw, hoarse bark, appetite and consciousness ilisturbed, wealaiess, with paralysis spreading in great majority of cases, and death four to five days after fu'st symptoms. There may be a number of cases showing transition types between these two forms. In Human Beings. — Furious Rabies. — The first definite symptoms are difficult and gasping breath with a feeling of oppression and difficulty in swallow- uig, the latter the most characteristic symptom. It is caused by convulsive contraction of the throat muscles. The attacks are brought out when attempt- ing to drink or swallow. The very thought of drinking may bring one on; and though there is no fear of water itself, there is fear of taking it because of the effect it produces. The convulsive attacks finally become more or less general over the whole body; in certain cases some parts are more affected by reflex excitation than others; for instance, there may be slight or no photophobia, while in exceptional cases, more frequently in dogs, the hydrophobia is also absent. Most of the special reflexes are increased. Pupils become irregularly' con- tracted and widened until thej' finally remain fixed. Human beings are seldom dangerous to the people about them; they do not make aggressive bites. In their convulsions they may bite things placed between their teeth, but not otherwise. At this time there is an increased flow of sahva, and one should avoid the contact of this with opened wounds. It may be so increased that the patient may try to get rid of it by taking it from the mouth with the hand and throwing it about. As a general thing, however, the patient has full possession of his senses between the convulsive attacks until very late ui the disease. The temperatme is increased from 38° to 40° C, at first with mornmg remis- sions. Just before death it may rise as high as 42.8° C. (In lower animals the temperature sinks below normal just before death.) The pulse is generally over 100 and is irregular. This stage lasts from one to four days. Death may occur during a convulsion, but more often there is a 'paralytic stage, which lasts from two to eighteen hours. The convulsions become less frequent and the patient becomes weaker until finally there is a complete paralj'sis. At the beginning of this stage the patient may be able to drink water better than formerly. Death maj' occur at anj' time through paralysis of the heart or respiratory centre. Paralytic Rabies. — This form occm's quite seldom in human beings, more fre- quently in dogs, but not so often as a convulsive form. It is supposed to occur in humans and dogs after a more severe infection. Instead of periods of con- vulsions, the various muscles simply tremble and become gradually weaker untU complete general paralysis supervenes. Sometimes paralysis develops very quickly and may be general before death from syncope or asphyxia occurs. This form generally lasts longer than ordinary rabies. Between these two tyjjical forms of rabies there are many different types, giving quite different pictures of the disease. Leng^th of the Disease. — The majority of the cases of furious rabies die on the thkd or fourth day after the symptoms show themselves. The limits of the reported cases are one to fifteen days, though there are reports of only one or two cases dying on any day over the ninth to the fifteenth. As the time when the symptoms really begin is difficult to notice, these statistics are probably only approximately correct. In paralytic rabies the average time in which death occurs is five days. 582 PATHOGENIC MICROORGANISMS Treatment.— The old treatment of rabies consisted simply in encouraging bleeding from the wound, or in first excising the wound and then encouraging bleeding by means of ligatures, warm bathing, cupping-glasses, etc.; the raw surface was then freely cauterized with caustic potash, nitric acid, or the actual cautery. It is doubtful whether the disease ever manifested itself after such heroic treatment if the wound were small and the treatment was begun soon after the bite; but when the wounds were numerous or extensive, the mortality was still high. As it was often impossible to apply cauterization to the wound rapidly or deeply enough to insure complete destruction of the virus, Pasteur and others were led to study the disease experimentally in animals with the hope of finding some means of immunization or even cure; these investigations finally resulted in the discovery of methods of preventive inoculation applicable to man. Pasteur's Method of Preventive Inoculation. — Pasteur's treatment is based upon the fact that rabic virus may be attenuated or intensified under certain conditions. He first observed that the tissues and fluids taken from rabid animals varied considerably in their virulence. Then he showed that the ^•irus may be intensified by successive passage through certain animals (rabbits, guinea-pigs, cats) and weakened in passing through others (monkeys). If successive inocu- lations be made into rabbits Avith virus, either from the dog or the monkey, the virulence may be so exalted beyond that of the virus taken from a street dog, in which the incubation period is from twelve to fourteen days, that at the end of the fiftieth passage the incubation period may be reduced to about six or seven days when it remains fixed. This ''fixed virus" was used by Pasteur and those after him in his preventive treatment because the dose could be more definitely regu- lated by subsec|uent attenuation or dilution. Original Method. — A series of spinal cords from rabbits dead from "fixed virus" infection are cut into segments and suspended in sterile glass flasks plugged with cotton stoppers and containing a quantity of some hygroscopic material, such as caustic potash; these are kept at a temperature of about 22° C. The cord when taken out at the end of the first twenty-four hours is found to be almost as active as the fresh untreated cord; that removed at the end of forty-eight hours is slightly less active than that remo\'ed twenty-four hours pre- viously; and the diminution in ^-irulence, though gradual, progresses regularly and surely until, at the end of the eighth day the virus is in- active. Pasteur began his treatment with an emulsion of the cord kept until the fourteenth day. A certain quantity of this was injected into the animal that had been bitten; this was followed by an injection of an emulsion of a twelve-day cord; and so on until the animal had been injected with a perfectly fresh and, therefore, extremely active cord, corresponding to the fixed virus. Animals treated in this way were found byPasteur to be absolutely protected, e\'en against subdural inoculation with consideable quantities of the most virulent virus, and thus Pasteur's protecti\'e inoculation against rabies became an RABIES 583 accomplished fact. As it would he undesirahle to inject any hut persons who had actually heen hitteii hy a rahid, or |)resuniahly rabid, animal Pasteur coutinueil his ex])eriments in order to see whetiier it would not he possible to cure a patient alri'ady liitten. lie carried cm, therefore, a series of experiments which led to the disc(i\ery tliat if the ]}rocess of inoculation be begim within fi\e days of the bite in animals in which tlie incubation period was at least fourteen days, almost e\'ery animal bitten can be saved; and that e\en if the treatment be commenced at a longer interval after the bite a certain proportion of recoveries can be obtained. Thus the application of this method of treatment to the human subject was not tried until it had been proved in animals that such protection could be obtained and that such protection would last for at least one year and probably longer. The chance of success in the human subject appears to be even greater than in the dog or rabbit. Man's period of incubation is comparatively prolonged. Thus there is an opportunity of obtaining immunity liy beginning the process of vaccination soon after the bite has been inflicted, the protection being complete before the incubation period has passed. Present Administration of Pasteur's Treatment in Hiunan Beings. — The original method of Pasteur in its entirety was soon adopted in many lands, and his results were corroborated. Before long, how- ever, a number of modifications were suggested by different observers, some slight, others more fundamental. Some have been widel^y used, such as Hogyes' dilution method; others have had a limited application in lower animals and are probably only of theoretic interest as regards man. Such are the intravenous inoculation of brain emulsions from street rabies into herbivora (Nocard and Roux, Protopopoff ) , and the intraperitoneal inoculations of large doses of fully virulent fixed virus into dogs, cats, or rabbits (Hellman, Heim, Remlinger). Immunity has been produced also in rats by allowing them to feed on rabid brains (Fermi, Repetto, Remlinger). Hogyes in Budapest was one of the first to use a different procedure. He claimed that the virus by Pasteur's method was attenuated only through the death of some of the specific organisms, that is, that there were simply fewer living organisms in the early doses given than in the later, and that therefore the same result might be obtained perhaps with even more accurate dosage by giving gradually decreasing dilutions of a fresh virulent cord. By diluting sufficiently he obtained a mixture which when inoculated did not produce rabies in the test animals, a result simi- lar to that following an 8- to 10-day dried cord. This dilution he used for the first inoculation and gradually stronger dilutions for the succeed- ing ones. Other methods of attenuating or diluting fixed virus have been used, such as exposure to the action of heat, cold, gastric juice, glycerin, or carbolic acid. The mixefl treatment with specific serum and \'accine has also been employed, chiefiy by Marie, by Remlinger, and by Babes. 5S4 PA THOGENIC MICROORGANISMS Methods of Attenuation by Gradual Drying.— Pasteur's classic method has undergone modifications in three general directions: (1) lengthening or shortening the period of treatment; (2) starting the inoculations with a less attenuated cord; (3) increasing or decreasing the amount given at each injection. The method of drying the cords, however, has remained essentially the same as that used by Pasteur. Fig. 201 One corner ut' eonwtaut temperature room siiowiiig drying bottle containing fixed virus cords being prepared for -vaccine. The cord is removed by a modification of the method of Oshida in the following manner: Strict asepsis is preserved. The rabbit when com- pletely paralyzed (seventh day) is killed by gas or chloroform and is dropped into a 5 per cent, solution of carbolic acid for 5 minutes. It is then removed, the excess of carbolic solution is drained off, and an incisi(m through the skin at the upper and inner part of the thigh is made. The skin is loosened by cutting around the lower portion of the trunk. It is then pulled by the hands toward the upper extremity of the animal and over the head to the ears, leaving the back exposed and sterile throughout the entire length of the spine. The spine is then divided transversely near each extremity b}'' bone-cutting forceps. The muscles are cut through about these areas so the spine may be more easily reached. With a long wire probe swabbed with cotton at one end the cord is pushed upward from its canal, freed from its nerves and membranes. The spine is steadied by lion-jawed forceps. The cord curls in a spiral as it emerges and rests on the sterile muscles of the neck. It is lifted with forceps, placed in a Petri dish and cut in two. A small jnece is cut from one end and is dropped into a tube RA BIES 585 of broth to test its purity. A ligature with one loii.i;- cud is ]>lace(i about each piece, both of which are then hung in a drying bottk' (Kig. 201), Dkyinc the Coud. — The drying bottles are sterile aspiration bottles with both openings plugged \\ itii I'otton. A layer one inch high of sticks of caustic potash covers the bottom, and the jneces of cord are suspended from the top cotton plug by their attached ligatures. The bottles are then labeled and placed in the constant temperature room (Fig. 201) or incubator, which is kept at a temperature of about 21° C. (70° F.). After 24 hours' drying the cord is known as 1-day cord; after two days, 2-day cord, etc. Pieces of cord cut off at any time and put into glycerin will retain about the same strength for several weeks. This procedure is followed in regions where there are few cases of rabies, and the daily killing of rabbits to keep up the vaccine would be a large expense. It may also be followed where treatment is sent by mail. The New York City Health Department used Pasteur's first schemata, with modifications, up to January, 1906, when they began treatment with a 10- and 9-day cord and finished with a 1-day. They continued with this until August, 1913. Since then they have been using the more intensi\-e method of the Flygienic Laboratory at Washington. From 1906 to 1913 inclusive they treated 4282 cases infected b.y rabid animals, with a total mortality of 0.54 per cent, and a corrected mor- tality of 0.19 per cent. They have had 7 cases of definite paralysis with two deaths; 6850 cases in all, including those not bitten by rabid animals, were treated. Since it had been found that fresh rabbit-fixed ^'irus inoculated sub- cutaneously into man is apparently harmless, the Berlin Institute, with the hope of obtaining an earlier immunization and a shorter treatment, began to give still earlier cords. In 1901 it began with the 8-day cord on the first inoculation, and was inoculating a 2-day cord on the eighth day of treatment. Its treatment lasted 21 days. This method was adopted at the Hygienic Laboratory in Washington in 1908, with slight variations for the difterent degrees of bites. Now only the intensi\'e schema is used for all cases as follows: Days. 1 i 2 3 4 5 6 7 8 9 10 11 12 13 j 14 16 16 17 18 19 20 21 Age of cord 8 7 6 4 3 5 4 3 3 2 2 1 5 4 4 3 1 3 2 2 1 I 4 3 2 3 2 1 Amount injected Adults 2.5 2.i 2.5 2.5 2.5 2.5 2.52.5 2.5'2.5 2.6 2.52.6 2.6 2.5 2.52.5 2.6 2.6,2.5 2.5 5 to 10 years 2.5 2.5 2.5 2.5 2.5 2 2.5,1.5 2.5 2.5 2.5 2.5;2.5 2.6 2.6 2.5 2.5 2.5 2.52.5:2.5 1 to 5 years 2.5 2 2,6 2 2 1.5 2 1 2.52.5 2.5| 2 ' 2 2 2 2.62.5 2 2 2.5, 2 1 Each dose contains ^ cm. of the indicated cord. The New York City Health Department has been using this schema since August, 1913, for severe head and face bites, but it reduces all doses by o"ne-fifth, i. e., ^ cm. of the indicated cord is emulsified in 3 c.c. of normal salt solution, and 2| c.c. of this emulsion is inoculated. It also substitutes a 2-day cord for the 1-day cord of the eighth and twenty- 386 PATHOGENIC MICROORGANISMS first day in other tlian severe cases. In cases with very slight wounds which have begun treatment immediately the inoculations are carried only as far as the fifteenth day. Treatment by Mail.— The New York City Health Department was the first to send out treatment by mail to physicians for their own patients. Full directions are sent in the mailing case. One-fourth per cent, of carbolic acid is added to the emulsions prepared as above for the first three days' treatment; 20 per cent, glycerin is added to all other emulsions. The carbolic acid and the glycerin are added as pre- servatives, and are therefore omitted when the vaccine is administered to patients at the laboratory. The results from the treatment sent in this way seem to be equally as good as those from the treatment administered at the laboratory. More Intensive Treatment. — In Berlin, where intensive treatment has been longest used, they began to employ even fresher cords for begin- ning doses because they continued to have late deaths, though not cjuite so often, after the more intensive methods they were using. Since 1910 Joseph Koch, the present chief of the Institute, has been using the following schema ; Days 1 2 3j4 5 6 7 8 9 10 11|12,13 14 15116 17|18 19 20 21 Age of cords .... 3 2 1 1 3 2 1 1, 3, 2| 1 1, 3j 2 1 1 1 3 2 1 1 1 The dose is 2 c.c. of cord emulsion (1 part of cord in 5 parts of sterile physiologic salt solution) inoculated once a day into the subcutaneous tissue of the abdomen. Children and adults receive the same dose. Simon gives the following statistics of the results of Berlin's increas- ingly intensive methods : Berlin Age of cord used for beginning inoculation. Statistics. Cases. ortality. Period. Paralysis. 1 M Per cent. I. 1898-1906 II. 1906-1909 III. 1909-1910 Chiefly 8-day cord 4-day cord. Sometimes S-day cord . 3-day cord for all cases. 2,896 1,490 819 2 3 21 7 .5 0.7 0.47 O.G Several other institutes are employing very intensive treatments, but tlieir cases are still too few for consideration. Other directors still use the older methods. Rapid Drying of Rabies Virus.— Recently Harris, of St. Louis, has published a new method of drying rabies virus and of regulating the dosage. 'J'echniqu?:. — The brain and cord are removed aseptically and ground uj) in a sterile mortar with a sufficient quantity of COo snow thoroughly RABIES 587 to freeze tlie tissue. The frozen nerve tissue and snow are tlien placed in a Scheibler jar o\'er H2SO4, the jar bein<; kept in a Fri<;'o apparatus. A vacuum of from 5 to 2 mm. is j)roduced in the jar, whicli is then kept at the temperature of 18° C. by an ice and salt mixture for a sufficient length of time to dry thoroughly the ue^^■e substance, whicli then api)ears as a dry powder. About two days are required for one brain and cord, which lose about one-half of their virulence in the process. The powder is then sealed in tul)es in vacuo and kept at a temperature below 0° C. until required for use. It has been found that by keeping the powder thoroughly dry and cold practically no further loss of virulence occurs for at least six months. Before storing the virus for use its strength in units is computed, the unit being the minimal infecting dose (M. I. D.) for a rabbit when injected intracerebrally. The advantages claimed for this method are: (1) the ease and econ- omy with which a large amount of virus can be prepared, it being neces- sary to prepare the ^'i^us for use even in large laboratories only at inter- vals of several months; (2) the possibility of more accurate dosage for the patients; (3) a shortened period of treatment; and (4) the inoculation of more ^'irus units. The required amount of powdered virus is weighed out each morning, and the necessary dilutions in salt solution for the various patients are made from this. Up to October 13, 1913, Harris had treated 240 cases exposed to infection from dogs, in which the diagnosis of rabies was either proved by laboratory methods or strongly probable from veterinary diagnosis. Of this number one patient, who had started treatment sLx days after the bite, died of rabies during the period of observation. No cases of paralysis have been reported. We must wait for further statistics before being able to judge of the efficiency of this method. Fixed Virus Modified by Dialysis. — Gumming, of Ann Harbor, has devised a method of antirabic vaccination, by which he uses fixed virus which has been rendered avirulent by dialysis. The emulsion of fixed virus is placed in collodion sacs (prepared by the Novy method and sterilized in the autoclave at 105° C. for 20 minutes) and dialyzed in distilled water for from 12 to 24 hours. The resulting vaccine does not produce rabies on intracranial inoculations, but does produce immunity on subcutaneous inoculations. Experiments by Gumming on rabbits show that whereas the original Pasteur method protects against only twice the minimum lethal dose (minute directions for obtaining the M. L. D. are given) injected intracerebrally, and the Hogyes method against one and one-half times the fatal dose, the dialj'sis method . protects against at least three times the fatal dose. He also claims that immunity is produced at an earlier date than by the other methods. Treatment (2 c.c. of the vaccine) is given daily for from 15 to 25 days. Gumming reports over 800 cases (62 per cent, bitten by animals proved to have been rabid) treated without a death and without complications. We must wait for further practical testing of this interesting method before deciding as to its comparative worth. 588 PATHOGENIC MICROORGANISMS Marie's Method.— For several years past the use of virus serum mixture has been in vogue at the Pasteur Institute iii Paris, the tech- nique of which is as fohows: 1 gm. of the mcdulhi of a rabbit dead of fixed virus is finely emulsified with 9 c.c. of 0.8 per cent, salt solution and filtered through linen. Two c.c. of this emulsion and 4 c.c. of antirabic serum (obtained from sheep, and inactivated at 56° C. for 30 minutes) are carefully mLxed after standing for a time. Six c.c. of this mixture, which contains an excess of A'irus, is injected into the patient. These injections are repeated on the next three days, after which the treatment proceeds according to the regular Pasteur schema, beginning with the use of a 6-day cord on the fifth day. The antirabic serum is obtained from sheep which have been subjected to a long and strong course of treatment wath fixed virus. It is claimed that a quicker immunity is produced by the serum-virus mixture than by the original Pasteur scheme, an advantage of especial value in the treatment of cases liable to become infected with a short incubation, such as bites on the head. Antirabic Serum. — The possibility that the serum of animals im- munized against rabies contains protective substances was suggested by Pasteur as early as 1889. The following year Babes recommended the use of the serum of vaccinated animals in combination with the Pasteur treatment. Since then the study of the amount and character of the anti- body content of animals immunized against rabies has been carried on more or less extensively both from the theoretic and the practical sides. It was hoped that a serum could be obtained that would effect a cure for developed rabies just as diphtheria antitoxin does for developed diph- theria. But such a definite applicability of the serum has not developed. It was soon found that, while serum of certain vaccinated animals pos- sessed the propertj' of neutralizing rabies ^■irus in vitro, it had only a slight inhibiting power when inoculated into the living animal, and apparently no action at all by any method of inoculation after the disease had become manifest. Babes still claims, however, that the serum has enough effect in tnvo to be used in treatment, and his serum treatment is based upon this claim. He gi\'es as his reason for em- ploying serum at the end of treatment that he wishes to introduce into the patient at the time he most needs it the largest amount of anti- bodies. He also claims that the serum so given will prevent or cure the occasional paralyses which occur during treatment. Those who did not agree with Babes were led to test the practical use of the serum combined with the beginning vaccine inoculations. Remlinger, Marie, and others showed that a serum-virus mixture with a slight excess of virus will protect an animal against infection into- the anterior chamber of the eye when inoculated during the three days following the vaccination. Thus Marie showed that immunity is pro- duced more quickly by these unsaturated mixtures of virus and serum than by the virus alone. If a surplus of serum is present the animals are not protected from a later infection. Marie, who has used the serum in his treatment of humans since 1904, RABIES 589 prepares it as follows: The brains of two rabbits dyinji' from fixed virus infection are finely rubbed up with physiologic salt solution in the pro- portion of 20 gni. in ISO c.c. This emulsion is filtered through fine cloth and heated for one-half hour at 37° C. Sheep are used for the inoculation. Each sheep receives intravenously 30 c.c. (3 gm. fixed virus) a week for six to eight weeks. Thirteen days after the last inocu- lation the first blood is drawn. Then in a period of two weeks, at 4 bleedings, 200 c.c. of blood are drawn. After a 14-day pause another series of inoculations are given and the animal is ready for another series of bleedings. From each animal yearly about 3 liters of antirabic serum are obtained. A strong serum is one that neutralizes 40 virus units in 1 c.c. A virus unit is 1 c.c. of five times the dilution of fixed virus that will surely kill a rabbit inoculated intracerebrally, e. g., the unit of a fixed virus that will surely kill a rabbit in 1 : 500 dilution is 1 c.c. of a 1 : 100 dilution. The nature of the antibodies in rabies serum has been the subject of many studies. Fermi and a few others claim that the antibodies are not specific. They say that they can obtain a similar serum after the inoculation of normal brain emulsions. Some even use normal brain emulsions in the treatment of their lighter cases. Certain investigators (Kraus, Marie, and others), while not able to corroborate all of these claims, have found that the sera of certain animals which are more or less refractory to rabies possess a small amount of rabicidal strength; e.g., 0.5 c.c. of normal chicken serum mixed with one unit of fixed virus (1 c.c. of 1 : 100 dilution) causes the latter to become neutral in eighteen hours. The neutralizing property is not due to a neurotoxic substance since animals stand very large doses of the serum without harm. All species of animals tried produce the specific antibodies, but not to an equal degree. Human beings and monkeys are said to have more antibodies after vaccination than rabbits. Centanni showed that immediately after vaccination the animal is not fully protected, though its serum may contain antirabic qualities, while later the animal is immune, though its serum may not be able to neutralize the rabies virus. These facts point to a cellular immunity. Results of Antirabic Treatment. — On the whole the results of pro- tective inoculations against rabies are marked. One has only to compare the statistics of mortality after bites from animals suffering from hydro- phobia with those given after any of the methods of treatment employed to see the benefit. As regards the best method to use, the case is dif- ferent. With many methods tried in many lands on a large number of cases, it would seem that we should be able by this time to determine their comparative worth. But the trouble is that the improvement on the whole is not great and the statistics are not kept uniformly or minutely enough to draw trustworthy comparisons. A slight decrease in mortality has been shown in the statistics from most of the antirabic institutes of the world. 590 PATHOGENIC MICROORGANISMS But these figures tell us little about the actual value of the different methods. In order to be able better to judge, the statistics should uni- formly give many more details. Some institutes give such details, others do not. tfntil some such scheme as the following is carried^ out by all, we must change cautiously a treatment that has already given good results. 1. Diagnosis of biting animal: (a.) Rabies, (b) probably rabies, (f) questionable, (d) not rabies, (e) nothing known. 2. Manner of making diagnosis: (o) B3' animal inoculation, {b) by microscopic examination, (<") by clinical diagnosis. ■3. Site and character of bites (e.g., number, depth, laceration, pro- tected by clothing, etc.) : (rt) Head, (fe) hands, (c) other parts of body. 4. Time elapsing between bite and beginning of treatment. 5. Method of treatment used. 6. Complications during or after treatment, particularly paralysis. 7. Character and time of death. That the time after the bite makes a great difference is shown by the following table : Time intervening between bite and beginning treatment. Babes . . 1 to 2 days . 3 to 5 days . 5 to 6 days . Diatroptuff . 1 week 2 weeks , 3 weeks . Immunity. — The immunity in human beings produced by the anti- rabic treatment apparently lasts a variable time. That it may not last more than fourteen months is shown by the history of one of our cases. The patient was an assistant in a hospital for dogs. He was given eighteen days' treatment after a light wound in the hand from a rabid dog. Four- teen months later he came down with typical hydrophobia. Since his treatment he had become very careless with cases of rabies, exposing wounded hands to saliva because he considered himself immune. He was warned that there might be danger. Six weeks before his death he put a wounded hand into the mouth of a rabid animal. There is little doubt but that this is a case of reinfection after loss of protection from the treatment rather than one of delayed hydrophobia. Marie has found complete immunity in dogs eighteen months after treatment. ni Effects of Treatment.— L(;)(:.\L. — There is only slight local dis- (■(iinfort, iucTcased a little if the emulsion (.'ontains glyceriji. ' During the second week an erytiicma often appears about the point of inocula- tion, which Stirason regards as a manifestation of hypersusceptibility to foreign nerve tissue. It disappears in a few days. Number of cases treated. Death. Percentages 3,406 2,541 809 3 2 1 0.088 0.077 0.124 4,602 961 313 26 16 10 0.560 1.660 3.190 RABIES 591 CoNSTiTUTiON.