Get the Best The New Standard DORLAND'S MEL A New and Dentistry, new and of Bacilli Operation ment, etc Pocket M full flexifc JUST issue: It conti This book tionary, and y finest quality, i and is just the stant reference defines hundret is especially fu great practical Anew feature them in colors, " I must ackn tively small space finding some of tl Professor of Prin " Dr. Dorland size. No errors cology, Johns Hopkins Umvei sity, Baltimore. MEDICAL >SC1HI©(DL LHEHBAIEY Gift of Dr# Gtto L. Gericke. ts' dic- r of the : leather, for con- :ds, and iary. It ldred of lethods. nany of ithin refo- rested in .l Park, 'Buffalo, mvenient of Gyne- W. B. SAUNDERS COMPANY, 925 Walnut St, Phila. London: 9, Henrietta Street, Covent Garden Fourth Edition, Enlarged With Complete Vocabulary THE AMERICAN POCKET MEDICAL DICTIONARY EDITED BY W. A. NEWMAN DORLAND, A. M., M. D., Assistant Demonstrator of Obstetrics, University of Pennsylvania. FOURTH EDITION, REVISED RECENTLY ISSUED. Bound in Full Leather, Limp, with Gold Edges. Price, $1.00 neti with Patent Thumb Index, $1.25 net. The book is an absolutely new one. It is not a revi- sion of any old work, but it has been written entirely anew and is constructed on lines that experience has shown to be the most practical for a work of this kind. It aims to be complete, and to that end contains practically all the terms of modern medicine. This makes an unusually large vocabu- lary. Besides the ordinary dictionary terms the book contains a wealth of anatomical and other tables. This matter is of particular value to students for memorizing in preparation for examination. " I am struck at once with admiration at the compact size and attractive ex- terior. I can recommend it to our students without reserve." — J AMES 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 COMPANY, 925 Walnut St., Phila. London: % Henrietta Street, Covent Garden ESSENTIALS OF BACTERIOLOGY Since the issue of the first volume of the Saunders Question=Compends, OVER 250,000 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 Nose-piece Coarse Adjustment Fine Adjustment BACTERIOLOGICAL MICROSCOPE (with Abbe and Blender in Position). SAUNDERS' QUESTION-COMPENDS, No. 20 ESSENTIALS BACTERIOLOGY CONCISE AND SYSTEMATIC INTRODUCTION TO THE STUDY OF MICRO-ORGANISMS /? vCbv M. V. BALL, M.D. Formerly Resident Physician, German Hospital, Philadelphia ; formerly Bacteriologist to St. Agnes' Hospital FIFTH EDITION, THOROUGHLY REVISED BY KARL M, VOGEL, M.D. Assistant in Pathology, College of Physicians and Surgeons, Columbia University, New York City With Ninety-Six Illustrations some in Colors, and Six Plates PHILADELPHIA AND LONDON W. B. SAUNDERS & COMPANY 1905 Set up, electrotyped, printed, and copyrighted October, 1891. Reprinted October, 1892. Revised, reprinted, and recopyrighted May, 1893. Reprinted June, 1894. Revised, reprinted, and recopyrighted November, 1896. Reprinted October, 1898. Revised, reprinted, and recopyrighted March, 1900. Reprinted May, 1903. Revised, reprinted, and recopyrighted August, 1904. Copyright, 1904, by W. B. Saunders & Company. Reprinted October, 1905. ELECTROTYPED BY WESTCOTT & THOMSON, PHILAOA. PRESS OF W. B. SAUNDERS & COMPANY. 1165- PREFACE TO THE FIFTH EDITION The progress in bacteriology during the last few years has involved more or less radical changes in many depart- ments of the science. Recent work on such subjects as immunity, tuberculosis, dysentery, yellow fever, the bu- bonic plague, and other infectious diseases has rendered obsolete many portions of any but the most modern books, and in countless minor details the teaching of to-day differs from that of even a few years ago. In this revision the attempt has been made to reflect as faithfully as possible the present status of bacteriology without overstepping the limits set by the scope of a book intended primarily as *an aid to students. Much assist- ance has been derived from the " Manual of Bacteriol- ogy" of Muir and Ritchie, and from F. C. Wood's "Laboratory Guide to Clinical Pathology." 7 5506r() PKEFAOE TO 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, bacteriologists. 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 : — Mac6: Trait6 pratique de Bacteriologie. Frankel: Grundriss der Bakterienkunde. Eisenberg: Bakteriologische Diagnostik. Crookschank, E. M.: Manual of Bacteriology. Gunther: Einflihring in das Studium der Bacteriologie, etc. Woodhead and Hare: Pathological Mycology. Salmonsen: Bacteriological Technique (English translation). M. V. BALL. Buffalo, N. Y., October 1, 1891. 62 Delaware Avenue. CONTENTS PART I. GENERAL CONSIDERATIONS AND TECHNIQUE. PAGE Introduction 15 Chapter I.— Classification, Structure, and Reproduction. 17 II.— Origin, Life, Growth, and Properties .... 23 " III.— Methods of Examination 26 " IV.— Staining of Bacteria 30 V— General Method of Staining Specimens ... 35 " VI.— Special Methods of Staining 39 " VII.— Methods of Culture 42 " VIIL— Nutrient Media 48 M IX.- -Solid Transparent Media 52 " X.— Inoculation of Gelatine and Agar 60 " XL— Growth and Appearances of Colonies ... 64 " XII. — Cultivation of Anaerobic Bacteria 67 " XIII.— Infection 70 " XIV.— Immunity 72 " XV.— Animal Experiments 76 PART II. SPECIAL BACTERIOLOGY. Chapter I.— Non-Pathogenic Bacteria 80 Bacillus Prodigiosus 80 Indicus 81 9 10 CONTENTS. PAGE Bacillus — Mesentericus Vulgatus 81 Megateriuni 82 Ramosus 82 Bacterium Zopfi 83 Bacillus Subtilia 83 Spinosus 84. Some Bacteria in Milk 84 Bacillus Acidi Lactici . . . 84 Butyricus 85 Amylobacter 85 Lactis Cyanogenus 86 Lactis Erythrogenes 86 Examination of Milk in Stained Specimens 87 Some Non-Pathogenic Bacteria of Water 87 Bacillus Violaceus * 87 Cceruleus 88 * Fluorescent Bacteria 88 Phosphorescent Bacteria 89 Leptothrix, Crenothrix, Cladothrix, and Beggiatoa 90 Micro-organisms found in Urine 91 Spirillum 92 Rubrum; Concentricum 92 Sarcina 92 Lutea 92 Aurantica Flava, Rosea, and Alba 93 Ventriculi , 93 s-Oppler Bacillus 93 Chapter II. — Pathogenic Bacteria • . 94 Bacteria Pathogenic for Man and Other Animals ... 94 Bacillus Anthracis 94 • Tuberculosis 97 Lepra Bacillus 107 Syphilis Bacillus 108 Bacillus of Glanders 108 CONTENTS. 11 PAGE Bacillus — of Diphtheria HO pseudo- 114 of Typhoid Fever 115 Paracolon or Paratyphoid . . . 121 Psittacosis 121 Coli Communis 122 Chapter III. — Pathogenic Bacteria — continued . . . . . 124 Spirillum Cholera 124 Bacteria Similar to Spirillum Cholerae ........ 127 Finkler-Prior . T\ .'. . ... r ..*... . . 127 Tyrogenum 128 Vibrio Metschnikovi 129 Bacteria of Pneumonia 130 Pneumobacillus of Friedlander ' . . 131 ofFrankel 132 Antitoxin of Pneumonia 134 Bacillus of Rhinoscleroma 135 Diplococcus Intracellularis Meningitidis 135 Micrococcus Tetragenus 135 Capsule Bacillus 136 Bacillus of Influenza 137 Micro-organisms of Suppuration , 137 Streptococcus Pyogenes 138 Staphylococcus Pyogenes Aureus 140 , Pyogenes Albus 141 Micrococcus Pyogenes Citreus ......... 141 Cereus Albus 141 Cereus Flavus 141 Pyogenes Tenuis 142 Bacillus Pyocyaneus 142 Micrococcus Gonorrhoeae 143 Microbes Similar to Gonorrhoea 145 Bacillus of Tetanus 147 12 CONTENTS. PAGE Bacillus — (Edematis Maligni ............. 150 Spirillum of Relapsing Fever 152 Bacillus of Soft Chancre 153 Icteroides ........ 154 of Bubonic Plague 155 # of Dysentery 157 Aerogenes Capsulatus 158 Micrococcus Melitensis 159 Pathogenic Protozoa . 159 Malarial Parasite 159 Amoeba Dysenterise 163 Small-pox and Vaccinia . 163 Trypanosomes 163 Chapter IV. — Bacteria Pathogenic for Animals, but not for Man 164 Bacillus of Symptomatic Anthrax 164 of Chicken Cholera . . . 165 Bacteria of Hemorrhagic Septicaemia, Swine Plague, Duck Cholera, etc 166 Bacillus of Erysipelas of Swine 167 Murisepticus 168 Micrococcus of Mai de Pis 169 Bacillus Alvei 169 Micrococcus Amylovorus 170 Bacterium Termo 170 Proteus Vulgaris 170 Mirabilis 171 Zenkeri 0 ....... . 171 APPENDIX. Yeasts 173 Oidiums 174 Moulds 175 CONTENTS. 13 PAGE Cladothrices and Strep tothrices 177 Streptothrix, or Cladothrix Actinomyces (Ray Fungus) . . 177 Madura . . 178 Farcinica 179 Examination of Air 180 of Water 184 of Soil 187 Bacteria of Milk and Other Foods 188 Examination of the Organs and Cavities of the Human Body 189 Tables of Chief Characteristics of the Principal Bacteria . . 194 Part I. Non-Pathogenic . 194 Part II. Pathogenic 218 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 in- sufficient to enable him to prove his theories. Anthony Van Leuwenhoeck, 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 loco- motion, and compared them in size with various grains of definite measurement. 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. Miiller, of Copenhagen, in 1786, made a classification composed of two main divisions — monas and vibrio ; and with the aid of the compound microscope was better able to describe them. Ehrenberg, in 1833, with still better instruments, divided bacteria into four orders: bac- terium, vibrio, spirillum, and spirochete. It was not until 1863 that any positive advance was made in connecting bacteria with disease. Rayer and Davaine had in 1850 15- 16 INTRODUCTION already found a rod-shaped bacterium in the blood of ani. mals 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 fer- mentation 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 an- thrax. 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, Pas- teur 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. Since then bacteriology has grown to huge proportions — become a science in itself — and thousands of earnest workers are adding yearly solid blocks of fact to the structure, which structure it will be our aim to briefly describe in the pages which are to follow. ESSENTIALS OF BACTERIOLOGY. PART I. GENERAL CONSIDERATIONS. CHAPTER I. 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 numerous unicellular vegetable organisms which form the lower limit of plant life as we know it multiply by fission and are hence called the Schizophyta, or splitting plants. This group is subdivided into two classes — (a) the Schizophycese, or fission alga?, and (b) the Schizomycetes, or fission fungi, or bac- teria, as we usually call them. Lately it has Become customary to subdivide the bacteria themselves somewhat arbitrarily into two classes — the lower bacteria and the higher bacteria. The lower bacteria are unicellular masses of protoplasm of microscopic size, multiplying by fission and existing without chlorophyll. Three main types are found : (1) Globular forms, called cocci ; (2) straight rod-shaped forms, called bacilli ; (3) curved or spiral rods, called spirilla. The higher bacteria show a tendency toward a more com- plicated mode of organization in two ways : (1) They consist of filaments made up of separate individuals, but which exhibit enough independence to foreshadow the rudiments of a physi- 2 (17) 18 ESSENTIALS OF BACTERIOLOGY. ological division of labor. (2) Certain elements may be differ- entiated for the purpose of reproduction. The Staphylococcus pyogenes, the anthrax bacillus, and the spirillum of relapsing fever are typical forms of the lower bac- teria, while the actinomyces, or ray-fungus, is the most impor- tant pathogenic member of the higher bacteria. Fig. 1. -V Micrococcus. Spirillum. Bacillus. Structure. Bacteria are cells ; they appear as round or cylin- drical of an average diameter or transverse section of 0.001 mm. (=1 micromillimeter), written 1 /*. The cell, as other plant- cells, is composed of a membranous cell- wall and cell-contents ; " cell-nuclei " can in some cases be seen by the use of special stains. Cell- Wall. The cell-wall is composed either of plant cellu- lose, or a form of albumin, since it is less permeable than cellu- lose membrane. The membrane is firm, and can be brought plainly into view by the action of iodin upon the cell-contents, which contracts them. Cell-Contents. The contents of the cell consist mainly of protoplasm, usually homogeneous, but in some varieties, finely granular, or holding pigment, chlorophyll, fat-droplets, and sul- phur in its structure. It is composed chiefly of mycoprotein. Gelatinous Membrane. The outer layer of the cell-membrane can absorb water and become gelatinoid, forming either a little envelope or capsule around the bacterium or preventing the separation of the newly-branched germs, forming chains and bunches, as strepto- and staphylo-cocci. Long filaments are also formed. Zooglcea. When this gelatinous membrane is very thick, irre- BACTERIA 19 gular masses of bacteria will be tunned, the whole growth being in one jelly-like lump. This is termed a zooglcea {$vt6v, plant. Parasites, napa, aside of, airoq, 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 Facultative Parasites. Conditions of Life and Growth of Bacteria. Influence of Tem- perature.— In general, a temperature ranging from 10° C. to 40° C. is necessary to their life and growth. Saprophytes take the lower temperatures ; Parasites, the tem- perature more approaching the animal heat of the warm-blooded. Some forms require a nearly constant heat, growing within very small limits, as the Bacillus of Tuberculosis. 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. 24 ESSENTIALS OF BACTERIOLOGY. A few varieties exist only at freezing point of water; and others again will not live under a temperature of 60° C. For the majority of Bacteria a temperature of 60° C. is de- structive ; and several times freezing and thawing very fatal. Influence of Oxygen. — Two varieties of bacteria in relation to oxygen. The one terobie, growing in air; the other, anserobic, living without air. Obligate serobins, those which exist only when oxygen is present. Facultative asrobins, those that live best when oxygen is present, but can live without it. Obligate or true anserobins, those which cannot exist where oxygen is. Facultative anwrobins, those which exist better where there is »^ oxygen, but can live in ito prcQcnce.'*"1^^^'-^ Some derive the oxygen which they require out of their nutri- ment, so that a bacterium may be aerobic and yet not require the presence of free oxygen. iErobins may consume the free oxygen of a region and thus allow the anserobins to develop. By improved methods of cul- ture many varieties of anserobins 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 only active 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 bac- teria. Effects of Electricity. — Electricity arrests growth. Effects of R'dntgen Rays. — Haye little or no effect on artificial cultures, but in the living tissues a pronounced bactericidal effect is produced, perhaps through the stimulation of the body-cells. Vital Actions of Microbes. Bacteria feeding upon organic com- pounds produce chemical changes in them, not only by the with- drawal of certain elements, but also by the excretion of these elements changed by digestion. Sometimes such changes are destructive to themselves, as when lactic and butyric acids are formed in the media. ORIGIN OF BACTERIA. 