Saunders' Qmstm Cmwmm mimi ipMiiiiiMifjiiiiK M. V. lALfc MJ, IffiKfilttfiW Bt!^'fiii^M M &Y New (7th) Edition \V^ DORLAND?S -;■ y«- AMERICAN ILLUSTRATED MEDICAL DICTIONARY This Edition Defines Over 5000 New, Words A Complete Dictionary of the terms tise^ in Medicine, Surgery, Dentistry, Pharmacy, Chemistry, Veterinary Medicine, ^a/i all kindred branches} together with new and elaborate Tables of Arteries* Mtfscles, Nerves, , 'Veins, etc.; of Bacilli, Bacteria, JSSicrococci, etc.; Eponymic Tables of Diseases, Operations, Signs and Symptoms, Stains, Tests, Methods of Treat^nent, etc. By W. A. N. DORtAND, M.D., Editor of the American Pocket Medical Dictionary. Large octavo, 1105 pages, bound in full flexitile leather.. Price, $4.50 net; :»^ith thttmb index, $5.p0flet. JUST READY-THE NEW (7th) EDITION CORNE LL UNIV ERSITY THE FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE Veterinary College 1897 This Volume is the Gift of l>r y, A Moore Jast Reacly Cornell University Library QR46.B181913 Edition Essentials of bacteriology; being a c^^^^ ^^ ^""1924 "boo 243 521 MEDICkiTTJKpapNSRY EDITED BY W. A. NEWMAisr DORLAND/A. M., U. D., Editor " Xmerican Illustrated Medical Dictionary" HUNDREDS OF NEW TERMS Bound in Full Leather, Limp, with Gold Edges. Price, St. 00 net: ~^ y with Patent Thumb Index, $1.25 net. The book is an absolutely new one. It is not a revi- sion of any old work, but it has been written entirely anew and is constructed on lipes that experience has shown to be the most practical for a; work of this kind. It aims to be complete, and to that end contains practically all the tern^s of modern medicine. This m^kes an urnjsually large vocabu- lary. Besides the ordinary dictionary terms the book contains a wealth qf anatomical and other tables. This matter is . of particular value to students for memorizing in preparation for examination, " I am struct at once with admiration at the compact size and attractive ex- ,4erior. I can recommend it to our students without reserve." ^James W. Bol- LAND,,M. D., of Jefferson Medical College. ' ' ' "This is aliandy pocket dictionary, which. is so full and complete that it puts to shame some of tfie more pretentious .volumes,;' ' — Journal of the American Medical Association,', ' ■ • -' ' \, ' ' i' ' ■ • ' - - "We have consulted it for the meaning of many new and rare terms, and have not met with a disappointment. The definitions are exquisitely clear and concise. We have never found so much information in so small a space. ''^-' publin Journal of Medical Science. '; ' ^ ■ ^^- ' "This is a handy little volume that, upon examin^tipn, seems fairly to fulfil the promise of its title, and to contain a vast amount of information in a very small space. '. . , It is somewhat surprising that it contains so many of the, rarer term§ used in medicine. ' ' — Bulletin Johns Hopkins Hospital, Baltimore. W. B. SAUNDERS CO., West Washington Square, Phila, London: 9, Henrietta Street, Govent Ga»aen Cornell University Library The original of tliis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000243521 ESSENTIALS BACTERIOLOGY. Since the issue of the first volume of the Saunders Question=Compends, OVER 342,000 COPIES of these unrivalled publications have been sold. This enormous sale is indisputable evidence of the value of these self-helps to students and physicians. aradvaird Draw tube Coarse Adiii^tf Wine Adjuster Objectives and Nose-piece Stage Svbstage wtth. Diaphragm and Condenser BACTEEIOLOGIC MICROSCOPE. SAUNDERS' QUESTION-COMPENDS, No. 20 ESSENTIALS BACTERIOLOGY CONCISE AND SYSTEMATIC INTRODUCTION TO THE STUDY OF BACTERIA AND ALLIED mCROORGANISMS BY M. V. BALL, M.D. member of the academy op natural sciences of philadelphia ; formerly instructor in bacteriology at the philadelphia polyclinic. Assisted By PAUL G. WESTON, M.D. PATHOLOGIST STATE HOSPITAL FOR INSANE AT WARREN, PA. SEVENTH EDITION, THOROUGHLY REVISED With its lUustrations, some in Colors PHILADELPftiA AND LONDON W. B. SAUNDERS COMPANY I9J4 Copyright, 1891, by W. B. Saunders. Reprinted October, 1892 Revised, reprinted, and recopyrighted May, 1893. Reprinted June, 1894. Revised, reprinted, and re- copyrighted November, 1896. Reprinted October, 1898. Revised, reprinted, and recopyrighted March, 1900. Reprinted May, 1903. Revised, reprinted, and recopyrighted August, 1904. Reprinted October, 1905, and August, 1907, Revised, entirely reset, reprinted, and recopyrighted September, 1908. Reprinted August, 1910, Revised, entirely reset, reprinted, and recopyrighted December, 1913. Copyright, 1913, by W. B Saunders Company. Reprinted January, 1914 13 2 / V K /' f ' PRINTED IN AMERICA PRESS OF W. B. SAUNDERS COMPANY PHILADELPHIA PREFACE TO THE SEVENTH EDITION This book has undergone a complete revision and many of the chapters have been rewritten in their entirety. Those which relate to immunity and infection have been carefully edited by Dr. Paul G. Weston, Pathologist at the State Hospital, Warren, Pa., who has also furnished the article on the Wassermann reaction. The author is likewise in- debted to him for valuable aid in other portions of the re- vision. The author realizes that compends of this nature must necessarily suffer in comparison with the larger and more elaborate works, and he trusts that the reviewers will bear this in mind in their criticisms. When this book first appeared in 1891 it was one of the first American publications on the subject, and only a few text-books had been issued in other countries. Although since then a great many excellent treatises have appeared, there still remains a place for this compend, and hence this new edition. The author hopes that he has succeeded in incor- porating all the newer established facts in bacteriology and in eliminating all that is obsolete and no longer in use. M. V. Ball. Wakren, Pa., December, 1913. PREFACE TO THE FIRST EDITION Feeling the need of a Compendium on the subject of this work, it has been our aim to produce a concise treatise upon the Practical Bacteriology of to-day, chiefly for the medical student, which he may use in his laboratory. It is the result of experience gained in the Laboratory of the Hygienical Institute, Berlin, under the guidance of Koch and Frankel; and of information gathered from the original works of other German, as well as of French, bac- teriologists. Theory and obsolete methods have been slightly touched upon. The scope of the work and want of space forbade adequate consideration of them. The exact measurements of bacteria have not been given. The same bacterium varies often much in size, owing to differences in the media, staining, etc. We have received special help from the following books, which we recommend to students for further reference: Mace: Traite pratique de Bacteriologie. Frankel: Grundriss der Bakterienkunde. Eisenberg: Bakteriologische Diagnostik. Crookshank, E. M.: Manual of Bacteriology. Gunther: Einfuhring in das Studium der Bacteriologie, etc. Woodhead and Hare: Pathological Mycology. Salmonsen: Bacteriological Technique (Enghsh transla- tion) . M. V. BALL. 11 CONTENTS PAGE Introduction 17 PART I GENERAL BACTERIOLOGY Chapter I — Structure and Development or Bacteria. .. . 21 Chapter II — Biologic and Chemic Activities 26 Chapter III — Infection 30 Chapter IV — Immunity 34 Chapter V — Methods of Studying Bacteria — Microscope 43 Chapter VI — ^Methods of Studying Bacteria (Continued), Solutions and Formulas for Staining 47 Chapter VII — General Method of Staining Specimens 54 Chapter VIII — Special Methods of Staining and Modifica- tions 58 Chapter IX — Cultivation of Bacteria 62 Chapter X — Preparation of Nutrient Culture-media .... 67 Chapter XI — Inoculation of Culture-media 78 Chapter XII — Cultivation of Anaerobic Bacteria 82 Chapter XIII — The Growth and Appearances of Colonies ... 87 Chapter XIV — Animal Inoculation.- 91 Chapter XV — Bacteeins (Vaccines) 95 PART II SPECIAL BACTERIOLOGY Chapter XVI — Some Common Bacteria Slightly Pathogenic. 97 Bacterium Prodigiosum 97 Bacillus Mesentericus Vulgatus 98 Bacillus Megaterium 99 Bacillus Ramosus 99 Bacterium Zopfii 100 Bacillus Subtilis (Hay Bacillus) 100 13 14 CONTENTS PAGE Boas-Oppler Bacillus loi Bacillus Violaceus 102 Microorganisms Found in Urine 102 Micrococcus Ureae 102 Spirilla . . . . r 103 Spirillum Rubrum 103 Sarcina 103 Sarcina Lutea 103 Sarcina Aurantiaca 104 Sarcina Ventriculi 104 Chapter XVII — Bacillus of Anthrax 105 Chapter XVIII — Bacillus Tuberculosis and Allied Organ- isms no Other Acid-fast Bacteria 116 Bovine and Human Tuberculosis iig Products of Tubercle Bacilli. 1 20 Lepra Bacillus 122 Smegma Bacillus of Alvarey and Tavel 1 23 BaciUus of Glanders (Bacillus Mallei; Rotz-Bacillus) 124 Chapter XIX — Diphtheria Bacillus 126 Chapter XX — ^The CoLON-TsnPHom Group 133 BaciUus Coli 134 Bacillus of Typhoid or Enteric Fever 135 Antityphoid Bacterins (Vaccines) 139 Differentiation Between Colon and Typhoid 142 Typhoid Bacilli from Blood 143 Paracolon or Paratyphoid BaciUi 143 Bacillus Botulinus 144 BaciUus Dysenteric 145 Bacterium Termo 147 BaciUus Proteus Vulgaris 147 Proteus Mirabilis 147 Proteus Zenker! 148 Chapter XXI — Cholera Bacteria 148 Spirillum Choleras (Comma Bacillus of Cholera) 148 Chapter XXII — Bacteria in Pneumonia 155 Diplococcus Pneumonije 157 Bacillus Pneumonias 159 Bacillus of Rhinoscleroma 159 BaciUus of Influenza 160 Koch- Weeks BaciUus 161 Bacillus of Pertussis (Whooping-cough) 161 BaciUus Melitensis ■ 162 Chapter XXIII — Pyogenic Cocci 163 Streptococcus Pyogenes; Streptococcus Erysipelatis 164 Staphylococcus Pyogenes Aureus 166 :ONTENTS 15 PAGE Staphylococcus Pyogenes Albus 168 Micrococcus Pyogenes Citreus 169 Micrococcus Cereus Albus 169 Micrococcus Cereus Flavus 169 Micrococcus Pyogenes Tenuis 169 Micrococcus Tetragenus 170 Morax-Axenf eld Diplobacillus of Conjunctivitis 171 Bacillus Pyocyaneus 171 Chapter XXTV — Gonococcus — Meningococcus 174 Micrococcus Gonorrhoeae 174 Allied Varieties 176 Diplococcus Intracellularis Meningitidis 177 BaciUus of Soft Chancre, Chancroid 178 Chapter XXV — ^Anaerobic Bacteria (Bacillus op Tetanus; Bacillus op Malignant Edema, Etc.) 180 Bacillus of Tetanus 180 Bacillus (Edematis Maligni; Vibrion Septique 184 Bacillus Aerogenes Capsulatus 186 BacUlus Enteritidis Sporogenes 187 Bacillus Chauvei 187 Chapter XXVI — Hemorrhagic Septicemia Group 190 Bacteria of Hemorrhagic Septicemia 192 Bacillus of Chicken Cholera 193 BaciUus of Erysipelas of Swine 194 Bacillus Murisepticus; Mouse Septicemia 195 Micrococcus of Mai de Pis 196 Chapter XXVTI — Protozoa 197 Entamoeba Histolytica; Amoeba Dysenteriae 198 Life Cycle of Malarial Sporozoa 199 Three Forms of Malarial Protozoa 201 Methods of Examination for Malarial Organisms 203 Trypanosomata 204 Trypanosoma Lewisi 205 Trypanosoma Brucei 206 Sleeping Sickness; Trypanosoma Ugandense Gambiense 207 Trypanosoma Evansi 208 Herpetomonas (Leishman-Donovan Bodies) 208 Piroplasma Bovis (Piroplasma Bigeminum) 208 Rabies or Hydrophobia — ^Negri Bodies 209 Chapter XXVIII — The Micro-organism op Syphilis and Al- lied Organisms 209 Spirochaeta Pallida 209 Wassermann Reaction 211 Noguchi Modification of Wassermann Reaction 214 Luetin Reaction 216 Yaws 217 Spirillum of Relapsing Fever 217 African Tick Fever 218 l6 CONTENTS PAGE Chapter XXDC — ^Filterable Organisms 218 Filterable or Ultra-microscopic Organisms 218 Small-pox and Vaccinia 218 Yellow Fever 219 Measles 219 Typhus Fever 219 Acute Poliomyelitis 219 Chapter XXX — Yeasts and Molds 220 Blastomycetes 220 Saccharomyces Cerevisise (Torula Cerevisiae) 220 Saccharomyces Rosaceus; S. Niger; S. Albicans 221 Saccharomyces Mycoderma 221 Oidium 221 Oiidium Lactis 222 Oidium Albicans (Soor; Thrush Fungus ) 222 Pathogenic Yeasts 222 Blastomycetic Dermatitis or Oidiomycosis 223 Hyphomycetes (True Molds) 223 Penicilliura Glaucum 223 Mucor Mucedo 224 Achorion Schonleinii 224 Trichophyton Tonsurans (Ring-worm) 225 Microsporon Furfur 226 Aspergillus Glaucus 226 Aspergillus Fumigatus 226 Examination o£ Yeasts and Molds 226 Cladothrices and Streptothrices 227 Crenothrix Kuhniana 227 Cladothrix Dichotoma 227 Leptothrix Buccalis 228 Beggiatoa Alba 2 28 Streptothrix or Cladothrix Actinomyces (Ray Fungus) 228 Streptothrix Madurae 230 Nocardia (Streptothrix) Farcinica; Bovine Farcin du Boeuf . . . 231 Plant Diseases Due to Bacteria 231 Chapter XXXI — Examination of Air, Soil, and Water 232 Chapter XXXII — Bacteria in Milk and Food 246 Chapter XXXIII — Bacteriologic Examination of the Organs and Cavities of the Hitman Body 257 Chapter XXXIV — Germicides, Antiseptics, and Antisepsis . . . 261 Tables of Cheef Characteristics of Principal Bacteria 268 Non-pathogenic 268 Pathogenic 286 Index 305 Essentials of Bacteriology INTRODUCTION History. — ^The microscope was invented about the latter part of the sixteenth century, and soon after, by its aid, minute organisms were found in decomposing substances. Kircher, in 1646, suggested that diseases might be due to similar organisms, but the means at his disposal were insuffi- cient to enable him to prove his theories. Anthony van Leeuwenhoek, of Delft, Holland (1680 to 1723), so improved the instrument that he was enabled thereby to discover micro-organisms in vegetable infusion, saliva, fecal matter, and scrapings from the teeth. He distinguished several varieties, showed them to have the power of locomotion, and compared them in size with various grains of definite measure- ment. It was a great service that this "Dutch naturaUst" rendered the world; and he can rightly be called the "father of microscopy." Various theories were then formulated by physicians to connect the origin of different diseases with bacteria; but no proofs of the connection could be obtained. Andry, in 1701, called bacteria worms. Miiller, of Copenhagen, in 1786, made a classification composed of two main divisions — monas and vibrio; and with the aid of the compound microscope was better able to describe them. Ehrenberg, in 1833, with still better instruments, divided bacteria into four orders: bac- terium, vibrio, spirillum, and spirochaste. It was not until 2 17 l8 ESSENTIALS OF BACTERIOLOGY 1863 that any positive advance was made in connecting bac- teria with disease. Rayer and Davaine had, in 1850, found a rod-shaped bacterium in the blood of animals suffering from splenic fever (sang de rate), but they attached no special significance to their discovery untU Pasteur made public his grand researches in regard to fermentation and the role bacteria played in the economy. Then Davaine resiuned his studies, and in 1863 estabUshed by experiments the bacterial nature of splenic fever or anthrax. But the first complete study of a contagious affection was made by Pasteur in 1869, in the diseases affecting silk-worms, — pebrine and flacherie, — which he showed to be due to micro-organisms . Then Koch, in 1875, described more fully the anthrax bacillus, gave a description of its spores and the properties of the same, and was enabled to cultivate the germ on arti- ficial media; and, to complete the chain of evidence, Pasteur and his pupils supplied the last link by reproducing the same disease in animals by artificial inoculation from pure cultures. The study of the bacterial nature of anthrax has been the basis of our knowledge of all contagious maladies, and most advances have been made first with the bacterium of that disease. Up to 1875 most medical men beheved that bacteria originated in pus and did not associate them with the cause of suppuration. Lister then began the practice of treating wounds and operating antiseptically, having formed the theory that inflammation and suppuration were due to the contamination of woimds by germs from the air, instruments, etc. From 1880 to 1890 the most important organisms were discovered and associated with disease. In 1890 the discovery of the blood-serum therapy, the antitoxin of Behring, established a new field of research, and much work was undertaken with a view to curing disease. The researches of Ehrlich and the endeavors of Metch- nikoff, Hankin and Ehrlich, to account for the phenomena of immunity, brought forth a great mass of literature and es- INTRODUCTION 1 9 tablished the "lateral-chain" theory and theory of phago- cytosis. These theoretic problems occupied the attention of the workers from 1890 to 1905 and are by no means ended. Laveran, in 188 1, had discovered the protozoa of malaria, and in 1903 Button had associated trypanosomes with sleeping sickness. In 1905 Schaudinn, by demonstrating the cause of syphilis to be a protozoon, gave added im- portance to this particular group of micro-organisms, and today investigators are looking in this branch of microbiology for the cause of cancer. The serxmi reactions of Wassermann and Noguchi, the tuberculins and other products of bacterial growth useful in diagnosis and treatment, have interested the whole medical world, and every physician must of necessity be familiar with some part of this knowledge. There is hope that the technic and the microscope will receive more attention in the next few years, so that the so- called ultramicroscopic and filterable organisms that are believed to exist will be definitely determined, and also the cause of such epidemic diseases as smallpox and scarlet fever be ascertained. PART I GENERAL BACTERIOLOGY CHAPTER I STRUCTURE AND DEVELOPMENT OF BACTERIA The bacteria occupy the lowest plane of plant life known to us, though they are by no means as primitive in their biology as was formerly supposed, and it is quite possible that still simpler forms may be discovered. The ultra- microscope gives promise of such minute organisms, and has made visible particles of matter ^hiT the size of our smallest known bacteria. The numerous unicellular vegetable organisms which form the lower limit of plant life multiply by fission and are -r/ a b c Fig. I. — ^Types of bacteria: a, Micrococcus; h, spirillum; c, bacillus. hence called the Schizophyta, or sphtting plants. This group is subdivided into two classes — (a) the Schizophycea, or fission algae, and (6) the Schizomycetes, or fission fungi, or bacteria, as we usually call them. Bacteria are unicellular masses of protoplasm of microscopic size, multiplying by fission and existing without chlorophyl. Three main types are found: (i) Globular forms, called cocci; (2) straight rod-shaped forms, called bacilli; (3) curved or spiral rods, called spirilla. (See Fig. i.) 22 ESSENTIALS OF BACTERIOLOGY Classifications. — Various ones have been proposed: Mor- phologic, as micrococci, spirilla, and bacilli. Physiologic, ac- cording to their activities and functions, as acid bacteria, alkali and indol bacteria; then subdivisions, according to motility or need for oxygen, but none are satisfactory. The tendency to place bacteria similar in their disease- producing manifestations in one group is growing, as, for in- stance, the colon group, the pus-producers, the pneumonic group, etc. Structure. — Bacteria are cells; they appear as roimd or cylindric, of an average diameter on transverse section of o.ooi mm. (=1 micromillimeter) , written M = TruTnr inch. The cell, as other plant-cells, is composed of a membranous cell- wall and cell-contents or cytoplasm. Cell-wall. — The cell-wall is composed either of hemi- cellulose, or a form of albumin, since it is less permeable than cellulose membrane. The membrane is firm, and can be brought plainly into view by the action of iodin upon the cell-contents, which contract them. Cell-contents. — The contents of the cell consist mainly of protoplasm, usually homogeneous, but in some varieties finely granular, or holding pigment, chlorophyl, fat-droplets, and sulphur in its structure. The protoplasm permits osmo- sis, and is like that of other plant-cells in its structure. Chemic Composition of Bacteria. — ^The ash is mostly phosphoric acid; potassium, chloiin, and calcium are present to a small extent; 80 to 90 per cent, is water. The bacteria resemble the lower animals, rather than plants, in chemic composition. Nuclein, hypoxanthin, and other nitrogen compounds are found in most bacteria. Varies with media in which grown; the proteids are about 10 per cent.; fats, i per cent.; ash, 0.75 per cent. Gelatinous Membrane.— The outer layer of the cell- membrane can absorb water and become gelatinoid, forming either a httle envelop or capsule around the bacterium or preventing the separation of the newly branched germs, STKUCTUEE AND DEVELOPMENT OF BACTERIA 23 forming chains and bunches, as streptococci and staphylococci. Long filaments are also formed. Zooglea. — When this gelatinous membrane is very thick, irregular masses of bacteria will be formed, the whole growth being in one jelly-like lump. This is termed a zooglea (f woj', animal, tXoios, glue). Locomotion. — Many bacteria possess the faculty of self- movement, carrying themselves in all manner of ways across the microscopic field-^some very quickly, others leisurely. Vibratory Movements. — Some bacteria vibrate in them- selves, appearing to move, but they do not change their /5^ 4 a c Fig. 2. — ^Types of flagella: a, Vibrio choleras, one flagellum at the end ■ — monotrichia type; b, Bacterium syncyaneum, tuft of flagella at the end, rarely at the side — ^lophotrichia type; c, Bacteriiun vulgare, flagella iged all about— peritrichia type (Lehtnann and Neumann). arran place; these movements are denoted as molecular or "Brown- ian," and are due to purely physical causes, such as may be obtained by suspending fine grains of carmin in water. Flagella. — ^Little threads or lashes are found attached to many of the motile bacteria, either at the poles or along the sides — sometimes only one, and on some several, forming a tuft. These flagella are in constant motion, and can probably be considered as the organs of locomotion; they have not been discovered upon all the motile bacteria, owing, no doubt, to our imperfect methods of observation. They can be stained and have been photographed. (See Fig. 2.) Flagella serve 24 ESSENTIALS OF BACTERIOLOGY sometimes to increase food-supply, and have been found on some species whichi are non-motile. Reproduction, — Bacteria multiply through simple divi- sion or fission, as it is called. Spore formation is simply a resting stage and not a means of multiplication. To accom- plish division the cell elongates, and at one portion, usually the middle, the cell-wall indents itself gradually, forming a septum and dividing the cell into two equal parts, just as occurs in the higher plant and animal cells. (See Fig. 3.) Successive divisions take place, the new members either existing as separate cells or forming part of a community or Fig- 3- — Division of bacteria: a, Division of a micrococcus; h, division of a bacillus (after Mac6). group. It has been computed that if division takes place every hour, as it often does, one individual in twenty-four hours will have 7,000,000 descendants. Spore Formations. — ^Two forms of sporulation, endo- sporous and arthrosporous. Endosporous. — First, a small granule develops in the pro- toplasm of a bacterium; this increases in size, or several httle granules coalesce to form an elongated, highly refractive, and clearly defined object, rapidly attaining its real size, and this is the spore. The remainder of the cell-contents has now disappeared, leaving the spore in a dark, very resistant mem- brane or capsule, and beyond this the weak cell-wall. The STRUCTURE AND DEVELOPMENT OF BACTERIA 25 cell-wall dissolves gradually or stretches and allows the spore to be set free. Each bacterium gives rise to but one spore. It may be at either end or in the middle (Fig. 4). Some rods take on a peculiar shape at the site of the spore, making the rod look like a drum-stick or spindle — clostridiima (Fig. 5). Spore Contents. — What the real contents of spores are is not known. In the mother-cell at the site of the spore little granules have been found which stain differently from the rest Fig. 4. — Sporulation (after De Bary). Fig. 5. — Clostridium. of the cell, and these are supposed to be the beginnings — the sporogenic bodies. The most important part of the spore is its capsule; to this it owes its resisting properties. It con- sists of two separate layers — a thin membrane around the cell, and a firm outer gelatinous envelop. Germination. — When brought into favorable conditions, the spore begins to lose its shining appearance, the outer firm membrane begins to swell, and it now assumes the shape and size of the cell from which it sprang, the capsule having burst, so as to allow the young bacillus to be set free. Requisites for Spore Formation. — It was formerly thought that when the substratum could no longer maintain 26 ESSENTIALS OF BACTERIOLOGY it, or had become infiltrated with detrimental products, the bacterium-cell produced spores, or rather turned itself into a spore to escape annihilation; but we believe now that only when conditions are the most favorable to the well-being of the cell does it produce fruit, just as with every other type of plant or animal life, a certain amount of oxygen and heat being necessary for good spore formation. The question is still unsettled, however. Asporogenic Bacteria. — Bacteria can be so damaged that they will remain sterile — not produce any spores. This con- dition can be temporary only or permanent. Arthrosporous. — In the other group, called arthrospores, individual members of a colony or aggregation leave the same, and become the originators of new colonies, thus assuming the character of spores. The micrococci furnish examples of this form. Some authorities have denied the existence of the arthro- sporous formation. Resistance of Spores. — Because of the very tenacious en- velop, the spore is not easily influenced by external measures. It is said to be the most resisting object of the organic world. Chemic and physical agents that easily destroy other life have very little effect upon it. Many spores require a temperature of 140° C. dry heat for several hours to destroy them. The spores of a variety of potato bacillus (Bacillus mesentericus) can withstand the application of steam at ioo° C. for four hours. CHAPTER II BIOLOGIC AND CHEMIC ACTIVITIES Origin of Bacteria. — As Pasteur has shown, all bacteria develop from preexisting bacteria or the spores of the same. They cannot arise out of nothing. Distribution. — The wide and almost universal diffusion of bacteria is due to the minuteness of the cells and the BIOLOGIC AND CHEMIC ACTIVITIES 27 few requirements for their existence. In a drop of water 1,700,000,000 cocci can find space. Very few places are free from germs; the air on the high seas and on the mountain-tops is said to be free from bacteria, but this is questionable. Specific Nature. — One kind of bacterium will not pro- duce another kind. A bacillus does not arise from a micro- coccus, or the typhoid fever baciUus produce the bacillus of tetanus. Saproph3rtes and Parasites. — Saprophytes : caTrpos, put- rid; (J}vt6v, plant. Parasites: irapa, aside of; crXros, food. Those bacteria which live on the dead remains of organic life are known as saproph3rtic bacteria, and those which choose the living bodies of their fellow-creatures for their habitat are called parasitic bacteria. Some, however, develop equally well as saprophytes and parasites. They are called faculta- tive parasites. All pathogenic (disease-producing) bacteria are parasites. Conditions of Life and Growth of Bacteria. — Influence of Temperature. — In general, a temperature ranging from 10° C. to 40° C. is necessary to the life and growth of bacteria. Saprophytes take the lower temperatures; parasites, the temperature more nearly approaching the animal heat of the warm blooded. Some forms require a nearly constant heat, growing within very small hmits, as the bacUlus of tubercu- losis. Some forms can be arrested in their development by a warmer or colder temperature, and then restored to activity by a return to the natural heat. A few varieties exist only at freezing-point of water, and others again will not live under a temperature of 60° C. and thrive in hot springs at a temperature of 89° C. For the majority of bacteria a temperature of 60° C. will prevent development, but steam under pressure at 125° C. is necessary to destroy spores. Ice may contain active bac- teria; frozen milk permits the growth of bacteria. Influence of Oxygen. — ^Two varieties of bacteria in relation 28 ESSENTIALS OF BACTERIOLOGY to oxygen — the one aerobic, growing in air; the other, anae- robic, living without air. Obligate aerobes, those which exist only when oxygen is present. Facultative aerobes, those that live best when oxygen is present, but can live without it. Obligate or true anaerobes, those which cannot exist where oxygen is; facultative anaerobes, those which exist better where there is no oxygen, but can live in its presence. Some derive the oxygen which they require out of their nutriment, so that a bacterium may be aerobic and yet not require the presence of free oxygen. Aerobes may consume the free oxygen of a region and thus allow the anaerobes to develop. By improved methods of culture many varieties of anaerobes have been discovered. Influence of Light. — Sunlight is very destructive to bacteria. A few hours' exposure to the sun has been fatal to anthrax bacilli and the cultures of Bacillus tuberculosis. The sun's rays, however, must come in direct contact with the germs, and are usually active only on the surface cultures. The rays at the violet end of the spectrum are the most active. The electric arc-light has much the same effect as sunlight on bacteria; the effect of sunlight is not due to heat-rays. Effects of Electricity. — Electricity arrests growth. Effects of Rontgen Rays. — ^Have Httle or no effect on arti- ficial cultures, but in the living tissues a pronounced bacteri- cidal effect is produced, perhaps through the stimulation of the body-cells. Moisture. — Water is necessary for the development of most bacteria; complete drying is usually destructive after a few days. Heat. — Dry heat is much less destructive than moist heat, steam under pressure most destructive. Biologic Activities. — Bacteria feeding upon organic com- pounds produce chemic changes in them, not only by the withdrawal of certain elements, but also by the excretion of these elements changed by digestion. Sometimes such BIOLOGIC AND CHEMIC ACTIVITIES 29 changes are destructive to the bacteria themselves, as when lactic and butyric acids are formed in the media. Oxidation and reduction are carried on by some bacteria. Ammonia, hydrogen sulphid, and trimethylamin are a few of the chemic products produced by bacteria. Nitrites in the soil are reduced to ammonia. Nitrification. — ^Albuminoids changed into indol, skatol, leucin, etc. ; then these into ammonia, ammonia into nitrites, nitrites into nitrates. Ptomains. — ^Brieger found a number of complex alkaloids closely resembling those found in ordinary plants, and which he named ptomains, from irTu/xa, corpse, because obtained from putrefying objects. These were at one time held to be the chief causes of bacterial disease, but are no longer con- sidered of much importance. Chemic Products. — Secretions, as, for instance, enz3Tnes, toxins. Excretions, pigments, indol, cell proteins, bacterins. Proteins. — The protein contents of the bacterial ceU may cause inflammation and fever. Producers of Disease. — ^Various pathologic processes are caused by bacteria, the name given to such diseases being infectious diseases, and the germs themselves called disease- producing or pathogenic bacteria. Those which do not form any pathologic process are called non- pathogenic bacteria. Fermentation. — This is an important property of bac- terial activity. Enzymes. — ^An enzyme or ferment is a substance capable of inaugurating a chemic reaction without entering into the reaction, and is a product of hving cells. Bacterial enzymes are closely related to the ferments of special cells of higher animals and plants, like ptyaHn and diastase. Ferments may be diastatic, changing starch into sugar, or proteolytic, transforming albumins into more soluble sub- stances, of which gelatin liquefaction is an example. Invert- ing, changing a sugar from one that does not undergo fermen- tation into one that does. 30 ESSENTIALS OF BACTEKIOLOGY Coagulating, fat-splitting, hydrolytic ferments are some of the other varieties. Toxins and toxalbumins are various albuminoids pro- duced in the animal organism and in culture-media which are very poisonous, and are considered the prime cause of disease. Putrefaction. — When fermentation is accompanied by development of offensive gases, a decomposition occurs which is called putrefaction, and this, in organic substances, is due entirely to bacteria. Pigmentation. — Some bacteria are endowed with the property of forming pigments either in themselves, or pro- ducing a chromogenic body which, when set free, gives rise to the pigment. In some cases the pigments have been isolated and many of the properties of the anilin dyes discovered in them. Phosphorescence. — Many bacteria have the power to form light, giving to various objects which they inhabit a characteristic glow or phosphorescence. Fluorescence. — An iridescence, or play of colors, devel- ops in some of the bacterial cultures. Gas-formation. — Many bacteria, anaerobic ones especi- ally, produce gases, noxious and odorless; in the culture- media the bubbles which arise soon displace the media. Odors. — Some germs form odors characteristic of them: some are pleasant and even fragrant; others, foul and nause- ous. Effect of Age. — ^With age, bacteria lose their strength and die. CHAPTER III INFECTION How Bacteria Cause Disease. — Many theories have been advanced to explain the action of bacteria in causing disease, but only a few of the more important ones can be discussed. Nearly all the changes found in the organs of the INFECTION 31 body are similar to those produced by drugs and can be reproduced by the injection of bacterial poisons. Infection is the successM invasion of an organism by microparasites, and implies an abnormal state resulting from the deleterious action of the parasite upon the host. Sources of infection may be exogenous or endogenous. Exogenous infections result from the successful invasion of the body by microparasites from sources entirely apart from the individual infected. Infection by the typhoid bacillus from water or milk, by the Spirochseta pallidum from dental instruments or drinking-cups, contraction of smallpox from fomites, and contraction of malaria from the bites of mosqui- tos are examples of exogenous infection. Endogenous infections result from the successful invasion of the body by microorganisms normally present on the body. The skin and mucous membranes furnish lodgment for a great variety of virulent pathogenic organisms which, when the resistance of the body is lowered, immediately become invasive. The pneumococcus is a normal inhabitant of the mouth and pharynx, but causes no infection until the body resistance is lowered. When this occurs, tonsillitis, pharyngitis, or lobar pnexmionia may follow. Pathogenesis. — ^The ability of a microorganism to do harm depends on its invasive powers and its ability to gener- ate toxins or both. Toxins. — ^Little is known of the chemic nature of toxins. Undoubtedly some are related to albumins. Others give no reactions common to compounds of this group. (A) Intracellular or Insoluble Toxins. — ^These are chiefly within the bodies of the bacteria, and are set free by disin- tegration of the organism. This group comprises most of the pathogenic bacteria. (B) Extracellular or Soluble Toxins. — These toxins are ap- parently excreted by the bacteria, and are foimd in the surroxmding medium. This group includes the diphtheria and tetanus bacilli. It has been shown that bacteria which apparently do not 32 ESSENTIALS OF BACTERIOLOGY produce toxins in artificial media may do so in the human body. These toxic substances are formed by the bacteria to combat the body defenses, and have been called by Bail aggressins. They have a paralytic action on phagocytes. A sublethal dose of bacteria, if injected along with aggressin, win cause death. Toxins are not stable, though tetanus toxin has been kept in powdered form for a number of years. They are soluble in water, destroyed by heat (thermolabUe), and precipitated by ammonium, sulphate. The Cardinal Conditions for Infection. — (i) The microorganism must he sufficiently virulent; (2) it must enter in sufficient numbers and by appropriate channels; and (3) the host must be susceptible. Virulence is a very variable quality, and depends on the ability of the micro-organism to invade or produce toxin or both. The virulence may be decreased by repeated trans- planting on artificial culture-media or by the action of heat. It may be increased by adding animal juices to the culture- medium, by inclosing the micro-organism in a coUodion sac, and placing the sac in the abdominal cavity of an animal, and by repeatedly passing it through animals. Infection Depends on Quantity of Bacteria. — Unless a sufficient number of bacteria enter the tissues no infection follows, because the body defenses immediately destroy the bacteria. The number necessary to cause infection depends on their virulence and the susceptibility of the host. Strep- tococci may become so virulent that a single coccus wUl cause death in a rabbit. It has been found that 820 tubercle bacilli are necessary to kUl a guinea-pig, and 1,000,000 staphylo- cocci to kUl a rabbit. The period of incubation can be ex- plained on the supposition that the organism requires a definite time to generate the amount of toxin necessary to produce symptoms. Avenues of Infection. — ^The organism must gain en- trance into the tissue or find lodgment on some part of the body that has been injured. Even when several avenues of infec- INPECTION 23 tion are open, the parasite most commonly invades through one that may, therefore, be regarded as the most appropriate for entrance; this channel furnishes the typical picture of the infection. Susceptibility of the Host. — Susceptibility varies in different species of animals, in different members of the same species, in the same individual at different times, and in the same individual to different organisms. Susceptibility may be natural, as in smallpox; acquired, as from exposure to conditions which lower the vitality, such as hunger, cold, intoxication, fatigue, inhalation of noxious vapors, and traumatic shock. Inherited susceptibility also occurs. The transmission of certain inherited character- istics, as narrow chest, predisposes to infection of the lungs. Mixed infections are the result of two or more micro- organisms successfully invading and intoxicating the host at the same time. Local Effects of Bacteria. — By mechanical obstruction from rapid growth of the bacteria, thrombosis, with its con- sequences, may occxir. Destruction of a part of the cells of a tissue with necrosis can arise from irritation, the bacteria acting as a foreign body. General Effects. — Bacteremia or septicemia occurs when bacteria proliferate and enter the whole system, as when anthrax and typhoid cause general disease. Toxemia. — ^When the poisons become widely distributed, though the bacteria remain few and localized, and never or seldom enter the circulation, as diphtheria and tetanus. Pyemia, a form of bacteremia, in which secondary or metastatic foci of suppuration occur throughout the body. Suppurative bacteria are those which give rise to inflam- mation and suppuration locally at the point of entrance, and secondarily through metastasis. Any organism may cause suppuration, but certain ones are peculiarly inclined to give rise to pus, and are known as pyogenic organisms. Specific Bacteria. — Infective bacteria are, as a rule, specific, the particular toxin having a specific action and caus- 3 34 ESSENTIALS OF BACTERIOLOGY ing a disease peculiar to the micro-organism. Thus typhoid fever is a disease distinctly different from tuberculosis; the infective organisms are distinct and the poisons they produce have specific characteristics. The Nature of Toxins. — ^Very similar to the venom of serpents; highly poisonous in minute doses d o^o s gram of tetanus toxin wUl kiU a horse weighing 600 kilos — 1200 pounds). At first toxins were called ptomains, or cadaveric alkaloids; but this term is applied now to such poisons as have a basic nature and arise in decomposing meat, cheese, and cream as a result of chemical change in the material, the bacteria causing the change. Then they were called toxal- bumins, and were supposed to belong to an albumin series; but when the bacteria are grown in non-albuminous media, the toxins correspond more in their chemical composition to a ferment, and therefore it is supposed that the albumin part of the toxin is furnished by the blood or albuminous media in which it is formed. The term toxin is to be preferred in speaking of bacterial poisons. CHAPTER IV IMMUNITY Ordinary Defenses to Bacterial Invasion. — The un- broken skin and the connective tissue imderneath prevent the passage of bacteria. The unbroken mucous surface of eye, nose, and mouth, because of the continuous washing, prevents the numerous bacteria that are constantly present in the discharge from finding suitable lodgment. The hairs and ciliated epithelium in upper respiratory tract retain many a dust particle and pathogenic cell on its way to the lungs. The acid gastric juice is destructive to most bacteria, and protects not only the stomach, but the intestines as well. IMMUNITY 35 The intestinal secretions are but mildly preventive of bac- terial growth, but peristalsis aids in dislodgment of micro- organisms. Immunity is the ability to resist infection and intoxica- tion. It is always relative and never absolute. C Natural Immunity Acquired Active. Passive. Natural immunity is a natural inherited resistance against infection or intoxication, peculiar to certain groups of animals, but common to all the iadividuals of these groups. It is peculiar to the kind of animal, not to the individual. Thus the field mouse is susceptible to glanders; the house mouse is slightly immune, and the white mouse is immime. Acquired immunity is resistance to infection or intoxica- tion possessed by certain animals of a naturally susceptible kind, in consequence of circumstances peculiar to them as individuals. Active acquired immunity arises from the activ- ities performed by the organism itself. It depends on infec- tion or intoxication, which may have been accidental or intentional; i. e., for the purpose of producing immunity. Some accidental infections, recovery from which renders the individual immune, are measles, scarlet fever, and smallpox. Other infections are followed by an immunity of short dura- tion, as typhoid fever and pneumonia. Immunity from intentional infection or intoxication is pro- duced by — {A) bringing about a different disease, as in the production of vaccinia to bring about immunity to small- pox. {B) Inoculation with killed bacteria, as in the protec- tive inoculation against typhoid fever or bubonic plague. (C) Inoculation with bacterial products, as diphtheria or tetanus toxin, ip) Inoculation with attenuated cultures of micro- organisms, as in Pasteur's anthrax vaccine or Hafikine's cholera vaccine. (£) Inoculation with virus of increasing viru- lence, as in the protective inoculations against hydrophobia. 36 ESSENTIALS OF BACTEKIOLOGY (F) Inoculation with sublethal doses of mrtdent bacteria, beginning with small doses, and gradually increasing their size. Guinea-pigs inoculated in this way have acquired a marked degree of immunity to tuberculosis. Passive acquired immunity is always artificially sup- plied to the animal. It follows when antibodies are supplied from an immimized animal to one normally susceptible. Immunization against diphtheria by the injection of diph- theria antitoxin is a good example. Theories of Immunity. — Phagocytic Theory of Metchni- koff. — Immunity is dependent on the action of the phago- cytes and their ferments. The phagocytes are of two kinds — macrophages, which include endothelia and connective-tis- sue cells, and microphages, the polymorphonuclear leukocytes. These phagocytes liberate ferments — macrocytase and micro- cytase respectively. Infecting organisms and their toxins are destroyed by the phagocytes and their ferments. This theory has been replaced by the lateral or side-chain theory of Ehrlich. Ehrlich's Lateral Chain Theory. — This derives its name from th e fact that it presents an analogy to what happens in the benzol ring of organic chemistry when its replaceable atoms of hydrogen are substituted by "side chains" of more or less complex nature. The molecule of protoplasm is sup- posed to consist of a central atom group, provided with a large number of side chains which subserve the vital processes of the molecule by combining with other organic molecules. These side chains are called receptors, and are of many dif- ferent kinds, so as to fit them for combination with many different varieties of extraneous groups. Three orders of receptors are described: Receptors of the first order, which concern themselves with the assimilation of simple substances (toxins, ferments, and other cell secre- tions), utilizing a single haptophore. Antitoxins, as an example. Receptors of the second order, which, in addition to the haptophore group, possess a second group, which affects the IMMUNITY 37 coagulation. Toxins may be regarded as receptors of the second order thrust off by the bacteria. Receptors of the third order, which possess two haptophore groups, one of which effects the union with the food-stuffs, whereas the other lays hold on certain substances circulating in the blood plasma, the complements, which cause ferment- like actions — cytolysins, as an example. The Formation of Antitoxin According to the Lateral Chain Theory. — The toxin molecule consists of two groups: (A) Fig. 6. — Graphic representation of receptors of the first order and of toxin uniting with the cell-receptor: a, Cell-receptor; h, toxin molecule; c, haptophore of toxin molecule; d, toxophore of toxin molecule; e, hapto- phore of the ceU-receptor (Ehrlich). The haptophore or combining group, by which the toxin molecule can join the receptor of the cell, and {B) the toxo- phore, or poisoning group, by which means it can attack the cell protoplasm after having been fixed to it by the hapto- phore group. The effect of the toxin depends on the number of mole- cules attached to the cell. A great number would bring 38 ESSENTIALS Or BACTERIOLOGY about death of the cell, while a few would act as an irri- tant. Weigert's Law. — ^When a cell is attacked by a few mole- cules of toxin, it reacts by forming new side chains or recep- tors, and, in accordance with the law of Weigert, always in excess. Repeated injections of toxins in increasing doses cause such an overproduction of receptors of the first order that they are thrust from the cell and float free in the blood- Fig. 7. — Graphic representation of receptors of the second order and of some substances imiting with one of them: c, Cell-receptor of the second order; d, toxophore or zymophorous group of the receptor; e, haptophore of the receptor; /, food substance or product of bacterial disintegration uniting with the haptophore of the cell-receptor (Ehrlich). stream. Here they can combine with toxin molecules, just as when they are attached to the cell. By thus combining, they prevent the toxin from reaching the cells. Antitoxins are specific in their action; that is, each anti- toxin will neutralize only a certain toxin. Thus diphtheria antitoxin wiU not neutralize tetanus toxin or snake venom, IMMUNITY 39 nor will tetanus antitoxin neutralize diphtheria toxin or snake venom. Lock and Key Theory. — ^This specific action is explained by supposing the molecule of toxin to have a shape peculiar to itself. The molecule of diphtheria toxin is of such shape that the haptophdre end will fit only on certain receptors of a cell; the molecule of tetanus toxin will fit on only certain other receptors. Fig. 8. — Graphic representation of receptors of the third order, and of some substance uniting with one of them: c, Cell-receptor of the third order, amboceptor; e, one of the haptophores of the amboceptor with which some food substance or product of bacterial disintegration, /, may unite; g, the other haptophore of the amboceptor with which complement may unite; k, complement; h, the haptophore, and z, the zymotoxic group of the complement (Ehrlich). An antitoxic serum is a suspension of receptors of the first order in blood-serum. Antitoxins for diphtheria and tetanus are the most common. Precipitins are bodies in serum which, when added to a protein in solution, will cause a precipitate to form. The precipitins are specific and act only with similar proteins. When a protein or food substance is injected into an 40 ESSENTIALS OF BACTERIOLOGY animal and becomes attached to the cell receptors of the second order by means of its haptophore group, the cell is irritated and new receptors are formed. Fiirther injection of larger amounts of protein stimulate the cell to such an excessive formation of these receptors that they are thrust free into the blood-stream. A precipitin serum is a suspension of receptors of the second order in blood-serum. The phenomenon of precipitation has found forensic application in the identification of blood-stains. Agglutinins are bodies present in a serum which, when added to bacterial cells, cause them to clmnp, and, if motile, to lose their motility. They are specific when diluted, and of value in diagnosis in such diseases as typhoid and Malta fever. Agglutinins are formed in response to the stimulus given the cells of a body by the union of antigenic cell-receptors with receptors of the second order of the cells of the animal re- ceiving the injection. Repeated injections stimulate the cells to the formation of such excessive quantities of these receptors that they are thrown from the cells into the blood- stream. Agglutinins bear no relation to the degree of immunity, and should never be used as an index to immunity. Cytolysins are bodies present in a serum which will dis- solve or destroy cells (corpuscles, bacteria, etc.). They are formed in the same manner as the agglutinins, except that receptors of the third order are involved. Recep- tors of the third order have a double combining affinity. One part attaches itself to the receptor of the cell injected, and the other combines with complement. Complement {alexin or cytase) is a thermolabile, ferment- like body found in all normal sera. Amboceptors, "substance sensibilatrice," fixateur, copula, and desmon are names given to receptors of the third order. Cytolytic sera are of little use in medicine. Sera have been prepared against staphylococci, pneumococci, streptococci, IMMUNITY 41 and others. Wassermann has made a very efficacious anti- meningococcus serum. Opsonins. — Opsonins are substances in the blood-serum which act on bacteria and prepare them for phagocytosis. Opsonins can be increased by whatever increases immunity. An increase is coincident with increased immunity. The most common method of bringing about an increase is by the injection of killed cultures of bacteria. Opsonins normally present in the serum are not specific. Opsonins resulting from reaction to infection or inoculation are specific. The opsonic index is the ratio between the number of bacteria ingested by Uving leukocytes when operating in the serum of a test and in normal serum respectively. After the injection of bacteria the opsonic index falls for a short time. This period is called the negative phase, and is followed by a rise in the index — the positive phase. The "estimation of the opsonic index is a very complicated way of finding out very little," and has been abandoned by the great majority of workers. Antigens. — ^Any substance that has, when injected into the body, power to produce an antitoxin or antibacterial body is called an antigen. The toxin of diphtheria, if injected, stimulates the normal cells to produce chemic substances (free receptors) which are at liberty to attach themselves to the active toxin mole- cules and thus save the body cells from being acted upon; toxin is, therefore, an antigen. Substances which have the power of destroying bacteria are called bactericides; those which dissolve them merely are called hacteriolysins. Hemolysis. — ^When the hemoglobin of the red blood-ceUs is Hberated, hemolysis is said to occur. This is brought about by the injection of certain substances, or hemolysins; these are present normally in some sera, and can be developed in others. Lysins and bactericidal substances seem to have two parts — one destroyed by heat (thermolabile), called 42 ESSENTIALS Or BACTERIOLOGY complement (the completor), and one, more resistant, called the amboceptor, or combiner, which unites with complement and with the cell. For lysis, therefore, it is necessary that ambocepter be united to the bacteria or cell, and that com- plement be present or added to join with amboceptor, com- pleting the circuit. Complement may be prevented from com- bining with amboceptor by "deviation of the complement." The amboceptor may be in excess, and the free group absorb or attach itself to all the available complement, leaving none to join the amboceptor; or anti-complements may be present to monopolize all this complement and leave none free to unite with amboceptor. This deviation prevents lysis. Fixation of Complement. — By adding a definite standardized complement to a mixture of antigen and amboceptor of a similar kind the complement is boimd or fixed, and none is left free. If the amboceptor is not like the antigen, the com- plement will not unite the two, will not be bound, and is free to unite with any other amboceptor that may be introduced. If this be a hemolytic amboceptor, and red corpuscles are added as an indicator, the cells will lose their hemoglobin, because hemolysis will occur from the completing of the re- action. The complement will unite to hemolytic ambocep- tor, since it is not fixed or bound by the other amboceptor, and the other amboceptor is not of the same nature as the antigen. This is the principle of the Wassermann serum re- action or test. Anaphylaxis or Allergy. — ^Under certain circumstances the second injection of a proteid as antigen instead of render- ing immune, produces hypersensitiveness. Behring, in 1892, noticed this with injections of antitoxin, and called it "hyper- susceptibility." Richet, in 1904, called a similar condition anaphylaxis, or the reverse of prophylaxis, and von Pirquet introduced the term "allergy," " altered reactivity," to express the same thing. Guinea-pigs may be rendered so sensitive by o.ooi c.c. of horse-serum that a second dose within a week or a few days produces fatal shock. METHODS or STUDYING BACTERIA 43 Other proteins, like beef-serum, egg-albumin, red blood- corpuscles, have produced similar results, varying doses and periods of incubation. Human beings may be sensitized by single injections of horse-serum. Hay-fever, asthma, puerperal convulsions, and sympathetic ophthalmia partake of the nature of anaphylactic reactions, and the peculiar intolerances to certain articles of food may be better explained by the same theory. The sudden attacks of collapse and death which have followed the injection of even small doses of antitoxins made from horse-serum are believed to come from this condition of hypersensitiveness. The use of globulins instead of the entire serum has lessened the danger from anaphylaxis. CHAPTER V METHODS OF STUDYING BACTERIA.