\L. — Ever since the beginning of treatment occasional non-fatal affections of the nervons system have been reported, which occurred during or shortly after the course of treatment. These have varied in degree all the way from a slight neuritis, through paraplegias to paralyses of various parts of the body. Very occasionally the paralyses are marked and the patient dies. Cases of true paralytic rabies which may occur within the period required for the establishment of im- munity by the treatment must be differentiated from cases occurring as a result of treatment. Simon pubhshed an extensive report of 84 cases occurring during the years ISSS to 1911 inclusive, and mentions a few others about which he could not get sufficient data. Simon classifies the cases collected by him according to the diagnosis of the biting animal, with the mortality in each group as follows: Positive group. Number Per cases. cent. Probable group. Number Per cases. cent. Questionable group. Number Per cases. cent. Negative group. Number Per eases. cent. Not known group. Number Per cases. cent 25 (2) 29.76 11 13.0 (4) 21 25 (5) 17 20.23 (3J 10 11.9 (5) Nineteen deaths occurred, as seen from the figures in parentheses, or 22 per cent, of the 84 cases. In analyzing the effect of different methods of treatment on paralyses, Simon gives the following summarj': Number of cases treated. Cases of paralysis. Proportion CLissic Pasteur method . Modified Pasteur method Hogyes method 32,676 8,657 51,417 6 16 3 1 : 5446 1: 541 1 : 171.39 It is seen that the number of paralyses following the Hogyes method are markedly less than those following the other methods. From the studies so far made of these paralyses the possibility of there being different causes for different cases cannot yet be ruled out. The chief theories advanced as to factors in producing the condition are six: 1. Due to "laboratory rabies" from the fixed virus vaccine inocu- lated. 2. Due to "modified rabies" resulting from the treatment on the street virus infection. 3. Due to a toxin produced by the rabies organisms. 4. Due to infection with extraneous organisms introduced with the virus during treatment. 5. Due to psychologic disorders. 6. Due to the inoculation of a foreign ])rotei(l with a suljsecpient anaphylactic reaction. The Cauterization of Infected Wounds. — We believe that in cases in which the Pasteur treatment cannot be applied great benefit may be tlerived from the correct use of cauterization with jwming nitric acid, 592 PATHOGENIC MICROORGANISMS even twenty-four hours after infection, and tliat even in cases in wliich tlie Pasteur treatment can be given, an early cauterization will be of great assistance as a routine practice and should be very valuable, as the Pasteur treatment is frequently delayed several days for obvious reasons, and then does not always protect. In the case of small wounds all the treatment probably indicated will be thorough cauterization with nitric acid within twelve hours from the time of infection. Our experience in dealing with those bitten by rabid animals goes to show that physicians do not appreciate the value of thorough cauterization of the infected womids. Preventive Measures in Animals. — Far more important than any treatment, curative or preventive, for hydrophobia in man is the preven- tion of rabies in dogs, through which this disease is usually conveyed. Were all dogs under legislative control and the compulsory wearing of muzzles rigidly enforced for two years where rabies prevails, hydro- phobia would practically be stamped out. This fact has been amply demonstrated by the statistics of rabies in countries {e. g., England) where such laws are now in force. New York City is now trying to enforce such laws. YELLOW FEVER. Yellow fever is an acute infectious disease of tropical countries with no characteristic lesions except jaundice and hemorrhage. Other lesions that exist are those common to toxemia. One attack usually produces complete immunity. Historical Note. — There have been many extensive studies on the etiology of this disease with numerous announcements of the discoverjr of its specific cause. Not one of the latter, however, has been corroborated. The Bacillus ideroides of Sanarelli (1897), found in the circulating lilood and in the tissues of most yellow fe^^er patients, was thought by many to be the real organism, and for some time it was the subject of most minute studies with the result that it, too, has been placed with the rejected organisms. The epoch-making investigations of the United States Ai'my Commission composed of Walter Reed, James Carroll, Aristides Agramonte, and Jesse W. Lazear (1901), established the truth, that this disease, like malaria, is carried from one infected person to another through the agency of a mosquito. Finley in 1881 was tlie first positively to assert that the mosquito was the transmitter of the disease. He was, however, unable to prove his theory, and it remained for the commission conclusively to show that a distinct species of mosquito carried the infection. The work of the American commission was fully corroborated by the French commission and by other worlcers. The principal facts established liy the commission have been summed 11]) by Goldberger as follows: 1. Yellow fever is transmitted, under natural conditions, only by the bite of a mosciuito {Aedes calopus) that at least twelve days before had fed on the blo(jd of a person sick with this disease during" the first three days of his illness. YELLOW FEVER 59:? 2. Yellow fever can be produced under artificial conditions by the subcutaneous injection of blood taken from the general circulation of a person sick with this disease during' the first three days of his illness. 3. Yellow fever is not conveyed by fomites. 4. Bacillus ideroides Sanarelli stands in no causative relation to yellow fever. Though the specific parasite remains yet undiscovered, facts have been brought out by these studies which give some idea of its character. 1. It seems to require two hosts (a mammal and an arthropod) for the completion of its life cycle (analogies, Plasmodium malariw, Babesia bigoniiium). 2. There is a definite time after the bite of the mosquito before the blood of the person bitten becomes infective (average five days), and a definite time that the blood remains infective (three days). 3. The blood during these three days is still infective after passing through the finest-grained porcelain filters (Chamberland B and F). 4. The blood loses its ^■irulence quickly (forty-eight liours) when exposed to the air at a temperature of 24° to 30° C. When protected from the air by oil and kept at the same temperature it remained virulent longer (five to eight days). Heated for five minutes at 55° C. it becomes non-virulent. 5. The bite of an infected mosquito does not become infectious until twelve days (at a temperature of 31° C.) after it has bitten the first patient. The cause of the disease still remains undiscovered, notwithstanding much study of human blood and other tissues and of infected mos- quitoes. The infective blood-filtrates show nothing with the dark- field illumination except small motile granules similar to those found in healthy persons. Certain facts relating to the disease seem to point to protozoa as the cause; for instance, the necessity for a second host and the long incubation time required before that host becomes infective after biting a yellow-fever patient. Seidelm's paraplasma has been shown by Agramonte to be tissue changes. The higher monkeys seem to be susceptible, though no complete experiments have been made with them. The Yellow-fever Mosquito (Figs. 202-21)7). — The name Stegomyia for this small tropical mosquito was suggested by the English entomologist Theobald, who separated this genus from the genus Culex, with which it was formerly classed. It was first given the specific name Fasciata, but Blanchard proved that this had already been used and the name Calopiis (Meigen, 1S18) was found to be the proper one. Later the genus Stegomyia was shown to be invalid and the organism now goes by the name .ledes caJoptis ^leigen. Salient characteristics of ^\edes are: (1) The palpi in the male are as long or nearly as long, as the proboscis; in the female the palpi are uniformly less than one-half 38 • m PA TIIOGENIC MICROURGA NISMS as loiii^'- (2) the legs are destitute of erect scales; and are alternately banded white and blaek; (3) the thorax is marked with hnes of silvery scales. Aedes calopus is spread over a wide range of territory, embrac- ing many varieties of climate and natural conditions. It has been found as far north as Charleston, S. C, and as far south as Rio de la Plata. There is no reason to believe that it may not be present at some time or other in any of the intermediate countries. In the United States specimens of Aedes calopus have been captured in Georgia, Fig. 202 The yollow-fuver iiiosquito (Aedvs cnlopus). Adult female. Rlueh enlarged. (Howard.) Louisiana, South Carolina, and eastern Texas. The island of Cuba is overrun A\'ith this insect. The fact that Aedes ccdopus has been known to exist at various times in Spain and other European countries may account for the spread of yellow fever which has f)ccurred there once or twice in former times; the same may be said of the country farther north in the United States, where Aedes calopus has not yet been reported, but which have suffered from invasions of yellow fever. The adult mosquito is so small that mosquito netting of 19 or YEI.LOW FEVER Fig. 203 595 Thu yellow -fi:'\'ur liiusquitu. Adult niak-. Much enlarged. (Huwuid.) Fig. 204 The yellow-fcvcr mos(iuito. Adult female, side view. Much eiilarj^ed. (Howard.) Fig. 20.5 The yello\v-fe\-er mosquito. Egg. Greatly enlarged. (Howard.) 596 PATHOGENIC MICROORGANISMS 20 strands or meshes to the inch is required to keep the insect from entering a place. Brackish water is unsuited for the development^ of Aedes larva;. The species Aedes calopus seems to select an}^ deposit of water which is comparatively clean. The defective drains along the eaves _ of tile roofs are a favorite breeding place in Havana and its suburbs; indoors they find an excellent medium in the water of cups of tin or china into which the legs of tables are usually thrust to protect the_ contents from the invasion of ants, a veritable pest in tropical countries. The same may be said of shallow traps, where the water is not frequently disturbed. Fig. 206 Fig. 207 The yellow-fever mosquito. Lar^'a. Much enlarged. (Howard.) The yellow-fever mosqviito. Pupa. Mueh enlarged. (Howard.) Like other Culicidce, it prefers to lay at night. It is eminently a town insect, seldom breeding far outside of the city limits. Agramonte never found Aedes calopus resting under bushes, in open fields, or in the woods; this fact explains the well-founded opinion that yellow fever is a domiciliary infection. The question of hibernation in the larval stage is important. Agra- monte failed to get larvse that could resist freezing temperature, and YELLOW FEVER T)!)? found that in the case of Acdcs calopiis tliis degree of cold was invariably fatal. The possibility of their being capal)le of life outside their natural element must also be considered from an epidemiologic point of view. The dry season in the countries where this species seems to abound is never so prolonged as completely to dry up the usual breed- ing places. Experimentally, adult larvre removed from the water and placed overnight upon moist filter paper could not be revived the following morning. The question of the life period of the female insect is of the greatest importance when we come to consider the apparently long interval which at times has occurred between the stamping out of an epidemic of yellow fever and its new outbreak without introduction of new cases. The fact is that Aedes calopus is a long-lived insect; one individual was kept by Agramonte in a jar through March and April into May, in all for seventj'-six days after hatching in the laboratory. These mosquitoes bite principally in the late afternoon, though they may be incited to take blood at any hour of the day. They are abundant from JNIarch to September, and even in November Agra- monte was able to capture them at will in his office and laboratory. Tlie mosquito is generally believed to be incapable of long flight unless very materially assisted by the wind. At any rate, the close study of the spread of infection of yellow fe\'er shows that the tendency is for it to remain restricted within very limited areas, and that when- ever it has travelled far beyond this, the means afforded (railway cars, vessels, etc.) have been other than the natural flight of the insect. Experiments have demonstrated that not all mosquitoes which bite a yellow-fe^•er patient become infected, but that of se\'eral which bite at the same time some may fail either to get the parasite or to allow its later development in their body. This condition is similar to that seen in Anopheles, with regard to malaria. The question as to the length of time infected mosquitoes remain dangerous to the non-immune community cannot be definitely answered at present; there is good presumptive evidence that the mosquito may harbor the parasite through the winter and be enabled to transmit in the spring an infection acquired in the fall. There is reason to believe that the mosquito, once infected, can transmit the disease at any time during the balance of its life. Freezing temperature, however, quickly kills the insect. Carrying out preventi\'e measures based on the knowledge gained by the splendid work of the American Army Commission, yellow fever has been practically wiped out of Cuba, the Isthmian Canal Zone, and other infected areas. References. Berry. The Complement Binding Test in Rabies. ,Jour. Exp., Med., 1910, vol. xii. Harris. A Method for the Staining of Negri Bodies. Jour, of Infect, Diseases, 1908, V, .566; also Treatment of Rabies. Hoayes, Lyssa. Nothnagel's Specielle Pathologie u. Therapie, Wien, 1S97. 598 PATHOGENIC MICROORGANISMS Howard. The Yellow Fever Mosquito, Farmers' Bulletin, .547, United States Depart- ment Agriculture, Washington, D, C. Kerr and Siimson, The Prevalence of Rabies in the United States. The .Jour, of the Amer. Med. A.s.soc., 1909, liii, 989. Kraus and Barbara. Dcut. med. Woeh., 1914, xl, 1.507. Marie. L'Etudc cxtn'rimentale de la Ka^e, Paris, 1909. Manouelian. Ann. Inst. Past., 1914, xxviii, 2.3.3. Noguchi. .Jour. Exp. Med., 1913, xviii, 314. OUo. Gelbfieber. In Kolle and Wassermann's Handbueli d. path. Mikroora, 1913, 2d ed., Jena. Poor and Sleinhardl. Jour. Inf. Dig., 1913, xiii, 203. Reed and Carroll. Jour. Exp. Med., 1900, v, 215. Heed and Carroll and Agramonte. Jour. Amer. Med. Assoc., 1901, xxxvi, 413. The Yellow Fever Institute Bulletin, No. 16, Yellow Fever, Etiology, Sjmiptoms, and Diagnosis, bj' Goldberger, gives a good resume with full literature to 1907. Volpino. Presse Med., 1914, p. 79. Williams. Rabeis, in Forscheimer's Therapeusis, 1914, vol. v. See. Ed., New York. With references. Williams and Lowden. Jour, of Infect. Dis., 1906, iii, 460, with full list of references to date on Negri bodies. PART Til. APPLIED MlUROBIOLOfJY CHAPTER XL VI. THE BACTERIOLOGICAL EXAMINATION OF WATER, AIR, AND SOIL. THE CONTAMINATION AND PURIFICA- TION OF WATER. THE DISPOSAL OF SEWAGE. The bacteriological examination of water is undertaken for the purpose of discovering whether any path(jgenic bacteria are liable to be present. The determination of the number of bacteria in water was for a time considered of great importance, then it fell into disrepute, and the attempt was made to isolate the specific germs of diseases which were thought to be water-bfirne. At first these attempts seemed very successful in that supposed typhoid bacilli and cholera sinrilla were found. Further study re^'ealed the fact that there were ccjmmon water and intestinal bacteria which were so closely allied to the above forms that the tests applied did not separate them. Even the use of a serum from an animal immunized to injections of the typhoid bacillus was found to agglutinate some other bacteria in high dilutions; so that the test as usually carried out was insufficient. With the latest tech- nique it is probable, but not certain, that absorption tests with the serum from an immunized animal will be sufficient to decide whether a suspected bacillus is the typhoid bacillus or not. The improbability of getting typhoid bacilli from suspected water except under unusually favorable conditions caused a return to the estimation of the number of bacteria in water and above all to the estimation of the number of intestinal bacteria. It is known that the group of colon bacilli ha\'e a somewhat longer existence than the typhoid bacilli, and as the colon bacilli come chiefly or wholly from the intestinal passages of men and animals, it was fair to assume that typhoid bacilli coukl not occur without the presence of the colon bacillus except in rare cases, as, for example, pollution with urine alone. The latter could, of course, occur abundantly without the typhoid bacillus. During the past few years the attention of sanitarians has been seriously devoted to the interpretation of the presence of smaller or larger numbers of colon bacilli in water, until at present, upon the quantitati\'e analysis (measuring, within certain limits, decom]xising GOO APPLIED MICROBIOLOGY organic matter) and the colon test (indicating more specifically pollution derived from intestinal discharges of man or animals), the bacteriological analysis of water is based. The determination of the ninnber of bacteria is also of value. Technique for Quantitative Analysis. — The utmost care is necessary to get reliable results. A speck of dust, a contaminated dish, a delay of a few hours, an improperly sterilized agar or gelatin, a too high or too low temperature, maj^ introduce an error or variation in results which would make a reliable test impossible. Collection of Samples. — The small sample taken must represent the whole from which it was drawn. If a brook-water, it must be taken some distance from the bank; if from a tap, the Avater in the pipes must first be run off, for otherwise the effect of metallic substances will invalidate the results; if from lake or pond, the surface scum or bottom mud must be avoided, but may be examined separately. The utensils by which the avater is taken should be of a good quality of glass, clean and sterile. From a brook the water can be taken directly into a bottle, the stopper being remoA'ed while it fills, avoiding the surface film and its attending excessive numbers of bacteria; from a river or pond it can be taken from the bow of a small boat, or from a bottle properly fastened on the end of a pole so as to avoid con- tamination; from a well a special apparatus has l)een deA'ised by Abbott, where a bottle with a leaded bottom is so held that when lowered to the proper depth a jerk will remove the cork and allow the bottle to fill. The same device or another accomplishing the same purpose can be rigged up readily by anyone. The sample of water should be tested as soon as possible, for the bacteria immediately begin to increase or decrease. In small bottles removed from the light preda- tory microorganisms and many bacteria begin to increase, and among these are the members of the colon group. Thus, the Franklands record a case in which in a sample of well-A\'ater kept during three days at a moderate temperature the bacteria increased from 7 to 49.5,000; while Jordan found that in a sample the bacteria in forty-eight hours fell from 535,000 to 54,500. In a sample we kept at room" temperature the colon bacilli during twenty-four hours increased from 10 to 100 per c.c. The only safe way to prevent this increase is to plate and plant the water in fermentation tubes within a space of one or two hours or to keep it at a temperature under 5° C. (41° F.). If it cannot be kept cold, it is far better to make the cultures in the open field or in a house rather than to wait six to twelve hours for the conveniences and advantages of the laboratory. The third matter of great importance is the adding of proper amounts of water to the broth in the fermentation tubes and the media for plant- ing. Usually 1 c.c, 0.1 c.c, and 0.01 c.c. are added to the fermenta- tion tubes and to 10 c.c. of the melted nutrient agar or gelatin. If possible, duplicate tests should always be made. When it is desired to know whether colon bacilli are ])resent in larger amounts than 1 c c (luantities as great as 10 or 100 c.c. can be added to bouillon, and then TECHNIQUE FOR QUANTITATIVE ANALYSIS (iOl lifter a few hours 1 c.c. added to fermentation tubes. Less than twenty colonies and more than two Inuidred on a })late gi\e inaccurate counts, the smaller number being too few to judge an average anil the larger number interfering -with each other. When as man\' as 10,000 colonies develop in the agar contained in one plate, it will be founil that there will develop in a second plate containing but one-tenth the amount of water from 20 to 50 per cent, as many colonies. This shows that the crowding of the colonies had prevented the growth or caused a fusion of all but one-fifth to one-half of them. The chemical composition of the medium on which the bacteria are grown affects the result of the analysis. Nutrient 1.5 per cent, agar gives slightly lower coimts than gelatin, but on account of its con- venience in summer and its greater uniformity it is being more and more generally used for routine quantitative work. There is an optimum reaction for e^'ery \'ariety of bacteria, and to ensure uniformity the committee of the American Public Health Association adopted a standard reaction of +1 per cent.,' which was as near as possible to the average optimum for water bacteria. Such a uniform standard is a necessity to secure comparability of results. At best only a certain proportion of bacteria develop, and it is only important that our counts represent a section through the true bacterial flora which fairly represents the cpuck-groM'ing sewage forms. Comparability is the vitally essential factor. The temperature at which the bacteria develop is of great importance, and they should be protected from light. The access of oxygen which prevents the growths of anaerobes must also not be forgotten. As a rule, the plate cultures are developed for four days at 20° to 21° C, and for twenty-four or forty-eight hours at incubator temperature. Some bacteria do not develop colonies in four days, but these are neglected. The number of bacteria growing at room temperature is usually much greater than those growing at 37°. As all the intestinal groups of bacteria grow at body temperature, while many of the water types do not, some investigators believe it important to develop the bacteria at both temperatures so as to compare the results. We have not found this to be of any advantage when tests are also made for the colon group of bacilli. The lactose broth is placed at 37° C. for the development of the colon bacilli. The fermentation tubes not showing gas are recorded as negative and usually discarded. Those showing gas are suspected to contain colon bacilli. To a number of tubes containing melted litmus-lactose agar at about 44° C. are added 1, 0.1, and 0.01 loop of the culture fluid. Plates are poured and the whole placed in the incubator. The Bacillus coli ferments lactose and thus produces acid, so that if colon bacilli are present we have a number of red colonies on a blue field. Later, if many colon bacilli were present, the whole medium becomes acid. At forty-eight hours, on account of alkali being produced by the formation of NH3, the blue may return. If > To phenolphthalein. cm APPLIED MICROBIOLOGY after inspection red colonies are seen, four or five are picked and planted into lactose bouillon and other media. Litnuis lactose agar is frequently used for the original plating of water sanii)les, the absence or presence of acid-producing colonies being thus inimediatel\' noted. The results of these tests are all more or less presumptive evidence of the bacilli belonging or not belonging to the intestinal colon bacillus group. Un- fortunately there are some types of bacilli growing in the soil which resemble them, if it is necessary to be more accurate. The colon-like cultures should be subjected to the Vosges reaction (page 120), and should be kept for one month at 20° C. in gelatin before a decision is made. Colon bacilli do not liquefy gelatin nor give the Vosges re- action. There are a few colon-like bacilli in the intestinal tract that give the Vosges reaction. For a more complete understanding of the technique and the interpretation of results of the bacteriological examination of water see Elements of Water Bacteriology — Prescott and Winslow. For the characteristics of the colon bacilli the INIassachusetts State Board of Health uses six media — gelatin, lactose agar, dextrose broth, milk, nitrate solution, and peptone solution, determining, respectively, absence of liquefaction, production of gas, turbidity, coagulation with- out liquefaction of the coagulum, production of nitrite, and of indol. Lactose-bile-peptone solution has been much used. In badly con- taminated waters this has a distinct advantage in that the bile inhibits many varieties of bacteria more than those of the colon-typhoid group. In good waters the results are very similar from the lactose-peptone and lactose-bile-peptone solutions. Significance of the Colon Bacillus. — The colon test has been received by the majority of engineers and practical sanitarians with great satis- faction, anfl has l)een applied with confidence to the examination not only of water, but of shell-fish and other articles of food as well. On the other hand, some have denied its value. Bacteriologists have found bacilli like certain members of the colon group in apparently unpolluted well-water. The discovery that animals have colon bacilli identical in the usual characteristics studied with those of man has complicated matters. Thus a fresh hillside stream may be loaded with colon bacilli from the washings of horse or cow manure put on the fields through which it runs or polluted by a stray cow or horse. Swine, hens, birds, etc., may contaminate in unsuspected ways. The number of colon bacilli rather than their presence in any body of surface water is therefore of importance. In well- and spring-water the presence of colon bacilli indicates contamination. The absence of colon bacilli in water proves its harmlessness so far as bacteriology can prove it. When the colon bacillus is present so as to be isolated from 1 c.c. of water in a series of tests, it is reasonable proof of animal or human pollution and the conditions should be investigated. Ten colon bacihi in I c.c. indicates serious pollution. Surface waters from inhabited regions will always contain numerous colon bacilli after a heavy rain, storm, or shower. The washings from roads and cidtivated fields con- tain necessarily large numbers. Winslow reports that in only two INTERPRETATION OF THE QUANTITATIVE ANALYSIS OOo out of fifty-eight samples of presumably non-polluted waters did he get eolon haeilli in the 1 c.e. sani]>les. Even in twenty-one stagnant pools he only found c'olon baeilli in five of the 1 c.e. samples. The experienee of all who ha\e studied the sulijeet i)raetieall\' is that in tlelieaey the eolon test surpasses chemieal anal.ysis: in eon- stancy and definiteness it also exeels the quantitative haeterial count. All these tests must, however, he supplemented by inspection. Interpretation of the Quantitative Analysis. — The older experi- menters attempted to establish arbitrary standards by which the salutary quality of water could be fixed automatically by the number of germs alone. This has been largely given up. Dr. Sternberg con- siders that a water containing less than 100 bacteria is presumably from a deep source and uncontaminated by surface drainage; that one with 500 bacteria is open to suspicion; and that one with over 1000 bacteria is presumably contaminated by sewage or surface drain- age. Even this conservative opinion must be applied M'ith caution. The source of the sample is of \'ital imj^ortance in the interpretation; thus, a bacterial count which would condemn a spring or well might be normal for a ri\'er. In woodland springs and lakes se^•eral hundred bacteria per c.e. are frequently found. In lakes the point at which the sample is taken is of great importance, as the bacterial count varies with the distance from the shore and with the depth. The weather also is an influence, since the wind causes currents and wa^'es which stir up the bottom mud, bringing up organisms which have been sedimented. Rains greatly influence streams by fltxxling them with surface water bringing a huge number of bacteria at times. The season of the year is an important factor. The counts are highest in the winter and spring months, and