25 Oxidation and reduction are carried on by some bacteria. Am- monia, hydrogen sulphide, and trimethylamin are a few of the chemical 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. Ni- trites into nitrates. Ptomaines. Brieger found a number of complex alkaloids, closely resembling those found in ordinary plants, and which he named ptomaines, from nrcbfia (corpse), because obtained from putrefying objects. Proteins. The components of the bacterial cell may cause inflammation and fever. Putrefaction. When fermentation is accompanied by devel- opment of offensive gases a decomposition occurs, which is called putrefaction, and this, in organic substances, is due entirely to bacteria. Producers of Disease. Various pathological processes are caused by bacteria, the name given to such diseases being in- fectious diseases, and the germs themselves called disease-pro- ducing or pathogenic bacteria. Those which do not form any pathological process are called non-pathogenic bacteria. Ferments are diastatic, changing starch into sugar ; proteolytic, transforming albumins into more soluble substances; gelatin liquefaction is an example. Inverting, changing a sugar from one that does not undergo fermentation into one that does. Coagulating, fat-splitting, hydrolytic ferments are some of the other varieties. Toxins and Toxalbumins are various albuminoids produced in the animal organism and in culture-media which are very poisonous, and are considered the prime cause of disease. Pigmentation. Some bacteria are endowed with the property of forming pigments either in themselves, or producing a thro- mogenic 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 aniline dyes discovered in them. Phosphorescence. Many bacteria have the power to form Zb ESSENTIALS OF BACTRIOLOGY. light, giving to various objects which they inhabit a character- istic glow or phosphorescence. Fluorescence. An iridescence, or play of colors, develops in some of the bacterial cultures. Gas Formation. Many bacteria, anaerobic ones especially, 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 nauseous. Effect of Age. With age, bacteria lose their strength and die. CHAPTER III. METHODS OF EXAMINATION. We divide the further study of the general characteristics of Bacteria into two portions : — First. The examination of bacteria by aid of the microscope. Second. The continued study through artificial cultivation. They both go hand in hand ; the one incomplete without the other. Microscopical. The ordinary microscope will not suffice for Bacteriologic research. Certain special appliances must first be added. 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 be- tween the object and the lens, do not enter the lens, and are lost. By interposing an agent which has the same refractive index as glass, cedar-oil, or clove-oil, for example, all the rays of light from the object enter directly into the lens. The " Homogeneous System/' or oil-immersion lens, consists 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. METHODS OF EXAMINATION. 27 Abbe's Condenser. The second necessary adjunct is a com- bination of lenses placed underneath the stage, for bringing wide rays of light directly under the object. It serves to intensify Fig. 7. the colored pictures 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. Together With it is Abbe's Condenser. usually found an instrument for shutting off part of the light — a blender or diaphragm. 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 Welsbach burner) is best for bacterial study : use the plane mirror for daylight and the concave mirror for artificial light. Iris Blender. 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 ocular. When using high power objective use weak ocular. A nose-piece will be found very useful, since it is sometimes neces- 28 ESSENTIALS OF BACTERIOLOGY. sary to change the objective on the same field, and this insures a great steadiness of the object. Great cleanliness is needed in all bacteriological methods ; but nowhere more so than in the microscopical 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 bichromate with 6 per cent, of strong sulphuric acid, washed in water, and kept in absolute alcohol. Examination of Unstained Bacteria. As the coloring of bac- Fig. 9. Platinum Needles. teria kills them and changes their shape to some extent, it is pre- ferable to examine them 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, we obtain a small drop from the liquid con- taining 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 passing all instruments, needles, etc., through the flame before and after each procedure ; it in- sures safety ; and once in the habit, it will be done automati- cally. From Solid Media. With a straight-pointed platinum needle, a small speck of the medium is taken and rubbed upon a glass METHODS OF EXAMINATION 29 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 immersion lens dipped gently down into it as close as possible to the cover-glass, the narrow blender shutting off the Abbe condenser, for this being an unstained specimen, we want but little light We now apply the eye, and if not in focus, use the fine adjust- ment or the coarse, but always away from the object — i. e. towards us — since the distance between the specimen and the lens is very slight, it does not require much turning to break the cover-glass and ruin the specimen. Having found the bacte- rium, we see whether it be bacillus, micrococcus, or spirillum ; discover if it be motile, or not. That is about all we can ascer- tain by this method. Fig. 10. Hanging Drop in Concave Glass Slide. Hanging Drop. 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 centre) 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 vaseline, and the slide inverted over the drop; the cover-glass sticks to the smeared slide, which, when turned 30 ESSENTIALS OF BACTERIOLOGY. 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 usefully), 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 bac- teria will usually be very thick in the centre 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 stages of the microscope. Agglutination as observed in Widal's test is best seen in the hanging drop. CHAPTER IV. STAINING OF BACTERIA. 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 ; and lastly, 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 bacteria. But now only a very few find general use, and with methyline 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, Methylin blue {not methyl blue), Thionin, Saffranin. And the acid colors to which eosin and acid-fuchsin belong. STAINING OF BACTERIA. 31 The basic dyes stain the bacteria and the nuclei of cells ; the acidE dyes stain chiefly the tissue, leaving the bacteria almost untouched. Carmine and Hematoxylin are also useful as con- trast 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 ; 1 part of this solution to about 100 parts of distilled water con- stitutes 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 chemical agents, the intensity of the aniline dyes can be greatly increased. Mordants. Agents that t(p 6iie" 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. 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. Carbol Fuchsin. Carbolic acid can be used instead of anilin oil, and forms one of the main ingredients of Ziehl's or Neelsen's solution, used principally in staining bacillus tuberculosis. Kuhne has a carbol- methylin blue made similar to the carbol fuchsin. Alkaline Stains. Alkalies have the same object as the above agents ; namely, to intensify the picture. Potassium hydrate, amnion, carbonate, and sodium hydrate are used. Loffier's alkaline blue and Koch's weak alkaline blue have in them potassium. 32 ESSENTIALS OP BACTERIOLOGY. Heat. Warming or boiling the stains during the process of staining increases their intensity. Decolorizing Agents. The object is usually over-colored in some part, and then decolorizing agents are employed. Water is sufficient for many cases ; alcohol and strong mineral acids com- bined are necessary in some. Iodin 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; prevents them from being decolorized, but allows all else to be faded. Then by using one of the acid or tissue dyes, a contrast 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 grammes of the powdered dye in a bottle and add 40 grammes of alcohol. Shake well and allow to settle. This can be used as the stock bottle. II. — Weak Solutions. Made best by adding about 1 part of number I. or stock solu- tion to 10 of distilled water. This is the ordinary solution in use. III. — Anilin Oil Water. Aniline oil 5 parts. Distilled water . . * . . 100 parts.— M. Shake wTell and filter. To be made fresh each time. IV.— Anilin Water Dyes. Sat. alcoh. sol. of the dye . . 11 parts. Aniline oil water .... 100 parts. Abs. alcohol 10 parts. — M. Can be kept 10 days. STAINING OF BACTERIA. 33 V.— Alkaline Methylin Blue. A. Loffler's. Sat. ale. Sol. methylin blue . . 30 Sol. potass, hydrat. (1-10,000) . 100— M. B. Koch's. Sol. potass, hydrat. (10 per eent.) 0.2 Sat. ale. sol. methyl, blue . . 1.0 • Distilled water .... 200.0— M. VI. — Carbolic Acid Solutions. A. Ziehl-Neelsen. Fuehsin (powd.) .... 1 part. Alcohol 10 parts. 5 per eent. sol. acid, carbolic . 100 parts.— M. Filter. The older the solution the better. B. Kilhne. Methylin blue . . . . 1.5 Alcohol 10.0 5 per cent. sol. ac. carbol. . . 100.0 Add the acid gradually. This solution loses strength with age. VII. — Gram's Iodin Solution. Iodine 1 Potass, iod 2 Aquae destillat 300.— M. VIII.— Loffler's Mordant (for flagella). Aq. sol. of tannin (20 per cent.) . 10 parts. Aq. sol. ferri sulph. (5 per cent.) . 1 part. Aqua? decoc. of logwood (1-8) . 4 parts. — M. Keep in well-corked bottle. IX. — Unna's Borax Methyl Blue. Borax 1 part. Methyl blue 1 part. Water 100 parts.— M. ■6 34 ESSENTIALS OF BACTERIOLOGY. X. — Gobbet's Acid Blue (rapid stain). Methylin blue .... 2 25 per cent, sulphuric acid . . 100.— M. XI. — Alkaline Anilin Water Solutions. Sodium hydrat. (1 per cent.) . . 1 Anilin oil water .... 100. — M. And add — Fuchsin, or methyl-violet powd. . 4 Cork well. Filter before using. XII. — Roux's Double Stain. Dahlia or gentian violet ... 0.5 gramme. Methyl green 1.5 " Distilled water 200.0 grammes. — M. Use as other stains, without acid. XIII. — Neisser's Stain. (For Diphtheria.) Solution I. Methylin blue .... Alcohol (96 per cent.) Dissolve and add water . Glacial acetic acid . 1 gramme. . 20 c.c. . 950 c.c. . 50 c.c— M. Solution II. Vesuvin Water 2 grammes. . 1000 c.c— M. Stain cover-glasses (1) three seconds in Sol. I. ; (2) wash in water; (3) three seconds in No. 2; (4) wash in water. Body of bacillus, brown ; oval granules at each end, blue. XIV. — Carbol-thionin. (Nicolle.) Sat. sol. thionin in ale (90 per cent.) 10 c.c. Aqueous sol. ac carbol. (1 per cent.) 100 c.c. — M. Stain sections, one-half to one minute. METHOD OF STAINING SPECIMENS. 35 XV. — Capsule Stain of Hiss. Use the following, heated until it steams : Sat. alcoholic solution of 1 K „ „ l . o c.c. gentian violet or fuchsin j Distilled water .... 95 c.c. Wash in 20 per cent, solution of cupric sulphate crystals. XVI. — Capsule Stain of Welch. (1) 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 ex- amine in salt solution 0.8-2.0 per cent. CHAPTER V. 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 speci- mens. 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 prevent- ing too great a degree of heat). Since most of the specimens contain a certain amount of albumenoid material, it is best in all cases to "fix" it, t, e., to coagulate the albumen. 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 doing so, 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. 11.) It prevents the flame or staining-fluid from reaching the fingers. 36 ESSENTIALS OF BACTERIOLOGY. The object is now ready for, staining. Staining.— A few drops of the staining solution are placed upon the cover-glass so that the whole specimen is covered, and it is left on a few minutes, the time depending upon the variety, the strength of stain, and the object desired. Instead Fig. 11. Author's Bent Forceps for Holding Cover-glass over Flame. of placing the dye upon the object, the cover-glass can be im- mersed in a small glass dish containing the solution ; or, if heat is desired to intensify or hasten the process, a watch- crystal holding the stain is placed over a Bunsen burner and in it the cover-glass ; and, again, the cover-glass can be held directly in the flame with the staining fluid upon it, which must be constantly renewed until the process is completed, or the cover-glass can be heated in a test-tube. Removing Excess of Stain. The surplus stain is washed off by dipping the glass in distilled water. 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 per- manently mounted by lifting the cover-glass off the slide (this is facilitated by letting a little water flow under it, one end METHOD OF STAINING SPECIMENS. 37 being slightly elevated). The water that still adheres is dried off in the air or gently over the flame, and when perfectly dry it 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 yourself 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 instrument. 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 specimens for staining, very thin sections of the tissue must be made. As with histological preparations, the tissue must be hardened before it can be cut thin enough. Alcohol is the best agent for this purpose. Fig. 12. I ' : Spatula for Lifting Sections. Pieces of the tissue one-quarter inch in size are covered with alcohol for 24 to 48 hours. When hardened it must be fixed upon or in some firm object. A paste composed of— Gelatine 1 part. Glycerine 4 parts. Water 2 parts. will make it adhere firmly to a cork in about 2 hours, or it can be imbedded in a small block of paraffine, and covered over with melted paraffine. Celloidin may be used as an imbedding agent, and formalin is useful to harden tissue quickly. Cutting. The microtome should be able to cut sections j^B inch in thickness ; this is the fineness usually required. The sections are brought into alcohol as soon as cut unless they have been imbedded in paraffine, when they are first washed in chloroform to dissolve out the paraffine. 38 ESSENTIALS OF BACTERIOLOGY. 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 speci- men. A very useful instrument for transferring the delicate sections from one solution to another is a little metal spatula, the blade being flexible. A still better plan, especially when the tissue is "crumbling/ is to carry out the whole procedure on the glass-slide. Fig. 13. Section Microtome. General Principles. The section is transferred from the alco- hol 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 5 to 15 minutes. Then- JJish II, containing 5 per cent, acetic acid (1 to 20); where it remains ^ to 1 min. 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 STAINING AND MODIFICATIONS. 39 section is then taken, avoiding the errors, if any ; and having reached this stage proceeded with as follows : — Dish IV, alcohol, 2 to 3 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 under neath will shine through). VI. Remove excess with filter-paper. VII. Mount in Canada balsam (xylol balsam). CHAPTER VI. SPECIAL METHODS OF STAINING AND MODIFICATIONS. Gram's Method of Double Staining. (For cover-glass speci- mens.)— I. A hot solution of anil, water gentian violet 2 to 10 minutes. II. Directly without washing, into Gram's solution of iod. potass, iod. 1 to 3 min. (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, picro-carmine, or bismark- brown. The bacteria will appear deep blue, all else red or brown on a very faint brown background. The following bacteria do not retain their color with Gram's method — are therefore not available for the stain : Bacillus of typhoid ; spirillum of cholera ; bacillus of chicken cholera, of hemorrhagic septicaemia, of malignant oedema, of pneumonia (Friedlander), and of glanders; diplococcus of gonorrhoea ; spi- rillum of relapsing fever. Gram's Method for Tissues (modified by Giinther). I. Stain in anil, water gent, violet . . 1 minute. II. Dry between filter paper. III. Iod. potass, iod. sol. .... 