— MICROSCOPE Microscope. — Most clinical instruments now on the mar- ket have all the necessary appliances for bacterial examina- tion. Three objectives are advisable — 16 mm. (^ inch); 4 mm. (}4 inch); 2 mm. (xV inch). It is not so much required to have a picture very large, as to have it sharp and clear. Oil-immersion Lens. — The penetration and clearness of a lens are very much influenced by the absorption of the rays of Ught emerging from the picture. In the ordinary dry system many of the light rays, being bent outward by the air which is between the object and the lens, do not enter the lens, and are lost. By interposing an agent which has the same refractive index as glass, cedar-oil or clove-oil, for example, all the rays of light from the object enter directly into the lens. The "homogeneous system," or oil-immersion lens, con- 44 ESSENTIALS OF BACTEKIOLOGY sists of a system of lenses which can be dipped into a drop of cedar-oU placed upon the cover-glass, and which is then ready for use. Abbe's Condenser. — The second necessary adjunct is a combination of lenses placed underneath the stage, for bringing wide rays of Ught directly imder the object. It serves to intensify the colored pic- tures by absorbing or hiding the unstained structure. This is very useful in searching a specimen for bacteria, since it clears the field of ever)^thing that is not stained. It is called Abbe's condenser (Fig. 9). Together with it is usually foimd an in- strument for shutting off part of the light — a blender or dia- phragm (Fig. 10). When the bacteria have been found, and their relation to the structure is to be studied, the "Abbe" is generally shut out by the iris blender, and the structure comes more plainly into view. A white light (dayhght or a Fig. 9. — Abbe's condenser. Fig. 10. — Iris blender. Welsbach burner) is best for bacterial study: use the plane mirror with the condenser. For all stained bacteria the oil-immersion lens and Abbe con- denser, without the use of blender. For unstained specimens, oU-immersion and the narrowed blender. When examining with low-power objective, use a strmg METHODS OF STUDYING BACTERIA 45 ocular. When using high-power objective use weak ocular. A revolving nose-piece will be found very useful, since it is sometimes necessary to change the objective on the same field, and this insures a great steadiness of the object. Great cleanliness is needed in aU bacteriologic methods, but nowhere more so than in the microscopic examination. The cover-glass should be very carefully washed in alcohol, and dried with a soft hnen rag. To remove the stains on the cover-glasses that have been used they should be soaked in hydrochloric acid or placed in a 6 per cent, aqueous solution of potassium dichromate with 6 per cent, of strong sulphuric acid, washed in water, and kept in absolute alcohol. Examination of Unstained Bacteria. — As the coloring of bacteria kills them and changes their shape to some extent, it b Fig. II. — ^Platinum needles for transferring bacteria, made from No. 27 platinum wire inserted in glass rods: a, Looped needle; b, straight- pointed needle (McFarland). is preferable to examine bacteria, when possible, in their natural state. We obtain the bacteria for examination either from liquid or sohd media. From Liquids. — With a long platinum needle the end of which is bent into a loop (Fig. 11, a) obtain a small drop from the liquid containing the bacteria, and place it on a cover- glass or shde, careful that no bubbles remain. Sterilize Instruments. — Right here we might say that it is best to accustom one's self to pass all instruments, needles, etc., through the flame before and after each proce- dure; it insures safety; and once in the habit, it will be done automatically. From Solid Media.— With a straight-pointed platinum needle (Fig. 11, 6) a small speck of the medium is taken and 46 ESSENTIALS Or BACTEEIOLOGY rubbed upon a glass slide with a drop of sterilized water or bouillon, and from this a little is taken on cover-glass, as before. The cover-glass with its drop is now placed on the glass slide, carefuUy pressing out aU bubbles. Then a drop of cedar-oil is laid on top of the cover-glass, and the oil-immer- sion lens dipped gently down into it as close as possible to the cover-glass, the narrow blender shutting of the Abbe conden- ser, for this being an unstained specimen, we want but little light. We now apply the eye, and if not in focus, use the fine adjustment or the coarse, but always away from the object — i. e., toward us — since the distance between the speci- Fig. 12. — A "concave slide" with "hanging drop" (McFarland). men and the lens is very slight, it does not require much turning to break the cover-glass and ruin the specimen. Having found the bacteriiun, we see whether it is bacillus, micrococcus, or spirillum, discover if it is motile or not. The phenomenon of agglutination is observed in this way. Hanging Drop (Fig. 12). — When the looped platinum needle is dipped into a liquid, a very finely formed globule will hang to it; this can be brought into a little cupped glass slide (an ordinary microscopic glass slide with a circular depression in the center) in the following manner: The drop is first brought upon a cover-glass; the edges of the concavity on the glass slide are smeared with vaselin, and the slide inverted over the drop; the cover-glass sticks to the smeared slide, METHODS OF STUDYING BACTERIA 47 which, when turned over, holds the drop in the depression covered by the cover-glass, thus forming an air-tight cell; here the drop cannot evaporate. Both slide and cover-glass should first be sterilized by heat. Search for the bacteria with a weak lens; having found them, place a drop of cedar-oil upon the cover-glass, and bring the oil immersion into place (here is where a nose-piece comes in very useful), careful not to press against the cell, for the cover-glasses are very fragile in this position. Search the edges of the drop rather than the middle; the bacteria will usually be very thick in the center and not so easily distinguished. Spores, automatic movements, fission, and cultivation in general can be studied for several days. This moist chamber can be placed in a brood-oven or on the ordinary warming stage attachment of the microscope. Hanging Block. — A small slice of agar containing some of the growth seared to the glass slide with a hot needle. Agglutination as observed in Widal's test is best seen in the hanging drop. CHAPTER VI METHODS OF STUDYING BACTERIA (Continued).— SOLUTIONS AND FORMULAS FOR STAINING Staining or coloring bacteria is done in order to make them prominent and to obtain permanent specimens. It is also necessary to bring out the structure of the bacteria, and serves in many instances as a means of diagnosis ; it would be well-nigh impossible to discover them in the tissues without staining. Anilin Colors. — Of the numerous dyes in the market, nearly all have, at one time or other, been used in staining 48 ESSENTIALS OF BACTERIOLOGY bacteria. But now only a very few find general use, and with methylene-blue and fuchsin nearly every object can be accomplished. Basic and Acid Dyes. — Ehrlich was the first to divide the aniHn dyes into two groups, the basic colors to which belong — Gentian-violet, or pyoktanin. Basic fuchsin. Methyl-violet, or dahlia. Bismarck-brown Methylene-blue {not methyl blue). Thionin. Safranin. And the acid colors to which eosia and acid fuchsin belong. The basic aniline dyes stain the bacteria and the nuclei of cells; the acid dyes stain chiefly the tissue, leaving the bac- teria almost untouched. Carmin and hematoxylin are also useful as contrast stains, affecting bacteria very slightly. The anil in dyes are soluble in alcohol or water or a mixture of the two. Staining Solutions. — A saturated solution of the dye is made with alcohol. This is called the stock or concentrated solution; i part of this solution to about lo parts of distilled water constitutes the ordinary aqueous solution in use or weak solution. It is readily made by adding to an ounce bottle of distilled water enough of the strong solution until the fluid is still opaque in the body of the bottle, but clear in the neck of the same. These weak solutions should be renewed every three or four weeks, otherwise the precipitates formed will interfere with the staining. Compound Solutions. — By means of certain chemic agents the intensity of the anilin dyes can be greatly increased. Intensifiers or Mordants. — Agents that "bite" into the specimen, carrying the stain with them, depositing it in the deeper layers, are called mordants or etchers. Various metallic salts and vegetable acids are used for such purpose. The mother liquid of the anilin dyes, anilin-oil, a member v of the aromatic benzol group, has also this property. METHODS or STUDYING BACTERIA 49 Anilin-oil Water. — Anilin-oil is shaken up with water and then filtered; the anilin water so obtained is mixed with the dyes, forming the " anUin- water gentian-violet" or anilin- water fuchsin, etc. Carbolfuchsin. — Carbolic acid or phenol can be used instead of anHin-oil, and forms one of the main ingredients of Ziehl's or Neelsen's solution, used principally in staining Bacillus tuberculosis. Kiihne has a carbol-methylene-blue made similar to the carbolfuchsin. Alkaline Stains. — Alkalis have the same object as the above agents, namely, to intensify the picture. Potassium hydroxid, ammonium carbonate, and sodium hydroxid are used. Loffler's alkaline blue and Koch's weak alkaline blue are made with potassium. Heat. — Warming or boUing the stains during the process of staining increases their intensity. Decolorizing Agents. — The object after staining is usu- ally overcolored in some part, and then decolorizing agents are employed. Water is suflScient in many cases; alcohol and strong mineral acids combined are necessary in some. lodin as Used in Gram's Method. — Belonging to this group, but used more in the sense of a protective, is tincture of iodin. It picks out certain bacteria, which it coats ; pre- vents them from being decolorized, but fades the rest of the picture. Then, by using one of the acid or tissue dyes, a con- trast color or double staining is obtained. Many of the more important bacteria are not acted upon by the iodin, and it thus becomes a very useful means of diagnosis. FORMULAS OF DIFFERENT STAINING SOLUTIONS I. Saturated Alcoholic Solution Place about lo grams of the powdered dye in a bottle and add 40 grams of alcohol. Shake well and allow to settle. This can be used as the stock bottle. 50 ESSENTIALS OF BACTERIOLOGY II. Weak Solutions Made by adding about i part of stock solution (I)^ to lo parts of distilled water. This is the ordinary solution in use. III. Anilin-oil Water Anilin-oil 5 P^rts Distilled water loo " — M. Shake well and filter. To be made fresh each time. IV. Anilin-oil Water Dyes Saturated alcoholic solution of the dye II parts Anilin-oU water loo " Absolute alcohol lo " — M. Can be kept ten days. V. Alkaline Methylene-blue A. Lqffler's: Saturated alcoholic solution methy- lene-blue 30 parts Solution potassium hydroxid (i per cent.) I part Water q. s. 100 parts — M. B. Koch's: Solution potassium hydroxid (10 per cent.) 2 parts Saturated alcoholic solution methy- lene-blue 10 " Distilled water 2000 " — M. VI. Phenol Solutions A. Ziehl-N eelsen: Fuchsin (powdered) i part Alcohol . 10 parts 5 per cent, solution phenol 100 " — M. Filter. The older the solution, the better. METHODS OP STUDYING BACTERIA 5 1 B. KUhne: Methylene-blue 1.5 parts Alcohol lo.o " 5 per cent, solution phenol loo.o " Add the phenol gradually. This solution loses strength with age. VII. Gram's lodin Solution lodin I part Potassium iodid 2 parts Distilled water 300 " — M. VIII. Loffler's Mordant (Jor Flagella) Aqueous solution of tannin (20 per cent.) 10 parts Aqueous solution ferric sulphate (5 per cent.) i part Aqueous decoction of logwood (1:8) 4 parts. — M. Keep in weU-corked bottle. IX. Unna's Borax Methyl-blue Borax i part Methyl blue i " Water 100 parts. — M. X. Gabbet's Acid Blue {Rapid Stain) Methylene-blue 2 parts 20 per cent, sulphuric acid 100 " — M. XI. Alkaline Anilin-water Solutions Sodium hydroxid (i per cent.) .... i part Anilin-oil water 100 parts. — M. And add — Fuchsin, or methyl-violet powdered 4 parts Cork well. Filter before using. 53 ESSENTIALS OF BACTERIOLOGY XII. Roux's Double Stain Dahlia or gentian-violet 0.5 part Methyl-green i-S parts Distilled water 200.0 " — M. Use as other stains, without acid. XIII. Neisser's Stain (for Diphtheria) Solution I Methylene-blue i part Alcohol (96 per cent.) 20 parts Dissolve and add — Water 950 parts Glacial acetic acid 50 " — M. Solution II Vesuvin 2 parts Water 1000 " — M. Stain cover-glasses — (i) Three seconds in solution I; (2) wash in water; (3) three seconds in No. II; (4) wash in water. Body of bacillus, brown; oval granules at each end, blue. XIV. Carholthionin (Nicolle) Saturated solution thionin in alcohol (90 per cent.) 10 parts Aqueous solution phenol (i per cent.) 100 " — M. Stain sections one-half to one minute. XV. Capsule Stain of Hiss Use the following, heated until it steams: Saturated alcoholic solu- tion of gentian-violet or f uchsin . . 5 parts Distilled water gj " — M. Wash in 20 per cent, solution of cupric sulphate crystals. METHODS or STUDYING BACTERIA S3 XVI. Capsule Stain of Welch (i) Pour glacial acetic acid on film. After a few seconds replace with anilin-water gentian-violet without washing in water. (2) Remove all acid by several additions of stain, and allow it to act for three to four minutes. (3) Wash and examine in salt solution 0.8-2.0 per cent. XVII. Romanowsky Stains A compound dye originally used for malarial parasites, but now employed in some of its modifications in staining blood- films, bacteria in tissues, and protozoa generally. The stain is difficult to prepare, and can be purchased of supply houses to better advantage. The chief modifications are: Leishman's stain, consisting of a i per cent, solution methyl- ene-blue, to which 0.5 per cent, sodium carbonate has been added and allowed to stand for twelve hours in incubator at 65° C, and then ten days at room temperature, and a solu- tion of eosin (i : 1000) in water. Equal parts of these solu- tions are mixed and allowed to stand for six hours. After it has been washed and dried, the precipitate is dissolved in methyl-alcohol. Giemsa Stain: Azur II. — eosin 3 parts Azur II 8 " Glycerin (pure) 250 " Methyl-alcohol 250 " — M. Azur is a mixture of methylene-blue and eosin prepared in a special way. Jenner's Stain. — 1.2 per cent, aqueous solution of water- soluble eosin; i per cent, aqueous solution methylene-blue (Grubler); equal parts of each. Mix; allow to stand twenty- four hours, wash the precipitate, dry it, dissolve 0.5 gm. in 100 c.c. methyl-alcohol. /. H. Wright's Stain. — ^Made in much the same way as Leishman's. The precipitate is not washed, but the satur- 54 ESSENTIALS OE BACTERIOLOGY ated methyl-alcohol solution is filtered and further diluted with methyl-alcohol. The stains are used in very dilute form. Where the blood-films or exudates are not first fixed in alcohol, the concentrated stain is allowed to cover the preparation for five to twenty seconds to fix; then water is poured on to dilute and from five to fifteen minutes allowed for staming, the excess removed with water. The stains can be purchased in powder or tablet form, and need only be mixed with methyl-alcohol to be ready for use. CHAPTER VII GENERAL METHOD OF STAINING SPECIMENS Cover-glass Preparations. — ^The material is evenly spread in as thin a layer as possible upon a cover-glass; then, to spread it still more finely, a second cover-glass is pressed down upon the first and the two slid apart. This also secures two specimens. Before they can be stained, they must be perfectly dry, otherwise deformities will arise in the structure. Drying the Specimen. — The cover-glass can be set aside to dry, or held in the fingers over the Bunsen burner (the fingers preventing too great a degree of heat) . Since most of the specimens contain a certain amount of albimiinoid mater- ial, it is best in all cases to "fix" — i. e., to coagulate the albumin. This is accomplished by passing the cover-glass (after the specimen is dry) three times through the flame of the burner, about three seconds being consumed in so doing, the glass being held in a small forceps, smeared side up. The best forceps for grasping cover-glasses is a bent one, bent again upward, near the ends (Fig. 13). It prevents the flame or staining fluid from reaching the fingers. The object is now ready for staining. Staining. — ^A few drops of the staining solution are placed GENERAL METHOD OF STAINING SPECIMENS 55 upon the cover-glass so that the whole specimen is covered, and the stain is left on a few minutes, the time depending upon the variety, the strength of stain, and the object de- sired. Instead of placing the dye upon the object, the cover- glass can be immersed in a small glass dish containing the solution; or, if heat is desired to intensify or hasten the proc- ess, a watch-crystal holding the stain is placed over a Bun- sen burner and in it the cover-glass ; the cover-glass may be held directly in the flame with the staining fluid upon it, which must be constantly renewed until the process is com- pleted, or the cover-glass can be heated in a test-tube, con- taining stain solution. Removing Excess of Stain. — The surplus stain is washed off by dipping the glass in distilled water. Fig. 13. — ^Author's bent forceps for holding cover-glass over iiame. The water is removed by drying between filter-paper or simply allowed to run off by standing the cover-glass slant- wise against an object. When the specimen is to be examined in water (which is always best with the first preparation of the specimen, as the Canada balsam destroys to some extent the natural appearance of the bacteria), a small drop of ster- ilized water is placed upon the glass shde, and the cover-glass dropped gently down upon it, so that the cover-glass remains adherent to the slide. The dry system or the oil immersion can now be used. When the object has been sufficiently examined, it can be permanently mounted by Ufting the cover-glass off the sHde (this is facilitated by letting a Httle water flow under it, one 56 ESSENTIALS OF BACTERIOLOGY end being slightly elevated). The water that still adheres is dried off in the air or gently over the flame, and when per- fectly dry, the cover-glass is placed upon the drop of Canada balsam which has been put upon the glass sUde. In placing the cover-glass in the staining solutions one must be careful to remember which is the spread side, by holding it between one's self and the window and scraping the sides carefully with the sharp point of the forceps, the side having the specimen on it will show the marks of the instru- ment. Little glass dishes, about one-half dozen, should be at hand for containing the various stains and decolorants. Tissue Preparations. — In order to obtain suitable speci- mens for staining, very thin sections of the tissue must be made. As with histologic preparations, the tissue must be hardened before it can be cut thin enough. Alcohol is the best agent for this purpose. Pieces of the tissue one-quarter inch in size are covered with alcohol for twenty-four to forty-eight hours. When hardened, it must be fixed upon or in some firm object. A paste composed of — Gelatin i part Glycerin 4 parts Water 2 " will make it adhere firmly to a cork in about two hours, or it can be embedded in a small block of paraffin and covered over with melted parafiin. Celloidin may be used as an embedding agent, and formalin is useful to harden tissue quickly. Cutting. — The microtome should be able to cut sections "ToTir inch in thickness; this is the fineness usually required. The sections are brought into alcohol as soon as cut, unless they have been embedded in paraflfin, when they are first washed in chloroform to dissolve out the paraffin. GENERAL METHOD OF STAINING SPECIMENS $7 Staining. — All the various solutions should be in readiness, best placed in the little dishes in the order in which they are to be used, as a short delay in one of the steps may spoil the specimen. A very useful instrument for transferring the delicate sections from one solution to another is a little metal spatula, the blade being flexible (Fig. 14). A stiU better plan, especially when the tissue is "crum- bling," is to carry out the whole procedure on the glass sHde. General Principles. — The section is transferred from the alcohol in which it has been kept into water, which removes the excess of alcohol, from here into — Dish I, containing the stain, where it remains five to fifteen minutes. Then — Dish II, containing 5 per cent, acetic acid (1:20), where it ■~r^ Fig. 14. — Spatula for lifting sections. remains one-half to one minute. The acid removes the excess of stain. Dish III, water, to rinse off the acid. The section can now be placed imder the microscope, covered with cover-glass to see if the intensity of the stain is sufficient or too great. A second section is then taken, avoiding the errors, if any; and having reached this stage, proceeded with as follows : Dish IV, alcohol, two to three seconds, to remove the water in the tissue. V. A few drops of oil of cloves, just long enough to clear the specimen to make it transparent (so that an object placed underneath will shine through). VI. Remove excess with filter-paper. VII. Mount in- Canada balsam (xylol balsam). Staining Blood Specimens. — ^A drop of blood is spread on 58 ESSENTIALS OE BACTERIOLOGY a cover-glass and stained with the ordinary dyes; but in order to eliminate the coloring-matter of the red corpuscles and bring the stained bacteria more prominently into view, Gunther recommends that the blood, after drying and fixing, should be rinsed in a dilute solution of acetic acid (i to 5 per cent.). The hemoglobin is thereby extracted, and the cor- puscles appear then only as faint outlines. Instead of "fixing" by heat, Canon employs alcohol for five minutes, especially in staining for influenza bacilli which have been detected in the blood. CHAPTER VIII SPECIAL METHODS OF STAINING AND MODIFICATIONS Gram's Method of Double Staining {Jfor Cover-glass Specimens). — I. A hot solution of anilin-water gentian-violet two to ten minutes. II. Directly, without washing, into Gram's solution of iodin potassium iodid one to three minutes (the cover-glass looks black) . III. Wash in alcohol 60 per cent, until only a light brown shade remains (as if the glass were smeared with dried blood). IV. Rinse off alcohol with water. V. Contrast color with either eosin, picrocarmin, or Bis- marck-brown. The bacteria will appear deep blue, all else red or brown on a very faint brown background. Gram's Method for Tissues (Modified by Gunther) I. Stain in anilin-water gentian- violet . . i minute II. Dry between filter-paper. III. Iodin potassium iodid solution 2 minutes IV. Alcohol ^ minute V. 3 per cent, solution hydrochloric acid in alcohol 10 seconds VI. Alcohol, oil of cloves, and Canada balsam. SPECIAL METHODS OF STAINING AND MODIFICATIONS 59 Behavior of the More Important Bacteria to Gram's Stain. — Positive means that the bacteria retain the primary color, or gentian- violet; negative, that they do not. Positive. Negative. Tubercle bacillus. Colon bacillus. Smegma bacillus. T5^hoid bacillus. Lepra bacillus. Cholera bacillus. Anthrax bacillus. Influenza bacillus. Tetanus bacillus. Friedlander's bacillus. Diphtheria bacillus. Plague bacillus. Pneumococcus. Diplococcus intracellularis. Streptococcus. Gonococcus. Staphylococcus. Koch-Weeks bacillus. Cocci of the urethra. Conjunctivitis bacillus of Morax. Loffler's Method for Tissues Alkaline methylene-blue S~3o minutes I per cent, acetic acid few seconds. Absolute alcohol, xylol, Canada balsam. Bacteria dark blue, nuclei blue, cell-bodies light blue. To Stain Spores. — Since spores have a very firm capsule, which tends to keep out all external agents, a very intensive stain is required to penetrate them, but once this object is attained, it is equally as difficult to decolorize them. A cover-glass prepared in the usual way, i. e., drying and passing the specimen through the flame three times, is placed in a watch-crystal containing Ziehl's carbolfuchsin solution, and the same placed upon a rack over a Bimsen burner, where it is kept at boiling-point for one hour, careful to supply fresh solution at short intervals lest it dry up. The bacilli are now decolorized in alcohol containing 0.5 per cent, hydrochloric acid. A contrast color, preferably methylene-blue, is added for a few minutes. 6o ESSENTIALS OF BACTEKIOLOGY The spores will appear as little red beads in the blue-stained bacteria, and loose spores lying about outside the cell- wall. Spore Stain {Modified). — I. Carbolfuchsin on cover-glass and heated in the flame to boiling-point 20 to 30 times. II. 25 per cent, sulphuric acid, two seconds; rinsed in water. III. Methylene-blue contrast. Alex. Klein recommends the following spore method: mix a Httle of the culture (potato) with three drops of physiologic salt solution, and heat gently with an equal quantity of carbolfuchsin for a period of six minutes. Spread then on cover-glasses, dry in the air, and fix by passing three times through Bunsen-burner flame. Decolorize in i per cent, sulphuric acid for one to two seconds; contrast in weak methylene-blue. Bowhill's Orcein Stain Saturated alcoholic solution of orcein . 15 c.c. 20 per cent, aqueous solution tannin 10 c.c. Distilled water 30 c.c. — M. Filter. Use orcein solution in watch-glass, float cover-glass in it, and heat gently, not boil, for ten minutes. Wash in water. Dry and mount in balsam. Five per cent, chromium trioxid appHed for fifteen minutes has been recommended in staining spores. This is followed by the carbolfuchsin stain as above. Sporogenic bodies stain quite readily, and in order to distin- guish them from spores Ernst uses alkaline methylene-blue, slightly warmed. Then rinse in water. Contrast with cold Bismarck-brown. The spores are colored bright blue, the spore granules a dirty blue, being mixed with the brown, which colors also the bacteria. Kuhne's Method. — In sections the alcohol used sometimes decolorizes too much. To obviate this Kuhne mixes the alco- SPECIAL METHODS OF STAINING AND MODIFICATIONS 6 1 hol with the stain, so that while the section is being anhy- drated, it is constantly supplied with fresh dye. Weigert uses anilin-oil to dehydrate instead of alcohol, and here, too, it can be used mixed with the dye. Capsule Stain {Buerger). — I. Spread culture by means of a drop of ascitic fluid on cover-glass. II. Fix in Miiller's fluid, which has been saturated with 5 per cent, bichlorid of mercury, and warm for three seconds. III. Wash quickly in water; rinse in alcohol. IV. Cover with tincture of iodin for one minute. V. Wash in alcohol and dry in air. VI. Stain in anilin-water gentian-violet for two seconds. VII. Wash in 2 per cent, salt solution. VIII. Mount in salt solution ringed with vaselin. Hiss' Method for Capsule. — Smear on cover-glass the organisms mixed with a drop of animal serum (beef-blood serum or ascitic fluid) . Dry in air. Fix by heat. Stain for few seconds in Hiss' stain (p. 52). Wash in 20 per cent, cop- per sulphate solution. Dry and mount. Capsule appears as faint blue halo about dark-purple cell. Flagella Stain, with Loffler's Mordant. — I. A few drops of the mordant stain (p. 51) are placed upon the spread ■ cover-glass and heated until it steams. II. Wash with water until the cover-glass looks almost clean, using a small piece of filter-paper to rub off the crusts which have gathered around the edges. HI. Anilin-water fuchsin (neutral) held in flame about one and one-half minutes. IV. Wash in water. If the stain is properly made, the bacteria are deeply colored and the flagella seen as little dark lines attached to them. Unna's Method for Fungi. — Especially useful for epi- dermic scales. Moisten horny scale or crust with acetic acid; macerate between two glass slides; dry in flame; wash out fat with ether and alcohol (equal parts) ; stain in borax methyl-blue 62 ESSENTIALS OF BACTERIOLOGY for ten seconds (over flame) ; bleach with glycerin and ether (equal parts) ; rinse in water, alcohol, dry, and mount. CHAPTER IX CULTIVATION OF BACTERIA Artificial Cultivation.— The objects of cultivation are to obtain germs in pure culture, free from all foreign matter, isolated, and so developed as to be readily used either for microscopic examination or animal experimentation. To develop bacteria properly we supply, as nearly as possi- ble, the conditions which hold for the especial germ in nature. With the aid of solid nutrient media the bacteria can be easily separated, and the methods have been gradually evolved from those originally devised by Pasteur and Koch. Sterilization of Culture-media, etc. — If we place our nutrient material in vessels that have not been properly dis- infected, we will obtain growths of bacteria without having sown any. If we have thoroughly cleaned our utensils and then not taken care to protect them from further exposure, the germs we have sown will be effaced or contaminated by multitudes of others that are constantly about us. We, therefore, have two necessary precautions to take: First, thoroughly to clean and sterilize every object that enters into, or in any way comes in contact with, the culture. Second, to maintain this degree of sterility throughout the whole course of the growth, and prevent, by proper containers, the entrance of foreign germs. Disinfectants. — Corrosive sublimate (bichlorid of mercury) which is the most effective agent we possess, cannot be gener- ally used because it renders the soil unproductive, and, there- fore, must be employed only in washing dishes, to destroy the CULTIVATION OF BACTERIA 63 old cultures. Even after washing a few drops of the solution may remain and prevent growth, so that one must be careful to have the glassware that comes in contact with the nutrient media free from the sublimate. Fig. 15. — ^Hot-air sterilizer. The gas-jets are inclosed witliin the space between the outer and middle walls, C, and can be seen at F. The heat ascends, wanning the air between the two inner walls, which ascends between the walls, K, K, then descends over the contents, /, and escapes through perforations in the bottom, B, to supply the draft at F, and eventually escapes again at S; R, gas regulator; T, thermometer. Heat. — ^Heat is the best agent we possess for general use. Dry heat and moist heat are the two forms employed, but these differ greatly in effectiveness. Thus Koch found that 64 ESSENTIALS OF BACTERIOLOGY while moist heat at ioo° C. killed the spores of the anthrax bacillus in one hour, it required three hours of dry heat at 140° C. to produce death. For obtaining dry heat — that is, a temperature of 150° C. (about 300° F.) — a sheet-iron oven (Fig. 15) is used which can be heated by a gas-burner. If it have double walls (air circulating between), the desired temperature is much more quickly obtained. A small opening in the top to admit a thermometer is necessary. These chests are usually about I foot high, \% feet wide, and ^ foot deep. In them glass- ware, cotton, and paper can be sterilized. When the cotton is turned slightly brown, it usually denotes sufficient steriliza- tion. All instruments, where practicable, should be drawn through the flame of an alcohol lamp or Bunsen burner. One hour in the oven at 170° C. usually sterilizes glassware, while the ordinary germs in liquids may be killed by boihng for five minutes if no spores are present. The boUing of any fluid at 100° C. for one and one-half hours nearly always insures sterilization. Moist Heat. — Steam at 100° C. in circulation has been shown to be a very efiective application of heat. The steam chest devised by Koch consisted of a long double boiler divided by a perforated shelf on which the material could rest while subjected to streaming steam. Arnold's steam sterilizer will answer every purpose of the Koch steam-chest. It is cheaper, also requiring less fuel to keep it going. The steam does not escape, but is condensed in the outer chamber. The autoclave (Fig. 16), which produces steam under pressure and allows a temperature of 120° C. to be obtained, is a most effective method of sterilization, but the higher temperatures are not suitable for gelatin or sugar solution. Gelatin loses its power of solidifying if the bo ilin g is pro- longed. Instead of sterilizing for a long time at once, successive steriHzation is practised with nutrient media, so that the albumin will not be too strongly coagulated. Fifteen minutes CULTIVATION OF BACTERIA 6S each day for three days in succession in the Arnold sterilizer, or one exposure ia the autoclave, five to fifteen minutes, at IS pounds pressure; 120° C. is sufficient to sterilize most culture-media. Fractional Sterilization of Tyndall. — Granted that so many spores originally exist in the object to be sterilized, it ^fcy Fig. 16. — Autoclave. Horizontal form. is subjected to 60° C. for four hours, in which time a part at least of those spores have developed into bacteria, and the bacteria destroyed by the further application of the heat. The next day more bacteria will have formed, and four hours' subjection to 60° C. heat will destroy them, and so, at the end of a week, using four hours' application each day, all the spores originally present will have germinated and the bacteria be destroyed. 66 ESSENTIALS OF BACTERIOLOGY As modified, and in use in most laboratories, fifteen minutes, sterilization in steam, at ioo° C, in the Arnold sterilizer on three successive days, has been found sufficient, while one sterilization in the autoclave at 120° C. for fifteen minutes wiU serve in most cases, especially if the medium is for imme- diate use, and does not contain gelatin or sugar. Cotton Plugs or Corks. — ^AU the glass vessels (test-tubes, flasks, etc.) must be closed with cotton plugs, cotton-wool, Fig. 17.— Wire cage. Fig. 18.— Cotton-plugged test-tubes. or a good quality of non-absorbent cotton), the cotton being easily sterilized and preventing the entrance of germs from the air. Tin-foil may be used to cover the cotton, or caps made of india-rubber. Test-tubes. — New test-tubes are washed with hydro- chloric acid and water to neutrahze the alkalinity often pres- ent in fresh glass, or in chromic acid cleaning mixture one hour. (Potassium dichromate, 6; water, 30; sulphuric acid, 46.) They are then well washed and rubbed with a brush' PREPAKATION OF NUTRIENT CULTURE-MEDIA 67 placed obliquely to drain, and when dry, corked with cotton plugs. Then put in the hot-air oven (little wire cages, Fig. 17, being used to contain them) for fifteen minutes, after which they are ready to be filled with the nutrient medium. (The cotton should fit firmly in the tube and extend a short space beyond it.) Test-tubes without flaring edges are more desirable, since the edges can easily be drawn out so as to seal the tube. Instead of test-tubes, ordinary 3-ounce panel medicine bottles can be used for retaining the nutrient media and cultures. According to investigations, the glass tubes become suffi- ciently sterile in the steam-chest without the preliminary sterilization in the dry oven. Sterilization by Filtration. — Germ Filters. — ^Kaolin or por- celain bougies, such as are used in the Berkefeld, Chamber- land, and Pasteur filters, restrain most bacteria, except those now known as ultramicroscopic. In the making of toxins this method is used, heat or disinfectants being undesirable. With the knowledge of smaller forms of life, the filter will need further improvement. CHAPTER X PREPARATION OF NUTRIENT CULTURE-MEDU Or the many different media recommended and used since bacteriology became a science, we can describe only the more important ones now in use. Each investigator changes them according to his taste. Potato as Medium. — ^The knowledge of bacteria and germs or molds settling and growing upon slices of potato 68 ESSENTIALS Or BACTERIOLOGY exposed to the air led to the use of soUd media for the artificial culture of the same. It was thus learned that each germ tends to form a separate colony and remain isolated, and so pure cultures were first obtained. Esmarch's Cubes.— The potato is first well cleaned and peeled. It is then cut in cubes y^ inch in size. These are placed, each in a little glass dish or tray, and then in steam-chest for one-half hour, after which they are ready for inoculation (the dishes first having been g I-. sterilized in hot-air oven). if m. Test-tube Potatoes.— Cones are cut out of the peeled potato and placed in test-tubes, which can then be plugged and easily pre- served. Roux's test-tube (Fig. 19), specially de- signed for potato cultures, consists of a tube with a small constricted portion at the bot- tom, in which water may be kept to keep the potato moist. Manner of Inoculating Potatoes. — ^With a platinum rod or a spatula (sterilized) the material is spread upon one of the slices, keeping free of the edges. The growth on this first, or original, potato will be quite lux- uriant, and the individual colonies often diffi- cult to recognize; therefore dilutions are made. From the original or first slice a small portion, including some of the meat of the potato, is spread upon the surface of a second slice, which is first dilution. From this likewise a small bit is taken and spread on a third slice, or second dilu- tion, and here usually the colonies will be sparsely enough settled to study them in their individuality. This is the principle carried on in all the cultivations. It is a physical analysis. Potato and Bread Mash. — These pastes are used chiefly in the culture of molds and yeasts. Peeled potatoes are mashed with distilled water until thick, and then sterilized Fig. 19. — ^Tube for potato cul- ture. PREPAKATION OF NUTRIENT CULTURE-MEDIA 69 in flasks three-quarters of an hour for three successive days. Bread Mash. — Bread devoid of crust, dried in an oven, and then pulverized and mixed with water until thick, and steril- ized as above. Solid transparent media are prepared from materials which are transparent and which can readily be converted into liquids. Such are the gelatiu and agar culture-media. Gelatin. — Gelatin is obtained from bones and tendons, and consists chiefly of chondrin and gluten. Agar-agar. — ^This agent, which is of vegetable origin, derived from sea-plants gathered on the coasts of India and Japan, has many of the properties of gelatin, retaining its solidity at a much higher temperature; it becomes liquid at 90° C. and congeals again at 45° C. (gelatin will liquefy at 35° C), whereas 38° C. is the temperature at which most pathogenic germs grow best. Agar cultures can be kept in incubator for days and weeks without liquefying. Agar is not affected very much by the peptonizing action of the bacteria. The crude agar should first be rinsed in water, and then in 5 per cent, acetic acid and clear water again, to rid it of im- purities. If agar is boiled thoroughly over a hot flame or in an autoclave, it can be filtered much more readily. The main point is to see that all the agar is dissolved. Glycerin-agar. — The addition of 4 to 6 per cent, of gly- cerin to nutrient agar greatly enhances its value as a culture- medium. Gelatin-agar. — ^A mixture of 5 per cent, gelatin and 0.75 per cent, agar combines in it some of the virtues of both agents. Blood-serum. — Blood-serum, being rich in albumin, co- agulates very easily at 70° C., and if this temperature is not exceeded, a transparent sohd substance is obtained upon which the majority of bacteria develop, and some with preference. 70 ESSENTIALS OF BACTERIOLOGY PREPARATION OF NUTRIENT CULTURE-MEDIA (After the recommendations of the American Public Health Association) Materials. — ^All water used should be distilled. Fresh meat. Dried peptone, Witte brand. Best French gelatin, as free as possible from impurities. Best commercial agar in threads. Sugars, dextrose, lactose, and saccharose, all chemically pure. Glycerin, double distilled. AzoHtmin in place of litmus. All other materials as nearly as possible chemically pure. Sterilization. — Preferably in the autoclave and in small containers, at 120° C.,with 15 pounds pressure for fifteen miautes. The sterilizer should be hot before the medium is put in. Intermittent. — For gelatin or sugar media a high tempera- ture is not suitable. The media are placed in streaming steam for thirty minutes on three successive days. Reaction. — One-half per cent, solution phenolphthalein (S grams to i liter alcohol) is needed as an indicator. The reaction should be +1 per cent., i. e., i per cent, alkaline solution required to make it neutral. Method of Obtaining Reaction. — To 5 c.c. of medium add 45 c.c. water. BoU one minute. Add i c.c. solution phenolphthalein. If the mixture is not tinted pink, the medium is acid or neutral and requires the gradual addi- tion of I : 20 normal sodium hydroxid solution until a faint pink color remains. The soda should be added while the mixture is hot or boiling. Calculate from the amount of alkali used for the 5 c.c. how much will be needed for the whole quantity of media and add the same, using normal solution instead of i : 20 normal. Example: If 2 c.c. - NaOH will neutralize 5 c.c. media, 2 c.c. Y NaOH will neutralize 100 c.c, or 20 c.c. ~ NaOH will neutralize 1000 c.c. media. If the medium is very alkaline, hydrochloric acid must be added to reduce to + i per cent. NUTRIENT CULTURE-MEDIA 71 Nitrate Broth. — One gram peptone to one liter water and add 0.2 gm. nitrite free potassium nitrate; place ten c.c. in test-tube, sterilize in autoclave. Nutrient Broth. — i. Cover i pound (500 gm.) chopped meat with 1000 c.c. water and place in refrigerator twelve hours. 2. Strain through Canton-flannel or cheese-cloth and add water to make 1000 c.c. 3. Add I per cent, peptone, warming until dissolved. 4. Heat over water-bath thirty minutes. 5. Restore loss of water. 6. Titrate and adjust reaction to +1 per cent, by adding alkali or acid. (See above.) 7. Boil two minutes over free flame. 8. Restore loss of evaporation. 9. Filter through absorbent cotton and Canton-flannel and refilter until clear. 10. Titrate and record final reaction. If it varies 0.2 per cent, from standard, readjust. 11. Tube, using 10 c.c. in each tube. 12. Sterilize. The nutrient broth as above prepared is used as a basis for most of the other media. It is practically the same as was devised by Lofiier in the early days of bacteri- ology. Sugar Broths. — Prepared as the standard broth with the addition of i per cent, dextrose, lactose, or other sugar just before final sterilization. Nutrient Gelatin. — ^Ten per cent, gelatin is added with the peptone to the meat- water infusion. Warm gently at 60° C. until dissolved, then adjust reaction. Heat over steam-bath for forty minutes. Restore loss of evaporation, readjust reaction, and boil five minutes. Make up loss from evaporation and record final reaction. Filter, tube, and sterilize fifteen minutes in autoclave at 120° C. Place at once in ice- water until solid and store in ice-chest. Nutrient Agar. — Boil 10 to 15 gm. thread agar in 500 c.c. 72 ESSENTIALS OF BACTERIOLOGY water for half -hour or digest in autoclave fifteen minutes. Restore loss by evaporation and allow to cool to 60 c.c. To meat-water infusion (500 parts meat to 500 c.c. water) add 2 per cent, peptone, also 500 c.c. agar solu- tion. Titrate after boiling one minute, and adjust reaction to -|-i. Heat in steam-bath forty minutes, and proceed as with nutrient gelatin, i. e., restore loss, read- just reaction, and filter and refilter until clear. The filtering should be done while the solution is hot. Pour into tubes or plates, sterilize in au- toclave, and finally. slant the tubes so as to obtain a larger surface. (Most agar tubes are used for stroke cultures.) The addition of the white of an egg will often clear it up; if this avails not, refiltering several times and at- tention to the few points mentioned will produce a clear solution. Lactose Litmus Agar. — One per cent, lactose added to nutrient agar just before sterilization. Reaction neu- tral. One per cent, azolitmin (Kahlbaum) boiled five minutes and added either to the tube before final sterilization or, if media used in plates, added at the time of plating. Preparation of Nutrient Blood-serum. — If the slaughter of the animal can be supervised, it were best to have the site of the woiuid and the knife sterilized, and sterUe flasks (Fig. 20) at hand to receive the blood directly as it flows. The blood is placed on ice forty-eight hours, and the serum is drawn out with sterile pipets into test-tubes, avoid- ing shaking of the jar. These are placed obliquely in an oven where the temperatiu-e can be controlled and main- tamed. (See Fig. 21.) Coagulation of Blood-serum.