lower from April to September. The following figures illustrate this point: Water. Observers. Year. Jan. New York City tap-water Houghton 1904 890 Boston tap-water . . . Whipple 1892 135 Merrimac River tap-water Clark 1899 4900 The winter and spring increases are not exceptions to the rule that high numbers indicate danger, but an indication of its truth; for it means a melting of the snow and a flow^ of surface water into the streams without the usual filtering soil filtration. A number of severe epidemics of typhoid fever have been produced in this w'ay. It is only the fact that typhoid fever is at a minimum in winter that prevents more frequent pollution. Although, as a rule, a series of tests are necessary to pass judgment on a water, a single test may be very important. A large increase in the number in tap-water a day after a storm points unerringly to surface pollution, and if towns exist in the water-shed, to street and sewer pollution. The Croton water frequently jumps from hundreds to thousands after such a storm. In a typhoid epidemic at Newport, Winslo^^- reports that a test of the water supplj' showed but oiJ-l bacteria per cubic centimeter. Feb. Mar. April. May. Juno 1100 650 240 350 370 211 102 52 53 86 .5900 0300 2901) 1900 3500 G04 APPLIED MICROBIOLOGY hut one from a well showed 6100. The suspicion arouseout thirteen times a year. _ Mechanical filtration plants find considerable favor where clarifica- tion as well as bacterial purification is desired. A coagulant such as sulphate of aluminum is employed and forms in the water a flocculent precipitate wliicli carries down with it all suspended matter; ] 25,000,000 or more gallons of water caTi lie filtered on an acre daily, l)ut the filters must be •washed air. Forests decrease the number of bacteria. On iiigh mountains and on the sea far from land i)acteria are very scarce. The bacteria that nuiltiply in the soil of street and country are almost entirely saprophytic types. Sunlight and drying rapidly destroy bacteria. In dwellings the bacterial content depends on many factors, of which the chief are the opening (tf windows to the outside dust-laden air, the cleanliness of the dwelling, and the amount of stirring up of the dust by sweeping. It is almost impossible to separate the eii'ect of the bacteria which we inhale from that of the dust particles which they accompany. Both probably act as slight irritants and so predispose to definite infections. Pathogenic bacteria are only occasionally present in the air. The practical results obtained from the examination of air for pathogenic bacteria have been slight. \Ye know that at times they must be in the air, but unless we purposely increase their numbers they are so few in the comparatively small .imount of air which it is practicable to examine that we rarely find them. Examination of dust, however, in hospital wards and sick-rooms, in places where only air infection was possible, have occasionally re\ealed tubercle bacilli and other pathogenic bacteria. Although very light they generally settle to the ground. The importance of air infection in the spread of infectious diseases is considered as much less than it was formerly thought. The simplest methoil of searching for the varieties of bacteria in the air and their number in any place is to expose to the air for longer or shorter periods nutrient agar spread upon the surface of the Petri dish. After exposure the plates are either put in the incubator at 37° C. or kept at room temperature. The more careful ciuantitative examination is made by drawing a given quantity of air through tubes containing sterile sand, which is kept in by pieces of metal gauze. When the operation is completed the sand is poured into a tube con- taining melted nutrient gelatin or nutrient agar, and after thoroughly shaking, the mi.xture is jjoured into a Petri dish and the bacteria allowed to develop, either at '^7° or 2o° C, according as the growth of the parasitic or saprophytic \'arieties is desired. Instead of agar or gelatin, ascitic broth or animals may be inoculated. Such examinations are occasionally made of the air of theatres, crowded streets in cities, etc. They gi^•e interesting, but hardly valuable, results. BACTERIOLOGICAL EXAMINATION OF THE SOIL. The subject from its agricultural side is considered on p. 627. Speci- mens of deep soil can be gathered in sterile, sharp-pointed, sheet-iron tubes. Through the examination, we wish to learn either the number of bacteria or the important \arieties of bacteria present. To estimate the number, snudl fractions of a gram are taken and planted in luitrient agar or in special media contained in Petri dishes. Anaerobic as well as aerobic cultures should be made. 39 610 . APPLIED MICROBIOLOGY According to Houston, uncultivated sand soil averages 10(),0(J0 bacteria per gram, garden soil 1,5()0,()()0, and sewage-polluted 115,- 000,000. The most important bacteria to be sought for are bacilli of the colon group and streptococci. Both of these suggest fairly recent excremental pollution. The period during which typhoid bacilli remain alive in soil is variable, since it depends on so many unknown factors and differs so in different places. The typhoid bacilli probably rarely increase in the soil and probably rarely survive a month in it. The main danger of soil bacteria is their being washed into water supplies by rains or carried to them by the wind. Reaction of Vosges and Proskauer. — Grow the culture in 1 per cent, glucose-peptone water in a fermentation tube for four days at 37° C. Add 1 c.c. of 50 per cent. KOII solution to the open end and allow the mixture to stand for two days at room temperature. With certain varieties of bacteria a red color like that of eosin de^'elops after twentj'- four to forty-eight hours. With true B. coll this color does not develop. CHAPTER XLVIT. THE BACTERIOLOGY OF MILK L\ ITS RELATION TO DISEASE. From the stand-point of the dairy many of the different varieties of baeteria found in milk are of importance, which have httle or no medical interest. We have space here only to consider the bacteri- ology of milk so far as it is related to health and disease. The saprophytic bacteria taken collectively have importance because one can determine from their number something as to the care taken in handling the milk and also because, when numerous, they produce chemical changes in the milk which are harmful for infants. Numerical Estimation of Bacteria. — The number of bacteria in a cubic centimeter of milk is usually estimated from the colonies de\'eIoping in nutrient agar plate cultures during a period of forty-eight hours when kept at a temperature of 37° C. This allows in market milk in which bacteria have de\'eloped at low temjjerature only a certain proportion of the varieties of bacteria to develop colonies. Sometimes fully twice as many colonies develop at 20° to 27° C. during three to five days as at 37° for two days. The a(h'antages of the shorter time and of uniformity ha\-e led to the arloption of the technicjue given in Part I. Any culture method necessarily imderestimates their number, as many of the bacteria remain after vigorous shaking in pairs or small groups. In order to overcome this and also to note the morphological types, the direct microscopical examination of smears of the sediment has been urged. A great practical objection to this is that, if a heated milk is examined, the dead as well as the living bacteria are counted. This method has, howe\'er, great ach'antages at the creamery or farm in that one can immediately tell M'hether a sample has few or many bacteria and also note the presence of streptococci and leukocytes. The microscopic count gives from two to ten times as many bacteria as the routine culture method. Smear Method for Direct Examination of Milk. — The Prescott- Breed method is the most accurate. That of Slack is also used. In the Slack method one cubic centimeter is withflrawn and put into a tube of small caliber ha\'ing two rubber corks and is centrifugalized for ten minutes. After centrifugalization the upper cork is removed and the supernatant cream and luilk are gently poured off; the Icnver cork which holds the sediment is then remo\'ed and the sediment is spread as e\'enly as possible on slides in areas of two square centimeters upon which a drop of sterile water has been previously placed. 612 APPLIED MICROBIOLOGY Prescott and Breed' explain their smear method briefly as follows: The sample of milk to be examined is shaken thoroughly and 0.01 c.c. is withdrawn by means of a specially constructed capillary pipette. The milk so obtained is spread evenly over an area of one square centi- meter on an ordinary glass slide. These areas may be easih* determined by placing the glass slide over paper or glass on which areas of this size have been accurately ruled out. More satisfactory results can be obtained by using circular instead of square areas. Duplicate smears should be made on the same slide. The milk is then dried with gentle heat, the fat dissolved out with xylol or other fat solvent, the smear again dried, immersed in alcohol for a few minutes to fix the film, again dried and stained with methylene blue or other stain. In the Slack a more simple method for fixing and staining is used in their laboratory. The smears are allowed to dry in the air, fixed in methyl alcohol and then stained with a watery solution of methylene blue. Alkaline stains or others which attack the casein and loosen the smear must be aA'oided. The glassware used must be cleansed but need not be sterilized as the bacteria have no chance to increase in number. The counting of the bacteria is done by a microscope and an oil immersion lens. If the diameter of the field is so adjusted, by means of the draw tube, that it equals O.Ki mm., then each field of the micro- scope covers approximately one-five-thousandth (0.0002) of a square centimeter. On this basis, each bacterium seen in a field taken at ran- dom represents 500,000 per c.c. if they are evenly distributed. However, it is impossible to distribute them evenly so that at least 100 fields of the microscope should be counted if accurate results are required. The total number of bacteria seen in ten fields multiplied by 50,000 or the total number seen in 100 fields multiplied by 5000 gives approxi- mately the total number of bacteria per cubic centimeter. Identification of Bacteria. — The milk is plated in a 2 per cent, lactose- litmus nutrient gelatin or agar, and the bacteria, after development of colonies, isolated and grown upon the usual identification media. The pathogenic properties of the dift'erent bacteria can be tested by intraperitoneal and subcutaneous inoculation in guinea-pigs with 2 c.c. of a forty-eight-hour broth culture, and by feeding young kittens for several days with ?> to c.c. daily of a twenty-four-hour broth culture by means of a medicine drojjper. Varieties. — Bacteria in milk can be divided into two great groups — those which get into the milk after it lea^'es the udder and those which come from the cow. The first group comprises bacteria from dust, hands, milking pails, strainers, etc. The extraneous bacteria are of importance because they produce changes in the chemical composition of the milk when they have develojjed in great munbers. The mmiber of bacteria in any sample of milk depends on three factors: the numlier deposited in the milk from the cow's udder, from tiie air, and utensils; the time during ' rcnljal, f. H;ik(., I'ani.silrij. n. Iiilrldionskraiikhcit.cu, 1911, Band xxx, Heft IG/IS. VARIETIES fil3 which they huvc (k'vclojH'd, and the teniiKTature at whicli the milk has stmul. The hist is periiaps the most imiH)rtaiit factor. The attcm])t was made duriiit;- a period of one year to connect illness in infants and children with special varieties of sajtrophytie hacteria, in milk. As a matter oi fact, no such connection was made out. From the milks altog'ether 2oi) varieties of hacteria were isolated and studied. These 2o9 varieties, having some cultural or other differences, were di\'ided into the .'31 classes, each class containing from 1 to 39 more or less related organisms. As to the sources of bacteria found in milk, we made sufficient exi)eri- ments to satisfy us that they came chiefly from outside the udder and milk-ducts. Bacteria were isolated from \arious materials which under certain conditions might be sources of contamination for the milk, and the cultures compared with those taken from milk. Thus there were obtained from 20 specimens of hay and grass, .31 varieties of bacteria; from 15 specimens of feces, manure, and intestinal contents, 28 varieties; from 10 specimens of feed, 17 varieties. Of these 76 varieties there were 42 which resembled closely those from milk — viz., 11 from grass or hay; 20 from manure; 5 from feed. During the investigation a number of the ^'arieties isolated from milk were shown to be identical with types commonly found in water. From the few facts quoted above and from many other observa- tions made during the course of the work, it would seem that the term "milk bacteria" assumes a condition which does not exist in fact. The expression would seem to indicate that a few varieties, especially those deri\-ed in some way from the cow, are commonly found in milk, which forms ha^"ing entered the milk while still in the udder or after its withdrawal, are so well fitted to develop in milk that they outgrow all other varieties. As a matter of fact, it was found that milk taken from a number of cows, in which almost no outside contamination had occurred, and plated immediately, contained, as a rule, very few bacteria, and these were streptococci, staphylococci, and other varieties of bacteria not often found in milk sold in New York City; the temperature at which milk is kept being less suitable for them than for the bacteria which fall into the milk from dust, manure, etc. A number of speci- mens of fairly fresh marked milk averaging 200,000 bacteria per cubic centimeter were examined immediately, and again after twehe to twenty-four hours. In almost every test the three or four predominant \'arieties of the fresher milk remained as the predominant varieties after the period mentioned. The above experiments seem to show that organisms which have gained a good percentage in the ordinary commercial milk at time of sale will be likely to hold the same relative place for as long a period as milk is usually kept. After the bacteria pass the ten or twenty million a change occurs, since the increasing acidity inhibits the growth of some forms before it does that of others. Thus some varieties of G14 APPLIED MICROBIOLOGY the lactic acid bacteria can increase until the acidity is twice as great as that which inhibits the growth of many bacteria. Before milk reaches the curdling ])()int, the bacteria may have reached over a billion to each culiic centimeter. For the most part specimens of milk from different localities showed a difference in the character of the bacteria present, in the same way that the bacteria from hay, feed, etc., varied. Even the intestinal contents of cows, the bacteriology of which might be expected to show common characteristics, contained, besides the predominating colon types, other organisms, which differed widely in different species and in different locahties. Cleanliness in handling the milk and the temperature at which it had been kept were also found to have a marked influence on the predominant varieties of bacteria present. Pathogenic Properties of the Bacteria Isolated. — Intraperitoneal injection of 2 c.c. of broth or milk cultures of about 40 per cent, of the varieties tested caused death. Cultures of most of the remainder produced no apparent deleterious effects e\en when injected in larger amounts. The filtrates of both cultures of a number of varieties were tested, but only one was obtained in which poisonous products were abundantly present. Death in guinea-pigs weighing 800 grams followed within fifteen minutes after an injection of 2 c.c. ; 1 c.c. had little effect. As bacteria in milk are swallowed and not injected under the skin, it seemed wise to test the effect of feeding them to very young animals. We therefore fed forty-eight cultures of 139 varieties of bacteria to kittens of two to ten days of age by means of a glass tube. The kittens received 5 to 10 c.c. daily for from three to seven days. Only one culture produced illness or death. Very young guinea-pigs were fed in the same manner with similar results. A full report on the identification of bacteria met with in this investigation can be found in an article by Dr. Letchworth Smith in the 1902 Annual Report of the Department of Health of New York City. After Hve years of effort to discover some relation between special varieties of bacteria found in milk and the health of children the con- clusion has been reached that neither through animal tests nor the isolation from milk given sick infants have we been able to establish such a relation. Pasteurized or "sterilized" milk is rarely kept in New York longer than thirty-six hours, so that varieties of bacteria which after long standing develop in such milk did not enter into our problem. The harmlessness of cultures given to healthy young kittens does not of course prove that they woukl be equally harmless in infants. Even if harmless in robust infants, they might be injurious when summer heat and previous disease had loM'ered the resistance and the digestive power of the subjects. Streptococci in Relation to Disease. — In an investigation by Dr. D. H. Bergey connection between diarrhea and pus and streptococci was sometimes foimd. The results f)f this investigation appear to warrant the following conclusions : DELETERIOUS EFFECT OF BACTERIA IN MILK ON INFANTS (115 1. The oceurrencc of an rxcrssixc ininilKT of ii'iikdcytcs in cows' milk is prohahly always associated with the picsciur in the uddci of some intlainmatory reaction hrousht ahont hy the presence of some of the ordinary pyogenic bacteria, esi)eciall.\ of streptococci. 2. When a cow's udder has once become infected with the pyogenic bacteria, the disease tends to persist for a long time, probably extending over several periods of lactation. •3. Lactation has no causati\-e influence per se upon the cellular and bacterial content of cows' milk, though it probably tends toward the aggravation of the disease Mdien the udder is once infected. It is impossible to differentiate in routine milk examinations the pathogenic streptococci of diseased cows from saprophytic varieties. Thus it happens that a milk which contains great numbers of strep- tococci may or may not be more dangerous than one which contains an equal number of other a])parently less harmful bacteria. The Deleterious Effect of Bacteria in Milk on Infants. — We have tested this ourselves in the following way: During each of the summers of 1902, 190o, and 1904 a special lot of milk was modified for a group of fift\- infants, all of whom were under nine months of age, and dis- tributed daily. To one half the milk was given raw; to the other half a similar milk heated at 60° for twenty minutes. Tlie modified milk was made from a fairly pure milk mixed with ordinary cream. The bacteria contained in the milk numbered on the average 45,000 per cubic centimeter, in the cream 30,000,000. The modified raw milk taken from the bottles in the morning averaged 1,200,000 bacteria per cubic centimeter, or considerably less than the ordinary grocery milk; the Pasteurized, about 1000; taken in the late afternoon of the same day thev had, respectively, about 20,000,000 and 50,000. Twentj'-one predominant ^^'lrieties of bacteria were isolated from six specimens of this milk collected on different days. The varieties represented the types of bacteria frequently found in milk. The infants were selected during the first week in June, and at first all were placed on Pasteurized milk. The fifty infants which had been selected were now separated into two groups as nearly alike as possible. On the fifteenth of June the milk was distributed without heating to one half the infants, the other half receiving as before the heated milk. In this way the infants in the two groups received milk of identically the same quality, except for the changes produced by heating to 165° F. for thirty minutes. The infants were observed carefully for three months and medical ad-s'ice was given when necessary. When se\'ere diarrhea occurred barley-water was substituted for milk. The first season's trial gave the following results: Within one week 20 out of 27 infants put on raw milk suffered from moderate or severe diarrhea; wliile during the same time only 5 cases of moderate and none of severe diarrhea occurred in those taking Pasteurized milk. Within a month 8 of the 27 had to l)e changed from raw back to heated milk, because of their contiiuied illness; 7, or 25 per cent., did well (■)] 6 APPLIED MICROBIOLOOY all summer on raw milk. On the other hand, of those recei^'ins the Pasteurized milk, 75 per cent, remained well, or nearly so, all summer, while 25 per cent, had one or more attacks of se^'e^e diarrhea. There were no deaths in either group of cases. During the second summer a similar test was made with 45 infants. Twenty-four M'ere put on raw modified milk; 13 of these had serious diarrhea, in 5 of whom it was so severe that they were put back upon heated milk; 10 took raw milk all summer without bad effects; 2 died, 1 from gross neglect on the part of the mother, the other from diarrhea. Of the 21 on Pasteurized milk, 5 had severe attacks of diarrhea, but all were kept on this milk except for short periods, when all food was omitted; 16 did well throughout the summer. One infant, marked!}' rachitic, died. The third summer's results have not been tabulated, but were similar to those of the first two tests. The outcome of these observations during the first two summers are summarized in the following table: Kinds of milk. Pasteurized milk, 1,000 to .50,- 000 bacteria per c.c. . . I 41 Raw milk, 1,200,000 to 20,000- 000 bacteria per c.c. . . I 51 Although the number of cases was not large, the results, almost identical during the three summers, indicate that even a fairly pure milk, when given raw in hot weather, causes illness in a much larger percentage of cases than the same milk given after Pasteurization. A considerable percentage of infants, however, do apparently quite as well on raw as on Pasteurized milk. Bacteria in Milk. Effect on Older Children. — The children over three years of age who received unheated milk, containing at different times from 145,000 to .350,000,000 bacteria per cubic centimeter, showed almost no gastro-intestinal disturbance. The conditions at three institutions wiU serve as examples. , In the first of these an average grade of raw milk was used which, during the summer, contained from 2,000,000 to 30,000,000 bacteria per cubic centimeter. This milk was stored in an ice-box until required. It was taken by children unheated and yet no case of diar- rhea of sufficient gravity to send for a physician occurred during the entire summer. This institution was an orphan asylum containing 650 children from three to fourteen years of age — viz., three to five years, 98; five to eight years, 162; eight to fourteen years, 390. ■ Thirteen of the fifty-one infants on raw milk were transferred before the end of the trial to Pasteurized milk because of serious illness. If these infants had been left on raw milk it is believed by the writers that the comparative results would have been even more unfavorable to raw milk. REsrn/rs WITH UK.\rRi) [mpvre and pure miek (;17 A second institution used an nnlieated hut very |)ure niillv whici: was olitained from its own Farm. This milk a^■eraged fjd.OOO hae- teria per cuhic centimeter. The inmates A\ere 70 children of ages ranging from three to fourteen years. In this institution not a single case of (Harrlical disease of any importance occurred during the summer. In a third institution an average grade of milk was used which was heated. This milk before heating contained 2,000,000 to 20,000,000 bacteria per cuhic centimeter. The institution was an infant asyhmi in which there were 120 children between the ages of two and five years. There were no cases of diarrhea during the summer. These clinical observations taken in connection with the bacterio- logical examination at the laboratory show that although the milk may come from healthy cattle and clean farms and be kept at a tem- perature not exceeding 60° F., a very great increase in the number of bacteria may occur. Furthermore, this may occur without the accumulation in the milk of sufficient poisonous products or living bacteria to cause appreciable injury in children over three years of age, even when such milk is consumed in considerable amount and for a period extending over several months. IMilk kept at tempera- tures somewhat above 60° F. was not met with in our investigations, but the histories of epidemics of ptomain poisoning teach that such milk may be very poisonous. It is also to be remembered that milk abounding in bacteria on account of its being carelessly handled is also always liable to contain pathogenic organisms derived from human or animal sources. Results with Very Impure Milk Heated vs. Those with Pure or Average Milk Heated. — During the summer of 1901 we were able to observe a number of bal)ies fed on milk grossly contaminated by bac- teria. In 1902 systematic supervision of all stores selling milk was instituted by the Health Department, so that the very worst milk was not offered for sale that summer. The observations upon the impure milk of 1901 are of sufficient importance to be given in detail, although already mentioned in the report of the observations upon infants of both summers which were fed on "store milk." A group of over 150 infants was so divided that 20 per cent, were allowed to remain on the cheapest store milk which they were taking at the time. To about the same number was given a pure bottled milk. A third group was fed on the same quality of milk as the second, but sterilized and modified at the Good Samar- itan Dispensary. A fourth group received milk from an ordinary dairy farm. This milk was sent to a store in cans aufl called for by the people. A few infants fed on breast and condensed milk were observed for control. In estimating the significance of the observations recorded in the tables, one should bear in mind that not only do different infants possess different degrees of resistance to disease, hut that, try as hard (ilS APPLIED MICROBIOLOGY as tlie physicians could, it was impossible to divide the infants into i>T(iups which secured equal care and were subjected to exactly' the same conditions. It was necessary to have the different groups in somewhat different parts of the city. It thus happened that the infants on the cheap store milk received less home care than the average, and that those on the pure bottled milk lived in the coolest portion of the city. Certain results were, however, so striking that their interpretation is fairly clear. It is to be noted that the number of infants included in each group is small. There is nothing in the observations to show that fairly fresh milk from healthy cows, living under good hygienic conditions and con- taining, on some days, when delivered, as many as 200,000 bacteria per cubic centimeter, had any bacteria or any products due to bac- teria that remained deleterious after the milk was heated to near the boiling point. Table Showing the Results of Feeding during July and August, 1901, in Tenement Houses, 'of 112 Bottle-fed Infants undeb one year of Age, and 'of 47 Bottle- fed Infants between One and Two Years or Age with Milk from Different Sources, and the Number of Bacteria Present in the Milk. Infants under one year. Infants over one year. Charafter of the milk. II 3.S 2; -Average weekly gain. Diarrhea. ci q o ^ 9 = 9 2: Average weekly gain. Diarrhea. 3 10 6 > m 8 2 ^ 6 a; > .1 1. Pure milk boiled and modified at dispensary or stations; given out in small bottles. Milk be- fore boiling averaged 20,000 bacteria per c.c; after boiling 2 per c.c. 41 3 oz. 11 2. Pure milk, 24 hours old, sent in] in quart bottles to tenements, heated and modified at home, !• 20,000 to 200,000 bacteria perl c.c. when delivered J 2.3 Hi oz. 8 5 24 4i oz. 8 2 3. Ordinary milk, 36 hours old, from a selected group of farms, kept cool in cans during trans- port; 1,000,000 to 25,000,000 bacteria per c.c, heated and modified at home before using. 