2 minutes. IV. Alcohol £ minute. V. 3 perct. sol. hydrochloric acid in alcohol 10 seconds. VI. Alcohol, ol. of cloves, and Canada balsam. 40 ESSENTIALS OF BACTERIOLOGY. To Stain Spores. Since spores have a very firm capsule, which tends to keep out all external agents, a very intensive stain is required to penetrate them, but once this object 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 carbol-fuchsin 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 £ per cent, hydrochloric acid. A contrast color, preferably methylin blue, is added for a few minutes. The spores will appear as little red beads in the blue bacteria, and loose ones lying about. Spore Stain (modified). — I. Carbol-fuchsin on cover-glass and heated in the flame to boiling point 20 to 30 times. II. 25 per cent, sulphuric acid, 2 seconds ; rinsed in water. III. Methylin blue contrast. Alex. Klein recommends the following spore method : mix a little of the culture (potato) with 3 drops of physiologic salt solution, and heat gently with an equal quantity of carbol- fuchsin for a period of 6 minutes. Spread then on cover-glasses, dry in the air, and fix by passing three times through Bunsen burner flame. Decolorize in 1 per cent, sulphuric acid for 1 to 2 seconds ; contrast in weak methylin blue. BowhilVs Orcein Stain. Sat. alcoholic solution of orcein . . .15 c.c. 20 per cent, aqueous sol. 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 10 minutes. Wash in water. Dry and mount in balsam. Five per cent, chromic acid applied for 15 minutes has been recommended in staining spores. This is followed by the carbol-fuchsin stain as above. STAINING AND MODIFICATIONS. 41 Flagella Stain, with Loffier's Mordant. — I. A few drops of the mordant (No. viii. p. 33) are placed upon the spread cover-glass and heated until it steams. II. Washed 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 1£ minutes. IV. Wash in water. If the stain is properly made, the microbes are deeply colored and the flagella seen as little dark lines attached to them. Sporogenic bodies stain quite readily, and in order to distin- guish them from spores Ernst uses alkaline methylin blue, slightly warmed. Then rinse in water. Contrast with cold bfemark- 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 de- colorizes too much. To obviate this Kulme mixes the alcohol with the stain, so that while the section is being anhydrated it is constantly supplied with fresh dye. Weigert uses aniline oil to dehydrate instead of alcohol, and here, too, it can be used mixed with the dye. Unna's Method for Fungi (especially useful for epidermic; scales). — Moisten horny scale or crust with acetic acid; mace- rate between two glass slides ; dry in flame ; wash out fat with ether and alcohol (equal parts) ; stain in borax methyl blue for ten seconds (over flame) ; bleach with glycerine and aether (equal parts) ; rinse in water, alcohol, dry, and mount. Behavior of the More Important Bacteria to Gram's Stain. Positive means that the bacteria retain the primary color, or gentian violet. Positive. Negative. Tubercle Bacillus, Colon Bacillus, Smegma Bacillus, Typhoid Bacillus, Lepra Bacillus, Cholera Bacillus, Anthrax Bacillus, Influenza Bacillus, 42 ESSENTIALS OF BACTERIOLOGY. Positive. Negative. 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 Moras. CHAPTER VII. METHODS OF CULTURE. 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 microscopical ex- amination or animal experimentation. To properly develop bacteria we supply as near as possible 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 are nearly perfect. Sterilization. If we place our nutrient material in vessels that have not been properly disinfected, 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 neces- sary precautions to take : — First. To thoroughly 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 con- tainers, the entrance of foreign germs. Disinfectants. Corrosive sublimate (bichloride of mercury), which is the most effective agent we possess, cannot be gene- rally used because it renders the soil unproductive and therefore METHODS OF CULTURE. 43 must only be employed in washing dishes, to destroy the 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 glass-ware that comes in contact with the nutrient media free from the sublimate. 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 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. Fig. 14. Hot Air Oven. For obtaining dry heat— that is, a temperature of 150° C, (about 300° F.)— a sheet-iron oven is used which can be heated by a gas-burner. If it have double walls (air circulating be- tween), the desired temperature is much more quickly obtained. A small opening in the top to admit a thermometer is neces- sary. These chests are usually about 1 foot high, 1^ foot wide, and f foot deep. In them, glassware, cotton, and paper can be sterilized. "When the cotton is turned slightly brown, it usually 44 ISSENTIALS OF BACTERIOLOGY, denotes sufficient sterilization. All instruments, where practi- cable, should be drawn through the flame of an alcohol lamp or Bunsen burner. One hour in the oven at 170° C. usually steri- lizes glass-ware, while the ordinary germs in liquids may be Fig. 15. Koch's Steam-chest. killed by boiling for five minutes if no spores are present. The boiling of any fluid at 100° C. for one and one-half hours nearly always ensures sterilization. Moist Heat. — Steam at 100° C. in circulation has been shown to be a very effective application of heat. METHODS OF CULTURE. 45 Koch's Steam-chest. Circulating steam is obtained by aid of Koch's apparatus. This consists, of a cylindrical tin chest .about 2£ feet high and about £ foofom diameter ; divided in its interior by a perforated diaphragtiJja, an upper chamber for the steam, c, and a lower one for ,, water, b. Two or more gas-burners placed underneath the chest, which stands on a Fig. 16. Arnold's Steam-sterilizer. tripod, supply the heat. In the cover is an opening for a ther- mometer. The chest is usually covered with felt. When the thermometer registers 100° C. the culture-medium or other sub- stance to be sterilized is placed in the steam and kept there from 10 to 15 minutes, or longer, as required. 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, (rig. 16.) 46 ESSENTIALS OF BACTERIOLOGY The autoclave of Chamberland allows a temperature of 120° C. to be obtained, and is much used in Pasteur's laboratory. Fig. 17. Chamberland's Autoclave with pressure. Instead of sterilizing for a long time at once, successive steri- lization is practised with nutrient media, so that the albumen will not be too strongly coagulated. Fifteen minutes each day for three days in succession. METHODS OF CULTURE, 47 Fractional Sterilization of Tyndall. Granted that so many spores originally exist in the object to be sterilized, it is sub- jected to 60° 0. 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° 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 destroyed. Fig. 18. Fig. 19. i1 1 i' ! i 1 1 i i ! LJ ■I !i Wire-Cage. Cotton plugged Test-Tubes. Cotton Plugs or Corks. All the glass vessels (test-tubes, flasks, etc.) must be closed with cotton plugs, the cotton being easily sterilized and preventing the entrance of germs. Tin-foil may be used to cover the cotton, or caps made of india-rubber. Test-tubes. New test-tubes are washed with hydrochloric acid and water to neutralize the alkalinity often present in fresh glass. They are then well washed and rubbed with a 48 ESSENTIALS OP BACTERIOLOGY. brush, placed obliquely to drain, and when dry corked with cotton plugs. Then put in the hot-air oven (little wire-cages being used to contain them) for fifteen minutes, after which they are ready to be filled with the nutrient media. (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 oz. panel medicine bottles can be used for retaining the nutrient media and cultures. According to late investigations, the glass tubes become suffi- ciently sterile in the steam-chest without the preliminary sterili- zation in the dry oven. CHAPTER VIII. NUTRIENT MEDIA. Of the many different media recommended and used since bacteriology became a science, we can only describe the more important ones now in use. Each investigator changes them according to his taste. Fluid Media. Bouillon (according to Loftier). A cooked infusion of beef made slightly alkaline with soda carbonate : 500 grammes of finely-chopped raw lean beef are placed in a wide-mouthed jar and covered with 1 litre of water ; this is left standing twelve hours with occasional shaking. It is then strained through cheese cloth, the white meat remaining being pressed until one litre of the blood-red meat-water has been obtained. The meat- water must now be cooked, but before doing this, in order to prevent all the albumen from coagulating, 10 parts of peptone powder and 5 parts of common salt are added to every 1000 parts meat-water. It is next placed in the steam-chest or water-bath for three-quarters of an hour. Neutralization. The majority of bacteria grow best on a neutral or slightly alkaline soil, and the bouillon, as well as NUTRIENT MEDIA. 49 other media, must be carefully neutralized with a sat. sol. of carbonate of soda. Since too much alkalinity is nearly as bad as none at all, the soda must be added drop by drop until red litmus paper commences to turn blue. The bouillon is then cooked another hour, and filtered when cold. The liquid thus obtained must be clearly alkaline, and not clouded by further cooking. If cloudiness occur, the white of an egg and further boiling will clear the same. To make bouillon, beef-extract can be used instead of fresh meat, 2 grammes to 1 litre of water. This is boiled with 5 grammes of salt and 10 of peptone, neu- tralized as above, and filtered when cold. Schultz's Method of Neutralization. — A more accurate method of obtaining the required reaction is to use an alcoholic solution (£ per cent.) of phenolphthalein as an indicator ; a few drops of this are mixed with 10 c.c. of the bouillon, and from a burette a solution of caustic soda 0.4 per cent, is added drop by drop until a faint red color appears. An average is taken from three different samples, and the amount of soda needed for the entire quantity of bouillon is calculated therefrom. Glucose broth, which is a good medium for anaerobic organisms, consists of bouillon to which 1 to 2 per cent, of grape-sugar has been added. Glycerin broth is bouillon to which 6 to 8 per cent, of glycerin has been added after filtration. Sterilization of the Bouillon. Erlenmeyer flasks (little conical glass bottles) or test-tubes plugged and properly sterilized are filled one-third full with the bouillon, and placed with their contents in the steam-chest. They are left in steam of 100° C. one hour for three successive days, after which the tubes and bouillon are ready for use. Solid Media. The knowledge of bacteria and germs or moulds settling and growing upon slices of potato exposed to the air, led to the use of solid media for the artificial culture of the same. It was also thus learned that each germ tends to form a separate colony and remain isolated. Potato-Cultures. A ripe potato with a smooth skin is the best. Several are brushed and scrubbed with water to get rid of the dirt and the "eyes" are cut out. 4 50 ESSENTIALS OF BACTERIOLOGY. Next placed in 1 to 500 solution of bichloride of mercury for £ hour. Then in the steam-chest for £ hour. In the meantime, a receptable is prepared for them. This is called the moist chamber. Fig. 20. Moist chamber for potatoes. The moist chamber consists of two large shallow dishes, one, the larger, as a cover to the other. These dishes are washed in warm distilled water. Fig. 21. Method of slicing potato. (After Woodhead and Hare.) A layer of filter paper moistened with a 15 to 30 drops of 1 to 1000 bichloride is placed in the bottom of the glass dish. The operator now prepares his own hands, rolling up his coat sleeves and carefully washing his hands, then taking a potato NUTRIENT MEDIA. 51 from the steam-oven and holding it between his thumb and index finger in the short axis, he divides the potato in its long axis with a knife that has been passed through the flame. The two halves are kept in contact until they are lowered into the moist chamber, when they of their own weight fall aside, the cut surface uppermost. They are then ready for inoculation. 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 ^ hour, after which they are ready for inocu- lation (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 preserved. Roux's test-tube (Fig. 22), specially designed Fig. 22. for potato cultures, consists of a tube with a small constricted portion at the bottom, in which water may be kept to keep the potato moist. Manner of Inoculation. 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 luxuriant, and the individual colonies often difficult to recognize; therefore dilutions are made. (Fig. 23.) 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 dilution, and here usually the colonies will be sparsely enough settled to study them in their indi- viduality. 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 moulds and yeasts. Peeled potatoes are mashed with distilled water until thick, and then sterilized in flasks f of an hour for three successive days. Tube for potato culture. 52 ESSENTIALS OP BACTERIOLOGY. Fig. 23. Method of inoculation. (Woodhead and Hare.) Bread Mush.— Bread devoid of crust, dried in an oven, and then pulverized and mixed with water until thick and sterilized as above. CHAPTER IX. SOLID TRANSPARENT MEDIA. Solid Transparent Media are materials which can be used for microscopical purposes and which can readily be converted into liquids. Such are the gelatine and agar culture media. Gelatine. Gelatine is obtained from bones and tendons, and consists chiefly of chondrin and gluten. The French golden medal brand is the one most in use, found in long leaves with ribbed lines crossing them. Koch-Loffler 10 per cent. Bouillon-Gelatine. To the meat- water as made for the bouillon are added 100 grammes gelatine, 10 " peptone, 5 " salt, to each 1000 grammes of the meat-water; or to the bouillon made from beef-extract the gelatine is added ; this is placed in a flask and gently heated until the gelatine is dissolved. Neutralization with the soda and then cooking in water-bath or carefully boiled over flame for 1 hour or more until the SOLID TRANSPARENT MEDIA, 53 liquid seems clear, then add white of an egg and boil £ hour longer ; the egg will produce a clearer solution and save much trouble. A small por- tion, while hot, is now filtered into a test-tube and tested for alkalinity, and then re-heated several times, watching if a cloudy ppt. forms. If the fluid remains clear upon cooling, the remainder of the material can be filtered. It must be accomplished while hot, else the gelatine will coagulate and prevent further filtration. This can be carried on either by keeping hot the so- lution continually in water- bath, and only filtering a small quantity at a time through the filter, or keeping the filter itself hot, either with a hot water filter or placing the filter in steam chest. (Fig. 24.) Clouding of Gelatine. If the gelatine does not come out clear, or becomes turbid on cooling, it may be due to several things— 1. The filter-paper too thin or impure. 2. Too strongly alkaline. 