b) Edition RY ords , Dentistry, d branches; les, Nerves, ic Tables of ts, Methods itor of the U05 pages, umb index, mimum space and at the lowest possible cost This edition is not a makeshift revision. The editor and a corps of expert ants have l>een working on it for two years. Result — a thoroughly down-to- thf-minute dictionary, unequalled for completeness and usefulness hy any other medical lexicon published. It meets your needs. It gives you all the ! in dictionary service nrrc wonts are what you want. Then, it has tuo- score other features that make it really a Medical' Encyclopedia. " I have very carefully looked the volume over, and note with pleasure the many chunyrs in the new edition. It h to UK- that the w.irk was > omplcu: before, but thi- • how much new in j ulx-n-d !'»r a great work of this kind."— JOHN }',. .Mi K- I-HY, M. I).. '.ool. norland's Dictionary is admirable. It is so well gotten up and of such convenient No errors have been found in my MSC of it."— HOWARD A. K.MLLY, J'ri'/.-s^or of Gjfie- i.'hns llapkir. mere. W. B. SAUNDERS CO., West Washington Square, Phila. London: 9, Henrietta Street, Covent Garden Just Read MEI th) Edition THE LIBRARY OF THE UNIVERSITY OF CALIFORNIA PRESENTED BY PROF. CHARLES A. KOFOID AND MRS. PRUDENCE W. KOFOID The b( not a revi- sion of ar irely anew and is con PRESENTED BY lown to be the most PROF. CHARLES A. KOFOID AND ims to be complete MRS PRUDENCE w. KOFOID l the terms of modern je vocabu- lary. Besii -k contains a wealth o s matter is of particul (reparation for examination. "~ y^: /v "-r;,: "" " I am struck at once with admiration at the compact size and attractive ex- terior. I can recommend it to our students without reserve." — JAMES W. HOL- LAND, M. D., of Jefferson Medical College. " This is a handy pocket dictionary, which is so full and complete that it puts to shame some of the more pretentious volumes." — Journal of the American Medical Association. " 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." — Dublin -Journal of Medical Science. " This is a handy little volume that, upon examination, 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 terms used in medicine." — Bulletin Johns Hopkins Hospital, Baltimore. W. B. SAUNDERS CO*, West Washington Square, Phiia. London: 9, Henrietta Street, Covent Garden ESSENTIALS OF BACTERIOLOGY SINCE the issue of the first volume of tha 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. Ocular Objectives and 'Nose-piece BACTERIOLOGIC MICROSCOPE. SAUNDERS' QUESTION-COMPENDS, No. 20 ESSENTIALS BACTERIOLOGY BEING A CONCISE AND SYSTEMATIC INTRODUCTION TO THE STUDY OF BACTERIA AND ALLIED MICROORGANISMS BY M. V, BALL, M.D. MEMBER OF THE ACADEMY OF 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 IIS Illustrations, some in Colors PHILADELPHIA AND LONDON W. B. SAUNDERS COMPANY J9J3 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. PRINTED IN AMERICA PRESS OF 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. WARREN, PA., December, 1913. M345650 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: Einfiihring in das Studium der Bacteriologie, etc. WOODHEAD and HARE: Pathological Mycology. SALMONSEN: Bacteriological Technique (English transla- tion) . M. V. BALL. 11 CONTENTS PAGE INTRODUCTION 17 PART I GENERAL BACTERIOLOGY CHAPTER I — STRUCTURE AND DEVELOPMENT OF BACTERIA .... 21 CHAPTER II — BIOLOGIC AND CHEMIC ACTIVITIES 26 CHAPTER III — INFECTION 3° 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— BACTERINS (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 101 Bacillus Violaceus 102 Microorganisms Found in Urine 102 Micrococcus Ureae 102 Spirilla 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 ,r. no Other Acid-fast Bacteria -. 1 16 Bovine and Human Tuberculosis 119 Products of Tubercle Bacilli 1 20 Lepra Bacillus 122 Smegma Bacillus of Alvarey and Tavel 1 23 Bacillus of Glanders (Bacillus Mallei; Rotz-Bacillus) 124 CHAPTER XIX — DIPHTHERIA BACILLUS 1 26 CHAPTER XX — THE COLON-TYPHOID GROUP 133 Bacillus 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 Bacilli 143 Bacillus Botulinus 144 Bacillus Dysenteriae 145 Bacterium Termo 147 Bacillus Proteus Vulgaris 147 Proteus Mirabilis 147 Proteus Zenkeri 148 CHAPTER XXI — CHOLERA BACTERIA 148 Spirillum Cholerae (Comma Bacillus of Cholera) 148 CHAPTER XXII — BACTERIA IN PNEUMONIA 155 Diplococcus Pneumonias 157 Bacillus Pneumoniae 159 Bacillus of Rhinoscleroma 159 Bacillus of Influenza 160 Koch- Weeks Bacillus 161 Bacillus of Pertussis (Whooping-cough) 161 Bacillus Melitensis 162 CHAPTER XXIII— PYOGENIC Cocci 163 Streptococcus Pyogenes; Streptococcus Erysipelatis 164' Staphylococcus Pyogenes Aureus 166 CONTENTS 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-Axenfeld Diplobacillus of Conjunctivitis 171 Bacillus Pyocyaneus 171 CHAPTER XXIV — GONOCOCCUS — MENINGOCOCCUS 174 Micrococcus Gonorrhoeae 174 Allied Varieties 176 Diplococcus Intracellularis Meningitidis 177 Bacillus of Soft Chancre, Chancroid 178 CHAPTER XXV — ANAEROBIC BACTERIA (BACILLUS OF TETANUS; BACILLUS or MALIGNANT EDEMA, ETC.) 180 Bacillus of Tetanus 180 Bacillus CEdematis Maligni; Vibrion Septique 184 Bacillus Aerogenes Capsulatus 186 Bacillus Enteritidis Sporogenes 187 Bacillus Chauvei 187 CHAPTER XXVI — HEMORRHAGIC SEPTICEMIA GROUP 190 Bacteria of Hemorrhagic Septicemia 192 Bacillus of Chicken Cholera 193 Bacillus of Erysipelas of Swine 194 Bacillus Murisepticus; Mouse Septicemia 195 Micrococcus of Mai de Pis 196 CHAPTER XXVII — PROTOZOA 197 Entamoeba Histolytica; Amoeba Dysenteric 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 or 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 1 6 CONTENTS PAGE CHAPTER XXIX— 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 Cerevisiae (Torula Cerevisiae) 220 Saccharomyces Rosaceus; S. Niger; S. Albicans 221 Saccharomyces Mycoderma 221 Oidium „-*-. . . 221 Oidium Lactis •; 222 Oidium Albicans (Soor; Thrush Fungus ) 222 Pathogenic Yeasts 222 Blastomycetic Dermatitis or Oidiomycosis 223 Hyphomycetes (True Molds) 223 Penicillium Glaucum 223 Mucor Mucedo 224 Achorion Schonleinii 224 Trichophyton Tonsurans (Ring- worm) 225 Microsporon Furfur 226 Aspergillus Glaucus 226 Aspergillus Fumigatus 226 Examination of Yeasts and Molds 226 Cladothrices and Streptothrices 227 Crenothrix Kiihniana 227 Cladothrix Dichotoma 227 Leptothrix Buccalis 228 Beggiatoa Alba 228 Streptothrix or Cladothrix Actinomyces (Ray Fungus) 228 Streptothrix Madurae 230 Nocardia (Streptothrix) Farcinica; Bovine Farcin du Bceuf . . . 231 Plant Diseases Due to Bacteria 23 1 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 HUMAN BODY 257 CHAPTER XXXIV — GERMICIDES, ANTISEPTICS, AND ANTISEPSIS . . . 261 TABLES OF CHIEF 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 naturalist" 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. Muller, 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 spirochaete. It was not until 2 I7 1 8 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 until Pasteur made public his grand researches in regard to fermentation and the role bacteria played in the economy. Then Davaine resumed his studies, and in 1863 established 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 believed 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 wounds 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 19 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 1881, 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 serum 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 •%%-$ 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 a b c Fig. i. — Types of bacteria: a, Micrococcus; &, spirillum; c, bacillus. hence called the Schizophyta, or splitting plants. This group is subdivided into two classes — (a) the Schizophycece, or fission algae, and (b) 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- phologicj 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 round or cylindric, of an average diameter on transverse section of o.ooi mm. (=i micromillimeter), written ju= 2sooO mcn- 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, chlorin, 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 little envelop or capsule around the bacterium or preventing the separation of the newly branched germs, STRUCTURE 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 o>oj>, animal, 7X0165, 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 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, Bacterium vulgare, flagella arranged all about — peritrichia type (Lehmann and Neumann). 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 which 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 ;-|t; Fig. 3. — Division of bacteria: a, Division of a micrococcus; &, 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, eiido- sporous and arthrosporous. Endosporous. — First, a small granule develops in the pro- toplasm of a bacterium ; this increases in size, or several little 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 — clostridium (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 J Fig. 4. — Speculation (after De Bary). Fig. 5. — Clostridium. of the cell, and these are supposed to be the beginnings — the sporo genie 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 100° 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 bacillus produce the bacillus of tetanus. Saprophytes and Parasites. — Saprophytes: aairpos, put- rid; VTOv, plant. Parasites: Trapa, aside of; crlros, food. Those bacteria which live on the dead remains of organic life are known as saprophytic 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 limits, as the bacillus 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 \vhere 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 Rb'ntgen Rays. — Have little 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 2p 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 Trrw/za, 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, enzymes, toxins. Excretions, pigments, indol, cell proteins, bacterins. Proteins. — The protein contents of the bacterial cell 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 living cells. Bacterial enzymes are closely related to the ferments of special cells of higher animals and plants, like ptyalin 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 BACTERIOLOGY 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 successful 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 pneumonia 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 found in the surrounding 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, will 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 (thermolabile), and precipitated by ammonium sulphate. The Cardinal Conditions for Infection.— (i) The microorganism must be 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 collodion 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 will cause death in a rabbit. It has been found that 820 tubercle bacilli are necessary to kill a guinea-pig, and 1,000,000 staphylo- cocci to kill 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- INFECTION 33 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 occur. 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 (TWO* gram of tetanus toxin will kill 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 wrere 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 underneath 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. [ Natural Immunity f I Acquired ( Natural immunity is a natural inherited resistance against infection or intoxication, peculiar to certain groups of animals, but common to all the individuals 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 immune. 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 — 04) 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. (D) Inoculation with attenuated cultures of micro- organisms, as in Pasteur's anthrax vaccine or Haffkine's cholera vaccine. (E) Inoculation with virus of increasing viru- lence, as in the protective inoculations against hydrophobia. 36 ESSENTIALS OF BACTERIOLOGY (F) Inoculation with sublethal doses of virulent 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 immunized 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- kojff. — 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 the 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; b, toxin molecule; c, haptophore of toxin molecule; d, toxophore of toxin molecule; e, hapto- phore of the cell-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 35 ESSENTIALS OF 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 uniting 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 will 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 haptophore 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. Further 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 clump, 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 sensibttatrice" 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 living 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 bacteriolysins. Hemolysis. — When the hemoglobin of the red blood-cells is liberated, 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 OF 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 maybe 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 bound 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 hyper sensitiveness. 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 OF 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. (>6 inch); 2 mm. (TV 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 light 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 dove-oil, for example, all the rays of light from the object enter directly into the lens. The "homogeneous system," or oil-immersion lens, con- ESSENTIALS OF BACTERIOLOGY sists of a system of lenses which can be dipped into a drop of cedar-oil 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 light directly under 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 everything that is not stained. It is called Abbe's condenser (Fig. 9). Together with it is usually found 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 (daylight 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, oil-immersion and the narrowed blender. When examining with low-power objective, use a strong 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 all 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 linen 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 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 solid media. From Liquids. — With a long platinum needle the end of which is bent into a loop (Fig. n, a) obtain a small drop from the liquid containing the bacteria, and place it on a cover- glass or slide, 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. n, b) a small speck of the medium is taken and 46 ESSENTIALS OF BACTERIOLOGY 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, carefully pressing out all 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 bacterium, 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 anilin 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 eosin 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 anilin 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 10 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 of the aromatic benzol group, has also this property. METHODS OF 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 " anilin-water gentian- violet " or anilin- water fuchsin, etc. Carbolfuchsin. — Carbolic acid or phenol can be used instead of anilin-oil, and forms one of the main ingredients of ZiehPs 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. LofHer's alkaline blue and Koch's weak alkaline blue are made with potassium. Heat. — Warming or boiling 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 sufficient 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 10 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. 5O ESSENTIALS OF BACTERIOLOGY II. Weak Solutions Made by adding about i part of stock solution (I) to 10 parts of distilled water. This is the ordinary solution in use. III. Anilin-oil Water Anilin-oil 5 parts Distilled water 100 " — 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-oil water 100 " Absolute alcohol 10 " — 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 OF STUDYING BACTERIA 51 B. Kiihne: Methylene-blue 1.5 parts Alcohol 10.0 " 5 per cent, solution phenol 100.0 " 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. Lqffler's Mordant (for Flagella) Aqueous solution of tannin (20 per cent.) 10 parts Aqueous solution ferric sulphate (5 per cent.) i part Aqueous decoction of logwood (i : 8) 4 parts. — M. Keep in well-corked bottle. IX. Unnd'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. 52 ESSENTIALS OF BACTERIOLOGY XII. Roux's Double Stain Dahlia or gentian- violet 0.5 part Methyl-green 1.5 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. Carbolthionin (Nicolle) Saturated solution thionin in alcohol (90 per cent.) 10 parts Aqueous solution phenol (i per cent.) TOO " — 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 95 " — M. Wash in 20 per cent, solution of cupric sulphate crystals. METHODS OF STUDYING BACTERIA 53 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 OF 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 staining, 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 albuminoid 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 flame. 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 slide, 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 lifting the cover-glass off the slide (this is facilitated by letting a little 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 slide. 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 paraffin. 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 -ginnr mcn m thickness; this is the fineness usually required. The sections are brought into alcohol as soon as cut, unless they have been embedded in paraffin, when they are first washed in chloroform to dissolve out the paraffin. GENERAL METHOD OF STAINING SPECIMENS 57 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 still better plan, especially when the tissue is " crum- bling," is to carry out the whole procedure on the glass slide. 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 77, containing 5 per cent, acetic acid (1:20), where it 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 under 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. F77. Mount in Canada balsam (xylol balsam). Staining Blood Specimens. — A drop of blood is spread on 58 ESSENTIALS OF 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 (For 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 ]4 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. Typhoid 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 5~3° minutes i per cent, acetic acid few seconds. Absolute alcohol, xylol, Canada balsam. Bacteria dark blue, nuclei blue, cell-bodies light blue. To Sjtain 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 Bunsen 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. 60 ESSENTIALS OF BACTERIOLOGY 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 little 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. BowhilFs 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 applied 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. Kiihne's Method. — In sections the alcohol used sometimes decolorizes too much. To obviate this Kiihne 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 Muller'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. III. 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 DC 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 within the space between the outer and middle walls, C, and can be seen at F. The heat ascends, warming 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; Tt 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 100° 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, 1^4 feet wide, and ^4 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 boiling for five minutes if no spores are present. The boiling of any fluid at 1 00° 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 effective 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 boiling is pro- longed. Instead of sterilizing for a long time at once, successive sterilization is practised with nutrient media, so that the albumin will not be too strongly coagulated. Fifteen minutes CULTIVATION OF BACTERIA 65 each day for three days in succession in the Arnold sterilizer, or one exposure in the autoclave, five to fifteen minutes, at 15 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 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. 5 66 ESSENTIALS OF BACTERIOLOGY As modified, and in use in most laboratories, fifteen minutes, sterilization in steam, at 100° 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 will serve in most cases, especially if the medium is for imme- diate use, and does not contain gelatin or sugar. Cotton Plugs or Corks. — All the glass vessels (test-tubes, flasks, etc.) must be closed with cotton plugs, cotton-wool, 1 I 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 neutralize the alkalinity often pres- ent in fresh glass, or in chromic acid cleaning mixture one hour. (Potassium dichrorrtate, 6; water, 30; sulphuric acid, 46.) They are then well washed and rubbed with a brush, PREPARATION 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-MEDIA OF 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 OF BACTERIOLOGY exposed to the air led to the use of solid 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 ^ 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 sterilized in hot-air oven). 