18 4 oz. 6 6 V 12 4 oz. 1 2 4. Cheap milk, 36 to 60 hours old, from various small stores, de- rived from various farms, some fairly clean, some very dirty; 400,000 to 175,000,000 bacteria per c.c. 21 i oz. 4 13 43 7 Vi oz. I 3 5. Condensed milk of difTerent] brands. Made up with hot water. As given, contained bac-[ t(Tia from 5,000 to 200,000 per c.c. 9 ioz. 5 2 3 4 3%oz. 1 3 6. Breast milk 16 21 oz. 5 2 ' This infant died from entpriti.s arid toxemia. - Tliis iiifant died of pneumonia. There had been no severe intestinal disorder noted. ■■ One of tlie four had pertussis, the reTTUiininp: three died from uncomplieated enteritis. BACTERIAL CONTAMINATION OF MILK 010 On the other hand, it is possible that certain varieties of bacteria may, under eonchtions that are unsanitary, find entrance to milk and survive moderate heat or may develop poisonous products resistant to heat in sufficient amount to be Iiarnifnl, e\'en when thc> have ac- cuuuilated to less than 20(),()0() per c.c. Turning now to the results of feeding with milk which has been heated and which before sterilization contained from 1,000,000 to 25,000,000 bacteria per cubic centimeter, averaging about 15,000,- 000, though obtained from healthy cows living under fairly decent conditions and although the milk was kept moderately cool in transit, we find a distinct increase in the amount of diarrheal diseases. Though it is probable that the excessive amount of diarrhea in this group of children was due to bacterial changes which were not neutralized by heat or to living bacteria which were not killed, yet it is only fair to consider that the difference was not very great and that the infants of this group were under surroundings not quite as good as those on the pure milk. Finally, we come in this comparison to the infants who received the cheap store milk after heating. This milk had frequently to be returned because it curdled when boiled, and contained, according to the weather, from 4,000,000 to 200,000,000 bacteria per cubic centimeter. In these infants the worst results were seen. This is shown not only by the death rate, but by the amount and by the severity of the diarrheal diseases, and the general appearance of the children as noted by the physicians. Although the average number of bacteria in the milk received by this group is higher than that re- ceived by the previous group, the difference in results between this group and the previous one can hardly be explained by the difference in the number of bacteria. The varieties of bacteria met with in this milk were more numerous than in the better milk, but we were miable to pro\'e that they were more dangerous. Probably the higher temperature at which the milk was kept in transit, and the longer interval between milking and its use, allowed more toxic bacterial products to accumulate. Bacterial Contamination of Milk — General Conclusions^ as to Rela- tive Importance. — 1. During cool weather neither the mortality nor the health of the infants observed in the investigation was appre- ciably affected by quality of the market milk or by the number of bac- teria which it contained. The different grades of milk varied much less in the amount of bacterial contamination in winter than in sum- mer, the store milk averaging only about 750,000 bacteria per cubic centimeter. 2. During hot weather, when the resistance of the children was lowered, the kind of milk taken influenced both the amount of illness and the mortality; those who took condensed milk and clieap store milk did the worst, and those who received breast milk, pure bottled ' These conclusions were drawn up by the writer in association with Dr. L. E. Holt, after a joint study of the results obtained in the studies above recorded. G20 APPLIED MICROBIOLOCY milk, and modified milk did the best. The efl'ect of hacterial con- tamination was very marked when the milk was taken without pre- vious heating; but, unless the contamination was very excessive, only slight when heating was employed shortly before feeding. 3. The nimiber of bacteria which may accumulate before milk becomes noticeably harmful to the average infant in summer differs with the nature of the bacteria present, the age of the milk, and the temperature at which it has been kept. When the milk is taken raw, the few^er the bacteria present the better are the results. Of the usual varieties, over 1,000,000 bacteria per cubic centimeter are certainly deleterious to the average infant. However, many infants take such milk without apparently harmful results. Heat of 145° F. for thirty minutes or of 170° F. for a shorter period not only destroys most of the bacteria present, but, apparently, some of their posionous products. No harm from the bacteria pre^'iously existing in recently heated milk was noticed in these observations unless they had amounted to many millions, but in such numbers they were decidedly dele- terious. 4. When milk of a\'erage quality was fed, sterilized and raw, those infants who received milk previously heated did, on the average, much better in warm weather than those who received it raw. The difference was so quickly manifest and so marked that there could be no mistaking the meaning of the results. 5. No special varieties of bacteria were found in unheated milk which seemed to ha\-e any special importance in relation to the sum- mer diarrheas of children. A few cases of acute indigestion were seen immediately following the use of Pasteurized milk more than thirty- six hours old. Samples of such milk were found to contain more than 100,(100,000 bacteria per cubic centimeter, mostly spore-bear- ing varieties. The deleterious effects, though striking, were neither serious nor lasting. 6. After the first tweh'e months of life, infants are less and less affected by the liacteria in milk derived from healthy cattle and the air. According to these observations, when the milk had been kept cool, the bacteria did not appear to injure the children over three years of age at any season of the year, unless in very great excess. 7. While it is true that even in tenements the results with the l)est bottle feeding are nearly as good as average breast feeding, it is also true that most of the bottle feeding is at present very badly done; so that, as a rule, the immense superiority of breast feeding obtains. This shoulfl, therefore, be encouraged by every means, and not dis- continued without good and sufficient reasons. The time and money required for artificial feeding, if expended by the tenement mother to secure better food and more rest for herself, would often enable her to continue nursing with advantage to her child. Influence of Temperature upon the Multiplication of Bacteria in Milk. — Few, even of the well informed, appreciate how great a difference a, few degrees of temperature will make in the rate of bacterial mul- MULTIPLICATION OF BACTERIA IN MILK (>21 s tiplication. Milk rapidly and sufficiently cooled keeps almost unaltered for thirty-six hours, while milk insufficiently cooled (leteriorates rapidly. The majority of the bacteria met with in milk grow best at tem- peratures above 70° F., but they also multiply sk)\vly even at 40° F.; thus, of ()0 species isolated by us, 42 developed good growth at the end of seven days at 39° F. Our observations have shown that the bacteria slowly increase in numbers after the germicidal prop- erties of the milk have disappeared, and the germs have become ac- customed to the low temperature. In fact, milk cannot be perma- nently preserved unaltered unless kept at .32° F. or less. The degree of cooling to which ordinary supplies of milk are subjected differs greatly in various localities. Some farmers chill their milk rapidly, by means of pipe coils over which the milk flows; others use deep wooden tanks filled with water into which the cans of milk are placed soon after milking. In winter these methods are very satisfactory for the water runs into the pipes or tanks at about 38° F. In warmer weather they are unsatisfactory, unless ice is used, as the natural temperature of the water may he as high as 55° F. A considerable quantity of milk is not cooled at all at the farms. It is sent to the creamery or railroad after two to six hours, and is then more or less cooled. These few hours in summer, when the milk is left almost at blood heat, allow an enormous development of bacteria to take place, as is shown in the table below. Table 1. — Showing the Development of Bacteria in Two Samples of Milk Main- tained AT Different Temperatures foe Twenty-four, Forty-eight, and Ninety-six Hours, Respectively. The first S.ample of Milk was Obtained UNDER THE BEST CONDITIONS POSSIBLE, THE SECOND IN THE UsU.iL WaY'. WhEN Received, Specimen, No. 1 Contained 3000 Bacteria per c.c, Specimen No. 2, 30,000 PER c.c. Time which elapsed liefore making test. lemperature. Fahrenheit. 24 hours. 48 hours. 06 hours. IG.S liours. 32° 240(J 2100 LS50 1400 30 ,000 > 27,000 24,000 19,000 39° 2500 3600 21S,(I00 4,209,000 38,000 56,000 4,300,000 38,000,000 42 o 2600 3600 500,000 11,200,000 43,000 210,000 5,760,000 120,000,000 40° 3njo 12,000 1,480.000 SO, 000, 000 42,000 360,000 12,200,000 300,000,000 .50° 11,600 540,000 300,000,000 1,000,000,000= 89,000 1,940,000 1,000,000,000= 5.5° IS.SOO 187,000 3,400,000 38,000,000 60° ISO, 000 900,000 2S, 000, 000 168,000,000 6S° 450,000 4,000,000 500.000,00(1 1,000,000,000= The figures referring to te.sts of the second sample are printed in heavy-f.acp type. ' Tliese figures signify the maximum growth and estimates only. (122 APPLIED MICROBIOLOGY Observation.s on Bacterial Multiplication in Milk at 90° V., A Tempebature Common in New York in Hot Summer Weather. Table II. — Number OF Bacteria per c.c. MUk I. Fresh and of good quality. Milk II. Fair quality from store. Milk III. Bad quality from store. ( iriginal numljer . . 5200 92,000 2,600,000 After two hours . . 8400 184,000 4,220,000 After four hours . . 12,400 470,000 19,000,000 After six hours . . 68,.500 1,260,000 39,000,000 After eight hours . 654,000 6,800,000 124,000,000 A sample of milk No. I removed after si.x hours and cooled to 50° F. contained 145,000,- 000 at the end of twenty-four hours. Some of this milk, kept coo! from the beginning, contained Ijut 12,800 bacteria per cubic centimeter at the end of twenty-four hours. Pasteurization of Milk. — The two dominant factors which control the temperature and time at which the milk should be heated are (1) the thermal death points of pathogenic bacteria, and (2) the thermolabile food constituents of the milk. The first factor is al- most equally important for milk used bj' persons of all ages, while the second factor is only important for milk used in very young children. The exposure of bacteria for a short time at a high temperature is equivalent to a longer time at a lower temperature. The ferments and other labile food constituents, on the other hand, are altered much more by the higher temperature. It is Avell, therefore, to choose the lowest possible temperature which will kill the non-spore-bearing patho- genic bacteria in a practicable length of time. Such an exposure is 60° C. (140° F.) for .30 minutes, 70° C. (158° F.) for 5 minutes. Very much shorter exposures, as one minute at 70° C, will kill the great ma- jority of pathogenic and other bacteria in the milk and add much of safety as seen in the tables below, but it is better to be on the safe side. Table Se lOWING E FFECT OF Heat Upon Tubercle Bacilli IN Milk Heated Instantly. Degree of heat. Time exposed. Amount milk. Result in guinea-pigs. 60° C. 15 min. 1 c.c. Infection 60° C. 20 min. 1 c.c. No infection 60° C. .30 min. 1 CO. No infection 70° C. 0.5^ min. 1 c.c. Infection' 70° C. 1 min. 1 c.c. No infection 70° C. 2 min. 1 c.c. No infection Control not hea ted .001 Infection This milk was infected by adding one-fifth of its quantitj- of sputum rich in tubercle bacilli. Development of Bacteria in Milk Which has been Heated. — There is a common idea that bacteria develop much more rapidly in milk that has been heated than in raw milk. This is only true for" freshly drawn milk which has slight bactericidal power. The table below shows the effect on bacteria in milk of heating to 70° C. for onc-lialf and one minute. Not only the immediate reduction in_ number is seen to be great, but tlic difference continues when the milk is kept cold for two days. ' Most of llie L'uiiiea-pigs were not infected by the milk heated for one-half minute. PASTEURI'ZATION OF MILK (123 Two Samples Mixed from lUU Samples of Inspectohs. Pastehhizeh at 100° F. Plates Made Same Day. S:uiii)U- I. Salijplu II. Before heating . (JOD.OUI) ( 'outiul .... 5,11)0,00(1 2 m 2000 ,; 111 . . . . . 7400 1 ni 1000 1 m (iOO Same Samples Kept in Ice-box Twenty-fouh Hours at 45° I'". (7° ('.). Colli rul '. Ill i ni . Control 2 ni 1 111 0,300,000 18,000 900 Control ■J ni . 1 111 . . In Ice-box Forty-eight Hours at 45° F. (7° C). Control 10,200,000 Control .... i ni 120,000 .; ni 1 m 10,000 i 111 In Room at 71° F. (22° C). 36,600,000 Control 5,460,000 J ni . . . 5,400,000 i m . . . 21,600,000 12,000 3600 63,000,000 276,000 90,000 150,000,000 4,500,000 3,600,000 Number of Bacteelv in Milk Produced under Different Conditions. 1 . The imnilDer of bacteria present at the time of milking and twenty-four, fortj'-eight, and seventy-two hours afterward in niillt obtained and l-cept under correct conditions. No preservatives were present in an}' of the following specimens: Pure inilk obtained where every reasonable means was taken to ensure cleanli- ness. The long hairs on the udder wei'e clipped; the cows roughly cleaned and placed in clean barns before milking; the udders were wiped off just previous to mrllving; the hands of the men were washed and dried; the pails used had small (six-inch) openings, and were thoroughly cleaned and sterilized by steam before use. MiUv cooled within one hour after milking to 4.5° F., and subsequently kept at that temperature. The first six specimens were obtained from individual cows; the last six from mixed milk as it flowed at different times from the cooler. Temperature of barns 5.5° F. Number of Bacteria in 1 c.c. of Milk. From Six Individuai Cow^i. hours after m ilking. After 24 hours. After 48 hours. After 72 houra 500 700 12,500 Not counted. 700 700 29,400 Not eounted. 19,900 5200 24,200 Not counted. 400 200 8600 Not counted. 900 1600 12,700 Not eounted. 13,600 3200 19,500 Not counted. 6000 1933 17,816 From iSam'pk-fi of Mixed Milk of Eiilirc Herd. 0900 12,000 19,800 494,000 6100 2200 20,200 550,000 4100 700 7900 361,000 1200 400 7100 355,000 6000 900 9800 446,000 1700 400 8700 3.89,000 A-s'eragc 4333 2766 10,583 329,000 Twenty-five samples taken separately from indi\'idual co-\\s on another day and tested immediately averaged 4550 bacteria per cubic centimeter and 4500 (124 APPLIED MICROBIOLOGY after twent^'-four hours. These twenty-five specimens were kept at between 45° and .50° F, 2 . Milk taken during winter in well-ventilated, fairly cleaUj but dusty barns. Visible dirt was cleaned off the hair about the udder before milking. Milkers' hands were wiped off, but not washed. Milk pails and cans were clean, but the straining cloths dusty. Milk cooled within two hours after milking to 45° F. Number OF B A.CTEHIA IN 1 C c. OF M ILK At time of milking. After 24 hours. After 4S liours 12,000 1.3,000 21,500 14,000 20,000 31,000 57,000 65,000 106,000 Average 15,500 21,660 76,000 Number in City Milk. •3 . The condition of the average raw city milk is very different, and is shown in the following tables. The twelve samples were taken late in March, 1912, bj- Iiisi)ectors of the Department of Health of New York City from cans of milk immediately ujion their arrival in the city. Raw milk at present gives similar counts. The temperature of the atmosphere averaged 50° F. during the previous t went jr-f our hours. The temperature of the milk when taken from the cans aver- aged 45° F. Much of this milk had been carried over two hundred miles. From the time of its removal from the cans, which was about 2 a.m., until its distribu- tion in nutrient agar, at 10 a.jm., the milk was kept at about 45° F. Fhom New Youk ,\nd Hud.son River Railro,4lD. From H.arlem Railroad. No. of bacteria No. of bacteria No. of sample. in 1 c.c. No. of sample. in 1 c.c. 50 35,200,000 48 6,200,000 51 13,000,000 49 2,200,000 52 ..... 2,.500,000 50 15,000,000 53 1,400,000 51 70,000 54 200,000 52 80,000 55 600,000 53 320,000 The results of the examination of many thousands of specimens last year indicated that most of the milk of grade A was f)f fair quality, hut that the raw milk of the other trades contained excessive numbers of bacteria before pasteurization. It must be kept in mind that milk averaging 3,000,000 bacteria per cubic centimeter will, when kept at the temperature common in the homes of the poor who comprise the larger part of the population, soon contain very largely increased numbers and show its dangerous condition by turning sour and curdling. Cleanliness Used in Obtaining Milk, and Its Influence. — The present conditions under wdiich much of tJie milk is obtained are not pleasant to consider. In winter, and to a less extent at other seasons of the year, the cows in many stables stand or lie down in stalls in the rear portion of which there is altogether too much of manure and urine. When milked the hands of the milkers are not cleansed, nor are the under portions of the cows, only visible masses of manure adhering to the hair about the udder being removed. Some milkers even moisten their hands with milk, to lessen friction, and thus wash oS the dirt of CLEANLINESS USED IN OHTAININO MILK (iLT) their hands and the cow's teats into the milk in tlie pails. Some may regard it as an unnecessary refinement to ask that farmers should roughly clean the floors of their stalls once each day, that no sweeping should lie done just before milking, and that the udders should he wijjed with a clean damp cloth and the milkers should thoroughly wash and wipe their hands before commencing milking. The pails and cans should not only be carefully cleansed, but afterward scalded out with boiling water. The washing of the hands would lessen the number of ordinary filth bacteria in the milk, and diminish risk of transmitting to milk human infectious diseases, like scarlet fever, diphtheria, and enteric fever, by the direct washing off of the disease germs from infected hands. It would also inculcate general ideas of the necessity of cleanliness and of the danger of transmitting disease through milk. The value of cleanliness in limiting the number of bacteria is demon- strated by the figures contained in the tables. General Conclusions. — Because of its location and its hairy covering, the cow's udder is always more or less soiled with dirt and manure imless cleaned. On account of the position of the pail and the access of dust-laden air it is impossible to obtain milk by the usual methods without mingling with it a considerable number of bacteria. With suitable cleanliness, however, the number is far less than when filthy methods are used, there being no reason why fresh milk should contain in each cubic centimeter, on the average, more than 12,000 bacteria per cubic centimeter in warm weather and 5000 in cold weather. Such milk, if quickly cooled to 46° F., and kept at that temperature, will at the end of thirty-six hours contain on the average less than 50,000 bacteria per cubic centimeter, and if cooled to 40° F. will average less than its original number. With only moderate cleanliness such as can be employed by any one without adding appreciably to his expense, namely, clean pails, with small openings, straining cloths, cans or bottles, and hands, a clean place for milking, and a decent condition of the cow's udder and Ijelly, milk when first drawn will not average in hot weather over 30,000, and in cold weather not over 25,000 bacteria per cubic centi- meter. Such milk, if cooled and kept at 50° F., will not contain at the end of twenty-four hours over 100,000 bacteria per cubic centi- meter. If kept at 40° F. the number of bacteria will not be over 100,000 per cubic centimeter after forty-eight hours. If, however, the hands, cattle, and barns are filthy and the pails are not clean, the milk obtained under these conditions will, when taken from the pail, contain very large numbers of bacteria, even up to a million or more per cubic centimeter. Freshly drawn milk contains a slight and variable amount of bac- tericidal substances which are capable of inhibiting bacterial growth. At temperatures under 50° F. these substances act efficiently (unless the milk is filthy) for from twelve to twenty-four hours, but at higher temperatures their eft'ect is very soon completely exhausted, and the bacteria in such milk will then rapidly increase. Thus the bacteria 40 (i2() APPLIED MICROBIOLOGY ill fresh milk which originally numbered 5000 per cubic centimeter decreased to 2400 in the portion kept at 42° F. for twenty-four hours, but rose to 7000 in that kept at 50° F., to 280,000 in that kept at 65° F., and to 12,500,000,000 in the portion kept at 95° F. As we have seen, the milk in New York City is found on bacterio- logical examination to contain, as a rule, excessive numbers of bac- teria. During the cold weather the raw milk in the shops averages over 300,000 bacteria per cubic centimeter, during cool weather about 1,000,000, and during hot weather about 2,000,000. The milk in other large cities is, from all accounts, in about the same condition. The above statement holds for milk sold at the ordinary shops, and. not that of the best of the special dairies, where, as previously stated, the milk contains only from 1000 to 30,000 bacteria, accord- ing to the season of the year. The question might be raised, Are even these enormous numbers of bacteria often found in milk during hot weather harmful? Our knowledge is probably as yet insufficient to state just how maiay bacteria must accumulate to make them noticeably dangerous in milk. Some varieties are undoubtedly more harmful than others, and we have no way of restricting the kinds that will fall into milk, except bjr enforcing cleanliness. We have also to consider that milk is not entirely used for some twelve hours after being purchased, and that during all this time bacteria are rapidly multiplying, especially where, as among the poor, no provision for cooling it is made. Slight changes in the milk which to one child would be harmless, would in another produce disturbances which might lead to serious disease. A safe conclusion is that no more bacterial contamination should be allowed than it is practicable to avoid. Any intelligent farmer can use sufficient cleanliness and apply sufficient cold, with almost no increase in expense, | cent per quart, to supply milk twenty-four to thirty-six hours old which will not contain in each cubic centimeter over 50,000 to 100,000 bacteria, and no milk containing more bacteria should be sold. The most deleterious changes which occur in milk during its trans- portation are now known not to be due to skimming off the cream or to the addition of water, but to the changes produced in the milk by nml- tiplication of bacteria. During this multiplication, acids and distinctly poisonous bacterial products are added to the milk, to such an extent that much of it has become distinctly deleterious to infants and invalids. It is the duty of health authorities to prevent the sale of milk rendered imfit for use through excessive numbers of bacteria and their products. The culture tests to determine the number of bacteria present in any sample of milk require at least forty-eight hours; so that the sale of milk found impure cannot l)e prevented. It will, however, be the purpose of the authorities gradually to force the farmers and the middle- men to use cleanliness, cold, and dispatch in the handling of their milk, rather than to prevent the use of the small amount tested on any one day. TRANSMISSION OF C0NTA(3I0US DISEASES THROUGH MILK 027 If the milk on the train or at the dealer's were found to contain excessive numbers of bacteria, the farmers would be cautioned and instructed to carry out the simple necessary rules, which would be furnished. Transmission of Contagious Diseases through Milk. — Pathogenic Bacteria in Milk. — Tuberculosis, typhoid fever, septic sore tliroat, scarlet fe^'er, and diphtheria are the chief diseases transmitted by means of milk in this locality. In other countries cholera, malta fever, and possibly other diseases may be due at times to milk infection The obscure disease trembles is also believed to be due to milk. The tubercle bacilli are in the majority of cases derived from the cow, l)ut may come from human sources, the typhoid bacilli are entirely from man, the contagion of true scarlet fever conveyed in milk is probably always from man, but the contagion of a disease closely allied to it is certainly given off by cows suffering from certain septic diseases as j'et not fully identified. Diphtheria bacilli are probably always of human origin, as animals, except cats, practically never suffer from the disease and these only under exceptional conditions. The streptococci exciting septic throats are probably usually from human cases of septic inflam- mation but may at times come from septic cows. As milk is usually kept below 60° F. the typhoid bacilli and the streptococci are the only pathogenic germs that we believe increase in any appreciable extent. The following epidemics and cases have been recorded in the bulletin of the Marine Hospital Service, as produced by cow's milk: Epidemics. Cases. Typhoid fever 179 6900 Scarlet fever 51 2400 Sore throat 7 1100 Diphtheria 23 900 Tuberculosis . . .... No case of measles, smallpox, chickenpox, whooping cough, or mumps has been clearly traced to milk. The Relation of the Typhoid Carrier to Milk Infection. — Many epi- demics of typhoid fever have until recently puzzled investigators be- cau,se, though evidently milk-borne, yet no case of typhoid fever could be found. The discovery that about 2 per cent, of those who have recovered from typhoid fever remain infected and continue during the rest of their lives to pass tj'phoid bacilli has cleared up the mystery. Epidemics due to these carriers have already been traced both in New York City and elsewhere. Many observers have already discussed the relation of typhoid cases to milk infection. Hands, water, flies, etc., may all aid in the transfer of the bacilli from the dejecta to the milk. Recently we traced over four hundred cases to infection of a milk supply by a typhoid carrier who had the disease forty-seven years ago. The Conveyance of Scarlet Fever by Means of MUk. — As wc do not know the organism which excites scarlet fever, we are not as clear as to the means b}' which it is spread as we are in the case of tuberculosis, typhoid fever, and diphtheria. We know, however, that the throat 628 APPLIED MICROBIOLOGY secretions are dangerous. Where the infection has been traced it has usually been found that the milker has suffered from an unrecognized ease or is convalescent. It seems as if the contagion must either increase in milk or be capable of infecting when greatly diluted, for cases have developed from milk after great dilution. A small number of epidemics have appeared to come from the milk of diseased cows. Many are skeptical about this, but after personal experience we cannot doubt it. The history of one case was as follows: The milk from a septic cow was delivered to two schools. About thirty of the boys who drank the milk developed the disease while none of the day scholars who went home to lunch did. Some of the cases developed at first only sore throats, others only the rash. On the second day the cases resembled very closely scarlet fever. There was no scarlet fever in the town. The milk contained immense numbers of long-chained strepto- cocci. Diphtheria and septic sore throats are occasionally produced by milk. The diphtheria bacilli usually originate from a mild case, the nature of which is not detected. Septic sore throats produced by milk are usually traced to contamination from a human source, but like cases develop- ing the scarlet rash the infection may come from cows suffering from some acute septic udder disease. The Grading of Milk. — The appreciation of the importance of the bacteria in milk has led to the pasteurization of all milk entering New York City, except that produced and transported under the very best conditions. Together with the farm conditions the bacterial content of milk is used to grade it. All milk in New York City is divided into grades A, B, and C. Grade A is raw and pasteurized. The raw is from cows which have successfully passed the tuberculin test. Grade B is all pasteurized. Grade C is all pasteurized. The grade A may contain 60,000 bacteria per cubic centimeter. The grade B may contain 1,500,000 when raw and 50,000 after pas- teurization. The grade C may contain any number before and 100,000 after pas- teurization. It is supposed to be used for cooking purposes only. The State of New York has adopted a similar grading except that it allows raw and pasteurized in all three grades. Manj-- cities and states are adopting such grades. A powerful influence in this direction was the report of the national committee of experts on milk standards. This was a commission appointed by the New York Milk Committee. CHAPTER XLVII 1. THE SOIL BACTERIA AND THEIR FUNCTIONS. SEWAGE BACTERIA. BACTERIA IN INDUSTRIES. The bacteria^ in tlie soil belong to many varieties. Some varieties are only accidentally present, being due to the contamination of the earth with the bacteria contained in animal feces and other waste products. The majority, however, pass their life and reproduce them- selves chiefly or wholly in the soil. Many of these varieties have most important functions to perform in continuing the earth's food supply. Without them plant food, and, therefore, animal food, would cease to exist. Some make available for plants the carbon, nitrogen, hy- drogen, and other compoimds locked up in the dead bodies of animals and plants. Others construct food for plants from the gases of the air and the inorganic elements of the earth which in their simpler forms were not available. The bacteria, together with the other somcA^-hat less important microscopic plants and animals, thus form a vital link in the earth's life cycle, plants and animals. The bacteria in the soil require for their activities food, moisture, and a proper temperature. They may be present to the extent of many millions in a single gram of rich loam, while in an ecpial quantity of sand they may be almost absent. The various species associated together in the soil flora influence each other. Thus anaerobic bacteria are enabled to grow because of associated aerobes using up the free oxygen, while other species make assimilable substances not usable by others. The Splitting up of Carbon Compounds. — The plants form starch, and from it cellulose, wood, fats, and sugar. These substances once formed cannot be utilized by other generations of plants. Some of these are transformed in the bodies of animals, but the largest per- centage await the activities of the microorganisms. The sugars and starches usually undergo an alcoholic fermentation excited by the yeasts and molds with the production of alcohol and carlDon dioxide, or an acid fermentation excited by bacteria with the production of acids and frequently of carbon dioxide. Cellulose which is so resistant to decay is attacked by certain varie- ties of bacteria which are abundant in the soil. They act both in the presence and absence of free oxygen. Molds also act on cellulose. Carbon dioxide, marsh gas, and other products are produced. Wood is apparently first attacked by the fungi and only later by the micro- organisms. These bacteria are carried into the intestines and a.ct upon cellulose and other substances. ' L. H. Bailey. "Bacteria in Relation to Country Life." G30 APPLIED MICROBIOLOGY The Decomposition of Nitrogenous Compounds. — Plants obtain their nitrogen chiefly in the form of nitrates. The small amount of usable nitrogen in the soil must be constantly replenished. This must either come from the nitrogen forming a part of ]:)rotei(l materials or from the free nitrogen in the air. The animals utilize the plant proteids and reduce them to much simpler compounds, such as urea, but even these are not suitable for l^lant use. We now know that microorganisms are employed to break compounds into simpler compounds and also to utilize the nitrogen of the air. Decomposition. — This process is to some extent carried out through the agency of yeasts, molds, and fungi, but it is chiefly due to the activities of bacteria. When this process is carried on in the absence of oxygen it is incomplete giving rise to substances with unpleasant odors, such as H2S, NH3 and CPI4. This is called putrefaction. When oxygen is freely accessible more complete decomposition occurs with such end products as CO2, N aiid H2O. These two processes, putre- faction and complete decay, cannot be sharply separated, as the second usually follows the first. The varieties of organisms causing these changes are many. Some groups will be found chiefly in decaying vegetable substances, others in animal tissues. They include all mor- phologic forms of bacteria as well as yeasts and higher fungi. These forms exist everywhere in nature, although in various degrees, so that every bit of dead organic matter is sure to be decomposed if only mois- ture and warmth are present. B. subtilis and B. froteus vulgaris are well-known laboratory bacteria that are commonly found among decomposing materials. B. proteus is described under Pathogenic Bacteria. B. subtilis (hay bacillus) has the following characteristics (Fig. 208): Source and Habitat. — Hay, straw, soil, dust, milk, etc. Morphology. — Short, thick rods with round ends, sometimes form threads; sometimes also chains of long rods, short rods, and coccus forms. 