3. Cooked too long or not long enough. The addition of the white of an egg, as before mentioned, will often clear it up ; if this avails not, re-filtering several times, and attention to the few points mentioned. Sterilizing the Gelatine. The gelatine is kept in little flasks or poured at once into sterile test-tubes, careful not to wet the neck where the cotton enters, lest when cool the cotton plug stick to the tube. The tubes are then placed in steam-chest for three successive Hot-water 54 ESSENTIALS OP BACTERIOLOGY. days, 15 minutes each day (or in water-bath 1 hour a day for three days). Then set aside in a temperature of 15° to 20° C, and if no germs develop and the gelatine remains clear, it can be used for cultivation purposes. Modifications. The amount of gelatine added to the meat- water can be variously altered, and instead of making gelatine bouillon milk, blood, serum, urine, and agar can be added. Glycerine (4 to 6%) is a common addition, and sometimes reducing agents to absorb the oxygen are mixed with it. 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 gelatine, retaining its solidity at a much higher temperature; it becomes liquid at 90° C. and con- geals again at 45° C. Gelatine will liquefy at 35° C. It is not affected very much by the peptonizing action of the bacteria — 38° C. is the temperature at which most patho- genic germs grow best. Preparation of Agar-Agar Bouillon or Nutrient Agar. The ordinary bouillon is first made, and then the agar cut in small pieces, added to the bouillon (15 grammes of agar to 1000 grammes bouillon. It is allowed to stand several minutes until the agar swells, and then placed in water-bath or steam-chest for six hours or more. It is then neutralized, very little of the alkali being sufficient. A white of an egg added, and boiled for several hours longer, when, even if not perfectly clear, it is filtered. The filtering process, very difficult because of the readiness with which the agar solidifies, must be done in steam-chest or with hot-water filter, and very small quantities passed through at a time, changing the filter-paper often. Cotton can be used instead of filter-paper, or filtering entirely dispensed with, simply decanting. A.s agar is seldom clear, a little more or less opaqueness will not harm. The test-tubes are filled as with the gelatine, and sterilized in the same manner. While cooling, some of the tubes can be placed in a slanting position, so as to obtain a larger surface to work upon. Water of condensation will usually separate and settle at the SOLID TRANSPARENT MEDIA. 55 bottom, or a little white sediment remain encysted in the centre ; this cannot easily be avoided, nor does it form any serious obstacle. The crude agar should first be rinsed in water, and then in 5% acetic acid and clear water again, to rid it of impurities. If agar is boiled thoroughly over a hot flame or in an auto- clave, it can be filtered much more readily. The main point is to see that all the agar is dissolved. It has been suggested to pour the hot agar into high cylin- drical glass vessels and allow it to cool slowly in the steam oven, the flame having been gradually lowered and then turned out. After a time the cloudy portion will form a sediment at the bottom ; the agar can then be shaken out as a long cylinder and the cloudy portion cut off. The Japanese Method. — Yokote prepares agar as follows : the meat is cooked in water over a sand bath 1? hours. Filtered, chopped agar is then added and the mixture cooked 1 hour longer ; peptone and salt added next. Neutralization. After the mixture has cooled to about 50° C. whites of 2 eggs are added and the mixture shaken thoroughly. Again the mixture is placed on the sand-bath and heated to 110° C. and over for lj to 2 hours, and then filtered through ordinary filter-paper. Yokote claims that by this procedure the agar can be filtered as easily as bouillon and without any loss. (The water evaporated in boiling must be added before filtering.) Glycerine Agar. The addition of 4 to 6% of glycerine to nutrient agar greatly enhances its value as a culture medium. Gelatine-Agar. A mixture of 5 per cent, gelatine and 0.75 per cent, agar combines in it some of the virtues of both agents. Blood Serum. Blood serum being rich in albumen coagulates very easily at 70° C, and if this temperature is not exceeded, a transparent, solid substance is obtained upon which the ma- jority of bacteria develop, and some with preference. 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 to receive the blood directly as it flows. It is placed on ice forty-eight hours, and the serum is drawn 56 ESSENTIALS OF BACTERIOLOGY. Fig. 25. out with sterile pipettes into test-tubes; these are placed ob- liquely in an oven where the temperature can be controlled and maintained at a certain degree. See Fig. 26. Incubators or Brood-ovens. Incubators or brood-ovens consist essentially of a double- walled zinc or copper chest, the space between the walls being filled with water. The oven is covered with some imperme- able material to prevent the action of the surrounding atmosphere. (Fig, 27.) It is supplied with a thermometer and a regu- lator. The regulator is connected with a Bunsen burner, and keeps the temperature at a certain height. There are several forms of regulators in use, and new ones are invented continually. The size of the flame in some is regulated by the expansion of mercury, which, as it rises, lessens the opening of the gas supply. The mercury contracting on cooling allows more gas to enter again. (Fig. 28.) Koch has invented a safety burner, by which the gas supply is shut oft* should the flame accidentally have gone out. Coagulation of Blood Serum. The tubes of blood serum having been placed in the oven, are kept at a tempera- ture of 65° to 68° C, until coagulation occurs ; then removed and sterilized. Sterilization of Blood Serum. The tubes are placed 3 to 4 days in incubation 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 the ordinary temperature of the room, it can be used for experi- mental purposes. Perfectly prepared blood serum is transparent, of a gelatine- 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. Blood serum Flask to receive blood serum. SOLID TRANSPARENT MEDIA. 57 may be prepared in a shorter way by coagulating the serum at a temperature short of boiling-point. Sterilization is completed in three days by exposing the tubes to a temperature of about 90° C. each day for five minutes. Tubes so prepared are opaque and white. Fro. 26. Thermostat for blood serum. Preservation of Blood Serum in Liquid State. Kirchner advises the use of chloroform. To a quantity of serum in a well-stop- pered flask a small amount of chloroform is added — enough to form about a 2 mm. layer on the bottom. 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 chloroform has been driven off (determined by absence of characteristic odor); the serum is then solidified and sterilized as in the ordinary way. 58 ESSENTIALS OP BACTERIOLOGY. Human blood serum derived from placenta, serum from as- citic fluid and ovarian cysts, is prepared in a similar manner to the above. Blood Coagulum, suggested by the author, is the blood itself (not the serum only) coagulated in test-tubes. It is dark brown in color and allows some colonies of bacteria to be more visi- ble. It requires less time to prepare, and is not so likely to become contaminated as when the serum is used. Fig. 27. Fig. 28. Babe's incubator. Thermo-regulators. Loffler's Blood Serum Mixture (see p. 111). Peptone Solution. (Dunham's.) Sodium chlorid, 0.5; pep- tone, 1 ; water, 100. Boil, filter, and sterilize. Useful to detect presence of indol. Other Nutrient Media. Milk, urine, decoctions of various fruits and plants, and lately for cultivating anaerobic bacteria, eggs. SOLID TRANSPARENT MEDIA. 59 Many combinations of the preceding are also in use, such as glucose-agar, glucose gelatin, blood- or serum-agar; and litmus is often added to media to show changes in reaction during bacterial growth. Dunham's Rosalie Acid Solution. Peptone sol. (Dunham) .... 100 c.c. 2 per cent. sol. rosalic acid . . .0.5 gr. Alcohol (80 per cent.) .... 100 c.c. M. To detect acids and alkalies. Eisner's Medium (for typhoid). (Iodo- potass. — Potato-gelatin.) Five hundred grammes of (peeled and washed) potatoes are mashed and pressed through a fine cloth. The juice is allowed to settle, is filtered, and after 1 hour's cooking has added to it 10 per cent, gelatin; then 2\ c.c. ^ normal sodic hydrate solu- tion, and finally 1 per cent, potassic iodid. Typhoid Medium of Hiss. This consists of a slightly acid mixture of gelatin and agar, beef-extract, sodium chloride, and dextrose, used in different proportions for plate and tube cul- tures. It is semi-solid in character and facilitates the identi- fication of the motile typhoid bacilli which produce a uniform clouding through the medium in tubes. Urine Media (Gonococci). Urine (sterile taken) . . . , .1 part. 2 per cent, agar solution . . . . 1 " Fresh Egg Cultures, after Huppe. 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 open- ing is covered with a piece of sterilized paper, and collodion. Boiled Eggs. Eggs boiled, shell removed over small portion, 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 prep- aration of bouillon, for the growth of special germs. The extracts of different organs have been added to the various media for experimentation. 60 ESSENTIALS OF BACTERIOLOGY, CHAPTER X. INOCULATION OF GELATINE AND AGAR. Glass Slide Cultures. Formerly the gelatine was poured on little glass slides such as are used for microscopical purposes, and after it had become hard, inoculated in separate spots as with potatoes. Fig. 29. Manner of holding tubes for inoculation: a, tube with material; b, tube to be inoculated ; c, cotton plugs. (After Woodhead and Hare.) Test Tube Cultures. The gelatine, agar, or blood serum having solidified in an oblique position, is smeared on the surface with INOCULATION OF GELATINE AND AGAR. 61 the material and the growth occurs, or the medium is punctured with a stab of the platinum rod containing the material. The first is called a stroke or smear culture, the second a stab or thrust culture. In removing the cotton plugs from the sterile tubes to carry out the inoculation, the plugs should remain between the lingers in such a way that the part which comes in contact with the mouth of the tube will not touch anything. After the needle has been withdrawn the plugs are re-inserted and the tubes labelled 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 inoculated with the material as follows . A looped platinum needle is dipped into the material and then shaken in the tube of liquid media, (gelatine, agar, etc.). This first tube is called original. From this three drops (taken with the looped platinum rod) are placed in a second tube, the rod being shaken somewhat in the gelatine 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. (Fig. 29.) The plugs of cotton must be replaced after each inoculation, and during the same must be carefully protected from contami- nation. To hasten the procedure and lessen the danger of contamina- tion, the tubes can be held in one hand aside of each other, each Fig. 30. Manner of holding plugs. plug opposite its tube. They are now ready for spreading on glass plates. 62 ESSENTIALS OF BACTERIOLOGY. Glass Plates. Fig. 31. z^Ek? The larger the surface over which the nutrient medium is spread the more isolated will the colonies be ; window glass cut in rec- tangular plates Gx4 inches in size is used ; about ten such plates are cleaned with dry towel and placed in a small iron box or wrapped in paper ; and sterilized in the hot-air oven at a temperature of 150° C. for ten minutes. (Fig. 31.) When the plates have cooled they are placed upon an apparatus designed to cool and so- lidify the liquid media, which is now poured upon the plates from the inocu- lated test-tubes. Nivellier Leveling and Cooling Apparatus. Ice and water are placed in a shallow round glass tray ; on top of this a square plate of glass, upon which the culture plate is placed, and cov- ering this a bell-glass. The whole is upon a low, wooden tripod, the feet of which can be raised or lowered, and a little spirit-level used to adjust it. (Fig. 32.) The glass plate taken out of the iron box is placed under the bell-glass. The tube containing the gelatine is held in the flame a second to singe the cotton plug to free it from dust, and the plug removed, the edges of the tube again flamed, the bell- glass lifted, and the inoculated gelatine carefully poured on the plate, leaving about one-third inch margin from the borders ; the Iron box for glass plates. Fig. 32. Nivellier leveling and cooling apparatus. lips of the tube being sterile can be used to spread the media INOCULATION OF GELATINE AND AGAR. 63 evenly. If the plate is at all cool, the fluid will solidify as it is being spread. The glass cover is replaced until the gelatine or agar is quite solid to prevent contamination. Fig. 33. Moist chamber with plates on benches. When the gelatine is congealed, the plate is placed upon a little glass bench or stand in the moist chamber. The Moist Chamber Prepared Out of Two Glass Dishes, as for the Potato- Cultures. • The glass benches are so arranged that one stands upon the other. In order to avoid confusion, a slip of paper with a number written on it is placed on the bench be- neath each plate. As the original or first plate would have the colonies developed in greatest profusion, it is placed the first day on the topmost bench ; but, since the colonies would be likely to overrun the plate and allow the gelatine to drop on the lower plates, it is best, as soon as evidences of growth appear, to place it below, and watch the third plate or second dilution for the characteristic colonies, forgetting not all this time to change the numbers accordingly. The date of culture and the name can be written upon the moist chamber. Petri Saucers. Agar hardens very quickly, even without any especial means for cooling, and it does not adhere very well to the glass. There- fore it is better to follow the method of Petri and use little shallow glass dishes, one covering the other. They are first sterilized by dry heat, and then the in- oculated gelatine or agar is poured into the lower dish, covered by the larger one, and placed in some cool place, different saucers being used for each dilution. Fig. 34. Petri saucers. 64 ESSENTIALS OF BACTERIOLOGY. This method is very useful for transportation ; the saucers can be viewed under microscope similar to the glass plates, and have almost entirely superseded them. Esmarch's Tubes, or Rolled Cultures. This method, especially used in the culture of anaerobic germs, consists in spreading the inoculated gelatine upon the inner walls of the test tube in which it is contained and allowing it to congeal. 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 gelatine around the cotton plug. The tubes can be placed directly under the microscope for further examination of the colonies. It is almost impossible to separate certain organisms, such as the tubercle bacillus and pneumococcus, from mixed cultures by ordinary plate methods, and the plan of producing the dis- ease in animals by inoculation, and then obtaining the organ- ism in pure culture, has to be employed. Spored organisms may also be separated from others by boil- ing the mixture for a few minutes, when all the non-spored forms will perish, and only the spores remain to germinate subsequently. CHAPTER XL THE GROWTH AND APPEARANCES OF COLONIES. Macroscopic. Depending greatly upon the temperature of the room, which should be about 65° C, the colonies develop so as to be visible to the naked eye in two to four days. Some require ten to fourteen days, and others grow rapidly, covering the third dilution in thirty-six hours. The plate should be looked at each day. The colonies present various appearances, from that of a small dot, like a fly-speck, to that resembling a small leaf. GROWTH AND APPEARANCE OF COLONIES. 