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, hi 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. PREPARATION 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 gelatin 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 solid 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. Azolitmin 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 minutes. 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 (5 grams to i liter alcohol) is needed as an indicator. The reaction should be +i 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. Boil 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. ^ NaOHwill neutralizes 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 4- i per cent. NUTRIENT CULTURE-MEDIA 7 1 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 +i 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 Loffler 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 -f-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 ^fore final sterilization or, if media usod 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 wound and the knife sterilized, and sterile 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 temperature can be controlled and main- tained. (See Fig. 21.) Coagulation of Blood-serum. — The tubes of blood-serum NUTRIENT CULTURE-MEDIA 73 having been placed in the thermostat, are kept at a temper- ature of 65° to 68° C. until 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 will 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 sterile 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 hi a fluid state. Human Blood-serum. — Blood-serum derived from placenta, serum from ascitic fluid and ovarian cysts, is prepared in a similar manner Fig. 22. — Incubator. 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. Loffler's Blood-serum Mixture. — To 3 parts clear serum add i per cent, glucose, beef infusion, and prepare as above; tube. NUTRIENT CULTURE-MEDIA 75 Hiss' Medium for Plating Agar 15 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 HC1. After filtration, dex- trose is added, then tubed and sterilized. Hesse's Medium for Typhoid Agar 5gm. Peptone 10 gm. Extract of beef 5 " Sodium chlorid 8.5 " Water 1000 c.c. Digest agar in 500 c.c. water, add the other ingredients dissolved in water. Mix and filter. Adjust reaction to i per cent, acid, tube, and sterilize in autoclave. Bile Salt Agar (MacConkey's) Sodium taupocholate 0.5 part Peptone 1.5 parts Lactose 3.5 " Agar.... 1.5 " Water q. s. 100.0 " Agar and peptone dissolved first. Lactose and bile salt added before tubing. Sterilize on three days intermittently. 76 ESSENTIALS OF BACTERIOLOGY (A) Conradi-Drigalski Medium Fresh meat 1500 gm. Water 2000 c.c. Mix and allow to stand twelve hours. Strain, boil one hour, and add — Peptone 20 gm. Nutrose 20 " NaCl 10 " Boil 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 30 gm. 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 plates. 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 2^ c.c. yV normal sodium hydroxid solution, and finally i per cent, potassium iodid. Endo Medium (Fuchsin-Lactose-Agar). — To 1000 c.c. agar add lactose, 10 grams; fuchsin (saturated alcoholic solution), 2 c.c.; solution sodium sulphite (10 per cent.), 25 c.c.; sterilize 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. Mix, sterilize in autoclave. B. Nutrient agar (neutral). Mix 3 parts A with 7 parts B, and pour 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 into 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. 78 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 various 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. — Manner 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 liquid 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. n, p. 45) are placed in a second tube, the rod being shaken somewhat in the 80 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 under 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 all 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 OF 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 will 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 Fig. 29. — Buchner's method of making anaerobic cultures (McFar- making anaerobic cultures (Mc- land). 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 "roll 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). Botkin's Method. — Petri dishes, uncovered, are placed on a rack under a large bell-jar, into which hydrogen gas is conducted. Alkaline pyrogallic acid is placed in the upper 30. — Wright's method for the cultivation of anaerobes. 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 solid 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 pyrogallic acid and i c.c. of 5 per cent, sodium hydroxid Fig. 31. — 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 medium 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, ^ dozen. Test-tubes, 100. One 1000 c.c. measuring glass. One 100 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 AND 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 four 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 (X4o) (Heim). small dot, like a fly-speck, to that resembling a small leaf. Some are elevated, some depressed, and some, like cholera, cup-shaped — umbilicated. 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 of— that one containing the colonies sufficiently 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, villous (Bacterium murisepticum) ; 5, arbor- escent (Bacillus mycoides). B, Liquefying: 6, Crateriform (Bacillus vulgare, twenty-four hours); 7, napiform (Bacillus subtilis, forty-eight hours); 8, infundibuliform (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 QO 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 Fig. 36.— Types of stroke cultures: i, Filiform (Bacillus coli); 2, echinulate (Bacterium acidi lactici); 3, beaded (Streptococcus pyogenes); 4, effuse (Bacillus vulgaris); 5, 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 prolific and easily kept, and is therefore a favorite animal for experiment. It lives well upon a little 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 young have opened their eyes, because the males, which have not been raised together, are apt to attack each other. Guinea-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 little 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. //. Through skin or mucous membrane. III. With the food. Method of Cutaneous Inoculation. — The ear of a mouse is best suited 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 hypodermic 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 suffice; 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 hydrophobia 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 nail or pin through each leg, and the head fixed with a pin 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 pericardium ex- posed. A platinum needle dipped into the heart after the pericardium 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- tablish immunity against infection. The best example of this is the immunization of armies and inmates of institu- tions against typhoid 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 bulb 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 reserved 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 under 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, 7 5 0,000,0000 (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 JJL 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. — A gar stroke: Growth limited to stroke; filiform, varying from a light 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 glistening, 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 liquid portion 7 97 98 ESSENTIALS OF BACTERIOLOGY containing small flakes of red pigment which settle at the bottom. Milk coagulated in twenty-four hours. A gar 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, slightly 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°-2$° 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. Fig. 38.— Colony of Bacillus Cancer Remedy. — Used in mesentericus vulgatus. Coley's treatment mixed with cultures of streptococci. Bacillus Mesentericus Vulgatus (Bacillus Vulgatus; Potato Bacillus of Flugge) (Fig. 38). Origin. — Surface of the soil, on potatoes, and in milk. Form. — Small thick rods with rounded ends, often in pairs. Very motile; produces abundant spores. Cultures. — Rapid growth; stain with Gram. A gar Colonies. — Round, with transparent center at first, then becoming opaque. The border is ciliated; little 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. SOME COMMON BACTERIA SLIGHTLY PATHOGENIC 99 Spores. — Are very resistant; are colored in the manner described in first part of the book for spores in general. Bacillus Megaterium (de Bary) (Fig. 39). — Origin. — Found on rotten cabbage and garden-soil. Form. — Large rods, four times as long as they are broad, 2.5/1. 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. subtilis. Stab-culture. — Funnel-shaped from above downward. Potato. — Thick growth with abundance of spores like B. subtilis. 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. A gar 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 Zopfii (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. A gar 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) (Ehrenberg). — 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 IOI long threads. Flagella are found on the ends. Spores of oval shape, strongly shining, very resistant. Very motile; Gram stain. Growth. — Rapid; strongly aerobic. Plate. — Round, gray colonies with depressed white center. Under microscope the center yellow; the periphery like a wreath, with tiny little rays projecting; very characteristic. A gar 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 like B. vulgatus. 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 subtilis 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 amounts 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. 102 ESSENTIALS OF BACTERIOLOGY Bacillus Violaceus (Schrater). — Origin. — Water. Synonym. — B. ianthinum (Zopf). Form. — A slender rod with rounded ends, three times as long as it is broad, often in threads. Spores. Motile, flagella. Stain. — With Gram and ordinary dyes. Cultures. — A gar stroke, moist, glistening, raised, at first yellow, then violet, inky colored. On Potato. — Violet black, moist, abundant growth. Gelatin Stab. — Rapidly liquefying 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 amount of H2S and indol. Pigment formed is in- soluble in water, slightly soluble in alcohol. Facultative anaerobe. Temperature. — 22°-25° 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 bacilli in 25 per cent, of patients affected 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 103 Properties. — Decomposes urea into ammonium carbon- ate; does not liquefy gelatin. Growth. — Grows rapidly, needing oxygen; can remain sta- tionary below o° C., growing again when a higher temperature is reached. Colonies on Plate. — On the surface like a drop of wax. Stab-cultures. — Looks like a very delicate 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 spirilla have been described. Spirillum 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 line; 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. 104 ESSENTIALS OF 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. A gar, 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 (XS3o) (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 10$ CHAPTER XVII BACILLUS OF ANTHRAX Bacillus Anthracis (Rayer and Davaine). — Rayer and Davaine, in 1850, first described this bacillus; but Pasteur, and later Koch, gave it the importance it now has. Synonyms. — Bactericie du charbon (Fr.); Milzbrand 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 Pfeiffer). Form. — Rods of variable length, largest of pathogenic or- ganisms 4 IJL to 10 /* 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 BACTERIOLOGY not occur in the circulating blood, but develop after death or in artificial media at 30° C. 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 I07 requires plenty of oxygen, but may be classed as a facultative anaerobe; grows well in all media. Colonies develop in two days; white shiny spots, which appear under microscope as slightly yellowish, granular, twisted balls, like a ball of yarn ; each separate string or hair, if looked at under high power, being composed of bacteria in threads. (See Fig. 44.) Fig. 45- Fig. 46. Figs. 45, 46. — Stab-cultures of anthrax in gelatin. Agar Stroke. — Grayish- white, slightly wrinkled layer with irregular edges. Gelatin Stab-cultures. — A white growth with thorn-like processes along the needle-track (like an " inverted fir tree"). Later on, gelatin liquefied, and flaky masses at the bottom. (See Figs. 45, 46.) 108 ESSENTIALS OF BACTERIOLOGY Potato. — A dry, creamy layer, and when placed in incu- bator, rich in spores. Staining. — Readily 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 bacilli 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 of 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 inocula- 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, liver, 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 ; wrhen 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 I Op have been infected, and through a scratch or slight 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 found, but a toxalbumin or proteid, called anthraxin, has been obtained. A certain amount 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 bacilli and spores temporarily. Hankin obtained an alexin from the blood and spieen of rats, they being naturally immune. It destroyed the anthrax bacilli in vitro, and used by injection in susceptible animals, made them immune. It is insoluble in alcohol or water. Protective Inoculation. — Animals have been rendered im- mune 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 found. The bacillus has never been found free in nature. 110 ESSENTIALS OF 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 public 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, slightly curved, 2 JJL 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; ERR 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 small 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 cigar-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 culture. Bits of tissue are placed on the surface, 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 113 " higher bacteria," mycobacteria, similar to actinomyces. Other acid-fast bacilli 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. Fig. 49. — Tubercle bacillus in sputum (Frank el and Pfeiffer). Bovine tubercle bacilli are about one-third smaller than hu- man tubercle bacilli. Resistance. — Bacilli in sputum, in dark, cool places may live several months. Dried sputum in sunlight and dust is infec- tive not more than ten days. The bacilli will resist in the dry state a temperature of 100° C. one hour. In moisture death occurs at 60° C. in a few minutes. Chemic Properties. — A waxy substance found in pure cul- tures, due to fatty acids. The fat-free substance is nucleo- 8 ESSENTIALS OF BACTERIOLOGY albumin, and the ash shows a large amount of phosphoric acid. Indol not found. Staining. — The tubercle bacilli require special methods to stain them, and a great number have been introduced. They are stained with great difficulty, but once stained, they are very resistant to decolorizing agents, hence called acid-proof or acid-fast. Upon these facts all the methods are founded. The resisting action of the bacil- lus to acids is supposed to be due to a peculiar arrangement of the albumin and cellulose of the cell, rather than to any particular cap- sule around it. A waxy substance, made up of fatty acid, has been found and supposed to account for this resistance. Others believe this substance to be an alcohol. It will be necessary to describe only those methods principally in use; and as the examination of sputum for bacilli is of so frequent an occurrence and so necessary, it is well to detail in particular the method of staining. Starting with the sputum, we search for little clumps or rolled-up masses; if these are not present, the most solid portions of the mucus are brought with forceps upon a clean cover-glass; very little suffices. With another cover-glass the mass is pressed and spread out evenly. Drawing one glass over the other, we obtain two specimens, and these are put aside or held high over the flame until dry. When the preparation is dry and has been fixed by passing through the flame three times, carbolfuchsin is dropped on Fig. 50. — Bacillus tuber- culosis; glycerin agar-agar culture, several months old (Curtis). BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS 115 the cover-glass and held over the flame until the stain boils; fresh stain is added, the boiling continued for a minute. Then the excess of stain is removed with edge of filter-paper. Decolorize in 25 per cent, nitric or 2 per cent, hydrochloric acid. The excess of acid is then washed out with 95 per cent, alcohol until no further color is imparted to the alcohol, and the smear is gray or light pink in color. The preparation is then washed with water and counterstained with aqueous methylene-blue for ten to thirty seconds. The Rapid Method (B. FrankeVs Method, Modified by Gab- bet). — The principle is to combine with the contrast stain the decolorizing agent; but the preparations are not permanent; the method, however, is very useful. Two solutions are required: one of Ziehl's carbolfuchsin; the other, Gabbet's acid methylene-blue. (See Formula No. x, on p. 51.) The cover-glass containing the dried sputum is passed three times through the flame, as described in the general directions. It is then placed in the carbolfuchsin solution five minutes (cold), or two minutes in the hot, immediately transferred to the second solution, the acid blue, where it remains one minute, then washed in water. The preparation is dried between filter-paper and mounted. Examined with oil-immersion. Slow Method. — The above method may also be used with- out heating, though in this case a much longer time is required before the bacilli take up the stain. The preparation is left in a small dish or beaker full of carbolfuchsin for eight to ten hours, and then decolorized and counterstained in the way described above. The method is less liable to produce artefacts than the quick method, but is not much used on account of the time it takes. Examination in Urine. — In urine, owing to the almost in- evitable contamination with the smegma bacillus, special methods are necessary to avoid error. The preparation may be left in 97 per cent, alcohol for eight hours, when the smegma bacillus will have become decolorized, or Pappenheim's Il6 ESSENTIALS OF BACTERIOLOGY method may be used: (i) Smear and fix as usual; (2) stain with hot carbolfuchsin for two minutes, pour off the surplus dye without washing; (3) counterstain and decolorize by pouring five times over the preparation the following solu- tion: A i per cent, alcoholic solution of corallin is saturated with methylene-blue and 20 parts of glycerin added. Wash in water, dry with blotting-paper, then in the air, and exam- ine. The tubercle bacilli are stained red, smegma bacilli, blue. Examination of Milk for Tubercle Bacilli. — Place a drop of the sample on a cover-glass and mix it with two drops of a i per cent, solution of sodium carbonate. The cover- glass is then gently warmed until evaporation is complete. The saponified fat is then stained, as the ordinary cover-glass preparation. Only a few tunes has any one succeeded in discovering the bacillus in milk. Other Acid-fast Bacteria. — The bacillus of leprosy re- sembles the tubercle bacillus in its staining properties, but gives up the carbolfuchsin more easily and is usually de- colorized by the acid and alcohol. It is colored blue by Pap- penheim's method. Acid-fast bacilli have also been obtained from timothy grass, butter, milk, manure, and the surfaces of animal bodies, but differ from the tubercle bacillus in cultural characteristics. Water has been found to contain acid-fast bacilli; care should be taken to test the water used previously to any im- portant examination for tubercle bacilli. Biederfs Method of Collecting Bacilli. — When the bacilli are very few in a great quantity of fluid, as urine, pus, abun- dant mucus, etc., Biedert advises to mix 15 c.c. of the fluid with 75 to 100 c.c. water and a few drops of potassium or sodium hydroxid, then boiling until the solution is quite thin. It is placed in a conical glass for two days, and bacilli with other morphologic elements sink to the bottom of the glass; when the supernatant liquid is decanted, the residue can be easily examined. In this way bacilli were found that had eluded detection examined in the ordinary manner. BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS 1 17 The centrifugal machine is used either in connection with Biedert's sediment method or without, to obtain the solids suspended in urine or serum. Antiformin Method. — A mixture of chlorin water and so- dium hydroxid; chlorin is liberated, and this dissolves most of the organisms in the sputum and the mucus, leaving un- altered the tubercle bacillus. Dilute thick sputum with dis- tilled water, add one-quarter volume antiformin, mix until solution is effected; add alcohol, equal volume, and allow mixture to stand eighteen hours. Prepare cover-slip prepa- rations from this. Staining Bacillus Tuberculosis in Tissue (Sections). — The general method of Gram can be used, but the better way is to use the following: Warm carbolfuchsin, fifteen to thirty minutes. 