0.8/j to 1.2;* broad, 1 . S^a to 3^ long. Often united in strings and threads. Staining Reaction. — Stains by Gram's method. Capsule, Flagella, Motility. — Bacilli possess a thin capsule and many flagella which are long and numerous; short forms actively motile; threads immotile. Spore Formation. — Oval spores formed in presence of air germinating at right angles to long diameter. Spores are set free in about 24 hours, size 1.2/n by 0.6m; widely distributed in nature, dust, air, excreta, etc. (Plate III, Fig. 23). Biology: Cultural Characters (Including Biochemical Features). — Bouillon. — Uniformly cloudy growth with marked pellicle, iwmkled and thick; copious spore formation. Gelatin Plates and Tubes. — Saucer-hke depressions; colonies have granu- lar centres and folded margins. Surface growth in stab cultures is whitish- gray; colonies sink on liquefaction of medium; liquefaction progresses in a cylindric form, and a thick white scum is formed. Agar Plates and Tubes. — Small, irregular, grayish-white colonies; moist glistening growth along needle track in stab cultures. The liacteria in taking certain atoms from the molecules utilized in their growth leave the other atoms to enter into new relations and DENITRIFICATION (131 form new eomiwuiuls. The actual products will depend on the decaying substance, the \ariety of bacteria and the conditions present. Nitrification. — This is a process of oxidation by which through bacterial activities ammonia comi)ountls arc changed to nitrates and thus rendered utilizable by plants. This change is accomplished in two stages: first, the ammonia is oxidized to nitrite and, second, to nitrate. The nitrates are taken up by the plant roots from the soil. The bacterial nature of these changes were discovered in 1877 by two French investigators, Schlosing and Muntz. They noted that fermenting sewage after a time lost its ammonia and gained in nitrates, but that if the sewage was treatetl with antiseptics, so that fermentation ceased, no such change occurred. Warrington first and Winogradsky later more thoroughly investigated the bacterial cause of these changes. The latter by means of silica jelly, which contained no organic matter, was able to isolate two varieties of cocci, one in Europe and the other in America, which were able to change ammonia to nitrites. He called Fig. 208 Bacillus sul:)tilis ^'ith spor(>s. Agar culture. .Stained with gentian violet. diameters. (Franke.) X 1000 the one nitrosomonas and the other nitrosococcus. They are capable of acting on almost any ammonia salt. One variety of organisms capable of changing nitrites to nitrates was isolated, and this bacillus he called nitrobacter. These are small, slightly elongated bacilli. These bacteria are remarkable in that in pure cultures very small amounts of organic matter in the media act as antiseptics. They appear to be able to depend on mineral substances for their food. These bacteria are extremely important, for the plants take up most of their nitrogen in the form of nitrates. These changes are mostly produced in the surface soil. If the reaction of the soil becomes acid growth ceases. Soil bacteriologists are studying the nitrifying power of different types of soil under identical conditions. The process being one of oxidation, the access of air is necessary. Denitrification. — This is a reducing process. The nitrate is made to yield up a part or all of its oxygen and thus becomes changed to nitrites and to ammonia and even to free nitrogen. The partial change 632 APPLIED MICROBIOLOGY does not rob the soil of its available nitrogen as does the total change, for the nitrites and ammonia may be changed by the nitrifying bacteria to nitrates. These bacteria exist normally in most soils and are especially abundant in manure. There are three different types of nitrogen reduction. (1) The reduction of nitrates to nitrites and ammonia. (2) The reduction of nitrates and nitrites to gaseous oxides of nitrogen. (3) The reduction of nitrites with the de^'elopment of free nitrogen gas. Nitrogen Fixing Bacteria. — Helbrigel in 1886 demonstrated that certain plants were able to use the nitrogen of the air and this ap- parently through the aid of bacteria growing in their roots. These root bacteria are named B. radicicola. They produce enlargements (tubercles) on the roots. According to Ball/ there is no reasonable doubt but that B. radicicola can and usually does remain active for very long periods in soil devoid of leguminous vegetation. Furthermore, the bacterium dift\ises at a very considerable rate through soils that are in proper condition; therefore, if a soil should be found lacking the organism, it is illogical to attempt to introduce it artificially without having first made the soil fit for the development of the bacteria. It has not been shown by anyone that increased powers of resistance to unfavorable conditions of certain varieties are at all correlated with their enhanced "greed for nitrogen." Moreover, it is far from being proven that any one race or "physiologic species" is really more virile than another. Greig-Smith- has shown that as many as three races are sometimes present in one and the same tubercle. Possibly, therefore, fixation of nitrogen may occur most rapidly only when two or more of these races are growing together. Buchanan^ has recently made a minute morphologic study of B. radicicola. Some of his conclusions are as follows: 1. Considerable variation in the morphology of B. radicicola may be induced in artificial media by the use of appropriate nutrients. Of the salts of the organic acids, sodium succinate brings about the most luxuriant development and the production of the greatest variety of bacteroids. 2. B. radicicola in the roots of the legumes shows the same type of bacteroids as may be found in suitable culture media. On the other hand, there is little or no correspondence between the type of bacteroid produced in culture media by a certain organism and that produced in the nodule by the same form. 3. It is probable that the term B. radicicola includes an entire group of closely related varieties or species, which differ from each other to some degree' in morphologic characters. 1 Ball, O. M. A nontribution to the Life History of B. radicicola Beij., Centralbl. f. Bakt., etc., 1909, II Abt., xxiii, 47. - Greig-Smith. Jour. Soc. Chem. Indust., 1907, No. 7. ^ Buchanan, R. E. The Bacteroids of Bacillus radicicola. Centralbl. f. Bakt., etc., 1!)0!), II A))t., xxiii, .59. BACTERIA IN SEWAGE 633 4. The luxhile orgiuiism resembles morphologically both the yeasts and the bacteria. The iliH'erence between this form and those ordi- narily included luider the terms Baeiihis and I'scudomonas justify the use of a separate generic name, lihizohltnii. In ISDo Wiuogradsky furnished proof that there are in the soil bac- teria which are outside of the plant roots performing the same function as those within the roots. These bacilli he called Clostridium pasteur- ianum. They are anaerobic and produce spores.. Their power to fix nitrogen is increased in presence of sugar and lessened in presence of nitrogenous substances. Beyerinck in 1901 described two aerobic species of nitrogen-fixing bacteria. Later Bailey described three additional species. These were called Azotobacter. These studies have already led to the inoculation of soils and to the investigation of the kind of soils and crops best fitted for the growth of these bacteria. Many impoverished soils have already been greatly improved. There are probably many other varieties of bacteria capa1)le of fixing nitrogen, because one can hardly examine the roots of any leguminous plants with outfinding tubercles different. The use of seed inoculated with the special variety of bacteria suitable for the plant and the soil is already largely practised. Bacteria and Soil Minerals. — Some of the bacterial products act upon the inorganic constituents of the soil. The carbonic dioxide and the organic acids act upon compounds of lime and magnesia, practically insoluble in water, to form more soluble substances. The same is true of the rock phosphates, the silicate of potassium, sulphates, etc. Scientific farming is beginning to make use of the knowledge already acquired, and there is reason to hope that great practical advantages will flow from the investigation of the relation of bacteria to soil exhaustion and replenishment. The effect of excessive bacterial development appears at times to be harmful to the soil. Each crop seems to favor the growth of certain varieties, and the exhaustion of the soil which follows the constant raising of the same crop is now suspected to be due in part at least to the continuance of a few restricted species of bacteria in the soil, which failing to produce all the necessary substances for the nutrition of the special crop, vegetation suffers, or again the bacteria finally entirely dissipate substances alreadj' in the soil necessary to growth. The application of manure not only adds food for plant life, but also countless numbers of bacteria which make the food more available. The greatest number of bacteria are contained a little below the sur- face of the soil, where they are protected from drying and sunlight and are in contact with oxygen and with the roots and other food of the superficial soil. Bacteria in Sewage. — The materials which flow from our sewers are a menace to public health, mainly because they so frequently contain pathogenic bacteria. The other products of men and animals are offensive, but rarely concentrated enough in drinking water to be (i34 APPLIED MICROBIOLOGY apprt'ciably deleterious. Sewage can be made harmless by being sterilized, but can be freed from offense only by the destruction of organic matter. This, except when chemical precipitants are used, is almost wholly obtained through bacterial processes. The putrefyijig value of soil has long been recognized. This is largely due to the action of the soil bacteria. In 1895, the Englishman, Cameron, introduced the "septic tank," which was a covered cemented pit. The sewage admitted at the bottom flowed out at the top, after about twenty-four hours' subjection to anaerobic conditions. The anaerobic bacteria during this time ferment the organic matter energetically, liquefy it, and develop abundant gas. The knowledge that soil and sand filters act not only mechanically but also and perhaps chiefly bacteriologically, having been acquired, intermittent soil filtration was established as one of the best means of bacteriologically purifying sewage. The sewage is conducted to the beds, allowed to pass through, and then after a few hours again poured on. This purification is based chiefly on the action of the aerobic bacteria in the upper layers of the soil or sand. The best practical results are obtained by combining the two processes; first the anaerobic treatment is used to break down the solid materials, and then the intermittent sand filtration, to oxidize the compounds and render these products harmless. With low temperatures the chemical changes are verj' much lessened and the filter beds act more like pure mechanical filters. The anaerobic bacteria change the proteid substances into simple chemical compounds, among which is ammonia. The carbohydrates are changed into gaseous compounds, acids, etc. The gases are mainly nitrogen, carbon dioxide, and marsh gas. The bacterial changes pro- duced in sewage poured on contact beds made of coarse coke, clinkers, or other material act much as in the sand filters after the filtration. Varieties of Bacteria in Filter Beds and Septic Tanks. — The septic tanks all contain spore-bearing bacilli which destroy cellulose, others that attack nitrogenous compounds. The cocci are in a minority. The filter beds have a number of small, non-spore-bearing bacilli; some of these change ammonia into nitrites and nitrates. There are also denitrifying bacteria. As before mentioned, the bacterial efficiency of the bed is increased with suitable temperature and much lessened with low temperature. Sewage Farming. — The action of bacteria is availed of in disposing of sewage over fields. The amount of sewage which can be poured on a certain area is limited. One acre of land can usually take care of the sewage from one hundred persons. If too much is poured on, it runs off impurified or clogs the soils and prevents the access of oxygen to aerobic bacteria. In warm weather evaporation and bacterial activities are much greater than in cold weather. So far as experience shows, those who eat vegetables from these small farms contract no (Hsease from them. BACTERIAL FERMENTATION 0.35 The Curing of Tobacco. — The curing of tobacco is apparently due partly to bacterial processes and partly to the action of leaf enzymes. The preservation of foods against decomposition by bacteria, yeasts, molds, and higher fungi is ojitained by using processes which will prevent the growth of microorganisms. Drying, exposure to wood smoke with consequent absorption of creosote, the addition of salt and sugar, of acids such as vinegar, spices, germicides such as boracic acid, formaldehyde, all are familiar methods of making foods unsuitable for bacterial growth. Instead of using food preserved by drying or chemicals, products may be kept at temperatures too low for bacterial growth. Cold storage of meats, eggs, vegetables, etc., is now common. The sterilization of food substances by heat with protection from infection afterward is made use of extensively in the canning of fruits and vegetables. Care must be taken that absolutely all bacteria are killed, for otherwise decomposition will finally occur. Vinegar Making. — Vinegar is made from some weak alcoholic solution by the union of alcohol with oxygen. This oxidation can be brought about by a purely chemical process. When vinegar is formed in the usual way bacteria are essential. The scum on the surface of the fermenting alcohol is a mass of microorganisms. The mother of vinegar was named mycoderma by Pearson. Kutzing showed that this was composed of living cells. Hansen proved these to be bacteria. We now know there are many varieties of bacilli capable of producing this fermentation. Each variety has its own optimum temperature and differs in the amount of acid it produces. Most of these have the peculiarity of growing at high temperatures into long threads without any traces of division. At low temperatures they produce long threads with swollen centres. The usual vinegar is made by using the variety of bacilli prevalent in the surroundings, but the custom is growing of adding to the pasteurized alcoholic solution the special variety desired in pure culture. Sauerkraut. — This is cabbage leaves shredded, slightly fermented, and prevented from decay by the lactic acid bacteria. At first both yeasts and bacteria increase together, but with the increase in acidity all growth ceases. Putrefaction is prevented by the same cause. The lactic acid bacteria are the same as those found in sour milk. Ensilage. — The fermentation here is believed to be due partly to enzymes in the corn tissues and partly to bacterial action. The first changes are due chiefly to the enzymes. The Bacterial Diseases of Plants. — These are probably as serious and varied for plants as for animals. The pear blight, the wilt disease of melons, the brown rot of tomatoes, the black rot of cabbages are examples. These plant diseases can be communicated by means of the pathogenic pure cultures of bacteria experimentally just as readily as animal diseases by their specific bacteria. Bacterial Fermentation in Relation to Miscellaneous Products.— Pasteur in 1857 explained the process of fermentation as due to the 036 APPLIED MICROBIOLOGY action of microorganisms. He demonstrated that the change of sugar into lactic acid only occurred when living hacilli were present. If the fluid was sterilized the ferjnentation ceased, lie stated that "or- ganic liquids do not alter until a living germ is- introduced into them." When the action is direct we speak of an organized ferment; when it is indirect, that is, due to the cell product, we call it an unorganized soluble ferment or enzyme. Similar enzymes are produced by the cells of the animal tissues, such as ptyalin, pepsin, and trypsin. Pas- teur's work led to the conclusion that the different fermentations were due to different varieties of organisms. The major part of fermenta- tion is due to yeast. Some important fermentations are due to bacteria and a few to the molds. Wines and Beers. — Alcoholic Fermentation. — If there is a develop- ment of the yeast cells in a solution of grape-sugar we have a fermenta- tion of the sugar with a final flevelopment of alcohol and carbon dioxide. It is thus that beers and wines are developed. When the carbohydrate is in the form of starch this is first converted into sugar and then later into the final products. If the sugar is in the form of saccharose, it is first changed by the yeast ferments to glucose. In all these three forms of fermentation the sugar is changed into alcohol and carbonic acid. When the alcohol reaches about 1.3 per cent, it stops further fermentation. These yeasts called saccharomyces comprise a number of distinct varieties, some of which are cultivated while others, called "wild yeasts," propagate themselves. The distillery, brewer^', and wine industries each make use of special yeasts and special conditions. The rising of bread is one of the most common uses of fermentation by yeast. The yeast acts upon the sugar made by the diastase from the starch. The resulting COo and alcohol create myriads of little bubbles in the dough. Diseases in Beer and Wines. — Hansen, Pasteur, and others demon- strated that the spoiling of beers and wines was due to the development of varieties of bacteria and yeasts which produce different kinds of fermentation from that desired. These produce alterations in flavor, bitterness, acidity. CHAPTER XLIX. THE DESTRUCTION OF BACTERIA BY CHEMICALS. PRACTICAL USE OF DISINFECTANTS. Many substances, when brought in contact witli bacteria, combine with their ceU substance and destroy the life of the bacteria. While in the vegetative stage bacteria are much more easily killed than when in the spore form, and their life processes are inhibited by substances less deleterious than those required to destroy them. Bacteria, both in the vegetative and in the spore form, differ among themselves considerably in their resistance to the poisonous eftectsof chemicals. The reason for this is not wholly clear, but it is connected with the structure and chemical nature of their cell substance. Chemicals in sufficient amount to destroy life are more poisonous at temperatures suitable for the best growth of bacteria than at lower temperatures, and act more quickly upon bacteria when they are sus- pended in fluids singly than when in clumps, and in pure water rather than in solutions containing organic matter. The increased energy of disinfectants at higher temperatures indicates in itself that a true chemical reaction takes place. In estimating the extent of the de- structive or inhibitive action of chemicals the following degrees are usually distinguished: 1. The growth is not permanently interfered with, but the patho- genic and zymogenic functions of the organism are diminished- attenuation. This loss of function is usually quickly recovered. 2. The organisms are not able to multiply, but they are not destroyed — antiseptic action. When transferred to a suitable culture fluid free of the disinfectant these bacteria are capable of reproduction. 3. The vegetative development of the organisms is destroyed, but not the spores — incomplete or complete sterilization or disinfection, according as to whether spores are present in the organisms exposed and as to whether these spores are capable of causing infection. 4. Vegetative and spore formation are destroyed. This is complete sterilization or disinfection} The methods employed for the determination of the germicidal action of chemical agents on bacteria are, briefly as follows: 1 Disinfection strictly defined is the destruction of all organisms and their products which are capable of producing disease. Sterilization is the destruction of all saprophytic as well as parasitic bacteria. It is not necessary in most cases to require disinfectants to be capable of sterilizing infected materials containing spores, for there are but few varieties of pathogenic bacteria which i^roduce spores. g:],s applied microbiology If it is desired to determine the minimum concentration of the chemi- cal substance required to produce complete inhibition of growth we proceed thus: A 10 per cent, solution of the disinfectant is prepared and 1 c.c, 0.5 c.c, 0..3 c.c, 0.1 c.c, etc., of this is added to 10 c.c. of liquefied gelatin, agar, or bouillon, or, more accurately 10 c.c. minus the amount of solution added, in so many tubes. The tubes then contain 1 per cent., 0.5 per cent., 0.3 per cent., and 0.1 per cent, of the disinfectant. The fluid media in the tubes are then inoculated with a platinum loopful of the test bacteria. The melted agar and gelatin may be simply shaken and allowed to remain in the tubes, and watched as to whether any growth takes place, or the contents of the tubes may be poured into Petri dishes, where the development or lack of development of colonies and the number can be observed. If no growth occurs in any of the dilutions, higher dilutions are tested. Bacteria that have been previousl.y injured in any waj' will be inhib- ited by much weaker solutions of chemicals than will vigorous cells. The same test can be made with material containing only spores. If it is desired to determine the degree of concentration required for the destruction of vegetative development, the organism to be used is cultivated in bouillon, and into each of a series of tubes is placed a definite amount of diluted culture from which all clumps of bacteria have been filtered ; to these a definite amount of watery solution of difl^erent percentages of the disinfectant is added. At intervals of one, five, ten, fifteen, and thirty minutes, one hour, and so on a small platinum loopful of the mixture is taken from each tube and inoculated into 10 c.c. of fluid agar or gelatin, from which plate cultures are made. Whenever it is possible that the antiseptic power of the disinfectant approaches somewhat the germicidal, it is necessary to inoculate a second series of tubes from the first so as to decrease still further the amount of antiseptic carried over. The results obtained are signified as follows: X per cent, of the disinfectant in watery solution and at x temperature kills the organism in twenty minutes, y per cent, kills in one minute, and so on. If there be any doubt whether the trace of the disinfectant carried over with the platinum loops may have rendered the gelatin unsuitable for growth, thus falsifying results, control cultures should be made by adding bacteria which have been somewhat enfeebled by slight contact with the disinfectant to fluid to which a similar trace of the flisinfectant has been added. If the strength of the disinfectant is to be tested for difl'erent substances it must be tested in these sub- stances or their equivalent, and not in water. The disinfectant to be examined should always be dissolved in an inert fluid, such as water; if, on account of its being difficultly solu- ble in water, it is necessary to use alcohol for its solution, control experi- ments may be required to determine the action of the alcohol on the organism. Sometimes, as in the case of corrosive sublimate, the chemi- cal unites with the cell substance to form an unstable compound, which inhibits the growth of the organism for a time before destroying it. If. this compound is not broken up in the media, it will probably not THE STANDARDIZATION OF DISINFECTANTS (ir,n be in the body. In some tests it is of interest to brealv up this union and note then whether the organism is ahve or dead. With corrosive subhmate the bacteria die in fifteen to thirty minutes after the union occurs. In the above determinations tlie absolute strengtli of the disinfect- ant required is considerably less when culture media poor in albumin are employed than when the opposite is the case. Cholera spirilla grown in bouillon containing no peptone or only 0.5 per cent, of pep- tone are destroyed in half an hour by 0.1 per cent, of hydrochloric acid; grown in 2 per cent, peptone-bouillon, their vitality is destroyed in the same time on the addition of 0.4 per cent. HCl. In any case the organisms to be tested should all be treated in exactly the same way and the results accompanied bj- a statement of the conditions under which the tests were made. It is becoming the custom to state the power of a disinfectant in terms of comparison with pure carbolic acid. A substance which had the same destructive power in a 1 to 1000 solution as carbolic acid in a 1 to 100 solution would be rated as of a strength ten times that of carbolic acid. The following table gives the results and methods used in an actual experiment to test the effect of blood-serum upon the disinfecting action of bichloride of mercury and carbolic acid upon bacteria: Test for the Difference of Effect of Bichloride of Mercury and Carbolic Acid Solutions on Xiphoid Bacilli in Serum and in Bouillon. Time. 1' 3' 5 10' 20' 30' 45' 1 hr. 1| hrs. hrs. A. Serum . 