65 Some are elevated, some depressed, and some, like cholera, cup- shaped — umbilicated. Fig. 35. Staphylococcus pyogenes aureus : colony two days old, seen upon an agar-agar plate; X 40 (Heim). Then they are variously pigmented. Some liquefy the gela- tine speedily, others not at all. The appearances of a few are so characteristic as to be recognized at a glance. Microscopic. We 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 culture plate 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 appearances 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 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 ; the pictures are very characteristic, and the majority of bacteria can be told thereby. (Fig. 32.) 66 ESSENTIALS OP BACTERIOLOGY. Impression or "Klatsch" Preparations. In order to more thoroughly 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 Microscopic appearances of colonies. Fig. 37. Klatsch preparations. stained or examined so. The Germans give the name oi "Klatsch" to such preparations. Many beautiful pictures can be so obtained. Fishing. To obtain and examine the individual members oi 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- 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 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 with- drawn, and the material thus obtained is further examined by staining and animal experimentation. The bacteria are then again cultivated by inoculating fresh gelatine, making stab and stroke cultures. CULTIVATION OF ANAEROBIC BACTERIA 67 It is necessary to transfer the bacteria to fresh gelatine about every six weeks, lest the products of growth and decay given off by the organisms destroy them. CHAPTER XII. CULTIVATION OF ANAEROBIC BACTERIA. Special methods are necessary for the culture of the anaerobic variety of bacteria in order to procure a space devoid of oxygen. Liborius's High Cultures. The tube is filled about | full with gelatine, which is then steamed in a water bath and allowed to cool to 40° C, when it is inocu- lated by means of a long platinum rod with small loop, the movement being a rotary vertical one, and the rod going to the bottom of the tube. The gelatine is next quickly solidified under ice ; very little air is present. The anaerobic germs will grow from the bottom upward, and any aerobins present will develop first on top, this method being one of isolation. From the anaerobic germs grown in the lower part, a stab culture is made into another tube containing f gelatine, the material being obtained by breaking test-tube with the culture. Hesse's Method. A stab culture having been made with anaerobic germs, gelatine in a semi-solid condi- tion is poured into the tube until it is full, thus dis- placing the air. (Fig. 39.) Esmarch's Method. Having inoculated a tube with Libo™s's the microbe, the gelatine is rolled out on the walls of the tube, a " roll culture," and the rest of the interior filled with gelatine, the tube being held in ice water. The colonies develop upon the sides of the tube and can be examined microscopically. Gases like Hydrogen to replace the Oxygen. Several ar- rangements for passing a stream of hydrogen through the culture: — 68 ESSENTIALS OP BACTERIOLOGY. Frankel puts in the test tube, a rubber cork containing two glass tubes, one reaching to the bottom and connected with a hydrogen apparatus, the other very short, both bent at right angles. When the hydrogen has passed through ten to thirty minutes, the short tube is annealed and then the one in connec- tion with the hydrogen bottle, and the gelatine rolled out upon the walls of the tube. (Fig. 40.) Hiippe uses eggs as described in Chapter IX. Fig. 39. Fig. 40. Fig. 41. Hesse's method. Frankel's method. Buchner's method. Use of iErobic Bacteria to remove the Oxygen. Roux inocu- lates an agar tube through a needle thrust after which semi- solid gelatine is poured in on top. When the gelatine has solidi- fied, 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. CULTIVATION OF ANAEROBIC BACTERIA 69 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 rubber stopper. (Fig. 41.) Botkin's Method. Petri dishes, un- covered, are placed on a rack under a large bell-jar, into which hydrogen gas is conducted. Alkaline pyrogallic acid is placed in the upper and lower dishes to absorb what oxygen remains. Wright's Method. Applicable to both fluid and solid media. After in- oculating the test-tube, the plug, which must be of absorbent cotton, is cut off flush with the extremity of the tube and pushed inward for a distance of 1 cm. It is then impregnated with 1 c.c. of a watery solution of pyrogallic acid and 1 c.c. of 5 per cent, sodium hydrate solution. A tightly fitting rubber stop- per is inserted, and the tube is then ready for incubation. 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 paraffme is then poured into the flask, which forms a layer over the medium and on congealing pro- vides an air-tight seal which does not adhere to the glass so closely as to pre- vent the escape of any gases formed by the bacterial growth. Fin. 42.— Wright's method for the cultivation of anae- robes. 70 ESSENTIALS OF BACTERIOLOGY. CHAPTER XIII. INFECTION. How Bacteria Cause Disease. Many theories have been put forward to explain the action of bacteria in causing dis- ease, but only a few of the more important ones can be taken up here. What are the Conditions Necessary to Produce Infection? First. As to the Infective Agent. The organism must have the power to produce disease. It must, in other words, be pathogenic. A non-pathogenic bacterium under certain conditions may cause disease, but this is not an infectious disease ; it is rather a tox- emia, and is due to the absorption of poisons generated outside of the body. It must be parasitic — have the power of growing within the body of an animal. Essentially an infectious disease is a toxemia, because it depends upon poisons or toxins produced in the body. Para- sitic or infectious bacteria cause disease by growing in the animal organism and generating products therein which are toxic. Saprophytic bacteria grow outside of the animal organism in dead matter, decaying particles, etc., and they may give rise to prod- ucts which also are toxic to the animal economy. Second. The toxins or poisons elaborated must be present in sufficient amount. Undoubtedly each animal organism is a law unto itself in regard to the amount of poison it will tolerate before disease is actually produced. The period of incubation can be explained on the supposition that the germ requires so much time to elaborate the amount of toxin necessary. This time period varies with different organisms, some carrying the toxin with them at the time of entry. Third. The animal infected must be susceptible. 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 the different forms of disease germs. Susceptibility may be natural to the race, it may be acquired, it may be inherited. Mice are naturally susceptible to anthrax. Acquired susceptibility occurs upon exposure to conditions INFECTION. 71 which lower vitality, as hunger, cold, advanced age, and sur- gical shock. Inherited susceptibility is a less important factor now than formerly. Many diseases were at one time considered inherited which now are known to be acquired during the life- time, of an individual. Still, certain physical characteristics, such as narrow chest, mouth-breathing, etc. — clearly inheritable characters — predispose to disease. Given a susceptible individ- ual and an infective microorganism producing toxins in suffi- cient amount, disease is certain to result. Local Effects of Bacteria. By mechanical obstruction from rapid growth, thrombosis, with its consequences, may occur. Destruction of a part of the cells of a tissue with necrosis can arise from irritation, as from a foreign body. General Effects. Sapremia, when toxic products of local suppuration are absorbed into the system. Septicemia, when the infective agent itself enters the blood-stream and causes general disturbance. Suppurative bacteria are those which give rise to inflamma- tion and suppuration locally at the point of entrance, and secondarily through metastasis. Any organism may cause suppuration, but a certain number are peculiarly inclined to give rise to pus, and are known as pyogenic organisms. Infective bacteria are as a rule specific, the particular toxin having a specific action and causing a disease peculiar to the microorganism. 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 ser- pents; highly poisonous in minute doses (y^ gramme of tetanus toxin will kill a horse weighing 600 kilos (1200 pounds,). At first toxins were called ptomaines, or cadaveric alkaloids ; but this term is applied now to such poisons as have a basic nature and arise in decomposing meat, cheese, and cream as a result of chemical change in the material, the bacteria causing the change. Then they were called toxalbumins, 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 72 ESSENTIALS OF BACTERIOLOGY. 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. Toxins may be of two sorts : (a) Chiefly within the bodies of the bacteria, so that they are set free by the disintegration of the organisms. This group comprises most of the pathogenic bacteria and must be combatted by the use of antibacterial sera. (b) The poisons seem to be excreted by the bacteria and are found in the surrounding medium. Antitoxic sera are applica- ble to this group, which includes the bacilli of diphtheria and tetanus. Welch has suggested that even bacteria which do not appear to form toxins in artificial cultures may do so in the human body. In the effort to adapt themselves to their environment and resist the hostile agencies of the body they produce the poisons we call toxins. (For method of produc- tion of an antitoxin, see article on Diphtheria.) CHAPTER XIV. IMMUNITY. Immunity, as distinguished from susceptibility, is merely a rela- tive term, as no animal is absolutely immune under all condi- tions. It is merely less susceptible, and some animals are by nature or can by artificial means be rendered so slightly sus- ceptible that to all practical purposes they are immune— that is, capable of resisting an attack of the particular disease against which they are said to be immune. Natural Immunity. The goat and dog are considered naturally immune to tuberculosis. Algerian sheep are resistant to anthrax, other varieties are susceptible. The field mouse is susceptible to glanders, the white mouse is ordinarily immune. House mice are susceptible to mouse septicemia, field mice are immune. Acquired Immunity. Immunity can be acquired in many ways. Active and passive immunity are varieties. Active immunity can be acquired from an attack of the disease; IMMUNITY. 73 such infectious diseases as measles, scarlatina, and whooping- cough usually confer immunity from future attacks. Some diseases render the individual immune for only a short period. Immunity from Inoculation with Attenuated or Weak- ened Cultures of Bacteria. Vaccination is an example. Haff- kine's cholera vaccines and Pasteur's vaccines of anthrax and chicken cholera are likewise examples of this method. Attenuation is produced as follows : Successive cultivation in artificial media destroys the virulence of bacteria. Old cultures are less virulent than fresh ones. Virulence is lessened by passing the cultures through animals that are less susceptible or entirely immune. The cautious use of chemicals and sun- light lessens virulence. Heat is an effective agent. An anthrax- culture exposed to a temperature of 42.6° C. for twenty days will prove destructive only to animals no larger than mice. Prolonged exposure to oxygen weakens the germs. Immunity Through Inoculations of Small Doses of very Virulent Microorganisms. A graduated resistance to the disease is reached somewhat after nature's method. By succes- sive inoculations with increased doses of the virus an immunity is often reached sufficient to withstand ten times the lethal dose. A poison-habit is thus acquired. Increased Virulence is produced as follows : The cultures may be greatly increased in virulence by successive cultivation through animals, and gradually changing from smaller animals to larger, until an amount of the culture that at the outset would not destroy a guinea-pig becomes finally virulent for chickens and dogs. Immunity Through Injections of the Sterilized Products of Bacteria. Cultures sterilized by heat or nitration through germ-filters still contain the chemical products of bacteria, the toxins ; and when these are injected in gradually increased doses the same immunity is obtained as with the bacteria themselves. Passive Immunity. The blood-serum and tissues generally of animals rendered immune in the" ways described above, when injected into susceptible animals render them immune against the same infection. This has been called passive immunity, but there is no strong reason why this term should be used. The 74 ESSENTIALS OP BACTERIOLOGY. blood-serum of immune animals is simply another means for immunization. It is less permanent than the other forms of immunization, but it appears very soon after the injection, and in a modified form has a curative action even when the symp- toms of the infection are already present in the system. Inherited Immunity. An immunity to disease acquired dur- ing the lifetime of the parents is probably never transmitted to the offspring, though the mother may transmit a temporary immunity to the child in utero or the child itself may have been subjected to the infection at the same time with its mother. But this cannot be called inherited. Theories of Immunity. Several older theories need only to be mentioned, as they are no longer tenable. They are the exhaustion theory of Pasteur, the retention theory, and the humeral theory. At present modifications of MetschnikofT's phagocytic theory and Ehrlich's side-chain theory seem the most plausible. Phagocytic or Cellular Theory. — Metschnikoff elaborated this after his study on inflammation. Phagocytosis occurs in animals when subjected to the action of an irritant. The leu- cocytes are attracted to the injured spot and envelop the irri- tating substance, be it bacteria or dead matter. The theory given out at first was that if the leucocytes conquer the bacteria, immunity results; if the bacteria eat up the leucocytes, disease occurs. Modified to suit other conditions, as, for instance, the germi- cidal properties of serum freed from its cellular elements, Metschnikoffnow states that at times phagolysis — that is, breaking up or solution of the phagocytes — takes place, and the fluids in which these cells are dissolved become charged with the powers originally present in the phagocytes. Chemotaxis is the term applied to the attraction of bacteria for the leucocytes, and is supposed to be chemical in its nature. The phagocytic cells comprise : (o) The polymorphonuclear leucocytes of the blood, termed microphages, and (b) a group called macrophages which includes all other cells having phagocytic properties, such as leucocytes other than the polymorphonuclears, endothelial cells, and connective-tissue corpuscles. When these cells are IMMUNITY. 