— The tubes of blood-seriun -Flask to blood-se- NUTRIENT CULTUEE-MEDIA 73 having been placed in the thermostat, are kept at a temper- ature of 65° to 68° C. mi to. coagulation occurs; then removed and sterilized by fractional sterilization. Sterilization of Blood-serum. — ^The tubes are placed three to four days in incubator at 58° C, and those tubes which show any evidences of organic growth are discarded. If, now, at the end of a week, the serum remains sterile at Fig. 21. — ^Thermostat or inspissator for blood-serum. the ordinary temperature of the room, it can be used for experimental purposes. Perfectly prepared blood-serum is transparent, of a gelatin- like consistence, and straw color. It wUl not liquefy by heat, though bacteria can digest it. Water of condensation always forms, which prevents the drying of the serum. Short Method. — Blood-serum may be prepared in a shorter 74 ESSENTIALS OF BACTERIOLOGY way by coagulating the serum at a temperature short of boil- ing-point. Sterilization is completed in three days by expos- ing the tubes to a temperature of about 90° C. each day for five minutes. Tubes so prepared are opaque and white. Preservation of Blood-serum in Liquid State. — Kirchner advises the use of chloroform. To a quantity of serum in a well-stoppered flask a small amount of chloroform is added — ^^enough to form about a 2 mm. layer on the bot- tom. If the chloroform is not allowed to evaporate, the serum remains sterUe for a long time. When needed for use, test-tubes are filled and placed in a water- bath at 50° C. until all chlo- roform has been driven off (determined by absence of characteristic odor); the se- rum is then solidified and sterilized as in the ordinary way, or may be used in a fluid state. Htiman Blood-sertun. — Blood-serum derived from placenta, serum from ascitic fluid and ovarian cysts, is prepared in a similar manner to the above. Blood coagulum, suggested by the author, is the blood itself (not the serum only) coagulated in test-tubes. It is dark brown in color and allows some colonies of bacteria to be more visible. It requires less time to prepare, and is not so likely to become contaminated as when the serum is used. LoflBler's Blood-serum Mixture. — ^To 3 parts clear serum add i per cent, glucose, beef infusion, and prepare as above: tube. ■Incubator. NUTRIENT CULTURE-MEDIA 75 Hiss' Medium for Plating Agar IS gm- Gelatin 15 " Meat extract 5 " Sodium chlorid 5 " Dextrose 10 " Distilled water 1000 c.c. Digest agar in autoclave, then add the other ingredients, except dextrose, which is added to the cleared and filtered product. No neutralization is necessary. Tube in regular way. For tube cultures this medium is modified by using agar 5 gm. and gelatin 80 gm. in place of the quantities given above. A careful titration is made and the reaction adjusted to 1.5 per cent, acid by adding HCl. After filtration, dex- trose is added, then tubed and sterilized. Hesse's Medium for Typhoid Agar 5gm. Peptone 10 gm. Extract of beef 5 " Sodiiun chlorid 8.5 " Water 1000 c.c. Digest agar in 500 c.c. water, add the other ingredients dissolved in water. Mis and filter. Adjust reaction to i per cent, acid, tube, and sterilize in autoclave. Bile Salt Agar (MacConkey's) Sodium taur-ocholate 0.5 part Peptone 1.5 parts Lactose 3.5 " Agar 1.5 " Water q. s. loo.o " Agar and peptone dissolved first. Lactose and bUe salt added before tubing. Sterilize on three days intermittently. y6 ESSENTIALS OF BACTERIOLOGY (A) Conradi-Drigalski Medium Fresh meat 15°° g^- Water 2000 c.c. Mix and allow to stand twelve hours. Strain, boil one hour, and add — Peptone 20 gm. Nutrose 20 NaCl 10 " BoU one hour, filter, then add — Agar 60 gm. Boil one hour in autoclave or until agar is dissolved. Render weakly alkaline to litmus, filter, and boil one-half hour. (B) Litmus solution (Kahlbaum) 300 c.c. Lactose 3° S^- Boil fifteen minutes. Mix with solution A, and make slightly alkaline with soda solution. Then add 4 c.c. 10 per cent, soda carbonate solution (hot sterile) and 20 c.c. of sterile i : 1000 crystal violet solution (Hochst B). Lactose-bile {Jackson). — Sterilized undiluted ox-gall, 98 parts; or dry bile, 10 per cent, solution; peptone, i part; lactose, I part. M. Filled into fermentation tubes, 40 c.c. each, sterilized fractional method. Blood-agar. — Human or other blood is obtained direct from the body under strict aseptic conditions, and a few drops smeared over the surface of agar in tubes or platjs. These are then placed in the incubator for a few days, and the contaminated ones are rejected. This medium is used for influenza bacilli and gonococci. Eisner's Medium (for Typhoid) (Potassium lodid — Potato-gelatin). — Five hundred grams of peeled and washed potatoes are mashed and pressed through a fine cloth. The juice is allowed to settle, is filtered, and after one hour's cooking has added to it 10 per cent, gelatin; then NUTRIENT CULTURE-MEDIA 77 2yi. c.c. -TO- normal sodium hydroxid solution, and finally i per cent, potassium iodid. Endo Meditim {Fuchsin-LcKtose-Agar). — To looo c.c. agar add lactose, lo grams; fuchsin (saturated alcoholic solution), 2 c.c; solution sodium sulpMte (lo per cent.), 25 c.c; sterUize in steam, and make acid, o.i per cent. Peptone Water (Modified Dunham) {Mother Solution): Dry peptone (Witte) 100 parts Sodium chlorid 100 Potassium nitrate i part Sodium carbonate i " Distilled water (95) q. s. ad 1000 parts — M. When wanted for use, dilute ten times with water. Dunham's rosalic acid solution consists of the following: Peptone solution (Dunham) 100 c.c. 2 per cent, solution rosalic acid ; 0.5 gm. Alcohol (80 per cent.) 100 c.c. — M. To detect acids and alkalis. Dieudonne's Medium: A. Normal solution potassium hydrate, defibrinated ox- blood, equal parts. MLx, sterilize in autoclave. B. Nutrient agar (neutral). Mix 3 parts A with 7 parts B, and poiu: into Petri dishes; allow to stand forty-eight hours at room temperature before using. Milk Culture-medium. — The milk used should be fresh and should be placed on ice for eight to ten hours to allow the cream to rise; the skimmed milk is siphoned off iato flasks or tubes and sterilized for three successive days. Litmus is often added, or sterile i per cent, azolitmin solution. Fresh Egg Cultures (After Hueppe). — The eggs in the shell are carefully cleaned, washed with sublimate, and dried with cotton. The inoculation occurs through a very fine opening made in the shell with a hot platinum needle; after inoculation, the opening is covered with a piece of sterilized paper and collo- dion. y8 ESSENTIALS OF BACTERIOLOGY Boiled Eggs.— Eggs boiled, shell removed over small por- tion, and the coagulated albumen stroked with the material. Guinea-pig Bouillon.— The flesh of guinea-pigs, as well as that of other experiment animals, is used instead of beef in the preparation of bouillon, for the growth of special germs. The extracts of different organs have been added to the \arious media for experimentation. Wertheim's Medium for Gonococcus: Nutrient agar 2 parts Human blood-serum or hydrocele fluid I part Melt agar and cool to 45° C; then add serum. Tube on slant or pour in Petri plate. Glycerin or glucose can be added to enrich. Solution Dried Blood Albumin (King) : Blood albumin (commercial) 15 parts Glucose bouillon 85 Dissolve, tube, inspissate, and sterilize as for blood-serum. CHAPTER XI INOCULATION OF CULTURE-MEDIA Glass Slide Cultures. — Formerly the gelatin was poured on little glass slides, such as are used for microscopic purposes, and after it had become hard, inoculated in separate spots as with potatoes. Test-tube Cultures. — The gelatin, agar, or blood-serum having solidified in an oblique position is smeared on the surface with the material, and the growth occurs along the smear, or the medium is punctured with a stab of the plati- num rod containing the material, and the growth follows the line of thrust. The former is called a stroke or smear culture, the latter a stab or thrust culture. INOCULATION OF CULTURE-MEDIA 79 Streaked Surface Plating. — The surface of the medium, hardened in a Petri dish, is scratched by a needle containing the inoculating material, three or more streaks being made without obtaining fresh material, so that the growth along the streak or scratches will represent varying amounts of the substance to be tested. In removing the cotton plugs from the sterile tubes to carry out the inoculation the plugs should remain between the fingers in such a way that the part which comes in contact with the mouth of the tube will not touch anything (Fig. 23). It is well to pass the mouth of the tube and the cotton plugs through a flame, scorching the latter before reinserting Fig. 23. — Maimer of holding plugs. Sterilizing Needle. — Sterilize needles by passing through the flame before and after each inoculation; also sterilize the glass part, as it is liable to become infected. After the needle has been withdrawn, the plugs are rein- serted and the tubes labeled with the kind and date of culture. Plate Cultures. — Several tubes of the culture-medium are made Hquid by heating in water-bath, and then inocu- lated with the material as follows. A looped platinum needle is dipped into the material and then shaken in the tube of liquid media (gelatin, agar, etc.). This first tube is called original. From this three drops (taken with the looped platinum rod, Fig. 11, p. 45) are placed in a second tube, the rod being shaken somewhat in the 8o ESSENTIALS OF BACTERIOLOGY gelatin or agar; this is labeled first dilution (a colored pencil is useful for such markings). From the first dilution three drops are taken into a third tube, which becomes the second dilution. The plugs of cotton must be replaced after each inocula- tion, and while being held must be carefully protected from contamination. Glass Plating. — The larger the surface over which the nutrient medium is spread, the more isolated will the colonies be; window glass cut in rectangular plates 6x4 inches in size was formerly used, but now Petri dishes consisting of 2 circular glass or porcelain dishes, one fitting over the other as a cover, are universally employed (Fig. 24). They are sterilized, the softened and inoculated agar or gelatin is poured from the test-tube into the dish with as much speed Fig. 24. — Petri dish for making plate cultures. as possible, and the lid replaced, avoiding contamination from the air and surroundings. They are labeled or marked with pencil, and placed in the incubator or kept at room temperature for further development. This method is very useful for transportation, and does away with the cooling apparatus and moist chamber for- merly employed; the saucers can be viewed imder micro- scope similar to the glass plates, and have entirely super- seded them. Esmarch's Tubes or Rolled Cultures.— This method, especially used in the culture of anaerobic germs, consists in spreading the inoculated gelatin upon the inner walls of the test-tube in which it is contained and allowing it to congeal. INOCULATION OF CULTURE-MEDIA 8l /■~\ The colonies then develop upon the sides of the tube without the aid of other apparatus. The method is useful whenever a very quick and easy way is required. The rolling of the tube is done under ice-water or running water from the faucet. The tube is held a little slanting, so as to avoid getting too much gelatin around the cotton plug. The tubes can be placed directly under the microscope for further examination of the colonies. Animals as Culture-media. — It is almost impossible to separate certain organisms, such as the tubercle bacil- lus and pneumococcus, from mixed cultures by ordinary plate methods, and the plan of producing the disease in animals by inoculation, and then obtaining the organism in pure cul- ture, has to be employed. Pure Cultures by Boiling. — Spored organisms may be separated from others by boiling the mixture for a few minutes, when aU the non-spored forms will perish, and only the spores remain to germinate subsequently. Fermentation Tube. — For show- ing the presence of gas or fermenta- tion the Smith tube (Fig. 25) or some of its modifications must be used. The closed end and part of the bulb are filled with the glucose or dextrose bouillon and sterilized at low temperatures for three succes- sive days, then inoculated and placed in the incubator. Gas forms gradually, displacing the fluid in the closed end. Fig. 25. — Smith's fer- mentation tube. 82 ESSENTIALS Or BACTERIOLOGY CHAPTER XII CULTIVATION OF ANAEROBIC BACTERIA Special methods are necessary for the culture of the ana- erobic variety of bacteria in order to procure a space devoid of oxygen. Liborius's High Cultures.— The tube is filled about three-quarters full with gelatin, which is then steamed in a water-bath and allowed to cool to 40° C, when it is inoculated Fig. 26. — Liborius's method. Fig. 27. — Hesse's method of making anaerobic cultures (McFarland). by means of a long platinum rod with small loop, the move- ment being a rotary vertical one, and the rod going to the bottom of the tube. The gelatin is next quickly solidified under ice; very little air is present. The anaerobic germs wiU grow from the CULTIVATION OF ANAEROBIC BACTERIA 83 bottom upward, and any aerobins present will develop first on top, this method being one of isolation. From the anaerobic germ grown in the lower part a stab culture is made into another tube containing three-quarters gelatin, the material being obtained by breaking test-tube with the culture. (See Fig. 26.) Hesse's Method. — A stab-culture having been made with Fig. 28. — Frankel's method of making anaerobic cultmres (McFar- land). Fig. 29. — Buchner's method of making anaerobic cultures (Mc- Farland). anaerobic germs, gelatin in a semisolid condition is poured into the tube until it is full, thus displacing the air (Fig. 27). Esmarch's Method. — ^Having inoculated a tube, the gela- tin is rolled out on the walls of the tube, a "roU culture," and the rest of the interior is filled with gelatin, the tube being held in ice-water. The colonies develop upon the sides of the tube and can be examined microscopically. 84 ESSENTIALS OF BACTERIOLOGY Gases like Hydrogen to Replace the Oxygen. — Several arrangements for passing a stream of hydrogen through the culture: Frankel puts in the test-tube a rubber cork containing two glass tubes, one reaching to the bot- tom and connected with a hydrogen apparatus, the other very short, both bent at right angles. When the hydrogen has passed through from ten to thirty minutes, the short tube is annealed and then the one in connection with the hy- drogen bottle, and the gelatin rolled out upon the walls of the tube (Fig. 28). Use of Aerobic Bacteria to Remove the Oxygen. — Roux in- oculates an agar tube through a needle-thrust, after which semi- solid gelatin is poured in on top. When the gelatin has solidified, the surface is inoculated with a small quantity of Bacillus subtilis or some other aerobic germ. The subtilis does not allow the oxygen to pass by, appropriating it to itself. Buchner's Method.— The test- tube containing the culture is placed within a larger tube, the lower part of which contains an alkaline solution of pyrogallic acid. The tube is then closed with a rub- ber stopper (Fig. 29). Petri dishes, uncovered, are placed on a rack under a large beU-jar, into which hydrogen gas is conducted. Alkaline pyrogallic acid is placed in the upper -Wright's method for the cultivation of anaerobes. Botkin's Method.- CULTIVATION OF ANAEROBIC BACTERIA 85 and lower dishes to absorb what oxygen remains. The Novy jar (Fig. 31) is used instead of a bell-jar, and sealed after the oxygen is displaced by hydrogen gas. Wright's Method. — ^Applicable to both fluid and soUd media. After the test-tube is inoculated the plug, which must be of absorbent cotton, is cut off flush with the ex- tremity of the tube and pushed inward for a distance of i cm. It is then impregnated with i c.c. of a watery solution of pyrogaUic acid and i c.c. of 5 per cent, sodium hydroxid ■Novy's jars for anaerobic cultures. solution. A tightly fitting rubber stopper is inserted, and the tube is then ready for incubation (Fig. 30). Park's Method. — An Erlenmeyer flask containing the mediimi to be used is boiled in a water-bath from ten to fifteen minutes to drive off dissolved oxygen, quickly cooled, and inoculated. Hot melted paraffin is then poured into the flask, which forms a layer over the medium, and on congeal- ing, provides an air-tight seal which does not adhere to the glass so closely as to prevent the escape of any gases formed by the bacterial growth. Requirements for a Small Laboratory Incubator, with thermostat and thermometers. Hot-air oven. 86 ESSENTIALS OF BACTERIOLOGY Arnold steam sterilizer. Autoclave. Bunsen burners. Erlenmeyer or liter glass flasks, yi dozen. Test-tubes, loo. One looo c.c. measuring glass. One loo c.c. measuring glass. One 5 c.c. pipet. One I c.c. pipet. One accurate buret. One-half dozen 20 c.c. porcelain capsules. Glass stirring rods. Normal soda solution. Hydrochloric acid. Lactose, dextrose, glucose, and phenolphthalein. A selection of dry stains, especially fuchsin, methylene- blue, and eosin. Gram's solution. Phenol. Alcohol, methyl alcohol. Cover-glasses, slides. Canada balsam, cedar-oil, xylol. A small microtome and embedding material. Cotton-wool for plugs. Twenty-five or more Petri dishes. Four platinum needles in glass handles. One-half dozen fermentation tubes. One-half dozen tubes for potato culture. One Novy jar. One animal holder. Three wire boxes for holding tubes. Test-tube rack. The materials must include what is needed for making culture-media: agar, gelatin, peptone, beef -extract, chemic- ally pure salt. And to this there will be added from time to time such other apparatus and material as occasion demands. THE GROWTH AND APPEARANCES OF COLONIES 87 CHAPTER XIII THE GROWTH APTO APPEARANCES OF COLONIES Macroscopic. — Depending greatly upon the temperature, which should be about 65° F. (20° C.) for gelatin, and 40° C. for agar, the colonies ordinarily develop so as to be visible to the naked eye in two to foiu: days. Some require ten to four- teen days, and others grow rapidly, covering the third dilu- tion in thirty-six hours. The plate should be looked at each day. The colonies present various appearances from that of a Fig. 32. — Staphylococcus pyogenes aureus: colony two days old, seen upon an agar-agar plate (X40) (Helm). small dot, like a fly-speck, to that resembling a small leaf. Some are elevated, some depressed, and some, like cholera, cup-shaped — umbUicated. Then they are variously pigmented. Some liquefy gelatin speedily, others not at all. The appearances of a few are so characteristic as to be recognized at a glance. Some produce gas-bubbles. 88 ESSENTIALS OF BACTERIOLOGY Microscopic— Use a low-power lens, with the Abbe nearly shut out— that is the narrowest blender. The stage of the microscope should be of such size as to carry a Petri saucer easily upon it. The second dilution or third plate is usually made use o±— that one containing the colonies suf&ciently isolated. These isolated ones should be sought for, and their appear- ance well noticed. There may be two or three forms from the same germ, the difference due to the greater or less amount of oxygen that Fig. 33- — Microscopic appear- ances of colonies. Fig. 34. — Klatsch preparations. they have received, or the greater or less amount of space that they have had to develop in. The microscopic picture varies greatly; now it is like the gnarled roots of a tree, and now like bits of frosted glass; some bacteria have quite characteristic colonies (Fig. 32). Impression or "Klatsch" Preparations. — In order more thoroughly to study a certain colony and to make a permanent specimen of the same, we press a clean cover-glass upon the particular colony, and it adheres to the glass. It can then be stained or examined. The Germans give the name of "Klatsch" to such preparations. Fishing. — To obtain and examine the individual members THE GROWTH AND APPEARANCES OF COLONIES 89 of a particular colony the process of fishing, as it is called, is resorted to. The colony having been placed under the field of the micro- Fig. 35. — Types of growth in stab-cultures: A, Non-liquefying: i, Filiform (Bacillus coli); 2, beaded (Streptococcus pyogenes); 3, echinate (Bacterium acidi lactici); 4, viUous (Bacterium murisepticum) ; 5, arbor- escent (Bacillus mycoides). B, Liquefying: 6, Crateriform (Bacillus vulgare, twenty-four hours); 7, napiform (Bacillus subtihs, forty-eight hours); 8, infundibuhform (Bacillus prodigiosus) ; 9, saccate (Micro- sporon Finkleri); 10, stratiform (Psorospermum fluorescens) (Frost). scope, a long platinum needle, the point slightly bent, is passed between the lens and the plate so as to be visible through the microscope, then turned downward until the colony is seen to be disturbed, and the needle is dipped into 90 ESSENTIALS OF BACTERIOLOGY the colony. This procedure must be carefully done, lest a different colony be disturbed than the one looked at, and an unknown or unwanted germ obtained. After the needle has entered the particular colony, it is withdrawn, and the material thus obtained is further exam- ined by staining and animal experimentation. The bacteria / §- \ •I > Fig. 36. — ^Types of stroke cultures: i, Filiform (Bacillus coli); 2, echinulate (Bacteriiun acidi lactici); 3, beaded (Streptococcus pyogenes); 4, effuse (Bacillus vulgaris); s, arborescent (Bacillus mycoides) (Frost). are further cultivated by inoculating fresh gelatin or agar, making stab- and stroke cultures. It is necessary to transfer the bacteria to fresh media about every six weeks, as the products of growth and decay given off by the organisms destroy them. Stroke and stab test- tube cultures are more characteristic than plate cultures, as the types in Figs. 35 and 36 show. ANIMAL INOCULATION - QI CHAPTER XIV ANIMAL INOCULATION Used: (i) For obtaining pure cultures; (2) to determine virulence; (3) to regain virulence of an organism that has become exhausted in artificial media; (4) to furnish a suit- able culture-medium for bacteria that have so far failed to grow on other media. The smaller rodents and birds are the ones usually employed for inoculation, as rabbits, guinea-pigs, rats, mice, pigeons, and chickens. These are preferred, because easily affected by the various bacteria, readily obtained, and not expensive. Monkeys have been used in recent years in connection with syphilis and meningitis. The white mouse is very prohfic and easily kept, and is therefore a favorite animal for experiment. It lives well upon a Httle moistened bread. A small box, perforated with holes, is filled partly with sawdust, and in this ten to twelve mice can be kept. When the female becomes pregnant, she should be removed to a glass jar until the yoimg have opened their eyes, because the males, which have not been raised together, are apt to attack each other. Gviinea-pigs. — ^When guinea-pigs have plenty of light and air, they multiply rapidly. Therefore it is best to have them in some large stall or inclosure. They can be fed upon all sorts of vegetables and grasses, and require but httle atten- tion. Methods of Inoculation. — I. Inhalation. — Imitating the natural infection, either by loading an atmosphere with the germs in question or by administering them with a spray. II. Through skin or mucous membrane. III. With the food. Method of Cutaneous Inoculation. — ^The ear of a mouse is best stilted for this procedure. A small abrasion is made with the point of a lancet or needle, which has been dipped in the virus or material to be inoculated. The animal is then sepa- 92 • ESSENTIALS OF BACTERIOLOGY rated from the rest and placed in a glass jar, which is partly- filled with sawdust and covered with a piece of wire gauze. Subcutaneous. — The root of the tail of a mouse is used for this purpose. The hair around the root of the tail is clipped off, and with a pair of scissors a very small pocket is made in the subcutaneous connective tissue, not wounding the animal any more than is absolutely necessary, avoiding much blood. The inoculating material is placed upon a platinum needle and introduced into the pocket; solid bodies, with a forceps. To hold the mouse still while the operation is going on a little cone made of metal is used. The mouse just fits in here. There is a slit along the top in which the tail can be fastened, and thus the animal is secure and immobile. Variously designed animal-holders are on the market and used in laboratories. Intravenous Injections. — Rabbits are very easily in- jected through the veins. Mice are too small. The ear of the rabbit is usually taken. It is first washed with I : 2000 bichlorid, which not only disinfects, but also makes the vessels appear more distinct. The base of the ear is compressed to swell the veins. Then a h3?podermic syringe, which can be easily sterilized, is filled with the de- sired amount of virus, which is slowly injected into any one of the more prominent veins present (Fig. 37). Intraperitoneal Injection. — This is used with guinea- pigs chiefly. The abdominal wall is pinched up through its entire thickness, and the needle of the syringe thrust directly through, so that it appears on the other side, then the fold let go, the needle withdrawn just far enough so as to be within the cavity. Inoculation in the Eye.— The anterior chamber and the cornea are the two places used. The rabbit is fixed upon a board, the eyelids held apart and head held still by an assist- ant. A few drops of cocain having first been introduced in the eye, a small cut is made in the cornea. The material is passed through the opening with a small forceps, and with a few strokes of a spoon it is pushed in the anterior chamber. ANIMAL INOCULATION 93 For the cornea a few scratches made in the corneal tissue will suiSce ; the material is then gently rubbed in. Inoculation of the Cerebral Membranes. — ^The skin and aponeurosis cut through where the skull is the thinnest. Then the bone carefully trephined, and the dura exposed. In rabies inoculation, the syringe containing the hydrophobic virus pierces the dura and arachnoid, and the virus is dis- charged beneath the latter. Fig. 37. — Method of making an intravenous injection into a rabbit. Observe that the needle enters the posterior vein from the hairy sur- face. Intratracheal. — The bacteria can be introduced directly into the trachea, thus coming in contact with the lungs. Intraduodenal. — Cholera germs are injected into the in- testines after they have been exposed by carefully opening the abdomen. This is done in order to avoid the action of the gastric juice. Celloidin sacs of small size are sometimes used to intro- 94 ESSENTIALS OF BACTERIOLOGY duce living cultures of bacteria into the bodies of animals without their coming into direct contact with the tissues. Obtaining Material from Infected Animals. — ^The ani- mal should be skinned, or the hairs plucked out, before it is washed — at least the portion where the incision is to be made. Then the entire body is washed in sublimate. Two sets of instruments are required — one for coarser and one for finer work: the one sterilized in the flame; the other, to prevent being damaged, heated in a hot-air oven. The animal, the mouse, for example, is stretched upon a board, a naU or pin through each leg, and the head fixed with a pia through the nose. The skin is dissected away from the belly without exposing the intestines. Then the ribs, being laid bare, the sternum is lifted up, and the pericardiiun ex- posed. A platinum needle dipped into the heart after the pericardiinn has been slit will give sufficient material for starting a culture. If the other organs are to be examined, further dissection is made. If the intestines are first to be looked at, they should be laid bare first. In this manner material is obtained and the results of inoculation noted. Frequent sterilization of the instruments is desirable. Koch's Rules in Regard to Bacterial Cause of Disease. — Before a microbe can be said to be the cause of a disease, it must — First, be found in the tissue or secretions of the animal suf- fering from, or dead with, the disease. Second, it must be cultivated outside of the body on ar- tificial media. Third, a culture so obtained must produce the disease in question when it is introduced into the body of a healthy animal. Fourth, the same germ must then again be found in the animal so inoculated. BACTERINS (VACCINES) 95 CHAPTER XV BACTERINS (VACCINES) Bacterins are sterilized suspensions of bacteria in normal saline solution. The term vaccines or bacterial vaccines is frequently but erroneously used in place of bacterins, as the word vaccine relates to a cow or calf. Bacterins are used in the treatment of localized infections, and especially those of a chronic nature, and have been employed extensively to es- tabhsh immimity against infection. The best example of this is the immmiization of armies and inmates of institu- tions against tjrphoid fever. Preparation. — ^The organism is grown on the surface of the most appropriate medium, usually agar-agar, until an abundant growth is present. This ordinarily requires twenty-four hours. The growth is then washed from the medium with sterile normal saline solution, and collected in a small sterilized flask or bottle containing glass beads and shaken to break up clumps. A sterilized glass bulb, drawn to a point (a test-tube drawn out answers as well), is filled with the resulting emulsion, the end sealed in a flame, and the bvdb immersed in a water-bath at 60° C. for one hour. The neck of the bulb is then broken, and a few drops of the emul- sion sown on culture-media to determine the presence or ab- sence of living organisms. Standardization. — The number of bacteria in a cubic cen- timeter of the mixture is determined as follows: a portion of the emulsion is reser\'ed unheated, and at once mixed with an equal volume of blood by aspirating into a capillary tube any quantity, usually a column 2.5 cm. long, of the emulsion, followed by an equal volume of blood. The blood and emulsion are then mixed on a glass slide and thin smears are made. After air drying, the films are fixed with satur- ated solution of bichlorid of mercury and stained with car- bolthionin. 96 ESSENTIALS OF BACTERIOLOGY Counting. — Two crossed hairs are placed on the dia- phragm in the eye-piece of the microscope and the slide examined mider the oil-immersion lens. The number of cor- puscles and bacteria in a number of fields are counted until at least 200 red corpuscles have been enumerated. As the number of corpuscles per cubic centimeter is 5,000,000,000 by simple proportion, the number of bacteria per cubic centi- meter can be determined. For example, 200 red corpuscles and 150 bacteria are counted in the same fields. Then — 200 corpuscles : 150 bacteria : : 5,000,000,000 : x x=3, 750,000,0000 (Number of corpuscles is to number of bacteria as the total number of corpuscles in a cubic centimeter is to the quantity to be determined.) Any number of bacteria per cubic centimeter can then be obtained by simple dilution with sterile normal saline solu- tion. When the final dilution is made, 0.2 per cent, of tri- kresol is added as a preservative. PART II SPECIAL BACTERIOLOGY CHAPTER XVI SOME COMMON BACTERIA SLIGHTLY PATHOGENIC Bacterium Prodigiosum (Ehrenberg). — This bacillus, formerly called micrococcus, is very common, and was one of the first noticed, because of the brilliant red pigment it forms on cooked vegetables and starchy substances. "The bleeding host" miracles are said to have been due to it. Morphology. — Short rods, often in filaments, resembling cocci, ends slightly pointed, i /x in size; spores absent. Facultative anaerobic, that is, it can grow without air; but the pigment requires oxygen for its development. Flagella and motion present in young bouillon cultures. Absent in older and those grown on potato. Stain easily with ordinary watery stains, but not with Gram. Cultural Features. — Agar stroke: Growth limited to stroke; fihform, varying from a Hght pink to dark purple in color, due to pigment (prodigiosin) formed by the growing colonies. Odor of trimethylamin present. Media colored brown under- neath growth. On potato, growth of pigment appears best. At first rose red, then in a few days dark purple, with a gHsterdng, green- gold luster, resembling the dry fuchsin dye. Odor more pronounced. Gelatin Stab. — In six hours liquefaction begins on surface, and spreading downward; funnel shape; the hquid portion 7 97 98 ESSENTIALS OF BACTERIOLOGY containing small flakes of red pigment which settle at the bottom. Alilk coagulated in twenty-four hours. Agar Colonies. — Small red points in thirty-six hours, irregu- lar in outline. Granular in structure. Gelatin Colonies. — On the surface, round, granular, smooth edges which soon liquefy and have depression in center. The edges then become irregular. Biologic Features. — The characteristic red pigment is in- soluble in water, shghtly soluble in alcohol and ether; alka- lies turn it orange, acids, violet red. Light fades it. Gases of methylamin and ammonia are produced. Gas and acid produced in sugar solutions. Indol feeble. Temperature, 22°-25° C; higher temperatures interfere with pig- ment. Pathogenic for small animals. When injected intraperitoneally, 1-2 c.c. has proved fatal; causes intoxication. Proteids of the cul- tures poisonous. Cancer Remedy. — Used in Coley's treatment mixed with cultures of streptococci. Bacillus Mesentericus Vulgatus {Bacillus Vulgatus; Potato Bacillus of Flugge) (Fig. 38). Ori^iw.— Surface of the soil, on potatoes, and in milk. Form. — Small thick rods with roimded ends, often in pairs. Very motile; produces abundant spores. Cultures. — ^Rapid growth; stain with Gram. Agar Colonies. — Round, with transparent center at first, then becoming opaque. The border is cihated; httle pro- jections evenly arranged. Potato.~A white covering at first, which then changes to a rough brown skin; the skin can be detached in long threads. Temperature. — Spores at ordinary temperature. Fig. 38. — Colony of Bacillus mesentericus vulgatus. SOME COMMON BACTERIA SLIGHTLY PATHOGENIC 99 S pores. ^Are very resistant; are colored in the manner described in first part of the book for spores in general. Bacillus Megateritom (de Bary) (Fig. 39). — Origin. — Found on rotten cabbage and garden-soil. Form. — ^Large rods, four times as long as they are broad, 2.5 ju. Thick, rounded ends. Chains with ten or more mem- bers often formed; granular cell contents. Abundant spore formation; very slow movement. Growth. — Strongly aerobic; grows quickly and best at a temperature of 20° C. Fig. 39. — Bacillus megaterium, with spores. Plate Colonies. — Small, round, yellow points in the depth of the gelatin. Under microscope, irregular masses like B. subtHis. Stab-culture. — Funnel-shaped from above downward. Potato. — Thick growth with abundance of spores like B. suhtilis. Bacillus Ramosus. — Synonyms. — Bacillus mycoides (Fliigge) ; Wurzel or root bacillus. Origin. — In the upper layers of garden or farm grounds and in water. Form. — Short rods, with rounded ends, about three times as long as they are thick; often in long threads and chains. 100 ESSENTIALS OF BACTERIOLOGY Immotile. Stain. — Gram. Agar Stroke. — Gray soft mass, gnarled and twisted; feath- ery extensions spreading over entire surface. Gelatin Stab. — Arborescent and plumose-parallel projections on either side of the stab; a thick skin on surface with slow liquefaction (Fig. 40). Colonies. — Twisted threads, like a bundle of hair; opaque center ; the threads or branches divide endlessly, forming coils. Growth. — At ordinary temperature, with plentiful supply of air. Staining. — Spores stain readily with the ordinary spore stain. Bacterium Zopiii (Kurth) (1883). — Origin. — Intestines of a fowl. Form. — Short thick rods forming long threads coiled up, which finally break up into spores, which were once thought to be micrococci. Properties.- — Very motile; does not dissolve or liquefy gelatin. Produces putrefaction in albuminous media, with gas formation. Growth. — In thirty hours abundant growth; aerobic; grows best at 20° C. Agar Plates. — Small white points which form the center of a very fine netting. With high power this netting is found composed of bacilli in coils, like braids of hair. Excellent impress or "Klatsch" preparations are obtained from these colonies. Staining. — Ordinary dyes and Gram. Bacillus Subtilis (Hay Bacillus) {Ehienheig) .—Origin. — Hay infusions; found also in air, water, soil, feces, and putrefying liquids. Very common, often contaminates cul- tures. Form. — Short, thick rods, three times as long as broad; slight roundness of ends; seldom found singly; usually in Fig. 40. — Bacillus mycoides (Frost). SOME COMMON BACTERIA SLIGHTLY PATHOGENIC lOI long threads. Flagella are found on the ends. Spores of oval shape, strongly shining, very resistant. Very motUe; Gram stain. Growth. — Rapid; strongly aerobic. Plate. — ^Round, gray colonies with depressed white center. Under microscope the center yellow; the periphery Hke a WTeath, with tiny little rays projecting; very characteristic. Agar Stroke. — Soft, round, smooth edges; gray. Gelatin Stab. — Gray on surface, sinks in thirty-six hours, shallow crater, in which small white particles are floating; as gelatin softens a skin forms on surface. Potato. — Thick, dirty-white growth, spreading over sur- face; dull, raised edges, wavy. Properties Uke B. mdgatus. Pathogenic. — ^Has been found present in eyeball suppura- tions, especially panophthalmitis. Injected in guinea-pigs it causes toxemia and death. Has been found in acute conjunc- tivitis, and may at times produce it. Staining. — Rods, ordinary stain; spores, spore stain. It is easily obtained by covering finely cut hay with dis- tilled water, and boiling a quarter of an hour. Set aside forty-eight hours. A thick scum will show itself on the sur- face, composed of the subtUis bacilli, whose spores alone have survived the heat. Was formerly considered a non-virulent form of B. anthrax. Boas-Oppler Bacillus. — ^Also known as the Bacillus geniculatus. Owing to the faculty possessed by this organ- ism of growing in the presence of amounts of lactic acid suf- ficient to check the development of all other lactic-acid form- ers, it usually predominates in stomach-contents containing large amounts of this substance. The parent type is com- posed of short rods, but in the presence of considerable amoimts of lactic acid these change to a longer form, which occurs singly or in long chains. It is stained brown by Gram's iodin solution. The bacillus affords confirmatory evidence of the presence of a new-growth, like cancer of the stomach, though it may occur in benign conditions. I02 ESSENTIALS OF BACTERIOLOGY Bacillus Violaceus (Schrater). — Origin.— W&ter. Synonym. — B. iantkinum (Zopf). Form. — ^A slender rod with rounded ends, three times as long as it is broad, often in threads. Spores. Motile, flageUa. Stain.— 'With. Gram and ordinary dyes. Cultures. — Agar stroke, moist, glistening, raised, at first yellow, then violet, inky colored. On Potato. — ^Violet black, moist, abundant growth. Gelatin Stab. — ^Rapidly Hquefying funnel-shaped masses of pigment along the stab. Colonies. — ^Hairy outer zone with liquid center, and small masses of opaque blue pigment floating about. Biology. — Acid formed in sugar bouillon. No gas. A moderate amovuit of H2S and indol. Pigment formed is in- soluble in water, shghtly soluble in alcohol. Facultative anaerobe. Temperature. — 22°-2S° C. Microorganisms Found in Urine. — ^When freshly passed, urine of a normal state contains no bacteria. By contact with the air and the urinary passages exposed to air, a great number of yeasts, molds and bacteria soon accumulate in the fluid. Bacteria also enter urine through the blood and dur- ing its secretion. A number of bacteria have the property of converting urea into carbonate of ammonia. The urine should be centrifuged and the deposit then exam- ined. The drying and fixing must proceed very slowly, since otherwise crystals of salts will be precipitated and mar the specimen. B. coli are frequently present, especially in acid urine. Typhoid bacilU in 25 per cent, of patients aflected with ty- phoid fever. Micrococcus Urese (Pasteur and Van Tiegham). — Origin. — Decomposed urine and in the air. Form. — Cocci, diplococci, and streptococci. SOME COMMON BACTERIA SLIGHTLY PATHOGENIC IO3 Properties. — Decomposes urea into ammoniuin carbon- ate; does not liquefy gelatin. Growth. — Grows rapidly, needing oxygen; can remain sta- tionary below 0° C, growing again when a higher temperature is reached. Colonies on Plate. — On the surface like a drop of wax. Stah-cidtures. — ^Looks hke a very deUcate thread along the needle- thrust. Other bacteria are found in urine in various pathologic proc- esses, such as tubercle bacilli, typhoid bacilli, gonococci, and other pyogenic organisms. Spirilla. — A number of non-pathogenic spirUla have been described. Spirilliun Rubrum (Esmarch). — Origin. — ^Body of a mouse dead with septicemia. Form. — Spirals of variable length, long joints, flagella on each end; no spores. Properties. — Does not liquefy gelatin; very motile; pro- duces a wine-red pigment, which develops only in absence of oxygen. Growth. — Can grow with oxygen, but is then colorless; grows very slowly; ten to twelve days before any sign; grows best at 37° C. Gelatin Roll-cultures. — Small, round ; first gray, then wine- red colonies. Stab-cultures. — A red-colored growth along the whole Una; it is deepest below, getting paler as it approaches the surface. Sarcina. — Cocci in cubes or packets of colonies. A great number have been isolated, many producing very beautiful pigments. The majority of them found in the air. Sarcina Lutea (Schroter). — Origin. — ^Air. Form. — Very large cocci in pairs; tetrads and groups of tetrads. Properties. — ^Liquefies gelatin slowly; produces sulphur- yellow pigment. Growth. — :Slowly, at various temperatures ; strongly aerobic. Plates. — Small, round, yellow colonies. I04 ESSENTIALS OE BACTERIOLOGY Stab-cultures. — Grows more rapidly, the growth being nearly all on the surface, a few separated colonies following the needle-thrust for a short distance. Agar, a very beautiful yellow, along the stroked surface. Sarcina Aurantica. — Flava, rosea, and alba are some of the other varieties. Many are obtained from beer. Sarcina Ventriculi (Goodsir) (Fig. 41). — Origin. — Stom- ach of man and animals. Fig. 41.— Sarcina ventriculi from stomach-contents (X 53°) (Van Valzah and Nisbet). Form. — Colorless oval cocci, in groups of eight and packets of eight. Properties. —Does not liquefy gelatin; shows tne reaction of cellulose to iodin. Growth. — ^Rapid. At end of thirty-six hours, round, yellow colonies, from which colorless cocci and cubes are obtained. Habitat. — They are found in many diseases of the stomach, especially when dilatation exists. Also normally; increased when fermentation occurs. BACILLUS OF ANTHRAX lOS CHAPTER XVII BACILLUS OF ANTHRAX Bacillus Anthracis (Rayer and Davaine). — ^Rayer and Davaine, in 1850, first described this bacillus; but Pasteur, and la.ter Koch, gave it the importance it now has. Synonyms. — Bactericie du charhon (Fr.) ; Milzhrand bacil- lus (German) ; bacillus of splenic fever or malignant pustule. Origin. — In blood of anthrax-suffering animals. Fig. 42.- -Bacillus anthracis, stained to show the spores (X 1000) (Frankel and Pteiffer). Form. — Rods of variable length, largest of pathogenic or- ganisms 4 At to 10 /i in length, nearly the size of a human blood- corpuscle; broad, cup-shaped ends; in bouillon cultures long threads are formed, with large oval spores (Figs. 42, 43). Spores. — Single, large, very resistant. Dry heat, 140° C, in three hours; steam in five minutes; necessary to kill. Do io6 ESSENTIALS OF BACTEEIOLOGY not occur in the circulating blood, but develop after death or in artificial media at 30° C. / \ I Fig. 43.— Anthrax bacilli in human blood (fuchsin staining) (Zeiss one- twelfth oil-immersion; No. 4 ocular) (taken from Vierordt). Fig. 44.— Bacillus anthracis, impression preparation, edge of colony Zettnow prep. (Kolle and Wassermann). Liquefies gelatin; immotile. Growth.— Grows, rapidly, between 12° C. and 45° C, and BACILLUS OF ANTHRAX 107 requires plenty of oxygen, but may be classed as a facultative anaerobe; grows weU in aU media. Colonies develop in two days; white shiny spots, which appear under microscope as slightly yellowish, granular, twisted balls, Uke a ball of yam ; each separate string or hair, if looked at imder high power, being composed of bacteria in threads. (See Fig. 44.) -^=^.i Fig. 45- Fig. 46. Figs. 45, 46. — Stab-cultures of anthrax in gelatin. Agar Stroke. — Grayish-white, slightly wrinkled layer wilii irregular edges. '^ Gelatin Stab-cultures. — A white growth with thom-Kke processes along the needle-track (Uke an "inverted fir tree"). Later on, gelatin liquefied, and flaky masses at the bottom; (See Figs. 45, 46-) Io8 ESSENTIALS OF BACTERIOLOGY Potato. — A dry, creamy layer, and when placed in incu- bator, rich in spores. Staining.— Rta.di\y take the anilin dyes with the ordinary methods. To bring out the cup-shaped concave extremities, a very weak watery solution of methylene-blue is best. Gram positive. Spores are stained by the usual method. When several bacilh are joined together, the place of their joining looks like a spore, because of the hollowed ends. Double staining will differentiate the spores. (See Fig. 42.) Sections 0} tissue are stained according to the ordinary methods, taking Gram's method very nicely. Pathogenesis. — ^When mice are inoculated with anthrax material through a wound in the skin, they die in twenty- four hours from an active septicemia, the point of inocvila- tion remaining unchanged. On autopsy will be found: Peritoneum. — Covered with a gelatinous exudate. Spleen. — Very much swollen, dark red, and friable. Liver. — Parenchymatous degeneration. Blood. — Dark red. The bacilli are found wherever the capillaries are spread out, in the spleen, hver, intestinal villi, and glomeruli of kidney, and in the blood itself. Only when the capillaries burst are they found in the tubules of the kidney. Mode of Entrance. — ^The bacilli can be inhaled, and then a pneumonia is caused, the pulmonary cells containing the bacilli ; when the spores are inhaled, a general infection occurs. Feeding. — The cattle graze upon the meadows, where the blood of anthrax animals has flowed and become dried; the resistant spores contaminate the grass and so enter the ali- mentary tract; here they then cause the intestinal form of the disease, ulcerating through the villi. Cattle are also in- fected by wading in streams which tannery washings have contaminated. Local Infection. — In man usually only a local action occurs ; by reason of his occupation — woolsorter, cattle-driver, tanner, etc. — he handles the hides or wool of animals that BACILLUS OF ANTHRAX I09 have been infected, and through a scratch or sUght wound he becomes infected, and local gangrene and necrosis set in, but death follows in the severer forms from a general pyemia; there is severe edema of the tissues in and about the wound, and pulmonary edema. Wounds about the face and neck are more fatal. Pneumonia by inhalation and intestinal infection also occur in man. Woolsorter's disease is the pulmonary form caused by in- halation of spores from infected wool. Susceptibility of Animals. — Dogs, birds, and cold-blooded animals affected the least; white mice, sheep, and guinea-pigs quickly and surely. Products of Anthrax Bacilli. — A basic ptomain has not been foimd, but a toxalbumin or proteid, called anthraxin, has been obtained. A certain amoimt of acid is produced by the viru- lent form, alkali by the weak. Attenuation and Immunity. — Cultures left several days in an incubator at a temperature between 40° and 42° C. soon become innocuous, and when injected into animals protect them against the virulent form. The lymph obtained from lymph-sac of a frog destroys the virulence of anthrax baciUi and spores temporarily. Hankin obtained an alexin from the blood and spicen of rats, they being naturally immune. It destroyed the anthrax bacOli in vitro, and used by injection in susceptible animals, made them inumme. It is insoluble in alcohol or water. Protective Inoculation. — Animals have been rendered im- mtme in various ways — ^by inoculation of successive atten- uated cultures; also with sterilized cultures — that is, cul- tures containing no bacilli, and with cultures of other bacteria. Immune Serum. — ^That obtained from animals rendered immune by attenuated cultures contains protective substances which seem to have some antitoxic action. Habitat. — In the serum about the wound and in the blood anthrax bacilli are readily foimd. The bacillus has never been found free in nature. no ESSENTIALS OP BACTERIOLOGY CHAPTER XVIII BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS This very important bacillus was first described, demon- strated, and cultivated by Robert Koch, who made his in- vestigations pubHc before the Physiological Society of Berlin on the twenty-fourth of March, in the year 1882. Synonyms. — Mycobacterium tuberculosis. ^ - *• , Fig. 47. — Tubercle bacilli in sputum; carbolfuchsin and methylene-blue (Zeiss one-twelfth oil-immersion). Origin. — In various tuberculous products of man and other animals and in the dust containing the discharges. Form. — ^Very slender rods, sUghtly curved, 2 /x to 4 ju in length, about one-quarter the size of a red blood-corpuscle's diameter, their ends rounded, usually solitary, often, how- ever, lying in pairs in such a manner as to form an acute angle. Sometimes they are S-shaped. In colored prepara- BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS III tions little oval spaces are seen in the rod which resemble spores, but have none of the properties of spores. (See Figs. 47, 48.) Properties. — Does not possess motility. Growth. — Requires special media for its growth, and a tem- perature varying but slightly from 37.5° C. It grows slowly, developing first after ten days, reaching its maximum in three weeks. It is facultative anaerobic. On gelatin it does not form a growth. The media should be slightly acid; Fig. 48. — Giant-cell containing bacilli (from a photograph made by Dr. Wm. M. Gray). growth mostly on surface. Subcultures grow more rapidly than those direct from lesions. Colonies on Blood-serum. — Koch first used blood-serum for culture, and obtained thereon very good growths. Stroke cultures or test-tubes inoculated with small bits of tubercu- lar tissue are placed in a well-ventilated and slightly humid incubator at 37° C. for ten to fourteen days, when smaU glistening white points appear, which then coalesce to form a dry, white, scale-like growth. Under microscope, composed of many fine lines containing the tubercle bacillus. Glycerin-agar. — By adding 4 to 6 per cent, glycerin to 112 ESSENTIALS OF BACTERIOLOGY ordinary agar-peptone medium, Nocard and Roux obtained a culture-medium upon which tubercle bacilli grow much better than upon blood-serum, especially after once obtained in pure cultvire. Bits of tissue are placed on the siuface, not rubbed in until after several weeks; then gently crushed and spread over surface; this hastens growth. Stroke cultures are used as with blood-serum. They are placed in incubator after inoculation, and remain there about ten days, at a temperature of 37° C. The cotton plugs of the tubes are covered with rubber caps, the cotton first having been passed through the flame, and moistened with a few drops of sublimate solution. The rubber cap prevents the evaporation of the water of condensation, which always forms and keeps the culture from drying up. The growth which occurs resembles the rugae of the stom- ach, and sometimes looks like moistened crumbs of bread. The impression or "Klatsch" preparation shows under the microscope a thick, curled-up center around which threads are wound in all directions. And these fine lines show the bacilli in profusion. Potato. — It can be cultivated on slices of potato which are placed in air-tight test-tubes to which glycerin has been added. Bouillon. — Bouillon containing 4 per cent, glycerin is a very good medium. Growth on the surface only. Pure Cultures from Sputum. — Kitasato recommends the thorough washing, changing the water ten times, of the small masses found in the sputum of tuberculous persons. When such specimens are examined, they show tubercle bacilli alone, and when inoculated in agar, give rise to pure cultures. Animal Inoculation for Diagnosis. — When the bacilli are so few in number in sputum or urine as to make their detec- tion difficult, and also when doubt exists as to the identity of acid-fast bacilli found, several guinea-pigs should be in- jected in the groin and smears and sections made from the enlarged glands resulting. Varieties. — Branching and other aberrant forms are not rare, and the tendency now is to class the organism with the BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS II 3 "higher bacteria," mycobacteria, similar to actinomyces. Other acid-fast baciUi exhibit similar types, and it is possi- ble that the bacillary parasitic form is only one stage in the life-history of the organism. Little granules, arranged like streptococci, which take the characteristic stain, and look as if the protoplasm had been destroyed that inclosed them, are frequently found in sputum. Some believe these " splinters " to develop into regular bacilli in cultures. rM- f /- C' VViUi Tetragenus (Koch; Gaffky). — Origin. — • microbe in the cavity of a tuberculous Ivmg. studied its pathogenic actions and gave it bears. Form. — Cocci which are gathered in the tissues in groups of four, forming a square — a tetrad. (See Fig. 80.) In artificial culture sometimes found in pairs. A cap- sule of light, gelatinous consistence sur- rounds each tetrad. Properties. — ^They are immobile; do not Kquefy gelatin. Growth. — They grow well on all nutrient media at ordinary temperatiu-e; are facul- tative aerobic. They grow slowly. Colonies in gelatin plates. In two days, little white spots, which, when on the surface, form little elevations of a porce- lain-like appearance; under low power they are seen very finely granulated. Stab-culture.