5 per cent, sulphuric acid, one minute. Alcohol, until a light-red tinge appears. Weak methylene-blue, three to five minutes. Alcohol, for a few seconds. Oil of cloves, until cleared. Canada balsam, to mount in. Instead of carbolfuchsin, alcoholic solution of fuchsin or anilin-water fuchsin can be used, but the sections must re- main in the stain overnight. Hardened Sputum and Sectioning. — Sputum can be hard- ened by placing it in 98 per cent, alcohol. Thin sections can be obtained by imbedding the hardened sputum in celloidin. The sections are then stained as ordinary tissue sections. To Preserve Sputum. — Sputum can be preserved for future use by placing it in alcohol, where it can be kept for months. Cover-glass preparations can then be made by softening the coagula with a small amount of liquor potassa. Pathogenesis. — When a guinea-pig has injected into its peritoneal cavity some of the diluted sputum containing tu- bercle bacilli, it perishes in about three weeks, and the follow- ing picture presents itself at the autopsy: at the point of inoculation there is a local tuberculosis — little tuberculous Il8 ESSENTIALS OF BACTERIOLOGY nodules containing the characteristic bacilli. In the lungs and the lymphatics similar tubercles are found — a general tuberculosis. If the animal lingers a few weeks longer, the tubercles becomes necrosed in the center and degeneration occurs, the periphery still containing active bacilli, cavities having formed in the center. Since the bacilli die hi course of time, killed by their own products, their number forms no correct guide of the dam- age present: even their absence in the sputum does not pre- clude the absence of a tuberculous process. It is their pres- ence only that warrants a positive declaration. The number of bacilli in a given specimen is no indication of the severity of the disease. They are found in the blood only when a vessel has come in direct contact with a tuberculous process through rupture or otherwise. They have been found occasionally hi other secretions — milk, urine, etc. Man is infected as follows.: Through Wounds. — Local tuberculosis. Through Nutrition. — Milk and meat of tuberculous ani- mals. Phthisical patients swallowing their own sputum and causing an intestinal tuberculosis. Inhalation. — This is the most usual way, probably consti- tuting the cause in nine-tenths of the cases in adults. The sputum of phthisical patients expectorated on the floors of dwelling-houses, in handkerchiefs, etc., dries, and the bacilli set free are placed in motion by the wind, or rising with the dust, are thus inhaled by those present. When the sputum is kept from drying by expectoration in vessels con- taining water, this great danger can be avoided. Intra-uterine or placental infection has been demonstrated, but is a great rarity. The ovum or human semen is seldom if ever infected, although tubercular infection of the testicle is common. Nearly all the cases of supposed heredity can be explained as follows: the young children, possessing very little resist- BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS Up ance, are constantly exposed to the infection through inhal- ation, and to intestinal infection through milk and other foods. Immunity. — No one can be said to be immune, though per- sons who have been greatly weakened offer less resistance than healthy individuals. Bovine and Human Tuberculosis. — Tuberculosis in Animals. — Tuberculosis is probably the most widely dis- seminated disease among domestic animals, and affects cattle, pigs, horses, dogs, cats, the smaller ruminants, birds, and even turtles and fish. The conclusion of Koch, made public in his address to the Tuberculosis Congress in 1901, that human and bovine tuberculosis are distinct and that in- fection of human beings from cattle occurs so seldom that no general regulations to restrict it are necessary, has found few adherents. In 1908 Koch reiterated his idea and chal- lenged his opponents to bring proofs to the contrary. Con- clusions at this writing seem to be that go per cent, of all pulmonary cases in adult man are not due to bovine infection. In children under five, however, 10 per cent, of the intestinal tuberculosis and cervical adenitis are due to the bovine type of infection through milk of diseased cows. It is true that certain differences exist between human and bovine tubercle bacilli, the latter appearing to be more virulent to animals, and it is a fact that cattle are very slightly suscepti- ble to, the human bacillus, but it is not likely that the con- verse is so. Children are particularly liable to infection through the gastro-intestinal tract, and it has been shown that the uninjured mucosa of the infant's intestine is per- meable to bacilli, so that the pulmonary disease in the young may often be the result of tuberculous bronchial nodes secondary to tuberculous glands of the mesentery. Various observations on animals have shown that the bacillus occurring in each species has acquired certain special characteristics regarding growth and virulence. The bacilli causing tuberculosis in the cold-blooded animals have de- parted farthest from the human type, those of birds to a less degree, and those of cattle least of all. 120 ESSENTIALS OF BACTERIOLOGY Products of Tubercle Bacilli. — The true nature of the tubercle toxin is not yet clear. It is not unlikely that several toxic bodies differing from one another in their properties are produced. Koch's tuberculin (1890), a bacterioprotein, was obtained by filtering through unglazed porcelain, concen- trated glycerin bouillon cultures of tubercle bacilli. It was speedily shown to be devoid of curative power, and is now used mainly for diagnosing the disease in cattle. In healthy animals little or no reaction is produced by the injection of 30 to 40 eg. of tuberculin, but if tuberculous, the temperature rises 2° to 3° F . in eight to twrelve hours, and remains elevated for a like period of time and may in larger doses prove fatal. It is dangerous unless used carefully. Tuberculocidin. — This is an albuminoid obtained from the original tuberculin by precipitation with alcohol. Klebs used it as a treatment for tuberculosis. Tuberculin residuum, an emulsion from the residuum, hence the name, T. R., is an extract made from dried and powdered living bacilli, and was recommended by Koch in place of the original or old tuberculin, O. T. Koch's bacillen emulsion (B. E.) is similar to tuberculin R, and is a glycerin emulsion of crushed bacteria, this being the entire substance instead of an extract. Theo. Smith recom- mends virulent uncrushed bacteria killed by moderate heat. Denys' B. F. (bouillon nitrate) tuberculin is a filtrate of liquid cultures to which 0.25 per cent, phenol has been added and allowed to stand two weeks. It is prepared in eight dilutions. Opsonic Treatment. — In recent years the use of tuberculin R has again been brought forward by Wright and others and curative claims made for it. It is used in very small doses — TT$Vo milligram at intervals of several days, and the effect on "he opsonic index carefully watched. Use of Tuberculin. — In the use of tuberculin severe reactions are to be avoided. The smallest dose possible is commenced with. Trudeau uses for afebrile cases a solution containing milligram, liquid measure, Koch's B. E., or Denys' B. F., BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS 121 increasing i decigram of the solution every three days until i c.c. of the pure nitrate can be injected without causing any reaction. A negative reaction sometimes occurs in well- advanced cases, and is, therefore, not a proof of the absence of disease. The reaction is due to the stimulation of irri- tating proteins. Yeast nucleins and other substances have a similar action. This treatment must extend over months. Tuberculin immunity does not last indefinitely. Under this careful treatment, associated with open air, proper food, and general hygiene, Trudeau and his followers have had some very good results. Von Pirquet Test. — Tuberculin applied to the abraded skin like a vaccination with cow-pox causes a local reaction in tuberculous infants and no reaction in healthy ones. It is not applicable to children over eighteen months of age. The test is so sensitive that it will be positive in the majority of instances, because the majority of people have at some time been affected with tuberculosis or exposed sufficiently to have within them sensitive bodies that are easily stimu- lated. Ophthalmic Tuberculin Reaction of Calmette. — A modified form of tuberculin is placed on the conjunctiva of an indi- vidual suspected of having tuberculosis. In a few hours a congestion, more or less severe, results, and lasts several days. In healthy persons no reaction occurs. The test is claimed to be harmless, though severe reactions have been reported in tuberculous patients, and even in healthy persons a second application to the same eye may cause an inflam- matory reaction. Moro's Test. — An ointment of tuberculin and lanolin, equal parts, rubbed in the skin of the arm. A crop of papules de- velops in twelve to twenty-four hours if test is positive. Agglutination. — Arloing and Courmont have describe^ an agglutination reaction for the tubercle bacillus similar to the Widal reaction of typhoid fever. (See p. 140.) It is very unreliable, however, and but little importance is at- tached to it. 122 ESSENTIALS OF BACTERIOLOGY Antituberculous Serum. — The attempts to produce an effective serum have so far been unsuccessful. Marmorek, by growing the bacillus on a special serum obtained by in- jecting calves with the leukocytes of guinea-pigs, has secured a toxin which he used to immunize horses, and the serum so obtained has been tried with encouraging results, but its value is still doubtful. Several other sera have been intro- duced, but none of them has shown any lasting virtues. Lepra Bacillus (Hansen). — Origin. — In 1880 Armauer Hansen declared, as the result of many years' investigation, that he found specific bacil- lus in all leprous processes. Form. — Small slender rods, somewhat shorter than tu- bercle bacilli, otherwise very similar in appearance. Neither in the form nor staining reactions can B. lepra be distinguished from B. tuberculosis. In the interior of the cell two or three oval spaces are usually seen, not believed tc be spores. They are immotile. Growth. — Bordoni-Uffred- uzzi have obtained growths upon blood-serum to which peptone and glycerin had been added, but the accuracy of this observation was doubted, and not until Clegg, in 1909, and Duval, in 1910, in work in the Philippine Islands devised special media was it possible to obtain readily initial and subcultures. The method depends upon supplying the organism with albumin partially metabolized. Clegg prepared this by planting the lepra bacilli on media containing ameba and bacteria; then, by short sterilization, destroying these, while the resistant B. lepra lived on. Duval, by adding trypsin to Fig. 51. — Pure culture of bacil- lus of leprosy, showing the charac- teristic morphology and arrange- ment of the bacilli (Duval). BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS 123 egg-albumin or blood-serum, was able to change the protein sufficiently without requiring ameba or bacterial digestion. The leprous nodule is cut in small slices and spread over an albumin slant or Petri plate, and the surface covered with i per cent, solution trypsin and placed in oven at 37° C. for ten days. The growth is moist and becomes yellow after several generations; it is on surface. Staining. — B. lepra resist the decolorizing action of acids, as the tubercle bacilli, but they are more easily stained, re- quiring but a few minutes more with the ordinary watery solu- tions. They take Gram's stain readily. Pathogenesis. — Arning inoculated a prisoner with tissue obtained from leprous patients and produced true leprosy, but this was a susceptible native and the evidence is not clear. Duval, by repeated injections of large amounts of pure culture, has produced leprosy in mice, guinea-pigs, and monkeys. Rabbits which have been infected through the anterior chamber of the eye showed the lepra nodules (containing the lepra bacilli) diffused through various organs. In man the skin and peripheral nerves are principally affected, but the lymphatic glands, liver, and spleen can also become the seat of the lepra nodules. The lepra cells which compose these nodules contain the bacilli in large numbers. By applying a vesicant to the leprous skin, the serum there- by obtained will contain great numbers of bacilli. This is a simple diagnostic test. Method of Infection. — Not yet determined; the air, soil, water, and food of leprous districts have been carefully ex- amined without result. The nasal secretion is very infectious. Intimate contact over a long period seems necessary, but the records of leper asylums show that very few cases ever de- velop among the attendants. Smegma Bacillus of Alvarey and Tavel. — Lustgarten, in 1885, through a certain staining process, found peculiar bacilli in syphilitic tissues which he thought had a direct 124 ESSENTIALS O-F BACTERIOLOGY connection with the disease. But this has been disproved and the cause of syphilis has been found in a protozoon which has been called Spirochaeta pallida, which see (p. 209). The smegma bacillus is found in and about the genital or- gans, is an acid-fast bacillus which resembles the tubercle bacillus in form, but is easily decolorized with alcohol, thus differing from the latter. It has no pathogenic properties, but is found at times in the throat and may be mistaken for B. Fig. 52. — Bacillus of glanders from a culture upon glycerin agar-agar (Xiooo) (Frankel and Pfeiffer). tuberculosis. Differentiated in staining, according to Pap- penheim's Method. Bacillus of Glanders (Bacillus Mallei (Loffler-Schiitz) ; Rotz-bacillus). — Origin. — In the "farcy buds" or little nodules of the disease, by Loffler and Schiitz, in 1882. Form. — Small slender rods, about the size of the tubercle bacillus. The ends rounded. Never appearing in large col- lections, usually singly (Fig. 52). Granules like spores appear in some cultures; branched forms are found. BACILLUS TUBERCULOSIS AND ALLIED ORGANISMS 12$ Properties. — They are not motile; do not produce gas; some pigment on potato. Growth. — The growth occurs between 25° and 40° C. — best at 37° C.; it is very sparse upon gelatin, but on glycerin-agar or blood-serum a very abundant growth occurs. Easily de- stroyed by heat, but cultures sealed and protected from light may live several months. Colonies. — On agar or glycerin-agar there appear in two to three days small white glistening drops, which under micro- scope seem as round granular masses with an even periphery, similar to young B. typhi colonies. Stroke Cultures. — On glycerin-agar and blood-serum small transparent drops of whitish or grayish color, which soon coalesce to form a broad band like B. coli. Potato. — An amber-colored, honey-like growth which grad- ually turns red, then brown, and greenish-brown around it. Weakly acid potatoes are a good medium and give the most typical growth. Staining. — Gram negative. Since the bacillus is very easily decolorized, some special methods have been recommended. Loffler's and Kiihne's solutions for cover-glass and sections. (See Staining.) Patho genesis. — If horses, field-mice, or guinea-pigs be inocu- lated subcutaneously with but a very small quantity of cul- ture, a local affection results, followed some time after by a general disturbance; ulcers form at the point of inocula- tion— little nodules, which then caseate, leaving scars and involving the lymphatics; metastatic abscesses then occur in the spleen and lungs, and death from exhaustion. Cattle, pigs, and rabbits are not easily affected; man is readily attacked. The bacilli gain entrance to the blood and urine. Nasal glanders occurs whatever the mode of inoculation. In the horse the type is more chronic than in the mule. A catar- rhal nasal discharge occurs, highly infectious. In the cutane- ous variety, the enlarged lymphatics or nodes which develop are called farcy-buds. Manner of Infection. — Glanders, being a highly contagious 126 ESSENTIALS OF BACTERIOLOGY disease, it requires but a slight wound to allow it to gain entrance. In horses the primary sore seems to be at the nasal mucous membrane. In man it affects those attending horses and is usually on the fingers, and terminates fatally within three weeks. Chronic glanders may last several years and end in recovery. Mullein. — A substance called mallein has been obtained from the cultures grown in glycerin bouillon. It gives a re- action when injected into cattle suffering from glanders, and is said to be useful in diagnosing the disease. The reaction is specific and never fails to reveal the presence of infection. The inoculation of a guinea-pig intraperitoneally with some of the suspected discharge will produce an orchitis if the glanders bacillus is present, which is quite characteristic and helpful in the diagnosis. CHAPTER XIX DIPHTHERIA BACILLUS Bacillus of Diphtheria (Klebs-Loffler) .— Origin.— Klebs found it in diphtheritic membrane in 1883; it was iso- lated by Lofller in 1884. Form. — Small, slightly curved rods about as long as tu- bercle bacilli and twice as broad; i n to 6 /z in length; the ends are at times swollen; spores have not been found. Their form is, however, very variable — sometimes much longer than usual, one end often greatly knobbed. Normal bacilli are found only in membrane. Stained forms are characteristic, since the ends are more eas- ily colored than the center, and usually the bacillus stains in segments, so that it seems to be made up of very short sec- tions or beaded. At first sight it appears like a chain of cocci. DIPHTHERIA BACILLUS 127 Small granules, the Babes-Ernst granules, are shown by the special staining of Neisser. Properties. — B. diphtherias is immobile; does not liquefy gelatin. Is not very resistant, being destroyed by a tempera- ture of 50° C., but may live on blood-serum for months. Acid is produced in sugar media. Growth. — Grow readily on all media, but best on blood- serum mixtures, between temperatures of 20° and 40° C. They are facultative anaerobic; they grow quite rapidly and ' -V .•** ,ill .^ • ' m Fig. 53- — Diphtheria bacilli from a culture on blood-serum, stained by Loffler's methylene-blue solution, showing deeply stained points (X20oo) (Wright and Brown). profusely. Egg cultures (Hueppe's method) give good growths. Passing currents of air increase the growth; on agar, growth is slow. Colonies on Agar Plates. — At 24° C. little round colonies, white under low power, granular center ; irregular borders. Stab-cultures. — Small, white drops along the needle-track. In glycerin-agar a somewhat profuse growth. Media should be slightly acid. Potato. — On alkaline surface, a grayish layer in forty-eight hours. 128 ESSENTIALS OF BACTERIOLOGY Blood-serum (After Loffler). — See p. 69 In a few hours (eight to sixteen) on the white opaque sur- face a slight moisture is noticeable which, if examined, is composed of bacilli. In twenty-four hours small round colonies are found which seem to ar- range themselves concentrically. The growth becomes more abundant, and the individual colonies larger and yel- lowish (Fig. 54). On Uood-coagulum (see p. 72) the growth is usually gray and the margins of the culture crenated. Often a diagnosis can be made in four hours if the serum-tubes are kept in the oven at 37° C. In milk, abundant growth, without curdling. Bouillon. — In bouillon an abundant growth takes place, and this medium is used to obtain the toxins. Staining. — Is positive by Gram's method. Stained best with Loffler's al- kaline methylene-blue. Neisser's double stain (see p. 52) shows granules, blue black, and body, brown. Pathogenesis. — By inoculation, ani- mals, which naturally are not subject to diphtheria, have had diphtheritic processes develop at the site of infec- tion; hemorrhagic edema then follows, and death. No agglutinins are developed in the serum. In rabbits paralyses develop, and when the inoculation occurs upon the trachea, all the prominent symptoms of diphtheria show themselves. Manner of Infection in Man. — The exact way is not yet known. It is supposed that the mucous membrane, altered in .—Bacillus diphtheriae; agar-agar culture (photograph by Dr. Henry Koplik). DIPHTHERIA BACILLUS I2Q some manner, the diphtheria bacillus then gains entrance and the disease develops. The bacilli may be found in healthy individuals who may act as a source of infection to sus- ceptible individuals without themselves becoming infected. They are seldom found in blood or other tissues; the symp- toms arise mainly from the absorption of the toxin. Prevalence of Bacillus Diphtheria. — Examinations made on a large scale of the throats of supposedly healthy individuals have shown that the Bacillus diphtherias is rather widely dis- tributed. Not only does it linger for many weeks in the throats of persons recently recovered from the disease, but it - 55- — Bacillus diphtherise, from a pure culture. is found in the caretakers, nurses, etc., and there are allied organisms, with more or less pathogenicity, that have been found in atrophic rhinitis, in conjunctivitis, and in the throats of unexposed normal individuals. Pseudodiphtheria. — The pseudobacillus of Hoffman is be- lieved by some investigators to be but a weakened diphtheria bacillus that has lost its toxic power, but its true relation is not settled. It is morphologically identical and at times is found side by side with the true bacillus. It grows well on agar, shows no granules with Neisser stain, and, contrary to B. diphtheria, does not produce as much acid in dextrose broth. 