75 injured they set free their digestive ferments, known as micro- cytases and macrocytases respectively, which correspond to the alexins of Ehrlich. EhrllcWs Side-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 hydro- gen are substituted by "side chains" of more or less complex nature. The molecule of protoplasm is supposed 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 different kinds so as to fit them for combination with many different varieties of extrane- ous groups. Bacterial toxins contain two groups : (1) the haptophores, by which the toxin molecule can become joined to the cell, and (2) the toxophores, by virtue of which it can attack the protoplasm after having been fixed to it by the hapto- phore. If the attack on the molecule is not too severe, this is stimulated into overactivity and throws out an abnormal num- ber of receptors, some of which (the haptins) become detached and are capable of uniting with free haptophores and prevent- ing their combination with the protoplasm of the molecule. In other words, they represent the antitoxin. Bacteriolysis is the destruction of the bacterial cells by the blood-serum, and is probably effected in a somewhat different manner. Antibacterial sera are effective through the combined activities of a destructive element, the "complement" (alexin or cytase), and an "immune body" (amboceptor) which serves the function of joining the complement to the bacterial mole- cule. These two bodies differ markedly in their properties — for example, the complement is destroyed at 60° C, while the immune body is very resistant. It is not stated what cells are the sources of these various anti-bodies, but probably any cell capable of being attacked by a toxin is also capable of responding by the production of anti- substances. Lysins. The substances producing destruction of bacteria are called lysins. Normal blood-serum is bacteriolytic to a 76 ESSENTIALS OF BACTERIOLOGY. slight degree, but during infection produces lysins specific for the germ in question. Agglutinins. These are bodies formed in the blood-serum in response to the stimulation of certain bacteria, such as the typhoid bacillus, Bacillus coli communis, Micrococcus meli- tensis, the bacillus of dysentery, the cholera spirillum, etc. When such a serum is added to cultures of the particular organism concerned, the bacteria become clumped in motion- less masses. A modified form of agglutination in which long strings of bacteria are formed is known as the " thread " reac- tion. Precipitins. Animals immunized to certain bacteria or to albumins of different sorts form bodies which cause the blood- serum to give a precipitate when added to cultures of these organisms or fluids containing the specific albumen. The phenomenon has found forensic application in the identifica- tion of blood-stains. CHAPTER XV. EXPERIMENTS UPON ANIMALS. The smaller rodents and birds are the ones usually employed for inoculation, as rabbits, Guinea-pigs, rats aud mice, and pigeons, and chickens ; sometimes monkeys. These are pre- ferred, because easily acted upon by the various bacteria, readily obtained, and not expensive. The white mouse is very prolific and easily kept, and is there- fore 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 EXPERIMENTS UPON ANIMALS. 77 some large stall or inclosure. They can be fed upon all sorts of vegetables and grasses, and require but little attention. Methods of Inoculation. L Inha lation.— Imitating the natural infection, either by loading an atmosphere with the germs in question or by administering them with a spray. II. Tlirougk Skin or Mucous Membrane. III. With the Food. Method of Cutaneous Inoculation. The ear of mice is best suited for this procedure. A small abrasion made with the point of a lancet or needle, which has been dipped in the virus. The animal is then separated 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 mice 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 absolutely necessary, avoiding much blood. The 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. Intravenous Injections. Rabbits are very easily injected through the veins. Mice are too small. The ear of the rabbit is usually taken. It is first washed with 1-2000 bichloride, 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 syringe, like the one used for the injection of " tuberculine," a Koch syringe, which can be easily sterilized, is filled with the desired amount of virus and slowly injected into any one of the more prominent veins present. (Fig. 43.) Intra-peritoneal Injection. This is used with Guinea-pigs mostly. 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. 78 ESSENTIALS OF BACTERIOLOGY. 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 assistant. A small cut is made in the cornea, a few drops of cocaine having first been introduced in the eye. 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. 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 Fig. 43. Manner of making intravenous injections in the rabbit. aponeurosis cut through where the skull is the thinnest. Then the bone carefully trephined, and the dura exposed. In Babies inoculation, the syringe containing the hydrophobic virus pierces the dura and arachnoid, and the virus is discharged beneath the latter. Intra-Tracheal. The bacteria can be introduced directly into the trachea, thus coming in contact with the lungs. Intra-duodenal. — Cholera germs are injected into the intes- EXPERIMENTS UPON ANIMALS. 79 tines 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 introduce living cultures of bacteria into the bodies of animals without their coming into direct contact with the tissues. Obtaining Material from Infected Animals. The animal 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 awa}r from the belly without exposing the intestines. Then the ribs being laid bare, the sternum is lifted up, and the pericardium exposed. A pla- tinum 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 were first to be looked at, they would be laid bare first. In this manner material is obtained, and the results of inocu- lation 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 artificial 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. PART II. SPECIAL BACTERIOLOGY. CHAPTER I. NON-PATHOGENIC BACTERIA. Special Bacteriology. Under this head the chief character- istics of individual bacteria will be detailed, pathogenic and non- pathogenic being the main divisions. It is usual to describe the non-pathogenic first. Non-Pathogenic Bacteria. There are 300 varieties of non- pathogenic bacteria, and the list is continually being added to. Bacillus Prodigiosus. (Ehrenberg.) This bacillus, formerly called a micrococcus, is very common, and one of the first noticed, because of the lively red color it forms on vegetables and starchy substances. "The bleeding host," miracles being due to it. Form. — Short rods, often in filaments, without spores. Immobile. — Has no automatic movements. Facultative anaerobic, that is, it can grow without air ; but the pigment requires oxygen to show itself. Growth. Gelatine. Liquefy rapidly. Colonies. — At first white, round points with smooth edge appearing brown under microscope, but soon changing to red. Stab Cultures. — The pigment develops on the surface, the growth occurring all along the line. Potato is well suited to the growth, the pigment developing after twelve hours. Agar and blood serum growths do well. Temperature.— Grows best at 25° C. Varieties. — By exposure to heat of brood-oven during several generations the power to produce pigment can be temporarily abolished. (80) NON-PATHOGENIC BACTERIA 81 Auto- red e Pigment. — A pigment-forming body is created by the bacillus, and the action of oxygen upon it produces the color. It is insoluble in water, slightly soluble in alcohol and ether ; acids fade it, alkalies restore the color. The pigment resembles fuchsin, presenting the same metallic lustre. Gases. — A trimethylamin odor arises from all cultures. Stain. — Takes all anilin dyes easily in the ordinary way. Bacillus Indicus. (Koch.) Syn. Micrococcus Indicus. Origin. — Found in the stomach of an Indian ape. Form. — Short rods with rounded ends. No spores, matic movements present ; faadtative ancerobin. Growth. Gelatine. — Liquefy rapidly. Colonies.— Round, or oval, granular margins ; brilliant pigment. Stab Cultures. — On the surface the pigment shows itself. Grows well on other media. Temperature.— Grows best at 35° C. Action on Animals. — In very large quantities, if injected into the blood, a severe and fatal gastro- enteritis can be produced. Stain.— Takes all dyes. Bacillus Mesentericus Vulgatus. The common potato bacillus of Fliigge. Habitat. — Surface of the soil, on potatoes, and in milk. Form. — Small thick rods with rounded ends, often in pairs. Properties. — Yery motile ; pro- duce abundant spores ; liquefy Fig. 44. gelatine ; diastolic action. Colony of Bacillus Mesentericus Vulgatus. Growth.— Rapid. Plate Colonies.— Round, with transparent centre at first, then becoming opaque. The border is ciliated ; little projections 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 temperatures. 6 82 ESSENTIALS OF BACTERIOLOGY. Spores. — Are very resistant ; are colored in the manner de- scribed in first part of the book for spores in general. Bacillus Megaterium (de Bary). Origin.— Found on cooked cabbage and garden soil. Form. — Large rods, four times as long as they are broad, 2.5 fi. Thick rounded ends. Chains with ten or more members often formed ; granular cell contents. Properties. — Abundant spore formation ; very slow movement ; slowly dissolves gelatine. Growth.— Strongly aerobic ; grows quickly, and best, at a tem- perature of 20° C. Plate Colonies.— Small, round, yellow points in the depth of the gelatine. Under microscope irregular masses. Fig. 45. Bacillus Megaterium, with spores. Stab Culture. — Funnel-shaped from above downwards. Potato. — Thick growth with abundance of spores. Bacillus Ramosus. Syn. Bac. 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,, NON-PATHOGENIC BACTERIA. 83 Properties. — Large, shining, oval spores ; a slight movement ; liquefy gelatine. Growth.— At ordinary temperatures, with plentiful supply of air. Plate Colonies. — Look like roots of an old tree gnarled together, radiating from a common centre. On surface soon liquid. Stab Culture.— Soon sl growth occurs along the needle track, and the whole resembles a pine tree turned upside down. The gelatine then becomes liquid, a thin skin floating on top, and small flakes lying at the bottom. Stroke Culture. — Feathery resemblance is produced. Staining. — Spores stain readily with the ordinary spore stain. Bacterium Zopfii. (Kurth.) 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 gela- tine. Growth. — In thirty hours abundant growth; cerobic; grows best at 20° C. Plates. — Small white points which form the centre 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. Bacillus Subtilis. (Hay Bacillus.) Ehrenberg. Origin.— Hay infusions ; found also in air, water, soil, faeces, and putrefying liquids. Very common, often contaminates cultures. Form. — Large rods, three times as long as broad ; slight roundness of ends, transparent; seldom found singly; usually in long threads. Flagella are found on the ends. Spores of oval shape, strongly shining, very resistant. Properties.— Yery motile ; dissolves gelatine. Growth. — Rapid ; strongly eerobic. Plate. — Round, gray colonies, with depressed white centre. 84 ESSENTIALS OF BACTERIOLOGY. Under microscope the centre yellow ; the periphery like a wreath, with tiny little rays projecting ; very characteristic. Potato. — A thick moist skin forms in twenty-four hours. Staining.— Rods, ordinary stain, spores, spore stain. It is easily obtained by covering finely cut hay with distilled water, and boiling a quarter of an hour. Set aside forty-eight hours. A thick scum will show itself on the surface composed of the subtilis bacilli, whose spores alone have survived the heat. Bacillus Spinosus. (Liideritz.) Called spinosus because small spine-like processes are formed by the colonies. Origin. — In the juices of the body of a mouse and guinea-pig which were inoculated with garden earth. Form.— Large rods, straight, some slightly bent, ends rounded ; often in long threads. Properties. — Large spores, the bacillus enlarging to allow the spores to develop ; very motile ; gelatine slowly liquefied. A gas is formed in the culture having an odor like Swiss cheese. Growth. — The growth occurs at ordinary temperatures only when the oxygen is excluded. Very strongly anaerobic. Glu- cose added to the gelatine (1 to 2 per cent.) increases the nutri- tive value. Colonies in roll cultures and high stab cultures appear as little spheres surrounded by a zone of liquefied gelatine. In the deeper growths thorn-like projections or spines develop pro- ceeding from a gray-colored centre. Staining.— With ordinary methods. This bacillus, being strongly anaerobic, must be cultured with the usual care taken with anaerobins. Some Bacteria found in Milk. Bacillus Acidi Lactici. (Huppe.) Belongs to the same group as the Bacillus coli com- munis. Origin. — In sour milk. Form.— Short thick rods, nearly as broad as they are long, usually in pairs. Properties.— Immotile. Spores large shining ones. Do not liquefy gelatine. Breaks up the sugar of milk into lactic acid and carbonic acid gas, the casein being thereby precipitated. Growth.Siow ; is facultative anaerobic. Grows first at 10° C. NON-PATHOGENIC BACTERIA. 85 Plate Colonies.— First small white points, which soon look like porcelain, glistening. Under microscope the surface colonies resemble leaves spread out. Stab Culture. — A thick dry crust with cracks in it forms on the surface after a couple of weeks. Attenuation. — If cultured through successive generations, they lose the power to produce fermentation. Several other bacteria will give rise to lactic acid fermentation ; but this especial one is almost constantly found, and is very wide spread. In milk, it first produces acidity, then precipitation of casein, and finally, formation of gases. A bacillus described by Grotenfeldt, and called Bacterium Acidi Lactici, forms alcohol in the milk. It was found in milk in Bavaria. Bacillus Butyricus. (Hiippe.) This bacillus causes butyric acid fermentation. Origin. — Found in milk. Form.— Short and long thin rods wfth rounded ends; large oval spores, seldom forming threads. Properties. — Very motile ; liquefies gelatine rapidly ; produces gases resembling butyric acid in odor. In milk it coagulates the casein, decomposes it, forming peptones and ammonia, with a bitter taste, and butyric acid fermentation. An alkaline reaction. Growth. — Quickly, at 35° to 40° C, with oxygen. Spons very resistant. Colonies. Plate. — Small yellow points which soon run together, becoming indistinguishable. Stab Culture.— A small yellow skin formed on the surface with delicate wrinkles ; cloudy masses in the liquefied portion. Staining. — With ordinary stains. Bacillus Amylobacter (Van Tiegham) ; or, Clostridium Buty- ricum. (Prazmowsky.) (Vibrion butyrique of Pasteur.) Origin. — Found in putrefying plant-infusions, in fossils, and conifera of the coal period. Form. — Large, thick rods, with rounded ends, often found in chains. A large glancing spore at one end, the bacillus becoming spindle-shape in order to allow the spore to grow ; hence the name Clostridium. 86 ESSENTIALS OF BACTERIOLOGY. Bacillus Amylobacter. Fig. 46. Properties. — Yery motile ; gases arise with butyric smell. In solutions of sugars, lactates and cellulose-containing plants, and vegetables, it gives rise to decomposi- tions in which butyric acid is often formed. Casein is also dissolved. Like granulose, a watery solution of iodine will color blue some portions of the bacillus ; therefore it has been called amy- lobacter. Growth. — It is strongly anaerobic, and fj\ 1 a # has not yet been satisfactorily cultivated. Bacillus Lactis Cyanogenus. Bacterium Syncyanum. (Hiippe.) Origin.