- — Small, round, separated colonies along the needle-track, and on the surface a button-like elevation — a form of "nail culture." (See Fig. 81.) Potato. — A thick, slimy layer which can be loosened in long shreds. Staining. — Colored with the ordinary anilin stains. Gram positive. Pathogenesis. — ^White mice and guinea- 1^'^' ^li^;- piss die in a few days of septicemia culture. Micrococ- '^ ° . . 1 . 1 , cus tetragenus. when mjected with the tetragenus cul- tures, and the micrococcus is then found in large numbers in the blood and viscera. Field-mice are immune. In the cavities of tuberculous limgs, in the sputum of phthisical and healthy patients, it is often found, but what action it has upon man has not yet been determined. PYOGENIC COCCI 171 Morax-Axenfeld Diplobacillus of Conjunctivitis. — This bacillus is foxmd in the greater number of cases of con- junctivitis. Form. — ^A short, plump bacillus, usually ia pairs and chains of pairs. Non-motile (Fig. 82). Growth. — ^With difficulty in blood-serum agar, it forms small pitted colonies or lacunae; liquefies. Staining. — ^Does not take Gram, but stains readily. Non-pathogenic for lower animals. Fig. 82. — ^Morax-Axenfeld diplobacillus from conjunctival exudate during coiurse of subacute conjunctivitis (obj. B. and L., one-twelfth oil- immersion) (Boston). Bacillus Pyocyaneus (Gessard). — Synonyms.— Bacillus fluorescens (Schroter) ; the bacillus of bluish-green pus. Origin. — Found in 1882 in green pus in pyemia. Has been found in water, in bandage material, in feces and street dust, in the mouth of healthy individuals, and in all suppurating conditions, especially in middle-ear discharge. Form. — Small slender rods with rounded ends, easily mis- taken for cocci. Often in groups of four and six, without spores. 172 ESSENTIALS OF BACTERIOLOGY Properties. — Very motile; liquefy gelatin rapidly; a pecu- liar sweetish odor and a blue pigment are produced in the cultures. GVoze)^A.— Develops readily at ordinary temperature, grow- ing quickly and mostly on the surface; it is aerobic. Agar plate: In two or three days a greenish iridescence appears over the whole plate. A bright green at first, causing fluorescence; then later a blue pigment in deeper portion. ■ ' -f^ y , '^^^^.' Fig. 83.— Bacillus pyocyaneus, from an agar-agar culture (X 1000) (Itzerott and Niemann). Gelatin Stab-cultures. — Mainly in upper strata, the lique- faction funnel shaped, the growth gradually settling at the bottom,- a rich green shimmer forming on the surface, and the gelatin having a deep fluorescence. Potato. — The potato is soaked mth the pigment, a deep fold of green occurring on the surface. Indol is produced. In ear abscesses pure cultures have been found. Bacillus fluorescens, found in water, is considered identical PYOGENIC COCCI 173 with Bacillus pyocyaneus and other fluorescent bacteria are believed to be varieties. Crystals develop on agar cultures in a short time. Staining. — With ordinary anilin dyes. Gram negative. Pathogenesis. — When animals are injected with fresh cul- tures in the peritoneal cavities or celliilar tissues, a rapidly spreading edema with general suppuration develops. The bacilli are found in the viscera and blood. If a small quantity is injected, a local suppuration occurs, Fig. 84.- -Koch- Weeks' bacillus from conjunctival exudate at third day of epidemic conjunctivitis (Boston). and if the animal does not die, it then can withstand large quantities. It is immune. The Pigment. — Pyocyanin. — When the pus, bandages, and dressings containing the Bacillus pyocyaneus are washed in chloroform, the pigment is dissolved and crystallizes from the chloroform in long needles. It is soluble in acidulated water, which is turned red thereby, and when neutralized, the blue color returns. It has no pathogenic action. It is an aromatic compound. The bacillus has no especial action on the wound, and is found sometimes in perspiration of healthy persons. 174 ESSENTIALS OF BACTERIOLOGY CHAPTER XXIV GONOCOCCUS.— MENINGOCOCCUS Micrococcus Gonorrhoeae (Gonococcus Neisser). — In 1879 Neisser demonstrated the presence of this germ in the secretion of specific urethritis. Form. — Cocci, somewhat triangular in form, found nearly always in pairs, the base of one coccus facing the base of the other and giving the appearance of a Vienna roll, hence the German name, Semmel (roll), form. Four to twelve such pairs are often found together. Immotile (Fig. 86). In pus usually within the cells. ^ Culture. — No growth on ordinary media ; on blood-serum or agar smeared with jT ■ y blood, cultures have been obtained. The ' ^^ temperature must be between 33° and 37° •■vf- ■ C., and the growth occurs very slowly and sparsely. Wertheim's medium {q. v., p. 78) has Fig. 85. given the best results. orrheal pus. Ani- Colonies. — Extremely delicate, translu- lin methy] -violet cent spots, separate, and of a slimy con- (X 050;. sistence, appearing in one to two days. Resistance. — The cultures live only a few days at room temperature, but in the ice-chest they last longer. A temperature of 45° C. destroys the gonococci and it is but slightly resistant to the ordinary chemic antiseptics. From the Blood. — In septicemic cases the gonococcus has been isolated from the blood direct by drawing 5 to 10 c.c. from a vein and adding it in equal parts of melted agar. The mixture is poured into Petri dishes and developed in the incubator at 37° C. Staining. — Colored easily with all ordinary anilin stains. Gram negative is one of its main diagnostic features. GONOCOCCDS. — MENINGOCOCCUS I7S The following method is recommended by Neisser: The cover-glasses, with some of the urethral discharge smeared upon them, are covered with a few drops of alcoholic solution of eosin, and heated for a few minutes over the flame. The excess of the dye is removed with filter-paper, then the cover-glass placed in concentrated methylene-blue (alcoholic solution) for fifteen seconds, and rinsed in water. The gonococci are colored dark blue, the protoplasm of the /" \' / Fig. 86. — Gonococcus in urethral pus (X looo) (Frankel and Pfeiffer). cell pink, and the nucleus a light blue, the gonococci lying in the protoplasm next to the nucleus (Fig. 85). Bacterial Diagnosis. — Other bacteria are similar to the gon- ococci in form; they are distinguished from the gonococcus in that they are positive with Gram's method. The points on which the diagnosis is to be made are the characteristic biscuit shape, the intracellular position of the organism, its failure to stain with Gram and very difficult to grow artificially on common media. 176 ESSENTIALS OF BACTERIOLOGY Pathogenesis. — The attempts to infect the experiment ani- mals with gonorrhea have so far been without success. In man, upon a healthy urethra a specific urethritis was pro- duced with even the twentieth generation of the culture. Gonorrheal ophthalmia contains the cocci in great niunbers, and endocarditis and gonorrheal rheumatism are said to be caused by the cocci. The micrococci have been found long after the acute attack, when only a very slight oozing remained, and the same were found very virulent. The specific inflammations of the generative organs of the female are due to this organism gaining entrance through the vagina. It is found chiefly in the superficial layers of the mucous membrane. Bacterins (Vaccines) . — A number of vaccines have been pre- pared in recent years for the treatment of gonorrhea and its complications. The bacterins are made as described under Bacterins. This method of treatment is still on trial. The best results have been obtained in gonorrheal rheumatism and epididymitis. Toxins. — ^True toxins not found, but the cells contain poi- sons that produce suppuration and death when injected into the mice and guinea-pigs. Allied Varieties. — ^A number of diplococci which resemble the gonococcus in form are foimd in the vaginal secretions and pus and may at times lead to a wrong diagnosis. The meningococcus is very sirmlar, but is easily cultivated and is not apt to be foimd in the same secretions as the gonococcus. Micrococcus citreus, albicans, and subflavus, described by Bumm, are all Gram positive and grow readily on gelatin and agar. The gonococcus is distinguished from all these similar micro- cocci by the tests enumerated above. These characteristics, taken in toto, form sufficient features for its ready recognition, and as it is often a serious question to decide, not so much because of the patient's health as because of his character, we should be very careful not to pronounce a GONOCOCCDS . — MENINGOCOCCUS 177 verdict until we have tested the micro-organism as above. When the germ is found which answers to the above descrip- tion, the process can be called gonorrhea without a doubt. Diplococcus Intracellularis Meningitidis (Weichsel- baum). — Synonyms. — Meningococcus; Micrococcus Meningi- tidis. Origin. — Found by Weichselbaum in epidemic cerebrospinal meningitis in 1887. Fig. 87. — Diplococcus intracellularis meningitidis in leukocytes. Cover-glass preparation from peritoneal exudate in a guinea-pig (X2000) (Wright and Brown). F'orm. — A small coccus occurring in pairs, flattened against each other, and contained within the leukocytes, resembling gonococcus. No capsule (Fig. 87). Properties. — Ferments sugars, with acid production. Growth. — Best on blood-agar, sertim-agar, and ascitic glu- cose-agar at body temperature; good growth in twenty-four hours. Sheep serum better mediimi. 178 ESSENTIALS OF BACTERIOLOGY Colonies. — Circular discs, whitisli, almost transparent, mar- gins, smootli. Stain. — ^With basic anilin. Gram negative. Jenner's blood-stain and Neisser stain best for spinal fluid specimens. Loffler's alkaUne methylene-blue a good stain. Resistance. — Organisms very perishable one to three days. Apparently destroyed by a self-elaborated ferment. Sim- light destroys in a few hours. Pathogenesis. — Causes epidemic cerebrospinal fever, prob- ably by infection through the nasopharynx; the organism is found in the spinal fluid and in other inflammatory exudates, and can be seen in fluid obtained by lumbar puncture. Ordinary laboratory animals immune, but Flexner has succeeded in inoculating monkeys. Agglutination. — On the fourth day; in dilution of i : 50 agglutination is had. By the use of large quantities of meningococci injected into a horse agglutinins, opsonins, and specific immune bodies (amboceptor) can be produced. Protective Serum. — Flexner has been able to obtain an anti- toxin from monkey serum that has therapeutic properties in man. Such an antiserum, when injected directly into the spinal canal, has a curative action, destroying the cocci. Bacterial Diagnosis. — By means of lumbar puncture the spinal fluid is obtained and allowed to settle. Smears made from sediment. Examined for bacteria. Gram-positive organisms are either pneumococci, strepto- cocci, or staphylococci. Meningococcus is Gram negative and within the leukocytes, and can be readily grown on blood-serum. If such an or- ganism is present, the disease is undoubtedly cerebrospinal meningitis. Bacillus of Soft Chancre, Chancroid (Ducrey-Unna, 1889). — ^A diplobacillus which is specific has been described by Ducrey as obtained from the secretion and in the depth and margins of the chancroid. Unna's bacillus is narrower and unbroken in the center (Fig. 88). GONOCOCCUS . — MENINGOCOCCUS 179 Cultivation. — Cultivation has occurred on blood-agar, the blood being added in the proportion of one to two. Colonies are small, round globules. Staining. — With borax, methylene-blue, decolorized with weak acetic acid. Pathogenesis. — Probably a mixed infection occurs in most Fig. 88. — Smear of pus of chancroid of penis (X 1500) (Davis) (photo- micrograph by Mr. L. S. Brown). chancroids, especially if buboes result. The bacillus of Ducrey is not found in unopened buboes, though often con- taminating the ulcerated ones. The disease has been reproduced by inoculation of the human subject. Laboratory animals are immune. l80 ESSENTIALS OF BACTERIOLOGY CHAPTER XXV ANAEROBIC BACTERIA (BACILLUS OF TETANUS; BACILLUS OF MALIGNANT EDEMA, ETC.) Similar in form and cultural requirements are a group of bacteria which are found as a result of injury or the infection of wounds. They vary greatly in the clinical symptoms produced. Bacillus of Tetanus (Nicolaier-Kitasato) . — Origin. — Nicolaier found this bacillus in the pus of a wound in one who had died of tetanus, describing it in 1884. Kitasato isolated and cultivated this germ (1889). Form. — A very slender rod. When the spores form, a small swelling occurs at the spore end, giving the bacUlus a driun-stick shape (Fig. 89). Properties. — Not very motUe, though distinctly so; lique- fies gelatin slowly. The cultures give rise to a foxil-smelling gas. Growth. — Develops very slowly, best at 36° to 38° C, and only when all oxygen is excluded — an obligatory anaerobin. In an atmosphere of hydrogen it flourishes. Colonies on gelatin plates in an atmosphere of hydrogen. Small colonies. After four days a thick center and radiating, wreath-like periphery, Hke the colonies of Bacillus subtilis. Pure cultures not easy to obtain (Fig. 90). High Stab-culture. — ^The gelatin having 2 per cent, glucose added and filling the tube. Along the lower portion of the needle-track, a thorn-like growth, little needle-like points shooting out from a straight line. The whole tube becomes clouded as the gelatin liquefies, and then the growth settles at the bottom of the tube (Fig. 91). Agar. — On agar, in the incubator, the growth is quite rapid, and at the end of forty-eight hours gas-bubbles have formed and the growth nearly reached the surface. ANAEROBIC BACTERIA l8l Bouillon. — Adding glucose to the bouillon gives a medium in which an abundant growth occurs. Stab-agar. — Inverted fir-tree appearance. Milk. — ^Acid reaction and slow coagulation. Inoculation of animals with suspected material may be necessary as preliminary step. Cultivation from Spores. — Kitasato, by exposing a portion of suspected material to a temperature of 80° C. for one hour, Fig. 8q. — Bacillus of tetanus with spores { X 1000) (Frankel and PfeifEer). killed off all the other bacteria, but the spores of tetanus escaped and these then vegetated. Staining. — All the ordinary stains, Gram's method also, the spores being colored in the usual way. Pathogenesis. — A small amount of the pure culture injected under the skin of experiment animals will cause, in two to three days, death from true tetenus, the tetanic condition starting from the point of infection. At the autopsy nothing characteristic or abnormal is found, and the bacilli have dis- l82 ESSENTIALS OF BACTERIOLOGY appeared, except near the point of entrance. This fact is explained as follows: Toxins. — Several toxic products have been obtained from the cultures, and they are produced in the body and give rise Fig. 90. — Bacillus tetani: cul- ture four days old in glucose-gela- tin (Frankel and Pfeiffer). Fig. 91. — Six days' culture of bacillus of tetanus in gelatin (deep stab) (Frankel and Pfeiffer). to the morbid symptoms. These have been isolated, and when injected singly, cause some of the tetanic symptoms. Tetanospasmin, the most important for man. ANAEROBIC BACTEEIA 1 83 Tetanolysin. — ^The blood and the urine contain the toxin and are fatal to animals. The virus enters the circulation, but does not remain in the tissues. The toxin is most virulent. It acts on the end- plates of the muscles, and then on the motor nerve-ceUs. The incubation period is from two to fourteen days after receipt of injury. The spores are very resistant to heat, drying, and chemicals. Burns and injuries from firearms, cartridges, powder, and fireworks, a common cause of tetanus. Immunity. — Kitasato, by inoculation of sterilized cultures, has caused immunity to the effects of virulent bacUU. An antitoxin obtained by Tizzoni and Cattani from the serum of animals made immune by sterilized cultures is used with curative effects in cases of tetanus ia man. It is a globuhn, but differs from the diphtheria antitoxin. By pre- cipitation with alcohol and drying in vacuo the antitoxin is obtained in a solid state. The aqueous solution is used for injection subcutaneously or subdurally through a trephine opening. Its injection into the spinal canal by lumbar punc- ture has also been recommended. Antitoxin is more beneficial in chronic cases than in acute. The dried antitoxin has been spread on the woimd with some curative action. The antitetanic serum, to be effective, must be given very, early and in large doses. Its greatest use is in preventing tetanus in wounds Uable to be infected. From 50 c.c. to loo c.c. of a biUion-imit serum should be given in divided doses; only sera with very high protective powers should be used. United States Government Unit for Tetanus Antitoxin. — "The inummity unit is ten times the least quantity of anti- tetanic serum necessary to save the Hfe of a 350-gram guinea- pig for ninety-sis hours against the official test dose of a standard toxin furnished by the Hygienic Laboratory at Washington." Habitat. — The bacillus is present in garden-earth, in man- ure, and it has been isolated even from mortar. 1 84 ESSENTIALS OF BACTERIOLOGY The earth of special districts seems to contain the bacilli in greater quantities. Spores of tetanus may gain access to animal sera, and if not properly destroyed, may produce tetanus during the use of these products. Previous testing for the tetanus bacU- lus should be made in the manufacture of all animal vac- cines, antitoxins, etc. Bacillus (Edematis Maligni (Koch, 1881) ; Vibrion Septique (Pastetir, 1875). — Synonym. — Bacillus (Edematis. Fig. 92. — Bacillus of malignant edema, from the body-juice of a guinea- pig inoculated with garden-earth (X 1000) (Frankel and Pfeiffer). Origin. — In garden-earth, foimd also in severe wounds in man when gangrene with edema had developed. Identical with the bacillus found in Pasteur's septicemia. Form. — Rods somewhat smaller than the anthrax bacillus, the ends roimded very sharply. Long threads are formed. Very large spores which cause the rods to become spindle shaped. Resembles in form and culture B. ckauvei (Fig. 92). ANAEROBIC BACTEEIA i8s rf. Properties. — ^Very motile; liquefies gelatin; gas is produced in cultures but very little in the body. Growth. — Grows rapidly, but only when the air is excluded, and best in incubator at 37° C. Roll Ctdtures {After Esmarch's Method). — Small, round colonies with fluid contents, under low power, a mass of motile threads in the center, and at the edges a wreath-like border. High Stai)-culture. — With glucose gelatin, the growth at first seen in the bottom of the tube, with a general liquefaction of the gelatin; gases de- velop and a somewhat unpleasant odor. Agar. — ^The gases develop more strongly in this medium, and the odor is more prominent. Guinea-pig Bouillon. — In an atmos- phere of hydrogen clouding of the en- tire culture-medium without any floc- culent precipitate until third day. Milk coagulated. Glucose media marked gas fermentation. Staining. — ^Are stained with the ordi- nary dyes, but Gram's method negative. Pathogenesis. — When experiment ani- mals, mice or guinea-pigs, are injected with a pure culture under the skin, they die in eight to fifteen hours, and the following picture presents itself at the autopsy : In guinea-pigs from the point of infection, spreading over a large area, an edema of the subcutaneous tissues and muscles, which are saturated with a clear red serous exudate, free from odor, and containing great quantities of baciUi. The spleen is enlargedj especially in mice. The bacilli are Fig. 93. — Bacillus of malignant edema growing in glucose- gelatin (Fraukel and Pfeiffer). l86 ESSENTIAXS OF BACTEEIOLOGY not found in the viscera, but are present in great numbers on the surface, i. e., in the serous coverings of the different organs; though when any length of time has elapsed between the death of the animal and the examination, they can be foxmd in the inner portions of the organs, for they grow well upon the dead body. In man they have been found in rapidly spreading gangrene foUowiag wounds. Habitat. — ^They are present in the soU, in putrefactions of various kinds, and in dirty water. Immunity. — ^Is produced by injection of the sterilized cul- tures, and also the filtered blood-serum of animals dead with the disease. Bacillus Aerogenes Capsulatus (Welch, 1891). — Synonym. — Bacillus Welchii; B. of Phlegmonous Emphysema (Frankel). Origin.- — The intestine of man and animals, soU, sewage, and water. Form. — A thick bacillus, 3 to 6 ^i in length, frequently capsulated. Properties. — Not motile, anaerobic, forms spores chiefly in cultures on blood-serum. Gram positive. Growth. — Best at 37° C. Gelatin. — ^Liquefied slowly or not at all. Bouillon. — Forms gas. Milh. — Coagulated and .becomes acid. Under anaerobic conditions. Potato. — Thin, gra3dsh-white growth with gas-production. Forms gas in abundance in dextrose, lactose, or saccharose media. Pathogenesis. — ^Is not usually pathogenic for rabbits and mice, though in guinea-pigs and birds it produces "gas phleg- mons." It is sometimes found in autopsies on human sub- jects, producing bubbles or cavities in the viscera (Schaum- organe), but this is probably due to postmortem migration of the germ from the intestine. It has been recovered from the blood during life, however, and is the most frequent cause of emphysematous gangrene. In man, infection of wounds, through dirt, with this bacUlus causes rapid emphy- ANAEROBIC BACTERIA 187 sema of the wound and a thin offensive discharge and fatal outcome. After death the bacillus develops rapidly and through the blood-vessels brings on general emphysema, with large accumulation of hydrogen gas in all the organs and subcutaneous tissue. Various foreign observers have de- scribed organisms having similar properties, and have given them such names as Bacillus perfringens, B. enteritidis sporo- genes, Granulobacillus immobUis, B. saccharobutyricus. Fig. 94. — Bacillus aerogenes capsulatus (from photograph by Professor Siraon Flexner). but they were probably dealing with the Bacillus aerogenes capsulatus. Bacillus Enteritidis Sporogenes (Klein, 1895).— Re- garded as identical with B. aerogenes capsulatus {q. v.). Bacillus Chauvei. — Synonyms. — Bacillus of Symptomatic Anthrax (BoUinger and Feser) ; Rauschbrand (German) ; Char- bon symptomatique (Arloing, Cornevin, and Thomas). Origin. — This bacillus, described in 1879, has been isolated, and by animal inoculation shown to be the cause of the "black-leg" or "quarter-evil" disease of cattle. l88 ESSENTIALS OF BACTERIOLOGY Form. — ^Large slender rods, which swell up at one end or in the middle for the spore (Fig. 95). Properties. — They are motile, and liquefy gelatin quite rapidly. A rancid odor is developed in the cultures. Cultures. — The growth occurs slowly, and only in an atmos- phere of hydrogen, being anaerobic; grows best at 38° C; under 15° C. no growth. Glucose-gelatin. — In a few days little round colonies develop. Fig. 95. — Bacilli of symptomatic anthrax, with spores ( X 1000) (Frankel and Pfeiffer). which, under low power, show hairy processes around a com- pact center. Stab-cultures in Full Test-tubes. — The first growth in the lower portion of the tube not very characteristic. Gases develop after a few days, and the gelatin becomes liquid. Agar at brood temperature, in twenty-four to forty-eight hours, an abundant growth with a sour odor and abundant gas-formation. ANAEROBIC BACTERIA 1 89 Staining. — Ordinary methods. Gram's method is nega- tive, but the spores can be colored by the regular double stain for spores. Sugar Media. — Gas production. Milk. — Rendered acid and coagulated. Variability. — Great variation in cultures. Toxin elaborated in fluid media fatal for rabbits when injected intravenously. Pathogenesis. — If a small amount of the culture be injected under the skin of a guinea-pig, in twenty hours a rise of tem- perature, pain at the site of injection, and a few hours later death, occur. At the autopsy, the tissues are found black- ened in color and soaked with a bloody, serous iiuid; in the connective tissue large collections of gas, but only in the neigh- borhood of the point of infection. The bacilli are found in great numbers in the serum, but only appear in the viscera some time after death, when spores have developed. The animals are usually infected through wounds on the extremities; the stalls or meadows having been soiled by the spore-containing blood of animals previously dead of the dis- ease. " Rauschbrand" is the German name; "Ckarbon symp- tomatique," the French, from the resemblance in its symp- toms to anthrax. Feeding experiments and infection from animal to animal negative. Dried virus inoculation practised by the United States Government as preventive. Immunity. — Rabbits, dogs, pigs, and fowl are immune by nature, but if the bacilli are placed in a 20 per cent, solution of lactic acid and the mixture injected, the disease develops in them. The lactic acid is supposed to destroy some of the natural resistance of the animal's cells. Immunity is produced by the injections of these weakened cultures, and also by some of the products which have been obtained from the cultures. 1 90 ESSENTIALS OF BACTERIOLOGY CHAPTER XXVI HEMORRHAGIC SEPTICEMIA GROUP Bacillus of Bubonic Plague (Yersin and Kitasato, 1894) . — Synonym. — Bacillus Pestis. — Bubonic plague or pest is an extremely infectious disease, more or less common in China and the East, and is believed to have its origin in man from rats and other rodents. It spreads with great rapidity. Fig. 96. — Bacillus pestis in smear from' rat's liver, showing bipolar staining (X 720) (Wherry). especially among those living under unsanitary conditions. The "Black Death" of the fourteenth century and the plague epidemics of the seventeenth century are said to have been the same disease. Nearly at the same time Yersin and Kitasato, working independently, discovered in the bubonic swellings and blood HEMORRHAGIC SEPTICEMIA GROUP IQI of affected persons a distinctive bacillus which has conformed to all the conditions necessary to make it the cause of the disease. Origin. — ^In the tissues and all the body-fluids and secre- tions of affected individuals. Form. — Short, thick rods with an indistinct capsule and rounded ends. Growing in chains in fluid media (Fig. 96) . Properties.— Immotile. Stains readily. No spores. Cul- tivated best in oxygen, but is facultative anaerobic. Stains stronger at the ends, producing bipolar appearance. Gela- tin not liquefied. Easily destroyed by sunlight and drying. Very resistant to cold. Growth. — ^Best at 30° C. ; aerobic. Gelatin. — ^At 22° C., in twenty-four hours, white, point- like colonies on the plates, with broad and flat surface, turn- ing gray and then brown. Milk not curdled; slightly acid. Stab. — Snow-white, spreading out on the surface to the edge, and fluorescent. Bouillon. — Granular precipitate, with clear fluid above. When covered with oil and kept at rest, filaments hang down from surface like stalactites. Agar and Blood-serum. — Glass-like colonies hke drops of dew at first, then growing larger with iridescent edges. Potato. — At 37° C. smaU white mass. Slow growth. No gas formation in glucose media. Staining readily with aU basic dyes. Gram negative. Capsule found in agar growths. Pathogenesis. — ^After subcutaneous injection in rats death follows in forty to sixty hours, with sjonptoms of severe toxe- mia and convulsions. The point of infection shows a local edema and inflammation of the Ijonphatics. AU the organs congested and surrounded by a bloody exudate. The charac- teristic bacilli in all the tissues and secretions. Nearly all the domestic animals are susceptible. Mosquitos and pigeons, however, are immune — flies are not; fleas are a very impor- tant element in the transmission, and the rat-flea may com- mimicate the disease to the rat from man or from the rat to 192 ESSENTIALS OF BACTERIOLOGY man. Infected ground squirrels are supposed to be a factor in spreading the disease. Animals protected from the flea may live near infected animals without danger. Direct in- fection by dust or other material seldom occurs. The sputum of patients having the pneumonic type is highly infectious. Close personal contact with the infected is a means of trans- mission. The main point of entrance is the skin. Fifty per cent, of wild rats immune and not easily affected. Products. — ^A toxin has been obtained and immunity has been effected; the serirni of immune animals has protective properties. The serum likewise shows agglutinating powers, and gives similar reactions to tjrphoid and cholera sera. Habitat. — Not foimd in water, but most likely spreads from the sou in damp and darkened areas. Rats become affected first, and then, through fleas, affect man and other animals. In man three forms of the disease are recognized according to the mode of infection and course of the disease — viz., bubonic, pulmonic, septicemic. Vaccines. — The vaccines of Haffkine and Terni and Bandi have been used extensively, and with some good results. Antitoxins. — ^The antitoxins of Yersin and of Lustig have been used, but without much result. Closely identified with Bacillus pestis is the group known as the hemorrhagic sep- ticemia bacteria Bacteria of Hemorrhagic Septicemia (Hueppe, 1886). — Under this heading Hueppe has gathered a number of bacteria very similar to the bacillus of chicken cholera, differ- ing from it and each other but very little. They have been described by various observers and found in different diseases. The bacteria of this group color themselves strongly at the poles, giving rise to the dumb-bell shape (Fig. 97). They do not take the Gram stain; they are without spores, and do not liquefy gelatin. They have been divided into three groups. Bacillus avi- septicus, as it appears in fowls; Bacillus hovisepticus , as it attacks cattle; Bacillus suisepticus, as it attacks swine. The prominent members of each group are: Bacillus of HEMOREHAGIC SEPTICEMIA GROUP I93 chicken cholera of Pasteur, bacillus of swine plague, and bacillus of cattle-plague or pleuropneumonia. Bacillus of Chicken Cholera (Perroncito, Pasteur, 1878). — Synonyms. — Micrococcus cholera gallinarum; Microbe en huit; avicidus bacillus; bacillus of fowl septicemia. Origin. — In 1879 Perroncito observed this coccus-like ba- cillus in diseases of chickens, and Pasteur, in 1880, isolated and reproduced the disease with the bacillus in question. Form. — At first it was thought to be a micrococcus, but it has been found to be a short rod, about twice as long as it is Fig. 97. — Bacillus of swine-plague (from photograph by E. A. de Schweinitz). broad, the ends slightly rounded. The center is very slightly influenced by the anilin colors, the poles easily, so that in stained specimens the bacUlus looks like a dumb-bell or a figure-of-8 (Microbe en huit) . Properties. — Does not possess self -movement; does not liquefy gelatin; no spores. Growth. — Occurs at ordinary temperature, requiring oxygen for development. It grows very slowly. 13 194 ESSENTIALS OF BACTERIOLOGY Gelatin Plates. — In the course of three days little round, white colonies, which seldom increase in size, having a rough border and very finely granulated. Stab-cultures. — ^A very delicate gray line along the needle- track, which does not become much larger. Agar Stroke Culture. — ^A moist, grayish-colored skin, more appreciable at brood-heat. Potato. — ^At 37° C, after several days, a very thin, trans- parent growth. Sugar Broth. — ^Acid fermentation, no gas. Indol is formed. Staining. — Methylene-blue gives the best picture. Gram's method is not applicable. As the bacillus is easily decol- orized, anOin-oil is used for dehydrat- ing tissue sections, instead of alcohol. Pathogenesis. — Feeding the fowls with the bacilli or injecting them under the skin will cause death in from twelve to twenty-four hours, the symptoms pre- Fig. 98.— Chick- ceding death being those of a severe en cholera in blood ,. (X 1000) (Frankel septicemia. and PfeiSer). The bacillus is then found in the blood and viscera and the intestinal discharges, the intestines presenting a hemorrhagic inflammation. Guinea-pigs and sheep are immune. Mice and rabbits are affected in the same manner as the fowls. Immunity. — Pasteur, by injecting different-aged cultures into fowls, produced in them only a local inflammation, and they were then immune. But as the strength of these cul- tures could not be estimated, many fowls died and the healthy ones were endangered from the intestinal excretions, which is the chief manner of infection naturally, the feces becoming mixed with the food. Bacillus of Erysipelas of Swine (Loffler, Schiitz). — Synonyms. — Schweinerotlauf bacillus (German); Rouget du Pore (French). Origin. — Found in the spleen of an erysipelatous swine by Loffler in 1885. HEMOREHAGIC SEPTICEMIA GROUP Ig5 Form. — One of the smallest forms of bacilli known; very- thin, seldom longer than i /i, looking at first like little needle- like crystals. Spores have not been foimd. Properties. — They are motile; do not liquefy gelatin. Growth at ordinary temperature very slowly, and the less oxygen, the better the growth. Gelatin Plate. — On third day little silver-gray specks, seen best with a dark background, coalescing after a whUe, pro- ducing a clouding of the entire plate. Stab-cultures. — In a few days a very light, silvery-like cloud- ing, which gradually involves the entire gelatin; held up against a dark object, it comes plainly into view. Staining. — ^AU ordinary dyes and Gram's method also. Tissue sections stained by Gram's method show the bacilli in the cells, capillaries, and arterioles in great numbers. Pathogenesis. — Swine, mice, rabbits, and pigeons are sus- ceptible; guinea-pigs and chickens, immune. When swine are infected through food or by injection, a torpidity develops with diarrhea and fever, and on the belly and breast red spots occur which coalesce, but do not give rise to any pain or swelling. The animal dies from exhaus- tion in twenty-four to forty-eight hours. In mice the lids are glued together with pus. At the autopsy the liver, spleen, and glands are enlarged and congested, little hemorrhages occurring in the intestinal mucous membrane and that of the stomach. BacUli are found in the blood and in all the viscera. One attack, if withstood, protects against succeeding ones. Immunity. — ^Has also been attained by injecting vaccines of two separate strengths. Bacillus Murisepticus (Koch) ; Mouse Septicemia. — Origin. — Found in the body of a mouse which had died from injection of putrid blood, and described by Koch in 1878. Form. — Differs in no particular from the bacillus of swine erysipelas, excepting that it is a very little shorter, making it the smallest known bacillus. Spores have been found, the cultures exactly similar to those of swine erysipelas. 196 ESSENTIALS OF BACTERIOLOGY The pathologic actions are also similar. Field-mice are immune, whereas for house and white mice the bacillus is fatal in two to three days. Micrococcus of Mai de Pis (Nocard). — Gangrenous mastitis of sheep. Origin. — In the milk and serum of a sheep sick with the "mal de pis." .,^ m ■'*-'.*H^- 4? ^i^ Fig. 99. — Bacillus of mouse septicemia, from the blood of a mouse ( X 1000) (Frankel and Pfeiffer). Form. — ^Very small cocci, seldom in chains. Properties. — ^Immotile; liquefying gelatin. Growth. — Growth occurs best between 20° and 37° C, is very rapid, and irrespective of oxygen. Plates of Gelatin. — ^White round colonies, some on the sur- PROTOZOA 197 face and some in the deeper strata, with low power, appearing brown, surrounded by a transparent areola. Stab-culture. — Very profuse along the needle-track, in the form of a cone after two days, the colonies having gathered at the apex. Potato. — A dirty gray, not very abundant, layer, somewhat viscid. Staining. — With ordinary methods; also Gram's method. Pathogenesis. — If a pure culture is injected into the mam- mary gland of sheep, a "mal de pis" is produced which causes the death of the animal in twenty-four to forty-eight hours. The breast is found edematous, likewise the thighs and perineum; the mammae very much enlarged, and at the nipples a blue-violet coloration. The spleen is small and black; other animals are less susceptible. In rabbits abscesses at the point of infection, but no general affection. CHAPTER XXVII PROTOZOA Protozoa are unicellular animal organisms, minute as bac- teria, and differing from bacteria in the methods of repro- duction. Their structure and functions are more complex, although the borderland is ill defined. A nucleus is usually present. Divisions. — There are four grand divisions of protozoa: (i) Sarcodina, containing 5500 species; (2) mastigophora, containing 500 species; (3) infusoria, containing 700 species; (4) sporozoa, containing 300 species. Sarcodina are chiefly marine forms, with processes change- able in shape. Examples: Ameba, foramnifera, entameba, parasitic for man. Mastigophora have undulating flagella and are known as flagellates; to this division the trypanosojnata belong. Example: Trypanosoma. 198 ESSENTIALS OF BACTERIOLOGY Infusoria have fine ciliary processes or numerous delicate flagella. Example: Balantidium. Sporozoa have no motile organs, and are reproduced by spores. To this division belong the coccidia of malaria and the organisms discovered by MaUory in scarlatina. Examples: Plasmodium, coccidium. Life-cycle. — The complete cycle of reproduction has been observed in only one of the pathogenic protozoa, namely, the protozoa of malaria. Methods of Cultivation. — Novy, Clegg and others have obtained pure cultures of protozoa by the use of blood-agar and animal tissue, or by cultivation with bacteria, on which the ameba and other protozoa live. Entamoeba Histolytica (Shaudinn, 1903). — ^Amoeba Dysenteriae. — Foimd in the intestinal ulcers, feces, and secondary liver abscesses in certain cases of dysentery. Kartulis, in 1886, definitely established the cause, although amebae were noted in feces by Lambl in i860. A non- pathogenic form. Amoeba coli, also occurs. The Amoeba dys- enteriae is a imicellular animal organism, measuring 25 to 35 n in diameter, though larger and smaller forms occur. A nu- cleus and a nucleolus are present; the protoplasm of the cell- body is vacuolated, and often contains red blood-ceUs and bacteria. In fresh, warm stools active ameboid motion may be observed. The non-pathogenic form is smaller and never contains red blood-cells. Examination for Ameba. — From the slimy part of the fresh feces a loopful is taken and diluted with salt solution and examined with moderate power on a warm stage. Look for contracting vacuole and motion. Staining with hematoxylin eosin or eosin methylene-blue after the film on a glass slide or cover-glass has been fixed in hot alcohol or methyl alcohol. Cultures. — On nutrient agar a loopful of feces is spread and examined from day to day, transplanting the young amoebae with their accompanying bacteria. Pathogenesis. — Inoculation experiments with monkeys and PROTOZOA 199 dogs produce dysentery and liver abscess. In man, 50 per cent, of human beings harbor non-pathogenic amebae, but the pathogenic variety is found mainly in tropical countries, where it produces serious lesions and often occurs in wide- spread epidemics. Source. — ^It is supposed to come from poor water supplies. Amebic dysentery difiers from the bacillary form in that no severe toxic symptoms are present and the amebic disease is more chronic. The Shiga bacillus, B. dysenteric, is found in the bacillary form of dysentery. Life Cycle of the Malarial Sporozoa. — ^According to its situation, the parasite exhibits two distinct phases of exist- ence : in the human blood it passes through an asexual repro- ductive cycle, known as schizogony, while in the body of the mosquito it undergoes an entirely different series of sexually reproductive changes, called sporogony. I. The AsexuM Cycle in Man. — An infected mosquito con- veys the parasites into the blood of man as minute hyaline bodies which enter the blood-cells. At first they are small, roimd, colorless bodies, exhibiting more or less active ameboid motion in the fresh blood. Sometimes, particularly in the estivo-autumnal form, a riag shape is assumed. Their size gradually increases and pigment-granules appear, whUe in stained specimens a nucleus containing chromatin granules is visible. As the parasite approaches maturity the chroma- tin becomes scattered, and finally the protoplasm or mother- cell, known as sporocyte, divides into six to twenty spores, daughter-cells or merozoites, each containing a portion of the chromatin. The number of spores formed and their arrange- ment before segmentation takes place differ in the three varieties and will be noted below. The spores burst through the envelop of the red corpuscle and become free in the blood, but speedily enter fresh corpuscles and pass through the same series of changes. The febrile stage is synchronous with sporulation and liberation of the young forms. Certain of the parasites do not, however, go on to segmenta- tion, but after reaching maturity, remain quiescent and form 2CX) ESSENTIALS OF BACTERIOLOGY the so-called gametes or sexual types. In the tertian and quartan varieties these are not very different from the mature A o Or© Human Phase- ■^ TMt EMDOOeHOUS p,0^ ASEXUAL, CyCLE., THE MOSQgiTO PHA-SE EXOQENOUS OR Sexual. cycuE— Fig. loo. — Schema showing the human and mosquito cycles of the malarial parasite: A, Normal red cell; B, C, D, E, red cells containing amebulas or myxopods; F, G, H, sporocytes; J', K', L', M', microgame- tocytes or male gametes; J", K", L", M", O, macrogametocytes, or female gametes; N', M', microgametes; P, traveling vermicule; Q, young zygote; R, S, zygotomeres; T, blastophore; U, mature zygote (modified from Blanchard's diagram illustrating life-cycle of Coccidium schubergi) (Rees, in "Practitioner," March, igoi). organisms, but the estivo-autumnal gametes are crescentic in shape and very. characteristic. PROTOZOA 20I 2. The^iiigtUial Cycle in the Mosquito. — The common mos- quito is known as Culex and does not harbor the malarial parasite. The anopheles species, spotted wings, is the true host; only the females are bloodsuckers and responsible for the spread of the disease. They take the infected blood containing the male element and which represents the male fertilizing element imicrogametes). These become detached, and, entering a female gamete {macro gamete), a true sexual fertilizing process takes place. In the alimentary canal of the mosquito these fertilized cells penetrate the stomach- walls and form cysts (oocysts) filled with a large number of filiform spores (sporozoites), which are extruded into the body cavity of the insect, and some of which reach the salivary glands, whence they are ejected when the mosqmto bites. This cycle of development takes seven or eight days. Three Forms of Malarial Protozoa. — i. Plasmodium Vivax, or The Tertian Form. — The adult forms are large, not very refractUe, and their outline is somewhat indistinct. There is an abundance of fine pigment-granules, and the ameboid motion is vigorous. Segmenting forms divide into fifteen to twenty merozoites ; the sexual forms or gametes are large. The red cell containing the organism is swollen and pale. Sporulation and, therefore, the malarial paroxysm occur every forty-eight hours. 2. Plasmodium MalaricE, the Quartan Form. — The organism is smaller, is more refractile, and its outline is more distinct. The pigment is coarse and situated at the periphery of the organism, while the protoplasmic motion is sluggish. Seg- mentation forms only six to twelve spores, and has the regular "daisy-head" appearance; the gametes are small. The red cells become dark in color, and the cycle requires seventy- two hours. 3. Plasmodium Falciparum, or Malignant Tertian, or Estivo- autumnal Form. — The adult forms are found mainly in the spleen and other viscera, and do not very often occur in the peripheral blood; their outline is sharp, and they are highly refractile. The pigment is scanty and fine; the motion is 202 ESSENTIALS OF BACTERIOLOGY J \ 4 >'- "•'5' r :\\ i'Ufi a 10 ./■ < -vJ // U J5 14 D ^/J'' 15 16 %5^ 7) XP-i& /f /5 i>0 i>/ i"-? a? ^# i>5 ">■ Fig. loi. PROTOZOA ^03 active. A variable niunber of merozoites is formed — usually six to twelve. The gametes are characteristic, beiog cres- centic in shape and very resistant to quinin. The red cell becomes shriveled and yellowish. The cycle usually takes forty-eight hours, though it is somewhat variable. Mixed infections with the different organisms or with two or more broods of the same organism may occur, so that quotidian and irregular paroxysms may be produced. Transmission. — Malaria is spread by means of a mosquito, the anopheles, in whose body the protozoon undergoes its highest development. Man is the intermediate host; the mosquito, the true host. Methods of Examination for Malarial Organisms. — I. Fresh preparations are made by placing a small drop of blood on a slide and a cover-glass over it, so that only a thin film is formed. A ring of vaselin is smeared over the edges of the cover-glass to prevent evaporation. This is the best method for studying flagellation and fertilization, but is less satisfactory for routine clinical work than — 2. Stained Smears. — ^These are made by spreading a drop of blood in a thin film over one slide with the edge of another, drying in the air, and staining. Many stains have been devised for the malarial organism, but Jenner's or Wright's is sufficient for ordinary use: (i) Jenner's Stain. — This is excellent for routine work, as no preparatory fixation is required. The smears are dropped into this stain for one to three minutes, without previous fixation, and at once rinsed in distOled water. The malarial parasites are stained blue, the cell-bodies a reddish brown. Fig. 10 1. — Various forms of malarial parasites (Thayer and Hewet- son): i-io inclusive, tertian organisms; 11-17 inclusive, quartan organ- isms; 18-27 inclusive, estivo-autumnal organisms. I, Young hyaline form; 2, hyaUne form with beginning pigmenta- tion; 3, pigmented form; 4, full-grown pigmented form; 5, 6, 7, 8, seg- menting forms; g, mature pigmented form; 10, flagellate form. II, Young hyaline form; 12, 13, pigmented forms; 14, fully devel- oped form; 15, 16, segmenting forms; 17, flagellate form. 18, 19, 20, Ring-like and cross-like hyaline forms; 21, 22, pigmented forms; 23, 24, segmenting forms; 25, 26, 27, crescents. 204 ESSENTIALS OF BACTERIOLOGY (2) Wright's Chromatin Stain. — This is the best of the chromatin stains. For its preparation, which is quite com- plicated, see Wright, Journal of Medical Research, vol. vii, 1902. It can be purchased already made. It is used as follows: 1. The stain is poured over the film and allowed to remain for one minute to secure fixation. 2. Add distUled water drop by drop until a metallic scum is formed on the surface. The staining now takes place and requires two to three minutes. Wash in distilled water until Fig. 102. — Pure culture of trypanosomes of mosquitos — Crithidia fasciculata. Multiplication reset showing large and small cells. Nine- day culture (Gen. i X 1500) (Novy, MacNeal, and Torrey). a pinkish tint appears in the thin portions of the smear. The body of the malarial parasite is stained blue, and its chromatin a lilac to red color. The red cells are orange-pink. If possible, examinations for malarial organisms should always be made before quinin is administered. Trypanosomata. — Trypanosomes are flagellate protozoa found in the blood of various animals, and causing a number of diseases, such as surra, dourine, and nagana, affecting horses and cattle, especially in tropical countries, and causing PROTOZOA 205 the sleeping sickness of Africa, which is very fatal for human beings. About 60 species have been described, and 10 dis- eases are believed to be due to this form of organism. Morphology. — A fusiform mass, containing at one end a flagellum (Fig. 103). In the living state these protozoa are very motile. In the stained specimen chromatin granules are found and two or more nuclei. From the smaller nucleus arises the undulatory membrane, which passes into the flagellum and assists in the wave-like motion. Fig. 103. — Pure culture of trypanosomes of mosquitos — Crithidia fasciculata. Part of roset of elongated crithidia witli flagella directed centrally (Gen. 39 X 1500) (Novy, MacNeal, and Torrey). In the body fluids division occurs, first of the nucleus and then of the protoplasm. Cultivation. — Novy and MacNeal have succeeded in culti- vating these protozoa on blood-agar, and multiplication goes on rapidly, so that rosettes are formed with the flagella ar- ranged around a common center. (See Figs. 102, 103, 104.) Trypanosoma Lewisi (Kent, 1878). — Found in rats by Lewis; not fatal to them, though often equaling the red cor- puscles in number. It was one of the first of this group to 2o6 ESSENTIALS OF BACTEKIOLOGY be described. The infection continues for two months with- out producing any illness, and the animal is then immune. Injection of infected rat blood into healthy rat causes the latter to become infected. The injection of serum from an immune rat will prevent the disease in normal rats. Cultivated best at 20° C. and is very resistant to cold. The rat is probably infected by the bite of a flea or louse. (See Fig. 105.) Fig. 104. — Pure culture of trypanosomes of mosquitos — Crithidia fasciculata. Elongated crithidia from same preparation as preceding (Novy, MacNeal, and Torrey). Trypanosoma Brucei (Plimmer and Bradford, 1894) causes nagana, or tsetse-fly disease, a disease affecting horses, cattle, and dogs in certain regions of South Africa. The trjTpanosome of Bruce is less motUe than that of Lewis. It has been cultivated at 25° C, and is less resistant to cold. All laboratory animals subject to infection. The rat dies in ten days. In the natural infection Bruce discovered that the tsetse- fly transmitted the disease, but that it did so by first biting some animal whose blood contained the trjrpanosome. The PROTOZOA 207 blood of infected animals contains the organism, and can, if injected, produce the disease without the agency of the fly. So far the tsetse-fly alone is responsible for the spread of the infection. Sleeping Sickness. — ^Trypanosoma Ugandense Gam- biense (Button, 1904).