2..j c.c.l HgChsol. l:1000 2.5o.c,l Typhoid broth culture. J B. Bouillon . 2.5 c.c.l HgChsol. 1:1000 2.5c.c.[ Typhoid broth culture. J C. Serum . 2. .5 c.o.1 Carbohosol. 5% 2.5 c.c.^ Typhoid broth culture. J D. Bouillon . 2.5 c.c.l r'arbolicsol. 5% 2.5e.c.f Tj'phoid broth culture. J + + + + + + - - [Solution ■j equals 1 : 2000 [ bichloride. Same. rSolutioM equals 2i% [ carbolic acid. Same. — Indicates total destruction of bacteria with no growth in media. + Indicates lack of destruction of bacteria with growth in media. THE STANDARDIZATION OF DISINFECTANTS. Rideal and Walker were the first to urge a useful method for doing this. In carrying out the test the various factors must be carefully con- trolled, thus: Time: this should be constant, the strength of the disinfectant being the variant. TcM organisms: a standard culture of (140 APPLIED MICROBIOLOGY the typhoid bacilhis (Hopkins' strain) is used to avoid any variations (kie to the different degrees of resistance of various strains. Tlie culture should be subinoculated three days before used. Medium: a standard meat-extract broth 1.5 per cent, acid to phenolphthalein ; 10 c.c. to a tube is employed. Temperature: this test is done at 20° C. This is important, as the germicidal activity increases with the temperature. Constant amount of culture used: 0.1 c.c. of the twenty- four-hour broth culture is added to 5 c.c. of the disinfectant solution. This is more accurate than the drop method. Aviount inoculated: it is essential that the same amount be inoculated from each dilution. Platinum loops made of 23 United States gauge wire, the loops being 4 mm. in diameter are employed. Several are used, being left on a rack after sterilization so as to be cold when needed. The loop is bent at an angle of 45 to the shank. The actual test is carried out as follows: A 5 per cent, carbolic solu- tion (phenol C. P.), is prepared and standardized by bromine titration. From this freshly prepared 1 to 90 to 1 to 100 and 1 to 110 dilutions are made as needed. The necessary dilutions of germicide are then prepared. Wide jumps in the dilutions are made and then narrowed as the limits of the disinfectant to be tested are determined. Five test-tubes are arranged in a row in a water bath at 20° C. and the solutions added in 5 c.c. amounts. Time must be allowed for the solution to reach 20° C. If the bath be large enough, little attention is needed to keep the temperature constant. The culture having been brought to 20° C. is then added in 0.1 c.c. amounts and the tubes shaken, an interval of .30 seconds allowed between each tube. Subinoculation of the first tube is then made after thirty seconds, which gives an interval two and one-half minutes after inoculation. The tubes are then sub- inoculated in order at thirty-second intervals, giving an interval for each of two and one-half minutes after inoculation, and starting at the first, gives an interval of five minutes, etc. It is possible to use ten tubes, as each step can be done in less than fifteen seconds if properly arranged, allowing a much wider range of each test. It is not necessary to keep the cotton plugs in the tubes during the operation nor to remove the tubes from the bath to obtain the loopful for inoculation. The loop is plunged to the bottom, care being taken not to touch the sides of the tubes, and care should also be taken that a loopful is carried away each time. The broth tubes are incubated for forty-eight hours and then examined for growth. The following are given as two examples: Time of exposure; 2..-) .5 7. .5 10 12. .5 1.^1 Phenol Sample. Dilution. niin. min. min. niin. min. inin. coefficient. Phenol 1 to 90 + - - - - - 1 to 100 + + + - - - 100)550 b.a Disinfectant A. . . 1 to 450 + 1 to 550 + 1 to 600 + Tlll<: STANDMiniZATION OF DISINFECTANTS M\ PIkmiuI cocni<;i(MlL. Til 10 of •XI osurc: 2.5 ■) 7. -5 10 \'lSi Sample. DiL ition. mill. mill. mill. mill. mill. PI flK 1 . 1 t ) 100 + + + - - 1 t ) 110 + + + + + D sill L'fUiat A. 1 t o 600 + + + + _ 1 t o 050 + + + + + 1 t o 700 + + + + + iio}ii.5o 5.191 These tables not only serve as an example, but also show that except many repetitions of the tests are made and averaged consider- able variations in the results may be obtained. A report of 5.5 or 5.1 is equally accurate in the test here reported of the Rideal-Walker method, even with certain improvements added. With practice, and by selection of the dilutions to be employed, the operator evidently can regulate the time factor so that fairly uniform results are obtain- able. On the other hand it not infrequently happens that if more than one carbolic dilution is employed, more than one time period is open to comparison. For these reasons Anderson and McCliutic have modified the test by setting two time limits two and one-half and fif- teen minutes and taking the average. The following is an example: Time of exiio.sure: 2. .5 5 7. .5 10 12..') 1.5 Phenol Sample. Dilution. min. niin. min. niin. min. min. eoefficient. Phenol .... 1 to 80 - - - - - - :-!75 + 650 1 to 90 + - - - - - • — 1 to 100 + + + - - - .SO 110 1 to 110 + + + + + = o Disiufeetant A. 1 to :j50 - - - 1 to 375 - - - 4.6(1 + 5.01 1 to 400 + - - - = 1 to 425 + + - - - - 2 1 to 450 + + 1 to 500 + + - - - - 5.30 1 to 550 + + + --- 1 to 600 + + + + 1 to 650 + + + + + 1 to 700 + + + + + + 1 to 750 + + + + + + Disinfectants vary widely in their germicidal properties, depending on whether organic matter is present or not. ks, under practical condi- tions organic matter is usually present, it is of some importance to know how far organic matter decreases the efficiency. For the purpose of obtaining comparable results, Anderson and McChntic have suggested the use of peptone 10 per cent, and gelatin 5 per cent, in distilled water. One part of the culture is mixed with 10 parts of the organic solution, 1.1 c.c. being then added to a series of dilution tubes containing 4 c.c. In determining the coefficient allowance must be- made for the added amount of organic matter. The modified methods of Anderson and McClintic^ are called the ' See Hygieiiie Laiioratory Bulletin No. S2, for further details ami apparatus for simplifying the steps of the test. 41 (142 APPLIED MICROBIOLOGY "hygienic laboratory phenol coefBcient," with or without organic matter. Any organic matter may be used in the test to approach the special conditions under which a disinfectant is to be used. In comparing the value of disinfectants the cost as well as the coefficient must be considered. This is best stated in terms of the relative cost of 100 units of efficiency as compared with pure phenol =100, thus: Cost of disinfectant per gallon _ ,„ , f \ ■ Coefficient of disinfe ctant . Cost of phenol per gallon ' Coefficient of phenol (1). = the efficiency, X 100= relative cost per 100 units. Antiseptic Value. — With certain disinfectants there is sufficient of the disinfectant carried over by the loop to exert antiseptic action and growth does not occur. If this is not taken in consideration a disinfectant will be given an excessively high coefficient. No satis- factory method has been devised to avoid this difficulty. The inocu- lated broth tube may be shaken and a loop or more inoculated from it to second broth tube, in this way diluting the disinfectant still farther. Chick^ has attempted to overcome the difficulty in the case of mercury-containing disinfectants by adding 0.2 c.c. of a saturated watery solution of hydrogen sulphide to each tube of broth. Many substances which are strong disinfectants become altered under the conditions in which they are used, so that they lose a portion or all of their germicidal properties; thus, quicklime and milk of lime act by means of their alkali and are disinfecting agents only so long as sufficient calcium hydroxide is present. If this is changed by the carbon dioxide of the air into carbonate of lime it becomes harmless. Bichloride of mercury and many other chemicals form compounds with many organic and inorganic substances, which, though still germicidal, are much less so than the original substaiices. Solutions of chlorine, peroxides, etc., when in contact with an excess of organic matter soon become inert because of the chemical compounds formed. The Disinfecting Properties of Inorganic Compounds. — Bichloride of Mercury. — This substance, which dissolves in 16 parts of cold water, when present in 1 part in 100,000 in nutrient gelatin or bouillon, inhibits the development of most forms of bacteria. In water 1 part in 50,000 will kill many varieties in a few minutes, but in bouillon twenty-four hours may be needed. With organic substances its power is lessened, so that 1 part to 1000 may be required. Most spores are killed in 1 : 500 watery solution within one hour. Corrosive sublimate is less effective as a germicide of alkaline fluids containing much albuminous substance than in watery solution. In such fluids, besides loss in other ways, precipitates of albuminate of mercury are formed which are at first insoluble, so that a part of the mercuric salt does not really exert any action. In alkaline solutions, such as blood, blood- serum, pus, sputum, tissue fluids, etc., the soluble compounds of mercury are converted into oxides or hydroxides. . 1 Journal of Hygiene, 190S, viii, 654. THE STANDARDIZATION OF DISINFECTANTS G4o For ordinary use, where corrosive sublimate is employed, solutions of 1 to 500 and 1 to 2000 will suffice, when brought in contact with bacteria in that strength, to kill the vegetative forms within from one to twenty minutes, the stronger solution to be used when much organic matter is present. Mercuric chloride volatilizes slowly and it is better to wash off walls after use of bichloride solutions. Solutions of this salt should not be kept in metal receptacles. Mercuric chloride solution has dis- advantages in that it corrodes metals, irritates the skin, and forms almost inert compounds with albuminous matter. In order to avoid accicients, solutions of this odorless disinfectant should be colored by some dye. Biniodide of Mercury. — This salt is very similar in its effect to the bichloride. Nitrate of Silver. — Nitrate of silver in watery solution has about one-fourth the value of the bichloride of mercury as a disinfectant, but nearly the same value in inhibiting growth. In albuminous solutions it is equal to bichloride of mercury. Compounds of silver nitrate and albuminous substances have been used because of the absence of irritative properties combined with moderate antiseptic power. Sulphate of Copper. — This salt has about 50 per cent, of the value of mercuric chloride. It has a quite remarkable affinity for many species of algie, so that when in water 1 to 1,000,000 it destroys many forms; 1 to 400,000 destroj^s typhoid bacilli in twenty-four hours when the water has no excessive amount of organic material. It is not known to be poisonous in this strength, so that it can be temporarily added to water supplies. Sulphate of Iron. — This is a much less powerful disinfectant than sulphate of copper. A 5 per cent, solution requires several days to kill the typhoid bacilli. It can only be considered as a mild antiseptic and deodorant. Zinc Chloride. — This is very soluble in water, but is a still weaker disinfectant than copper sulphate. Sodium Compounds. — A 30 per cent, solution of NaOH kills anthrax spores in about ten minutes, and in 4 per cent, in about forty-five minutes. One per cent, kills vegetative forms in a few minutes. Sodium carbonate kills spores with difficulty even in concentrated solution, but at 85° C. it kills spores in from eight to ten minutes. It is used frequently to cover metallic instruments. A 5 per cent, solution kills in a short time the vegetative forms of bacteria. Even ordinary soapsuds have a slight bactericidal as well as a marked cleansing effect. The bicarbonate has almost no destructive effect on bacteria. Calcium Compounds. — Calcium hydroxide, Ca(0H)2, is a powerful disinfectant; the carbonate, on the other ha]id, is almost without effect. The former is prepared by adding one pint of water to two pounds of lime (quicklime, CaO). Exposed to the air the calcium hydrate slowly becomes the inert carbonate. A 1 per cent, watery solution 644 APPLIED MICROBIOLOGY of the hydroxide kills bacteria which are not in the spore form within a few hours. A 3 per cent, solution kills typhoid bacilli in one hour. A 20 per cent, solution added to equal parts of feces or other filth and mixed with them will completely sterilize them within two hours. ECect of Acids. — An amount of acid which equals 40 c.c. of normal hydrochloric acid per liter is sufficient to prevent the growth of all varieties of bacteria and to kill many. Twice this amount destroys most bacteria within a short time. The variety of acid makes little difference. Bulk for bulk, the mineral acids are more germicidal than the vegetable acids, but that is because their molecular weight is so much less. A 1 to 500 solution of sulphuric acid kills typhoid bacilli within one hour. Hydrochloric acid is about one-third weaker, and acetic acid somewhat weaker still. Citric, tartaric, malic, formic, and salicylic acids are similar to acetic acid. Boric acid destroys the less resistant bacteria in 2 per cent, solution and inhibits the others. Gaseous Disinfectants. — The germicidal action of gases is much more active in the presence of moisture than in a dry condition. Sulphur Dioxide (SO2). — Numerous experiments have been made with this gas owing to the fact that it has been so extensively used for the disinfection of hospitals, ships, apartments, clothing, etc. This gas is a much more active germicide in a moist than in a dry condition; due, no doubt, to the formation of the more active disinfecting agent — sulphurous acid (H2SO3). In a pure state anhydrous sulphur dioxide does not destroy spores, and is not certain to destroy bacteria in the vegetative form. Sternberg has shown that the spores of the Bacillus anthracis and Bacillus suhtilis are not killed by contact for some time with liquid SO2 (liquefied by pressure). Koch found that various species of spore-bearing bacilli exposed for ninety-six hours in a dis- infecting chamber to the action of SO2, in the proportion of from 4 to 6 per cent, by volume, were not destroyed. In the absence of spores, however, the anthrax bacillus in a moist condition, attached to silk threads, was found by Sternberg to be destroyed in thirty minutes in an atmosphere containing 1 volume per cent. As the result of a large number of experiments with SO2 as a disin- fectant it has been determined that an "exposure for eight hours to an atmosphere containing at least 4 volumes per cent, of this gas in the presence of moisture' will destroy most, if not all, of the patho- genic bacteria in the absence of spores. Four pounds of sulphur burned for each 1000 cubic feet will give an excess of gas. Peroxide of Hydrogen (H2O2).— This is an energetic disinfectant, and in 2 per cent, solution (about 40 per cent, of the ordinary commercial article) will kill the spores of anthrax in from two to three hours. A 20 per cent, solution of a good commercial hydrogen peroxide solution will quickly destroy the pyogenic cocci and other spore-free bacteria. It combines with organic matter, becoming inert. It is prompt in its action and not poisonous, but apt to deteriorate if not properly kept. THE STANDARDIZATION OF DISINFECTANTS 045 Chlorine. — Chlorine is a powerful gaseous germicide, owing its activity to its affinity for hydrogen and tlie consec(uent release of nascent oxygen when it comes in contact witli microorganisms in moist condition. It is, therefore, a much more active germicide in the presence of moisture than in a dry condition. Thus, Fischer and Proskauer found that dried anthrax spores exposed for an hour in an atmosphere containing 44.7 per cent, of dry chlorine were not destroyed; but if the spores were previously moistened and were exposed in a moist atmosphere for the same time, 4 per cent, was effective, and when the time was extended to three hours 1 per cent, destroyed their vitality. The anthrax bacillus, in the absence of spores, was killed by exposure in a moist atmosphere containing 1 part to 2500 for twenty-four hours. In watery solutions 0.2 per cent, kills spores within five minutes and the vegetative forms almost immediately. One part in one million is sufficient to destroy typhoid bacilli in a water containing little organic matter in the course of a few hours. In water with much organic matter a much greater amount is required. Chlorinated Lime (Called "Chloride of Lime"). — Chlorinated lime is made by passing nascent chlorine gas over unslaked lime. It should not contain less than 10 per cent, of available chlorine, and can now be obtained containing .30 per cent. It should have a strong odor of chlorine. Its efficacy depends on the chlorine it contains in the form of hypochlorites. The calcium hypochlorite is readily broken up into hypochlorous acid. A solution in water of 0.5 to 1 per cent, of chlorin- ated lime will kill most bacteria in one to five minutes, and 1 part in 100,000 will destroy typhoid bacilli in twenty-four hours. A 5 per cent, solution usually destroys spores within one hour. Chlorinated lime not only bleaches, but destroys fabrics. The Hypochlorites (Labarraque's Solution). — Solutions of hypo- chlorites are practically the same as solutions of chlorinated lime and are much more expensive. Bromirie and iodine are of about the same value as chlorine for gaseous disinfectants, in the moist condition; but, like chlorine, they are not applicable for general use in house disinfection, owing to their poisonous and destructive properties; thej' have a use in sewers and similar places. Trichloride of iodine in 0.5 per cent, solution destroys the vegetative forms of bacteria in five minutes. Organic Disinfectants. — Alcohol in 10 per cent, solution inhibits the growth of bacteria; absolute alcohol kills bacteria in the vegetative form in from several to twenty-four hours. According to Epstein, 50 per cent, alcohol (in water) has more germicidal power than any other strength, the power gradually diminishing with both stronger and weaker solutions. Formaldehyde. — Formaldehyde, or formic aldehyde, was isolated by von Hoffmann in 1867, who obtained it by passing the vapors of methyl-alcohol mixed with air over finely divided platinum heated to G46 APPLIED MICROBIOLOGY redness. Tlie methyl-alcohol is oxidized and produces formaldehyde as follows: (•H3OH + O = r'HjO + H,(). Formaldehyde is a gaseous compound possessed of an extremely irritating odor. At a temperature of 68° F. the gas is polymerized — that is to say, a second body is formed, composed of a union of two molecules of CH2O. This is known as a paraformaldehyde, and is a white, soapy body, soluble in boiling water and in alcohol. Formal- dehyde is sold in commerce as a clear, watery liquid containing from 33 to 40 per cent, of the gas and 10 to 20 per cent, of methyl-alcohol, its chief impurity. If the commercial solution — ordinarilj^ known in the trade as "formalin" — is evaporated or concentrated above 40 per cent., paraformaldehyde results; and when this is dried in vacuo over sulphuric acid a third body — trioxymethylene — is produced, consisting of three molecules of CH2O. This is a white powder, almost soluble in water or alcohol, and giving off a strong odor of formaldehyde. The solid polymers of formaldehyde, when heated, are again reduced to the gaseous condition; ignited, they finally take fire and burn with a blue flame, leaving but little ash. When burned they have no germicidal properties. Formaldehyde has an active affinity for many organic substances, and forms with some of them definite chemical combinations. It combines readily with ammonia to produce a compound called hexa- methylene tetramine, which possesses neither the odor nor the anti- septic properties of formaldehyde. This action is made use of in neutralizing the odor of formaldehyde when it is desired to dispel it rapidly after disinfection. Formaldehyde also forms combinations with certain aniline colors — viz., fuchsin and safranin — the shades of which are thereby changed or intensified. These dyes are tests for aldehydes. These are the only colors, however, which are thus af- fected, and as they are seldeom used in dyeing, owing to their liability to fade, this effect is of little practical significance. The most deli- cate fabrics of silk, wool, cotton, fur, leather, etc., are unaffected in texture or color by formaldehyde. Iron and steel are attacked, after long exposure, by the gas in combination with watery vapor; but copper, brass, nickel, zinc, silver, and gilt work are not at all acted upon. For- maldehyde unites with nitrogenous products of decay — fermentation or decomposition — forming true chemical compounds, which are odor- less and sterile. It is thus a true deodorizer in that it does not replace one odor by another more powerful, but forms new chemical compounds which are odorless. Formaldehyde has a peculiar action upon albu- min, which it transforms into an insoluble and indecomposable sub- stance. It renders gelatin insoluble in boiling water and most acids and alkalies. It is from the property of combining chemically with the albuminoids forming the protoplasm of bacteria that formaldehyde is supposed to derive its bactericidal powers. Formaldehj'de is an excel- lent preser\'ative of organic products. It has been proposed to make THE STANDARDIZATION OF DISINFECTANTS (;47 use of this action for the preservation of meat, milk, and other food products; but, according to Trillat and other investigators, formalde- hyde renders these substances indigestible and unfit for food. It has been successfully emplo}'ed as a preservative of pathologic and histohjgic specimens. There are no exact experiments recorded of the physiologic action of formaldehyde on the human subject when taken internally. A 1 per cent, solution has been taken in considerable quantity without serious results; and trioxy methylene has been given in doses up to 90 grains as an intestinal antiseptic. The vapors of formaldehyde are extremely irritating to the mucous membrane of the eyes, nose, and mouth, causing profuse lacrimation, coryza, and flow of saliva. Aronson reports that in many of his experiments rabbits and guinea- pigs allowed to remain for twelve and twenty-four hours in rooms which were being disinfected with formaldehyde gas were found to be perfectly well when the rooms were opened. On autopsy the ani- mals showed no injurious effects of the gas. Others have noticed that animals, such as dogs and cats, which have accidentally been confined for any length of time in rooms undergoing formaldehyde disinfection occasionally died from the effects of the gas. Many observers, how- ever, have reported that insects, such as roaches, flies, and bedbugs, are not, as a rule, affected. The result of these observations would seem to indicate that although formaldehyde is comparatively non-toxic to the higher forms of animal life, nevertheless a certain degree of caution should be observed in the use of this agent. It is important to remem- ber that formaldehyde is practically inert as an insecticide except in extremely great concentrations. The researches of Pottevin and Trillat have shown that the germi- cidal power of the gas depends not only upon its concentration, but also upon the temperature and the condition of the objects to be steril- ized. As with other gaseous disinfectants — viz., sulphur dioxide and chlorine — it has been found that the action is more rapid and complete at high temperatures— i. e., at 35° to 45° C. (95° to 120° F.)— and when the test objects are moist than at lower temperatures and when the objects are dry. Still, it has been repeatedly demonstrated by actual experiment in rooms that it is possible to disinfect the surface of apartments and articles contained in them, under the conditions of temperature and moisture ordinarily existing in rooms even in winter, by an exposure of a few hours to a saturated atmosphere of formalde- hyde gas. The results of numerous experiments have shown that in the air 2.5 per cent, by volume of the aqueous solution, or 1 per cent, by volume of the gas, are sufficient to destroy fresh virulent cultures of the common non-spore-bearing pathogenic bacteria in a few minutes. Stahl has shown that bandages and iodoform gauze can be kept well sterilized by placing in the jars containing them pieces of a prepa- ration of paraformaldehyde in tablet form containing 50 per cent, of formaldehyde. The same experimenter has also succeeded in making carpets and articles of clothing germ-free by spraying them (148 APPLIED MICROBIOLOrSY witli 0.5 to 2 per cent, solution of formaldehyde for fifteen to twenty minutes witJiout tlie color of the fabrics being in any way affected. The investigations of Trillat, Aronson, Pottevin, and others have shown that a concentration of titi'iikt of the aqueous soluti(jn (40 per cent.), equal to TTj-cnyiT of pure formaldehyde, was safe and sufficiently powerful to retard bacterial growth. A 2 per cent, watery solution of formalin destroys the vegetative forms of bacteria within five to thirty minutes. In our experiments formalin has upon the vegetative forms about one-half the strength of pure carbolic acid. Chloroform (CHCI3). — This substance, even in pure form, does not destroy spores, although it kills bacteria in vegetati-\'e form, even in 1 per cent, solution. Chloroform is used practically as an antiseptic in antitoxic sera and in blood-serum for culture purposes. The chloro- form is expelled from the serum by heating it to 55° C. Iodoform (CHI3). — This substance has but very little destructive action upon bacteria; indeed, upon most varieties it has no appreciable effect whatever. When mixed with putrefying matter, wound dis- charges, etc., the iodoform is reduced into soluble iodine compounds, which act partly destructivelj' upon the bacteria and partly by uniting with poisons already produced. Carbolic Acid (CeHsOH). — Pure phenol crystallizes in long, colorless crystals. In contact with air it deliquesces. It has a penetrating odor, a burning taste, and is a corrosive poison. It is soluble at ordinary temperatures in about 15 parts of water. Carbolic acid dissolves in water with some difficulty and should be therefore thor- oughly mixed. It is not destructive to fabrics, colors, metals, or wood, and does not combine as actively with albuminous matters as bichloride of mercury. It is therefore more suitable for the disinfection of feces, etc. A solution having 1 part to 1000 inhibits the growth of bacteria; 1 part to 400 kills the less resistant bacteria, and 1 part to 100 kills the remainder. A 5 per cent, solution kills the less resistant spores within a few hours and the more resistant in from one day to four weeks. A slight increase in temperature aids the destructive action; thus, even at 37.5° spores are killed in three hours. A 3 per cent, solution kills streptococci, staphylococci, anthrax bacilli, etc., within one minute. Carbohc acid loses much of its value when in solution in alcohol or ether. An addition of 0.5 HCl aids its activity. Carbolic acid is so permanent and so comparatively little influenced by albumin that it is rightly widely used in practical disinfection even in place of more powerful substances. Cresol. — Cresol [C6H4(CH3)OH] is the chief ingredient of the so-called "crude carbolic acid." This is almost insoluble in water, and therefore of restricted value. Many methods are used for bring- ing it int(j solution so as to make use of its powerful disinfecting properties. With equal parts of crude sulphuric acid it is a powerful disinfectant, but it is, of course, strongly corrosive. An alkaline emulsion of the cresols and other products contained in "crude" car- THE STANDARDIZATION OF DISINFEf'TANTS r.4n l)olic' acid witli soiq) is eallcil creplin. Jt is used in 1 to 5 per cent, emulsions. Tt is fully as powerful as ])ure earholie acid. I.ysol is similar to creolin, except that it has more of tlic cresols and less of the other prt)ducts. It and creolin arc of about the same value. Tricresol. — Tricresol is a refined mixture of the three cresols (meta- cresol, paracresol, and orthocresol). It is soluble in water to the extent of 2.5 per cent, and its disinfecting power is about three times as great as that of carbolic acid. Creolin. — Creolin contains 10 per cent, of cresols held in solution by soap. Lysol. — Lysol contains about .50 per cent, of cresols. It mixes with water in all dilutions. Oil of Turpentine, 1 to 200, prevents the growth of bacteria. Camphor has very slight autiseptic action. Creosote in 1 to 200 kills many bacteria in ten minutes; 1 to 100 failed to kill tubercle bacilli in twelve hours. Essential Oils. — Cardeac and Meumir found that the essences of cinnamon, cloves, thyme, and others killed typhoid bacilli within one hour. Sandalwood required twelve hours. Thymol and encalyptol have about one-fourth the strength of car- bolic acid (Behring). Oil of peppermint in 1 to 100 solution prevents the growth of bacteria. Tables of Antiseptic Values.