— Found in blue milk. 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 gelatine ; form spores usually in one end. A bluish-gray pigment is formed outside of the cell, around the medium. The less alkaline the media the deeper the color. It does not act upon the milk other- wise than to color it blue. Growth. — Grows rapidly, requiring oxygen. Colonies on plate. Depressed centre 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 gelatine 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. Bacillus Lactis Erythrogenes. Bacillus of Bed Milk. (Hiippe and Grotenfeldt.) Origin. — Found in red milk, and in the faeces of a child. Form. — Short rods, often in long filaments, without spores. Properties. — Does not possess self-movement. Forms a nause- ating odor ; liquefies gelatine. Produces a yellow pigment which can be seen in the dark, and a red pigment in alkaline media, NON-PATHOGENIC BACTERIA. 87 away from the light. In milk it produces the yellow cream on top of the blood-red serum, or, fluid in the centre, and at the bottom the precipitated casein. Growth. — Grows rapidly in bouillon and on potatoes ; slower on the other media; Plates. A cup-like depression in the centre of the colony, with a pink coloration around it, the colony itself being slightly yellow. Stab Culture. — The growth mostly on surface. The gelatine afterwards colored red and liquefied. Potato. — A golden yellow pigment formed at 37° C, after six days. Examination of Milk in Stained Specimen. A drop of milk diluted with a drop of distilled water is dried on the cover-glass and fixed by heat. Chloroform methyl blue, prepared by mix- ing 12 to 15 drops of saturated alcoholic solution of methyl blue with 3 or 4 c.c. of chloroform, is used for staining. The chlo- roform is then evaporated by exposing the specimen for a few minutes to the air. Bacteria blue ; rest of field unstained. Another method is to mix a drop of milk with two or three drops of a 1 per cent, solution of sodium carbonate on a cover- glass. Saponification of the fat occurs on heating the mixture to evaporation. The preparation is then stained in the ordinary manner. Some Non-Pathogenic Bacteria found in Water. The bacteria found here are very often given to producing pigments or phos- phorescence, and are in great number. The more common ones only will be described. Bacillus Violaceus. Origin. — Water. Form.— A slender rod with rounded ends, three times as long as it is broad, often in threads ; middle-sized spores. Properties. — Very motile ; forms a violet-blue pigment, which is soluble in alcohol, and depends upon oxygen for its growth. Rapidly liquefies gelatine, but not agar. Growth.— Grows fairly quick, is facultative anaerobic. Cultures on Plate.— At first the colonies look like inclosed air- bubbles. Low power shows irregular masses, with a centre containing the pigment and a hairy-like periphery. growth —Terr fine little rods ; no —Motile 5 fbnns gelatine. Rapid onhrafc ordinary the periphery the needle thrust ; the MOX-PATHOOEfrlC BACTERIA, $$ Plate*.— Bound colonies, cup-shaped depressions, the solid geb:ine that remains becoming colored witb greenish-yellow Stab Culture.— ()n the surface, air-bobble depressions; toe white colonies in the bottom of these depressions, and the solid gelatine aroond the inoculation shining with the fluorescence. Phosphorescent Bacteria, Six varieties of phosphorescent bacteria bare been described ; they are found usually in sea* water, or upon objects firing in the sea, Bacillus Photphoreseens Indieus. (Fischer,) Origin.— Tropical waters. Form.— Thick rods, with rounded ends, sometimes forming bag Uueada Properties.— Very motile ; liquefying gelatine at a tempera* tare of 2KP to Sf/"- C, with oxygen and a little moisture, and in the dark, a peculiar electric-bine light develops a phosphores- cence. Growth.— Slowly ; must bare oxygen ; does not grow under 1CPC, ororerSCPC, Plates,— Little round, gray points, which under low power appear as green colonies with reddish tinge around them. Cooked fish, when smeared upon the surface with a little of the culture, show the phosphorescence most marked. Grows well on potatoes aid Wood-serum. Bacmusl^jsphcresceiis Indigents (Fischer,) Origin.— Waters in the northern part of Germany, It differs from the Indian bacillus, in that it grows at a temperature of IP C, and does not develop upon potatoes or blood-serum, BaciUiuPbospbcmseeiuGetidiiA (Fdrster,) Origin — Surfaces of salt-water fish. Form.— Short, thick rods, looking oral sometimes ; zoogkea arc often formed. Properties.— Motile ; does not liquefy gelatine ; a beautiful phosphorescence from the surface of fish; it can be photographed by its own light Colonies.— Grows best between (P and 2

2~ * * » ^ Tubercle bacilli in sputum, carbol-fuchsin, and methylin blue. Zeiss 1:12 oil immersion. the size of a red corpuscle's diameter, their ends rounded, usu- ally solitary, often, however, lying in pairs in such a manner as to form an acute angle. Sometimes they are S -shaped. In colored preparations little oval spaces are seen in the rod, which resemble spores ; but the question of the existence of spores is still undecided. Properties. — Does not possess self-movement. Growth.— Requires special media for its growth, and a temper- ature varying but slightly from 37.5° C. It grows slowly, de- veloping first after ten days, reaching its maximum in three weeks. It is facultative anaerobic. On gelatine it does not form a growth. PATHOGENIC BACTERIA. 99 Colonies on Blood Serum. — Koch first used blood serum for culture ground, and obtained thereon very good growths. Test- tubes with stroke culture were placed in the brood oven at 37° C. for ten to fourteen days, when small glistening white points ap- peared which then coalesced to form a dry, white, scale-like growth. Under microscope composed of many fine lines con- taining the tubercle bacillus. Glycerine Agar.— By adding four to six per cent, glycerine to ordinary agar-peptone medium, Nocard and Koux obtained a- culture ground upon which tubercle bacilli grew much better than upon blood serum. This is now almost exclusively used. Stroke cultures are here used as with blood serum. They are placed in brood-oven after inoculation, and remain there about ten days, at a temperature of 37° C. p10> 52 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 dry- ing up. The growth Which OCCUrs resembles Tubercle bacilli in human the rusas of the stomach, and some- liver 500 X- (Frankei and times looks like crumbs of bread moist- ened. The impression or"Klatsch" preparation shows under the microscope a thick curled-up centre 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. Bouillon. — Bouillon containing four per cent, glycerine is a verv good nurture ground. Varieties. — Branching and other aberrant forms are not rare, and the tendency now is to class the organism with the " higher bacteria." Other acid-fast bacilli exhibit similar types and it is 100 ESSENTIALS OF BACTERIOLOGY. possible 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 enclosed them, are frequently found in sputum. Fig. 53. Klatsch preparation. Bovine tubercle-bacilli are about one-third smaller than human tubercle bacilli. 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. Upon these facts all the methods are founded. PATHOGENIC BACTERIA. 101 It will only be necessary to describe 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 it is pressed and spread out evenly ; drawing one glass over the other, we obtain two specimens, and these put aside or held high over the flame until dry. If we desire to examine the specimen quickly, or make a hurried diagnosis, we use the rapid method, with hot solutions ; Fig. 54. Growth on Agar. otherwise we let it stay, in cold solution until the next mornin<* the advantages of which will be later on described. Tlie Eapid Method.— (B. Frankel's 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 carbol-fuchsin ; the other Gabbet's acid methylin blue. (See No. X., on page 34. ) 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 carbol-fuchsin solution five minutes (cold), or two minutes in the hot, immediately then transferred to the 102 ESSENTIALS OP BACTERIOLOGY. second solution, the acid blue, where it remains one minute, then washing in water. The preparation is dried between filter-paper, and mounted best first in water. Examined with oil-immersion. A somewhat longer, but preferable, method is to decolorize the carbol-fuchsin with weaker acid. The smear is treated with 5 per cent, nitric or 10 per cent, sulphuric acid until, after washing with water, a bright pink remains. The excess of color is then washed out with 95 per cent, alcohol until no further color is imparted to the alcohol and the smear is a pinkish gray. The preparation is then washed writh water and counterstained with aqueous methylin-blue for ten to thirty seconds. A mechanical stage is of great assistance in the search for the bacilli, as it permits every portion of the prepa- ration to be inspected systematically. In urine, owing to the almost inevitable 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 de- colorized, or Pappenheim's method may be used : (1) Smear and fix as usual ; (2) stain with hot carbol-fuchsin for two minutes, pour off the surplus dye without washing ; (3) counter- stain and decolorize by pouring five times over the preparation the following solution: A 1 per cent, alcoholic solution of corallin is saturated with methylin-blue and 20 parts of gly- cerine added. Wash in water, dry with blotting-paper, then in the air, and examine. The tubercle bacilli are stained red, smegma bacilli, blue. The bacillus of leprosy resembles the tubercle bacillus in its staining properties, but gives up the carbol-fuchsin more easily and is usually decolorized by the acid and alcohol. It is colored blue by Pappenheim'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. Slow Method. — The stain may also be used without 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 PATHOGENIC BACTERIA. 103 dish or beaker full of carbol-fuchsin for eight -to ten hours, and then decolorized and counterstained in the usual way. The method is less liable to produce artefacts than the quick method, but is not much used on account of the time it takes. BiederVs Method of Collecting Bacilli, when the bacilli are very few in a great quantity of fluid, as urine, pus, abundant mucus, etc., Biedert advises to mix 15 c.cm. of the fluid with 75 to 100 c.cm. water and a few drops of potassium or sodium hydrate, then boiling until the solution is quite thin. It is placed in a conical glass for two days, and bacilli with other morphological 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. The centrifugal machine is used either in connection with Biedert's sediment method or without, to obtain the solids sus- pended in urine or serum. When the bacilli are so few in number in sputum or urine as to make their detection difficult, and also when doubt exists as to the identity of acid-fast bacilli found, several guinea-pigs should be injected in the groin and smears and sections made from the enlarged glands resulting. Carbolic Acid to Sediment Sputum. — Pure carbolic acid added to sputum (about 1 part of the acid to 6 parts of sputum) will in a few hours produce a coagulation and allow the sputum to be spread evenly on the cover-glass, showing greater collections of bacilli. Without cover-glass. — Sputum can be spread and stained on the glass slide without the use of a cover-glass, the oil of cedar being placed directly on the stained sputum, and the oil immer- sion lens dipping into it. It is a rapid and cheap way ; and when a given case is to be studied daily the method is useful. Pure Cultures from Sputum. — Kitasato recommends the tho- rough washing, changing the water ten times, of the small masses found in the sputum of tubercular persons. When such speci- mens are examined they show tubercle bacilli alone, and when inoculated in agar give rise to pure cultures. Staining Bacillus Tuberculosis in Tissue (sections). — The general 104 ESSENTIALS OF BACTERIOLOGY. method of Gram can be used, but the better way is to use the following : — Carbol-fuchsin, 15 to 30 minutes. 5 per cent, sulphuric acid, 1 minute. Alcohol, until a light-red tinge appears. Weak methylin blue, 3 to 5 minutes. Alcohol, for a few seconds. Oil of cloves, until cleared. Canada balsam, to mount in. Instead of carbol-fuchsin, alcoholic solution qffuchsin or aniline water fuchsin can be used, but the sections must remain in the stain over night. Hardened sputum and sectioning. — Sputum can be hardened l»v placing it in 98 per cent, alcohol. Thin sections can be obtained by imbedding the hardened sputum in collodion. 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. The resisting action of the bacillus to acids is supposed to be due to a peculiar arrangement of the albumen and cellulose of the cell rather than to any particular capsule around it. Pathogenesis. — When a guinea-pig has injected into its peri- toneal cavity some of the diluted sputum containing tubercle bacilli it perishes in about three weeks, and the following picture presents itself at the autopsy : at the point of inoculation a local tuberculosis shows itself, little tubercular nodules contain- ing 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 become necrosed in the centre and degeneration occurs, the periphery still containing active bacilli, cavities having formed in the centre. Since the bacilli die in course of time, killed by their own pro- ducts, their number forms no correct guide of the damage present. Even their absence in the sputum does not preclude the ab- sence of a tubercular process. It is their presence only that PATHOGENIC BACTERIA. 105 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 tubercular process through rupture or otherwise. They have been found in other secretions, milk, urine, etc. Man is infected as follows : — Through wounds. —Local tuberculosis. Through nutrition.— Milk and meat of tuberculous animals. Phthisical patients swallowing their own sputum and causing an intestinal tuberculosis. Inhalation. — This is the most usual way, probably constitu- ting the cause in ^ of the cases, except in children. 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 containing water, this great danger can be avoided. Nearly all the cases of heredity can be explained in this man- ner; the young children, possessing very little resistance, are constantly exposed to the infection through inhalation and are especially prone 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 would offer less resistance than healthy individuals. Tuberculosis in animals. Tuberculosis is probably the most widely disseminated 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 infection of human beings from cattle occurs so seldom that no general regulations to restrict it are necessary, has found few adherents. 