— (T. Castellani, T. Hominis, T. Neprevi.) — Sleeping sickness, or human trypanosomiasis, is a disease peculiar to some parts of Africa. It is accompanied by periods of fever, anemia, and, finally, a lethargy deepening Fig. 105. — Trypanosome from blood of gray rat; stained with a 2 per cent, aqueous solution of methylene-blue (Boston). into coma and death. The disease may be rapid, and it may last with recurrences for many yeirs. ' Tj-ypanosomes identical with those found in nagana disease have been found in the blood of infected persons, and described by various observers, and given different names. Monkeys, when inoculated with cerebrospinal fluid from affected persons, develop a similar disease, and the parasites are found in the blood. So far the organism has not been cultivated. 2o8 ESSENTIALS OF BACTERIOLOGY A blood-sucking fly, known as the Glossina palpalis, is con- sidered the means of infection. The fly is closely related to the Glossina morsitans, or tsetse fly. The sleeping sickness in man is most likely the same thing as the nagana of cattle. Methods of Examinations. — From Blood. — A patient search may fail to detect the organisms — a large amount of blood, 10 c.c, obtained by venesection — is centrifuged and the white cells examined in hanging drop or stained smear. Cerebrospinal fluid will at times give results. Animal Inoculation. — The blood of suspected person in- jected into monkeys or rats and the resulting infection stu- died by above methods. Staining. — The organism is best stained by Giemsa stain or the Romanowsky method. Trypanosoma Evansi (Steel, 1880). — Pathogenic for all animals. Discovered by Evans in the blood of horses suffering from surra, a disease prevalent in India and the Philippine Islands. The disease resembles nagana. T. equiperdum and T. Rougetii are names given to similar organisms found in do urine, a disease affecting horses in southern France and Spain. Trypanosomes are found in fish, oysters, birds, and frogs, and many varieties have been described. Herpetomonas (Leishman, 1903) {Leishman-Donovan Bodies). — A disease called variously kala-azar, dum-dum fever, tropical splenomegaly, is considered to be due to an or- ganism somewhat related to the trypanosomes. Smears are stained after fixation by the Wright or Roman- owsky stains. Cultivation has succeeded on blood-media made acid with citric acid. The bedl^ug is considered instrumental in transmitting the organism. Piroplasma Bovis (P. Bigeminum) (T. H. Smith, 1893). — Origin.— In the blood of ariimals suffering from Texas cattle-fever. Form. — ^A pear-shaped protozoon, found in pairs in the red THE MICRO-ORGANISM OP SYPHILIS AND ALLIED ORGANISMS 209 cells of the blood, the smaller ends of pear in opposition; coarse ameboid movement. Transmission. — ^An insect or tick (Boophilus bovis) be- comes infected, and by its bite infects other animals. Other similar sporozoa have been found in animal diseases and in man in Rocky mountain fever. The P- hominis has been described, but not definitely determined. Rabies or Hydrophobia. — Negri Bodies (Negri, 1903). — Origin. — Found in the nervous system of animals dying of rabies (hydrophobia). Form. — ^Round and oval, hyaline bodies, with a sharp out- line and containing a nucleolus. The plasma is slightly granular. They are regarded as protozoa. Staining. — ^A smear from brain tissue is made on a cover- glass and fixed in methyl-alcohol for five minutes; then stained by Giemsa; stain for half -hour to three hours. All mammals susceptible; man chiefly from bite of dog. Only a small percentage of persons bitten by rabid dog be- come infected — 16 per cent. The virus resides in the saliva, and also in the central nervous system. The Pasteur preventive is an accepted fact, and depends for its power on a form of active immxmization. The virus used is obtained from dried spinal cord of infected rabbits, gradually increasing the virulence, older cords first used and then cords exposed to drying for lesser time. CHAPTER XXVIII THE MICRO-ORGANISM OF SYPHILIS AND ALLIED ORGANISMS Spirochaeta Pallida (Schaudinn, 1905). — Spironema Pallidum; Treponemal Pallidum. — Found in hereditary syph- Uis in all organs, in chancre, and lymphatic glands, and in secondary lesions, mucous patches, in the internal organs, 14 210 ESSENTIALS OE BACTERIOLOGY and likewise in the tertiary lesions, the very latest being the brain, and cerebrospinal fluid in cases of general paralysis, and establishing the identity of this disease with cerebral syphilis. Form. — A minute, spiral-shaped organism, with 6 to 20 curves, ends tapering. Actively motile in fresh specimen (Fig. 106), intracellxilar, and affecting glandular epithelium. Staining. — The organism requires special staining, and a number of complicated methods have been introduced by different investigators. The Giemsa stain is said to give the best results. (See Staining Fluids, p. 47.) The slide is fixed, dried in air, hardened in absolute alcohol twenty - five minutes, stained with dilute stain (i drop to i c.c. of water) for ten min- utes, washed in water, and mounted. In tissues the organ- ism can be shown by fixing with silver ni- trate after the manner of Ramon y Cajal. The tissue is — (i) Hardened in formalin for twenty-four hours (the sections should be thin); (2) washed in water for one hour; (3) alcohol, twenty-four hours; (4) i-|- per cent, silver nitrate solution in incubator at 37° C, three days; (5) washed in water twenty minutes; (6) placed in mixture of pyrogallic acid, 4 parts; formalin, 5 parts; distilled water, to make 100 parts, and kept in dark bottle for forty-eight hours; (7) washed in water and alcohol and then embedded in paraffin and sectioned. Spirochastag black, tissues, pale yellow. Or counterstain of fuchsin can be employed. Fig. 106.— Spirochseta pallida. Micro- photograph made by Dr. R. E. Lavenson from a specimen prepared by H. Fox (Stengel). THE MICRO-ORGANISM OP SYPHILIS AND ALLIED ORGANISMS 211 The India Ink Method. — A drop of fluid from a lesion is mixed with a drop of India ink upon a clean glass slide and allowed to dry. Examine with oil-immersion lens. The spirilla appear dark in a mass of carbon particles. By using dark ground illumination, the organism appears brightly refractive. Culture Methods. — Noguchi, by using a serum water (i part sheep or horse serum, 3 parts water, and adding a piece of sterile rabbit's kidney or testicle), under strict anae- robic conditions at 35° C. succeeded in cultivating the organ- ism direct from lesions in man. After several transfers the organism wUl grow on agar containing the bit of tissue. Inoculation Experiments. — Pure cultures inoculated into rabbits and monkeys produce lesions resembling the primary sores, and the blood of such animals gives a Wassermann reaction. Cutaneous inoculation on eyebrows and genitals of material from primary and secondary lesions produces results in from fifteen to fifty days. Wassermann Reaction. — In 1906 Wassermann, Neisser, and Bruck described a method of making the diagnosis of syphilis by demonstrating in the blood and spinal fluid of a patient complement-binding substances not present in normal blood. Technic. — The following reagents are employed: (i) Syphi- litic antigen; (2) serum to be tested; (3) fresh guinea-pig serum; (4) washed sheep corpuscles and antisheep ambo- ceptor. The antigen is an alcoholic extract of liver from a congenital syphilitic, and is prepared by extracting the ground-up liver with five volumes of absolute alcohol for ten days and then filtering. Complement is normal guinea-pig serum. Antisheep amboceptor is obtained by injecting into a rabbit 2, 4, 6, 8, and 12 c.c. of washed sheep corpuscles on the first, tenth, nineteenth, twenty-eighth, and thirty-seventh days respectively. Nine days after the last injection the animal is bled to death from the carotid and the blood collected in 212 ESSENTIALS OF BACTERIOLOGY sterile test-tubes. After clotting has taken place the clear serum is removed. This is the amboceptor serum. Washed sheep corpuscles are obtained by centrifuging de- fibrinated sheep blood, pipeting off the serum, replacing it with normal salt solution, shaking, and again centrifuging. This is repeated three times. Patient's serum obtained from blood from the patient's arm is heated thirty minutes at 56° C. to destroy complement. Titration or Testing of Reagents. — Titrate amboceptor. One c.c. of a 5 per cent, suspension of washed sheep cor- puscles in salt solution and o.i c.c. of fresh guinea-pig serum are added to a series of test-tubes. The amboceptor serum is then added so that each tube receives more than the preceding one. Salt solution is added to make 5 c.c. and the tubes incubated for two hours at 37° C. with occasional shaking. That tube in which complete hemolysis has taken place in just two hours contains \ unit of amboceptor. Titration of Complement. — Into each of a series of tubes place I c.c. of the corpuscle suspension and ^ unit of ambo- ceptor. Next add 0.6, 0.7, 0.8, 0.9, i, i.i, 1.2 c.c. of fresh guinea-pig serum respectively and incubate for two hours, shaking occasionally. Those tubes which show complete hemolysis in just two hours contain i unit of complement. Titration of Antigen. — Two-tenths c.c. of serum, pre- viously heated to 56° C. for a half-hour, from a known, un- treated case of secondary syphilis, and i unit of complement are added to each of a series of test-tubes. Antigen is now added, so that each tube contains more than the preceding one, and salt solution added and brought to 3 c.c. The mixture is incubated for one hour at 37° C, at the end of which time 2 units of amboceptor and i c.c. of corpuscle suspension are added and the tubes returned to the incubator. After a short period the tube containing the smallest amount of antigen will show complete hemolysis. As the dose of antigen is increased the amount of hemolysis is decreased untU a point is reached at which no hemolysis takes place even after twenty-four hours. The first tube in the series THE MICRO-ORGANISM OF SYPHILIS AND ALLIED ORGANISMS 213 which shows no hemolysis after twenty-four hours contains I unit of antigen provided twice that amoiuit will not prevent hemolysis when no serum is added. Having found out the exact amount of guinea-pig serum (complement) necessary to unite with hemolytic amboceptor (rabbit serum) in order to hemolyze blood-corpuscles, this amount is mixed with sjrphilitic antigen plus the suspected syphilitic serum amboceptor, and incubated for one hoxir at 37° C. If the amboceptor is syphilitic, it will combine with the antigen and guinea-pig complement. To find out if the complement has been bound, the hemolytic amboceptor and its antigen sheep corpuscles are added to the mixture, and if no hemolysis takes place, the complement is fixed and the patient's serum contains the syphilitic antibodies or amboceptors. To Set Up Test. — Nine tubes needed for Wassermann reac- tion and control. Into each tube i c.c. diluted complement guinea-pig serum. Into tubes i, 2, and 9, 0.2 c.c. of patient's serum. Into tubes 3 and 4, control, syphilitic serum 0.2 c.c; in 5 and 6, normal serum as control, 0.2 c.c. ; antigen extract, I imit placed in i, 3, 5, and 7. To each tube is now added sufficient normal salt solution to make 3 c.c. Tubes gently shaken and placed in incubator at 37° C. one hour. At end of the hour to each tube is added 1 unit of suspension sheep corpuscles, and to all but No. 9 2 units of standard amboceptor, in i c.c. saline. The tubes again placed in incubator for one hour, readings taken, and then placed in ice-box twenty-four hours, when final results noted. If Wassermann positive — No. I. No hemolysis. No. 2. Complete hemolysis. No. 3. No hemolysis. No. 4. Complete. No. 5. Complete. No. 6. Complete. No. 7. Complete. No. 8. Complete. No. 9. No hemolysis. 214 ESSENTIALS OF BACTERIOLOGY "WASSERMANN SCHEME § 1 y ^ s 1 « 6 Is Vi ii 1 D Q 1 + Resuit I ZU3 1 o < I I c.c. 0.2 C.C. I No hemolysis. + 2 I c.c. 0.2 C.C. o M g Complete hemolysis. 3 I c.c. 0.2 C.C. I & o No hemolysis. 4 I c.c. 0.2 C.C. U ^1 + Complete hemolysis. S I c.c. 0.2 C.C. I Complete hemolysis. 6 I c.c. 0.2 C.C. 3 s ^ Complete hemolysis. 7 I c.c. I 3 Complete hemolysis. 8 I c.c. 1—3 1 Complete hemolysis. 9 I c.c. 0.2 C.C. As a rule, no hemolysis. Noguchi modification of the Wassennann reaction consists in using human corpuscles and antihuman ambocep- tor, and, as antigen, acetone insoluble lipoids. Antigen. — Extract a finely ground ox-heart with lo vol- umes of absolute alcohol at 37° C. for several days; filter and evaporate the extract (using an electric fan and not heat) almost to dryness. Extract the residue with ether; decant, evaporate the ether, and redissolve in the smallest quantity of pure water-free ether. To this ethereal solution add 5 volumes of water-free acetone. A precipitate forms which is the antigen. The precipitate is dissolved in purest methyl- alcohol in the proportion of 3 per cent. For use, i c.c. of this alcoholic solution is mixed with 9 c.c. of salt solution. Titration of Antigen. — (i) Hemolytic Action. — A tube con- THE MICRO-ORGANISM OF SYPHILIS AND ALLIED ORGANISMS 21 S taining 0.4 c.c. of the antigen emulsion, o.i c.c. of 10 per cent, suspension of corpuscles, and 0.6 c.c. of salt solution should show no hemolysis after two hours at 37° C. (2) Anticomplementary Bodies. — ^A tube containing 0.4 c.c. of antigen, o.i c.c. of a 40 per cent, dilution of complement, 2 units of amboceptor, and 0.6 c.c. of salt solution is incu- bated for one hour and 0.1 c.c. of 10 per cent, corpuscle sus- pension added. In two hours there should be complete hemolysis. (3) Antigenic Properties. — After incubating for one hour a tube containing 0.02 c.c. antigen, 0.02 c.c. of a known syphilitic serimi, 0.1 c.c. of a 40 per cent, dilution of complement, 2 units of amboceptor, and 0.8 c.c. of salt solution, 0.1 c.c. of a 10 per cent, corpuscle suspension is added, and the tube returned to the incubater. At the end of two hours there should be no hemolysis. (4) Amboceptor and complement are titrated the same as in the Wassermann reaction, except that a i per cent, sus- pension of human corpuscles and 0.02 c.c. of complement and antihuman amboceptor are used. NOGUCm SCHEME d la a w W 1 z ■ < _\ ApTEE Ten Hours I I . . .1. • T.T J tissue yellow. Fig. 114. — btreptotnrix MadurEe oiixi.' lurj in a section of diseased tissue (Vin- btreptothnx Madurse cent). (Vincent). — Origin. — .Found in the disease known as Madura foot, or mycetoma, an ulceration affecting the feet, especially of individuals living in the tropics. Two varieties, the pale and the black, have been described. Form. — Branched fUaments resembling the actinomyces streptothrix. In the mycelia spores are seen (Fig. 114). *l * v.~«4 YEASTS AND MOLDS 23I Cultivation. — ^In liquid media containing vegetable infu- sions growth occurs best. Temperature of 37° C. most suited. The colonies near the siurface become colored red. Agar. — Glazed colonies, at first colorless, then rose-colored, about the size of a pea, with the central part umbilicated and pale. Gradually the rose color fades. Acid Potato. — A slow and meager growth. Pathogenesis. — Only local reaction has been caused by inoculation in animals. In man the disease usually follows a slight injury and attacks the leg or foot, slowly forming a nodular growth, which in the course of months or a year begins to soften and ulcerate, and with the seropus are dis- charged numerous little granules, some black, some pink, containing mycelia. The limb becomes much deformed, the tissue vascularized, and the degenerated area filled with the strep to thrix filaments. Staining. — The organism itself stained with ordinary stains. Gram's method for the tissue. Nocardia (Streptothrix) Farcinica.(Nocard) ; Bovine Farcin du Boeuf . — Origin. — ^A disease affecting cattle, and giving rise to tubercle-like lesions in the lungs, liver, and spleen. Common in France. Form. — Small interwoven mass of threads arranged in tufts found in the centers of the tubercles. Culture. — At body-temperature in various media. Bouillon. — Colorless masses, irregular in size and shape. Agar and Gelatin. — Small, rounded, opaque colonies, thicker at the periphery. Potato. — ^Rapid growth of pale-yellow, dry scales, consist- ing of many spores. Pathogenesis. — Pure cultures introduced into the perito- neum of guinea-pigs give rise in nine to twenty days to tubercle-like lesions. Subcutaneous injections cause abscesses with secondary involvement of the lymphatics, ending in recovery. Dogs, horses, and rabbits are immune. Staining. — Wright's double stain for tissues; also Gram's. Plant Diseases due to Bacteria. — ^There are a great 232 ESSENTIALS OE BACTERIOLOGY variety of blights, rots, and new-growths, such as galls attack- ing plants, which are seemingly due to bacteria. About 30 varieties have so far been more or less accurately described, but only a few of the organisms have been definitely asso- ciated with the disease. The pear Might is due to Bacillus amylovorus. Crown gaU, which affects a great many plants and trees, is supposed to be due to Bacterium tumefaciens; the black rot of cabbage to a pseudomonas. There is much left to be done to place this part of bacteriology on a par with that devoted to animals and man. CHAPTER XXXI EXAMINATION OF AIR, SOIL, AND WATER Air. — Many germs are constantly found in the atmosphere about us. Bacteria unaided do not rise into the air and fly about; they usually become mixed with small particles of dirt or dust and are moved with the wind. The more dust, the more bacteria, and, therefore, the air in summer contains a greater number than the air in winter, and aU the other dif- ferences can be attributed to the greater or less quantity of dust and velocity of the wind. By the use of balloons, living bacteria have been foimd at an altitude of 4000 meters. Methods of Examination. — ^The simplest method is to expose a Petri dish with gelatin or agar in a dust-laden atmos- phere or in the place to be examined. In the course of twenty- four to forty-eight hours colonies wUl form wherever a germ has fallen. But this method wUl not give any accurate results in regard to the number of bacteria in a given space; for such purposes somewhat more complicated methods are used, so that a definite amount of air can come in contact with the nutrient medium at a certain regulated rate of speed. EXAMINATION OF AIR, SOIL, AND WATER 233 This form of analysis, however, has not yielded any very practical results, and is not much resorted to. Hesse's Method. — ^Hesse's method requires an apparatus called an aeroscope, which, by means of siphoning bottles (aspirator), sucks air through a cylinder lined with gelatin, i'rir^ and by regulating the rate of flow an approximate idea of the number of bacteria per liter of air can be obtained. A less complicated method is known as Petri's method. Fig. 115. — Petri's sand-filter for air-examination (McFarland). Fig. 116. — Sedgwick's expanded tube for air - examination (Mc- Farland). Sand is sterilized by heating to redness, and while still warm placed in test-tubes, which are then plugged. 234 ESSENTIALS OF BACTERIOLOGY The tube and its contents, the ends having first been plugged with cotton, are sterilized in a hot-air oven at 150° C. One end of the tube is then fitted with a rubber cork through which passes a glass tube, which is connected with an aspirator (a hand-pump with a known capacity). If 100 liters of air pass through the tube in fifteen min- utes, the germs should all be arrested in the first sand-filter. And when the filters are removed, each filter for itself, and thoroughly mixed with gelatin, there should be no colonies developed from the second filter, i. e., the one nearest the aspirator. Sedgwick-Tucker Method. — ^A special form of tube is used, called an aerohioscope. It consists of a neck 2.5 cm. in length, an expanded portion 15 cm. long, and a long narrow tube of 15 cm. After sterilization the tube is partly filled with granulated sugar, which is the filtering material. By means of a vacuum gage and an air-pump, or ordinary aspirat- ing bottles, the volume of air passing through the apparatus can be determined. After the air has been passed through, the sugar is gently shaken from the narrow tube into the expanded portion, and 20 c.c. of liquefied gelatin is poured in. The sugar dissolves, and the mixture is then roUed on the inner side of the glass as an Esmarch tube. This part of the apparatus is divided into squares to make the counting of colonies easy. The aerobioscope is very highly recommended. Varieties Found in Air. — ^The only pathogenic bacteria found with any constancy are the Staphylococcus aureus and citreus; but any bacterium can, through accident, be lifted into the atmosphere, and imder certain conditions may be always present — the Bacillus tuberculosis, for example, in rooms where consumptives are living. Typhoid fever, influenza, pneumonia, and diphtheria may be conveyed through the air by the cough and expectora- tion of affected persons. Non-pathogenic. — The micrococci predominate. Sarcinae, yeasts, and molds constantly contaminate cultures. EXAMINATION OF AIR, SOIL, AND WATER 235 In the ordinary habitations the average number of germs to the liter of air does not exceed five. Aroxmd water-closets, where one would imagine a great number to exist, but few will be found, owmg to the undis- turbed condition of the air. Sewer air seldom, if ever, contains bacteria, and neither typhoid fever, malaria, nor diphtheria has ever been traced to the escape of so-called sewer-gas. Ezamination of Water. — ^The bacteriologic examination of water is today of as much importance as the chemical analysis, and must go hand in hand with it. A water containing thousands of germs to the cubic centi- meter is far less dangerous than one containing but two germs, if one of these two be a typhoid bacillus. It is not the number that proves dangerous, it is the kind. If a natural water contains more than 500 germs to the cubic centimeter, it were well to examine its source, and consider it with suspicion. As a means of diagnosis the examination is of but little use. An epidemic of typhoid fever occurs, the water is suspected, an examination is undertaken; but the period of incubation and the days passed before the water is analyzed have given the tjrphoid germs, if any had been "present, ample time to disappear, since in water that contains other bacteria they live a few days only. Again, the water tested one day may be entirely free and the next day contain a great number, and before the typhoid germ can be proved to be present in that particular water the epidemic may be past. Human sewage contamination is determined by finding the colon bacillus, and if this is found in the course of an epidemic of typhoid the water containing it may well be suspected as being the cause. Purity of Waters. — ^The purest water we have is the natural spring-water — water that has slowly filtered its way through various layers of gravel and sand and comes finally clear and sparkling from the ground. It is free from bac- teria, but let such a water stand walled up in cisterns or 236 ESSENTIALS OF BACTERIOLOGY wells, or run through the wood, gathering the washings from pastures and farm lands, it becomes, as surface water, open to all sorts of impurities, and the bacterial nature of it changes every moment. Artesian or Driven Well. — The driven well will secure to a certain extent a pure water. It is the only form of well or cistern that will insure this, since the water does not become stagnant in it; but it may connect with an outhouse — the soil being very loose — and thus bacteria and refuse water find their way into the well. The casing may not be water-tight and surface water can be sucked in. Filtered Water. — Dangerous as surface water is, the greater quantity used is such, the inhabitants of larger towns and cities using chiefly the rivers and other large waters which course near them for drinking purposes. A purification or filtration can, to a certain extent, render these waters harmless. FUtration is carried on on a large scale in the water-works of cities and towns, and bacteriologic examination is here of great service to determine if a water which has been filtered and may have a very clear appearance, and give no harmful chemical reaction, is entirely free, or nearly so, from germs; in other words, if the filter is a germ-filter or not; daily tests are necessary in order to insure safety, and if it is performing this function regularly, a good filter plant should show 99.8 per cent, efficiency, removing nearly all the bacteria. Filter Materials. — When waters are muddy or when rapid filtration is wanted, mechanical filters are employed. The water is first treated with coagulants, like alum, which forms a flocculent precipitate and carries down with the suspended matter much of the bacterial content. This is then filtered through sand and gravel. Sedimentation and filtering slowly through gravel and sand is known as the slow process; the other as the rapid, filtration. Charcoal sponge and asbestos, the materials formerly in use, are objectionable because germs readily develop on them and clog them, so that they require frequent renewal. In EXAMINATION OF AIR, SOIL, AND WATER 237 very large filters, sand and gravel give the best results; the number of bacteria in a cubic centimeter is reduced to forty or fifty and kept at that number. This is a very pure water for a city water, though, as we stated before, not a safe one, for among those forty germs very dangerous ones may be found. It is then necessary for the users to refilter the water, before drinking it, through a material which will not allow any germs to pass, or, in the presence of an epidemic, to boil all water used for drinking purposes. Fig. 117. — Flask fitted with porcelain bougie for filtering large quantities of fluid. Pasteur-Chamberland Filter. — This very perfect filter consists of a piece of polished porcelain in the form of a cylinder closed at one end and pointed at the other. It is placed in another cylinder of glass or rubber, and the pointed portion connected with a bottle containing the water, or directly with the faucet of the water-pipe. The water courses through the porcelain very slowly and comes out nearly free from germs; pipe-clay, bisque, infusorial earth, and kaolin are also good filters. The only disadvantage is the long time it takes for the water to pass through. Pressure in the form 238 ESSENTIALS OF BACTERIOLOGY of an aspirator or air-pump is used to accelerate the pas- sage. These porcelain cylinders can easily be sterilized and the pores washed out. All the cylinders or bougies are not germ proof, so that they must be tested, and most of them must be cleaned every fourth day. In recent years a number of organisms have been suspected of being so minute as to pass through a Berkefeld or Pasteur filter. At least the poison or virus is filterable, and, therefore, we cannot regard these as abso- lutely safe. Boiling as a Means of Purifying. — ^The only safe measure in times of epidemics and with waters of unknown composi- tion is boiling, not only of the drinking water, but aU water used for domestic purposes; and this should especially be done in times of tjrphoid and cholera epidemics. Varieties Found in Water. — The usual kinds found are non-pathogenic, but, as is well known, typhoid, cholera, and dysentery are principally spread through drinking-water, and many other germs may find their way into the water. Some of the common varieties give rise to fluorescence or produce pigment. Eisenberg gives 100 different varieties as ordinarily found. Other intestinal diseases besides those mentioned above are supposed to be water borne. Diarrheas in epidemic form may come from suddenly changing a public supply, and the presence of the Bacillus coli communis means sewage contamination or fecal contamination; such contamination may come from the droppings of birds or other animals and need not necessarily imply human sewage, but 10 colon bacUli in i c.c. water is a serious pollution. Ice supplies require the same supervision as water supplies, for many bacteria, like the typhoid bacillus, retain their vitality for weeks after freezing. Method of Examination.— (^//er that suggested by the American Public Health Association, igi2 report.) — Since the germs rapidly multiply in stagnant water, an examina- EXAMINATION OF AIR, SOIL, AND WATER 239 tion must not be delayed longer than possible after the water has been collected. Every precaution must be taken in the way of cleanliness to prevent contamination; sterilized flasks with glass stoppers, pipets, and plugs must be at hand, glassware sterilized in autoclave at 120° C. for fifteen minutes, or dry heat at 160° C. for one hour, and the gelatin tubes or agar dishes be inoculated on the spot. If this cannot be done, the sample should be packed in ice until it arrives at the laboratory. If it is necessary to send the sample by raU, the bottle containing the sample should be wrapped in steril- ized cloth, or the neck covered with tinfoil and the bottles placed in tin boxes (about 4 ounces — 100 c.c. — ^is sufficient for bacterial analysis), and then packed in cotton or paper to prevent breakage and surrounded by plenty of ice untU it reaches its destination. As soon as it arrives at the lab- oratory the sample is placed in a sterilized glass flask, and the flask then closed with a sterile cotton plug. A sterilized pipet is then dipped into the flask, and i c.c. of the water withdrawn and added to a Petri dish. To a second dish, a dilution of i c.c. of the sample with sterile distilled water is added, and other dilutions made if desired. To each plate 10 c.c. of standard agar at a temperature of 40° C. is added. Mix the water and media thoroughly by tipping the dish back and forth, and place in incubator at 37° C. for twenty- four hours. The incubator should be in a dark, weU-venti- lated, and moist place. Then count all the colonies present on each plate, which wiU give the number per cubic centi- meter. Water that is very rich in germs requires dilution with sterilized water fifty to one himdred times. Fewer colonies win be found on agar than on gelatin, even at the same tem- perature. Special Media and Preparation. — In the preparation of media for water analysis, sodium chlorid must not be used. The reaction of most culture-media should be -|-i per cent, to phenolphthalein. Sugar broths should be neutral, and must be sterilized care- 240 ESSENTIALS OF BACTERIOLOGY fully in steam and not overheated, so as to prevent inversion of the sugar. Examination for Bacillus Coli and Sewage Bacteria. — Instead of examining for typhoid bacilli, sewage contamina- tion is best indicated by the presence of the colon group of organisms, although their abimdance rather than mere pres- ence is to be considered. There are many closely related bacteria which give reactions similar to the Bacillus coli, but they are chiefly of fecal origin, and for practical purposes they can be included in the colon group. General Characteristics of Colon Group. — i. Fermentation of dextrose and lactose with gas-production. 2. Short bacillus, non-liquefying. Gram negative. The committee of the Public Health Association recom- mends the following procedure: Two Methods. — Method a. — ^Applicable for sewage waters. Preparation of an agar plate with a known volume of water, using lactose litmus-agar and incubating at 40° C. Bacillus coli wUl show its presence by red colonies (acid fermentation of the sugar) ; further testing is then needed to fully identify. Not all red colonies Bacillus coli. Method h. — Cultivation, at 40° C, of a measured quantity of water in a fermentation tube containing a sugar broth. If gas appears, a portion of the liquid is plated as in method a. Additional Details. — If in twenty-four hoinrs no red colonies appear in the agar-lactose litmus Petri dishes. Bacillus coli is considered absent, providing the sample was a polluted one, so that the bacilli, if present, would be in a concentrated form. Only i or 2 c.c. of water can be used, because the ordinary water-bacteria spread rapidly and contaminate the other bacteria. // acid-forming colonies are found, five or six are fished for subcultures on slanted agar, in fermentation tubes, milk, gelatin, peptone solution, and nitrate broth. If the water is not strongly contaminated, an underground water, for instance, or a moimtain stream, the better way is to inoculate two or three lactose or dextrose bouillon fermen- EXAMINATION OF AIR, SOIL, AND WATER 241 tation tubes and place in an incubator at 40° C. Note the presence of gas, if any, at the end of twelve, twenty-four, thirty-six, and forty-eight hours. // no gas forms, sewage bacteria are absent. U S'^^ forms, plate at once a portion of the sediment as above on lactose litmus-agar. Test the other fermentation tubes for acidity, and the nature of the gas, whether any, and how much is absorbed by a 2 per cent, solution of sodium hydroxid. Bacillus coli should produce between jo and 70 per cent, of gas, of which about one-third is CO2 and is ab- sorbed by the alkali; the remainder is hydrogen. The other broth culture can be tested for the presence or absence of unfermented sugar by Fehling's solution. Diagnostic Points of Colon Bacillus. — Microscopic. — Non-spore-bearing motUe bacillus. Gelatin. — Non-liquefactive. Dextrose Broth. — Fifty per cent, gas; one-third absorbed, CO2; two- thirds, hydrogen. Milk (litmus) coagulated in forty-eight hours and rendered acid; litmus colored red. Peptone Solution. — Production of Indol. — (A peptone solu- tion tube is inoculated with the cultiire and kept together with a control four days at 37° C. Then 2 drops of concentrated sulphuric acid and i centimeter of a o.oi per cent, solution of sodiimi nitrate are added. The appearance of a pink color at the end of thrity minutes denotes the presence of indol.) Presumptive Test. — ^If a water from a well or spring pro- duces gas in the sugar broth and forms acid colonies on litmus- lactose agar, the presumption is strong that there is sewage contamination. If gas-production continues in a series of samples carefully collected for several days or weeks, there can be no doubt of a contamination, and especially if the well or spring is protected from surface water. Algae which grow in service pipes, reservoirs, and deep wells may give rise to non-acid gas fermentation, but all well-water that, without further testing, forms acid colonies on litmus-agar lactose plates and ferments sugar broth, is open to suspicion, and if 16 242 ESSENTIALS OF BACTERIOLOGY there is evidence of the presence of typhoid fever or diarrheal diseases, the water should be boiled and subjected to careful analysis daUy. There may be serious contamination and the chemical tests show no appreciable increase in the chlorids. Bile Media. — In recent years bile salts or fresh bile mixed with lactose have been extensively used, as the bile inhibits the action of many bacteria and allows the colon and ty- phoid group to develop readily. The Jackson bile media (see formula for media. Chap. X) is placed in fermentation tubes of 40 c.c. capacity, and in- oculated with varying proportions of the water to be tested. Incubated at 37° C, and presence of gas looked for in twelve hours, twenty-four hours, and forty-eight hours, and the quantity and time noted. In sewage and contaminated waters the lactose-bUe gives better results than any other medium. The Presumptive Test {Modified). — Plant yV; ^j ^^^ i<^ c.c. of water into liver broth tubes. Transplant from these into lactose bile in six and twelve hours. By using implan- tations of both lactose bUe and liver broth, and then trans- planting the liver-broth cultures into other lactose bile, we have in the original bile the vigorous Bacillus coli. The liver- broth dilutions give all the gas formers, strong and weak, and the difference between the original and the transplanted gives an idea of the attenuated Bacillus coli present. Thus all the gas formers are cultured. Bacillus Typhosus. — By the use of bile media and other special media as enrichment and then transplanting on Hesse Agar, Conradi-Drigalski, or Endo media, the Bacillus typhosus are increased in number and the possibilities of diagnosing them made much easier. The Widal test is used to differentiate Bacillus typhosus from Bacillus coli. Quantitative Tests.- — ^The number of acid colonies in i c.c. and in 5 c.c. of water is taken as a measure of pollution, to- gether with the total number of colonies of all bacteria present. Thus in i c.c. on the gelatin plate at 20° C. there may be EXAMINATION OF AIR, SOIL, AND WATER 243 fifty colonies; on the agar plate at 37° C. ten colonies, five of which were acid-formers, or presumably Bacillus coli. To count the colonies which develop upon the plates, a special apparatus has been designed, known as — Wolfhugel's Counter. — A glass plate divided into squares, each a centimeter large, and some of these subdivided. This plate is placed above the dish with the colonies, and the num- ber in several quadrants taken, a lens beLag used to see the smaller ones. It is best to count aU the colonies on the plate or dish. Bacterial Treatment of Sewage. — Where sewage is to be rendered innocuous before being allowed to flow into streams, the process of nature has been imitated by the construction of septic tanks in which the sewage remains excluded from the air and subject to the action of the anaerobic bacteria present in the sewage. The organic nitrogen is reduced, and compounds of hydrogen and sulphur are formed. The effluent is then filtered through coke-beds, where the aerobic bacteria assist in further purification and over sand filters, or exposed to the air on contact beds. No method of sewage purification is very practical or safe. Pure water should not depend on the efficiency of sewage filtration, but should be obtained from a reasonably pure source. Sewage is also treated by sedimentation with alum and filtration of the effluent over larger beds, or allowed to per- colate through the soil, which is thereby enriched and utilized for agriculture. It is also dried and sold in a compressed form for fertilizer. The Examination of the Soil. — The upper layers of the SOU contain a great many bacteria, but because of the diffi- culty in, analyzing the same, the results are neither accurate nor constant. The principal trouble lies in the mixing of the earth with the nutrient medium; little particles of groimd will cling to the walls of the tube, or be embedded in the gelatin, and may contain within them myriads of bacteria. As with water, the soil must be examined immediately or very soon after it is collected, the bacteria rapidly multiplying in it. 244 ESSENTIALS OF BACTERIOLOGY When the deeper layers are to be examined, some precau- tions must be taken to avoid contamination with the other portions of the soil. One method, very laborious and not often practical, is to dig a hole near the spot to be examined and take the earth from the sides of this excavation. Frankel's Borer. — Frankel has devised a small apparatus in the form of a borer, which contains near its lower end a small cavity, which can be closed up by turning the handle, or opened by turning ia the opposite direction. It is introduced with the cavity closed, and when it is at the desired depth, the handle is turned, the earth enters the cavity, the handle again turned, incloses it completely, and the borer is then withdrawn. The earth can then be mixed with the culture-medium in a tube, and this gelatin then rolled on the walls of the tube after the manner of Esmarch, or it can be poured upon a plate, and the colonies developed therein. Another method is to wash the earth with sterilized water, and the water then mixed with the culture-medium, as many of the germs are taken up by the water. The roll-cultures of Esmarch give the best results, many of the varieties usually found being anaerobic. Animals inoculated with the soil around Berlin are said to die almost always of malignant edema, and the soil of other towns produces tetanus. Many of the germs found are nitro- gen formers and play a great role in the economy of the soil. Bacteria and Soil Fertility. — Nitrifying organisms are found in the superficial layers of the earth. Organic matters found in sewage and in the fecal evacuations of animals form the basis for their activity, whereby nitrates, ammonias, and nitric acid result. The nitrogen necessary for the growing plant is thus produced. The nitromonas of Winogradsky belongs to this group. The soil tends to destroy ordinary disease-bacteria in a short time, but spores may remain dor- mant for a number of years, as, for instance, the spores of anthrax. As bacteria are instrumental in transforming organic EXAMINATION OF AIR, SOIL, AND WATER 245 matter, their influence in making the soil more useful for agricultural purposes has been the subject of much research. The richer the soil, the greater the number of bacteria. Most bacteria are found under the surface between i and 2 inches. The rod-shaped organisms predominate. From an agricultural standpoint the most important bacteria are those capable of liberating nitrogen and break- ing up protein substance. Carbohydrates are added to soil by manure, by the growth of grasses and crops, and these are decomposed by bacteria and methane and hydrogen produced. Ammonia Production. — Most soil bacteria can produce ammonia; a few, the so-called urea bacteria, are capable of rapid transformation — nitrification. Ammonia, oxidized into nitrites or nitrates, is possible through the agency of a group of micro-organisms given especial prominence by Winogradski. Moisture conditions and the presence of lime and mineral carbonates influence the nitrif3ring organisms. The character of the growing crop affects the accumulation of nitrates; legumes assimilate nitrogen more rapidly than non-legumes. Denitrification. — ^The reduction of nitrates to nitrites and ammonia is accomplished by a number of bacteria. Nitrate reduction is of little importance in the field, but under exces- sive manuring it may become so. Bacteria play the impor- tant part of making available to vegetation the nitrogen of the air. Azofication. — Certain bacteria can fix atmospheric nitrogen and make it serve, but the energy necessary must be fur- nished by carbohydrates. The enrichment of the soil by the growth of legumes has been shown to be due to the bacteria contained in the nodules or tubercles of the plant, these bacteria having the power to fix nitrogen and deriving their energy from the plant juices, 246 ESSENTIALS OF BACTERIOLOGY the plants in turn utilizing the nitrogen compounds created by the bacteria. Soil Inoculation. — Artificial help to soils deficient in nitro- gen-fixing organisms has been the subject of much experiment. Nitragin. — Pure cultures of legume bacteria under the above name have been tried. Dried cultures imder the name of nitro-bacterine have likewise been marketed, but neither of these methods has proved valuable; the matter is still in the experimental stage. CHAPTER XXXII BACTERIA IN MILK AND FOOD The Bacteria of Milk. — Milk as secreted is sterile, but at every step in its passage from the cow to the consumer it is liable to contamination. Even the lower portion of the teat is a source of infection, owing to the presence of stag- nated milk from the former milking, and, as mUk ready for consumption usually contains thousands to millions of bac- teria to the cubic centimeter, sterilization or pasteurization and supervision of the dairies should always be enforced for nulk used for infant feeding. A standard milk should he free from pus and should not contain more than 10,000 bacteria to the cubic centimeter. Leukocytes are normally fovmd in milk, and only when their number exceeds one million and pyogenic organisms are also present can pus be said to exist. Pasteurization of un- clean nulk sometimes renders it more dangerous as a food than untreated milk, because, by preventing the action of lactic-acid formers, other bacteria are permitted to develop and produce pathogenic toxins. Pure Milk. — A pure mUk is one that is obtained from a healthy cow, well groomed, in a clean room, by a healthy, clean person, in clean cans or bottles, and transported to the BACTERIA IN MILK AND FOOD 247 consumer in as short time as possible without further hand- ling, keeping the container in the mean time at a low tem- perature and protected from the air. Such treatment is safer than any form of sterilization. Classification of Milk. — {Abstract of resolutions adopted by the Commission on Milk Standards at Richmond, Va., May 2-3, 1913.) : Milk shall be divided into three grades, which shall be the same for both large and small cities and towns. Gkade a. — Raw milk. — Milk of this class shall come from cows free from disease as determined by tuberculin tests and physical examinations by a qualified veterinarian, and shall be produced and handled by employees free from disease as determined by medical inspection of a qualified physician, under sanitary conditions such that the bacteria count shall not exceed 100,000 per cubic centimeter at the time of de- livery to the consumer. It is recommended that dairies from which this supply is obtained shall score at least 80 on the United States Bureau of Animal Industry score card. Pasteurized Milk. — Milk of this class shall come from cows free from disease as determined by physical examinations by a qualified veterinarian and shallbe produced and handled imder sanitary conditions such that the bacteria count at no time exceeds 200,000 per cubic centimeter. All rmlk of this class shall be pasteurized under official supervision, and the bacteria coimt shall not exceed 10,000 per cubic centimeter at the tim^e of delivery to the consumer. It is recommended that dairies from which this supply is obtained should score 65 on the United States Bureau of Animal Industry score card. The above represents only the minimum standards under which milk may be classified in grade A. Geade B. — Milk of this class shall come from cows free from disease, as determined by physical examinations, of which one each year shall be by a qualified veterinarian, and shall be produced and handled under sanitary conditions such that the bacteria count at no time exceeds 1,000,000 per 248 ESSENTIALS OP BACTERIOLOGY cubic centimeter. All milk of this class shall be pasteurized under oflScial supervision, and the bacteria count shall not exceed 50,000 per cubic centimeter when delivered to the consumer. It is recommended that dairies producing grade B mUk should be scored and that the health departments or the controlling departments, whatever they may be, strive to bring these scores up as rapidly as possible. Grade C. — Milk of this class shall come from cows free from disease as determined by physical examinations and shall include all mUk that is produced under conditions such that the bacteria count is in excess of 1,000,000 per cubic centimeter. All milk of this class shall be pasteurized, or heated to a higher temperature, and shall contain less than 50,000 bac- teria per cubic centimeter when delivered to the customer. It is recommended that this rmlk be used for cooking or manu- facturing purposes only. Whenever any large city or community finds it necessary, on account of the length of haul or other peculiar conditions, to allow the sale of grade C milk, its sale shall be surrounded by safeguards such as to insure the restriction of its use to cooking and manufacturing purposes. Classification of Cream. — Cream should be classified in the same grades as milk, in accordance with the require- ments for the grades of mUk, excepting the bacterial standards, which in 20 per cent, cream shall not exceed five times the bacterial standard allowed in the grade of milk. Ice Cream. — Made and handled under sanitary conditions it contains mostly Bacillus lactis acidi type, not dangerous; but if made from mUk and cream containing putrefactive bacteria, freezing will not prevent further growth and bac- terial poisons may be developed, causing sickness and death. An examination of specimens collected gave as the lowest count 50,000 bacteria per cubic centimeter, and the highest 150,000,000 per cubic centimeter. JJAUTKKIA IJM MILK AND rOOD 249 SOME BACTERIA FOUND IN HILK Fermentation of Milk. — Lactic Acid Lactose. — Fermenta- tion of mUk is due to the conversion of milk-sugar into lactic acid. This can be accomplished by a number of different bacteria, such as Bacillus coli, streptococci and staphylo- cocci, which are apt to be present about the dairy. The lactic-acid bacteria are commonly present in sour irdlk, and are chiefly concerned with fermentation. There are several varieties, but principally three groups. The first group, like the Streptococcus pyogenes, is called the Bacterium lactis acidi group. Milk is curdled within twenty-four hours without gas-formation. The milk has a mild acid taste and agreeable odor. The curd is even, a true lactic fermentation. The second group resembles the Bacillus coli — Bacillus laciis aerogenes. Indol and hydrogen sulphid often formed. MUk curdles, but the curd shrinks. Not easily emulsified. This fermentation undesirable. The third group, true lactic bacteria — Bacterium bulgari- cum; exclusively lactic acid; curd easily broken. Bacterium Acidi Lactici (Hiippe) . — Belongs to the same group as the Bacillus coli communis (see page 134). Synonyms. — Bacillus acidi lactici; B. lactis aerogenes (Escherich). Origin. — In sour milk. Form. — Short thick rods, nearly as broad as they are long, usually in pairs, resembling B. coli. Properties. — ImmotUe. Does not liquefy gelatin. Breaks up the sugar of milk into lactic acid and carbonic acid gas, the casein being thereby precipitated. The fermentation of milk produced by this group is offensive; taste undesirable. Curd is firm. Stain. — Does not take Gram. Growth. — Rapid and abundant; is facultative anaerobic. Grows at 10° C. Grows in all media and in absence of car- bohydrates. Stab-culture. — ^A thick -dry crust with cracks in it forms on the surface after a couple of weeks. 