9 130 ESSENTIALS OF BACTERIOLOGY Methods of Diagnosis. — A small piece of exudate or some secretion from pharynx, tonsil, or nares is obtained on a ster- ile cotton swab and transferred, as soon as possible, to the sur- face of two or more blood-serum tubes (if these are not avail- able, the swab should be placed in a sterile test-tube or bottle, and sent to the laboratory at once). The inoculated tubes are placed in the incubator at 37° C., and examined in twelve hours. If a growth is visible, a slide is made and stained ;( ; MW »i »i / IV /'* *-' 4? . •"••' & r , ^ . •x/w'.'/^.--^» v '•LA-** &' V l^..- :#*&«. 1 \i!v ..<• /- Fig. 56. — Bacillus diphtheriae, from a culture upon blood-serum (Xiooo) (Frankel and Pfeiffer). with Loffler's and Neisser's stain, and if bacilli are present, with characteristic granules, the diagnosis of diphtheria is most probable. Negative results are not to be depended on. The use of antiseptics, gargles or the failure to obtain a portion of the exudate may give a negative culture result in a case of diphtheria. If the symptoms are suggestive, it is best to use antitoxin and isolate the patient, notwithstanding a negative report from the laboratory. If there are no clinical signs, the growth should be tested for toxicity by inoculating a DIPHTHERIA BACILLUS 13! guinea-pig ; it should be grown in alkaline sugar bouillon and tested in two days for acid. The xerosis and Hoffman's bacilli are not pathogenic for guinea-pigs. Products. — But it is not the mere presence of the bacillus that gives rise to trouble : certain products which generate it get into the system and produce the severe constitutional symptoms. Toxins of Diphtheria. — Roux and Yersin, in 1888, discov- ered the toxin and showed that the injection of the filtered culture bouillon (that is, freed of all diphtheria bacilli) gave rise to the same palsies as when the bacilli themselves were introduced. The toxins may be separated from three-weeks-old bouillon cultures by filtration. They are not albumins and are very complex. Ehrlich claims three forms: one he calls toxone; the other, toxin; the toxone produces paralytic symptoms and appears to be less affected by antitoxin; the third, toxoid, combines with antitoxin. The toxins are highly poisonous — o.ooi c.c. may be sufficient to kill a guinea-pig in less than twenty-four hours. The substance is unstable, losing its toxic power gradually. Heating at 58° C. for two hours is destruc- tive, but drying renders it more stable; Direct sunlight destroys its power in a few hours. Boiling in five minutes. If kept cold and in the dark, it may remain active two years. Alcohol and calcium chlorid precipitate the toxic element. Antitoxin. — Behring, in 1890, found that animals rendered immune had a principle in their blood that was antagonistic to the development of the toxin. Immunity. — Brieger and Frankel, by injecting 10 to 20 c.c. of a three-weeks-old culture of diphtheria bacilli which had been heated at 70° C. for one hour, produced an immunity in guinea-pigs against the virulent form. This active principle is unknown chemically, but has been called antitoxin. The toxin generated by the germ is supposed to be neutral- ized by the antitoxin and prevented from injuring the body tissues. The value of antitoxin in diphtheria seems to be established beyond a doubt, and it is the claim of eminent 132 ESSENTIALS OF BACTERIOLOGY sanitarians that the death-rate from this disease has been re- duced from 66 per 100,000 to 19 per 100,000 since the use of antitoxin (Park). The strength commonly employed in human beings is 5000 units, and as much as 120,000 units may be given without detriment in severe cases. If this amount is injected sub- cutaneously and even intravenously into a child suffering from diphtheria in the earlier stages (second to third day), the disease is often arrested. The membrane begins to dis- appear, and in two or three days has vanished. The con" stitutional symptoms are likewise greatly influenced by the injection. For prophylaxis and immunizing well persons 1000 to 3000 units are employed. In such conditions as«asthma severe and fatal results have followed the use of the serum, and some cases of peculiar sen- sitiveness to horse serum (see Anaphylaxis) have been re- ported, fatal results having occurred, but fortunately such mishaps are exceedingly rare. The antitoxin has no influence on the bacteria themselves; their virulence and length of residence in the body are not lessened. Preparation of Antitoxic Serum. — Horses are rendered immune by gradually increased doses of diphtheria toxin, the power of the toxin having first been standardized by its neu- tralization with some standard antitoxin in powdered form. Preparation of Toxin. — The bacillus is grown in veal broth with an alkaline reaction. (Acids prevent toxin formation.) There should be a free supply of oxygen, and, therefore, large shallow flasks are used. The maximum toxicity is developed in seven to ten days. The strength should be 3-^ c.c., fatal for 5oo-gram guinea-pig. The toxin is at first injected subcutaneously, then intraven- ously, and after several months' treatment a resistance is ob- tained that will withstand 300 to 500 times the original lethal dose. The horse is then bled, and from five to nine liters withdrawn; this is then allowed to coagulate, and under very careful precautions the serum is placed in sterile packages, THE COLON-TYPHOID GROUP 133 its strength having first been compared with a standard fur- nished by the United States Government. Unless kept in the dark and at low temperature, it loses strength rapidly. Antitoxic Unit. — An immunity unit, according to Ehr- lich, is the amount of antitoxic serum which will neutral- ize 100 times the minimum lethal dose of toxin, when serum and toxin mixed and injected into a 250-gram guinea-pig does not cause death in four days. Thus, if the serum will protect in doses of -5-$ c.c., then each cubic centimeter has 50 units' power, and 20 c.c. will contain 1000 units, or will be sufficient to neutralize an amount of. toxin that would be fatal for 25,000 kilos (12,500 pounds) of guinea-pigs, or 100,000 pigs weighing 250 grams each. The serum is con- centrated by precipitation and separation from the blood- serum of the pseudo-globulins containing the antitoxic prin- ciple, so that 10 c.c. contain more units than formerly. The doses given now are much larger than when first introduced. As much as 100,000 units have been employed in a single case. (Sera containing 1000 units to i c.c. are now being marketed.) Streptococcus in Diphtheria. — Streptococci have been found quite constant in diphtheria, but they resemble the Streptococcus pyogenes, and have no specific action. CHAPTER XX THE COLON-TYPHOID GROUP IN this group are placed a variety of organisms similar in form and growth and having many biologic properties in common, but differing in pathogenesis. The more impor- tant members of this group are: Bacillus coti, B. typhosus, B. enteritidis, B. dysenteries. Another closely related organism is the B. suipestifer (hog cholera). The form is usually a plump rod with rounded ends. Gram-negative. No spores. 134 ESSENTIALS OF BACTERIOLOGY Motile, all possessing flagella, on gelatin surface, a leaf -shaped, thin colony. They all reduce nitrate to nitrite. Gelatin not liquefied. Ferment sugar broth ; some produce acid in milk, some do not. Some form gas in sugar, some not. Bacillus Coli (Escherich). — Synonyms. — Bacterium coli commune; Colon Bacillus. — Found (1886) in human feces, in- testinal canal of most animals, in pus and water. Form. — Short rods, with very slow movement, often asso- ciated in little masses, resembling the typhoid germ, flagel- \jy» x>U >*;^» Fig. 57. — Bacillus coli communis, from an agar-agar culture (X 1000) (Itzerott and Niemann). lated, not forming spores (Fig. 57). Very short round ends; oval forms are found in animal tissues. Properties. — Does not liquefy gelatin, causes fermentation in saccharine (dextrose) solutions in the absence of oxygen, forming gas. Two parts hydrogen to i part carbon dioxid. Produces acid fermentation in milk; coagulates; its optimum temperature for growth is 37° C.; causes formation of indol in peptone solutions. In bouillon, forms cloudiness with slimy precipitate. Some cultures non-motile. THE COLON-TYPHOID GROUP 135 Growth. — On potato a thick, moist, yellow-colored growth ; on agar a gray- white growth; on gelatin a growth similar to typhoid. It can also develop on phenol-gelatin, and with- stands a temperature of 45° C. Staining.— Ordinary stains; does not take Gram. Pathogenesis. — Inoculated into rabbits or guinea-pigs, death follows in from one to three days, the symptoms being those of diarrhea and coma; after death tumefactions of Peyer's patches and other parts of the intestine; perforations into peritoneal cavity, the blood containing a large number of bacilli. The colon bacillus by many writers is held responsible for most of the complications of typhoid fever, such as peri- tonitis, cholangitis, etc. Epidemics of a cholera or dysentery nature, called by Esche- rich colitis contagiosa, and due to infection of water and food, have been noted by a number of writers. The onset is very sudden and prostrating, though not fatal. Many other forms of suppuration are associated with the presence of Bacillus coli. It is supposed to give rise to cystitis, infecting the bladder either through the urethra or the blood. The urine is then acid. Distribution. — The bacillus has been found very constant in acute peritonitis and in cholera nostras. All normal persons harbor the B. coli in the intestine, where, under ordinary con- ditions, it produces no disturbance. After death it multiplies rapidly, invading the tissues. In Water. — The presence of B. coli in surface waters is natural, owing to contamination with the fecal discharges of man and other animals. In well-water its presence denotes sewage or surface contamination, and such a well should be condemned until free from coli. (See Water Analysis.) Bacillus of Typhoid or Enteric Fever (Eberth-Gaffky) . — Origin. — Eberth, in the year 1880, found this bacillus in the spleen and lymphatic glands of persons dying of typhoid, and Gaffky isolated and cultivated the organism four years later. 136 ESSENTIALS OF BACTERIOLOGY Form. — Rods with rounded ends about three times as long as they are broad. Usually solitary in tissue-sections, but in old artificial cultures found in long threads. Flagella on all sides (Fig. 58). Properties. — Very motile. Spores have not been found; they do not liquefy gelatin. Growth. — They are facultative anaerobic; grow best at 37° C., but can also develop at ordinary room temperature. They develop chiefly on the surface, and very slowly. Repeated Fig. 58. — Bacillus typhi, from an agar-agar culture six hours old, showing the flagella stained by Loffler's method (Xiooo) (Frankel and Pfeiffer). freezing and thawing do not affect the vitality of the germ, and phenol in i to 2 per cent, solution has no effect on it. A ten-minute exposure to 60° C. is invariably fatal. Colonies on Gelatin Plates. — Two forms: the ones near the surface spread out like a leaf, transparent, with bluish fluor- escence. The deeper ones resemble whetstone crystals of uric acid, with the same yellowish tinge (Fig. 59). In five days they attain to 3 millimeters in diameter THE COLON-TYPHOID GROUP 137 Bile Salt Media. — Rapid growth without gas formation ; a number of special media suited for the growth of typhoid, namely, Jackson's, Hesse, Hiss, Conradi-Drigalski, etc. (See Water Analysis and formula for Media.) Stab-cultures. — Mainly on the surface, a pearly layer. A gar Stroke Cultures. — A transparent thick layer. Potato. — The growth here is quite characteristic. At 37° C. in forty-eight hours a moist, transparent film is formed over the whole surface, but so transparent that it can hardly be seen without close observation. If a small portion of this is placed under a microscope, it will be seen swarming with bacilli (Fig. 60). The growth never becomes more prominent; the potato must have a neutral or acid reaction. On Potato Gelatin. — The colo- nies do not have the yellow color; they are transparent; later on they become dark brown with green iridescence. Milk. — The bacteria grow very well in milk, producing a slightly acid reaction, but no coagulation. Fermentation Tube. — In sugar broth, in the fermentation tube, acids are formed without gas. Glucose Gelatin. — In glucose gelatin there is no gas-produc- tion. Indol is likewise not generated by the typhoid bacillus, whereas it is by the colon bacillus. Staining. — Colored with the ordinary anilin dyes, when they are warmed; since they are easily decolorized, acids should be avoided. Gram negative. Distribution. — Outside of the body it is rarely found. Typhoid or enteric fever is a general infection, but affecting chiefly the Peyer's patches of the intestine. The bacilli are found in the intestinal glands and in the enlarged and deeply Fig. 59. — Colonies of typhoid bacilli three days old (Xioo) (Frankel and Pfeiffer). 138 ESSENTIALS OF BACTERIOLOGY congested spleen. Metastatic abscesses form in various parts of the body, and here likewise the organisms abound. They occur in the feces only in small numbers, more commonly in the urine. The urine may contain active bacilli for weeks after recovery from the fever. Typhoid Bacilli in Water. — Although all evidence shows that the water-supply is a frequent source of infection, very few persons have ever isolated the typhoid bacillus from such Fig. 60. — Bacillus typhosus. Impression preparation from gelatin plate. Fuchsin (Xiooo) (Hicks). an infected source. The earlier reports show that no account was taken of Bacillus coli, which is usually present in pol- luted waters. (See Water Analysis.) Persistence in Water. — Franckland kept bacilli alive in water, sterilized by heat, seventy-five days; in filtered water at 19° C., five days; at 6° C., twelve days. In ordinary water they are likely to be destroyed in a short time by the over- growth of other bacteria. Under ordinary conditions they do not multiply, but decrease steadily in numbers. In soil THE COLON-TYPHOID GROUP 139 they are more persistent. Sewer-gas or air is never a source of infection. Mode of Infection. — The bacilli in the dejecta of the dis- eased person find their way into drinking-water, milk, or dirty clothes, and so into the alimentary tract of a person predisposed to the disease. Flies act as conveyors by infect- ing food. The bacilli enter the blood through the lymphatics, and so become lodged in various organs. They are quite resistant, living for some time in the soil and water, and are more resistant than other organisms to the action of phenol. An epidemic has been traced to the eating of oysters taken from contaminated wrater. Milk-cans washed in polluted water may be the origin of an epidemic. Ice is rarely a cause. Pathogenesis. — Lower animals do not have enteric fever, though their death has been caused by injection of the bacilli into the veins of the ear and peritoneum due to toxic substance. In man the bacillus has been found in the urine, blood, spu- tum, milk, intestinal discharges, roseolar spots, and in various organs, as spleen, liver, lymphatic glands, and intestinal villi. It is found in secretions several days after the attack has subsided. It is found in this disease only. Typhoid Carriers. — Some individuals retain a culture of the bacilli in the gall-bladder for years, and manufacture, or at least expel, true virulent bacilli through the feces and urine intermittently. Such persons' have infected other individuals without suffering any inconvenience themselves. Some forms of chronic inflammation, as cholecystitis and appen- dicitis, have been caused by the typhoid bacillus, though more often the colon bacillus is found. Products. — Brieger found a substance in the cultures, which he named typhotoxin, with the formula CgHnNC^. It has no specific action. A toxalbumin insoluble in water has also been isolated, but, as experiment animals are immune to the disease, no definite actions have yet been determined. The cultures, when old, show an acid reaction. Antityphoid bacterins (vaccines) have been used very ex- tensively in armies and institutions as a prophylactic or pro- 140 ESSENTIALS OF BACTERIOLOGY tective. Bacterins are made from a weakly virulent culture. The results so far obtained would indicate that this inocula- tion has some value, but the evidence is far from conclusive and the statistics on the subject require more careful study before they can be accepted as positive proof. An eighteen- hour agar surface growth is washed in sterile salt solution and killed by heating at 56° C. one hour. It is then diluted so as to contain one billion bacilli to i c.c. Tricresol, 0.25 per cent., is added to preserve, and animals tested for purity of the vaccine. A slight local reaction follows the inoculation; about three injections of jE/2, i, and one billion bacilli at ten- day intervals, render the subject immune. General symp- toms rarely occur. The Gruber-Widal Blood-serum Test. — In 1896 Widal and Griinbaum, working separately, developed what is now spoken of as the "Widal serum test," or "Widal reaction," or aggluti- nation test. It consists in testing a drop of blood of a patient suspected of having typhoid fever, by mixing a dilution of it with a drop of a fresh bouillon culture of typhoid bacilli, and examining the mixture in a hanging drop under the microscope. Within fifteen minutes to an hour the motility of the bacilli will cease, and they will have arranged them- selves into clusters, as if stuck or glued together (Fig. 61). If this reaction occurs within an hour, and with the proper dilution of the serum, the patient has or has had typhoid fever. Widal first used the serum of the blood; this has been modified so that a drop of dried blood is sufficient. Method of Test. — The method as applied in city laboratories is as follows: The physician is told to clean the finger of the patient with water (no germicides) , and with a needle draw a drop of blood on to a piece of ordinary note-paper. This is then sent to the laboratory; the paper with the dried blood is soaked for a few minutes in a watch-glass containing 4 drops of clean water, thus obtaining a dilution of i : 5. One drop of this is then mixed with one drop of a bouillon culture of typhoid bacilli of about twenty-four-hours' growth, and examined under the microscope in the hanging drop. Weaker THE COLON-TYPHOID GROUP 141 dilutions of the serum have been recommended (i : 50), and this should be used in cases of doubt. So far, about 95 per cent, of the cases examined, and which clinically were con- sidered typhoid fever, have given a positive reaction. It is not often present until the fifth day of the fever, and dis- appears usually within a year, though in some individuals it has been found ten years after an attack of the disease. The agglutinating properties have been found in nearly all the secretions of the body — tears, urine, milk, pleuritic effu- sions serous fluid from blisters, etc. Fig. 61. — The Widai agglutination reaction (Slater and Spitta). There is no relation between the reaction and the bacteri- cidal power of the serum; the agglutination is not a destruc- tion. The agglutinating power is active, though the blood be dried and sealed up for months. It seems to have no direct relation with the question of immunity, since it occurs at the height of the disease, and intense agglutinating serum may be had in severe cases and in cases with relapses. A negative result does not exclude typhoid. The test is quantitative — i. e., it depends upon the dilution of the blood-serum, since the serum of healthy persons in strong dilution will cause agglutination and loss of motility. 