^ .A.lum . 1 to 222 Mercuric chloride 1 to 14,300 Aluminum acetate . 1 to 6000 Mercuric iodide . 1 to 40,000 Aramoniuin fhloride . 1 to 9 Potassium bromide 1 to 10 Boric acid .... 1 to 14.3 Potassium iodide 1 to 10 Calcium chloride . . 1 to 2.5 Potassium permanganate 1 to 300 Calcium hypochlorite 1 to 1000 Pure formaldehyde 1 to 2.5,000 CarboUc acid . . - . . 1 to 333 Quinine sulphate 1 to 800 Chloral hydrate . 1 to 107 Silver nitrate 1 to 12,500 Cupric sulphate . . 1 to 2000 Sodium borate 1 to 14 Ferrous sulphate . . 1 to 200 Sodium chloride . 1 to 6 Formaldehyde (40%) . 1 to 10,000 Zinc chloride .... 1 to 500 Hydrogen peroxide . . 1 to 20,000 Zinc sulphate 1 to 20 ^ These figures are approximately correct, and represent the percentage of disinfection required to be added to a fluid containing considerable organic material, in order perma- nently to prevent any bacterial growth. Solutions of half the given strength will inhibit the growth of most bacteria and i>revent the growth of many varieties. CHAPTER L. PRACTICAL DISINFECTION AND STERILIZATION (HOUSE, PERSON, INSTRUMENTS, AND FOOD). STERILIZATION OF MILK FOR FEEDING INFANTS. DISINFECTANTS AND METHODS OF DISINFECTION EMPLOYED IN THE HOUSE AND SICKROOM. Disinfection and Disinfectants. — Sunlight, pure air, and cleanliness are always very important agents in maintaining health and in protect- ing the body against many forms of illness. When, however, it becomes necessary to guard against such special dangers as infectious material from communicable diseases the additional protection of disinfection becomes essential. In order that disinfection shall afford complete protection it must be thorough; and perfect cleanliness is better, even in the presence of contagious disease, than filth with incomplete disinfection. In order that as few articles as possible shall be exposed to the germs causing the communicable diseases, and thus become possible carriers of infection, it is important when conditions allow of it that all articles not necessary for the care and comfort of the sick person, especially upholstered furniture, carpets, and curtains, should be removed from the room before placing the sick person in it. Agents for Cleansing and Disinfection. — Too much emphasis cannot be placed upon the importance of cleanliness, both as regards the person and the dwelling, in protecting the body from all kinds of infectious disease. Sunlight and fresh air should be freely admitted, and personal cleanliness should be attained by frequently washing the hands and body, replacing fabrics infected by expectoration, bowel dis- charges, etc. By these means most of the bacteria are removed or discharged. Cleanliness in dwellings, and in all places where men go, may, under ordinary circumstances, be well maintained by the use of the two following solutions : 1. Soapsuds Solution. — For simple cleansing, or for cleansing after the method of disinfection by chemicals described below, one ounce of common soda should be added to twelve quarts of hot soapsuds (soft soap and water). 2. Strong Soda Solution. — This, which is a stronger and more effective cleansing solution and also a fairly efficient disinfectant, is made by dissolving one-half pound of common soda in three gallons of hot water. The solution thus obtained should be applied by scrubbing with a hard brush. DISINFECTION IN THE HOUSE AND SICK-ROOM 051 When it becomes necessary to prevent the spread of connnnnicable diseases by surely kilHng the Hving germs which cause them, more powerful agents must be employed than those required for simple cleanliness, and these are commonly called disinfectants. Tlie following are some of the most reliable ones: 1. Heat. — Complete destruction by fire is an absolutely safe method of disposing of infected articles of small value, but continued high temperatures not as great as that of fire will destroy all forms of life; thus, boiling or steaming in closed vessels for ten minutes will absolutely destroy all disease germs except spores. 2. Carbolic Acid Solution. — Dissolve six ounces of carbolic acid in one gaUon of hot water (200 grams in 4000 c.c). This makes approxi- mately a 5 per cent, solution of carbolic acid, which, for most purposes, may be diluted witli an equal cjuantity of water. The commercial "soluble crude carbolic acid" which is cheaper and twice as effective as the carbolic acid, can be used for privies and drains.' It makes a white emulsion on account of its not entering readily into solution. Care must be taken that the pure acid does not come in contact with the skin. 3. Bichloride Solution (bichloride of mercury or corrosive sublimate). — Dissolve sixty grains of pulverized corrosive sublimate and two table- spoonfuls of common salt in one gallon of hot water. This solution, which is approximately 1 : 1000, must be kept in glass, earthen, or wooden vessels (not in metal vessels). For safety it is well to color the solution. The carbolic and bichloride solutions are very poisonous when taken by the mouth, but are harmless when used externally. 4. Milk of Lime. — This mixture is made by adding one quart of dry, freshly slaked lime to four or five quarts of water. (Lime is slaked by pouring a small quantity of water on a lump of quicklime. The lime becomes hot, crumbles, and as the slaking is completed a white powder results. The powder is used to make milk of lime.) Air-slaked lime (the carbonate) has no value as a disinfectant. 5. Dry Chlorinated Lime, "Chloride of Lime." — This must be fresh and kept in closed vessels or packages. It should have the strong, pungent odor of chlorine (see page 643). 6. Formalin (this is a watery solution containing 40 per cent, of formaldehyde). — Add 1 part of formalin to 10 of water. This equals in value the 5 per cent, carbolic acid solution. 7. Creolin, Tricresol, and Lysol. — The first is of about the same value as pure carbolic acid, the latter two about three times as powerful. The proprietary disinfectants, which are so often widely advertised and whose composition is kept secret, are relatively expensive and often ' The cost of the pure carbohc acid solution is much greater than that of most of the other solutions, but except for the disinfection of the skin, which in some persons it irritates, and of woodwork, it is generally much to be preferred by those not thoroughly familiar with disinfectants, as it does not deteriorate, and is rather more uniform in its aetioii than some of the other disinfectants. 052 APPLIED MICROBIOLOGY unreliable and inefficient. It is important to remember that substances which destroy or disguise bad odors are not necessarily disinfectants, and that there are very few disinfectants that are not poisonous when taken internally. Their value should be stated in the circular in comparison with pure carbolic acid, so that their strength may be known. Methods of Disinfection in Infectious and Contagious Diseases. — The diseases to be commonly guarded against, outside of surgery, by disinfection are scarlet fever, measles, diphtheria, tuberculosis, small- pox, typhoid, bubonic plague, and cholera. 1. Hands and Person. — Dilute the 5 per cent, carbolic solution with an equal amount of water or use the 1 : 1000 bichloride solution without dilution. Hands soiled in caring for persons suffering from contagious diseases, or soiled portions of the patient's body, should be immediately and thoroughly soaked with one of these solutions and then washed with soap and water, and finally immersed again in the solutions. The nails should always be kept clean. Before eating, the hands should be first washed in one of the above solutions, and then thoroughly scrubbed with soap and water by means of a brush. 2. Soiled clothing, towels, napkuis, bedding, etc., should, on removal, be immediately immersed in the 2.5 per cent, carbolic solution, in the sick-room, and soaked for one or more hours. Articles such as beds, woollen clothing, etc., which cannot be washed, should be thoroughlj' exposed to formaldehyde gas, as noted later (see page 654). This is not necessary after measles. 3. Food and Drink. — Food thoroughly cooked and drinks that have been boiled are free from disease germs. Food and drinks, after cooking or boiling, if not immediately used, should be placed when cool in clean dishes or vessels and covered. In the presence of an epidemic of cholera or typhoid fever, milk and water used for drinking, cooking, washing dishes, etc., should be boiled before using, and all persons should avoid eating uncooked fruit and fresh vegetables. 4. Discharges of all kinds from the mouth, nose^ bladder, and bowels of patients suffering from contagious diseases should be received into glass, metal, or earthen vessels containing the carbolic solution, or milk of lime, or they should be removed on pieces of cloth, which are immediately immersed in one of these solutions or boiled or destroyed by fire. Special care should be observed to disinfect at once the vomited matter and the intestinal discharges from cholera patients. In typhoid fever the urine and the intestinal discharges, and in diphtheria, measles, and scarlet fever the discharges from the throat and nose all carry infection and should be treated in the same manner. The volume of the solution used to disinfect discharges should be at least twice as great as that of the discharge, and should completely mix with it and cover it. After standing for an hour or more the discharges with the exception of the feces may be thrown into the water-closet. Masses of feces are extremely difficult to disinfect except on the surface, for it takes disinfectants such as the carbolic acid solution DISINFECTION IN THE HOUSE AND SICK-ROOM 053 some twelve hours to penetrate to tlieir interior. If fecal masses are to be thrown into places where the disinfectant solution covering them will be washed off, it will be necessary to be certain that the disinfectant has previously penetrated to all portions and destroyed the disease germs. This can be brought about by stirring them with the disinfectant and allowing the mixture to stand for one hour, or by washing them into a pot holding soda solution which is already at the boiling temperature, or later will be brought to it. 5. Sputum from Consumptives. — The importance of the proper dis- infection of the sputum is still underestimated. Consumption is an infectious disease, and is always the result of transmission from the sick to the healthy or from animals to man. The sputum contains the germs which cause the disease, and in a large proportion of cases is the source of infection. After being discharged, unless properly disposed of, it may become dry and pulverized and float in the air as dust. This dust contains the germs, and is a common cause of the disease, through inhalation. In all cases, therefore, the sputum should be disinfected when discharged. It should be received in covered cups containing the carbolic or milk-of-lime solution. Handkerchiefs soiled by it should be soaked in the carbolic solution and then boiled. Dust from the walls, mouldings, pictures, etc., in rooms that have been occupied by consumptive patients, where the rules of cleanliness have not been carried out, contain the germs and will produce tuberculosis in ani- mals when used for their inoculation; therefore, rooms should be thoroughly renovated or disinfected before they are again occupied. If the sputum of all consumptive patients were destroyed at once when discharged a large proportion of the cases of the disease would be prevented. 6. Closets, Kitchen and Hallway Sinks, etc. — The closet should never be used for infected discharges until they have been thoroughly dis- infected; when done, one quart of carbolic solution or of 5 per cent, solution of formalin should be poured into the pan (after it is emptied) and allowed to remain there. Sinks should be flushed at least once daily. 7. Dishes, knives, forks, spoons, etc., used by a patient should, as a rule, be kept for his exclusive use and not removed from the room. They should be washed first in the carbolic solution, then in boiling hot soapsuds, and finally rinsed in hot water. The remains of the patient's meals may be burned or thrown into a vessel containing the carbolic solution or milk of lime, and allowed to stand for one hour before being thrown away. 8. Rooms and Their Contents. — Rooms which have been occupied by persons suffering from contagious disease should not be again occu- pied until they have been thoroughly ;) Hypodermic and Other Syringes.— These when not boiled are steril- ized l)y drawing np into them boiling water a lunnljer of times and then finally a 5 per cent, solution of carbolic acid, the acid after three minutes to be washed out by boiling water. If cold water is used the carbolic solution should remain in the barrel for ten minutes. Great care should be taken to wash out all possible organic matter before using the carbolic acid or boiling to sterilize. Syringes made entirely of glass or of glass and asbestos can be boiled in soda solution. The Sterilization of Milk.— Complete sterilization destroys all the germs in milk, and so, if no new ones gain entrance, prevents per- manently fermentative changes. This requires boiling for fifteen to forty-five minutes on two or three consecutive days, according to the presence or absence of certain spores. Milk is best sterilized by heat, for nearly all chemicals, such as boric acid, salicylic acid, and formalin, are not only slightly deleterious themselves but also make the milk less digestible, and, therfore, less fit for food. Formalin is the least objectionable of the three. Milk may be sterihzed at a high or low temperature — that is, at the boiling temperature — or at a lower degree of heat, obtained by modifying the steaming process. Milk heated at as high a temperature as 100° C. is not altogether desirable for prolonged use for infants, as the high tem- perature causes certain changes in the milk which make it less suitable as a food for them. Pasteurization. — These changes are almost altogether avoided if a temperature below 70° C. is used. It is recommended, therefore, that the lowest temperature be used for partial sterilization which will keep the milk wholesome for twenty-four hours in the warmest weather and kill the tubercle, typhoid, and other non-sporebearing bacilli. Raising the milk to a temperature of 60° C. for twenty minutes, 65° C. for fifteen, 70° for five, 75° for two, or 80° for one will accomplish this. Exposure for even one minute at 70° destroys 98 per cent, of the bacteria which are not in the spore form. Fully 99 per cent, of tubercle bacilli are destroyed. This subject is considered more fully in the chapter on milk. One of the many forms of apparatus is the following: (a) A tin pail or pot, about ten inches deep by nine inches in diameter, provided with the ordinary tin cover which has been perforated with eight holes each an inch in diameter. (6) A wire basket, with eight nursing bottles (as sold for this pur- pose in the shops). (c) Rubber corks for bottles and a bristle brush for cleaning them. Directions (Koplik). — Place the milk, pure or diluted (as the physi- cian may direct), in the nursing bottles and place the latter in the wire basket. Put only sufficient milk for one nursing in each bottle. Do not cork the bottles at first. Having previously^ poured about two inches of water in the tin pail or pot and brought it to the boiling point, lower the basket of nurs- ing bottles slowly into the pot. Do not allow the bottles to touch the water or they will crack. Put on the perforated cover and let the steaming continue for ten minutes; then remove the cover and firmly 664 APPLIED MICROBIOLOGY cork each bottle. x\fter replacing the cover, allow the steaming to continue for fifteen minutes. The steam must he allowed to escape freelj' or the temperature will rise too high. The process is now complete. Place the basket of bottles in a cool, dark place or in an ice-chest. The bottles must not be opened until just before the milk is to be used, and then it may be warmed by plunging the bottle in warm water. If properly prepared the milk will taste but little like boiled milk. The temperature attained under the conditions stated above will not exceed in extreme cases 87° C. (188° F.). A different but admirable method is the one devised by Dr. Free- man.^ Here a pail is filled to a certain mark with water, and then placed on the stove until the water boils. It is then removed, and immediately a milk-holder, consisting of a series of zinc cylinders, is lowered with its milk bottles partially full of milk. The cover is again applied. The heat of the outside water raises the temperature of the milk in ten minutes to about 65° C. (150° F.), and holds it nearly at that point for some time. After twenty minutes the milk is removed, placed in cold water, and quickly cooled. The milk is kept in the ice- chest until used. When milk is pasteurized in great quantities it should always be done by the "holding process" as "flash" pasteurization is unreliable. Milk should be pasteurized when it is as fresh as possible, and only sufficient milk for twenty-four hours should be pastuerized at one time. If after nursing the infant leaves some milk in the bottle this should be thrown away. Care of the Bottles. — After nursing, the bottles should be filled with a strong solution of washing soda, allowed to stand twenty-four hours, and then carefully cleaned with a bristle (bottle) brush. The rubber corks and nipples after using should be boiled in strong soda solution for fifteen minutes and then rinsed and dried. After sterilizing milk should never be put into unsterilized bottles, as this will spoil it. GENERAL CONCLUSIONS ON DISINFECTION. The previous pages have shown that it is comparatively easy to destroy microbes by germicides or heat when access to them is attain- able. Foods, instruments, clothing, bedding, the excreta, the surface of the body, etc., can })e readily disinfected, but when we try to disin- fect the mucous membranes of the living person we fail. The Importance of Disinfection After Recovery or Death from Infec- tions Diseases. — Year by year knowledge is accumulating which indi- cates that nearly all eases of spread of infection are due to the immediate transfer from a living carrier to the person who contracted the disease. The carrier may be diseased or may simply harbor the germs. If cleanliness is maintained throughout the disease there is, as a rule, little need of disinfection after recovery. ' AkciiI for I'liKtcurizor, JuiDi'S DonEhcrly, 411 W. .59th St. INDEX OF AUTHOIIS. A Bignami, 548, 549 Billroth, 22 Abbott, 59S Binger, 488 Abel, 354, 470 Blanchard, 591 Abrami, 237 Blandford, 513 Agramonte, 590, 591, 594, 595 Blochmann, 530 Albrecht, 275 Bookman, 660 Ambroz, 34 Boeckman, 660 Amoss, 492 Boerner, 377 Anderson, 224, 492, 493, 039 Boinet, 363, 422 Andrews, 249 von Bokay, 227 Anthony, 252, 494 Bolduan, 265 Aristotle, 590 Bolhnger, 477 Arloing, 412 Bolton, 325, 489 Arning, 423 Bonhoff, 276 Aronson, 645, 64G Boni, 34 Arrhenius, 109, 170 Bonomc, 427 Arthus, 224 Booker, 387 Ashburn, 492 Bordet, 159, 160, 162, 165, 193 Atkinson, 16S, 169, 173, 174 Borget and Gengou, 177, 441, 442 A very, 340 Borrel, 422, 448, 517, 565 Axenfeld, 438 Bosquillon, 570 Ayer, 657 Bossowski, 247 B Bostroem, 477, 478, 480 Bouchard, 384, 425 Babes, 558, 581, 586 Braun, 64 Baelz, 541 Breinl, 512, 526, 559, 560 Baeslack, 490 Bretonneau, 292 Bail, 174 Brieger, 149, 150, 324 Bailey, 627, 631 Brill, 493 Baker, 503 Bioeden, 503 Balbiani, 544 Brooks, 547 Baldwin, 340 Brown, 300, 374 Balfour, 526 Bruce, 290, 503, 508 Ball, 630 Bruck, 523 Banzhaf, 146, 168, 173, 174, 227 Brunner, 387 Barbagallo, 533 Buchanan, 630 Barbara, 535 Buchner, 56, 60, 160, 163 Bard, 292 Buermann, 236 Baroni, 576 Bumm, 283 Bass, 106, 553 Bunge, 80 Bassi, 22 Burri, 74 Bateman, 508 Buschke, 239, 521 Baumgarten, 388 Busse, 30, 237, 239 Beattie, 544 Blitschh, 19, 34 Beebe, 301, 517 Buxton, 374 von Behring, 154, 165, 172, 173, 310, 325, 404, 647 C Beijerinck, 489 Cahn, 341 Bendick, 340 Calkins, 496, 518, 532, 537, 560, 564, 571 Bentley, 558 Callison, 363 Bergey, 612 Calmette, 408, 448, 564 Bernberg-Gossler, 658 Cameron, 632 Berry, 103, 576 Canon, 432 Bertarelli, 521, 564 Capitan, 425 , Bertram, 546 Cardiac, 647 Besredka, 222, 225, 363 Carle, 320 Beyerinck, 631 Carhsle, 526 Bienstock, 335, 343 Carroll, 590 Biggs, 308 Carter, 526 OGG INDEX OF .1 UTHORS Casagrandi, 492, 533 von Dusch, 21 CastcUani, 503, 523 Dutton, 503, 500, 511, 526, 527 Catrin, 494 Duval, 420 Colli, 533 E Centanni, 587 Eaton, 363 Cernovodeanu, 327 Eberth, 355 Chagas, 503, 506 Elders, 385 Chamberland, 456 Ehrenberg, 515, 516 Chan'in, 57, 193, 384, 386, 425 EhrUch, 22, 160, 161, 162, 165, 166, 169, Chevrcul, 21 170, 172, 173, 193, 471, 512, 513 Chick, 640 Eichhorn, 513 Chowning, 494 Elser, 276 Christophers, 500, 558, 502 Emmerich, 52, 53, 245, 384, 469 CUairmont, 270 Endo, 104 Clausen, 470 Engelmann, 19 Clegg, 420, 481, 509, 533, 535, 537, 538 Eppmger, 477, 482, 486 Cohan, 522 Epstein, 643 Cohen, 436, 441 Ernst, 385 Cohendy, 338 Escherich, 335, 343 Cohn, 21, 324, 517 Esmarch, 659 Cole, 270, 271 von Esmarch, 488 Coley, 255 Evans, 503 Collins, 199, 270 Ewing, 492, 517, 521 Conner, 494 Conradi, 341, 360, 380 F Conte, 501 Fagdet, 487 Councilman, 532, 533, 548, 564, 566 Famuleuer, 227 (^urmont, 412 Fantham, 506, 544, 562 Craig, 492, 533, 535, 536, 537, 538, 541 Fehleisen, 255 Cramer, 50 Feinberg, 34 CuUinan, 364 Fenger, 427 Cumming, 577, 585 Fermi, 587 Cunningham, 533 Ferre, 487 Curtis, 238 Field, 332, 492, 493 Gushing, 360 Finger, 522 Cj'gnaeus, 358 Finkler, 464 D Fiocca, 533 Dale, 57 Firth, 364 Dallera, 227 Fisch, 325 Danyz, 377 Fischer, 34, 521, 643 Darling, 500, 526, 540, 660 Flexner, 83, 276, 277, 378, 482, 491, 492, D'Arsonval, 57 533 Davaine, 22, 450, 497 Fliigge, 320, 397 Davis, 303 Foley, 526 Dean, 212 Fontaine, 238 Deneke, 464 Ford, 336 Denys, 208 Foulerton, 476 Dieudonne, 56 Fox, 362, 371 Dochez, 270, 271 Frankel, 262, 244, 526 Dock, 541 Franklands, 598 Doerr, 173, 226 Freeman, 662 Doflein, 496, 547 Friedberger, 147, 224, 225. 226, 576 Dold, 226 Friedlander, 262, 353 Donitz, 328 Friedmann, 415 Donne, 530 Frosch, 360, 489 Donovan, 500 Fullerton, 40 Doptor, 276 Fiirbinger, 229, 660 Dorset, 489 Douglass, 209 G Doutrelepont, 422 Dreyer, 194 Gabritchewskt, 260 Di'igalski, 360 Gaffky, 247, 355 Ducrey, 443 Gage, 73 Dujartlin, 19 Galli-Valerio, 559 Dunliani, 100, 378, 463 Gamprecht, 326 Durham 193, 374, 513 Garr<;, 246 !NDJ growtli of special, 103-107 bacteria in, 609, 618 Bordet-Gengou, 103 lactic acid milk, 337 for cholera, 105 number of bacteria in, 609 for protozoa, 106 pasteurization of, 620, 661 synthetic, 104 pathogenic properties of bacteria for toxin production, 105 isolated from, 612 for typhoid-colon groups, 104 smear method of estimating num- for yeasts and molds, 106 ber of bacteria, 609, 610 storage of, 95 sterihzation of, 661 various kinds of, 95-107 streptococci in, 612 Meningitis, bacteriologic diagnosis of, 280 time required for multiplication of vai ious oi-ganisms exciting, 280 bacteria in, 609 Meningococcus, 273-281 transmission of disease through, agglutination, 275 625 GENERAL INDEX C)79 Molds, pathogonio, 27, 229. See Hypho- mycetes. Monotricha, 35 Mosfiuilocs as agenis o( inl'i'i-lioii in malaria., rAH, 552, 553 ill yellow fever, 592-595 (lypanosdiue.s in, 497 INIonla'nts, 77 Woi'phology of bacteria, 30 of molds, 27 of protozoa, 42 of yeasts, 29 Mosaic diseases of tobacco, 4S9 Motility' of bacteria, 36 organs of, 35 of protozoa, 44 organs of, 42 Mucor, 229, 230 INIumps, 494 Mycetoma, 481 Mycomycetes, 28 Myxosporidia, 544 N Nagana, 503 Negri bodies in rabies, 570, 571, 573 morphology of, 573 smear method of demonstrating, 572 staining method for, S3 Neurorycetes h^'drophobia-, 571 Nitrification, 629 Nit.rifj'ing bacteria, 629 Nitrogen fixing bacteria, 630 Nocardia, 42, 482 animal inoculation, 486 cultivation, 485 morphology, 486 pathology, 482-485 treatment of infection, 487 Nosema, 543 bombyois, 545 lophii, 645 rat virus, 490 OiDiuM, 235 Opsonic index, 211 accui-acy of, 212 determination of, 212 diagnostic value, 216 dilution method, technique, 212 Simon's method, technique, 212 test, 211-214 variation of, during treatment by inoculation, 214 variation in healthy persons, 215 Wright's method, technique, 211 Opsonins, 208, 216, 217 distribution in body, 217 PAKAnYKHN'I'KltV liacilli, 379 I'araiueniiigococcus, 276 Paratyphoid group, 374 bacilli, 374 type A, 374 type B, 374 type C, 377 diagnosis, 377 enteritidis types, 374, 377 Pasteur treatment of labies, 580 Pasteurization, 661, 663 Pellagra, 495 PeniciUium, 229, 230 Peritricha, 35 Pest (bubonic plague), 446 Phenolphthalein as indicator, SS Phycomycetes, 28 Pigment production by microbes, 61 Piroplasma (Babesia), 558 bigeminuin, 558 blood organisms similar to, 562. morphology, 559 pathogenicity, 561 prognosis, 561 prophylaxis, 561 symptoms, 561 ticks as carriers of, 560 treatment, 561 canis, 559 staining, 559 V. Pirquet cutaneous ( iiberculin tost, 408 Pityriasis versicolor, 234 Plague, bubonic, 446 bacillus, 447 biology, 447 morphology, 447 pathogenicity, 447 staining, 447 bacteriologio diagnosis, 449 historic, 446 immunity, 448 occurrence, 449 resistance, 449 vaccines, 449 Plants, bacterial disease of, 633 Plasmodium (Hemameba), 543 malarice, 543, 548, 551 gestivo-autumnal parasite, 547 classification, 551 cultivation, 553 cycle in man, 549 in mosquito, 552 examination of blood for, 549 pathogenicity, 555 quartan parasite, 549 staining methods for, 73, 74, 78, 81, 82 tertian parasite, 549 prophylaxis, 555 toxin production, 555 vivax, 547 Pleuropneumonia, contagious, of cattle, 490 G80 GENERAL INDEX Pneumobacillus. See Bacillus mucosus Rabies, Negri bodies"in,'572-575 capsulatus. Pasteur's treatemnt, 580 Pneumococcus, 262-272 pathogenesis, 577 agglutination reaction, 270 preparation of virus, 582, 584, 586 biologic charactei'istics, 263 prevention, 590 cultural reactions, 264, 265 preventive inoculation against, 580 immunity to infection by, 271 resistance of virus, 576 morphology, 262 serum, antirabic, 586 mucosus, 270 smear method, 572 special media for cultivation, 265 symptoms, 578 staining, 263 treatment, 580, 584, 587, 588, 689 pathogenicity, 266-270 Rat leprosy, 423 resistance, 265 virus, Novy's, 490 therapeutic experiments, 271 Reaction of media, correction of, 88 toxin production, 266 Vosges' and Proskauer's test in, 608 vaccines, 272 Relapsing fever, spirillum of, 492, 524 varieties of, 270 Rhinosporidium kinealjd, 544 virulence, 266 Rinderpest, 489 Poisons, nature of microbal, 147 Rocky Mountain spotted fever, 494 vegetable and animal, 147 Russell's double sugar medium, 104 Poliomyehtis, epidemic, 490 Polymastigida, 528, 530 Polyvalent serum, 164 S Precipitins, 206, 207 Proteins, bacterial, 134, 135 Saccharomyces, 237 Proteosoma, 557 Busse, 237 Protista, 24 Sarcinas, 32 Protozoa, 42 Sarcocystis, 543 blepharoplast, 42 muris, 546 centrosome, 43 Sarcoma, chicken, 490 chromidia, 43 Sarcosporidia, 543, 545 chromatin, 43 Sarcosporidiosis, 594 classification, 24 Sauerkraut, 633 cyst-formation, 45 Scarlet fever, 492 cytoplasm, 42 conveyance of, by milk, 625 developmental cycle, 45 streptococci in, 260 ectoplasm, 42 Schizogony, 549 entoplasm, 42 Schizomyoetes, 25 general characteristics of, 42 Sections, preparation of, 84 groups, 45 LofHer's staining method for, 185 growth, 44 Sera, antitoxic, 168, 175 karyosome, 43 bactericidal, 155, 158 motility, 44 bacteriolytic, 160 nucleus, 42 cytolytic, 160 locomotor or kinetic, 43 demonstration of nature of, 160 nutrition, 44 therapeutic value, 155 physiologic characteristics of, 43 multipartial, 164 relationship to other microorgan- polyvalent, 164 isms, 24, 25 precipitating, production of, 207 reproduction, 44 Serum, sickness, 226 respiration, 44 Sewage, bacteria in, 031 sexual phenomena, 44 disposal of, 005 Pseudodiphtheria bacilli, 303 farming, 632 Ptomains, 149 Schick test, 309 Pyogenic cocci, 241 Silkworm disease, 545 Sleeping sickness, 503. See Trypano- somiasis R Smallpox, 492, 563 etiology, 564 "^ Rabies, 489 historical, 563 complement, binding test, 576 pathogenicity, 566 cultivation of organism, 575 relation to vaccinia, 563 diagnosis of, 575 virus, preparation of, 566 fixed virus, 580 Smear method in diagnosis of rabies, 572 material and methods for study, 572 for direct examination of milk, methods of immunization, 580" 609 GENERAL INDEX GSl Soft chancre, 443 Stains, blue, 75, 77, 78 Soil, bacteria in, 607, 627 brown 75 examination of, 607 eosin-methylenc-blue, 83 South African horse