250 ESSENTIALS OF BACTERIOLOGY Attenuation. — ^If grown through successive generations, it loses power to produce fermentation. Streptococcus Acidi Lactici (Grotenfeld) (1889). — Widely distributed in nature. Synonyms. — Bacterium lactis acidi; Bact. Guntheri. Origin. — In sour milk. Appearance. — Very short cells, often as large as oval cocci, in pairs or small chains, outer ends pointed. Properties. — ^Immotile. Stain with Gram. Growth best at 3o°-3S° C. Growth. — Facultative anaerobic. Delicate, opaque, re- sembling dewdrops. Bouillon containing glucose grows cloudy. Gelatin not liquefied. MUk coagulated. Strong acid reaction. Curd is soft and easily mixed within twenty- four hours. Gas is not formed. In lactose-agar stab no surface growth, but aU along the line. Potato. — Scant growth. Origin. — Almost always in sour milk, and the chief cause of lactic acid formation. Found at times in combination with B. acidi lactici and other bacteria. Sauer-kraut fermentation is due to streptococcus of lactic acid and yeasts, the latter producing gas. Bacterium Bulgaricum. Synonym. — Bacterium caucasicus (v. Freudenreich). Origin. — Present in milk. Thought to be a product of eastern countries, but now recognized as universal. Arises from aHmentary tract. Properties. — Produces large amount of acid at higher tem- perature ; non-motHe. Form. — Slender rods, 2 ;u to 4 /* long, tending to form threads. Staining. — Gram positive. Growth. — ^Best growth at 40° C. Very meager colonies, hardly visible. Curdling homogeneous, changed later into soluble products. Gelatin not liquefied. Used to produce artificial buttermilks. •BACTEMA IN MILK AND POOD 251 Potato. — Growth wrinkled and many-folded, gray changing to brown, extending over the entire surface as a thick cover- ing or skin. Agar Stroke. — ^Abundant, grayish, fatty, later on wrinkled skin. Gelatin Stab. — On surface, grajdsh, fatty exudate covered with skin which slowly sinks as the media liquefy. Gelatin Uquefied. No gas in sugar bouillon; acid is formed; no indol. Has been found in ropy or gelatinous bread and is considered the cause. Bacillus Butyricus (Hiippe). — This bacillus causes bu- tyric-acid fermentation. Supposed to be identical with Bacillus mesentericus. Bacillus Amylobacter (Van Tieg- ham). — Synonyms. — Clostridium butyri- cum (Prasmowsky) ; Vibrion butyrique of Pasteur; Bacterium saccharobutyricus (Klecki) (Fig. ii8). — Origin. — Found in putrefying plant-infusions, in fossils and conifera of the coal period, in cheese, water, earth. Form. — ^Large, thick rods, with rounded ends, often found in chains, t- „ t, ■„ A 1 1 ■ ^ J ^1. *^'g- 118. — Bacillus A large glancmg spore at one end, the amylobacter. baciUus becoming spindle shaped in or- der to allow the spore to grow; hence the name, Clostridium. Properties. — Very motile; gases arise with but3Tic smell. In solutions of sugars, lactates, and cellulose-containing plants and vegetables it gives rise to decompositions in which butyric acid is often formed. Casein is also dissolved. A watery solution of iodin will give the starch reaction and color blue some portions of the bacillus; therefore it has been called amylobacter. Growth in Glucose Agar. — Rapid at 37°. Small indefinite colonies with gas-bubbles. No growth in gelatin. Bacillus Cyanogenes {Bacterium Syncyanum) (Hiippe). — Origin. — Found in blue milk. 252 ESSENTIALS OF BACTERIOLOGY Form.- — Small narrow rods about three times longer than they are broad; usually found in pairs. The ends are rounded. Properties. — ^They are very motile; do not liquefy gelatin. A bluish-gray pigment is formed outside of the cell, around the medium. The less alkaline the medium, the deeper the color. It does not act upon the milk otherwise than to color it blue. Growth. — Grows rapidly, obligate aerobe. Colonies on Plate. — Depressed center, surrounded by ring of porcelain-like bluish growth. Dark-brown appearance under microscope. Stab-culture. — Grows mainly on surface; a nail-like growth. The surrounding gelatin becomes colored brown. Potato. — ^The surface covered with a dirty blue scum. Attenuation. — ^After prolonged artificial cultivation loses the power to produce pigment. Staining. — By ordinary methods. Gram positive. Red milk and yellow milk are due to other chromogenic organisms, as, for instance, B. erythrogenes. Examination of Milk. — American Standard. — Some bac- teria are found in all milk as ordinarily handled. Strepto- cocci and colon group, when present, always regarded with suspicion. A high-cell leukocyte coimt, when accompanied by chain bacteria, is an indication of udder disease. There should be several samples taken one week apart and an average made. Bacteria present may be counted in one of three ways. Stewart^Slack Method. — Centrifuge i to 2 c.c. of milk; smear sediment on slide, and stain with Jenner or Wright stain and count bacteria in field. Prescott-Breed Method. — ^In a special capillary tube y^ c.c. of milk is sucked up and spread over a square centimeter on a microscopic slide, dried and fixed with methyl-alcohol. Flood with xylol to dissolve fat, stain with methylene-blue or Jenner, and decolorize slightly with alcohol. Focus 15 mm. of the specimen and count bacteria and cells present. BACTERIA IN MILK AND POOD 253 Multiply by 5000. This equals the number in y-5-5- c.c. Count several fields and average the result. The Plate Method. — Microscopic examination, while not to be relied upon wholly, gives valuable and quick informa- tion as to the general character of bacteria, their apparent number, the presence or absence of barn-dirt and chain bacteria. The microscopic count differs greatly from plate count, because dead cells as well as living are shown. Certified milk should have less than 10,000 bacteria to the cubic centimeter. According to the average taken from a count of four specimens, a rating is given to the milk, and this rating is to be interpreted only as other conditions are considered, such as cleanliness of the cattle and stalls, and chemic composition and method of handling the product. Temperature. — Milk kept at 10° F. or lower will not allow ordinary bacteria to develop to any considerable extent; kept at a higher temperature, bacteria develop rapidly. Separating or centrifuging permits the bacteria to be con- centrated, and top-milk and cream contain more bacteria per cubic centimeter than whole milk. Time, an elemsnt. MUk freshly drawn, imder proper precautions, may con- tain but few bacteria, but in forty-eight to seventy-two hours on ice bacteria will increase enormously. Market milk as ordinarily found in cities may contain mUlions of bacteria per cubic centimeter. Pasteurization. — Milk heated to 60° C. for twenty minutes is called pastemized. This increases the keeping quality and tends to destroy the vegetative forms of pathogenic bacteria. To kill lactic acid, the instantaneous method, higher tem- perature, a few seconds only for pathogenic organisms is required. Pastemrization is beneficial only when there are supervision and inspection of original supply. Milk as Source of Contagion. — Harmless Varieties. — Sour milk contains the Bacterium lactis acidi and is not dangerous, and is even considered beneficial, as, for instance, buttermilk. 254 ESSENTIALS OF BACTERIOLOGY Neutral Forms. — Many species of air and chromogenic varieties found in milk have no pathogenic properties, neither do they affect the composition of the milk. Injurious Organisms. — ^Human diseases, like typhoid, diphtheria, and scarlet fever, may be conveyed through mUk, the infection coming from some one concerned in handling the particular supply. The milk acts as a favorable medium for the pathogenic organisms that accidentally iind their way into it. Animals wading in infected water have infected the mUk. Utensils washed in polluted water have been found to be the cause in some epidemics of typhoid. Carriers, persons who harbor the diphtheria and tjrphoid bacteria, but who are not affected with illness, may likewise start epidemics of a kind, especially if working about dairies. Bacteria may enter milk from the animal, as Bacillus tuber- culosis from diseased udder. Infantile diarrheas from the putrefactive Bacillus coli group, streptococcic sore throat from udder disease, are other forms of disease originating in mUk. Butter and Cheese. — Butter is milk-fat separated by creaming and churning, and as such partakes somewhat of the bacterial nature of the milk from which it is derived. The flavor of butter is due to the character of the acid bac- teria used in souring the milk. By eliminating the gas-form- ing bacteria and by keeping his starting cultures pure the butter-maker can control and develop flavors as easfly as the wine-maker. Pure cultures of lactic acid are supplied to butter-makers and used in creameries to inoculate sweet cream and mUk. Bacteria coming from unclean utensils, polluted water, or dirty milk undoubtedly affect the flavor and often produce a poor quality of butter. Disease bacteria are not often conveyed through butter, although it is claimed that Bacillus tuberculosis has been found in salted butter. Cheese. — ^The fat and casein salts and sugar-of-milk sepa- rated by curdling from the bulk of soluble portion of milk constitutes cheese. The curdling is accomplished by acid bacteria normally in milk, so-called acid curd cheeses, or by BACTERIA IN MILK AND FOOD 255 the use of rennet to form a curd, rennet curd cheese, to which, all the important varieties belong. MOk for cheese should be free from Bacillus coli or other deleterious bacteria. The milk for cheese cannot be pasteurized as for butter. Testing Milk for Bacillus Coli. — ^A sample of nulk is incu- bated at 35° C. for a few hours, noting the curd, whether firm or soft and gassy. Wisconsin Test. — Milk cvirdled by rennet; curd cut and drained and jars kept at 30° C. to 40° C. The curd should have clean acid odor and taste. After the curd has been formed, the cheese is allowed to ripen, and this is due to acid-forming bacteria, which permit the pepsin in the rennet to act. Various molds, notably PenicUliimi and Oi'dium lactis, are used to give certain foreign cheeses their characteristic flavor. Condensed milk has few bacteria in it. The sugar and condensation heat tend to prevent further growth of micro- organisms. Concentrated unsweetened milk is a form of pasteurized milk which is reduced in volume one-fourth. It is not always sterile, and bacteria may develop in it if exposed to warmth and air. Buttermilk and similar fermented drinks depend on Bacillus lactis acidi and added yeasts. Bacillus bulgaricus gives more acid and allows partial sterOization. Foods as a Source of Infection. — Foods eaten after little or no cooking, such as fruits, salads, and the like, and also oysters, are possible soiurces of bacterial diseases, and the so-called ptomain poisoning observed after the consumption of ice-cream, sausage, canned meats, etc., is the result of the action of bacteria or their products. Oysters and fish from sewage-polluted waters have pro- duced typhoid. Vegetables grown in manured ground or sprinkled with polluted water may be a possible source of disease. The practice of exposing meats and other food to street dust and flies is no doubt responsible for some disease. Alcohol and Vinegar Fermentations. — On grapes are to 256 ESSENTIALS OE BACTERIOLOGY be found all forms of air bacteria as well as molds and yeasts, some beneficial, some harmful. The acid of grape-juice de- stroys many of the harmful forms, but some persist and must be dealt with by the wine-maker. The various yeasts produce alcohol from the sugar of the grape. Vinegar bacteria likewise form a small amount of acetic acid. The wine-maker's success lies in obtaining a clean, unhruised grape, aiding the work of the wine yeasts, and preventing the injurious forms from working. The grapes are crushed and the juice allowed to settle. Pure cultures of tested yeast are used as starters of fermentation. Fermentation is regulated by burning sulphur, which in- hibits the growth of molds and harmful bacteria. After fermentation is completed the wine is cleared and freed from all organisms and kept as nearly as possible in a sterile con- dition. Beer. — The fermentation is produced by yeasts and with a mixture of grains. Barley or other grains which have been allowed to germinate produce malt. The malt contains the enzymes which change starch into sugar. Then, by boiling, the enzyme is destroyed and fermentation by yeasts is per- mitted. The yeasts in modern breweries are pure cultures. Wild yeasts or lactic-acid bacteria may contaminate beer. Alcohol, brandy, and whisky are likewise the product of yeast fermentation, some sugary substance furnishing the material. ORGANS AlsTD CAVITIES OE THE HUMAN BODY 257 CHAPTER XXXIII BACTERIOLOGIC EXAMINATION OF THE ORGANS AND CAVITIES OF THE HUMAN BODY The body, on account of its constant contact with the sur- rounding air, is necessarily exposed to infection, and we would be likely to find on the skin and in the oral, anal, and nasal cavities the varieties of microorganisms commonly around us. Through the water and food the body is also contaminated, but some organisms by predilection inhabit the mouth, intes- tine, and other cavities, and form there a flora distinctly their own. The Skin. — ^The majority of microorganisms met with on the skin are non-pathogenic, although underneath the nails and in the hair pus-forming microorganisms often occur, producing sometimes serious abscesses on other parts of the body. In the sweat-glands and the sebaceous glands various organisms have been found. The Staphylococcus pyogenes seems to be present constantly. In foul-smelling perspiration of the feet Rosenbach found microorganisms pathogenic for rabbits. Micrococcus cereus albus and flavus, Diplococcus liquefa- ciens albus and flavus. Staphylococcus pyogenes aureus, and Streptococcus pyogenes are found underneath the nails. In eczema, Diplococcus albicans tardus, Diplococcus citreus liquefaciens, Diplococcus flavus liquefaciens, and Ascobacillus citreus. In colored sweat. Micrococcus hasmatoides, Bacillus pyocyaneus. A diplococcus is found in acute pemphigus. The lepra bacillus, the tubercle bacillus in lupus, and the t)T3hoid bacillus in the eruption of tjqihoid fever are a few of the specific germs found on the skin during the disease stage. 17 258 ESSENTIALS OF BACTERIOLOGY Infection results through some damage of the superficial layers. The injury may be very slight — an expanded hair- follicle may suffice to permit entrance of suppurative organ- isms. The Conjunctiva. — The micrococcus of trachoma, the Koch-Weeks bacillus, considered to be the specific cause of acute catarrhal conjunctivitis, or "pink eye," and the Bacillus xerosis, are special germs found on the conjunctiva; the other varieties of air- and water-organisms, and those usually present on the skin, are also found. Loffler's bacillus and the pneumococcus have been found in some forms of con- junctivitis. The Koch- Weeks bacillus is the most contagious. A special diplobacillus, known as the bacillus of Morax- Axenfeld, produces a stubborn form of conjunctivitis. The gonococcus is found in ophthalmia of the new-born. The Mouth. — The mouth is a favorite seat for the devel- opment of bacteria. The alkaline saliva, the particles of food left in the teeth, the decayed teeth themselves, aU fur- nish suitable soil for their growth. Quite a number of germs have been isolated and their properties partly studied. Many have some connection with the production of caries of the teeth, as Miller has well shown in his careful studies. The Leptothrix buccalis, found in nearly all mouths, is a long chain or filamentous bacillus which stains blue with iodin. It was formerly considered the cause of tartar on the teeth. The Spirillum sputigenimi, Spirochaeta dentium. Micro- coccus gingivae pyogenes. Bacillus dentalis viridans. Bacillus pulpse pyogenes, micrococcus of sputum septicemia, and Micrococcus salivarus septicus are a few of the organisms cultivated by Miller and Biondi from the mouth and sup- posed to be separate varieties. Besides these, the pneumo- bacteria, diphtheria bacillus, and tubercle bacillus are often met with, the first two in the mouths of healthy persons. The expired air in quiet respiration is free from bacteria, but in coughing, sneezing, etc., large numbers of organisms are violently ejected and the atmosphere about tubercular ORGANS AND CAVITIES OF THE HUMAN BODY 259 patients is often saturated with tubercle bacilli. The bac- teria may enter the system from the pharynx to the tonsils and cervical glands by means of the lymphatics. Ear. — ^In the middle ear of new-born infants no pathogenic organisms have been found, but quite a number of non- pathogenic ones. In affections of the ear the pneumobacillus and the Staphylococcus pyogenes are most frequent. When the streptococcus is present in acute suppurations, there is great danger of mastoiditis. In chronic otitis the gas-forming bacteria, as well as Bacillus pyocyaneus, is often found. Nasal Cavity. — The nasal secretion, containing as it does dead cells and being alkaline in reaction, forms a good soil for the growth of germs. Diplococcus coryzae, Micrococcus nasalis. Bacillus foetidus ozsenee, BacUlus striatus albus et flavus. Bacillus capsvilatus mucosus, and Vibrio nasalis are some of the organisms de- scribed by various observers. Stomach and Intestine. — The secretion of the stomach is in its normal state not a favorable soil for the development of bacteria, yet some germs resist the action of the gastric juice and flourish in it. When the acids of the stomach are diminished in quantity or absent altogether, the conditions for the growth of bacteria are more favorable. The alimen- tary canal of the new-born infant is sterile, but in a few hours after birth microorganisms begin to appear. Some gastric bacteria normally present are Sarcina ventric- uli. Bacterium lactis aerogenes. Bacillus subtilis. Bacillus amylobacter. Bacillus megaterium. The intestinal organisms are more numerous, and the mucous lining of the intestines and the secretions there present are favorable to germ-growth. Bacillus geniculatus Boas considers a sign of carcinoma of the stomach, and is always present, he claims, when the con- tents contain lactic acid. Some investigators consider digestion dependent on micro- bic activity, but experiments with animals have shown that 26o ESSENTIALS OF BACTERIOLOGY life and digestion can proceed in a perfectly sterile condition. Food and air sterilized will not develop bacteria in the feces. In the feces of the young a great many bacteria have been found that are supposed to stand in close relation with the intestinal disorders common to nurslings. The majority of bacteria usually present in the intestines are non-pathogenic. The following varieties may be met with in the feces: Micro- coccus aerogenes, Bacillus subtUis, Bacillus butyricus, BacU- lus putrificus coli, Bacillus lactis aerogenes, BacUlus coli commune, Bacillus subtUiformis, and the bacteria of cholera, dysentery, and typhoid, besides many yeast-ceUs. Genito-urinary Passages. — In vaginal secretion Bumm has been able to find a number of organisms, some of which closely resemble the gonococcus; thus, there is the Diplococ- cus subflavus. Micrococcus lacteus faviformis, Diplococcus albicans amplus, and the vaginal bacillus. In the urethra of healthy persons bacteria are sometimes found, usually having entered from the air. In the normal secretions around the prepuce a bacillus called the smegma bacillus has been discovered. The spiro- chaete of syphilis can be obtained from lesions about the genitalia. From urethral pus a number of diplococci other than the gonococci have been isolated. From the urine itself a great number of bacteria have been obtained, but mostly derived from the air, finding- in the urine a suitable soil. The colon and typhoid bacilli gain entrance into the bladder, possibly by way of the urethra, and produce cystitis. In a larger number of typhoid fever patients the bacilli are foimd in the urine. Microorganisms of the Blood. — Many of the bacteria described in this book are found in the blood of the animal infected; anthrax bacilli are always found in the blood. When animals are subcutaneously injected with pneumo- cocci they are found in large quantities in the blood. The diseases of a hemorrhagic nature affecting fowls and swine usually show the presence of bacteria in the vascular system. GERMICIDES, ANTISEPTICS, AND ANTISEPSIS 261 Bacteria may be recovered from the blood in all forms of septic infection, such as general sepsis, malignant endocar- ditis, puerperal sepsis, and typhoid fever. Tubercle bacUli are rarely if ever obtained from the blood. Staining Blood Specimens. — A drop of blood is spread on a cover-glass and stained with the ordinary dyes; but in order to eliminate the coloring-matter of the red corpuscles and bring the stained bacteria more prominently into view, Gunther recommends that the blood, after drying and fixing, should be rinsed in a dilute solution of acetic acid (i to 5 per cent.). The hemoglobin is thereby extracted, and the cor- puscles appear then only as faint outlines. Instead of "fixing" by heat. Canon employs alcohol for five minutes, especially in staining for influenza bacilli, which have been detected in the blood. Blood Cultures. — ^As large a quantity of blood as pos- sible—never less than 10 c.c. — is taken from a superficial vein, the median basilic, for example, by means of a sterUe antitoxin syringe, a small incision being made through the skin over the vein in order to avoid skin infection. The blood so obtained is immediately transferred to culture-tubes, where the organisms are allowed to develop, and are then studied in the customary manner. CHAPTER XXXIV GERMICIDES, ANTISEPTICS, AND ANTISEPSIS Sunlight, pure air, and ordinary soap and water are effec- tive disinfectants. Too often the burning of chemicals and the dipping of hands into antiseptic solutions partake of the nature of religious sacrifice, and the more nauseous the odor, the more effective is the incense supposed to be. Much of the perfunctory fumigation by the boards of health after the 262 ESSENTIALS OF BACTERIOLOGY minor contagious diseases, instead of teaching the people a lesson, create a false impression of security, and permit them to neglect the commoner means of ordinary cleanliness because of this assumed virtue of fumigation. The whole subject of fumigation and quarantine regulation needs more careful investigation and study. A germicide is an agent capable of destroying bacterial life. An antiseptic solution or substance is one that can inhibit or prevent the growth of bacteria without necessarily de- stroying them. A disinfectant must be germicidal. A deodorant may have no germicidal or antiseptic properties. Preservatives are substances which prevent fermentation, but they are not always germicides. In considering the value of a germicide, the strength in which it acts is the main consideration. Some very weak chemicals will inhibit and destroy the growth of bacteria if used in sufficiently concentrated solutions. Some bacteria will die in an acid medium; others are destroyed by too much alkali. Some bacteria are very readily destroyed in pure cultures, but are resistant to a considerable degree in the body tissues. Again, a germicide may be ideal in laboratory experiments, but wholly impractical at the clinic. A I : 300,000 solution of mercuric chlorid (corrosive sub- limate) will prevent the development of anthrax spores, but a I : 1000 solution is needed to destroy them. Germicides are tested by action in various dilutions or in gaseous form on threads impregnated with virulent and spore- forming organisms. The length of time is noted that it takes to destroy anthrax bacilli or pyogenic organisms. The infected material is subjected to the solution and then inoculated on media and compared with control, or tested for virulence on animals. Spore-forming organisms are very resistant to the most potent agents. Heat is perhaps the best general germicide. For all articles that can be subjected to boiling or the direct flame there is no safer agent. GERMICIDES, ANTISEPTICS, AND ANTISEPSIS 263 Superheated steam, or steam under pressure, is now in general use in sterilizing surgical dressings and instruments, and requires less time than ordinary steam. The salts of metals of high atomic weights come next in order. Bichlorid of mercury and cyanid of mercury are the most powerful of chemical germicides, but in the human body they can be used in dilute solutions only, and in contact with highly albuminous solutions, insoluble and inert albuminates are liable to form, lessening the germicidal value. A i : 200 solution combined with an acid like citric will destroy the spores of anthrax in one hour, but much weaker solutions will destroy the anthrax bacilli in the blood, and for all practical purposes a i : 2000 solution is sufficient, destroying bacterial life in a few minutes. One per cent, solution soda lye, NaOH, kills most bacteria in a few minutes, and, therefore, hot soapsuds is quite effec- tive as a germicide. Phenol in 5 per cent, solution will destroy most of the bac- teria in less than five minutes. Tricresol, a combination of cresols, has three times the disinfecting power of phenol. Formaldehyd, in gaseous form or in a Uquid spray, is a very efficient germicide, and from the fact that it is not destruc- tive to fabrics or paper has come into general use as a dis- infectant. In combination with potassium permanganate or in suitable generators it is employed in houses after infec- tious diseases. It has no effect on insects, and where it is necessary to destroy these, other agents, known as insecti- cides, must be used in connection with the gas. The gas should be in a moist state — ^from 6 to 16 oxmces for an ordi- nary room are needed; the room should be made as air-tight as possible, and the gas evolved as speedily as possible. In the permanganate method 8 oimces (by weight) of potassium permanganate crystals are placed in a large tin vessel ten times the capacity of the disinfectant used. One pint of formaldehyd solution is quickly poured over the crystals. Formaldehyd gas is thereby generated at once. 264 ESSENTIALS OF BACTERIOLOGY This will produce enough gas for disinfection of 1000 cubic feet. Solid formaldehyd in the form of candles is useful for small rooms, and some health boards employ it exclusively. Sulphur dioxid, or sulphurous acid gas, is a germicide and insecticide, and is much used in disinfecting ships after yellow fever and malaria. It is obtained by burning sulphur in a pan over water, and about 3 pounds to 1000 cubic feet are necessary. Copper sulphate, i part to 1,000,000 of water, is effective in destroying algae, and is useful in large reservoirs as a tem- porary disinfectant. Alcohol, iodin, chlorin, potassium permanganate, hydrogen dioxid, the salts of silver, lead, and zinc, salicylic acid, boric acid, anilin dyes (methyl-violet and methylene-blue) , naphthalin, and creosols are a few of the substances in use as antiseptics and germicides in surgery. Their power varies with the strength of the solution and all have limitations. In surgical operations more dependence is placed today on securing and maintaining a germ-free or aseptic condition than on the attempt to destroy germ life by chemicals. The irritation of antiseptics in some instances prevents the natural body defenses (phagocytes) from acting, and in abdominal operations, where no pus has been encountered, the blood- serum is stifficient or normal salt solution is alone used. Sterilization of Hands, etc. — ^It has been shown by elaborate experiments that the skin, the hair, and clothing harbor many bacteria, some of a pathogenic nature. The surgeon who is anxious to secinre good results should carefully attend to his toilet; the use of operating gowns, rubber gloves, operating shoes, face guards is now universal. The toUet of the hands of the surgeon is as important as that of the field of operation, but with the use of rubber gloves the painstaking directions as to the emplojnnent of a half-dozen or more cleansing agents and germicides are no longer followed. Soap is an eflEicient germicide, the lye being in most cases powerful enough to prevent the growth of germs. GERMICIDES, ANTISEPTICS, AND ANTISEPSIS 265 Filtration.- — In the laboratory, and on a larger scale in the management of water-works, filtration is a method of steril- ization, acting as it does by mechanically separating bacteria from a solution. General Measures for Disinfection. — For discharges — urine, feces, sputum, vomitus — solution of phenol, 5 per cent., also fresh milk of lime, i part lime to 4 parts water. Lime is of value only when sufficient alkali present. Blankets, woolen clothing, soiled handkerchiefs, liaen, boiling in steam, for- maldehyd gas, or hot-air exposure. Articles of little value shoiild be burned. Books can be sub- jected to formaldehyd vapor or immersed in gasolene. The hands and body washed in strong soapsuds and then in I : 1000 mercuric chlorid solution. Tincture of iodin, for the skin and hairy parts, painted over the field of operation, has come into vogue as a very efficient antiseptic. Woodwork and floors should be washed with soapsuds and I : 1000 solution of mercuric chlorid, the room itself subjected to formaldehyd vapor. Testing the Value of Disinfectants. — Rideal-Walker Standard. — For comparing one disinfectant with another, they are compared with phenol solutions of known strength in their action on a culture of some microorganism (the Bacillus typhosus is now used in most laboratories). A standard temperature of 20° C. has been adopted by the workers of the United States Hygienic Laboratory, and some changes have been made by them in the Rideal-Walker method, so that it is referred to as the "Hygienic Labor- atory Phenol Coefficient." The medium is made of beef-extract, according to the American Health Association standard, and must have a reaction of -|-i-S in test-tubes containing 10 c.c. each of the mediiun. The organism is a twenty-four-hour-old filtered broth culture of the Bacillus typhosus. Temperature of cultures and dilutions must be brought up to 20° C. 266 ESSENTIALS OE BACTERIOLOGY One-tenth of a cubic centimeter of the culture is added to 5 c.c. of the disinfectant dilution. The phenol control is made of different dilutions, from 5 to 10 strengths being employed. The disinfectant to be tested is likewise diluted, depending on the solubility, etc. An accurately graduated pipet distributes x5' c.c. of the culture to each one of the dilutions, both of the phenol control and the test, and the tubes are then shaken gently three times. At intervals of two and one-half minutes a platinum loopful (the loop 4 mm. in diameter) is transferred from each tube and planted in the tube of broth medium. The inoculated tubes are then placed in an incubator at 37° C. for forty- eight hours, and at the end of this time results are recorded. The coefficient is determined and recorded as in the ex- ample here given. Example. Name of disinfectant to be tested, A. Temperature, 20° C. Culture used. Bacillus typhosus, o.i c.c. to 5 c.c. disin- fectant. Time Exposed in Minutes Phenol: I : 80 . . I : 90 . . I : 100. I : no. Disinfectant I :3So- I :37s- I : 400 . I : 500 . I :650. -f- -I- + + + + + iH + + + + + -t- + IS The weakest disinfectant dilution that kills within two and one-half minutes (1-375) is divided by the weakest phenol GERMICIDES, ANTISEPTICS, AND ANTISEPSIS 267 dilution (i:8o), thus -^ = 4.69, and the same is done for the Strength that kills in fifteen minutes, namely: -^= 5-91 no The average of these, ^ — ^— — = 5.30, is called the co- efficient. In other words, disinfectant A has a value of 5.30 times that of phenol. A disinfectant with a phenol coefficient less than 1 is of very low germicidal value. 268 CHIEF CHARACTERISTICS CHIEF CHARACTERISTICS PART I.— This classification into non-pathogenic and pathogenic is not strictly correct, as special Name. Genus. Biology. Product ACETI. Bacillus. Short motile rods in zooglea; aerobic. Ferment. ACIDI LACTICI. Bacillus. Short, immotile rods; aerobic. ACIDI LACTICI. Streptococcus. Short, immotile, oval cocci. ACTINOBACTER. Bacillus. Immotile rods with capsule; facul. an- aerob. Aerogenes. Bacillus. Identical with B. acid lactici. Aerophilus. Bacillus. Slender rods in threads ; immotile; oval spores; aerobic. Agilis. Micrococcus. Mobile diplococci with fine flagella. Red pigment. Alba. Beggiatoa. Cocci and spirals with sulphur. Small cocci in packets Alba. Sarcina. White pigment. Albicans amplus. Micrococcus. Large cocci and dip- lococci. Albicans tardissi- Micrococcus. Diplococci colored by MUS. Gram. Albicans tardus. Micrococcus. Diplococci not motile. Allii. Bacillus. Very small rods. Alkaloid pigment. Amyliferum. Spirillum. Rigid spirilla with spores; turns blue with iodin. Amylobacter. Bacillus. See Buiyricum, with which it is identical. Aquatilis. Micrococcus, Very small cocci in ir- regular groups. Arachnoidea. Beggiatoa. Very thick filaments containing sulphur; motile. Arborescens. Bacillus. Thin rods, with round- ed ends in threads, andsingly ;immotile. Yellow pigment. Attenuatum, Spirillum. Threads with narrow- ed ends. Aurantiaca. Sarcina. Small cocci in pairs Orange-yellow pig- and tetrads; strongly ment. aerobic. Aurantiacus. Bacillus. Motile, short thick Orange-yellow pig- rods, often in long ment. threads. OF THE PRINCIPAL BACTERIA 269 OF THE PRINCIPAL BACTERIA. NON-PATHOGENIC BACTERIA. many of the non-pathogenic varieties have disease-producing properties under conditions. Culture Characters. Actions. Habitat. Discoverer. Not Uquefy; membran- Produces acetic-acid Air. Kiitzing. ous growth. fermentation. Not liquefy; small white Lactic-acid fermenta- Air; sour milk Pasteur. points, porcelain-like ; tion; precipitates slow. casein. Growth faster than above Alcohol is formed af- Sour milk. Grotenfeldt. appearance same. ter the lactic-acid fermentation. Causes fermentation Air. Duclaux. with gas and alcohol. Miller. Liquefy rapidly; small Old cultures. Liborius. yellow-gray colonies. Slowly liquefying, form- Drinking-water. Ali-Cohen. ing a cone with rose- red color. Sulphur springs. Vauch. Slow growth in small Air and water. Zimmerman. white colonies. Slowly hquefy; gray col- Is colored by Gram's Vaginal secretion. Bumm. onies; growth fairly method. rapid. Small white points, not Urethral pus. Bumm. liauefying; very slow growth. Grows slowly on surface. Skin in eczema. Unna, the boundary raised; Tommasoli. twice as large as above. Bright-green pellicle on Decomposes albumin. Green shme of Griffiths. agar. onions. Water. Van Tiegham. Light-yellow colonies ; Old distilled water. Bolton. serrated edges. Sulphur water. Agardh. Colonies, radiating from London Water- Frankland. an oval center like works. roots ; later on colored yellow; slowly liquefy. Stagnant water. Warming. Rapidly liquefy; little Air and water. Koch. orange-yellow colonies. not growing in high temperature. Slowly growing; nail cul- Water. Frankland. tures; shining and orange-yellow; not li- quefy. 270 CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. Biology. Product. AURANTIACUS. Micrococcus. Oval cocci in pairs and Orange-yellow pig- singly ; immotile. ment in water, al- cohol, and ether; insoluble. Aurea. Sarcina. Cocci in packets. Golden-colored pig- ment; soluble in alcohol. Aureus. Bacillus. Straight motile rods Golden-yellow pig- lying parallel. ment. Balticus. Bacillus. Short rod. Phosphorescence. BlENSTOCKII. Bacillus. See Putrificus, coli. BiFIDUS. Bacillus. Slender diplococcus, pointed ends, non- motile, anaerobic. BiLLROTHII. Micrococcus Groups of cocci sur- (ascococcus). rounded with cap- sule; zooglea, aero- bic. Brunneus. Bacillus. Motile rods. Brown pigment. Butyric-acid fer- Bacillus. Large, slender motile Diastase. mentation. rodsinpairs;spores; facul. anaerobin. BuTYRicuM (amy- Clostridium. Thick motile rods en- Amyloid substance. lobacter). 1 a r g i n g for the spores; obligate aerobic. C^RULEUS. Bacillus. Rods in long chains. Blue pigment, not sol- uble in water, alco- hol, or acid. Candicans Micrococcus. Masses of cocci. • . . • (Candidas). Carotarum. Bacillus. Threads of rods that bend in various di- rections; oval spores Catenula. Bacillus. Motile rods with spores. Caucasicus. Bacillus. Motile rods, with spores in each end. Cerasinus siccus. Micrococcus. Very small cocci, singly and in pairs; aerobic. Cherry-red pigment. Cereus albus. Micrococcus. Cocci in short chains and bunches, colored by Gram. Cereus flavus. Micrococcus. Staphylococcus and streptococcus, and in zooglea, colored by Gram. Chlorinus. Bacillus. Large rods, motile. Green pigment, sol- green-colored , due uble in alcohol. to chlorophyll; aerobic. OF THE PRINCIPAL BACTERIA 271 Bacteria. — {Continued.) Culture Characters. Actions. Habitat. Discoverer, Round orange-yellow col- Water. Cohn. onies, mostly on sur- face; slow growth; not liquefying. JLiquefy; bright golden Exudate of pneu- Mace. layer on potato. monia. Slow-growing, chrome- Water and skin of Adametz and yellow, whetstone in eczema. Unna. shape; not liquefy. Do not liquefy; require Baltic Sea. Fischer. glucose for growth. Oval colonies after three Feces of infants Tissier. days on glucose agar. breast-fed. Creamy layer on surface Putrid broth. Cohn. of gelatin. Maize. Schroter. Liquefy rapidly; gray veil Casein precipitates Air, Hueppe. on surface of potato. and changed into butyric acid ; am- monia set free. Not cultivated. Forms butyric acid in Air, earth, and Prazmowski presence of lactic water. and Van acid. Tiegham, Liquefy; a deep-blue lay- Water. Smith. er on potato. Not liquefy; nail-shaped Air around old cul- Fliigge. in test-tube. tures. Rapidly liquefy on sur- Cooked carrots A. Koch. face, a network center and beets. on potato; round, light gray; grow rapidly. Causes albumin to ferment. Old cheese. Duclaux. Ferments milk, pro- Kefir; grain. Kern. ducing the kefir drink. On potato; rapidly form- Water. List. ing cherry-red scum. not developed on gela- tin. Not liquefy; small, wax- Pus. Passet. Hke drops; thick gray layer on potato; growth rapid. Not liquefy; dark-yellow Pus. Passet. colonies; wax-like ap- pearance. Liquefy; greenish-yellow Water. Engelman. colonies. 272 CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. Biology. Product. Chlorinus. Micrococcus. Cocci in zooglea. Green pigment, sol- uble in alcohol and water. CiNNABAREUS. Micrococcus. Large oval cocci in Brown-red pigment ■ pairs ; aerobic. foul odor. CiTREUS. Bacillus (asco- Straight and bent Citron - yellow pig- coccus). rods in bundles; motile. ment. CiTREUS. Micrococcus. Large round cocci in Cream-colored pig- chains of eight and ment. more. CiTREUS CONGLOM- Micrococcus. Diplococci and tet- ERATUS. rads; aerobic. Claviformis. Bacillus Small rods; spores; (tyrothrix) . true anaerobin. CLOAC-E. See Proteus. CONCENTRICUM. Spirillum. Thick motile spirals with flagella; aerobic. CORONATUS. Micrococcus. Cocci singlyandstrep- tococci; aerobic. CORYZM. Micrococcus. Large diplococci with rounded ends, the contact surfaces flat. Crepesculum. Micrococcus. Round and oval cocci, singly and in zo- oglea. Cyaneus. Micrococcus. Oval cells. Blue pigment. Cyanogenus (blue Bacillus. Motile rods in chains; Alkali and a pigment milk). spores; aerobic. deepened by acids. DiCHOTOMA. Cladothrix. Various forms — rods, spirals, and cocci, in long threads. DiFFLUENS. Micrococcus. Oval cocci; aerobic. Fluorescent pigment, soluble in water. DiSTORTUS. Bacillus Motile rods; spores; Alkali. (tyrothrix) . aerobic. Dysodes. Bacillus. Long and short rods; An odor resembling spores. peppermint and turpentine. Endoparagogicum. Spirillum. Dry motile spirals, joined in peculiar shapes. Erythrosporus. Bacillus. Motile rods and Greenish-yellow pig- threads; spores, ment. slender. FiGURANS Bacillus. Large motile rods; (mycoides). spores ;long threads; aerobic. FlUFORMIS. Bacillus Short motile rods; (tyrothrix). spores in one end. OF THE PRINCIPAL BACTERIA Bacteria. — {Continued.) 273 Culture Characters. Actions. Habitat. Discoverer. Yellow-green layer on Boiled eggs. Cohn. gelatin. Not liquefy; slow growth; Air and water. Fliigge. bright-red points. Slow growth; after two Skin in eczema. Unna and weeks small yellow Tommasoli. points which take va- rious shapes on potato; citron-yellow layer; growth more rapid. Dirty, cream-colored col- Water. List. onies, which are raised and moist. Lemon-yellow colonies. Dust and blennor- rhagic pus. Bumm. Ferments milk, giving Fermenting albu- Duclaux. rise to alcohol. min. Not liquefying; concen- Putrefying blood. Kitasato. trically disposed colon- ies; very slow growth; not growing on potato. A halo formed around Air. Fliigge. the colonies. White, raised glassy col- No pathogenic action. Acute coryzal se- Hajek. onies, at first like pneu- cretion. mococci, later culture flattened; not lique- fying. Putrefying infu- Cohn. sions. Bluish-green colonies. Cooked potatoes. Cohn. Not liquefying; small Changes milk to deep- Air of certain Fuchs. white colonies. blue color. countries. Cultivated in infusion of Water. Cohn. plants. Do not liquefy; small Air. Schroter. granular, yellow col- onies; green fluores- cence. Milk made viscid and Air. Duclaux. casein precipitated. Bread and yeast. Zopf. Trunk of worm- Sorokin. eaten tree. Does not liquefy; greSn Air and putrefying Cohn. fluorescence; white col- substances. onies. Liquefying; root-like pro- Garden-earth. Fliigge. cesses extending in the gelatin; feather form in test-tube. Causes casein to be Duclaux. precipitated from milk. 18 274 CHIEF CHAEACTEEISTICS Non-Pathooenic Genus. FiSCHERI. Bacillus. Fitzianus. Bacillus. Flava. Sarcina. Flavus. Bacillus. Flavus desidens. Streptococcus. Flavus liquefa- CIENS. Flavus tardi- GRADUS. Fluorescens fce- TIDUS. Micrococcus. Micrococcus. Micrococcus. Fluorescens liquefaciens. Fluorescens niva- lis. Bacillus. Bacillus. Fluorescens pu- TRIDUS. Bacillus. KOERSTERI. Cladothrix. Fcetidum. Clostridium. fcetidus. Fuscescens. FULVUS. Fuscus limbatus. Fusiforme. Geniculatus. GiGANTEUS URE- THRiE. Grass. See Tim- othy i Graveolens. HiEMATODES. Micrococcus. Sarcina. Micrococcus. Bacillus. Bacillus. Bacillus (tyrothrix) . Micrococcus. Bacillus. Biology. Short rods in threads ; spores as large as the rods. Small cocci in pack- ets. Small rods; immotile. Cocci and diplococci in chains; aerobic. Cocci and diplococci in zooglea. Cocci in short chains, and diplococci. Small diplococci. Short motile rods; very thin. Short rods; motile. Motile rods; short, with rounded ends. Threads twisted in spirals; very irreg- ular. Rods of varying length; very motile; a large spore in one end; anaerobic. See Crepesculum, with Round cocci. Short rods; very mo- tile; facultatively anaerobic. Spindle-shaped, with pointed ends. Rods variable length; spores. Streptococci in thick knots. Small rods, nearly as broad as they are long. Micrococcus. Cocci in little ^ooglea Product. Phosphorescence. Pigment. Pigment. Yellow-brown pig- ment. Pigment. pig- Chrome-yellow ment. Blue-green pigment: acids turn red. Green fluorescent pigment. Blue-green pigment. Green fluorescent pigment. Strong gas-produc- tion; very foul odor. which it is identical. Brown pigment. A bitter substance. Foul gas. Red pigment. or THE PRINCIPAL BACTERIA Bacteria. — {Continued.) 275 Culture Characters. Not liquefying; requires peptone for growth. Transparent on surface; dark center in the deep ; not liquefying. Liquefying. Liquefying ; yellow viscid colonies ; foul odor. Yellow porcelain-white colonies. Liquefying rapidly; yet- low colonies. Softens gelatin; yellow beads, isolated. Little button-like colon- ies that later on sink in, surrounded by vio- let-green color; lique- fying; growth rapid. Liquefying; white, sunk- en, iridescent colonies. Quickly liquefying; growth rapid; small white points; later on, surrounded by blue- green fluorescence. Not liquefying; transpar- ent at first, then green fluorescence and urin- ary odor. Liquefying; growth rapid; small colonies that soon become filled up with fluid and assume a spherical form. Conic rusty-red colonies. Small brown colonies along needle-track little branches; not liquefy, No growth on gelatin; on agar, thin drops; nearly transparent; very slow growth; in bouillon, a flaky precipitate. Liquefying; irregular grayish, later green- ish, colonies, with very foul odor. Grows best on white of egg at 37° C. ; red layer, Actions. Produces ethylic alco- hol in meat extract. Colors the glacial wa- ters green. Habitat. Unboiled hay-in- fusion. Vomited matter. Drinking-water. Air and old cul- tures; water. Air and old cul- tures; water. Air. Post-nasal space. WE^ter and air; conjunctival sac, In snow and ice of Norway. All putrefactions. Lacrimal canal. Old cheese and se- rum of mice in- oculated with garden-earth. Excrement of horse, In foul eggs. Spongy layer on sea-water. Air and mUk. Normal urine and urethra. Skin between toes. Sweat of man. Discoverer. Beyerinck. Zopf. de Bary. Mace. Fliigge. Fliigge. Fliigge. Klamann. Fliigge. Schmolck. Fliigge. Cohn. Liborius. Cohn. Scheiben- zuber. Warming. Duclaux. Lustgarten. Bordoni- Uffreduzzi. Zopf. 276 CHIEF CHARACTERISTICS Non-Pathogenic Name. Hansenii. Hay. See SubUlis. Hoffman's. See Hyacinth r. Hyalina. Ianthinus. Indicus. Intestinalis. Jequirity. KtJHNIANA. Lacteus favifor- MIS. Lactis erythrog- ENES. Leptomitiformis. Leucomel^num. LiNEOLA. LlODERMOS. Litoralis. LiTOREUS. LiVIDUS. LUTEA. LUTEUS. LUTEUS. Genus. Bacillus. Pseudodiphiheri Bacillus. Sarcina. Bacillus. Bacillus. Sarcina. Bacillus. Crenothrix. Micrococcus. Bacillus. Beggiatoa Spirillum. Bacillus. Bacillus. Merismopedia. Bacillus. Bacillus. Sarcina. Bacillus. Micrococcus. Biology. Product. Medium large rods. Yellow pigment; in- soluble. a. Short rods in dumb- bell shapes. Round cocci in groups of 4 to 34- See Bacillus violaceus. Short, motile rods; no Scarlet pigment al- spores; anaerobin tered by heat. facul. Very regular packets of cocci, eight in each. Medium-sized rods; Ferment called spores. abrin. Long threads, break- ing up into cocci. Theyareensheathed . Diplococci; not decol- orized by Gram. Short immotile rods; Yellow pigment and round ends. red pigment. Filaments medium size. Two or three spirals ; dark granular con- tents; clear spaces between. Short motile rods in zooglea, with flagel- la. Short motile rods; rounded ends. Cocci in groups of fours, containing sulphur- Oval rods, never in chains or zooglea. Medium-sized rods; Deep blue-black motile. pigment. Cocci singly and in Pigment citron-yel- fours. low. Short immotile rods. Pigment; soluble in with large oval water; acids inten- spores. sify. Oval cocci. Pigment, not acted upon by acid or al- kali. OF THE PRINCIPAL BACTERIA Bacteria. — {Continued.) 277 Culture Characters. Actions. Habitat. Discoverer. On potato, a yellow growth which changes with age. Yellow skin of nutrient infu- sions. Rasmussen. Slime of diseased hyacinth-bulbs. Marshes. Wakker. Kutzing. Liauefying; oval colon- ies; scarlet-colored. Intestine of mon- key. Koch. Intestine of fowls. Zopf. Colonies brick - colored from oxid of iron. Ferment causes oph- thalmia. Infusion of jequir- ity bean. Drinking-water of wells. Sattler. Rabenhorst. Not liquefying; white col- onies ; grow well on po- Mucus of vagina and uterus. Bumm. tato. Small, round yellow dots, later on cup-shaped, with rose-colored pe- riphery; liquefying. In red milk and feces. Sulphur waters. Grotenfeldt. Trgvisan. Water over rot- ting plants. Perty. Slimy layer on potatoes. Stagnant water. MuUer. Liquefying; transparent, then thick layer on po- tato; like gum. Air and potatoes. Sea-water. Flugge. Oersted and Rabenhorst. Sea-water. Warming. Ink-spot at first, slowly liquefying; blue-violet colored later on; slow Berlin Water- works. Flugge and Proskauer. growth. Not liquefying; little ele- vations; citron-yellow center; yellow layer on potato. Not liquefying; irregu- lar in form; golden- yellow colored. Do not liquefy; small citron-yellow colonies on potato. Air. Air. Air. Schroter. Fliigge. Schroter. 278 CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. LUTEUS. Micrococcus. Maid IS. Bacillus. Marsh. Spirillum. Megaterium. Bacillus. Melanosporus. Bacillus. Merismopedi- Bacillus. OIDES. M esentericus fus- cus (potato). Bacillus. MaSENTERICUS VULGATUS(pOtato) . Bacillus. Mesenteeoides. Leukonostoc. Miller's. Bacillus. MiNUTA. Sarcina. MlRABILIS. Beggiatoa. MULTIPEDICULOSUS Bacillus. Mycoides. See Rasmosus. Nasalis. Micrococcus. Navicui,a. Bacillus. NiTRIEICANS. Micrococcus. NiVEA. Beggiatoa. NODOSUS PARVUS. Bacillus. Biology. Diplococci very mo- tile. Rods with pointed ends, very motile; seldom in threads; oval spores. See Plicatile. Large motile rods ; spores; aerobic. Rods; aerobic. Threads of rods which are formed from cocci-like spores ; zooglea in packets. Small motile rods with spores. Thick motile rods in threads; spores. Masses of cartilagin- ous zooglea, com- posed of rods and cocci; arthrospores. Delicate rods, sUghtly curved; immotile. Cube-shaped packets. Very wide threads, rounded ends and curled; sulphur granules. Long, slender rods. Diplococci, motile; also streptococci. Spindle-shaped rods. Small cocci. Very thin filaments. Rods formed at an- gles; immotile. Product. Yellow pigment, turn- ing brown-red. Black pigment, not acted upon by acids or alkalis. Diastase. Amyloid material. Forms saltpeter. OF THE PRINCIPAL BACTERIA Bacteria. — {Continued.) 279 Culture Characters. Actions. Discoverer. Round, light-yellow col- onies, growing larger in a few days ; on pota- to a slimy covering with moldy odor; slowly liquefying. Gray points in deep, veil- like on surface; lique- fying; on potato, a wrinkled skin of brownish color. Yellow irregular masses; thick layer on potato. First gray, then black, pellicle. Liquefying; white colo- nies, ray-like periphery brown layer on potato. Yellow colonies, dark cen- ter, ciliary processes at periphery ; brown layer on potato, penetrating the substance. Liquefies; not growing on the surface. Grows slowly; reacts to iodin, turning blue. Insect-shaped colonies. Grayish points, raised, opaque; rapid growth; not liquefying. White flakes. Slow growth at ?,1° C; in agar a white line, which in the center becomes porous. In solutions of sugar an aldehyd is pro- duced. Coagulates milk and forms diastase out of starch. Converts molasses in- to a gelatinous mass. In maize and in pellegra; feces. Cooked cabbage. Air and potatoes. Stagnant water. Air and old pota- toes. Beet-root juice. Paltauf and Heider. De Bary. Eidam. Zopf. Fliigge. Flugge. Cienkowski. Caries of teeth. Miller. Sour milk. De Bary. Sea-water. Cohn. Flugge. Nasal space and secretion. Potatoes. Soil. Sulphur waters. Urethral secretion. Hack. Reinke and Berthold. VanTiegham Rabenhorst. Lustgarten. 