142 ESSENTIALS OF BACTERIOLOGY A serum in a dilution of i : 100 causing complete clumping in half an hour is undoubtedly typhoid. The culture must be kept in a vigorous condition by fre- quent subplanting, and must be tested occasionally with normal serum. Cultures kept in an incubator for a long time tend to agglutinate spontaneously. Macroscopic Agglutination Test. — Where laboratory facili- ties are not available, the sedimentation test is practical. It consists in adding the diluted blood or serum to be tested to a suspension of dead typhoid bacilli in salt solution. If the reaction is positive, a flocculent precipitate forms which consists of masses of agglutinated bacilli. A control tube con- taining normal serum and the suspension should remain opaque and show no flocculi. Differentiation Between Colon and Typhoid. — The colon bacillus and the typhoid bacillus resemble each other so closely that much attention has been paid to methods of differentiation. Points of Resemblance Between Bacillus Typhi and Bacillus Coli Communis. — First, microscopic appearance; second, agar and gelatin cultures; third, sometimes growth on potato the same; fourth, staining peculiarities; fifth, resistance to phenol. Points of Difference: Colon Bacillus. Typhoid Bacillus. Bile media, gas. None. Less motile. Actively motile. Gelatin colonies • develop Develop slowly. more rapidly. Produces gas on dextrose or Does not. lactose media. Coagulates milk. Does not. Produces indol. Does not. Growth on potato visible. Invisible. Changes neutral red to Does not reduce neutral red. yellow. Endo-fuchsin red. Not. THE COLON-TYPHOID GROUP 143 Differences are also noted in the growth on special media, such as those of Hiss and Eisner. On Eisner's potato-gelatin the colon bacillus and the typhoid bacillus both grow read- ily. The medium of Hiss is of some assistance in isolating the germ. (See p. 75.) Hiss Media. — Shows B. coli, large colony, even borders. B. typhi, small colony, hairy and fringy threads. (See p. 75.) Endo-fuchsin-lactose A gar (see p. 77). — Incubation on plates of this media shows B. coli red; typhoid as clear, color- less drops. Malachite green added to agar permits the growth of B. typhi, but not B. coli. The dye must be as nearly neutral as possible. Bile Salt Media. — Fresh bile or sodium taurocholate added to lactose glucose agar or broth permits the rapid growth of both B. typhi and B. coli, fermentation with gas formation denoting B. coli, growth without fermentation meaning typhoid. Drigalski and Conradi Media. — Petri plates filled with this media are inoculated on the surface only. Placed in incubator sixteen to twenty-four hours. Typhoid colonies small, transparent, and blue. Colon colonies red, coarser, and larger. Typhoid Bacilli from Blood. — Conradi, Busquet, Coleman and Buxton, and others have found the bacilli in the blood of every patient by the following method: A mixture of ox-bile, 90 c.c., glycerin, 10 c.c., and peptone, 2 gm., is distributed into 20 c.c. flasks and sterilized. Ten cubic centimeters of blood is drawn from the elbow into a glass syringe and divided among three flasks. These are incubated, and in twenty-four hours litmus-lactose- agar plates are inoculated on the surface by a stroke from the flasks. A growth is obtained in five or six hours. If the growth is a bacillus which has not reddened the medium, it is tested for the Widal reaction with immune se- rum. The diagnosis has been made as early as the second day. Paracolon or paratyphoid bacilli are members of the 144 ESSENTIALS OF BACTERIOLOGY colon group described by Widal, Gwyn, Schottmiiller, and others. They are of importance, since they produce fevers clinically resembling a mild form of typhoid, but which are rarely fatal. They may be the sole cause of the disease, and also occur together with the typhoid bacillus in mixed and secondary infections. Morphologically, they resemble the typhoid bacillus, but differ from it culturally and give their own serum reactions with the blood of affected patients. They ferment glucose, but not lactose or saccharose; litmus milk at first becomes acid, but later grows alkaline and is not Fig. 62. — Bacillus botulinus, with spores. Pure culture on sugar- gelatin. Van Ermengem prep. (Kolle and Wassermann). coagulated. On potato a slight visible growth occurs; in- dol is usually not formed. Typhoid sera do not agglutinate paracolon bacilli, and vice versa; also different paracolon infections may not agglutinate each other. Bacillus Botulinus (Van Ermengem) . — An anaerobic ba- cillus cultivated by Van Ermengem in 1896 from ham wrhich had caused poisoning. Form. — A large bacillus with rounded or spindle-shaped ends, and often with oval terminal spores, motile, with lateral flagella (Fig. 62). THE COLON-TYPHOID GROUP 145 Staining. — Gram positive, easily stained with ordinary dyes. Growth. — Strictly anaerobic. Forms abundant gas in glu- cose, gelatin, and liquefies cultures, producing butyric acid odor. Best temperature between 20° and 30° C. Patho genesis. — Produces a powerful toxin in the tissues, like the tetanus bacillus. This toxin may be present in the affected meat without causing decomposition, and thus give rise to poisoning. Bacillus Dysenteriae (Shiga, 1898). — The term dysen- tery is applied to an intestinal disease displaying more or less Fig. 63. — Bacillus dysenteriae from agar culture. Fuchsin stain. Zett- now prep. (Kolle and Wassermann). constancy in its clinical manifestations, but having, as is now known, a variety of causative agents. It is fairly certain that one type is the result of infection with an ameba, while non- amebic forms can probably be produced by several bacteria. Chief among these is the bacillus first described by Shiga in Japan, and since then found by Kruse in Germany, by Flex- ner, Strong, and Harvie in the Philippine Islands, and by Vedder and Duval in the United States. The fact that it is constantly present in the feces in one type of dysentery, that 146 ESSENTIALS OF BACTERIOLOGY such cases give a positive agglutination reaction, the produc- tion of a curative serum by the immunization of animals with pure cultures, and the results on experiment animals, leave little doubt as to the specificity of the organism. Origin. — The dejecta of dysenteric patients. Form. — A plump bacillus with rounded ends, resembling the typhoid and colon bacilli (Fig. 63). Properties. — Motility doubtful, but numerous flagella have been demonstrated. Does not form spores. Staining. — Stains readily, negative to Gram; facultative anaerobe. Growth. — Best at 37° C. Killed by ten minutes' exposure to 55° C. Gelatin. — A white line of growth along puncture; super- ficial growth slight. Bouillon. — Uniform clouding. Indol usually not produced ; milk not coagulated. A gar. — Resembles typhoid bacillus. Potato. — Thin whitish layer, turning light brown. No gas-formation in glucose or lactose media. Acid is formed. Pathogenesis. — Mice and guinea-pigs die in one or two days after intraperitoneal inoculation. Rabbits usually recover, though lesions analogous to those of human dysentery have been produced. Dogs die in five or six days, with well- marked diarrhea. Products. — The patient's blood-serum agglutinates the ba- cillus in cases in which it can be cultivated from the stools. The reaction is absent from other cases. Shiga has reduced the mortality from 34.7 to 19 per cent, by means of a serum obtained from immunized horses, but in more extensive tests the antidysenteric serum proved of little value. In man the organism or some of its varieties is associated with dysentery and is found chiefly in the stools; abscesses are seldom found; the amebic dysentery forms liver abscess, not in other organs. Polluted water is responsible for its spread in epidemic form. THE COLON-TYPHOID GROUP 147 In the summer diarrhea of infants associated with mucus, B. dysenteric has been found, and is considered a causative agent. Bacterium Termo (Cohn). — This was a name given to a form of microorganism found in decomposing albuminous material, and was supposed to be one specific germ. Hauser, in 1885, found three different distinct bacilli which he grouped under the common name of proteus, which have the putrefy- ing properties ascribed to Bacillus termo. Bacillus Proteus Vulgaris (Hauser, 1885). — Origin. — In putrid animal matter, in the feces, and in water. Form. — Small rods, slightly curved, of varying lengths, often in twisted chains, having long cilia or flagella. Properties. — Very motile, and very soon liquefying gelatin; forms hydrogen sulphid gas ; causes putrefaction in meat. Growth. — Growth very rapid, best at 24° C.; is facultative aerobic. Gelatin Plates. — Yellowish-brown, irregular colonies, with prolongations in every direction, forming all sorts of figures; an impression preparation shows these spider-leg processes to consist of bacilli in regular order. Stab-culture. — The gelatin soon liquid, a gray layer on the surface, but the chief part of the culture in small crumbs at the bottom. A gar. — Rapid, moist, gray growth. Milk. — Acid coagulation. Dextrose Broth. — -Gas-production. Pathogenesis. — Rabbits and guinea-pigs injected subcutane- ously die quickly; a form of toxemia, hemorrhagic condition of lungs and intestines, present. When neurin is injected previously, the animals do not die. This ptomain is sup- posed to be generated by the Proteus vulgaris. In man these or similar bacteria have been associated with food-poisoning epidemics, infantile diarrhea, infectious jaun- dice (Weil's disease). Proteus Mirabilis (Hauser). — Differs from Proteus vul- 148 ESSENTIALS OF BACTERIOLOGY garis in that the gelatin is less rapidly liquefied. Found also in putrid material. Proteus Zenkeri (Hauser). — Does not liquefy gelatin; otherwise similar to the other two. CHAPTER XXI CHOLERA BACTERIA Spirillum Cholera (Koch) (Comma Bacillus of Chol- era) . — Synonym, Vibrio Cholera. — Origin. — Koch, as a mem- ber of the German expedition sent to India in 1883 to study cholera, found this micro- organism in the intestinal contents °f cn°lera patients, and by further experiments identified it with the disease- Form. — The spirillum as seen or- dinarily appears as a short, arc-like body, about half the size of a tuber- Fig. 64.-Comma bacillus, de bacillus, but when seen in large pure culture (x 600). groups, spirals are formed, each little arc appearing then as but a segment, a vibrio. Each arc is about three times as long as it is broad, and possesses a flagellum at one end. Old agar cultures show straight forms; S-shaped forms not uncommon, made of two vibrios end to end (Fig. 64). Properties. — The spirilla are very motile; liquefy gelatin. They are easily affected by heat and dryness. Spores have not been found. Growth. — At ordinary temperatures on all nutrient media that have an alkaline or neutral reaction. Strongly aerobic. Colonies, Gelatin. — After twenty-four hours, small white points which gradually come to the surface, the gelatin being CHOLERA BACTERIA 149 slowly liquefied, a funnel-shaped cavity formed, holding the colony in its narrow part, at the bottom, and on the fifth day all the gelatin is liquid. If the colonies of three days' growth are placed under microscope, they appear as if composed of small bits of frosted glass with sharp, irregular points. Stab-culture. — After thirty hours a growth can be distin- guished along the needle-track, and on the surface a little cavity is formed, filled by a bubble of air, and this liquefaction Fig. 65. — Cholera colonies after thirty hours (Xioo) (Frankel and Pfeiffer). proceeds until, on the sixth day, it has reached the sides of the tube, tapering, funnel-shaped, to the bottom of the tube. After several weeks the spirilla are found in little collections at the bottom of the fluid gelatin. In eight weeks the bacilli have perished. A gar. — Stroke cultures. A shiny white layer which lasts many months. Alkaline Agar. — Plates at 37° C. Flat discs, transparent, grayish blue. 150 ESSENTIALS OF BACTERIOLOGY Potato.— A yellow, honey-like, transparent layer if the potato is kept at animal heat. Bouillon. — A wrinkled scum is soon formed in bouillon. The spirilla live well and grow in sterilized milk and sterilized water, remaining virulent in the latter for many months. Fig. 66.— Cholera Fig. 67.— Cholera Fig. 68.— Cholera bacillus (forty-eight bacillus (sixty hours; -bacillus (seventy- hours; 5 per cent. 5 per cent, gelatin). two hours; 15 per gelatin). cent, gelatin). Figs. 66-68.— Tube-cultures (from United States Government Report on Cholera. — Shakespeare). In ordinary water the bacteria present are destructive to the comma bacilli, and they die in a few days. Dunham's Peptone Solution. — Useful for the development of nitrites and the indol reaction. (See p. 77.) Also for the rapid development of the cholera vibrio. In four hours after inoculation of peptone water pure cultures may be obtained; CHOLERA BACTERIA 151 best to make several plantings from the peptone to agar after six hours' growth. Dieudonne's Medium. — (See p. 77.) In this cholera vibrio grow abundantly; other intestinal bacteria very scantily. This medium valuable mostly for feces, less for infected water. Staining. — They are colored well with watery anilin solu- tions. The flagella can be well seen by staining according to the flagella stain or Giemsa. Pathogenesis. — Experiment animals are not subject to cholera Asiatica, but, by overcoming two obstacles, Koch produced choleraic symptoms in guinea-pigs. Nicati and Rietsch prevented peristalsis and avoided the acidity of the stomach-juices by direct injection into the duodenum, after tying the gall-duct. Koch alkalinized the gastric juice with 5 c.c. of 5 per cent, solution of sodium carbonate, and then injected 2 grams of opium tincture for every 300 grams of weight into the peritoneal cavity, paralyzing peristalsis. The cholera culture then introduced through a stomach-tube, the animals die in forty-eight hours, presenting the same symp- toms in the appearance of the intestines as in man, the serous effusion containing great numbers of spirilla. Rabbits in- jected into the ear veins with cholera cultures die very quickly and present intestinal lesions. The vibrio is met with in the layer of flaky mucus which coats the surface of the intestine. It may invade the biliary passages. Manner of Infection in Man. — Usually through the alimen- tary tract, with the food or drink, the intestinal discharges of cholera patients having found entrance into the source of drinking-water. Soiled clothes to fingers, fingers to the mouth, etc.; torpid catarrhal affection of the digestive tract predisposing. The spirilla are not found in the blood or any organ other than the intestines — the tissue of the small intes- tines. They are also found in the vomit and the intestinal contents. Toxins. — From broth cultures soluble toxins which have a hemolytic action have been isolated. The toxin is easily destroyed by heat (thermolabile). 152 ESSENTIALS OF BACTERIOLOGY Products — "Cholera red." Indol Reaction. — Present in peptone water cultures containing nitrates. The indol is shown by the addition of a few drops of pure sulphuric acid, the solution turning red — the so-called "cholera red" Once thought distinctive, but other bacteria also give rise to indol, and the same reaction. Serum Agglutination Test. — The agglutination test is made in the same way as the Widal test for typhoid fever. Ag- glutinins appear in the blood five to ten days after infection. -, *\ V ^ N. Fig. 67. — Comma bacillus in mucus, from a case of Asiatic cholera. Cultures in serum dilutions of 1:1000 up to i : 10,000 are agglutinated. Detection of Cholera Organisms in Drinking-water. — When a few bacteria are supposed to be present in fecal matter or drinking-water, it is best to add a large quantity of the ma- terial (200 c.c. of drinking-water) to about 10 c.c. of bouillon or peptone-water, and place the mixture for twenty-four hours in an incubator, which will cause rapid reproduction, and then the organisms can be readily discovered. CHOLERA BACTERIA 153 From Feces. — The following technic is recommended: 1. Examine mucous flakes in stained preparations and hanging drop. 2. Isolate on agar media at 37° C. (a) Plant plates of alkalinized agar and Dieudonne's medium with particles of feces. (b) Plant in 50 c.c. peptone solution i c.c. fecal matter. After six hours or longer in the incubator at 37° C. take several loopsful from the surface and plant on several plates Dieudonne's and ordinary alkaline agar. (c) Investigate agglutination reaction, using drops from isolated colonies and secure pure cultures. 3. Demonstrate the reaction of Pfeiffer and agglutination with the pure colonies. Protective Bacterins. — Virulent cultures killed by heat have shown protective power and were used extensively during an epidemic in Japan. Haffkine has obtained a great reduction in mortality in cholera regions by the use of anticholera bacterins as a pro- tective measure. Serum therapy has not been successful. Carriers. — In some recent examinations of persons exposed to cholera, carriers of typical cholera vibrio have been found. The vibrio may be found, as in typhoid fever, in the gall- bladder. Pfeiffer's Reaction and Agglutination. — The serum of an animal (a rabbit) made immune against cholera by the in- jection of sterile or living cultures, intravenously, three times at intervals of a week, has an action against the cholera spiril- lum. It first precipitates the bacteria outof an emulsion, leav- ing a clear liquid (agglutination), and then dissolves the bac- teria (bacteriolytic action), leaving only spheric granules. This action is specific, i. e., the cholera immune sera will affect only cholera vibrio. Such serum is not antitoxic, it is bacteriolytic. For diagnostic purposes an agglutination in dilution of i : 1000 by a serum with an activity of i : 4000 is suspicious of cholera. The blood-serum of convalescents 154 ESSENTIALS OF BACTERIOLOGY and cholera-vaccinated individuals contains the same bac- tericidal substances. Allied Varieties. — Many vibrios resembling the spirillum of cholera have been isolated from drinking-waters and from the stools of persons suffering with diarrhea, and some bac- teriologists are inclined to consider them as varieties of the true cholera spirillum, which under certain conditions become pathogenic. Among these are Spirillum berolinense, S. dun- barii, S. danubicum, S. of Wernicke, S. bonhoffii, S. weibeli, S. schuylkilliensis, S. milleri, S. aquatilis. The last two are non-pathogenic for experiment animals, also the Finkler- Prior vibrio, vibrio Metchnikovii, and tyrogenum, which have historic interest because of their close identity with the cholera organism, but with the agglutination tests and Pfeiffer phenomenon they have been shown to be dissimilar. Conclusions of International Committee of Public Hygiene, Adopted October p, 1911. — Every choleriform vibrio can be considered as truly choleraic which presents agglutination in the proportion of at least 1:1000 by a cholera serum of 1:4000 activity, or a positive Pfeiffer reaction, and every choleriform affection in which is encountered such a vibrio should be considered as a case of cholera. Method of Pfeiffer. — i. Secure immune serum by injecting into peritoneal cavity of a rabbit an entire agar culture which has been killed by heating for one hour at 56° C. Four- teen days after collect the blood-serum. 2. Dilute the suspected vibrio by adding one loopful of an eighteen-hour-old agar culture to i c.c. meat water. 3. Add to the above about i milligram of immune serum and inject this into peritoneal cavity of a guinea-pig. 4. At the same time a second guinea-pig is inoculated with diluted culture (2), but without the serum. 5. A third guinea-pig is inoculated with a similar dilution of culture to which has been added about 10 milligrams normal rabbit serum. 6. At the end of twenty minutes, and again at the end of one hour, some of the peritoneal fluid is examined from each BACTERIA IN PNEUMONIA 155 pig, under strong magnification, in hanging drop and dark field illumination. 7. The reaction is positive if in the fluid from No. i pig the vibrios are dissolved, while in that from No. 2 and No. 3 the vibrios are very motile and active and form well pre- served. It is necessary that the vibrio be of good virulence. Method of B or del. — As experiment animals are not always available, Bordet has elaborated a test-tube method. The immune serum is diluted 1:50, 1:100, 1:500, and 1:1000. Into a series of test-tubes there are poured 5 drops of a guinea- pig serum, 5 drops of a mixture of suspected culture (one loop- ful of an eighteen-hour-old agar culture to i c.c. salt solution), and enough of immune serum and salt solution to make the necessary dilution and up to 20 drops. A series of controls is made with normal serum and the same amount of microbic culture and guinea-pig serum. After eighteen hours the cholera vibrios will be active in the control, but dissolved and clumped up in the tubes containing the immune serum. CHAPTER XXII BACTERIA IN PNEUMONIA KLEBS in 1875 called attention to the presence of bacteria in pneumonia, and in 1882 Friedlander developed a bacillus from the lung tissue of a pneumonic person which he thought was a coccus, and called it pneumococcus. In 1886 A. Frankel and Weichselbaum proved that this organism was not constant — in fact, was rare. A. Frankel obtained in the majority of cases of pneumonia an organism that he had described in 1884 under the name of sputum-septicemia micrococcus. Weichselbaum called this Diplococcus pneumonia, and be- 156 ESSENTIALS OF BACTERIOLOGY Fig. 70. — Bacillus pneumonias of Friedlander, from the expectoration of a pneumonia patient (Xiooo) (Frankel and Pfeiffer). Fig. 71. — Diplococcus pneumonias in exudate from human lung; anilin- water-fuchsin; Weichselbaum prep. (Kolle and Wassermann). BACTERIA IN PNEUMONIA 157 lieved it to be the real cause of pneumonia. It is the generally accepted organism of the disease, and can be isolated from nearly all cases of acute croupous pneumonia. It is found in about three-quarters of all cases of pneumonia. Diplococcus Pneumonias (Frankel and Weichselbaum, 1 886) . — Synonyms. — Streptococcus Lanceolatus; Pneumococcus; Diplococcus Lanceolatus; M. of Sputum Septicemia; Fr anker s Pneumococcus. Origin. — Found it in the sputum of pneumonic patients. It has been found in many other serous inflammations, and also in the mouths of healthy persons. Fig. 72. — Diplococcus of pneumonia in blood of rabbit (Xiooo) (Frankel and Pfeiffer). Form. — Large, lancet-shaped cocci. Usually found in pairs, sometimes in filaments of three and four elements. In the material from the body a capsule surrounds each coccus. In the artificial cultures this is not found (Figs. 71 and 72). Properties. — Variable in form, approaching the bacillary type. Do not liquefy gelatin. There are no spores. Non- motile. Growth. — Best between 27° C. and 41° C., seldom below 25° C. Facultative anaerobic. The culture-media must be slightly alkaline; the growth is slow. 158 ESSENTIALS OF BACTERIOLOGY Colonies. — Glucose or Glycerin A gar Plates. — Growth slow, of small, round, moist colonies, separated. Stab-cultures. — Along the needle-track small separate white granules, one above the other, like a string of beads. Blood Bouillon. — Bouillon containing one- third blood-serum or ascitic fluid favors the growth. They grow better here than in the other media, remaining alive a longer period of time. Blood-serum or Blood-agar. — Growth more vigorous. A good growth on blood-serum or blood-agar. Fig- 73.