sicl^ncss, 490 Giemsa, 81 Species, permanence of, 2() Goldhorn, 82 Specimens, preservation of, 84 Gram, 78 Spirilla, general characteristics of, 32 green, 75 Spirillum of Asiatic cholera, 464. Hcc Heidenhain, 84 Cholera. Hiss, 79 of Finkler and Prior, 475 Jenner, 82 of Metchnikoff, 475 Koch-Ehrhch, 78 of relapsing fever, 524. See Spiro- Kilhne methylene-blue, 78 cheta obermeieri. Leishman, 82 Spirocheta, 25, 515 LofHer's, 77, 80 balantidis, 516 MacNeal, 81 balbiani, 516 Mallory, 83 buccahs, 517 Moeller, 79 carteri, 526 Neisser, 295 dentium, 517 Nooht-Romanowsky, 81 duttoni, 526 red, 75 gallinarum, 518 Ross, 83 obermeieri (recurrentis), 524 violet, 75 biologic characteristics, 525 Van Gieson, 83 morphology, 524 Welch, 79 pathogenicity, 625 Williams, 83 paDida, 518. See Treponema pal- Wright, 82 lidum. Ziehl-Neelsen, 77 pertenuis, 523 Staphjdocoocus, 241, 247. See Cocci. refringens, 517 Sterilization, 91 macrodentium, 518 fractional, 93 microdentium, 518 intermittent, 94 vincenti, 517 of glassware, 86 Spirochetae, 515 of milk, 661 cultivation, 516, 519 Sterihzer, dry heat, 89 in frambo?sia tropica, 523 steam, 93 in mouth, 517 Stock vaccines, 223 methods for study, 515 Streptococci, 249, 261. See Cocci. miscellaneous, 518 Streptothrix. See Nocardia. pathology, 520, 522 Sulphur-dioxide gas in house disinfec- refringens, 517 tion, 642 in relapsing fever, 524 Symbioses, 52 staining, 516 Symptomatic anthrax, 457 in tick fever, 526 ■ Syphilis, 518, 520, 623. See also Tre- treponema pallidum, 518 ponema pallidum. in tumors, 517 immunity, 522, 623 in Vincent's angina, 517 Wassermann reaction in, 623 Spirochetes from relapsing fever in America, 526 Spores, 39, 41, 49 T arthrospores, 40 f endospores, 40 Tabardillo, 493 v<^- formation, 39 Tetanolysin, 324 germination of, 41 Tetanospasmin, 324 resistance to heat, 55 Tetanus, 320 protozoan, 49 antitoxin, 325 staining of, 79 therapeutic value, 332 Sporotricha, 236 method of administration of, Sporozoa, 49 329 life cycle, 45 persistence in blood, 326 Spotted fever. Rocky Mountain, 494 production of, for therapeutic Staining acid-fast bacteria, 79 purposes, 325 capsules, 79 results of use of, for immuniza- flagella, 80 tion, 331 principles underlying, 76 technique of testing, 325 spirochetes, 82, 83 unit, 325 spores, 79 bacillus, animal experiments, 322 GENE UAL INDEX 673 Bacillus, pseudo-influenza., 430 putrificus, 343 ^-pyocyaneus, 383 radicicola, 630 rat leprosy, 422 of rhinoscloroma, 354 of smegma, 424 of soft chancre, 443 subtilis, 96 suisepticus, 445 of swine plague, 445 of tetanus, 320 of timothy grass, 424 of tuberculosis, 388 of typhoid, 340 welchii, 343, 462 xerosis, 304 Bacteria, 30 adaptation to environment of, 55 aerobic 53, 115 anabohc power of, 82 in air, 606 anaerobic, 53, 115, 450 basic forms of, 31 botanical relationship of, 24 capsules, 33 carriers, 143 characteristics of, 30 chemical effects of, 59-64, 627-631 composition of, 50 classification of, 24, 30 definition of, 30 degeneration forms of, 38 dentrification, 629 effect of agitation, 57 electricity, 57 pressure, 57 radium, 57 surrounding forces upon, 51 a;-rays, 57 examination of, in hanging drops, 72 in tissues, 84 fermenting, 627 ferments, 60, 634 general characteristics of, 30 granules, 35 growth and reproduction, 37 habital, 50 higher forms of, 41, 476 in Hodgkin's disease, 421 in industries, 632 influence of one species upon another, 52 of quantity in infection, 137 intestinal, 334 prevalent, 340 regional distribution of, indi- gestive tract, 335 substitution of one variety for another, 336 involution forms, 38 katabohc power of, 62 lactic acid, 337-340 loss of capacity to be agglutinated or absorbed, 199 in which they excite 30 Bacteria, manner disease, 132 membrane, 33 mesophilic, 54 morphology and structure of, motflity of, 36, 36 niti-ification by, 63, 629 non-agglutinable strains, 200 nuclear substances in, 34 oxygen and other gas rcciuiremcnls, 53 putrefactive, 628 psychrophilic, 54 physiologic characteristics of, 36 products of growth, 59 reproduction of, 37 saprophytic, 51 in sewage, 605, 632 shape, 31 size, 31 in soil, 627 species, permanence of, 26 spore formation of, 39 staining of, 76 structure of, 33 substance, 34 thermic effects of, 59 thermophilic, 54 varieties of, in milk, 610 in water, 600 Bacterial proteins, 147 Bactericidal sera, 155, 158 power, 158, 200 substances, origin of, 163 Bacteriolysis, 158 Bacteriolytic sera, nature of, 160 Bacterium, oaucasicum, 340 Balantidium, 25, 50 coli, 546 minutum, 547 Nyctootherus faba, 547 Beggiotoa, 50 Bichloride of mercury, 640 Bile media,, 105 Black-leg, 457 Black-water fever, 558 Blastomycetes, 25, 28, 29, 229 classification, 29 pathogenic, 237 complement fixation, 237 cultivation, 109, 240 staining, 240 Blepharoplast, 43 Blister fluid, 201 Blood, bactericidal properties of, 156 flagellates, 496 germicidal properties of, 158 Blue pus, baciUus of, 383 Bodo, 528 lacertaj, 528 urinarius, 528 Bordet theory of antibodies, 162 Bordet-Gengou phenomenon, 177 Brownian movement, 36 Bubonic plague, 446 bacillus of, 446 674 GENERAL INDEX Bubonic plague, bacillus of, biologj' of, 447 morphology of, 447 pathogenicity, 447 resistance, 449 staining, 447 vaccines, 449 diagnosis of, 449 immunity against, 448 Calmette's oplithalmo-tubei'culin test, 408 Capsules of bacteria, 33 staining of, 79 Carbohydrates, action of Ijacteria on, 61, 629 Carbolic acid as disinfectant, 646 Carbon dioxides, produclion of, by colon bacillus, 347 Carcinoma, treatment of, 255 Carriers, 143, 360, 375, 474, 508, 527 Cell membrane, 33 substance, 34 Cellulitis, streptococci in, 254 Cellulose, fermentation of, 627 Centrosome, 43 Cercomonas hominis, 497 Cerebrospinal meningitis, 273 Chemotaxis, 58 Chicken sarcoma, 490 Chlamydozoa, 439 Chloride of lime, as disinfectant, 643 Chlorine, as disinfectant, 643 Chloroform, as disinfectant, 646 Cholera, Asiatic, 464 carriers, 474 lesions in man, 469 serum therapj', 472 si^irillum, 4(j4 allied oi'ganisms of, 474 biology of, 465 distribution in body, 469 immunity against, 471 isolation, 472, 474 morphology of, 464 pathogenicity of, 468 prophylaxis, 471 resistance and vitality of, 467 staining, 465 toxin of, 470 spread of, 469 Chromidia, 43 Ciliata, 49, 546 Cladothrix, 41, 476 Classification of microorganisms, 24-27 Claviccps purpurea, 229 Closlridium pasteurianum, 631 Cocci, characters of, 31 enterococcus, 342 micrococcus tetragenus, 247 staphylococcus epidermidis (albus (Welch), 246 pyogenes aureus, 241 Cocci, staphylococcus, biology, 241 cultivation, 241 immunity, 246 morphology, 241 occurience, 245 pathogenicity, 244 pigment formation, 243 staining, 241 toxin formation, 243 vaccine therapy, 246 staphylococcus (pyogenes) albus, 246 staphylococcus (pyogenes) citreus, 247 streptococcus, pyogenes, 250 biology, 251 bacteriologic diagnosis, 260 complement fixation, 190 cultural reaction, 251 development of hemolysis, 252 of pigment, 252 effect on tumors, 255 immunity against, 256 morphology, 250 occurrence, 254, 255 pathogenicity, 253 resistance, 253 serum, 257 Coccidia, 543 Coccidium bigeminum, 544 cuniculi, 543 Coley's streptococcus toxins, 255 Colon bacillus, 344 association with other bacteria, 348 behavior toward carbohydrates, 347 biology of, 346 chemical activities, 347 cultivation of, 346 in cystitis, 351 in diarrhea, 350 differential diagnosis from ty- phoid bacillus, 352, 356, 373 as disease producer, 349 flagella, 346 gas production, 347 group of, 344 growth of, with other Ijacteria, 348 immunity, 352 immunization against, 352 in inflammations of bile tract, 352 isolation, 352 morphology of, 345 occ\uTenc(^ in man ami animals, 349 in other inflanmiations, 352 of pancreas, 351 passage through walls of intes- tines during life, 349 pathogenicity, 349 in peritonitis, 350 as pus formers, 352 reduction processes, 348 GENERAL INDEX 675 Culon bacillus resistance, 34S in sepsis, 350 significance of, in water, 600 staining, 346 toxins, 348 ticatment, 352 of urinary tract, 351 vaccine treatment, 352 group, 344 Colonies, characteristics of, 109 counting of, 110-113 study of, in plate cultures, 109 various forms of, 1 10, 111 Complement, 101-163, 181 Bordet-GengoTi phenomenon, 177 fixation of, 177-192 test for glanders, 190 gonococcus infection, 189 meningitis, 192 pertussis, 190 streptococcus infection, 190 syphihs, 187, 523 tubercvilosis, 191 typhoid, 192 multiplicity of, 103 origin of, 163 Conidia, 28 Conjunctivitis, influenza-like bacilh in, 437 Koch-Weeks bacillus of, 437 Conradi and Drigalski medium, 104 Copper sulphate, as germicide, 641 capsule stain, 79 Corrosive sublimate, 640 Cover-glass, preparations of, how made, 73 how stained, 74 how cleaned, 73 Cowiiox, etiology of, 664 relation to smallpox, 563 Creolin, 647 Creosote, 647 Crescent bodies in malaiia, 552, 556 Cresol, 646 Crithidia, 497 Culex in malaria, 557 Cultivation of microorganisms, 86 of protozoa, 106 Culture, anaerobic, 116 block and hanging mass, 73 media, inoculation of, 113, preparation of, 91 reaction of, 88 special, 103 plate, making of, 107 piu-e, 86, 118 storage of, 95 titration of, 88 Cyst formation, 45 Cytolytic serum, 160 Dark ground examination, Delhi boil, 499 Dengue, 492 Dilution methods, 112 Diphtheria, agglutinin dcveldpmeut, 312 antitoxin, 306 deleterious effects, 309 globulin preparation, 311 neutralizing charactertstics of, 307 persistence of, in blood, 312 res>dt of, treatment of, 308 results of use of refined, 311 treatment, danger of, 311 use of, in treatment and im- munization, 307 bacillus, 292-306 biology of, 296 causal relationship of, to diph- theria, 292 cultivation, 296-298 exudate due to, contrasted with that due to other bacteria, 313 in healthy throats, 301 morphology, 292, 295 non-virulent forms of, 302 pathogenicity, 298 persistence of, in throats, 301, 30(i of characteristics in t3fpes of, 304 psGudodiphtheria, 303, 304 resistance to heat, drying, and chemicals, 305 staining of, 293 varieties of, 305 virulence in cases of diphtheria, 300 virulent, in healthy throats, 301 causes of death in, 299 comparative virulence of different cultures, 300 diagnosis, baoteriologic, 315-319 relation of bacteriology to, 314 historic, 292 -like bacilli, pathogenic for guinea- pigs, but not producing diph- theria toxin. 302 mixed infection in, 313 pseudomembranous exudative in- flammations due to bacteria other than diphtheria bacillus, 313 susceptibility to and immunity against, 306 tissue changes in natural human infection, 298 toxin, 299 as index of virulence, 300 production of, in culture media 299 transmission of, 306 Diploooccus gonorrhoea. iS'ee Gonococcus intracellularis meningitidis. See Meningococcus, of pneumonia. See Pneumoeoccus. Disease in beer and wines, 634 Disinfectants, 635-047 gaseous, 642 (■>76 GENERAL INDEX Disinfectants, inorganic, 640 organic, 643 standardization of, 637 Disinfection, 648-663 agents for, 648-650 of booiis, 654 of clothing, 650, 652 in contagious diseases, 650 of discharges, 650 by formaldehyde, 652, 658 by heat, 649 practical, of house, person, instru- ments, and food, 650, 651, 652 657 preventive, 652 rooms, etc., 651 by steam, 658, 659 by sulphur dioxide, 657, 658 for surgical operation, 659 DrigaJski and Conradi medium, 104 Drying, effects of, on bacteria, 58 Dunham's peptone solution, 96 Dysentery, amebic, 532. See also Amcbse, bacillary, 378 bacilli, group of, 378 agglutination characteristics, 382 differential diagnosis of, 382 fermentation reaction, 379 historical note of, 378 morphology of, 378 pathogenicity of, 379 pathology, 380 resistance, 381 staining, .378 toxins, 380 types of, 378, 382 E Ehrlich's side-chain theory, 165 Eimeria schubergi, 544 stiedae, 543 Elimination of microorganisms through milk, 144 through skin and mucous mem- brane, 145 Endo medium, l04 Elndospores, 40 Ensilage, 633 Entamoeba. See also Amebte. buccalis, 5.33 coli, .532 histolytica, 532 Enterococcus, 342 Enzymes, 60 Eumycetes. See Hyphomycetes. Examination, of air, 606 of hanging dro]), 72 of soil, 607 of water, 597 Farcy, 482 Favus, 233 Fermentation, 60 alcoholic, 634 by bacteria, 627, 633 tube, 88 of urea, 61 Ferments, characteristics of, 60 diastatic, 61 inverting, 61 proteolytic, 60 rennin-like, 61 Film preparations, 73 Filter beds, 605 Filters, 91-93, 488 Filtrable viruses, 488 Group I, of unknown morphology, 489 Group II, visible, 490 Group III, of questionable filtra- bility, 492 Filtration, "technique of, 488 of water, 604, 605 Fixatives, 74, 84 Fixation of complement. See Comple- ment . Flagella, 35 staining of, 80 Flagellata, 25, 496 classification, 24 general characteristics of, 45 life cycle of, 47 materials and mathods, 496 Flies as carriers, relation to trypanoso- miasis, 508 Foot and mouth disease, 489 Formaldehyde as disinfectant, 643 permanganate of potash method, 6.55 Wilson's rapid generator, 656 Frambcesia tropica, 523 Fungi, pathogenic varieties, 25, 28, 229- 237 of pityriasis, 234 of ringworm, 230 of sporotrichosis, 236 of thi-ush (soor), 29, 235 G Gas production by bacteria, 64 test for, 119 Gelatin media, 96 Germicidal action, method of determina- tion, 158, 1.59 Germination of spore, 41 Glanders bacillus, 425 biology of, 425 cultivation of, 426 pathogenicity, 426 staining, 425 diagnosis of, 190, 428, 429 by agglutination test, 430 by complement [fixation, 190, 431 by ophthalmo-reaotion, 429, 430 immunity against, 427 mode of spread, 427 GENERAL INDEX ('.77 Glanders, lest for (niallcin), 428 vaccine, 4'-'8 Glassware, preparation of, 8(i, 179 Globulin inanliloxic sera., 17o, 174 Cilossina in relation to trypanosoniiasis, 508, 510 (lonococcus, 283-289 agglutination, 287 bacteria resembling, 289 bacteriologic diagnosis, 288 cultivation, 286 diseases excited by, 286, 287 in endocarditis, 287 morphology, 283 occmrence of, 286 serum and vaccine therapy, 287 staining reactions, 284 toxins of, 286 Gonorrhea, bacteriologic diagnosis of, 288 Gram-negative and Gram-positive mi- crobes, list of, 128-131 stain, 78 Grass bacillus, 628 Group agglutinins, 195 Gruber-Widal reactio]^i|i^i06 of bacteria, 72 Halteridium, 557 Hanging drop for mass or block, Hemameba (see Plasmodium malariaj), falciparum, 549 malarisB, 549 vivax, 549 Hemoglobinophilic bacilli, 432 HemolysiDs, Ehrlich's studies on, 160 Hemolysis, 161, 177 Hemolytic sera, 160, 161 Hemoproteus, 557 Herpetomonas, 497 Histoplasma capsulatum, 500 Historical sketch of microbiology, 17 Hodgkin's disease, 421 Hog cholera, 489 Hollow shde, 72 Hydrogen peroxide, 642 Hydrophobia {see Rabies), 570 Hypersensitiveness, 224. See also Ana- phylaxis. Hyphomycetes, 27, 229 agglutination, 237 classification, 27 complement fixation, 237 cultivation, 236 pathogenicity, 237 Ice, bacteria in, 55 Immune body, 161 multiplicity of, 163 Immunity, active, 161, 219 passive, 154 Immunity, specific, 153 Increase of toxicily and virulences of bacteria, 138 Incubators, 113-115 low t.(>iiii)ei'alurcs in, 114 India-ink method of examining l)acteria, 74 Indol, 120 Infection, 134 influence of quantity in, 137 localized, vaccine therapy for, 219, 220 mixed, 139 protection afforded by skin and mucous membranes, 140, 145 spread of, 142 Influence of one species upon growtli of another, 52 Influenza bacillus, 432 agglutination of, 437 bacteriologic diagnosis, 435 biology, 433 complement fixation, 434 cultivation 433 distribution in the body, 434 epidemiology, 435 examination of sputum for, 436 immunity to, 434 morphology of, 432 pathogenicity, 434 resistance, 433 serum therapy, 437 staining characteristics, 433 in tuberculosis, 435 vaccines, 437 Inoculation, methods of, 122 Insect carriers, 527. See also Carriers and Vermin. Intestines, anaerobic conditions in, 335 development of bacteria in, 334 methods used in examination of feces, 336 regional distribution of bacteria in, 335 significance of bacteria in, 334 Isolation of microorganisms, 107-109 Kala-aza^ 498 Koch's original tuberculin, 403 phenomenon of, 175 407 Lamblia, 530 intestinalis, 531 Laverania malaria?, 549 Leishmania donovani, 499 bedbugs as carriers, 501 cultivation, 501 morphology, 501 pathology, 499, 501 prophylaxis, 502 078 ' GENERAL INDEX Ijoprosy bacillus, 420-424 : Meningococcus, biicteriologic diagnosis. difff'icntial diagnosis of, 42)! 280 iiioipliology of, 420 morphology, 273 paihogpnicity of, 422 cocci resembling, 2S1 Leptomonas, 497 lialhogenicitjr, 274 Leptolhrix, 41, 470 pri'sentu: in nares of both sick and Leukocytes, extract of, 217 healthy pei-sons, 275 part played by, in immunity, 208 resisf.an(te, 274 production of exudates rich in, 217 serum treatment, 27() for testing phagocytosis, 124 staining, 273 Light, production of, by bacteria, 59 Metachromatic granules, 35 infiueuce of, on bacteria, 56 Metchnikoff, spirillum of, 475 Litmus, 88 Microbiology, applied, 579 Lophotrioha, 36 historical sketch, 17 Lyssa (see Rabies), 570 Micrococcus catarrhalis, 282 gonorrhcea, 283-289 intracellularis, 273-281. See Men- M ingococcus, lanceolatus, 262-272. Sec Pneu- Madura foot, 481 mococcus. Malaria, 548. See also Plasmodium mclitensis, 290 malaria. tetragenus, 247 diagnosis 555 biology, 248 estivo-autumnal, 549 growth on media, 248 historical note, 548 morphology, 247 immunity, 555 pathogenicity, 248 infection, how acquired, 549 staining, 248 -like organisms in other animals, zymogens, 291 557 Microorganisms, chemical composition. materials and methods for study, 549 50 mo.squitoes in relation to, 552, 554 cla,ssification, 24-27 parasites, 549 effects of surrounding forces upon, prophylaxis, 555 51-59 quartan, 549 general characteristics, 24-65 technique of blood examination in, group characteristics, 27-50 83, 555 products of growth of, 59-64 tropical, 549 Microscope, different parts of, 66 Malignant edema, bacillus of, 460 Microscopic methods, 71 Mallei, bacillus, 425. See Glanders. Microsporon, 234 Mallein, preparation of, 428 Milk, bacterial contamination of, G17 test for gland eis, 429 bacteriology of, in relation to dis- Malta fever, 290 ease, 609, 613, 614, 617 spread by goats' milk, 290 development of bacteria in heated, Material for baoteriologic examination. 620 procuring of, from those suf- elimination of bacteria through. fering from disease, 125 144 routine technique of examina- examination of, 609 tion, 127 grading of, 626 Measles, 492 heated vs. raw, in feeding, 615 Media, preparation and sterilization of. identification of bacteria in, 610 86 influence of cleanliness on, 622 reaction of, 52, 06 of temperature or> growth of special, 103-107 bacteria in, 609, 618 Bordet-Gengou, 103 lactic acid milk, 337 for cholera, 105 number of bacteria in, 609 for protozoa, 100 pasteurization of, 620, 661 .synthetic, 104 pathogenic properties of bacteria for toxin produciion, 105 isolated from, 612 for typhoid-colon groups, 104 smear method of estimating nmn- for yeasts and molds, 106 ber of bacteria, 609, 610 storage of, 95 sterilization of, 661 various kinds of, 95-107 streptococci in, 612 Meningitis, bacteriologic diagnosis of, 280 time required for multiplication of vaiious organisms exciting, 2S0 liacteria in, 009 Meningococcus, 273-281 transmission of disease through, agglutination, 275 625 GENERAL INDEX 079 Molds, pathogcnio, 27, 229. See Hypho- myoetes. Monotviclui, 35 ]\Io.s(|ni(oes as agcnis ul' inrci-lidii in iiuilaria, MS, 052, 553 ill yi-Uow fever, 592-595 (rypanosdiucs in, 497 Monlauls, 77 Moi'phology of bacteria, 30 of molds, 27 of protozoa, 42 of yeasts, 29 Mosaic diseases of tobareiiii)ei'alurcs in, 114 India-ink method of examining l)acteria, 74 Indol, 120 Infection, 134 influence of quantity in, 137 localized, vaccine therapy for, 219, 220 mixed, 139 protection afforded by skin and mucous membranes, 140, 145 spread of, 142 Influence of one species upon growtli of another, 52 Influenza bacillus, 432 agglutination of, 437 bacteriologic diagnosis, 435 biology, 433 complement fixation, 434 cultivation 433 distribution in the body, 434 epidemiology, 435 examination of sputum for, 436 immunity to, 434 morphology of, 432 pathogenicity, 434 resistance, 433 serum therapy, 437 staining characteristics, 433 in tuberculosis, 435 vaccines, 437 Inoculation, methods of, 122 Insect carriers, 527. See also Carriers and Vermin. Intestines, anaerobic conditions in, 335 development of bacteria in, 334 methods used in examination of feces, 336 regional distribution of bacteria in, 335 significance of bacteria in, 334 Isolation of microorganisms, 107-109 Kala-aza^ 498 Koch's original tuberculin, 403 phenomenon of, 175 407 Lamblia, 530 intestinalis, 531 Laverania malaria?, 549 Leishmania donovani, 499 bedbugs as carriers, 501 cultivation, 501 morphology, 501 pathology, 499, 501 prophylaxis, 502 078 GENERAL INDEX Leprosy bacillus, 420-424 (iiffc'icntial diagnosis of, 42o iiioiiihology of, 420 l)aih(igeiiir-ify of, 422 Lci)toiiioiias, 407 Loptollirix, 41, 47(i Leukocytes, exti-acf cjf, 217 part plaj'pd by, in immunity, 208 production of exudates rich in, 217 for testing phagocytosis, 124 Light, production of, by bacteria, 59 influeuce of, on bacteria, 56 Litmus, 88 Lophotricha, 36 Lyssa (see Rabies), 570 M Madura foot, 481 Malaria, 548. >S'ee also Plasmodium malaria;, diagnosis 555 estivo-autumnal, 549 historical note, 548 immunity, 555 infection, how acriuired, 549 -like organisms in other animals, 557 materials and methods for study, 549 mosquitoes in relation to, 552, 554 parasites, 549 prophylaxis, 555 quartan, 549 technique of blood examination in, S3, 555 trojncal, 549 Malignant edema, l^acillus of, 400 Mallei, bacillus, 425. See Glanders. Mallein, preparation of, 428 test for glanders, 429 Malta fever, 290 spread by goats' milk, 290 Material for baoteriologic examination, procuring of, from those suf- fei-ing from disease, 125 routine technique of examina- tion, 127 Measles, 492 Media, preparation and sterilization of. 86^ reaction of, 52, 06 special, 103-107 Bordet-Gengou, 103 for cholera, 105 for protozoa, 100 .synthetic, 104 for toxin produclion, 105 for typhoid-colon groups, 104 for yeasts and molds, 106 storage of, 95 various kinds of, 95-107 Meningitis, bacteriologic diagnosis of, 280 various organisms exciting, 2S0 Meningococcus, 273-281 agglutination, 275 Meningococcus, bacteriologic diagnosis, 280 morphology, 273 cooci resembling, 2S1 ])athogenicity, 274 priisence in nai-(;s of both sick and healthy pei-sons, 275 resistances, 274 serum treatment, 276 staining, 273 Metachromatic granules, 35 Metchnikoff, spirillum of, 475 Microbiology, applied, 579 historical sketch, 17 Micrococcus catarrhalis, 282 gonorrhoea, 283-289 intracellularis, 273-281. -^ee Men- ingococcus, lanceolatus, 262-272. iS'ec Pneu- mococcus. melit.ensis, 290 tetragenus, 247 biology, 248 growth on media, 248 morphology, 247 pathogenicity, 248 staining, 248 zymogens, 291 Micro(irganisms, chemical composition, 50 classification, 24-27 effects of surrounding forces upon, 51-59 general charactcrislics, 24-05 group characteristics, 27-50 products of growth of, 59-64 Microscope, different parts of, 66 Microscopic methods, 71 Microsporon, 234 Milk, bacterial contamination of, 617 bacteriology of, in relation to dis- ease, 609, 613, 614, 617 development of bacteria in heated, 620 elimination of bacteria through, 144 examination of, 609 grading of, 626 heated vs. raw, in feeding, 615 identification of bacteria in, 610 influence of cleairliness on, 622 of temperature on growth of bacteria in, 609, 61S lactic acid milk, 337 number of bacteria in, 609 pasteurization of, 620, 661 pathogenic properties of bacteria isolated from, 612 smear method of estimating niun- ber of bacteria, 609, 610 sterilization of, 661 streptococci in, 612 time required for multiplication of bacteria in, 609 transmission of disease through, 625 GENERAL INDEX ()7< Molds, pathogonic, 27, 229. See Tlypho- mycetes. Monotvlcha, 35 Mosquitoes as agcnis of infeclion in iiuilavia, 548, 552, 553 in y(>llo\v fpvcr, 592-505 tvypanosonu's in, 497 Mordants, 77 Morphology of bacteria, 30 of molds, 27 of protozoa, 42 of yeasts, 29 IMosaic diseases of tobacco, 4S9 Motility of bacteria, 36 organs of, 35 of protozoa, 44 organs of, 42 Mucor, 229, 230 Mumps, 494 Mycetoma, 481 Mycomycetes, 28 Myxosporidia, 544 N Nagana, 503 Negri bodies in rabies, 570, 571, 573 morphology of, 573 smear method of demonstrating, 572 staining method for, 83 Neurorycetes hydrophobia', 571 Nitrification, 629 Nitrifying bacteria, 629 Nitrogen fixing bacteria, 630 Nocardia, 42, 482 animal inoculation, 486 cultivation, 485 morpholog}', 486 pathology, 482-485 treatment of infection, 487 Nosema, 543 bombycis, 545 lophii, 645 rat virus, 490 OiDiUM, 235 Ojosonic index, 211 accm-acy of, 212 determination of, 212 diagnostic value, 216 dilution method, technique, 212 Simon's method, technique, 212 test, 211-214 variation of, during treatment by inoculation, 214 variation in healthy persons, 215 Wright's method, technique, 211 Opsonins, 208, 216, 217 distribution in body, 217 I'auadvsiontbiiv bacilli, :;79 ParanicTiiiigococcus, 276 Paratyphoiil group, 374 bacilli, 374 type A, 374 typo B, 374 type C, 377 diagnosis, 377 enteritidis types, 374, 377 Pasteur treatment of labies, 580 Pasteurization, 661, 663 Pellagra, 495 Penicillium, 229, 230 Peritricha, 35 Pest (bubonic plague), 446 Phenolphthalein as indicator, 88 Phycomycetes, 28 Pigment production by microbes, 61 Piroplasma (Babesia), 558 bigeminum, 558 blood organisms similar to, 562. morphology, 559 pathogenicity, 561 prognosis, 561 prophylaxis, 561 symptoms, 561 ticks as caiTiers of, 560 treatment, 561 canis, 559 staining, 559 V. Pirquet cutaneous tuberculin test, 408 Pityriasis versicolor, 234 Plague, bubonic, 446 bacillus, 447 biology, 447 morphology, 447 pathogenicity, 447 staining, 447 bacteriologic diagnosis, 449 historic, 446 immunity, 448 occurrence, 449 resistance, 449 vaccines, 449 Plants, bacterial disease of, 633 Plasmodium (Hemameba), 543 malarite, 543, 548, 551 sestivo-autumnal parasite, 547 classification, 551 cultivation, 553 cycle in man, 549 in mosquito, 552 examination of blood for, 549 pathogenicity, 555 quartan parasite, 549 staining methods for, 73, 74, 78, 81, 82 tertian parasite, 549 prophylaxis, 555 toxin production, 555 vivax, 547 Pleuropneumonia, contagious, of cattle, 490 680 GENERAL INDEX Pneumobaoillus. See Bacillus mucosas Rabies, Negri bodies"in,'572-575 capsulatus. Pasteur's treatemnt, 580 Pneumococous, 262-272 pathogenesis, 577 agglutination reaction, 270 preparation of virus, 582, 584, 586 biologic characteristics, 263 prevention, 590 cultural reactions, 264, 265 preventive inoculation against, 580 immunity to infection by, 271 resistance of virus, 576 morphology, 262 serum, antirabic, 586 mucosus, 270 smear method, 572 special media for cultivation, 265 symptoms, 578 staining, 263 treatment, 580, 584, 587, 588, 589 pathogenicity, 266-270 Rat leprosy, 423 resistance, 265 virus, Novy's, 490 therapeutic experiments, 271 Reaction of media, correction of, 88 toxin production, 266 Vosges' and Proskauer's test in, 608 vaccines, 272 Relapsing fever, spirillum of, 492, 524 varieties of, 270 Rhinosporidium kinealyi, 544 virulence, 266 Rinderpest, 489 Poisons, nature of microbal, 147 Rocky Mountain spotted fever, 494 vegetable and animal, 147 Russell's double sugar medium, 104 Poliomyehtis, epidemic, 490 Polymastigida, 528, 530 Polyvalent serum, 164 s Precipitins, 206, 207 Proteins, bacterial, 134, 135 Sacchabomyces, 237 Proteosoma, 557 Busse, 237 Protista, 24 Sarcinse, 32 Protozoa, 42 Sarcocystis, 543 blepliaroplast, 42 muris, 546 centrosome, 43 Sarcoma, chicken, 490 chromidia, 43 Sarcosporidia, 543, 545 chromatin, 43 Sarcosporidiosis, 594 classification, 24 Sauerkraut, 633 cyst-formation, 45 Scarlet fever, 492 cytoplasm, 42 conveyance of, by milk, 625 developmental cycle, 45 streptococci in, 260 ectoplasm, 42 Schizogony, 549 entoplasm, 42 Schizomycetes, 25 general characteristics of, 42 Sections, preparation of, 84 groups, 45 LofHer's staining method for, 185 growth, 44 Sera, antitoxic, 168, 175 karyosome, 43 bactericidal, 155, 158 motility, 44 bacteriolytic, 160 nucleus, 42 cytolytic, 160 locomotor or kinetic, 43 demonstration of nature of, 160 nutrition, 44 therapeutic value, 155 physiologic characteristics of, 43 multipart! al, 164 relationship to other microorgan- polyvalent, 164 isms, 24, 25 precipitating, production of, 207 reproduction, 44 Serum, sickness, 226 respiration, 44 Sewage, bacteria in, 631 sexual phenomena, 44 disposal of, 605 Pseudodiphtheria bacilli, 303 farming, 632 Ptomains, 149 Schick test, 309 Pyogenic cocci, 241 Silkworm disease, 545 Sleeping sickness, 503. See Trypano- somiasis R Smallpox, 492, 563 etiology, 564 Rabies, 489 historical, 563 complement, binding test, 576 pathogenicity, 566 cultivation of organism, 575 relation to vaccinia, 563 diagnosis of, 575 virus, preparation of, 566 fixed virus, 580 Smear method in diagnosis of rabies, 572 material and methods for study, 572 for direct examination of milk, methods of immunization, 580" 609 684 GENERAL INDEX Water, typhoid bacilli in, 602 Weigert's law of overproduction, 167 Whooping cough, 441 Widal reaction. See Gruber-Widal, 201- 206 Wilson, apparatus for formaldehyde disinfection, 656 method for anaerobic cultures, 116 Wolffhiigel's apparatus, 113 Wool-sorters' disease, 450. See Anthrax. Yaws, 523 Yeasts. See Blastomycetes. Yellow fever, 489 etiology, 590, 591 mosquitoes in, 591