28o CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. Biology. Product. Oblongus. Micrococcus. Motile cocci, singly and in filaments; aerobic. OCHROLEUCUS. Micrococcus. Cocci in pairs and packets; spores. Yellow pigment. Paludosa. Sarcina. Spheric, transparent, colorless cocci. Pasteurianus. PflOgeri. Bacillus. Bacillus. Differs from Bacillus aceti in that the cells contain an amyloid matter. Short rods in threads. Phosphorescence. Phosphorescens GELrous. Bacillus. Motile; round, short rods; aerobic. Phosphorescence. Phosphorescens INDICUS. Bacillus. Large motile rods. Phosphorescence. Phosphorescens, North Sea. Bacillus. Motile rods. Phosphorescence. Photometricus. Plicatile. polymyxa. Prodigiosus. Bacillus. Spirillum. Clostridium. Bacillus. Motile, red-colored rods. Long motile, thin spirals; round ends. Motile rods in threads with spores. Short motile rods; aerobic. Sulphur and red pig- ment caused by light. Amyloid colored blue by iodin. Red pigment, sol- uble in alcohol trimethylamin. Proteus mirabilis. Bacillus. Very motile, short rods; aerobic. Proteus vulgaris. Bacillus. Rods sometimes curved as spirillum. Proteus Zenkeri. Bacillus. Motile rods. PSEUDO-DIPHTHE- RLE (Hoffman) . Bacillus. Small rods, similar to the true bacillus; immotile. Putrificus coli. Pyogenes tenuis. Bacillus. Micrococcus. Slender motile rods; long threads; spores. Radiatus. Bacillus. Motile rods with rounded ends; an- aerobic oval spores. Strong-smelling gas. OF THE PRINCIPAL BACTERIA 281 Bacteria . — {Continued . ) Culture Characters. Habitat. Discoverer. Grows best in cultures to which glucose and am- monium tartrate have been added. Liquefying; slow growth; thin yellow membrane; sulphurous odor. Not liquefying; requires glucose; grows well on potato. Not liquefying; grows best with glucose and salt. Liquefying; grows best at 30° C. Liquefying; colonies look as if punched out; grows best at 15° C. Movements depend upon light. Thick skin on potato. Little red colonies; lique- fying rapidly; espe- cially abundant on potatoes. Liquefying slowly; opaque center, irregu- lar processes. Liquefying quickly. Not liquefying; thick white layer on potato. Grows at ordinary tem- perature, rapidly form- ing on surface a brown- ish growth; pin-head colonies raised above surface; not Uquefymg, On agar, a glassy growth. Liquefying; growth rap- id; colonies like molds, from center radiating in all directions and through the gelatin; the air must be ex- cluded. Causes gluconic fer- mentation. Urine. Water from sugar- factory. Heavy beers. Putrid meat and fish. I Tropical seas. Water around Kiel. Causes fermentation in dextrin solutions. Not virulent. Decomposes albumin. Not pathogenic. Stagnant water. Bread and pota- toes. Putrefaction. Putrefaction. Putrefaction. In diphtheric membrane and normal pharynx. Human feces. Closed abscesses. In serum of white mice inoculated with earth. Prove. Schroter. Hansen. Ludwig. Forster. Fischer. Fischer. Engelman. Ehrenberg. PrEizmowski. Ehrenbeig. Hauser. Hauser. Hauser. Wellenhof. (Hoffman.) Bienstock. Rosenbach. Liideritz. 282 CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. Biology. Product. Radiatus. Streptococcus. Small cocci in chains. Ramosus liquefa- Bacillus. Motile rods. CIENS. Reitenbachii. Merismopedia. Cocci in packets or plates; colorless cell- w a 1 1 containing chlorophyll. ROSACETTS. Micrococcus. Large cocci in pairs and tetrads. Spheric cocci in cubi- Red pigment. Rosea. Sarcina. cal packets. Rosea perseina. Beggiatoa. Long rods with cocci- Pigment called bac- shaped bodies in teriopurpurjn. them, containing sulphur and a red pigment. ROSEUM. Spirillum. Very short curved Pigment soluble in al- rods; motile and cohol. spores. Ruber. Bacillus. Motile rods in groups. Brick-red pigment. RUBRUM. Spirillum. Motile; short spirilla; aerobic. Pale-rose pigment. RUFUM. Spirillum. Long motile spirals. Red-rose pigment. Rugula. Spirillum Motile rods, in long (vibrio) . spirals, singly and in chains, with flag- ella and spores ; an- aerobic. Saprogenes. Bacillus. Large rods, terminal spores ; facultatively anaerobic. SCABER. Bacillus Short motile rods in Tyrosin and leucin (tyrothrix) . chains; spores; aerobic. are formed. Scheurlen's. Bacillus. Short motile rods ; spores. Septicus. Bacillus. Non-motile rods in threads and spores ; anaerobic. Serpens. Spirillum. Long, lively threads, with three windings. SiMILIS. Bacillus. Immotile rods; trans- parent spores. Spinosus. Bacillus. Large motile rods; spores; true anaero- bin. SUBFLAVUS. Micrococcus. Diplococci colored by Gram's fluid. SUBTILIFORMIS. Bacillus. Immotile rods in threads; transparent! spores. ' OF THE PRINCIPAL BACTERIA 283 Bacteria. — {Continued.) Culture Characters. Actions. Habitat. Discoverer. Liquefying; white colon- Air. Fliigge. ies with greenish tinge; funnel-shaped in test- tube. Liquefying ; concentric Air. Flugge. colonies ; funnel-shaped in test-tube. Caspary. Not liquefying; small red Air. Flugge. knobs, with fecal odor. Marshes. Schroter. Marshes. Zopf. Not liquefying; thick Blennorrhagic pus. Mace. violet colonies; deep red on potato. . Boiled rice. Frank. Not liquefying; grows Dead mice. Esmarch. slowly; pale-rose col- onies. Stagnant water. Perty. Liquefying rapidly ;round Causes cellulose to Vegetable infu- Miiller. yellow dots with zone; ferment. sions and tartar fecal odor. of teeth. Grows slowly; foul odor. Putrefaction, Rosenbach, Duclaux. Growth best at 39° C; In carcinomatous Scheurlen. slowly liquefying on and normal potato ; a yellow mamma. wrinkled skin, under- neath whichared color. Putrid blood. Klein. Stagnant water. Miiller. Grows rapidly. Human feces. Bienstock, Liquefying; spiny periph- Albuminous decom- Garden-earth. Liideritz. ery; foul odor due to position. methylmercaptan . Liquefying; yellow dots. Vaginal secretion and lochia! dis- cbarges. Bumm. Grows best at 37° C. Human feces. Bienstock. 284 CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. Biology. Product. SuBTiLis (hay ba- Bacillus. Large motile rods, cillus). three times longer than broad, in threads, with flag- ella and spores; aerobic. Syncyaneus. Bacillus. Same as Cyanogenus. Synxanthus (yel- Bacillus. Short , thin motile Yellow pigment, solu- low milk) . rods. ble in, water; simi- lar to anilin colors. Tenue. Spirillum. Large motile spirals with fiagella. Tenuis. Bacillus Motile rods in long (tyrothrix) . chains; spores. Termo. Bacillus. Short motile, cocci- like rods in zooglea. TUMESCENS. Bacillus. Shortrods with spores. TURGIDUS. Bacillus Short immotile rods Carbonate of ammo- (tyrothrix). in long chains; spores; aerobic. nium. Ulna. Bacillus. Very large rods in chains and singly; not very motile; large spores. Undula. Spirillum. Long motile spirals, with fiagella. UREiE. Bacillus. Short rods; spores; Ferment, propyla- aerobic. min. Urin^. Sarcina. Small cocci in fami- lies. Cylindric motile rods Urocephalus. Bacillus (tyrothrix) . with spores; anae- robic. VENTRICtJLA. Sarcina. Cubic packets of 8 to 64 cocci. Ventriculi. Bacillus. Rods motile, often in bundles of four. Versicolor. Micrococcus. Small cocci. ViOLACEUS. Bacillus. Motile rods, round Violet pigment, sol- end; spores. uble in alcohol. Viola CEus. Bacillus. Immotile rods, form- Violet pigment, like ing large spores. anilin. Virens. Bacillus. Straight rods ; spores; Supposed to contain immotile; green chlorophyll. tinged. or THE PRINCIPAL BACTERIA Bacteria.- — (Continued . ) 28s Culture Characters. Liquefying; gray center, wreath - like border; thick layer on potato. In boiled milk a yellow pigment is formed. Liquefying; opaque cen- ter, yellow layer next, and the periphery lobed ; funnel-shaped in test-tube. On boiled carrots a wrin- kled, gelatinous disk. A pellicle formed on sur- face of milk; a heavy precipitate beneath. On boiled egg little zoog- lea. Resembling a globule of fat ; grows well in mu- cous urine. Not liquefying. Round colonies with dark center; slow growth; not liquefying. Not liquefying; irides- cent yellow surface. Not liquefying; center deep violet; color re- mains on agar a long time. Liquefying; transparent colonies, surrounded by violet zone. Actions. Precipitates casein; forms a pellicle on milk. Splits urea into am- monii carbonas. Peptonizes albumin. Habitat. Soil and dust, hay, etc. Boiled milk and potatoes. Stagnant water. Fermenting cheese and milk. Connected with putrefaction of plants. Boiled carrots. Fermenting milk and cheese. Putrefying water and boiled eggs. Discoverer. Ehrenberg. Ehrenberg. Ehrenberg. Duclaiix. Dujardin. Zopf. Duclaux. Cohn. Vegetable infu- Miiller. sions. Stale urine. Miquel. Bladder. Welcker. Fermenting milk Duclaux. Contents of stom- ach. Stomach of dogs fed on meat. Goodsir. Raczynssky. Air. Fliigge. Water. Zopf. Boiled potato and water. Schroter. Stagnant water. VanTiegham 286 CHIEF CHARACTERISTICS Non-Pathogenic Name. Genus. Biology. Product. ViRESCENS. Bacillus. Short motile rods Deep-green pigment, with flagella very turning yellow- broad. brown. ViRGULA. Bacillus Slender immotile (tyrothrix) . rods; spores aerobic. ViRIDIS. Bacillus. Little immotile rods; oval spore, which is tinged green. Viscosus. Bacillus. Motile rods, rounded ends, usually in pairs. Green pigment. Viscosus. Micrococcus. Streptococci of glob- Gummy substance ular cells. called viscosa, and ferment. VlTICULOSUS. Micrococcus. Oval cocci in large groups. \'OLUTANS. Spirillum. Long spirals with flagella. ZOPFII. Bacillus. Long motile rods, breaking up into spores like cocci. PART II,— Name. Genus. Biology. Product. Aerogenes Capsu- Bacillus. Usuallyfound in pairs. Gas with character- LATUS. resembling diplo- cocci , capsulated ; obligate anaerobe. istic odor. Bacillus. Rods like colon and Produces alkali in typhoid, motile. mannite and milk. Alvei. Bacillus. Rods with large spores. Amylovorus. Micrococcus. Oval cells, never in chains. Forms butyric acid. Anthracis Symp- Bacillus. Large slender rods Rancid odor. TOMATICI. with swellings at spore; anaerobic. Anthracis. Bacillus. Straight rods, slightly concave ends; im- motile; aerobic ; spores. T^xalbumin. Articulorum Micrococcus. Oval cocci in long (diphtheriticus). chains, identicEil with pyogenes. OF THE PRINCIPAL BACTERIA Bacteria. — {Concluded.) 287 Culture Characters. Actions. Habitat. Discoverer. Deep round colonies, the Green sputum. Frick. vicinity colored green; grows on surface; slow growth ; not liquefying. Milk. Duclaux. Water. VanTiegham. Rapid growth, liquefying ; Water and earth. Frankland. small hair-like pro- cesses from colonies; later on, viscid and in threads, with green fluorescence. Mucoid fermentation Beer and wine. Pasteur. in wine and beer. Not liquefying; a fine Air. Flugge. network in the colony; mucoid layer on potato. Marshes. Ehrenberg. Not liquefying; forms Intestinal c n- Kurth. thick coils like braided tents of fowls. hair. PATHOGENIC BACTERIA. Culture Characters Actions. Habitat. Discoverer. Acid reaction in litmus Causes fermentation; Intestinal con- Welch. milk; coagulates casein can produce gas tents; earth; with cavity-formation from proteid alone. water; raw due to gas- foods. Colonies like typhoid. Feces and water. Petruschky. Liquefying; growths ra- Produces a disease in Larvae of bees. Cheshire and diating from center bees called "foul Cheyne. downward; on potato brood." a dry yellow layer. "Fire-blight" in pear Burrill. trees. Liquefy gelatin; grow Causes quarter evil in Blood and tissues. Bollinger- only in atmosphere of hydrogen. Liquefying; granular col- animals. Causes splenic fever in Found in tissues Rayer and onies with irregular animals; malignant and excreta of Davaine. border; on potato a pustule in man. diseased animals. dry, creamy layer; in test-tube a thorny, prickly track. Grows well on gelatin; Fatal in mice and rab- Mucous membrane Loffler and pale-gray colonies; not bits. of diphtheria. Cohn. liquefying; slow growth on potato. 288 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. AviSEPTicus. See Hemorrhagic Se pticemia. BOMBYCIS. Micrococcus. Oval cocci in chains and zooglea; motile. Bordet-Gengou. See Whooping- cough. BOTULINUS. Bacillus. Large rounded ends; Butyric acid; and a motile; flagellated; powerful toxin. anaerobic. BoviSEPTicus. See Hemorrhagic Se pticemia. Bubonic Plague Bacillus. Short thick rods with (Pestis). indistinct capsule. BUCCALIS. Leptothrix. Long threads in thick bundles, containing masses of cocci and spirals. Catarrhaus. Micrococcus. Diplococci at times resembling gono- coccus. Cattle Plague See Hemorrhagic Sep ficemia and Swine (Texas fever). Cavicida. Bacillus. Little rods twice as Propionic acid long as broad. through decomposi- tion of sugar. Chauv^i. See Symptomatic A nthrax (Rauschbra nd). Cholera Asiatics. Spirillum. Motile, spiral-shaped Ptomain-like mus- rods, often in chains; carin and toxalbu- very short flagella min, soluble in on ends, and strictly water. aerobic; spores have not been found. Cholers galli- Bacillus. Immotile, cocci-like Toxalbumin. narum (chicken rods ; without spores ; cholera) . strictly aerobic. Cholera nostras Spirillum. M otile , comma-shaped (Finckler). rods; strictly aero- bic. COLI COMMUNIS. Bacillus. Short motile rods, slightly curved. without spores; fac- ultatively anaerobic. Crassus sputig- Bacillus. Short, thick rods with ENUS. rounded ends. Decalvans. Micrococcus. Spheric cells in great numbers. OF THE PRINCIPAL BACTERIA 289 BACT:E.mA.~~{ConHnued.) Culture Characters. Actions. Habitat. Discoverer. Causes "flacherie" in Intestines of silk- B6champ. silkworms. worms. Gelatin colonies appear Sausage and meat Intestine of pig. Van Ermen- as small semi-trans- poisoning. gera. parent spheres. Does not liquefy gelatin; Causes bubonic plague Tissues, body Yersin and white, point-like col- fluids, and secre- Kitasato. onies turning gray and tions of plague then brown. patients. Causes dental caries. Teeth sHme. Robin. At 37° on agar, round, Not pathogenic. Mucous secretions R. Pfeiffer. gray colonies, serrated healthy persons. edges. Plague. Not liquefying; irregular Kills guinea-pigs. Human feces. Brieger. scale-like colonies, mak- ing the gelatin viscid. Liquefying slowly, small Causes cholera Asia- Feces of cholera ' Koch. depressed scars giving tica in man and a patients. a frosted appearance, sim.ilar trouble in or like ground glass; animals. on potato, atthin brown layer; in test-tube, a funnel-shaped lique- faction, with a bubble of air in the top, the funnel taking six or seven days to form well. Not liquefying; small iso- Causes chicken chol- Blood and feces of Pasteur. lated white disks; in era in fowls; not diseased fowls. test-tube, a granular acting on man. track; very faint. Liquefying rapidly; col- Harmless in man; fatal Feces of cholera Finckler and onies yellow-brown to guinea-pigs. nostras and ca- Prior. thick masses; in test- ries pf teeth. tube, funnel formed in twenty-four hours, dissolving all gelatin in two days; profuse gray mass on potato. Not liquefying; dark cen- Fatal to guinea-pigs Feces of nursing E^cherich. ter, undulated per- and rabbits; causes infants; . water; iphery; green-colored diarrhea in man; choleraic stools. layer on potato; milky ferments sugar. layer on surface of test-tube. Not liquefying;, oval. Mice and rabbits die Sputum. Kreibohm. grayish, slimy colonies; in forty-eight hours nail-shaped growth in with gastro-enteritis. test-tube. Causes alopecia area- ta. In roots of hair. Thin, 19 290 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. Dentalis viri- DANS. Bacillus. Slightly curved rods, ■ round ends. Gray pigment. Diarrhea of In- Bacillus. Motile, medium-sized Toxalbumin. fants. rods; spores; aero- bic. Diarrhea of Meat-poisoning (Enteritidis sporo- genes) . DIPHTHERIvE. Diphtheria of Calves (Vitu- lorum). Diphtheria in Pigeons (Colum- barum) . DiPLOBACILLUS OF Conjunctivitis. Bacillus. Rods in groups of two and singly; round ends; spores. Bacillus. Bacillus. Bacillus. Bacillus. Immotile, middle- sized rods, rounded ends; facultative anaerobic. Long rods in threads. Short rods in groups. Non-motile; usually occurs in pairs. Toxalbumin. Duck Cholera. Bacillus. Similar to chicken cholera bacillus; immotile. Dysenteric. Dysentery (epi- demic). Bacillus. Bacillus. Resembles typhoid bacillus. Short motile rods; very thin. First slightly acid, then alkaline. Enteritidis. Bacillus. Resembles typhoid bacillus. Enteritidis sporo- GENES. See Diar Erysipelas of Swine C Rot hlauf ; rouget du pore). rhea of Meat Pot Bacillus. soning. Small, slender motile rods ; facultatively anaerobic. Two vaccines, which give immunity. FCETIDUS OZJENJE. Bacillus. Short rods, very mo- tile; in pairs and chains. Foul gas. Frog Plague. Gangrene. Bacillus. Micrococcus. See Swine Plague. Oval cocci in zooglea. Gigantea. Leptothrix. Long rods, cocci and short rods in one; thread also spiral. or THE PRINCIPAL BACTERIA B ACTEia A . — {Continued. ) 291 Culture Characters. Actions. Habitat. Discoverer. Not liquefying; round, sharply outlined col- onies, \vith bluish gray opalescence. Not liquefying; green colonies with foul odor. Not liquefying; little yel- lowish colonies; a mem- branous layer on po- tato. Whitish patches. Addition of blood-serum to media necessary; liquefying. Small round yellow col- onies like wax-drops; not liquefying. Resembles typhoid bacil- lus in many respects. Not liquefjang; concen- trically arranged colon- ies; dry yellow mem- brane on potato. Resembles typhoid ex- cept that it ferments dextrose. Very delicate silver-gray clouds on the gelatin, like bone-cells; not liq- uefying; in test-tube a very faint clouding. Small greenish colonies which soon become liquefied and indistin- guishable; a foul odor produced. Grayish colonies with foul odor. Septic processes and death in mice and pigs. Causes green diarrhea in animals when in- travenously injected, and is the cause of green diarrhea in in- fants. Causes death in ani- mals, with symptoms of septicemia. Gives rise to diphthe- ria in man and ani- mals. When inoculated in mice causes death. Necrosis in pigeons and other animals. Found in subacute conjunctivitis. Fatal for ducks, but not for chickens or pigeons; less active than chicken chol- era; causes diarrhea and exhaustion. Produces one variety of dysentery. The cause of epidemic dysentery in man; enteritis in guinea- pigs. Produces enteritis in man and animals. Causes erysipelas in swine and other ani- mals; the German ' ' Rotlauf , ' ' French "rouget du pore." Mice are killed by in- jection; rabbits af- fected with progres- sive gangrene. Causes caries of teeth. In caries of teeth. Feces of infants suffering from green diarrhea. Blood and juices of choleraic diar- rhea. Diphtheric exu- date. Diphtheric mem- brane of calf. Diphtheric mem- brane in pigeons, Conj unctival se- cretion. Blood of diseased ducks. In dysenteric stools. In feces and mes- enteric glands. Intestinal con- tents; its toxin in meat of dis- eased animals. Blood and organs of diseased ani- mals. Secretion of per- sons suffering from ozena. Gangrenous tissue. Diseased teeth of animals. Lesage. Klein. Loffler (Klebs). LofEer. Comil and Toupet. Shiga. Chantemesse and Widal. Hajek. Eberth. Miller. 292 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. Gingivae p y g- Bacillus. Short thick rods with ENES. rounded ends. Glanders (Rotz, Bacillus. Slender, im motile Mallei). rods; usually singly; spores; facultative- ly anaerobic. GONORRHCE^ Micrococcus. Diplococci kidney- (Gonococcus) . shaped; motile; do not color with Gram. Grouse Disease.. Bacillus. Small rods and oval cocci in chains; im- motile. H^MATOCOCCUS Diplococcus. Cocci seldom in BOVIS. chains; surrounded by a pale zone. HiEMOPHILIA NEO- Micrococcus. NATORUM. Hemorrhagic Bacillus. Short rods, twice as Septicemia (In- long as broad; im- fectious Pleuro- motile. pneumonia, Wild Plague, German Swine Plague, Cattle Plague, Steer Plague, Rabbit Septice- mia). Hog Cholera Bacillus. Very motile oval rods, Peptonizes milk with. (Swedish swine similar to hemor- out coagulation. plague) . rhagic septicemia. Icteroides. Bacterium. Once supposed to be the cause of yellow fever. Identicalwth Sanarelli. Influenza. Bacillus. Very minute rods or in clumps. INSECTORUM. Micrococcus. Oval cells in chains and zooglea; strep- tococci. Intracellularis Diplococcus. Resembles gonococ- Meningitidis. cus in morphology and arrangement in interior of leuko- cytes. Koch-Weeks. Bacillus. Resembles influenza bacillus. Lactisaerogenes. Bacillus. Short, thick immotile rods. OF THE PRINCIPAL BACTERIA Bacteria. — {Contin tied.) 293 Culture Characters. Growth rapid; liquefying; round colonies, visible to naked eye in twenty- four hours. Light yellow, like honey, colonies, turning red- brown in a few days. Grow on blood-serum. Not liquefying; small scales wMch turn gray in a few days, the edges serrated. Best at 38° C; not lique- fying ; small white points; sparse growth on potato; transparent. White isolated pinhead points, not growing on potato; best at 37° C., not liquefying. Very good growth on gel- atin and potatoes; a yellow-brown color. Grow best on blood-agar colonies very small, almost transparent. Transparent colonies, forming thin layer on LofHer's blood-serum and glycerin agar. Rarely grows, except on serum agar. Small, porcelain-like disks with depressed center; funnel-shaped in test-tube with gas. Actions. Fatal to mice, with septic processes. Glanders is caused by the bacillus in man and animals. Gonorrhea in man. Fatal for mice and guinea-pigs. Fatal for rabbits and rats; hyperemia of lungs and spleen ; blood - exudate in peritoneal cavity. Supposed to be the cause of the disease. A disease having dif- ferent names in dif- ferent animals.char- acterized by edema, hemorrhage, and septicemia. In experiment, ani- mal's death in four to eight days; bac- teria in little emboli in capillaries. Produces epidemic in- fluenza. A contagious disease in the chinch-bug. Causes epidemic cere- brospinal meningi- tis. Most common cause of acute contagious conjunctivitis. Fatal to guinea-pigs and rabbits; coagu- lates milk; decom- poses sugary solu- tions. Habitat. Suppurating pulp of tooth. In epithelium and ulcerated glands. Gonorrheal pus; in pus-cells and epithelium. In blood and or- gans of diseased grouse. Blood and organs of animals dis- eased with hemo- globinuria. Found in this dis- ease. Blood and serum of diseased ani- mals. Not spread through tissue.butin cap- illaries of dis- eased swine. Secretions of res- piratory tract. Stomach of chinch-bug. In cerebrospinal fluid and nasal secretions. Conjunctival se- cretion. Feces of nursing infants and of cholerine. Discoverer. Miller. Lofiler. Neisser. Klein. Babes. Klebs. Hiippe. Salmon and Selander. Pfeiffer, Kitasato, Canon. Burrill. Weichsel- baum. Koch and Weeks. Escherich. 294 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. Lepr^. Bacillus. Slender, immotile rods with pointed ends. LiQUEFACIENS CON- JUNCTIVA. Micrococcus. Single cocci; never in threads. Lupus. Bacillus. Same as Tuberculosis. Malignant Edema (Gangrenous Sep- ticemia, Vibrio Septique). Bacillus. Large, slender rods, rounded ends, often in threads; motile, with flagella and spores ; strongly anaerobic. Soluble vaccine. Mammitis of Cows. Micrococcus. Oval cocci in chains; streptococci; facul- tatively anaerobic. Mammitis of Sheep. Micrococcus. Streptococci and in fours. Melitensis (Malta Fever). Micrococcus. Sju in diameter; occurs singly or in chains of two or more; said to be flagellated. Metchnikovi Spirillum (vibrio). Motile spirals with flagella; aerobic. An alkaline vaccine which will cause im- munity. Neapoutanus. Bacillus. Small immotile rods, with rounded ends; no spores; faculta- tively anaerobic. Produces acids in gel- atin cultures. NOMiE. Bacillus. Small rods, with rounded ends, grow- ing often in long threads. OhKM. Bacillus. Motile aerobic; does not Uquefy gelatin. Alkali in milk. OXYTOCUS PERNI- CIOSUS. Bacillus. Short rods with round ends. Paratyphoid. Bacillus. Resembles typhoid bacillus. Indol sometimes pro- duced. Perfringens. See Aerogenes capsu \atus. Pestis. See Bubon ic Plague. OF THE PEINCIPAL BACTERIA Bacteria. — {Continued.) 295 Culture Characters. Actions. On blood-serum round white plaques with ir- regular borders. Liquefying; growth rap- id ; colonies on surface, with little radiating branches from a dark center; those in deep, berry-shaped. Liquefying; thick center, radiating periphery; in high culture in test- tube, gas-bubbles arise, with foul odor. Not liquefying; brown, round granular colon- ies; grows slowly; in test-tube, heavy de- posit along the needle's track. Liquefying; round cen- ters with zone of lique- faction; cone-shaped in test-tube. Small, round, slightly raised disks; do not hquefy. Grows quickly; colonies, some like cholera As- iatica, others like chol- era nostrEis; Uquefying. Not liquefying; thin pearl like scales in several layers; wrinkled and mucous layers on po- tato. Granular spheric colonies in the deep, flat on the surface; not liquefying; growth rapid; best at 35° C. Whitish growth on cul- ture-media. Small yellow granular colonies; nail-culture in test-tube. Ferments glucose, but not lactose or saccharose; does not coagulate milk. Causes leprosy in man and animals. On cornea of rabbits causes slight cloud- ing. Animals quickly die with extensive gan- grene and edema. Causes contagious mammitis in cows: coagulates milk. Causes contagious gan- grenous mammitis in sheep. Causes Malta fever. Causes vibrion septi- cemia in guinea-pigs and pigeons. Causes death in some animals; not the cause of cholera. No action on mice or rabbits. Causes olive-gall on olive plant. Intravenous injection causes death in mice and rabbits; turns milk acid. Causes continued fevers. Habitat. Leprous tissue. Normal human conjunctiva. Garden-earth. Mammary gland. Found in the milk of diseased sheep. Best obtained from spleen. Feces of fowls. Cholera epidemic of Naples, 1884. In necrotic tissue of noma. Sour milk. Intestinal c on- tents. Discoverer, Hansen. Gombert. Pasteur. Nocard and Mollereau. Nocard. Bruce. Gamaleia. Emmerich. Schimmel- busch. Wyssokow- itsch. Widal, Gwyn, Schott- miiller. 296 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. Pneumonia (Pneu- Bacillus. Short, immotile rods, mococcus of Fried- singly or in diplococ- lander). ci, surrounded with capsule; no spores; not colored with Gram; facultatively anaerobic. Pneumonia (Pneu- Bacillus. Short, oval rods, of- mococcus of Fran- ten in chains ;,immo- kel; Micrococcus tile ; no spores ; in the of Pasteur). tissue surrounded with capsule, col- ored with Gram ; facultatively anae- robic. Pneumonicis Bacillus. Short, thick motile AGILIS. rods in pairs. Proteus septicus. Bacillus. Slightly curved rods, swelled in portions, sometimes in long threads; motile. Foul gas. PsiTTACi (perni- Micrococcus. Streptococci and ciosus) . zooglea. Pyocyaneus. Bacillus. Thin, motile rods; fa- Pyocyanin, a non- cultatively anaero- poisonous pigment. bic. Pyocyaneus /3. Bacillus. Forms a brown- yel- low pigment; other- wise identical with above. Pyogenes (Strepto- Micrococcus. Streptococci and coccus erysipela- zooglea. tis — Fehleisen). Pyogenes albus. Micrococcus. Staphylococci and streptococci; facul- tatively anaerobic. Pyogenes aureus Micrococcus. Staphylococci and Ptomain, toxalbumin, (micrococcus of zooglea; faculta- and pigment. osteomyelitis, tively anaerobic. Becker). Pyogenes citreus. Micrococcus. Same as Pyogenes au- reus. Pyogenes fcetidus. Bacillus. Short motile rods in pairs. Pyogenes tenuis. Micrococcus. Cocci without defin- ite arrangement. Relapsing Fever Spirillum. Long, wavy spirals; (Obermeier). motile. OF THE PRINCIPAL BACTERIA Bacteria. — (Continued.) 297 Culture Characters. Actions. Habitat. Discoverer. Does not liquefy; grows quickly; a button-like colony; in test-tube, as if a nail driven in the gelatin with head on surface. Does not liquefy; grows slowly; small, well-de- fined masses; in test- tube, little separate globules, one above the other. Liquefying; dark granu- lar colonies ; thick sed- iment in test-tube. Growth rapid; liquefy- ing ; colonies have foul odor, are small, thick branches, but soon all liquid. Liquefying; large, flat colonies with greenish fluorescence; on po- tato, yellow-green skin; deeply coloring the pulp. Not liquefying; round punctif orm colonies ; slow-growing. Liquefying; white opaque colonies. Liquefying; small colon- ies with a yellow- orange pigment in cen- ter; yeast-like smell; a moist layer on potato. Colonies, citron-yellow color. Not Uquefying; mucous layer on potato; very thick; in test-tube, a slight layer on surface, and small points along the track. On surface, transparent thin growth; grows slowly. Cannot be cultivated. An accompaniment of pneumonia, not a cause; animals not affected. Causes pneumonia in man, septicemia in animals; also serous inflammations in man, as pleurisy, peritonitis, etc. Pneumonia in rabbits, Fatal for mice in one to three days. Causes disease in gray parrots. Fatal for animals ; col- ors the dressings green. Suppuration and sep- ticemia in animals. Suppuration and ab- Causes abscesses and suppuration in man and animals. Suppuration. Fatal to animals. Causes fever in man and animals, and is the cause of relaps- ing fever, Pneumonic and other sputum, and lung tissue. Sputum of lung affections and se- rous inflamma- tions. From rabbits' pneumonia. From a child dy- ing of intestinal gangrene. In blood of par- rot's disease. Pus. Pus. Pus. Pus. Pus. Pus. Friedlander. A. Frankel. Schon. Wolff. Gessard. Ernst. Rosenbach. Rosenbach. Rosenbach. Passet. Pus of abscesses. Rosenbach. Blood of man dur- ing an attack of the disease. Obermeier. 298 CHIEF CHARACTERISTICS Pathogenic Name. Rhinoscleroma. Salivarius pyog- Salivarius septi- cus. Salivarius septi- cus. Saprogenes No. XL Saprogenes No. III. Saprogenes fceti- DUS. Senile Gangrene. Septicemia after Anthrax. Septicemia Mice. Septicemia of Rabbits (Cuni- culicida). Septicus acumina- tus. Septicus agrig- enus. Septicus liquefa- CIENS. Septicus ulceris. Genus. Bacillus. Micrococcu3. Bacillus. Micrococcus. Bacillus. Bacillus. Bacillus. BacillL Micrococcus. Bacillus. Bacillus. Micrococcus. Bacillus. See Pneumococcus of Very small round coc- ci and staphylococ- ci. Short, immotile rods, encapsulated in pairs, sometimes long chain; aerobic. Cocci singly and in zooglea; aerobic. Short rods; faculta- tively anaerobic. Very short rods ; fac- ultatively anaero- bic. Immotile rods; spores. Thin rods; immotile; singly and in pairs; ends somewhat thickened; aerobic; spores. Motile streptococci. Smallest bacillus known; immotile. See Hemorrhagic Septi Thin , lancet-shaped rods; very slender. Very short rods. Streptococci and dip- lococci. Oval rods; motile. Product, Friedlander, with Foul gas. Foul gas. Foul gas. Gas ; no odor. OF THE PRINCIPAL BACTERIA Bacteria. — {Continued.) 299 Culture Characters. Actions. Discoverer. which it is identical. Slowly liquefying; small white opEilescent col- onies. Not liquefying; gray cir- cular colonies; trans- parent zone; in test- tube, separated. Not liquefying; round colonies; separated dots in test-tube. Grows quickly; on agar, hyaline drops which quickly coalesce, and form a mucoid layer with a foul odor, that of perspiring feet. Forms a fluid gray band on agar; odor of pu- trefaction. Not liquefying; thin, transparent layer; pu- trid odor. Round yellow colonies; liquefying in thirty-six hours; best growth at 37° C. In bouillon virulence de- stroyed. Not liquefying; small flocculent masses in the deep; grows. very slowly; in the test- tube producing a faint cloud. - At 37° C. on blood-serum small transparent plates; later on. turn- ing yellow. Not liquefying; brown center, a ring, the^i yellow zone. Liquefying; a thin gran- ular streak, the surface sunken in; later, cone- hke, the walls covered with leaf-shaped col- onies. Liquefying; yellow coU onies, taken up with gas later on. Local abscess in ani- mals. Fatal to animals. Fatal to animals. Produces septicemia in rabbits. Suppuration in rabbit, Rabbits killed with large doses. Causes gangrene in mice, similar to se- nile gangrene of man. Septicemia in rabbits, but not in chickens or guinea-pigs. Septicemia in house- mice, but not field- mice. Saliva. Frischl. Biondi. Saliva of healthy Biondi. persons. Saliva of puer- peral women. Sweat df feet. Putrid marrow of bone. Mesenteric glands of swine with erysipelas and of healthy swine. In gangrenous tis- sue and blood of senile gangrene. Blood of animal dead from an- thrax. Putrefying liquids. Pathogenic for rabbits and guinea-pigs; fev- er; and bacilli in blood and organs. Septicemia in mice and rabbits. Pathogenic for mice and rabbits, produc- ing edema, in the serum of which the cocci abound. An ulcer in inoculated In blood of child animals, followed by with gangrenous paralysis and death. | ulcer. Navel stump of child dead of septicemia. Earth of recently plowed fields. Blood and organs of child dying of septicemia. Biondi. Rosenbach. Rosenbach. Schottelius. Tricomi. Koch. Babes. Babes. 300 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. Septicus vesicae. Bacillus. Rods always single; very motile; oval spores. Smegma. Bacillus. Slender curved rods, identical with what was known as syph- ilis bacillus of Lust- garten. Soft Chancre. Bacillus. Minute oval rods, chiefly in groups or chains. Sputigenum. Spirillum. Curved, comma- shaped rods ; motile. SUBFLA\TJS. Micrococcus. Diplococci like gono- cocci; colored by Gram. Swine Plague Bacillus. Motile, oval rods, Causes casein precipi- (American and similar to that of tate in milk and acid French). hog cholera. formation. SyCOSIFERUS FtETI- Bacillus. Short, straight immo- On potatoes a foul DUS. tile rods, often in threads. odor. Syphilis (Spiro- Spirocheta. Small delicate spirals, cheta pallida). difficult to stain. Tetanus. Bacillus. Large, slender motile Ptomains, tetanin, rods, with spores in tetanotoxin, spas- one end, drumstick motoxin; alsoatox- shape, often in albumin. threads; true anae- robic. Tetragenus. Micrococcus. Large round cells, uni- ted in groups, usual- ly of four, and sur- rounded by a cap- sule; immotile; aerobic. Timothy Grass. Bacillus. Extremely acid-fast; resembles tubercle bacillus; in cultures may show club for- mation and branches Toxicatus. Micrococcus. Cocci singly and in pairs. OF THE PRINCIPAL BACTERIA Bacteria. — (Continued.) 301 Culture Characters. Actions. Habitat. Discoverer. Not liquefying ; small pin- Pathogenic for mice In urine of cys- Clado. head colonies, growing and rabbits, produc- titis. slowly; never larger; a ing death. brown center, yellow periphery. Not cultivated. Normal preputial Alvarez and secretions. Tavel. Has not been cultivated. Produces soft chancre. In the sore. Ducrey. Not cultivated. Causes death in ani- In caries of teeth Lewis. mals. and saliva. Growth slow; liquefying; No result on mucous Normal secretion Bumm. on tenth day yellow membrane; injected of vagina and points with thready under skin, abscess urethra. boundary; on potato, a results. brown, thread - like growth aftertwo weeks. Not liquefying; growth Found in American Found in capil- Billings, similar to typhoid and French swine laries in little Rietsch, and germ ; on potatoes plague, in frog emboli ; not Eberth. good growth. plague, and Texas spread in organs fever- animals af- of diseased ani- fected locally. mals. Slow growth; not liquefy- On human skin causes From sycosis of Tommasoli. ing; after four days, lit- eruption, vesicular the beard. tle white points, which around hairs, then do not change for sev- it becomes pustular; eral weeks, then the similar to sycosis. superficial ones are mucus-like; nail growth; on potatoes. rapid growth. On blood-serum thin Supposed to cause In tissue and se- Schaudinn. growth. syphilis. cretions of syph- ilitics. Liquefy gelatin slowly; Produces tetanus in Earth and manure. Nicolaier and colonies have radiated man and animals. Kitasato. appearance; a thorny growth along the track in test-tube. Not liquefying; little por- Fatal to guinea-pigs Found in cavern- Gaffky. celain-like disks ; thick and white mice. ous phthisical slimy layer on potato. lungs. Colonies visible in thirty- May produce tuber- Infusions of tim- Moeller. six hours, scale-like cles. othy grass. and grayish white. Supposed to be the Found in the Rhus Burrill. cause of Rhus (pois- toxicodendron. on ivy) poisoning. 302 CHIEF CHARACTERISTICS Pathogenic Name. Genus. Biology. Product. Tuberculosis. Bacillus. Slender rods, usually in pairs; not motile; spores not definitely determined; facul- tatively anaerobic. Kochin or paratolin, a glycerin extract of the pure culture (tu- berculin). Typhoid. Bacillus. Slender motile rods, sometimes in threads ; flagella, but no spores ; facultatively anae- robic. Typhotoxin and toxalbumin. Typhoid of Swine (swine plague) . Tyrogenum. Spirillum (vibrio) . See Swine Plague. Spiral-shaped rods ; aerobic. Whooping-cough. Bacillus. Short oval. Welchii. Xerosis. See AUrogenes Bacillus. capsulatus. Similar to diphtheria bacillus. OF THE PRINCIPAL BACTERIA Bacteria . — {Concluded . ) 303 Culture Characters. Actions. Grows best on blood-ser- um and glycerin agar at 37" C, forming little white crumbs on the surface; under micro- scope a hairy, ma,tted coil is seen; growths on potatoes when air- tight have been ob- tained. Not liquefying; little whetstone-shaped yel- low colonies inthedeep. and leaf-shaped ones on the surface; on po- tato, a very transpar- ent, moist layer. Liquefy rapidly; small round colonies; dark funnel-shaped liquefac- tion in test-tube. Blood agar. White growth on surface. Differs from diphtheria bacillus in not produc- ing acid in bouillon. Causes tuberculosis, local and general, in man and lower ani- mals. Gives rise to enteric or typhoid fever in man. Several animals have died from inocula- tions. Produces spasmodic cough in animals. In all organs and secretions of tu- bercular persons. Found in dejecta and spleen and urine of typhoid patients. From old cheese. Found in whoop- ing cough. Found in patho- logic conditions of conjunctiva, sometimes in normal eye. Discoverer. Koch. Eberth. Bordet- Gengou. Kuschbert and Neisser. INDEX Abb£'s condenser, 44 Achorion schonleinii, 224 Acid, boric, 264 curd cheese, 254 dyes, 48 salicylic, 264 Acid-fast bacteria, 116 Actinomyces, cladothrix, 228 streptothrix, 228 Actinomycosis, 228 Aerobes, 28 facultative, 28 obligate, 28 Aerobioscope, 234 African tick fever, 218 Agar^ bile salt, MacConkey's, 75 blood-, 76 fuchsin-Iactose, 77 glycerin-, 69 lactose litmus, 72 nutrient, 71 Agar-agar, 69 Age, influence of, on bacteria, 30 Agglutination test for tubercle ba- cilli, 121 in cholera, 152 in typhoid fever, 140 Pfeiffer's cholera, 153, 154 Agglutinins, 40 Aggressins, 32 Air, bacteria in, 232 Hesse's method of collecting, 233 Petri method of collecting, 232. 233 Sedgwick-Tucker method of collecting, 234 varieties, 234 examination of, 232 sewer, bacteria in, 235 Albumin, blood, solution dried, 78 Alcohol, 264 fermentation, 255 Alcoholic solution, saturated, 49 Alexin, 40 Algae, fission, 21 Alkaline anilin- water solutions, 51 methylene-blue, 50 stains, 49 Alkaloids, cadaveric, 34 Allergy, 42 Altered reactivity, 42 Alum, 236 Amboceptors, 40, 42 Amebae, examination for, 199 Ammonia production, 245 Amceba coli, ig8 dysenterise, 198 Anaerobes, 28 facultative, 28 obhgate, 28 true, 28 Anaerobic bacteria, 180 cultivation, 82 Botkin's method, 84 Buchner's method, 84 Esmarch's method, 83 Frankel's method, 84 Hesse's method, 83 Liborius's method, 82 Park's method, 85 Roux's method, 84 Wright's method, 85 Anaphylaxis, 42 Anilin dyes, 47, 264 acid, 48 basic, 48 Anilin-oil, 48 water, 49, 50 dyes, so 30s 3o6 INDEX Animal inoculation, 91 celloidin sacs, 93 cerebral membranes, 93 cutaneous, 91 eye, 92 guinea-pigs, gi intraduodenal, 93 intraperitoneal, 92 intratracheal, 93 intravenous, 92 methods, 91 obtaining material after, 94 subcutaneous, 92 Animals as culture-media, 81 tuberculosis in, 119 Anthrax 105 symptomatic, 187 Anthraxin, 109 Antiformin method of collecting tubercle bacilh, 117 Antigens, 41 Antisepsis, 261, 262 Antiseptics, 261, 262 Antistreptococcic bacterins, 166 serum, 166 vaccines, 166 Antitoxic serum, 39 unit, 133 Antitoxin, formation of, accord- ing to lateral-chain theory, 37 of diphtheria, 131 specific action, 38 tetanus, 183 unit for, 183 Antituberculous serum; 122 Antityphoid bacterins, 139 vaccines, 139 Arnold's steam sterilizer, 64 Artesian well water, 236 Arthrosporous bacteria, 26 Artificial cultivation, 62 Asbestos, 236 Asexual cycle in man, 199 Aspergillus flavus, 226 fumigatus, 226 glaucus, 226 Asporogenic bacteria, 26 Autoclave, 64, 65 Avicidus bacillus, 193 Azofication, 245 Bacillaey dysentery, 199 Bacillus acidi lactici, 249 aerogenes capsulatus, 186 amylobacter, 251 amylovorus, 232 anthracis, 105 avicidus, 193 avisepticus, 192 Boas-Oppler, loi botulinus, 144 bovisepticus, 192 butyricus, 251 capsule, of PfeifEer, 159 chauvei, 187 coli, 134 diagnostic points of, 241 in milk, Wisconsin test for, 255 in urine, 102 in water, examination for, 240 colon, 134 bacillus typhosus and, differ- entiation, 142 comma, of cholera, 148 cyanogenes, 251 diphtheria, 126 products of, 131 dysenterias, 145, 199 products, 147 enteritidis, 135 sporogenes, 187 fluorescens, 171 hay, 100 icteroides, 219 Klebs-Loffler 126, Koch-Weeks, 161 lactis aerogenes, 249 lepra, 122 mallei, 124 megaterium, 99 melitensis, 162 mesentericus vulgatus, gS Milzbrand, 105 murisepticus, 195 mycoides, 99 oedematis, 184 maligni, 184 of anthrax, 105 of bluish-green pus, 171 of bubonic plague, igo of chancroid, 178 INDEX 307 Bacillus of chicken cholera, 193 of cholera, 148 of diphtheria, 126 products, 131 of dysentery, 145 products, 146 " of enteric fever, 135 of erysipelas of swine, 194 of fowl septicemia, 193 of glanders, 124 of influenza, 160 of lepra, 122 of malignant pustule, 105 of pertussis, 161 of phlegmonous emphysema, 186 of rhinoscleroma, 159 of soft chancre, 178 of splenic fever, 105 of symptomatic anthrax, 187 of tetanus, 180 of typhoid fever, 135 paracolon, 143 paratyphoid, 143 perfringens, 187 pestis, 190 pneumonias, 159 potato, of Flugge, 98 proteus vulgaris, 147 pyocyaneus, 171 ramosus, 99 root, 99 rotz-, 124 saccharobutyricus, 187 Shiga, 199 smegma, 123 subtilis, loj suipesti'jr, 133 suiseri-icus, 192 tubercle, no products of, 120 tuberculosis, no products of, 1 20 typhosus, 13s colon bacillus and, differentia- tion, 142 from blood, 143 in water, 138 diagnostic points, 242 products, 139 violaceus, 102 Bacillus vulgatus, 98 Welchii, 186 Wurzel, 99 X, 219 Bacteremia, 33 Bacteria, acid-fast, 116 aerobic, 28 anaerobic, 28, 180 cultivation of, 82 Botkin's method, 84 Buchner's method, 84 Esmarch's method, 83 Frankel's method, 84 Hesse's method, 83 Liborius's method, 82 Park's method, 85 Roux's method, 84 Wright's method, 85 and soil fertility, 244 arthrosporous, 26 atsporogenic, 26 avenues of entrance, 32 biologic activities, 26, 28 Brownian movements, 23 causing disease, 29 chemic activities, 26 composition, 22 products, 29 chief characteristics, 268-303 cholera, 148 classification, 22 colonies of, growth and appear- ances, 87 colon-typhoid group, 133 cultivation, 62 defenses to invasion, 34 development of, TJi diseases from, 29 distribution, , 26 effects of, general, 33 local, 33 [ endosporous, 24 facultative, 27 fermentation by, 29 filterable, 218 fluorescence of, 30 gas-forming, 30 general effects, 33 growth of, 27 how cause disease, 30 in air, 232 3o8 INDEX Bacteria in air, Hesse's method of collecting, 233 Petri method of collecting, 232,. 233 Sedgwick-Tucker method of collecting, 234 varieties, 234 in artesian well water. 236 in blood, 260 in butter, 254 in buttermilk, 255 in cheese, 254 in condensed milk, 255 in conjunctiva, 258 in cream, 248 in ear, 259 in feces, 260 in filtered water, 236 in food, 246 in genito-urinary passages,- 260 in ice-cream, 248 in intestine, 259 in milk, 246, 249 in mouth, 258 in nasal cavity, 259 in pneumonia, 155 in sewer air, 235 in skin, 257 in soil, 232, 243 in stomach, 259 in urethra, 260 in urine, 102, 260 in vagina, 260 in water, niethod of exatninEjtion, 238^ varieties, 238 in, well water, 2^6 infective, 33 influence of age on, 30 of electricity on, 28 pf heat on, 28 of hght on, 28 of moisture on, 28 of oxygen on Ufe and growth of, 27 of Rontgen rays on, 28 of temperature on life and growth, 27 local effects, 33 locomotion, 23 methods of studying, 43 Bacteria, nitrification by, 29 non-pathogenic, 29 table of, 268-287 odors from, 30 of hemorrhagic septicemia, 192 origin, 26 oxidation by, 29 parasitic, 27 pathogenic, 29, 97 table of, 287-303 phosphorescence by, 30 pigmentation by, 30 plant diseases due to, 231 pyogenic, 33 . j , quantity of, infection depend- ing on, 32 reduction by, 29 reproduction, 24 sewage, examination for, 240 specific, 33 nature, 27 spore contents, 25 ^ formations, 24 staining of, 47 sltjucture of, 21, 22 su^ura-tive, 33 tableV of, 268-303 types, V '■ ultra-mitroscopic, 218 unstained^-, exammation of, 45 vibratory ^lovements, 23 Bacterial vacciines, 95 treatment oft sewage, 243 Bactericides, 4r Bactericie du dSaFbon, 105 Bacterins, 95 anticholera, 153 antistreptococcic, 166 antityphoid, 139 counting of, 96 preparation of, 95 standardization of, 95 Bacteriologic examination of or- gans and cavities, 257 Bacteriolysins, 41 Bacterium acidi lactici, 249 bulgaricum, 250 caucasicus, 250 coli commune, 134 guntheri, 250 lactis acidi, 250 INDEX 309 Bacterium prodigiosum, 97 as cancer remedy, g8 saccharobutyricus, 251 syncyanum, 251 termo, 147 tumefaciens, 232 Zopfii, 100 Basic dyes, 48 Beer fermentation, 256 Beggiatoa alba, 228 Bichlorid of mercmry, 62 Biedert's method of collecting tubercle bacilli, ii5 Bile, lactose-, 76 salt agar, MacConkey's, 75 Biologic activities, 26, 28 Black-leg, 187 Blastomyces, 223 Blastomycetes, 220, 222 Blastomycetic dermatitis, 223 Blender, 44 Blood albumin, solution dried, 78 bacillus typhosus from, 143 bacteria in, 260 coagulum, 74 cultures, 261 specimens, staining, 57, 261 Blood-agar, 76 Blood-serum, 69 coagulation of , 7 2 human, preparation of, 74 mixture, Loffler's 74 nutrient, preparation of, 72 preservation, in liquid state, 74 sterilization of, 73 test, Gruber-Widal, 140 Bluish-green pus, bacillus of, 171 Boas-Oppler bacillus, loi Boiled eggs, 78 Boiling as means of purifying wa- ter, 238 Bordet's cholera test, 155 Borer, Frankel's, 244 Boric acid, 264 Botkin's method of cultivating an- aerobic bacteria, 84 Bouillon filtrate, Denys', 120 guinea-pig, 78 Bovine farcin du bceuf, 231 tuberculosis, human tuberculo- sis and, relation, 1 19 Bowhill's orcein stain, 60 Bread mash, 69 Brill's disease, 219 Broth, nitrate, 70, 71 nutrient, 71 sugar, 71 Brownian movements, 23 Bubonic plague, 190, Buchner's method of cultivating anaerobic bacteria, 84 Budding fungi, 220 Buerger's method of staining cap- sule, 61 Butter, bacteria in, 254 Buttermilk, bacteria in, 255 Cadaveric alkaloids, 34 Calmette's ophthalmic tuberculin reaction, 121 Cancer remedy, bacterium prodigi- osum as, 98 Capsule bacillus of Pfeiffer, 159 of spore, 25 stain, 61 Buerger's method, 61 Hiss' method; 52, 61 of Welch, S3 Carbolfuchsin, 49 Carbolthionin stain, 52 Carriers, cholera, 153 typhoid, 139 Catarrhal conjunctivitis, 258 Cattle-fever, Texas, 208 Cell-contents, 22 Celloidin sacs for animal inocula- tion, 93 Cell- wall, 22 Cerebral membranes, animal in- oculation by, 93 Cerebrospinal meningitis, epi- demic, 177 Chancre, soft, 178 Chancroid, 178 Charbon symptomatique, 187, 189 Charcoal sponge, 236 Cheese, bacteria in, 254 Chemic activities, 26 composition, 22 products of bacteria, 29 Chicken cholera, 193 3IO INDEX Chlorin. 