— Pneumobacillus in blood (Xiooo) (Frankel and Pfeiffer). Staining. — Takes Gram's method and the other anilin stains very readily. The capsule stained by Hiss method (p. 61) or Welch. Resistance. — Cultures in sugar media must be frequently transplanted, as the organism is destroyed hi a few days by the acid generated. In albumin alkaline media (blood-serum, etc.) the cultures can be kept active two weeks or more. In sputum the pneumococcus may survive several days. When dried but exposed to sunlight, death occurs in a few hours. BACTERIA IN PNEUMONIA 159 Patho genesis. — Rabbits and guinea-pigs, if subcutaneously injected, die in the course of a couple of days with septicemia (o.i c.c. of a fresh bouillon culture suffices). Autopsy shows greatly enlarged spleen and myriads of micrococci in the blood and viscera, the lungs not especially affected. If injected into the trachea, a pneumonia occurs. In man they are found in 90 per cent, of croupous pneumonia, and usually only during the existence of the rusty sputum, i. e., the first stage. Found in the tissue of the inflamed lung, and in the blood in nearly all cases of lobar pneumonia. The pneumococcus has also been found in pleuritis, peri- tonitis, pericarditis, meningitis, and endocarditis. It stands in some intimate relation to all infectious inflammations of the body. Their presence in healthy mouth secretion does not speak against this, it requiring some slight injury or low- ered resistance to allow this ever-present germ to produce a pneumonia from an infectious disease like measles or in- fluenza. Toxins and antitoxins have not been separated or demon- strated. The poisons are probably endotoxins, and closely connected with the cell-body. Agglutination properties of pneumonia blood serum, if any, are very weak — i : 50. Immunity and Serum Therapy. — One attack produces no immunity; and no immune serum has been found of any value. By growing in an acid medium, the organism has been ren- dered less virulent. Bacillus Pneumoniae (Friedlander, 1882).— Synonym. — Capsule Bacillus of Pfei/er. — Once supposed to be a cause of pneumonia. It grows readily on ordinary media; is Gram negative; in form and capsule formation it sometimes re- sembles the pneumococcus (Fig. 70). Bacillus of Rhinoscleroma (Frisch, 1882) . — It was found in the tissue of a rhinoscleroma, but resembles the Fried- lander bacillus in nearly every respect, and as the disease rhinoscleroma is not reproduced by the inoculation of the bacillus in animals, it can be considered identical. The 100 ESSENTIALS OF BACTERIOLOGY growth, cultures, and properties are the same as the pneumo- bacillus of Friedlander. Bacillus of Influenza (Pfeiffer, 1892). — Origin. — One of the smallest of the known bacilli, 1.5 // by 0.3 /j, about one- half the size of the bacillus of mouse septicemia, and ar- ranged in chain form. It develops upon blood-serum agar. It is aerobic, without movement (Fig. 74). Stain. — It is best stained with diluted carbol-fuchsin, the . ,«r \ . -• •?* .. ' ;.""w*. Fig. 74. — Bacillus influenzae, from a gelatin culture (Xiooo) (Itzerott and Niemann). contrast-stain being Loffler's methylene-blue; does not take the Gram stain. Growth. — Upon blood-agar or glycerin-agar, over which a drop of blood has been spread, in an incubator at 37° C. at the end of twenty-four hours a very delicate growth occurs which resembles condensed moisture. Very small colonies, never larger than a pinhead, feebly resistant. Subcultures must be made every few days. Pathogenesis. — It is found in the sputum and in the bron- chial and nasal secretions and blood of influenza patients. It BACTERIA IN PNEUMONIA l6l has been transmitted to monkeys; other animals are not suscep- tible. It has never been found outside the body. Its resistance is very feeble ; in water, the bacilli die in twenty-four hours, but sputa containing the germs may be ejected for days and weeks. Influenza bacilli are found accompanying broncho- pneumonia, tuberculosis, meningitis, and other inflammations. The bacillus is found in healthy individuals, to a consider- able extent in the nasal secretions, and it is probably spread in the fine droplets of mucus expelled in sneezing and coughing. Koch-Weeks Bacillus (1883-87). — Cause of epidemic conjunctivitis, or "pink eye"; found in the secretion. Form. — Very minute bacillus, resembling the influenza bacillus; non-motile. (See Fig. 84, p. 173.) Growth. — They grow best on blood-serum agar, but very sparsely in minute transparent colonies; non-liquefying. Stains. — With carbolfuchsin, and is often intracellular. Does not take Gram. Pathogenesis. — Very contagious, found in 10 per cent, to 20 per cent, of all cases of conjunctivitis. Not infectious for lower animals, and not causing any other form of disease. Bacillus of Pertussis (Whooping-cough) (Bordet- Gengou, 1906). — It has been shown that very minute bacilli resembling the influenza bacillus occur in the cilia of the cells lining the trachea and bronchi of persons affected with whooping-cough ; these bacilli interfere with the normal movement of the cilia, and cause an irritation producing symptoms peculiar to the disease. Morphology. — Very minute bacilli with rounded ends (Fig- 75)- Cultures. — On potato-blood-agar, after twenty-four hours, slight growth, sticky, grayish; subcultures made on blood- serum and veal-agar grow readily. Staining. — Gram-negative, stain lightly with ordinary dyes. Pathogenesis. — By inhalation inoculation young rabbits were made to develop a spasmodic cough, and the bacillus was recovered from the trachea and from bronchi in pure cul- tures. In the sputum of persons affected with whooping- ii 1 62 ESSENTIALS OF BACTERIOLOGY cough the bacillus is found in large numbers. The recent work of Mallory, Henderson, and Horner (Jour. Med. Re- search, March, 1913) seems to establish this organism as the real cause of pertussis. Bacterins made from the culture have been recommended to allay the spasmodic cough. Bacillus Melitensis (Bruce, 1887). — Synonym. — Micro- coccus Melitensis. — Malta fever, also known as Mediterra- nean fever, occurs in the region from which it derives its Fig. 75. — The Bordet-Gengou bacillus of whooping-cough. Twenty-four- hour-old culture upon solid media containing blood (Bordet-Gengou). name, but has been observed in India, the Philippine Islands, and Porto Rico. Bruce cultivated an organism from the spleen and proved its specificity. Origin. — Is found most abundantly in the spleen. Form. — Rounded or oval, very small, coccus-like bacilli, 0.5 [i in diameter, singly, in pairs, or short chains. Properties. — Non-motile, though flagella said to be present; grows slowly, best at body-temperature. Gelatin. — Not liquefied; growth very slow. PYOGENIC COCCI 163 Bouillon. — Turbid, with sediment. A gar. — Pearly white growths. Potato. — Slight invisible growth. Stained by ordinary anilin dyes. Gram negative. Glucose broth, unfermented. Milk made alkaline. The disease may be produced in monkeys by even small amounts of pure culture. In man a chronic, remittent febrile disease is produced, with sweating and arthritis. The mor- tality is 2 per cent. A reaction can be obtained and is diagnostic. Agglutination — 1:30 dilution of serum will give positive result, but the complement-fixation test considered more cer- tain (which see). Flies an agency for transmission. Mode of Transmission. — Zammitt found that 50 per cent, of the goats of Malta gave the agglutination reaction to the micrococcus, and it was present in the milk in 10 per cent. Monkeys fed on the milk contracted the disease. Preventive measures instituted in 1906 have borne out the theory that the milk of goats is the cause of Malta fever, and since the practice of importing goats from Malta has stopped, the disease has disappeared from Gibraltar. In Malta, among the troops, the fever has been greatly reduced by eliminating milk from the dietary. CHAPTER XXIII PYOGENIC COCCI NEARLY all micro-organisms can produce suppuration, but in the acute abscesses occurring in the skin and lymphatics and accompanying all pus affections are found groups of micro- cocci so regularly that they have been designated as the pus- forming or pyogenic cocci. The two most important mem- 164 ESSENTIALS OF BACTERIOLOGY bers of this group are the Staphylococcus pyogenes, and the Streptococcus pyogenes, so named from the mode of division, the former being found usually in clusters or bunches, the lat- ter in chains. Streptococcus Pyogenes (Rosenbach) : Streptococcus Erysipelatis (Fehleisen). — Origin. — Fehleisen in 1883 dis- covered this microbe in the lymph- atics of the skin in erysipelas, and he thought it the cause of the same. Under the name Streptococcus pyogenes, Rosenbach described an identical coccus which has been found in nearly all suppurative conditions. • Fig. 76. — Streptococcus pyogenes; culture upon agar- agar two days old (Frankel and Pfeiffer). Fig. 77. — Streptococcus pyogenes (Jakob). Spores Form. — Small cocci singly and in chain-like groups, have not been found (Fig. 77). Properties. — They are immotile; do not liquefy gelatin. Growth. — They grow slowly, usually on the surface, and best at higher temperatures. PYOGENIC COCCI 165 Colonies. — In three days a very small grayish speck, which hardly ever becomes much larger than a pin-head; under microscope, looking yellowish, finely granular, the edges well defined. Stab-cultures. — Along the needle-track little separated col- onies, like strings of beads, which after a time become one solid white string. Stroke-culture on A gar. — Little drops, never coalescing, having a bluish tint, very transparent. Potato. — No apparent growth. Bouillon. — At 37° C. clouds are formed in the bouillon, which then sink to the bottom, and long chains of cocci found in this growth. Lqffler's Blood-serum and Serum Bouillon. — Development more abundant in serum media. Milk. — Good growth; produce lactic acid and coagulate milk. Preservation of Cultures. — In ice-chest, the cultures may be kept alive several weeks at room temperature; they usually die out in ten days. Staining. — Easily colored with the ordinary stains. Gram's method is also applicable. Pathogenesis. — Inoculated subcutaneously in the ear of a rabbit, an erysipelatous condition develops in a few days, rapidly spreading from point of infection. The micro-organism acts variously, depending upon the nature of the lesion from which it originally was obtained. Injected into the circulation, septicemia results. The more virulent the affection, the more virulent the strain. In man, inoculations have been made to produce an effect upon carcinomatous growths, and erysipelas has always re- sulted. When it occurs upon the valves of the heart, endo- carditis results. Puerperal fever is caused by the microbe infecting the endometrium, the Streptococcus puerperalis of Frankel being the same germ. In scarlatina, variola, yellow fever, cerebrospinal menin- gitis, and many similar diseases, the microbe has been an 1 66 ESSENTIALS OF BACTERIOLOGY almost constant attendant. It is often associated with the diphtheria bacillus in true diphtheria, and is the cause of many of the diphtheritic complications. It is associated with the influenza bacillus in acute ear suppurations; with pneu- monia bacteria; with tubercle bacilli, and in such instances usually causes high fever. In osteomyelitis and mastoiditis it is usually the sole cause. Streptococci in Milk. — In milk streptococci are often found, but it is not considered an absolute indication of udder in- flammation. Protective Sera. — An antistreptococcic serum has been used as a curative agent in puerperal fever, scarlatina, and other diseases supposed to be due to this germ. The antistrepto- coccic sera have been given an extensive trial in a variety of suppurative and inflammatory diseases, but the results are still under discussion. Coley's Fluid. — A mixture of a culture of pyogenes and prodigiosus has been used as an injection, with apparent benefit, in inoperable cases of sarcoma, and is known as Coley's fluid. Immune Bodies. — Neither antitoxic nor bactericidal bodies have been found in the blood of animals made resistant by the injection of dead or attenuated cultures. Polyvalent (Vaccines} Bacterins. — As it is possible that there are several varieties of streptococci, and varying in patho- genic properties, bacterins made from several strains have been used as injections against suppurative processes, and with some degree of success. Autogenous bacterins are more reliable. Distribution. — Streptococci can often be found in air, dust, on the skin, on all the mucous surfaces, pharynx, conjunctiva, tonsils. Staphylococcus Pyogenes Aureus (Rosenbach).— Origin. — Found commonly in pus (80 per cent, of all suppura- tions), in air, water, and earth; also hi sputum of healthy persons. Form. — Micrococci in clusters like bunched grapes, hence PYOGENIC COCCI 167 the name staphylo, which means grape. They never form chains. Spores have not been found, though the cocci are very resistant (Fig. 78). Properties. — Immotile; liquefying gelatin. Giving rise to an orange-yellow pigment in the various cultures. Growth. — It grows moderately fast at ordinary tempera- ture, and can live without air, a facultative aerobin and an- aerobin. Colonies on Gelatin. — On second day small dots on the sur- face, containing in their center an orange-yellow spot. The Fig. 78. — Staphylococcus pyogenes albus (Jakob). gelatin all around the colony is liquefied; the size is never much greater than that attained the second day. Colonies on A gar. — The pigment remains for a long time. Stab-culture. — At first, gray growth along the track, which, after three days, has settled at the bottom of the tube in a yellow, granular mass, the gelatin being all liquid (Fig. 79). Stroke-culture on A gar. — The pigment diffused over the sur- face where the growth is in moist masses. Potato. — A thin white layer which gradually becomes yel- low and gives out a doughy smell. i68 ESSENTIALS OF BACTERIOLOGY Staining. — Very readily colored with ordinary stains; also with Gram's method. Pathogenesis. — When rabbits are injected with cultures of this microbe into the knee-joint or pleura, they die in a day. If injected subcutaneously, only a local action occurs, namely, abscesses. If directly into circulation, a general phlegmonous condi- tion arises, the capillaries become plugged with masses of cocci, infarcts occur in kidney and liver, and metastatic abscesses form in viscera and joints. Garre, by rubbing the culture on his forearm, caused carbuncles to ap- pear. Several varieties of the pyogenic staph- ylococci are recognized according to their color-producing properties and slight vari- ations of growth. Of these, the Staph- ylococcus pyogenes aureus is the most virulent, and is considered the type of the group. They are always present on the surface of the body, beneath the nails, in the nose and mouth, in the dust of streets, and on the floors of houses, and are found in nearly all suppurative processes, whether on the surface or internally. Staphylococcus pyogenes albus dif- fers from the preceding only in the absence of pigment and in its slight virulence. Welch describes a variety constantly found both on the skin and in its deeper layers, which he calls the Staphylococcus epidermidis albus. Specific Therapy. — Sera have been found of no special value. Bacterins (Vaccines). — Twenty-four-hour-old agar surface culture killed by heating at 60° C. is emulsified with normal saline solutions and injected for the treatment of boils, ab- scesses, and acne. The cultures should be autogenous, i. e.y Fig. 79. — Stab- culture. Micro- coccus pyogenes aureus. PYOGENIC COCCI 1 69 derived from the person affected, although stock vaccines have been used with some success. The Opsonic Index. — It was proposed by Wright that the opsonic index should be obtained before treatment with vac- cines, although most of the treatment is now given without such control. Micrococcus Pyogenes Citreus (Passet). — This lique- fies gelatin less rapidly than the pyogenes aureus, and forms a citron-yellow pigment instead of the orange-yellow of the aureus. Micrococcus Cereus Albus (Passet). — Differs from the Fig. 80. — Micrococcus tetragenus in sputum (tubercle bacillus also). pyogenes albus in the form of colony. A white, shiny growth, like drops of wax; hence the name, cereus. Micrococcus Cereus Flavus (Passet) . — A lemon-yellow colored growth after a short time, otherwise not differing from cereus albus. Micrococcus Pyogenes Tenuis (Rosenbach) .— Origin. — Found in the pus of large inclosed abscesses. Form. — Cocci, without any especial arrangement. Properties. — Not much studied. Growth. — Cultivated on agar, it forms clear, thin colonies; along the needle-track an opaque streak, looking as if var- nished over. ESSENTIALS OF BACTERIOLOGY \\ Micrococcus Tetragenus (Koch; Gaffky). — Origin.— Koch found this microbe in the cavity of a tuberculous lung. Gaffky, in 1883, studied its pathogenic actions and gave it the name it now 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 liquefy gelatin. Growth. — They grow well on all nutrient media at ordinary temperature; 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- pigs die in a few days of septicemia when injected 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 lungs, in the sputum of phthisical and healthy patients, it is often found, but what action it has upon man has not yet been determined. Fig. 81.— Stab- culture. Micrococ- cus tetragenus. PYOGENIC COCCI 171 Morax-Axenfeld Diplobacillus of Conjunctivitis. — This bacillus is found in the greater number of cases of con- junctivitis. Form. — A short, plump bacillus, usually in 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 course 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. Growth. — 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. * '•' % ' 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 with 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. Patho genesis. — When animals are injected with fresh cul- tures in the peritoneal cavities or cellular 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 Gonorrhoeas (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 , •rv-. blood, cultures have been obtained. The ' IA& temperature must be between 33° and 37° V* C., and the growrth occurs very slo\vly and sparsely. Wertheim's medium (q. v., p. 78) has Fig. 85.— given the best results. Gonococci in gon- ' _ . ^ . . .. . orrheal pus. Ani- Colonies. — Extremely delicate, translu- lin methyl-violet cent spots, separate, and of a slimy con- 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. GONOCOCCUS. — MENINGOCOCCUS 175 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 1000) (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 numbers, 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 found in the vaginal secretions and pus and may at times lead to a wrong diagnosis. The meningococcus is very similar, but is easily cultivated and is not apt to be found 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 GONOCOCCUS . — 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- 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 (Xsooo) (Wright and Brown). Form. — 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, serum-agar, and ascitic glu- cose-agar at body temperature; good growth in twenty-four hours. Sheep serum better medium. 178 ESSENTIALS OF BACTERIOLOGY Colonies. — Circular discs, whitish, almost transparent, mar- gins, smooth. Stain. — With basic anilin. Gram negative. Jenner's blood-stain and Neisser stain best for spinal fluid specimens. Loffler's alkaline methylene-blue a good stain. Resistance. — Organisms very perishable one to three days. Apparently destroyed by a self-elaborated ferment. Sun- light destroys in a few hours. Pathogenesis. — Causes epidemic cer^brospinal 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 I79 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) (phot< 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. l8o 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 bacillus a drum-stick shape (Fig. 89). Properties. — Not very motile, though distinctly so; lique- fies gelatin slowly. The cultures give rise to a foul-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 nourishes. Colonies on gelatin plates hi an atmosphere of hydrogen. Small colonies. After four days a thick center and radiating, wreath-like periphery, like 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). A gar. — 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. 89. — Bacillus of tetanus with spores (X 1000) (Frankel and Pfeiffer). 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- 182 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 BACTERIA 183 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-cells. 