264 Cholera, 148 bacteria, 148 carriers, 153 chicken, 193 comma bacillus of, 148 hog, 133 red, 152 Chromatin stain, Wright's, for ma- larial organisms, 204 Chromium trioxid for staining spores, 60 Cladothrices, 227 Cladothrix actinomyces, 228 dichotoma, 227 Classification, 22 Clostridium butyricum, 251 Coagulating ferments, 30 Coagulation of blood-serum, 72 Coagulum, blood, 74 Cocci, pyogenic, 163 Coley's fluid, 166 Colitis contagiosa, 135 Colon bacillus, 134 bacillus typhosus and, differ- entiation, 142 Colon-typhoid group, 133 Colonies, growth and appearances, 87 impression of, 88 macroscopic appearance, 87 microscopic appearance, 88 Combiner, 42 Comma bacillus of cholera, 148 Complement, 40, 42 deviation of, 42 fixation of, 42 Completer, 42 Concentrated staining solutions, 48 Condensed mUk, bacteria in, 255 Condenser, Abbe's, 44 Conjunctiva, bacteria in, 258 Conjunctivitis, catarrhal, 258 diplobacillus of, 171 epidemic, 161 Conradi-Drigalski medivun, 76 Copper sulphate, 264 Copula, 40 Corrosive sublimate, 262 Cotton plugs or corks, 66 Cover-glass preparations, 54 Cream, classification of, 248 Crenothrix kuhniana, 227 Cresols, 264 Crown gall, 232 Cultivation, 62 artificial, 62 of anaerobic bacteria, 82 Botkin's method, 84 Buchner's method, 84 Esmarch's method, 83 Frankel's method, 84 Hesse's method, 83 Liborius's method, 82 Park's method, 85 Roux's method, 84 Wright's method, 85 Culture-media, inoculation of, 78 nutrient, preparation of, 67, 70 reaction, 70 solid transparent, 69 sterilization of, 62, 70 Cultures, blood, 261 fresh egg, 77 glass slide, 78 plate, 79 pure, by boiling, 81 rolled, 80 stab, 78 stroke, 78 test-tube, 78 thrust, 78 Cutaneous inoculation of animals, Cutting sections, 56 Cytase, 40 Cytolysins, 40 Cytolytic serum, 40 Decolorizing agents, 49 Denitrification, 245 Denys' B. F. tubercuhn, 120 Deodorants, 262 Dermatitis, blastomycetic, 223 Dermo-tuberculin reaction of von Pirq;iet, 121 Desmon, 40 Development, 21 Deviation of complement, 42 Diastatic ferments, 29 INDEX 3" Dieudonne's medium, 77 for spirillum cholerse, 151 Diphtheria, 126 antitoxin of, 131 methods o£ diagnosis, 130 Neisser's stain for, 5 2 streptococcus in, 133 toxins of, 131 DiplobaciUus of conjunctivitis, 171 Diplococcus intracellularis men- ingitidis, 177 lanceolatus, 157 pneumoniae, 155, 157 Discharges, disinfection of, 265 Disinfectants, 62, 262 testing value of, 265 Disinfection, general measures for, 265 of discharges, 265 Distribution, 26 Dourine, 204 Dry heat, 64 Drying specimens, 54 Dum-dum fever, 208 Dunham's modified peptone wa- ter, 77 peptone solution for spirillum cholerse, 150 rosalic acid solution, 77 Dyes, acid, 48 anilin, 47, 264 anilin-oil water, 50 basic, 48 Dysentery, 145, 198 bacillary, 199 Ear, bacteria in, 259 Edema, malignant, 184 Eggs, boiled, 78 fresh, cultures, 77 Ehrlich's lateral-chain theory of immunity, 36 Electricity, influence of, on bac- teria, 28 Eisner's typhoid medium, 76 Emphysema, phlegmonous, 186 Emphysematous gangrene, 186 Endo medium, 77 Endogenous infection, 31 Endosporous bacteria, 24 Entamoeba histolytica, 198 Enteric fever, 135 Epidemic cerebrospinal meningi- tis, 177 conjunctivitis, 161 Erysipelas, 164 of swine, 194 Esmarch's cubes, 68 method of cultivating anaerobic bacteria, 83 tubes, 80 Estivo-autumnal form of mala- rial protozoa, 201 Exogenous infection, 3 1 Extracellular toxins, 31 Eye, inoculation of animals by, 92 Facultative aerobes, 28 anaerobes, 28 bacteria, 27 Farcin du boeuf, bovine, 231 Farcy-buds, 125 Fat-splitting ferments, 30 Favus, 225 Feces, bacteria in, 260 cholera bacteria in, 153 Fermentation, 29 alcohol, 255 beer, 256 tube, 81 vinegar, 255 Ferments, 29 coagulating, 30 diastatic, 29 fat-spUtting, 30 hydrolytic, 30 inverting, 29 proteolytic, 29 FertiUty, soil, bacteria and, 244 Filter materials, 236 Pasteur-Chamberland, 237 Petri's sand, 233 Filterable organisms, 218 Filtered water, 236 Filters, 67 Filtration, 265 steriUzation by, 67 Finkler-Prior vibrio, 154 312 INDEX Fishing, 88 Fission algse, 21 fungi, 21 Fixateur, 40 Fixation of complement, 42 Flagella, 23 Loffler's mordant for, 51 stain, with Loffler's mordant, 61 Flagellates, 197 Flask to receive blood-serum, 72 Fliigge's potato bacillus, 98 Fluorescence, 30 Food, bacteria in, 246 Foods as source of infection, 255 Foot, Madura, 230 Formaldehyd, 263 sohd, 264 Fowl septicemia, 193 Fractional sterilization of Tyn- dall, 65 Frankel's borer, 244 method of cultivating anaerobic bacteria, 84 of staining tubercle bacilli, 115 pneumococcus, 157 Fresh egg cultures, 77 Fuchsin-lactose-agar, 77 Fungi, budding, 220 fission, 21 ray-, 228 staining, Unna's method, 61 thrush, 222 Gabbet's acid blue, 51 modification of Frankel's method of staining tubercle bacilli, 115 Gametes, 200 Gangrene, emphysematous, 186 Gangrenous mastitis of sheep, 196 Gas phlegmons, 186 Gas-formation, 30 Gelatin, 69 nutrient, 71 potato, 76 Gelatinous membrane, 22 Genito-urinary passages, bacteria in, 260 Germicides, 261, 262 Germination, 25 Giemsa's stain, 53 Glanders, 124 Glass plating, 80 slide cultures, 78 Glossina morsitans, 208 palpalis, 208 Glycerin-agar, 69 Gonococcus, 174 aUied varieties, 176 Neisser, 174 Neisser's stain for, 175 Wertheim's medium for, 78 Gram's iodin solution, 5 1 method of double staining, 58 of tissue staining, 58 GranulobaciUus immobilis, 187 Growth, 27 Gruber-Widal blood-serum test, 140 Guinea-pig bouillon, 78 Guinea-pigs, 91 Giinther's stain for blood speci- mens, 261 Hands, sterilization of , 264 Hanging block, 47 drop, 46 Haptophore group, 37 Hay bacillus, 100 Heat, 49 as disinfectant, 63 as germicide, 262 dry, 64 influence of, on bacteria, 28 moist, 64 Hemolysins, 41 Hemolysis, 41 Hemorrhagic septicemia, 190 bacteria of, 192 Herpes tonsurans, 225 Herpetomonas, 208 Hesse's medium for typhoid, 75 method of collecting bacteria from air, 233 of cultivating anaerobic bac- teria, 83 Hiss' capsule stain, 52, 61 medium for plating, 75 Hoffman, pseudobacillus of, 1 29 Hog cholera, 133 Homogeneous system, 43 INDEX 313 Host, susceptibiKty of, 33 Hot-air sterilizer, 63 Hueppe's fresh egg cultures, 77 Hydrogen dioxid, 264 Hydrolytic ferments, 30 Hydrophobia, 209 Hygienic laboratory phenol coeffi- cient, 265 Hypersensitiveness, 42 Hypersusceptibihty, 42 Hyphomycetes, 223 Ice cream, 248 Immunity, 34, 35 acquired, 35 active, 35 passive, 36 Ehrlich's lateral-chain theory, 36. from intentional infection or in- toxication, 35 lock and key theory, 39 Metchnikoff 's phagocytic theory of, 36 natmral, 35 theories of, 36 unit, 133 Impression of colonies, 88 Incubator, 74 Index, opsonic, 41 negative phase, 41 positive phase, 41 India ink method of identifying spirochseta pallida, 211 Indol reaction in cholera, 152 Infantile paralysis, epidemic, 218, 2ig Infection, 30, 31 avenues of, 32 cardinal conditions for, 32 depending on quantity of bac- teria, 32 endogenous, 31 exogenous, 31 foods as source of, 255 mixed, 33 pathogenesis, 31 sources of, 31 susceptibihty to, 33 virulence of, 32 Infective bacteria, 33 Influenza, 160 Infusoria, 197, 198 Inoculating potatoes, manner of, 68 Inoculation, animal, 91. See also Animal inoculatwn. of culture-media, 78 Insecticides, 263 Insoluble toxins, 31 Inspissator for blood-serum, 73 Instruments, sterilization of, 45 Intensifiers, 48 Intestine, bacteria in, 259 Intracellular toxins, 31 Intraduodenal animal inoculation, 93 Intraperitoneal injections of ani- mals, 92 Intratracheal inoculation of ani- mals, 93 Intravenous injections of animals, 92 Inverting ferments, 29 lodin, 264, 265 as used in Gram's method, 49 solution, Gram's, 51 Iris blender, 44 Jackson bile media, 242 Jenner's stain, 53 for malarial organisms, 203 Kala-azar, 208 King's solution dried blood albu- min, 78 Klatsch preparations, 88 Klebs-Loffler baciUus, 126 Klein's method of staining spo.'es, 60 Koch's alkahne methylene-blue, 50 bacillen emulsion, 1 20 rules in regard to bacterial cause of disease, 94 Koch-Weeks bacillus, 161 Kiihne's method of staining spores,. 60 stain, SI 314 INDEX Laboratory, small requirements of, 8s Lactose litmus agar, 72 Lactose-bile, 76 Lateral-chain theory of immunity, Ehrlich's 36 Law, Weigert's, 38 Leishman-Donovan bodies, 208 Leishman's stain, 53 Lens, oil-immersion, 43 Lepra bacillus, 122 Leprosy, 122 LeptothrLx buccalis, 228 gigantea, 228 innominata, 228 maxima, 228 Liborius's method of cultivating anaerobic bacteria, 82 Life cycle of malarial sporozoa, 199 of protozoa, 198 Light, influence of, on bacteria, 28 Lime, 265 Litmus agar, lactose, 72 Lock and key theory of immunity, 39 Locomotion, 23 Loffler's alkaline methylene-blue, SO blood-serum mixture, 74 mordant for flageUa, 51, 61 stain for tissues, 59 Luetin reaction, 216 Lye, soda, 263 Lysis, 42 MacConkey's bile salt agar, 75 Macrocytase, 36 Macrogamete, 201 Macrophages, 36 Madura foot, 230 Mai de pis, 196 Malarial organisms, methods of examination for, 203 protozoa, estivo-autumnal form, 201 forms of, 201 quartan form, 201 tertian form, 201 sporozoa, life cycle of, 199 Malignant edema, 184 pustule, 105 tertian form of malarial pro- tozoa, 201 Mallein, 126 Malta fever, 162 Mash, bread, 69 potato, 68 Mastigophora, 197 Mastitis, gangrenous, of sheep, 196 Measles, 219 Media, culture-, inoculation of, 78 nutrient, preparation of, 67 preparation of, 70 reaction of, 70 soUd transparent, 69 sterilization of, 70 Mediterranean fever, 162 Membrane, gelatinous, 22 Meningitis, epidemic cerebro- spinal, 177 Meningococcus, 174, 177 Mercuric chlorid, 262 Mercury, bichlorid of, 62 Merozoites, 199 Metals, salts of, 263, 264 Metchnikoff's theory of immunity, 36 Methylene-blue, alkaline, 50 Microbe en huit, 193 Micrococcus albicans, 176 cereus albus, 169 flavus, 169 cholera gallinarum, 193 citreus, 176 gonorrhoeae, 174 melitensis, 162 meningitidis, 177 of mal de pis, 196 of sputum septicemia, 157 pyogenes citreus, 169 tenuis, 169 subflavus, 176 tetragenus, 170 ureae, 102 Microcytase, 36 Microgametes, 201 Microphages, 36 Microscope, 43 Microsporon furfur, 224, 226 INDEX 315 Milk as source of contagion, 253 bacillus coli in, Wisconsin test for, 25s bacteria in, 246, 249 classification of, 247 condensed, bacteria in, 255 culture-medium, 77 examination of, 252 for tubercle bacilli, 116 pasteurized, 247, 253 - pure, 246 raw, 247 red, 252 yellow, 252 Milzbrand bacillus, 105 Mixed infection, :is Moist heat, 64 Moisture, influence of, on bac- teria, 28 Molds, 220, 221 examination of, 226 true, 223 Morax-Axenfeld diplobacillus of conjunctivitis, 171 Mordant, LofHer's, for fiagella, 51 for flagella, 61 Mordants, 48 Moro's tuberculin test, 121 Mouse septicemia, 195 Mouth, bacteria in, 258 white, 222 Mucor mucedo, 224 Mycetoma, 230 Mycobacterium tuberculosis, no Nagana, 204, 206 Naphthalin, 264 Nasal cavity, bacteria in, 259 Needles, platinum, 45 steriUzation of, 79 Negri bodies, 209 Neisser's gonococcus, 174 stain for diphtheria, 52 for gonococcus, 175 Nicolle's stain, 52 Nitragin, 246 Nitrate broth, 70, 71 Nitrification, 245 by bacteria, 29 Nitro-bacterine, 246 Nocardia farcinica, 231 Noguchi's luetin reaction, 216 Novy's jars, 85 Nutrient agar, 71 blood-serum, preparation of, 72 broth, 71 culture-media, preparation, 67, 70 gelatin, 71 Obligate aerobes, 28 anaerobes, 28 Odors from bacteria, 30 Oidiomycosis, 223 Oidium, 221, 223 albicans, 222 coccidioides, 223 lactis, 222 Oil-immersion lens, 43 Oocysts, 201 Ophthalmic tuberculin reaction of Calmette, 121 .Opsonic index, 41 negative phase, 41 positive phase, 41 Opsonins, 41 Orcein stain, Bowhill's, 60 Origin, 26 Oxidation by bacteria, 29 Oxygen, influence of, on Hfe and growth of bacteria, 27 Pappenheim's method for staining tubercle bacilU in urine, 115 Paracolon bacillus, 143 Paralysis, epidemic infantile, 218, 219 Parasites, 27 Parasitic bacteria, 27 thrush, 222 Paratyphoid bacilh, 143 Park's method of cultivating an- aerobic bacteria, 85 Pasteur-Chamberland filter, 237 Pasteurized milk, 247, 253 Pathogenic bacreria, 29, 97 table of, 287-303 yeasts, 222 Pear blight, 232 3i6 INDEX Penicillium glaucum, 223 Peptone water, modified Dunham, 77 Pertussis, 161 Pest, 190 Petri dish, 80 method of collecting bacteria from air, 232, 233 sand-filter, 233 Pfeiffer's capsule bacillus, 159 cholera reaction and agglutina- tion, 153, IS4 Phagocytes, 36 Phagocytic theory of immunity, Metchnikoff's, 36 Phenol, 263 coefficient, Hygienic Labora- tory, 26s solutions, so Phlegmonous emphysema, 186 Phlegmons, gas, 186 Phosphorescence, 30 Pigmentation, 30 Pink eye, 161, 258 Piroplasma bigeminum, 208 bovis, 208 hominis, 209 Pityriasis versicolor, 226 Plague, bubonic, 190 Plant diseases due to bacteria, 231 Plants, splitting, 21 Plasmodium falciparum, 201 malariae, 201 vivax, 201 Plate cultures, 79 method of examining milk for bacteria, 253 Plating, glass, 80 . streaked surface, 79 Platinum needles, 45 Pneumococcus, 157 Frankel's, 157 Pneumonia, 15s bacteria in, 155 Poliomyelitis, infantile, 218, 219 Potassium iodid medium, 76 permanganate, 264 Potato as culture-media, 67 as medium, 67 bacillus of Fliigge, 98 mash, 68 Potatoes, manner of inoculating, 68 test-tube. 68 Potato-gelatin, 76 Precipitin serum, 40 Precipitins, 39 Prescott-Breed method of exam- ining milk for bacteria, 252 Preservatives, 262 Protein contents of bacterial cell, 29 Proteolytic ferments, 29 Proteus mirabilis, 147 Zenkeri, 148 Protozoa, 197 life-cycle of, 198 malarial, estivo-autumnal form, 201 forms of, 201 quartan form, 201 tertian form, 201 Pseudobacillus of Ho£Eman, 1 29 Pseudodiphtheria, 129 Ptomains, 29, 34 Puerperal fever, 165 Pure cultures by boiling, 81 Pus, bluish-green, bacillus of, 171 Pustule, malignant, 105 Putrefaction, 30 Pyemia, 33 Pyocyanin, 173 Pyogenic bacteria, 33 cocci, 163 Quartan form of malarial pro- tozoa, 201 Quarter-evil, 187 Rabies, 209 Rauschbrand, 187, 1,89 Ray-fungus, 228 Reaction. See Test. of culture-media, 70 Reactivity, altered, 42 Receptors, 36 free, 41 of first order, 36 of second order, 36 of third order, 37 INDEX 317 Red milk, 252 Reduction by bacteria, 29 Relapsing fever, 217 Removing excess of stain, 55 Rennet curd cheese, 255 Reproduction, 24 Rhinoscleroma, 159 Rideal-Walker standard for dis- infectants, 265 Ring-worm, 225 Rolled cultures, 80 Romanowsky's stain, 53 Rontgen rays, influence of, on bac- teria, 28 Root bacillus, 99 Rosalie acid solution, Dunham's, 77 Rotz-baciUus, 124 Rouget du pore, 194 Roux's double stain, 52 method of cultivating anaerobic bacteria, 84 test-tube, 68 Saccharomyces albicans, 221 cerevisia;, 220 mycoderma, 221 niger, 221 rosaceus, 221 Saccharomycetes, 220 Salicylic acid, 264 Salts of metals, 263, 264 Sand-filter, Petri's, 233 Saprophytes, 27 Sarcina, 103 aurantica, 104 lutea, 103 ventriculi, 104 Sarcodina, 197 Schizogony, 199 Schizomycetes, 21 Schizophyceas, 21 Schizophyta, 21 SchweinerotlaufbaciUus, 194 Sedgwick's expanded tube for air- examination, 233 Sedgwick-Tucker method of col- lecting bacteria from air, 233 Sedimentation test in typhoid fever, 142 Septicemia, 33 fowl, 193 hemorrhagic, 190 bacteria of, 192 mouse, 195 sputum, micrococcus of, 147 Serum, antistreptococcic, 166 antitoxic, 39 and tuberculous, 122 blood-, 69 coagulation of, 72 human, preparation of, 74 nutrient, preparation of, 7 2 preservation of, in liquid state, 74 sterilization of, 73 cytolytic, 40 precipitin, 40 test, Wassermann, 42 Sewage bacteria, examination for, 240 bacterial treatment of, 243 Sewer air, bacteria in, 235 Sexual cycle in mosquito, 201 Sheep, gangrenous mastitis of, 196 Shiga bacillus, 199 Side-chain theory of immunity, Ehrhch's, 36 Skin, bacteria on, 257 Sleeping sickness, 205, 207 Small-pox, 218 Smear culture, 78 Smegma bacillus, 1 23 Smith's fermentation tube, 81 Soap, 264 Soda lye, 263 Soil bacteria in, 232, 243 examination of, 232, 243 fertility, bacteria and, 244 Soluble toxins, 31 Soor, 222 Spatula for lifting sections, 57 Specimens, drying, 54 Spirillum, 103 aquatilis, 154 berolinense, 154 bonhofiii, 154 cholerae, 148 allied varieties, 154 products, 152 danubicum, 154 3i8 INDEX Spirillum dunbarii, 154 milleri, 154 obermeieri, 217 of relapsing fever, 217 of Wernicke, 154 rubrum, 103 schuylkiUiensis, 154 weibeU, 154 Spirochjeta pallida, 209 Spironema pallidum, 209 Splenic fever, 105 Splenomegaly, tropical, 208 Splitting plants, 21 Sporangium, 224 Spore contents, 25 formations, 24 requisites for, 25 Spores, resistance of, 26 staining, 59 Klein's method, 60 Kiihne's method, 60 Weigert's method, 61 Sporidium vaccinale, 219 Sporocyte, 199 Sporogenic bodies, 25 Sporogony, 199 Sporozoa, 197, 198 malarial, life cycle of, 199 Sporozoites, 201 Sputum septicemia, micrococcus of, 157 Stab culture, 78 Stain, alkaline, 49 BowhiU's orcein, 60 capsule, 61 Buerger's method, 61 Hiss' method, 61 carbolthionin, 52 fiageUa, with Loffler's mordant, 61 Frankel's, for tubercle bacilli, IIS Giemsa's, 53 Giinther's, for blood specimens, 261 Hiss' capsule, 52, 61 Jenner's, 53 for malarial organisms, 203 Koch's, so Kiihne's, 51 Leishman's, 53 Stain, Loffler's, 50 for tissues, 59 Neisser's, for diphtheria, 52 for gonococcus, 17s Nicolle's, 52 removing excess of, SS Romanowsky's, 53 Roux's double, 52 Welch's capsule, 53 Wright's, 53 for malarial organisms, 204 Ziehl-Neelsen, 50 Staining, 47 blood specimens, 57, 261 fungi, Unna's method, 61 general method, 54 Gram's double method, 58 for tissues, 58 malarial organisms, 203 of tissue sections, 54, 56, 57 solutions, 48 compound, 48 concentrated, 48 formulas of, 49 stock, 48 special methods, 58 spores, 59 Klein's method, 60 Kiihn's method, 60 Weigert's method, 61 tubercle bacilli in sputum, 117 Staphylococcus, 23 epidermidis albus, 168 pyogenes, 164 albus, 168 aureus, 166 Steam, superheated, 263 Stegomyia fasciata, 219 Sterilization by filtration, 67 fractional, of Tyndall, 65 of blood-serum, 73 of culture-media, 62, 70 of hands, 264 of instruments, 45 of needles, 79 Sterilizer, Arnold's steam, 64 hot-air, 63 Stewart-Slack method of examin- ing milk for bacteria, 252 Stock staining solutions, 48 Stomach, bacteria in, 259 INDEX 319 Streaked surface plating, 79 Streptococcus, 23 acidi lactici, 250 erysipelatis, 164 in diphtheria, 133 lanceolatus, 157 puerperahs, 165 pyogenes, 164 Streptothrices, 227 Streptothrix actinomyces, 228 farcinica, 231 madurse, 230 Stroke culture, 78 Structure, 21, 22 Subcutaneous inoculation of ani- mals, 92 Substance sensibilatrice, 40 Sugar broths, 71 Sulphur dioxid, 264 Sulphurous acid gas, 264 Suppurative bacteria, 33 Surra, 204 Susceptibility, 33 acquired, 33 inherited, 33 natural, 33 Swine, erysipelas of, 194 Symptomatic anthrax, 187 Syphilis, 209 luetin reaction in, 216 Wassermaim reaction in, 211 results, 216 Noguchi modification, 214 Temperature, influence of, on life and growth of bacteria, 27 Tertian form of malarial protozoa, 201 Test, agglutination, for tubercle ba- cilli, 121 in cholera, 152 in typhoid fever, 140 Pfeiffer's cholera, 153, 154 Bordet's cholera, 15s Gruber-Widal blood-serum, 140 luetin, 216 Pfeiffer's cholera, 153, 154 sedimentation, in typhoid fever, 142 Test, tuberculin, Calmette's oph- thalmic, 121 Moro's, 121 von Pirquet's, 121 Wassermanu, in syphilis, 211 Noguchi's modification, 214 results, 216 Widal, in typhoid fever, 140 Wisconsin, for bacillus coli in milk, 255 Test-tube, 66 cultures, 78 potatoes, 68 Roux's, 68 Tetanolysin, 183 Tetanospasmin, 182 Tetanus, 180 antitoxin, 183 unit for, 183 Texas cattle-fever, 208 Thermostat for blood-serum, 73 Thrush fimgus, 222 parasitic, 222 Thrust-culture, 78 Tick fever, African, 218 Tinea, 225, 226 Tissue preparations, 56 Torula cerevisiae, 220 Toxalbumins, 30, 34 Toxemia, 33 Toxins, 30, 31, 34 of diphtheria, 131 extracellular, 31 insoluble, 31 intracellular, 31 nature of, 34 soluble, 31 Toxoid of diphtheria, 131 Toxone of diphtheria, 131 Toxophore group, 37 Treponema palUdum, 209 Trichophyton tonsurans, 224, 225 Tricresol, 263 Tropical splenomegaly, 208 Trypanosoma, 204 brucei, 206 castellani, 207 equiperdum, 208 Evansi, 208 hominis, 207 lewisi, 205 320 INDEX Trypanosoma neprevi, 207 Rougetii, 208 ugandense gambiense, 207 Trypanosomes, 204 Trypanosomiasis, human, 207 Tsetse fly, 208 Tsetse-fly disease, 206 Tubercle bacillus, no products of, 120 Tuberculin B. E., 120 Denys' B. F., 120 Koch's B. E., 120 R, 120 reaction, ophthalmic, of Cal- mette, 121 residuum, 120 test, Moro's, 121 von Pirquet's, 121 use of, 120 Tuberculocidin, 120 Tuberculosis, no bovine, human tuberculosis and, relation, 119 in animals, 119 Tubes, Esmarch's, 80 Tjmdall's fractional sterilization, 6S Types, 22 Typhoid carriers, 139 fever, 135 sedimentation test in, 142 Widal reaction in, 140 medium, Eisner's, 76 for typhoid, 75 Typhotoxin, 139 Typhus fever, 219 Ultra-microscopic organisms, 218 Unna's borax methyl-blue, 51 method for staining fungi, 61 Urethra, bacteria in, 260 Urine, bacillus coli in, 102 bacteria in, 102, 260 examination of, for tubercle ba- cilli, ns Vaccines, 95 antistreptococcic, i65 Vaccines, antityphoid, 139 bacterial, 95 Vaccinia, 218 Vagina, bacteria in, 260 Variola, 218 Vibratory movements, 23 Vibrio cholerse, 148 Finkler-Prior, 154 Metchnikovii, 154 tyrogenum, 154 Vibrion butyrique of Pasteur, 251 septique, 184 Vinegar fermentation, 255 Virulence of infection, 32 von Pirquet's dermo-tubercuUn test, 121 Wassermann reaction in syphiUs, 211 Noguchi modification, 214 results, 216 Water, artesian well, 236 bacillus coli in, examination for, 240 typhosus in, diagnostic points of, 242 bacteria in, method of examina- tion, 238 varieties, 238 boiling of, as means of purifying, 238 cholera bacillus in, 152 examination of, 232, 235, 238 filtered, 236 purity of, 235 well, 236 Weak solutions, 50 Weigert's law, 38 method of staining spores, 61 Welch's capsule stain, 53 Well water, 236 Wernicke's spirillum, 154 Wertheim's medium for gonococ- cus, 78 White mouth, 222 Whooping-cough, 161 Widal reaction in typhoid fever, 140 Wire cage, 66 INDEX 321 Wisconsin test for bacillus coU in milk, 255 Wolfhugel's counter, 243 Woolsorter's disease, 109 Wright's chromatin stain for ma- larial organisms, 204 method of cultivating anaerobic bacteria, 85 stain, S3 Wurzel bacillus, 99 Yaws, 217 Yeasts, 220 examination of, 226 pathogenic, 222 Yellow fever, 219 milk, 25 Ziehl-Neelsen stain, 50 Zooglea, 23 SAUNDERS' BOOKS GYNECOLOGY and OBSTETRICS W. B. SAUNDERS COMPANY West Washington Square Philadelphia 9, Henrietta Street Covent Garden, London Our Handsome Complete Catalogue will be Sent on Request Norris' Gonorrhea in Women Gonorrhea in Women. By Charles C. Norris, M. D., Instructor in Gynecology, University of Pennsylvania. With an Introduction by John G. Clark, M. D., Professor of Gynecology, University of Pennsylvania. Large octavo of 520 pages, illus- trated. Cloth, ^6.00 net ; Half Morocco, ^7.50 net. FOR PHYSICIAN, SURGEON. SPECIALIST. AND SOCIOLOGIST Dr. Norris has neglected no phase of his subject. First you get an in- tensely interesting Historic Narrative ; then the Bacteriology and Pathogenesis, giving you the best cultural and staining methods. The trzvzc side is very fully considered, giving you the various methods employed by the govern- ments of Europe and the Orient for the limitation and suppression of the social evil, and the situation as it is to-day in the United States. So down to the minute is the book that the work of the Chicago Vice Commission and the even more recent municipal instigations of New York and Philadelphia are included. Both operative and medieinal treatments are taken up, including a noteworthy discussion of serum and vaccine therapies. A special chapter is devoted to the drugs used in treating gonorrhea, giving formulas, solutions, etc. DISEASES OF WOMEN DeLee's New Obstetrics Text=Book of Obstetrics. By Joseph B. DeLee, M. D., Professor of Obstetrics at Northwestern University Medical School, Chicago. Large octavo of 1060 pages, with 913 illus- trations, 150 in colors. Cloth, $8.00 net; Half Morocco, iJIg-So net. AGAIN REPRINTED You will pronounce this new book the most elaborate, the most superbly illustrated work on Obstetrics you have ever seen. Especially will you value the 913 illustrations, all, with but few exceptions, original, and the best work of leading medical artists. Some 150 of these illustrations.are in color. Such a magnificent collection of obstetric pictures^-and with really practiced value — has never before appeared in one book. You will find the text extremely practical throughout. Diagnosis is fea- tured, and the relations of obstetric conditions and accidents to general medi- cine, surgery, and the specialties are brought into prominence. Regarding Treatment: You get here the verj- latest advances in this field, and you can rest assured every method of treatment, every step in operative technic, is just right. Dr. DeLee' s twenty-one years' experience as a teacher and obstetrician guarantees this. Worthy of your particular attention are the descriptive legends under the illustrations. These are unusually full, and by studying the pictures serially with their detailed legends you are better able to follow the operations than by referring to the pictures from a distant text — the usual method. These illustrations, with their explanatory legends, you will find particularly valu- able for the quick reference work your daily practice demands. The subject-matter of the book is divided into four parts : The Physiology of Pregnancy, Labor and the Puerperium ; the Conduct of Pregnancy, Labor and the Puerperium ; the Pathology of Pregnancy, I^abor and the Puer- perium ; and Operative Obstetrics. This arrangement, you see, is extremely practical. GYNECOLOGY. Cullen's Uterine Adenomyoma uterine Adenomyoma. By Thomas S. Cullen, M. D., Associate Professor of Gynecology, Johns Hopkins University. Octavo of 2 75 pages, with original illustrations by Hermann Becker and August Horn. Cloth, ^5.00 net. ILLUSTRATED BY BECKER AND .HORN Dr. Cullen's large clinical experience and his extensive original work along the lines of gynecologic pathology have enabled him to present his subject with originality and precision. The work gives the early literature on adenomyoma, traces the disease through its various stages, and then gives the detailed findings in a large number of cases personally examined by the author. Formerly the physician and surgeon were unable to determine the cause of uterine bleeding, but after following closely the clinical course of the disease, Dr. Cullen has found that the majority of these cases can be diagnosed clinically. The results of these observations he presents in this work. The Lancet, London "A good example of how such a monograph should be written. It is an excellent work, worthy of the high reputation of the author and of the school from which it emanates." Cullen's Cancer of the Uterus Cancer of the Uterus. By Thomas S. Cullen, M. B., Associate Professor of Gynecology, Johns Hopkins University. Large octavo of 693 pages, with over 300 colored and half-tone text-cuts and eleven lithographs. Cloth, ^7. 50 net; Half Morocco, ^8.50 net. Howard A. Kelly, M. D. Professor of Gynecologic Surgery, Johns Hopkins University. " Dr. Cullen's book is the standard work on the greatest problem which faces the sur- eical world to-day. Any one who desires to attack this great problem must have this book." SAUNDERS' BOOKS ON Kelly and Cullen's Myomata of the Uterus Myomata of the Uterus. By Howard A. Kelly, M. D., Professor of Gynecologic Surgery at Johns Hopkins University; and Thomas S. Cullen, M. B., Associate in Gynecology at Johns Hopkins Universit)-. Large octavo of about 700 pages, with 388 original illustrations by August Horn and Hermann Becker. Cloth, ^7.50 net; Half Morocco, ^9.00 net. A MASTER WORK ILLUSTRATED BY AUGUST HORN AND HERMANN BECKER This monumental work, the fruit of over ten years of untiring labors, will remain for many years the last word upon the subject. Written by those men who have brought, step by step, the operative treatment of uterine myoma to such perfection that the mortality is now less than one per cent., it stands out as the record of greatest achievement of recent times. The illustrations have been made with wonderful accuracy in detail by Mr. August Horn and Mr. Hermann Becker, whose superb work is so well known that comment is unnecessary. For painstakmg accuracy, for attention to every detail, and as an example of the practical results accruing from the associa- tion of the operating amphitheater with the pathologic laboratory, this work will stand as an enduring testimonial. Surgery, Gynecology, and Obstetrics " It must be considered as the most comprehensive work of the kind yet published. It will always be a mine of wealth to future students." New York Medical Journal " Within the covers of this monograph every form, size, .variety, and complication of uterine fibroids is discussed. It is a splendid example of the rapid progress of American professional thought." Bulletin Medical and Chirurgical Faculty o{ Maryland " Few medical works in recent years have come to our notice so complete in detail, so well illustrated, so practical, and so far reaching in their teaching to general practitioner, specialist, and student alike." GYNECOLOGY. Bandler's Medical Gynecology Medical Gynecology. By S. Wyllis Bandler, M. D., Adjunct Professor of Diseases of Women, New York Post- Graduate Medical School and Hospital. Octavo of 702 pages, -with 150 original illustrations. Cloth, ^5.00 net; Half Morocco, %(>.<,o net. SECOND EDITION This new work by Dr. Bandler is just the book that the physician en- gaged in general practice has long needed. It is truly the practitionei' s gynn- cology — planned for him, written for him, and illustrated for him. There are many gynecologic conditions that do not call for operative treatment ; yet, because of lack of that special knowledge required for their diagnosis and treatment, the general practitioner has been unable to treat them intelligently. This work gives just the information the practitioner needs. American Journal of Obstetrics " He has shown good judgment in the selection of his data. He has placed most emphasis on diagnostic and therapeutic aspects. _He has presented his facts in a manner to be readily grasped by the general practitioner." Bandler's Vaginal Celiotomy Vaginal Celiotomy. By S. Wyllis Bandler, M. D. Octavo of 450 pages, with 148 illustrations. Cloth, ^5.00 net. SUPERB ILLUSTRATIONS The vaginal route, because of its simplicity, ease of execution, absence of shock, more certain results, and the opportunity for conservative measures constitutes a field which should appeal to all surgeons, gynecologists and obstetricians. Posterior vaginal celiotomy is of great importance m the re- moval of small tubal and ovarian tumoi-s and cysts, and is an important step " the performance of vaginal myomectomy, hysterectomy, and hystero- myomectomy. Anterior vaginal celiotomy witli thorough separation of tie bladder is the only certain method of correcting cystocele. The Lancet. London , , v >. 1 " Dr Bandler has done good service in writing this book which gives a very clear description of aU the operations which may be undertaken through the vagina. He makes out a strong case for these operations." SAUNDERS' BOOKS ON Ashton's Practice of Gynecology The Practice of Gynecology. By AV. Easterly Ashton, M.D., LL.D., Professor of Gynecology in the Medico-Chirurgi- cal College, Philadelphia. Handsome octavo volume of iioo pages, containing 1058 original line drawings. Cloth, 1^6.50 net; Half Morocco, ^8.00 net. THE NEW (5th) EDITION Among the important new matter may be mentioned the De Keating-Hart fulguration treatment, Coley's mixed toxins for sarcoma of the genito-urinary organs, the cutireaction of von Pirquet in the diagnosis of tuberculosis, " 606 " for syphihs, the hormone theory, the Fowler- Murphy treatment of suppurative peritonitis, tincture of iodin in sterilization, and Baldy's new round ligament operation for retrodisplacement. Notliing is left to be taken for granted, the author not only telling his readers in every instance what should be done, but also precisely 'how to do it. A distinctly original feature of the book is the illustrations, numbering 1058 line drawings made especially under the author's personal supervision. From its first appearance Dr. Ashton's book set a standard in practical medical books ; that he /las produced a work of unusual value to the medical practitioner is shown by the demand for new editions. Howard A. Kelljr, M. D.. Professor of Gynecologic Surgery, yohns Hopkins Uniziersity "It is different from anything that has as yet appeared. The illustrations are particu- larly clear and satisfactory. One specially good feature is the pains with which you describe so many details so often left to the imagination." Charles B. Penrose, M. D., Formerly Professor of Gynecology, University of Pennsylvania. " I know of no book that goes so thoroughly and satisfactorily into all the details of everything connected with the subject. In this respect your book differs from the others." George M. Edebohls, M.D. Professor of Diseases of Women, New York Post-Graduate Medical School. " I have looked it through and must congratulate you upon having produced a text' book most admirably adapted to teach gynecology to those who must get their knowledge, even to the minutest and most elementary details, from books." .DISEASES OF WOMEN. Webster's Diseases qf Women Diseases of Womien. By J. Clarence Webster, M. D. (Edin.)j F. R. C. p. E., Professor of Gynecology and Obstetrics in Rush Medical College. Octavo of 712 pages, with 372 illus- trations. Cloth, ^7.00 net ; Half Morocco, J8.50 net. FOR THE PRACTITIONER Dr. Webster has written this -work especially for the general practitioner, discussing the cHnical features of the subject in their widest relations to general practice rather than from the standpoint of specialism. The magni- ficent illustrations, three hundred and seventy-two in number, are nearly all original. Drawn by expert anatomic artists under Dr. Webster' s direct super- vision, they portray the anatomy of the parts and the steps in the operations with rare clearness and exactness. Howard A. Kelly, M.D., Professor o/Gynecologk Surgery, Johns HopkinsUniversUy. " It is undoubtedly one of the best works which has been put on the market within recent years, showing from start to finish Dr. Webster's well-known thoroughness. The illustrations are also of the highest order." Webster's Obstetrics A Text=Book of Obstetrics. By J. Clarence Webster, M. D. (Edin.), Professor of Obstetrics and Gynecology in Rush Medical College. Octavo of 767 pages, illustrated. . Cloth, ^5.00 net; Half Morocco, ^6.50 net. Medical Record, New York '* The author's remarks on asepsis and antisepsis are admirable, the chapter on eclamp- sia is full of good material, and . . . the book can be cordially recommended as a safe guide." SAUNDEMS' BOOKS ON Hirst's Obstetrics The New (7th) Edition A Text-Book of Obstetrics. By Barton Cooke Hirst, M. D., Professor of Obstetrics in the University of Pennsylvania. Handsome octavo, 1025 pages, with 900 illustrations, 46 in colors. Cloth, Js-oo net; Half Morocco, ^6.50 net. INCLUDING RELATED GYNECOLOGIC OPERATIONS Immediately on its publication this work took its place as the leading text- book on the subject. Both in this country and abroad it is recognized as the most satisfactorily written and clearly illustrated work on obstetrics in the language. The illustrations form one of the features of the book. They are numerous and the most of them are original. In this edition the book has been thoroughly revised. Recognizing the inseparable relation between ob- stetrics and certain gynecologic conditions, the author has included all the gynecologic operations for complications and consequences of childbirth, together with a brief account of the diagnosis and treatment of all the path- ologic phenomena peculiar to women. British Medical Journal " The illustrations in Dr. Hirst's volume are far more numerous and far better exe- cuted, and therefore more instructive, than those commonly found in the works of writers on obstetrics in our own country." Hirst's Diseases of Women A Text=Book of Diseases of Women. By Barton Cooke Hirst, M. D. Octavo of 745 pages, 701 illustrations, many in colors. Cloth, ;^s.oo net; Half Morocco, $6.50 net. SECOND EDITION As diagnosis and treatment are of the greatest importance in considering diseases of women, particular attention has been devoted to these divisions. The palliative treatment, as well as the radical operation, is fully described, enabling tlie general practitioner to treat many of his own patients without referring them to a specialist. Medical Record, New York " Its merits can be appreciated only by a careful perusal. . . Nearly one hundred pages are devoted to technic, this chapter being in some respects superior to the descrip- tions in other text-books." GYNECOLOGY. Kelly and Noble's Gynecology and Abdominal Surgery Gynecology and Abdominal Surgery. Edited by Howard A. Kelly, M. D., Professor of Gynecology in Johns Hopkins. University; and Charles P. Noble, M.D., formerly Clinical Professor of Gynecology in the Woman's Medical College, Phila- delphia. Two imperial octavo volumes of 950 pages each, con- taining 880 illustrations, mostly original. Per volume : Cloth, $8.00 net; Half Morocco, $9.50 net. BOTH VOLUMES NOW READY WITH 880 ORIGINAL ILLUSTRATIONS BY HERMANN BECKER AND MAX BRODEL In view of the intimate association of gynecology with abdominal surgery the editors have combined these two important subjects in one work. For this reason the worlc will be doubly valuable, for not only the gynecologist and general practitioner will find it an exhaustive treatise, but the surgeon also will find here the latest technic of the various abdominal operations. It possesses a number of valuable features not to be found in any other publication cover- ing the same fields. It contains a chapter upon the bacteriology and one upon, the pathology of gynecology, dealing fully with tlie scientific basis of gyne- cology. In no other work can this information, prepared by specialists, be found as separate chapters. There is a large chapter devoted entirely to- medical gynecology^ written especially for the physician engaged in general practice. Heretofore the general practitioner was compelled to search through an entire work in order to obtain the information desired. Abdominal sur- gery proper, as distinct from gynecology, is fully treated, embracing operations upon the stomach, upon the intestines, upon the liver and bile-ducts, upon the pancreas and spleen, upon the kidney, ureter, bladder, and the peritoneum. Special attention has been given to modern technic. The illustrations are the work of Mr. Hermann Becker and Mr. Max Brodel. American Journal of the Medical Sciences " It is needless to say that the work has been thoroughly done : the names of the authors and editors would guarantee this ; but much may be said in praise of the method of presen- tation, and attention mav be called to the inclusion of matter not to be found elsewhere." SAUNDERS' BOOKS ON GET A«r»^«.5r««k*i '''"^ ^^^ THE BEST /inieriCall standard Illustrated Dictionary Just Out— The New (7th) Edition, Reset The American Illustrated Medical Dictionary. A new and complete dictionary of the terms used in Medicine, Surgery, Dentistry, Pharmacy, Chemistry, Veterinary Science, Nursing, and all kindred branches ; with over i oo new and elaborate tables and many handsome illustrations. By W. A. Newman Borland, M.D., Editor of " The American Pocket Medical Dictionary." Large octavo, 1105 pages, bound in full flexible leather. Price, ^4.50 net; with thumb index, |is.oo net. A KEY TO MEDICAL LITERATURE Gives a Maximum Amount of Matter in a Minimum Space ENTIRELY RESET— 5000 NEW WORDS This edition is not a makeshift revision. The editor and a corps of expert assistants have been working on it for two years. Result — a thoroughly down- to-the-minute dictionary, unequalled for completeness and usefulness by any other medical lexicon published. It meets your wants. It gives you all the new words, and in dictionary service new words are what you want. Then, it has two-score other features that make it really a Medical Encyclopedia. PERSONAL OPINIONS Howard A. Kelly, M. D.. Professor of Gynecologic Surgery, fohns Hopkins University , Baltimore, " Dr. Borland's dictionary is admirable. It is so well gotten up and of such conve- nient size. No errors have been found in my use of it." J. Collins Warren, M.D., LL.D.. F.R.C.S. (Hon.) Professor of Surgery, Harvard Medical School. *' I regard it as a valuable aid to my medical literary work. It is very complete and of convenient size to handle comfortably. I use it in preference to any other." GYNECOLOGY AND OBSTETRICS. Penrose's Diseases of Women Sixth Revised Edition A Text=Book of Diseases of Women. By Charles B. Penrose, M. D., Ph. D., formerly Professor of Gynecology in the University of Pennsylvania ; Surgeon to the Gynecean Hos- pital, Philadelphia. Octavo volume of 550 pages, with 225 fine original illustrations. Cloth ^3.75 net. ACCURATE Regularly every year a new edition of this excellent text-book is called for, and it appears to be in as great favor with physicians as with students. Indeed, this book has taken its place as the ideal work for the general prac- titioner. The author presents the best teaching of modern gynecology, un- trammeled by antiquated ideas and methods. In every case the most modern and progressive technique is adopted, and the main points are made clear by excellent illustrations. Howard A. Kelly, M.D.. Processor of Gynecologic Surgery, Johns Hopkins University, Baltimore. " I shall value very highly the copy of Penrose's ' Diseases of Women * received. I have already recommended it to my class as the best book." Davis* Operative Obstetrics operative Obstetrics. By Edward P. Davjs, M.D., Pro- fessor of Obstetrics at Jefferson Medical College, Philadelphia. Octavo of 483 pages, with 264 illustrations. Cloth, ^5.50 net. INCLUDING SURGERY OF NEWBORN Dr Davis' new work on Operative Obstetrics is a most practical one and no pxpense has been spared to make it the handsomest work on the subject, as well Every step in every operation is described minutely, and the technic shown by beautiful new illustrations. Dr. Davis' name is sufficient guarantee for something above the ordinary. 12 SAUNDERS' BOOKS ON Dorl&nd's Modern Obstetrics Modern Obstetrics : General and Operative. By W. A. Newman Borland, A. M., M. D., Professor of Obstetrics at Loyola University, Chicago. Handsome octavo volume of 797 pages, with 201 illustrations. Cloth, ^4.00 net. Second Edition, Revised and Greatly Enlarged In this edition the book has been entirely rewritten and very greatly enlarged. Among the new subjects introduced are the surgical treatment of puerperal sepsis, infant mortality, placental transmission of diseases, serum- therapy of puerperal sepsis, etc. Journal of the American Medical Association " This work deserves commendation, and that it has received what it deserves at the hands of the profession is attested by the fact that a second edition is called for within such a short time. Especially deserving of praise is the chapter on puerperal sepsis." Davis* Obstetric and Gynecologic Nursing' Obstetric and Gynecologic Nursing. By Edward P. Davis, A. M., M. D., Professor of Obstetrics in the Jefferson Medical College and Philadelphia Polyclinic ; Obstetrician and Gynecologist, Philadelphia Hospital. i2mo of 480 pages, illus- trated. Buckram, ^1.75 net. JUST READY— NEW (4th) EDITION This volume gives a very clear and accurate idea of the manner to meet the conditions arising during obstetric and gynecologic nur.sing. The third edition has been thoroughly revised. The Lancet, London '* Not only nurses, but even newly qualified medical men, would learn a great deal by a perusal of this book. It is written in a clear and pleasant style, and is a work we can recommend.* GYNECOLOGY AND OBSTETRICS 13 Garrigues* Diseases of Women TWrd Edition A Text-Book of Diseases of Women. By Henry J. Garrigues, A. M., M. D., Gynecologist to St. Mark's Hospital and to the German Dispensary, New York City. Handsome octavo, 756 pages, with 367 engravings and colored plates. Cloth, ^4.50 net; Half Morocco, $6.00 net. Thad. A, Reamy, M. D,, Prof essor 0/ Gynecology , Medical College of Ohio. " One of the best text-books for students and practitioners which has been published in the English language ; it is condensed, clear, and comprehensive. The profound learning and great clinical experience of the distinguished author find expression in Macfarlane's Gynecology for Nurses (zd) Edmon A Reference Hand-Book of Gynecology for Nurses. By Cath- arine MacfarlANE, M. D., Gynecologist to the Woman's Hospital of Philadelphia. l6mo of 156 pages, with 70 illustrations. Flexible leather, |Si.25 net. A. M. Seabrook, M. D., Woman's Medical College of Philadelphia. "It is a most admirable little book, covering in a concise but attractive way the sub- ject from the nurse's standpoint." American Text-Book of Gynecology EdXn American Text-Book of Gynecology. Edited by J. M. Baldy, M. D. Imperial octavo of 718 pages, with 341 text-illustrations and 38 plates. Cloth, ;jS6.oo net. American Text-Book of Obstetrics second Edition The American Text-Book of Obstetrics. In two volumes. Edited by Richard C. Norris, M. D. ; Art Editor, Robert L. Dick- inson, M. D. Two octavos of about 600 pages each ; nearly 900 illus- trations, including 49 colored and half-tone plates. Per volume : Cloth, IS3.50 net. Matthew D. Mann, M. D., Professor of Obstetrics and Gynecology^ University of Buffalo. " I like it exceedingly and have recommended the first volume as a text-book. It is certainly a most excellent work. I know of none better." 14 SAUNDEJiS' BOOKS ON Schaffer and Webster's Operative Gynecology Atlas and Epitome of Operative Gynecology. By Dr. O. Schaffer, of Heidelberg. Edited, with additions, by J. Clarence Webster, M. D. (Edin.), F. R. C. P. E., Professor of Obstetrics and Gynecology in Rush Medical College, in affili- ation with the University of Chicago. 42 colored lithographic plates, rhany text-cuts, a number in colors, and 138 pages of text. In Saunders' Hand-Atlas Series. Cloth, J3.00 net. Much patient endeavor has been expended by the author, the artist, and the hthographer in the preparation of the plates for this Atlas. They are based on hundreds of photographs taken from nature, and illustrate most faithfully the various surgical situations. Dr. Schaffer has made a specialty of demon- strating by illustrations. Medical Record, New York "The volume should prove most helpful to students and others in grasping details usually to be acquired only in the amphitheater itself." De Lee's Obstetrics for Nurses Obstetrics for Nurses. By Joseph B. DeLee, M. D., Professor of Obstetrics in the Northwestern University Medical School, Chicago ; Lecturer in the Nurses' Training Schools of Mercy, Wesley, Provident, Cook County, and Chicago Lying-in Hospitals. i2mo of 508 pages, fully illustrated. Cloth, ;?2.5o net. JUST READY— NEW (4th) EDITION While Dr. DeLee has written his work especially for nurses, the practi- tioner will also find it useful and instructive, since the duties of a nurse often devolve upon him in the early years of his practice. The illustrations are nearly all original and represent photographs taken from actual scenes. The text is the result of the author' s many years' experience in lecturing to the nurses of five different training schools. J. Clifton Edgar, M. D„ Professor of Obstetrics and Clinical Midwifery , Cornell University^ New York. " It is far and away the best that has come to my notice, and I shall take great pleasure- in recommending it to my nurses, and snidents as well." GYNECOLOGY AND OBSTETRICS. ij Schaffer and EdgarV Labor and Operative Obstetrics Atlas and Epitome ol Labor and Operative Obstetrics. By Dr. O. Schaffer, of Heidelberg. From the Fifth Revised and Enlarged German Edition. Edited, with additions, by J. Clifton Edgar, M. D., Professor of Obstetrics and Clinical Mid- wifery, Cornell University Medical School, New York. With 14 lithographic plates in colors, 139 other illustrations, and iii pages of text. Cloth, J 2. 00 net. In Saunders' Hand-Atlas Series. This book presents the act of parturition and the various obstetric opera- tions in a series of easily understood illustrations, accompanied by a text treating the subject from a practical standpoint. Americem Medicine *' The method of presenting obstetric operations is admirable. The drawings, repre- senting original work, have the commendable merit of illustrating instead of confusing." Schaffer and Edg'arV Obstetric Diagnosis and Treatment Atlas and Epitome of Obstetric Diagnosis and Treat= ment. By Dr. O. Schaffer, of Heidelberg. From the Second Revised German Edition. Edited, with additions, by J. Clif- ton Edgar, M. D., Professor of Obstetrics and Clinical Mid- wifery, Cornell University Medical School, N. Y. With 122 colored figures on 56 plates, 38 text-cuts, and 315 pages of text. Cloth, ^3.00 net. In Saunders' Hand- Atlas Series. This book treats particularly of obstetric operations, and, besides th« wealth of beautiful lithographic illustrations, contains an extensive text of great value. This text deals with the practical, clinical side of the subject. New York Medical Journal " The illustrations are admirably executed, as they are in all of these atlases and tne text can safely be commended, not only as elucidatory of the plates, but as expoundmg the scientific midwifery of to-day." 16 SAUNDERS' BOOKS ON GYNECOLOGY AND OBSTETRICS. American Pocket Dictionary , Just Ready '' New (8th) Edition The American Pocket Medical Dictionary. Edited by W. A. Newman Dorland, A. M., M.D. With 677 pages. Full leather, limp, with gold edges, Si.oo net ; with patent thumb index, $1.25 net. James W. Holland, M. D., Professor of Chemistrj/ and Toxicology, at the Jefferson Medical College. Philadelphia. " I am struck at once with admiration at the compact size and attractive exterior. I can recommend it to our students without reserve." Cragin's Gynecology Seventh Edition Essentials of Gynecology. By Edwin B. Cragin, M. D., Pro- fessor of Obstetrics, College of Physicians and Surgeons, New York. Crown octavo, 240 pages, 62 illustrations. Cloth, ^I.OO net. In Saunders' Question- Compend Series. Galbraith's Four Epochs of Woman's Life Edition The Four Epochs of Woman's Life: A Study in Hygiene. Maidenhood, Marriage, Maternity, Menopause. By Anna M. Gal- ERAITH, M. D. With an Introductory Note by John H. Musser, M. D. , University of Pennsylvania. l2mo of 247 pages. Cloth, Si. 50 net. Schaffer and Norris' Gynecology Saunders' Atlases Atlas and Epitome of Gynecology. By Dr. O. Schaffer, of Heidelberg. Edited, with additions, by Richard C. Norris, A. M., M. D., Assistant Professor of Obstetrics, University of Pennsylvania. 207 colored illustrations on 90 plates, 65 text-cuts, and 272 pages of text. Cloth, ^3.50 net. Ashton's Obstetrics Seventh Edition Essentials of Obstetrics. By W. Easterly Ashton, M. D., Pro- fessor of Gynecology in the Medico-Chirurgical College, Philadelphia. Crown octavo, 252 pages, 109 illustrations. Cloth, $1.00 net. In Saunders' Question-Compend Series. Southern Practitioner '*An excellent little volume, containing correct and practical knowledge. An admir* able compend, and the best condensation we have seen." Barton and Wells' Medical Thesaurus A Thesaurus of Medical Words and Phrases. By Wilfred M, Barton, M. D., Assistant to Professor of Materia Medica and Thera- peutics, Georgetown University, Washington, D. C. ; and Walter A. Wells, M. D., Demonstrator of Laryngology, Georgetown University, Washington, D. C. i2mo of 534 pages. Flexible leather, ^2.50 net; with thumb index, $3.00 net. Saunders' Compends SAUNDERS' Question Compends^ arranged in question- and-answer form, are the latest, most complete, and best illustrated series of compends ever issued. They are now recognized as the standard authorities , in medical literature with students and practitioners in every city of the^ United States aiid Canada. Since the first appearance of these in- • valuable student-helps there have been sold over 342,500 copies. The entire series has been kept thoroughly revised , and enlarged when necessary, many of them being in their fifth and sixth editions. A COMPLETE LIST OF VOLUMES Cloth, $1.00 net per copy, unless otherwise noted I. ESSENTIALS OF PHYSIOLOGY. 3d edition. By Sidney P. BUDGETTj.M.P. . , . i?. ESSENTIALS OF SURGERV::' :?th ed. 90 illustrations. By Edward Martin, M.D. , i 3. ESSENTIALS OF ANATOMY. 7th ed.r 151 illustrations. By C. B. Nancrede, M.D, . . 4. ESSENTIALS OF MEDICAL CHEMISTRY. 7th ed. By Law--- RENCE Woi-FF, M.D. Revised by A. Ferree Wither, Ph.D. 5. ESSENTIALS OF OBSTETRICS, 7thed,:i09iIlustra{ions. By W. Easterly AsHTON) M.D. Revised by J. A. McGlinn, M.D. ft. ESSENTIALS OF PATHOLOGY AND MORBID ANATOMY. By H. Harlow Brooks, M.D. A new. worki Preparing.' '\ 7. ESSENTIALS OF MATERIA MEDICA. THERAPEUTICS, AND PRESCRIPTION- WRITING. ^ l'^ ed. By Henry ' Morris, M.D. Revised Tjy, W. AJ BAsTedOj Ph.G., M.D. 8,9. ESSENTIALS OF PRACTICE OF MEDICINE. By W. R, Williams; M.D. (Double number, II1.7S net.) Saun3ers^ Compends 10. ESSENTIALS OF GYNECOLOGY. Jthed. With 57 illustrations. -By Edwin B. GrAgin, M.D. Revised ty Frank S. Mathews, M.D. 11. ESSENTIALS, OF DISEASES OF THE SKIN, yth edition. 80 illustrations. By H. W. Stelwagon, M.D. "' 12. ESSENTIALS OF MINOR SURGERY, BANDAGING, AND VENEREAL DISEASES. 2d ed. 78 illustrations. By Edward Martin, M.D. \y. ESSENTIALS OF GENITO-URINARY AND VENEREAL DIS- EASES. By S. S, Wilcox, M. D. New (2d) edition, fully illustrated. . 14. ESSENTIALS OF DISEASES OF THE EYE. 4* ed., illus- trated. By Edward Jackson, M.D. ^ 15. ESSENTIALS OF DISEASES OF CHILDREN. 3d ed. By. Wm. M. Powin, M.D. 17. ESSENTIALS. OF DIAGNOSIS. 2d ed. By S. Solis-Cohbn, M.D., and A. A. EsHNER, M.D. 19. ESSENTL^LS OF NOSE AND THROAT. 4* ed., illustrated: By E. B. GleAson, M.D. 20. ESSENTIALS OF BACTERIOLOGY. 7* ed. 100 illustrations > and6piates. By M. V. Bali,, M.D. 21. ESSENTIALS OF NERVOUS DISEASES AND INSANITY. 4tli ed. S3 illustrations. By John C. Shaw, M. D. Revised by Smith Ely Jelliffe, M. D. 24 ESSENTIALS OF DISEASES OF THE EAR. 3^ ed., fllus- trated. By E. Baldwin GleaSon, M. D. ' 25. ESSENTIALS OF HISTOLOGY. 4th ed. iic illustrations. By Ix)uis Leroy, M. D. W. B. SAUNDERS CO., West Washington Square, PMa. / /