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 bacilli. 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 in man. It is a globulin, 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 wound 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 liable to be infected. From 50 c.c. to 100 c.c. of a billion-unit 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 immunity unit is ten times the least quantity of anti- tetanic serum necessary to save the life of a 35<>gram guinea- pig for ninety-six 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. 104 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 bacil- lus should be made in the manufacture of all animal vac- cines, antitoxins, etc. Bacillus (Edematis Maligni (Koch, 1881) ; Vibrion Septique (Pasteur, 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, found 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 rounded very sharply. Long threads are formed. Very large spores which cause the rods to become spindle shaped. Resembles in form and culture B. chauvei (Fig. 92). ANAEROBIC BACTERIA I8S r 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 Cultures (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 Stab-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. A gar. — 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 bacilli. The spleen is enlarged, especially in mice. The bacilli are Fig. 93. — Bacillus of malignant edema growing in glucose- gelatin (Frankel and Pfeiffer). 1 86 ESSENTIALS OF BACTERIOLOGY 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 found 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 following wounds. Habitat. — They are present in the soil, 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, soil, sewage, and water. Form. — A thick bacillus, 3 to 6 ju 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. Milk. — Coagulated and becomes acid. Under anaerobic conditions. Potato. — Thin, grayish-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 bacillus 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 immobilis, B. saccharobutyricus, Fig. 94. — Bacillus aerogenes capsulatus (from photograph by Professor Simon 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 (Bellinger 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. 1 88 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 growrth 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. A gar at brood temperature, in twenty-four to forty-eight hours, an abundant growth with a sour odor and abundant gas-formation. ANAEROBIC BACTERIA 189 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 fluid; 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. " Ratischbrand" is the German name; "Charbon 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. 190 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. A gar and Blood-serum. — Glass-like colonies like drops of dew at first, then growing larger with iridescent edges. Potato. — At 37° C. small white mass. Slow growth. No gas formation in glucose media. Staining readily with all basic dyes. Gram negative. Capsule found in agar growths. Patho genesis. — After subcutaneous injection in rats death follows in forty to sixty hours, with symptoms of severe toxe- mia and convulsions. The point of infection shows a local edema and inflammation of the lymphatics. All 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- municate the disease to the rat from man or from the rat to IQ2 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 serum of immune animals has protective properties. The serum likewise shows agglutinating powers, and gives similar reactions to typhoid and cholera sera. Habitat. — Not found in water, but most likely spreads from the soil 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 bovisepticus, as it attacks cattle; Bacillus suisepticus, as it attacks swine. The prominent members of each group are: Bacillus of HEMORRHAGIC SEPTICEMIA GROUP 1 93 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 hull; 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 bacillus 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 IQ4 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. A gar 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, anilin-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 77° . (X 1000) (Frankel septicemia. and Pfeiffer). 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. HEMORRHAGIC SEPTICEMIA GROUP 195 Form. — One of the smallest forms of bacilli known; very thin, seldom longer than i /*, looking at first like little needle- like crystals. Spores have not been found. 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 while, 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. — All 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. Bacilli 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 "maldepis." 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 denned. 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 trypanosomata belong. Example: Trypanosoma. IQ8 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 Mallory 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. — Found 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 1860. A non- pathogenic form, Amoeba coli, also occurs. The Amoeba dys- enteriae is a unicellular animal organism, measuring 25 to 35 ju 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-cells 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. Patho genesis. — 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 differs from the bacillary form in that no severe toxic symptoms are present and the amebic disease is more chronic. The Shiga bacillus, B. dysenteriae, 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 Asexual 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, round, colorless bodies, exhibiting more or less active ameboid motion in the fresh blood. Sometimes, particularly in the estivo-autumnal form, a ring shape is assumed. Their size gradually increases and pigment-granules appear, while 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 200 ESSENTIALS OF BACTERIOLOGY the so-called gametes or sexual types. In the tertian and quartan varieties these are not very different from the mature THE MOSQUITO PHASE Fig. 100. — Schema showing the human and mosquito cycles of the malarial parasite: A, Normal red cell; B, C, D, E, red cells containing amebulasormyxopods; F, G, H, sporocytes; ]', 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, 1901). organisms, but the estivo-autumnal gametes are crescentic in shape and very characteristic. PROTOZOA 201 2. The Sexual 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 (micro gametes}. 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 (sporozo'ites), which are extruded into the body cavity of the insect, and some of which reach the salivary glands, whence they are ejected when the mosquito 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 refractile, 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 Malaria, 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 IS IB J3 24- Fig. 101. PROTOZOA 203 active. A variable number of merozoites is formed — usually six to twelve. The gametes are characteristic, being 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 distilled 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, hyaline form with beginning pigmenta- tion; 3, pigmented form; 4, full-grown pigmented form; 5, 6, 7, 8, seg- menting forms; 9, mature pigmented form; 10, flagellate form. n, 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 chroma tin 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 distilled 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 roset 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 with 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 Lewis! (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 206 ESSENTIALS OF BACTERIOLOGY 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 trypanosome of Bruce is less motile 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 trypanosome. 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. Neprem.) — 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 years. Trypanosomes 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. 208 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 dourine, 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- owrsky stains. Cultivation has succeeded on blood-media made acid with citric acid. The bedbug is considered instrumental in transmitting the organism. Piroplasma Bovis (P. Bigeminum) (T. H. Smith, 1893). — Origin. — In the blood of animals suffering from Texas cattle-fever. Form. — A pear-shaped protozoon, found in pairs in the red THE MICRO-ORGANISM OF SYPHILIS AND ALLIED ORGANISMS 2OQ 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 immunization. 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 Spirochseta Pallida (Schaudinn, 1905). — Spironema Pallidum; Treponema Pallidum. — Found in hereditary syph- ilis in all organs, in chancre, and lymphatic glands, and in secondary lesions, mucous patches, in the internal organs, 14 2IO ESSENTIALS OF 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), intracellular, 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- Fig. 106 .— Spirochaeta pallida. Micro- ism can be shown by photograph made by Dr. R. E. Lavenson r • •, v «i from a specimen prepared by H. Fox fixmg Wlth sllver m~ (Stengel). 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. Spirochaetae black, tissues, pale yellow. Or counterstain of fuchsin can be employed. THE MICRO-ORGANISM OF 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 will 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. s 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 folio wing 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 until 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 amount 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 syphilitic antigen plus the suspected syphilitic serum amboceptor, and incubated for one hour at 37° C. // 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 unit 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. 2I4 ESSENTIALS OF BACTERIOLOGY WASSERMANN SCHEME g g | M p 1 1 M 1 I8 i 7 i 09 _w II 2 t/3 M H D 1 + RESULT s^ 1 i Q H ^ s o < i fc K ± *"« fe M 7. I I C.C. 0.2 C.C. I I I No hemolysis. d 2 I C.C. 0.2 C.C. . CS O I I .§ Complete "1 g hemolysis. 3 I C.C. 0.2 C.C. X •g I I o Fig. 118. — Bacillus A large glancing spore at one end, the amylobacter. bacillus becoming spindle shaped in or- der to allow the spore to grow; hence the name, clostridium. Properties. — Very motile; gases arise with butyric 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 A gar. — 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 count, 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^-jj 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 FOOD 253 Multiply by 5000. This equals the number in y^- 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 element. Milk freshly drawn, under 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 millions of bacteria per cubic centimeter. Pasteurization. — Milk heated to 60° C. for twenty minutes is called pasteurized. 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. Pasteurization 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 milk, 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 find their way into it. Animals wading in infected water have infected the milk. Utensils washed in polluted water have been found to be the cause in some epidemics of typhoid. Carriers, persons who harbor the diphtheria and typhoid 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 milk. 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 easily as the wine-maker. Pure cultures of lactic acid are supplied to butter-makers and used in creameries to inoculate sweet cream and milk. 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. Milk 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 milk is incu- bated at 35° C. for a few hours, noting the curd, whether firm or soft and gassy. Wisconsin Test. — Milk curdled 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 Penicillium and Oidium 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 sterilization. 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 sources 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 OF 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, unbruised 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 AND CAVITIES OF THE HUMAN BODY 257 CHAPTER XXXIII BACTERIOLCGIC 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 haematoides, Bacillus pyocyaneus. A diplococcus is found in acute pemphigus. The lepra bacillus, the tubercle bacillus in lupus, and the typhoid bacillus in the eruption of typhoid 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, all 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 sputigenum, Spirochaeta dentium, Micro- coccus gingivae pyogenes, Bacillus dentalis viridans, Bacillus pulpae 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 fcetidus ozaenae, Bacillus striatus albus et flavus, Bacillus capsulatus 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 260 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 subtilis, Bacillus butyricus, Bacil- lus putrificus coli, Bacillus lactis aerogenes, Bacillus coli commune, Bacillus subtiliformis, and the bacteria of cholera, dysentery, and typhoid, besides many yeast-cells. 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 found 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 26l 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 bacilli 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 sterile 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. Formaldehydj in gaseous form or in a liquid 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 ounces 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 ounces (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 sufficient 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 secure good results should carefully attend to his toilet; the use of operating gowns, rubber gloves, operating shoes, face guards is now universal. The toilet 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 employment of a half-dozen or more cleansing agents and germicides are no longer followed. Soap is an efficient 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, nitration 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, linen, boiling in steam, for- maldehyd gas, or hot-air exposure. Articles of little value should 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 +1.5 in test-tubes containing 10 c.c. each of the medium. 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 OF 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 -^ 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. DILUTION TIME EXPOSE 0 IN Ml NUTES 2^ 5 7K IO I2# 15 Phenol: 80 QO. . + IOO + + + __ no 4- 4- + + + Disinfectant: 3^0 •27? 400 + 500 6^0. . + + + + + + -f — 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 (1:80), thus ^ = 4.69, and the same is done for the strength that kills in fifteen minutes, namely: 650 — ^— = S-Qi no The average of these, -'• — ^~~==5-3o> 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 i 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 i^Arac*. vjc>AMja. | .UlUlAJllX. .TKUIJLH-J ACETI. Bacillus. Short motile rods in Ferment. zooglea; aerobic. ACIDI LACTICI. 1 Bacillus. Short, immotile rods; aerobic. ACIDI LACTICI. Streptococcus. Short, immotile, oval cocci. ACTINOBACTER. Bacillus. Immotile rods with capsule; facul. an- aerob. ABROGENES. Bacillus. Identical with B. acid lactici. AfiROPHlLUS. Bacillus. Slender rods in threads ; immotile; oval spores; aerobic. AGILIS. Micrococcus. Mobile diplococci Red pigment. . with fine flagella. ALBA. Beggiatoa. Cocci and spirals with sulphur. ALBA. Sarcina. Small cocci in packets White pigment. ALBICANS AMPLUS. Micrococcus. Large cocci and dip- i 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- Yellow pigment. ed ends in threads. andsingly ;immotile 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 liquefy; membran- Produces acetic-acid Air. Kutzing. 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 liquefy; gray col- Is colored by Gram's Vaginal secretion. Bumm. onies; growth fairly method. rapid. Small white points, not Urethral pus. Bumm. liouefying; 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 slime 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 singly ; immotile. Orange-yellow pig- ment in water, al- cohol, and ether; insoluble. AUREA. Sarcina. Cocci in packets. Golden-colored pig- i ment; soluble in : alcohol. AUREUS. Bacillus. Straight motile rods Golden-yellow pig- lying parallel. ment. BALTICUS. Bacillus. Short rod. Phosphorescence. BlENSTOCKII. Bacillus. See Puirificus, coli. BlFIDUS. Bacillus. Slender diplococcus, pointed ends, non- motile, anaerobic. BlLLROTHII. 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. rods in pairs ;spores; facul. anaerobin. BUTYRICUM (amy- lobacter). Clostridium. Thick motile rods en- 1 a r g i n g for the Amyloid substance. spores; obligate aerobic. C/ERULEUS. Bacillus. Rods in long chains. Blue pigment, not sol- uble in water, alco- hol, or acid. CANDICANS Micrococcus. Masses of cocci. (candidus). 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, Cherry-red pigment. singly and in pairs; aerobic. 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 BACTERIA. — (Continued.) 271 CULTURE CHARACTERS. ACTIONS. HABITAT. DISCOVERER. Round orange-yellow col- Water. Cohn. onies, mostly on sur- face; slow growth; not liquefying. Liquefy; 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 Old cheese. Duclaux. ferment. Ferments milk, pro- ducing the kefir Kefir; grain. Kern. *x drink. On potato; rapidly, form- Water. List. ing cherry-red scum, not developed on gela- tin. Not liquefy; small, wax- Pus. Passet. like 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. ClNNABAREUS. Micrococcus. Large oval cocci in Brown-red pigment; pairs; aerobic. , foul odor. ClTREUS. Bacillus (asco- Straight and bent Citron - yellow pig- coccus). ; rods in bundles; ment. • motile. ClTREUS. Micrococcus. Large round cocci in Cream-colored oig- chains of eight and ment. more. ClTREUS CONGLOM- Micrococcus. Diplococci and tet- ERATUS. rads; aerobic. CLAVIFORMIS. Bacillus Small rods; spores; (tyrothrix). true anaerobin. CLOACA. See Proteus. CONCENTRICUM. Spirillum. Thick motile spirals .... with flagella; aerobic. CORONATUS. Micrococcus. Cocci singly and strep- tococci; aerobic. CORYZ.E. 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. DlCHOTOMA. Cladothrix. Various forms — rods, spirals, and cocci, in long threads. DlFFLUENS. Micrococcus. Oval cocci; aerobic. Fluorescent pigment, soluble in water. DlSTORTUS. Bacillus Motile rods; spores; Alkali. (tyrothrix). aerobic. DYSODES. Bacillus. Long and short rods; An odor resemblins 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. FlLIFORMIS. Bacillus Short motile rods; (tyrothrix). spores in one end. OF THE PRINCIPAL BACTERIA 273 BACTERIA. — (Continued.) 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- Bumm. rhagic pus. 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- onies, at first like pneu- No pathogenic action. Acute coryzal se- cretion. Hajek. mococci, later culture flattened; not lique- fying. Putrefying infu- Cohn. sions. Bluish-green colonies. Not liquefying; small Changes milk to deep- Cooked potatoes. Air of certain Cohn. 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; green fluorescence; white col- Air and putrefying substances. Cohn. onies. Liquefying; root-like pro- cesses extending in the Garden-earth. Flugge. gelatin; feather form in test-tube. Causes casein to be .... Duclaux. precipitated from milk. 18 274 CHIEF CHARACTERISTICS NON-PATHOGENIC NAME. GENUS. BIOLOGY. PRODUCT. FlSCHERI. Bacillus. Phosphorescence. FlTZIANUS. Bacillus. Short rods in threads;) .... spores as large as the rods. FLAVA. Sarcina. Small cocci in pack- Pigment. ets. FLAVUS. Bacillus. Small rods; immotile. Pigment. FLAVUS DESIDENS. Streptococcus. Cocci and diplococci Yellow-brown pig- in chains; aerobic. ment. FLAVUS LIQUEFA- Micrococcus. Cocci and diplococci Pigment. CIENS. in zooglea. FLAVUS TARDI- Micrococcus. Cocci in short chains, Chrome-yellow pig- GRADUS. and diplococci. ment. FLUORESCENS F illustrated. Cloth, $1.50 net. THE NEW (5th) EDITION New York Medical Journal " We are confident if this little work could find its way into the hands of every trained nurse and of every mother, infant mortality would be lessened by at least fifty per cent." NURSING. Sanders' Nursing Modern Methods in Nursing. By GEORGIANA J. SANDERS, formerly Superintendent of Nurses at the Massachusetts General Hospital. i2mo of 88 1 pages, with 227 illustrations. Cloth, $2.50 net. COMPLETE Miss Sanders' book gives only modern methods. Then it gives the details of nursing operation cases, both in the hospital and in the home. The thorough way in which ward work is taken up makes her book indispensable for teaching purposes. In giving directions for mustard baths, poultices, etc., the quantities are given exactly. This is an important point often overlooked. Stoney's Nursing' Practical Points in Nursing : for Nurses in Private Practice. By EMILY M. A. STONEY, formerly Superintendent of the Training School for Nurses at the Carney Hospital, South Boston, Mass. 495 Pages> fully illustrated. Cloth, $1.75 net. THE NEW (4th) EDITION In this volume the author explains the entire range of private nursing as distinguished from hospital nursing, and the nurse is instructed how best to meet the various emergencies of medical and surgical cases when distant from medical or surgical aid or when thrown on her own resources. An especially valuable feature will be found in the direction how to improvise everything ordinarily needed in the sick-room. Stoney's Technic for Nurses Bacteriology and Surgical Technic for Nurses. By EMILY M. A. STONEY, Carney Hospital, South Boston. Re- vised by FREDERIC R. GRIFFITH, M. D., Surgeon, N. Y. i2mo, 3 1 1 pages, illustrated. $1.50 net. New (3d) Edition SAUNDERV BOOKS ON Hoxie and Lap tad's Medicine for Nurses New (2d*Vdit!on MEDICINE FOR NURSES AND HOUSEMOTHERS. By GEORGE HOWARD HOXIE, M.D., Physician to the German Hospital, Kansas City; and PEARL L. LAPTAD, formerly Principal of Training-School, University of Kansas. 1 2mo of 351 pages, illustrated. Cloth, #1.50 net. This work is truly a practice of medicine for the nurse, enabling her to recognize any signs and changes that may occur between visits of the physician, an. I, if neces- sary, to combat them until the physician's arrival. This information the author pre- sents in a way most acceptable, particulaily emphasizing the nurse's part. There Jare also special chapters on the diseases of the eye, ear, nose, and throat, venereal dis- eases, nervous and mental diseases, surgical nursing, emergency measures. " This book has our unqualified approval." — Trained Nurse and Hospital Review. McCombs' Diseases of Children for Nurses DISEASES OF CHILDREN FOR NURSES. By ROBERT S. MCCOMBS, M. D., Instructor of Nurses at the Children's Hospital of Philadel- phia. lamo of 460 pages, illustrated. Cloth, $2.00 net. Dr. McCombs has given a short but clear description of each disease found in infancy and childhood, so that the nurse will be enabled to know what symptoms to expect and what complications to guard against. " We have needed a good work on children's diseases adapted for nurses' use, and this volume admirably fills the want." — National Hospital Record. Wilson's Obstetric Nursing SemA Edmon A REFERENCE HAND-BOOK OF OBSTETRIC NURSING. By W. REY- NOLDS WILSON, M. D., Visiting Physician to the Philadelphia Lying- in Charity. 321110 of 258 pages, illustrated. Flexible leather, $1.25 net. " Every page emphasizes the nurse's relation to the case/' — American Journal of Obstetrics. Fruhwald and Westcott on Children DISEASES OF CHILDREN. By PROF. DR. F. FRUHWALD, of Vienna. Edited by THOMPSON S. WESTCOTT, M.D., University of Pennsylvania. Octavo, 533 pages, 176 illustrations. Cloth, $4.50 net. Boyd's State Registration for Nurses STATE REGISTRATION FOR NURSES. By LOUIE CROFT BOYD, R.N., Graduate Colorado Training-school for Nurses. Octavo of 42 pages. 50 cents net. Aikens' Primary Studies for Nurses New (2d) Edition PRIMARY STUDIES FOR NURSES : A Text-Book for First-year Pupil Nurses. By CHARLOTTE A. AIKENS, formerly Director of Sibley Mem- orial Hospital, Washington, D. C. I2mo of 437 pages, illustrated. Cloth, $1.75 net. This work brings together in concise form well-rounded courses of lessons in all subjects which, with practical nursing technic, constitute the primary studies in a. nursing course. Trained Nurse and Hospital Review " It is safe to say that any pupil who has mastered even the major portion of this work would be one of the best prepared first-year pupijs that ever stood for examina- tion." Aikens' Clinical Studies for Nurses New /2dx Edition CLINICAL STUDIES FOR NURSES. By CHARLOTTE A. AIKENS, for- merly Director of Sibley Memorial Hospital, Washington, D. C. !2iiK> of 569 pages, illustrated. Cloth, $2.00 net. This new work is written along the same lines as Miss Aikens' earlier work on " Primary Studies," to which it is a companion volume. It takes up all subjects taught during the second and third years and takes them up in a concise, forceful way. Dietetic and Hygienic; Gazette " There is a large amount of practical information in this book which the experienced nurse, as well as the undergraduate, will consult with profit. The illustrations are- numerous and well selected." Aikens' Training-School Methods and Head Nurse HOSPITAL TRAINING-SCHOOL METHODS AND THE HEAD NURSE. By CHARLOTTE A. AIKENS, formerly Director of Sibley Memorial Hospital, Washington, D. C. I2mo of 267 pages. Cloth, #1.50 net. Trained Nurse and Hospital Review " There is not a chapter in the book that does not contain valuable suggestions." Aikens' Hospital Management HOSPITAL MANAGEMENT. By CHARLOTTE A. AIKENS, formerly Di- rector of Sibley Memorial Hospital, Washington, D. C. I2mo of 488 pages, illustrated. Cloth, $3.00 net. The Medical Record " Tells in concise form exactly what a hospital should do and how it should be run, from the scrubwoman up to its financing. A valuable addition to our literature on this subject." io SAUNDERS' BOOKS ON Register's Fever Nursing A TEXT-BOOK ON PRACTICAL FEVER NURSING. By EDWARD C. REGISTER, M. D., Professor of the Practice of Medicine in the North Carolina Medical College. I2mo of 352 pages. Cloth, #2.50 net. " Nurses will find this book of great value in this practical branch of their work." — Trained Nurse and Hospital Review. Hecker, Trumpp, and Apt on Children ATLAS AND EPITOME OF DISEASES OF CHILDREN. By Dr. R. HECKER and Dr. J. TRUMPP, of Munich. Edited, with additions, by ISAAC A. APT, M. D., Professor of Pediatrics, Northwestern Medical School, Chicago. With 48 colored plates, 144 text-cuts, and 453 pages of text. Cloth, $5.00 net. Aikens' Home Nurse's Hand- Book HOME NURSE'S HAND-BOOK. BY CHARLOTTE A. AIKENS. i2mo of 276 pages, illustrated. Cloth, #1.50 net. The point about this work is this : It tells you and shows you just how to do those little but important things often omitted from other nursing books. " Home Treatments " and " Points to be Remembered " — terse, crisp reminders — stand out as particularly practical. Just the book for those who have the home-care of the sick. Lewis' Anatomy and Physiology Second Edition ANATOMY AND PHYSIOLOGY FOR NURSES. By LEROY LEWIS, M.D., formerly Surgeon to and Lecturer on Anatomy and Physiology for Nurses at the Lewis Hospital, Bay City, Michigan. I2mo of 375 pages, with 150 illustrations. Cloth, $1.75 net A demand for such a work as this, treating the subjects from the nurse's point of vinv has long existed. Dr. Lewis has based the plan and scope of this work on the methods employed by him in teaching these branches, making the text unusually simple and clear. " It is not in any sense rudimentary, but comprehensive in its treatment of the sub- jects in hand. The application of the knowledge of anatomy in the care of the patient is emphasized." — The Nurses Journal of the Pacific Coast. Friedenwald and Ruhrah's Dietetics Second Edition DIETETICS FOR NURSES. By JULIUS FRIEDENWALD, M. D., Pro- fessor of Diseases of the Stomach, and JOHN RUHRAH, M. D., Pro- fessor of Diseases of Children, College of Physicians and Surgeons, Baltimore. I2mo volume of 395 pages. Cloth, £1.50 net. This work has been prepared to meet the needs of the nurse, both in the training school and after graduation. It aims to give the essentials of dietetics, considering briefly the physiology of digestion and the various classes of foods and the part they play in nutrition. "It is exactly the book for which nurses and others have long and vainly sought. A simple manual of dietetics, which does not turn into a cook-book at the end of the first or second chapter." — American Journal of Nursing. NURSING. Macfarlane's Gynecology for Nurses New (2d) Edition A REFERENCE HAND-BOOK OF GYNECOLOGY FOR NURSES. By CATHARINE MACFARLANE, M. D., Gynecologist to the Woman's Hos- pital of Philadelphia. i6mo of 156 pages, with 70 illustrations. Flexible leather, $1.25 net. " This is a nut-shell book, and the flexible leather covers are full of meat."— Dietetic and Hygienic Gazette. Galbraith's Personal Hygiene and Physical Training for Women PERSONAL HYGIENE AND PHYSICAL TRAINING FOR WOMEN. By ANNA M. GALBRAITH, M.D., Fellow New York Academy of Medicine. I2mo of 371 pages, with original illustrations. Cloth, $2.00 net. Dr. Galbraith's book is just what has long been needed — a simple manual of hy- giene and physical training along scientific lines. Just Ready De Lee s Obstetrics for Nurses New (4th) Edition OBSTETRICS FOR NURSES. By JOSEPH B. DE LEE, M. D., Pro- fessor of Obstetrics in the Northwestern University Medical School. i2mo volume of 507 pages, fully illustrated. Cloth, $2.50 net. " 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 students as well." — J. CLIFTON EDGAR, M. D., Cornell Medical School, N. Y. Just Issued Davis* Obstetric Nursing New (4th) Edition OBSTETRIC AND GYNECOLOGIC NURSING. By EDWARD P. DAVIS, A. M., M. D., Professor of Obstetrics, Jefferson Medical College and Philadelphia Polyclinic. i2mo of 480 pages, illustrated. Buckram, $1.75 net. " 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." — The Lancet, London. Beck's Hand-Book for Nurses New (3d) Edition A REFERENCE HAND-BOOK FOR NURSES. By AMANDA K. BECK, of Chicago, 111. 321110 of 230 pages. Flexible leather, $1.25 net. This little book contains information upon every question that comes to a nurse in her daily work, and embraces all the information that she re- quires to carry out any directions given by the physician. " Must be regarded as an extremely useful book, not only for nurses, but for phys- icians."— Boston Medical and Surgical Journal. 12 SAUNDEKS' BOOKS ON StoneyV Materia Medica for Nurses Practical Materia Medica for Nurses, with an Appendix containing Poisons and their Antidotes, with Poison-Emergencies ; Mineral Waters ; Weights and Measures, etc. By EMILY M. A, STONEY, formerly at the Carney Hospital, South Boston, Mass. i2mo, 300 pages. $1.50 net. THE NEW (3d) EDITION In this work the consideration of the drugs includes their names, their sources and composition, their various preparations, physiologic actions, directions for handling and administering, and the symptoms and treatment of poisoning. Journal of the American Medical Association " So far as we can see, it contains everything that a nurse ought to know in regard to drugs. As a reference-book for nurses it will without question be very useful." Bolduan and Grund's Bacteriology for Nurses APPLIED BACTERIOLOGY FOR NURSES. By CHARLES F. BOLDUAN, M. D., Assistant to the General Medical Officer, and MARIE GRUND, M. D., Bacteriologist, Research Laboratory, Department of Health, New- York City. I2mo of 155 pages, illustrated. Cloth, $1.25 net. We were fortunate in getting these practical physicians to write this work. It gives particular emphasis to the immediate application of bacteriology to nursing, only the really practical being included. A study of all the modes of infection transmission is presented. At the end of each chapter are suggestions for practical demonstration. Bohm and Painter's Massage just Ready MASSAGE. By MAX BOHM, M. D., of Berlin, Germany. Edited, with an Introduction, by CHARLES F. PAINTER, M. D., Professor of Ortho- pedic Surgery at Tufts College Medical School, Boston. Octavo of 91 pages, with 70 practical illustrations. Cloth, $1.75 net. Manhattan Hospital Eye, Car, Nose, Throat Nursing NURSING IN DISEASES OF THE EYE, EAR, NOSE, AND THROAT. By the Committee on Nurses of the Manhattan Eye, Ear, and Throat Hos- pital. I2ino of 260 pages, illustrated. Cloth, $1.50 net. NURSING AND CHILDREN. 13 Paul's Fever Nursing New (2d) Edition NURSING IN THE ACUTE INFECTIOUS FEVERS. By GEORGE P. PAUL, M. D., formerly Assistant Visiting Physician to the Samaritan Hospital, Troy, N. Y. I2mo of 246 pages. Cloth, #1.00 net. Dr. Paul has taken great pains in the presentation of the care and management of each fever. The book treats of fevers in genera ">, then each fever is discussed indi- vidually, and the latter part of the book deals with practical procedures and valuable information. " The book is an excellent one and will be of value to those for whom it is intended. It is well arranged, the text is clear and full, and the illustrations are good." — The London Lancet. Paul's Materia Medica for Nurses New (2d) Edition MATERIA MEDICA FOR NURSES. By GEORGE P. PAUL, M.D., formerly Assistant Visiting Physician to the Samaritan Hospital, Troy. I2mo of 240 pages. Cloth, $1.50 net. Dr. Paul arranges the physiologic actions of the drugs according to the action of the drug and not the organ acted upon. An important section is that on pretoxic signs, giving the warnings of the full action or the beginning toxic effects of the drug, which, if heeded, may prevent many cases of drug poisoning. The nurse should know these signs. " This volume will be of real help to nurses : the material is well selected and well arranged, and the book is as readable as it is useful." — The Medical Record. Pyle's Personal Hygiene The New (5th) Edition A MANUAL OF PERSONAL HYGIENE : Proper Living upon a Physio- logic Basis. By Eminent Specialists. Edited by WALTER L. PYLE, A.M., M.D., Assistant Surgeon to \Vills Eye Hospital, Philadelphia. Octavo volume of 515 pages, fully illustrated. Cloth, $1.50 net. To this new edition there have been added, and fully illustrated, chapters on Domestic Hygiene and Home Gymnastics, besides an appendix containing methods of Hydrotherapy, Mechanotherapy, and First Aid Measures. There is also a Glos- sary of the medical terms used. " The work has been excellently done, there is no undue repetition, and the writers have succeeded unusually well in presenting facts of practical significance based on sound knowledge." — Boston Medical and Surgical Journal. Galbraith's Four Epochs of Woman's Life second Edition THE FOUR EPOCHS OF WOMAN'S LIFE. By ANNA M. GALBRAITH, M.D. With an Introductory Note by JOHN H. MUSSER, M.D., Univer- sity of Pennsylvania. I2mo of 247 pages. Cloth, $1.50 net. " We do not as a rule care for medical books written for the instruction of the public ; but we must admit that the advice in Dr. Galbraith's work is in the main wise and wholesome." — Birmingham Medical Revieiv, England. Spratling on Epilepsy EPILEPSY AND ITS TREATMENT. By WILLIAM P. SPRATLING, M. D., formerly Professor of Physiology and Nervous Diseases, College of Physicians and Surgeons, Baltimore. Octavo of 522 pages, fully illus- trated. Cloth, $4.00 net. 14 SAUNDERS' BOOKS ON Peterson and Haines' Legal Medicine & Toxicology A Text-Book of Legal Medicine and Toxicology. Edited by FREDERICK PETERSON, M. D., Professor of Psychiatry in the Columbia University (College of Physicians and Surgeons), New York; and WALTER S. HAINES, M. D., Professor of Chemistry, Pharmacy, and Toxicology, Rush Medical College, in affiliation with the University of Chicago. Two imperial octavo volumes of about 750 pages each, fully illustrated. Per volume: Cloth, $5.00 net; Sheep or Half Morocco, $6.50 net. Sold by Subscription. TWO VOLUMES The object of the present work is to give to the medical and legal profes- sions a comprehensive survey of forensic medicine and toxicology in moderate compass. An interesting and important chapter is that on '• The Destruction and Attempted Destruction of the Human Body by Fire and Chemicals." A chapter not usually found in works on legal medicine is that on "The Medicolegal Relations of the X-Rays." Columbia Law Review " For practitioners in criminal law and for those in medicine who are called upon to give court testimony in all its various forms ... it is extremely valuable." Fiske's Human Body Structure and Functions of the Body. By ANNETTE FISKE, A.M., Graduate of the Waltham Training School for Nurses. i2mo of 221 pages, illustrated. Cloth, $1.25 net. PRESENTED IN A NEW WAY The way in which this book presents anatomy and physiology is a depart- ure from the usual method — a departure, however, of a very practical kind. Miss Fiske has woven the physiology in with the anatomy, thus making her work a most readable one. It is an extremely practical book — one that can be readily understood. LEGAL MEDICINE 15 Draper's Legal Medicine A TEXT-BOOK OF LEGAL MEDICINE. By FRANK WINTHROP DRAPER, A. M., M. D., late Professor of Legal Medicine in Harvard University. Octavo of 573 pages, illustrated. Cloth, $4.00 net. , Golebiewski and Bailey's Accident Diseases ATLAS AND EPITOME OF DISEASES CAUSED BY ACCIDENTS. By DR. ED. GOLEBIEWSKI, of Berlin. Edited, with additions, by PEARCE BAILEY, M. D., Consulting Neurologist to St. Luke's Hospital, New York. With 71 colored illustrations on 40 plates, 143 text-illustrations, and 549 pages of text. Cloth, $4.00 net. In Saunders1 Hand' Atlas Series. Chapman's Medical Jurisprudence Third Edition MEDICAL JURISPRUDENCE, INSANITY, AND TOXICOLOGY. By HENRY C. CHAPMAN, M. D., late Professor of Institutes of Medicine and Med- ical Jurisprudence in Jefferson Medical College, Philadelphia. I2mo of 329 pages, fully illustrated. Cloth, $1.75 net. Hofmann and Peterson's Legal Medicine ATLAS OF LEGAL MEDICINE. By DR. E. VON HOFMANN, of Vienna. Edited by FREDERICK PETERSON, M. D., Professor of Psychiatry in the College of Physicians and Surgeons, New York. With 120 colored figures on 56 plates and 193 half-tone illustrations. Cloth, $3.50 net. Jakob and Fisher's Nervous System ATLAS AND EPITOME OF THE NERVOUS SYSTEM AND ITS DISEASES. By PROFESSOR DR. CHR. JAKOB, of Erlangen. Edited, with additions, by EDWARD D. FISHER, M. D., University and Bellevue Hospital Medical College, New York. With 83 plates and copious text. Cloth, $3.50 net. Crothers* Morphinism and Narcomania MORPHINISM AND NARCOMANIA. By T. D. CROTHERS, M. D. 12010 of 35 1 pages. Cloth, $2.00 net. Abbott's Transmissible Diseases Second Edition THE HYGIENE OF TRANSMISSIBLE DISEASES : Their Causes, Modes of Dissemination, and Methods of Prevention. By A. C. ABBOTT. M. D., Professor of Hygiene and Bacteriology, University of Pennsyl- vania. Octavo of 351 pages, illustrated. Cloth, $2.50 net. 16 SAUNDERS* BOOKS ON NURSING. American Pocket Dictionary New AMERICAN POCKET MEDICAL DICTIONARY. Edited by W. A. NEWMAN BORLAND, M.D. Containing the pronunciation and definition , of the principal words used in medicine and kindred sciences, with 64 extensive tables. With 677 pages. Flexible leather, with gold edges, $1.00 net; with patent thumb index, $1.25 net. Morrow's Immediate Care of Injured IMMEDIATE CARE OF THE INJURED. By ALBERT S. MORROW, M. D., Attending Surgeon to the New York City Hospital for the Aged and In- firm. Octavo of 360 pages, with 242 illustrations. Cloth, $2.50 net. Dr. Morrow's book on emergency procedures is written in a definite and decisive style, the reader being told just what to do in every emergency. It is a practical book for every day use, and the large number" of excellent illustrations can not but make the treatment to be pursued in any case clear and intelligible. Physicians and nurses will find it indispensible. Starr's Diets for Infants and Children DIETS ^FOR INFANTS AND CHILDREN IN HEALTH AND IN DISEASE. By Louis* STARR, M. D., Consulting Pediatrist to the Maternity Hospi- tal, Philadelphia. 230 blanks (pocket-book size). Bound in flexible Morocco, $1.25 net. Grafstrom's Mechano-Therapy 2d Revbed Edition A TEXT-BOOK OF MECHANO-THERAPY (Massage and Medical Gym- nastics). By AXEL V. GRAFSTROM, B. Sc., M.D., Attending Physician to the Gustavus Adolphus Orphanage, Jamestown, New York. I2mo, 200 pages, illustrated. Cloth, #1.25 net. Shaw on Nervous Diseases and Insanity Fourth Edition, Revised ESSENTIALS OF NERVOUS DISEASES AND INSANITY : their Symptoms and Treatment. A Manual for Students and Practitioners. By the late JOHN C. SHAW, M. D., Clinical Professor of Diseases of the Mind and Nervous System, Long Island College Hospital, New York. I2mo of 204 pages, illustrated. Cloth, $1.00 net. In Saunders1 Question- Compend Series. Powell's Diseases of Children 3d Edition, Revised ESSENTIALS OF THE DISEASES OF CHILDREN. By WILLIAM M. POWELL, M. D. Revised by ALFRED HAND, JR., A. B., M. D., Dis- pensary Physician and Pathologist to the Children's Hospital, Philadel- phia. I2mo volume of 259 pages. Cloth, $l.oo net. In Saunders' Question- Commend Series. RETURN TO the circulation desk of any University of California Library or to the NORTHERN REGIONAL LIBRARY FACILITY Bldg. 400, Richmond Field Station University of California Richmond, CA 94804-4698 ALL BOOKS MAY BE RECALLED AFTER 7 DAYS 2-month loans may be renewed by calling (415)642-6233 1-year loans may be recharged by bringing books to NRLF Renewals and recharges may be made 4 days prior to due date DUE AS STAMPED BELOW LIBRARY USE JAM 15 '87 Saunders' Compends 10. ESSENTIALS OF GYNECOLOGY. 7^ ed. With 57 illustrations. By EDWIN B. CRAGIN, M.D. Revised by FRANK S. MATHEWS, M.D. 11. ESSENTIALS OF DISEASES OF THE SKIN. 7th 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. 13. 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. 4th ed., illus- trated. By EDWARD JACKSON, M.D. 15. ESSENTIALS OF DISEASES OF CHILDREN. 3d ed. By WM. M. POWELL, M.D. 17. ESSENTIALS OF DIAGNOSIS. 2d ed. By S. SOLIS-COHEN, M.D., and A. A. ESHNER, M.D. 19. ESSENTIALS OF NOSE AND THROAT. 4th ed., illustrated. By E. B. GLEASON, M.D. 20. ESSENTIALS OF BACTERIOLOGY. 7th ed 100 illustrations and 6 plates. By M. V. BALL, M. D. 21. ESSENTIALS OF NERVOUS DISEASES AND INSANITY. 4th ed. 53 illustrations. By JOHN C. SHAW, M. D. Revised by SMITH ELY JELLIFFE, M. D. 24 ESSENTIALS OF DISEASES OF THE EAR. 3^ ed., illus- trated. By E. BALDWIN GLEASON, M. D. 25. ESSENTIALS OF HISTOLOGY. 4* ed. lie illustrations. By Louis LEROY, M. D. W. B. SAUNDERS CO., West Washington Square, Phila.