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 106 ESSENTIALS OF BACTERIOLOGY. slightly susceptible to the human bacillus, but it is not likely that the converse is so. Children are particularly liable to in- fection through the gastrointestinal tract, and it has been shown that the uninjured mucosa of the infant's intestine is permeable to bacillus, 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 charac- teristics regarding growth and virulence. The bacilli causing tuberculosis in the cold-blooded animals have departed farthest from the human type, those of birds to a less degree, and those of cattle least of all. Products of Tubercle Bacilli. The true nature of the tuber- cle toxin is not yet clear. It is not unlikely that several toxic bodies differing from one another in their properties are pro- duced. Koch's tuberculin (1890) was obtained by filtering, through unglazed porcelain, concentrated glycerine 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 tubercular, the temperature rises 2° or 3° F. in eight to twelve hours, and remains elevated for a like period of time. In man the use of tuberculin as a diagnostic measure is falling into dis- favor, as it is both dangerous and unreliable. Tuber culocidin. — This is an albuminoid obtained from the original tuberculin by precipitation with alcohol. Klebs used it as a cure for tuberculosis. Tuberculin R. is an extract made from dried and powdered living bacilli, and was recommended by Koch in place of the original tuberculin, but it has likewise proved useless. Agglutination. Arloing and Courmont have described an agglutination reaction for the tubercle bacillus similar to the Widal reaction of typhoid fever. It is very unreliable, how- ever, and but little importance is attached to it. Antituberculous Serum. The attempts to produce an effec- tive serum have so far been unsuccessful. Marmorek, by grow- PATHOGENIC BACTERIA. 107 ing the bacillus on a special serum obtained by injecting calves with the leucocytes 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 doubt- ful. Examination of Milk for Tubercle Bacilli. Place a drop of the sample on a cover-glass and mix it with 2 drops of a 1 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 ver}r few persons have succeeded in discovering the bacillus in milk. Lepra Bacillus. (Hansen.) Origin. — In 1880 Armauer Hansen declared, as the result ot many years' investigation, that he found a bacillus in all leprous processes. Form.— Small slender rods somewhat shorter than tubercle bacilli, otherwise very similar in appearance. In the interior of the cell two to three oval spaces are usually seen, not known if spores or otherwise. Properties.— They are immotile, do not liquefy the nutrient media. Growth.— Bordoni-Uftreduzzi have obtained growths upon blood serum to which peptone and glycerine had been added, but the accuracy of this observation is very doubtful. Staining. — They resist the decolorizing action of acids as the tubercle bacilli, but they are easily stained, requiring but a few minutes with the ordinary watery solutions. They take Gram's stain readily. Pathogenesis.— Arning has inoculated prisoners with tissue obtained from leprous patients, and produced true leprosy. Rabbits which had been infected through the anterior chamber of the eye showed the lepra nodules (containing the lepra bacilli) diffused through various organs, but here again the results are not wholly satisfactory. 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 108 ESSENTIALS OF BACTERIOLOGY. nodules contain the bacilli in large numbers. By applying a vesicant to the leprous skin the serum thereby obtained will con- tain 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 examined with- out result. The nasal secretion is very infectious. Syphilis Bacillus of Lustgarten (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 connection with the disease. Van Niessen, Joseph and Piorkowski, DeLisle and Jullien all describe other organisms that they have found in syphilitic lesions, and Schuller mentions a protozoon-like body he has seen in many cases, but these results still lack confirmation. Metschnikoff, Roux, and Lassar have lately succeeded in inocu- lating chimpanzees with what appears to be true syphilis. The question yet remains an open one, what relation the syphilis or the smegma bacillus bears to syphilis, and will remain so until the bacillus can be cultivated, which so far has not been accomplished. Bacillus of Glanders. {Bacillus Mallei, Loffler-Shutz.) Botz bacillus. Origin. — In the " farcy buds" or little nodules of the disease, by Loffler and Shiitz in 1882. Form. — Small slender rods, about the size of the tubercle bacillus. The ends rounded. Never appearing in large collec- tions, usually singly. Spores are said to exist, but this is doubtful. Properties. — The rods are very resistant, living in a dried state for three months and longer without any spores present. They are not motile ; possess, however, great molecular vibration. Growth. — The growth occurs between 25° and 40° C, best at 37° C. ; it is very sparse upon gelatine, but on glycerine-agar or blood serum a very abundant growth occurs. Colonies. — On agar or glycerine-agar there appear in two to three days small white glistening drops, which under microscope seem as round granular masses with an even periphery. Stroke Cultures. — On glvcerine-asrar and blood serum small PATHOGENIC BACTERIA 109 transparent drops of whitish or grayish color, which soon coalesce to form a broad band. Potato. — An amber-colored honey-like growth which gradually turns red, then brown, and greenish-brown around it. Weakly acid potatoes are a good medium and give the most typical growth. Fig. 55. Bacillus of Glanders. Staining. — Since the bacillus is very easily decolorized, some special methods have been recommended. Lbffler's.— (For cover-glass preparations.) 1. Alkaline methylin blue (Loffler's). 5 minutes. 2. Acetic acid with a few drops of tropaeolin. 1 second. 3. Washed in water. For Sections. — Instead of tropseolin acetic acid, the following mixture is used : — $.— Oxalic acid 5 per cent. . . . gtt. j. Cone, sulphuric, acid gtt. ij. Aq. destill. 3ij. — M. The sections are kept in this 5 seconds. Kuhne^s method. Coverglass. 1. Warm carbol-blue 2 min. 2. Decolorized in weak sol. of muriatic acid (10 parts to 500). 3. Washed in water. 110 ESSENTIALS OF BACTERIOLOGY. Sections of Tissue. 1. Carbol-blue, £ hour. 2. Decolorized in £ per cent, muriatic acid. 3. Washed in distilled water. 4. Dehydrated in alcohol 1 second. 5. Aniline oil with 6 gtt. of turpentine. 5 min. 6. Turpentine, xylol, Canada balsam. If contrast stain, add 5 gtt. of safranin (Bismark-brown) to turpentine, and use it after the xylol. Pathogenesis. — If horses, field mice, or guinea-pigs be inocu- lated subcutaneously, with but a very small quantity of culture, a local affection results, followed some time after by a general disturbance ; ulcers form at the point of inoculation ; little nodules, which then caseate, leaving scars and involving the lymphatics ; metastatic abscesses then occur in the spleen and lungs, and death arises from exhaustion. Cattle, pigs, and rab- bits are not easily affected ; man is readily attacked. The ba- cilli gain entrance to the blood and urine. Nasal glanders occurs whatever the mode of inoculation. Manner of Infection.— Glanders being a highly contagious dis- ease, 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 is usually on the fingers. Boiling water or 1-10,000 sublimate solution will quickly destroy the virulence of this bacillus. Mallein. A substance called mattein has been obtained from the cultures grown in glycerin bouillon. It gives a reaction when injected into cattle suffering from glanders, and is said to be useful in diagnosing the disease. Bacillus of Diphtheria. (Klebs-Loffler.) Origin. — Klebs found it in membrane in 1883; it was isolated by Loffler in 1884. Form. — Small, slightly curved rods about as long as tubercle bacilli and twice as broad; 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 easily PATHOGENIC BACTERIA. Ill colored than the centre, and usually the bacillus stains in seg- ments, so that it seems to be made up of very short sections. At first sight it appears like a chain of cocci. Properties.— They do not possess any movement; do not liquefy gelatine. They are not very resistant, being destroyed by a temperature of 50° C, but they have lived on blood-serum five months. Growth.— Grow readily on all media, but best on blood-serum mixtures, between temperatures of 20° and 40° C. They are fac- ultative anaerobic ; they grow quite rapidly and profusely. Egg cultures (Hueppe's method) give good growths. Passing cur- rents of air increase the growth. Colonies on Gelatine Plates.— At 24° C. little round colonies, white under low-power, granular centre; irregular borders. Stab Cultures.— Small, white drops along the needle track. In glycerine-agar a somewhat profuse growth. Potato.— On alkaline surface, a grayish layer in 48 hours. Blood-Serum (after Loftier). — Blood serum 3 parts, and bouil- lon 1 part ; the bouillon contains peptone, 1 per cent., chloride of sodium, £ per cent., and dextrin (or glucose), 1 per cent. In a few hours (eight to sixteen) on the white opaque surface a slight moisture is noticeable, which, if examined, is composed of bacilli. In twenty -four hours small round colonies are found which seem to arrange themselves concentrically. The growth becomes more abundant, and the individual colonies larger and yellowish. On blood-coagulum 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 a brood oven. Serum- Agar. — Joos finds serum-agar better than Loffler's serum : 300 c.c. blood-serum mixed with 50 c.c. normal soda solution and 150 c.c. water, heated in water bath for 2 to 3 hours at 60° to 70° C, then raised to 100° O, or in steam chest I hour. Then 500 c.c. peptone bouillon (slightly alkaline) and 20 gm. agar. When the agar is dissolved by heat, avoiding prolonged boiling, the mixture is filtered and sterilized \ hour at 100° to HO" C. in autoclave ; then poured into petri dishes. Strepto- cocci do not grow on this medium, whereas diphtheria bacilli will grow in from 6 to 12 hours. 112 ESSENTIALS OP BACTERIOLOGY. Bouillon. — In bouillon an abundant growth takes place, and this medium is used to obtain the toxins. .f<; Fig. 50. W i «nv <^* /&%< V ttrM it* : Bacillus diphtheria?, from a pure culture Fig. 57. & Sis" V i «' Bacillus diphtherise, from a culture upon blood-serum ; X 1000 (Fraukel and Pfeiffer). Staining. — Is not colored by Gram's method. Stained best with Loffler's alkaline methylin-blue. PATHOGENIC BACTERIA. 113 Pathogenesis. — By inoculation, animals, which naturally are not suhject to diphtheria, have had diphtheritic processes de- velop at the site of infection ; hemorrhagic oedema then follows, and death. In rahhits 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 some man- ner, 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 susceptible individuals with- out themselves becoming infected. Products. — But it is not the mere presence of the bacillus that gives rise to all trouble ; certain products which they generate get into the system and produce the severe constitutional symp- toms. Roux and Yersin, in 1888, discovered 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. Toxins of Diphtheria. — Brieger and Frankel filter the bouillon culture, evaporate (in vacuo at 27° C.) to i volume, then treat with 10 volumes of alcohol and acetic acid, the precipitate re- dissolved in water and reprecipitated with the acidulated alco- hol until a clear aqueous solution is obtained ; this is then dialyzed for 72 hours, and again precipitated with alcohol, and dried ; a white amorphous body results, giving all the reactions of an albumen, and called by them toxalbumen. The toxin of diphtheria, first demonstrated by Roux and Yersin, is not an albumen. It is obtained by growing virulent bacilli in bouillon for three or four weeks at 37° C. After a sufficient alkalinity has been produced the cultures are fil- tered, and the filtrate should have a toxicity that will destroy a 500-gramme guinea-pig in twenty-four hours when 0.1 c. cm. of the toxin is injected. Antitoxin. Behring found that animals rendered immune 8 114 ESSENTIALS OF BACTERIOLOGY. 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.cm. 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 important discovery has been utilized in a practical Avay. Horses are made immune by gradually increased doses of the toxin until 300 c. cm. can be borne without bad effect. This may require several months' time. The serum of such an im- munized animal is now possessed of antitoxic properties. Behring has standardized the strength of antitoxic serum, so that we say a serum has an immunizing strength of 60 units or 100 units, which means that 0.1 c. cm. of the serum would protect against 1 c. cm. of the toxin when injected together into guinea-pigs. 1 cubic centimetre of this is the unit. The strength commonly employed in human beings is 1500 units in 10 c. cm. If this amount is injected into a child suffering from diphtheria in the earlier stages (second to third day), the disease is often arrested. The membrane begins to disappear, and in two or three days has vanished. The constitutional symptoms are likewise greatly influenced by the injection. If a smaller dose is injected into persons who have been exposed to contagion, the disease is prevented from appearing. The antitoxin has no influence on the bacteria themselves; their virulence and length of residence in the body is not lessened. The toxin generated by the germ is supposed to be neutral- ized by the antitoxin and prevented from injuring the body tissues. Pseudo-diphtheria bacilli, so called, differ from the true organism in certain cultural and morphological characteristics, and do not produce a toxin, but their true status is still uncer- tain. Site of Bacilli. — Bacilli are usually found in the older portions of the pseudo-membrane very near to the surface. The secre- tions of the throat of a diphtheritic child produced bacilli three weeks after the temperature was down to normal. PATHOGENIC BACTERIA. 115 Streptococcus in Diphtheria. Streptococci have been found quite constant in diphtheria, but they resemble the strepto- coccus pyogenes, and have no specific action. Fig. 58. Bacillus typhi, from an agar-agar culture six hours old, showing the flagella stained by Loffler's method ; X 1000.(Frankel and Pl'eifier.) Pseudo-diphtheritic bacillus is probably a weakened or a vir- ulent form of the true bacillus. Bacillus of Typhoid or Enteric Fever. (Eberth-Gaffky.) Origin. — Eberth found this bacillus in the spleen and lym- phatic glands in the year 1880, and Gaff ky isolated and cultivated the same four years later. Form. — Rods with rounded ends about three times as Ions as they are broad. Usually solitary in tissue-sections, but in arti- ficial cultures found in long threads. Flagella on the side. Properties. — They are very motile ; they take the aniline dyes less deeply than some similar bacilli. Spores have not yet 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. All 116 ESSENTIALS OF BACTERIOLOGY. nutrient media can be used as culture ground. They develop chiefly on the surface, and very slowly. Repeated freezing and thawing do not affect the vitality of the germ, and carbolic acid in 1 to 2 per cent, solution has no effect on it. A ten-minute exposure to 60° C. is invariably fatal. Colonies on Gelatine 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. In five days they attain to 3 millimetres in diameter. On Potato Gelatine. — The colonies do not have the yellow color, they are transparent, later on they become dark brown with green iridescence. Stab Cultures.— Mainly on the surface a pearly layer. Stroke Cultures. — A transparent thick layer. Potato. — The growth here is quite characteristic. At 37° C. Fig. 59. Fig. 60. Typhoid fever bacillus in pure cul- Colonies of typhoid bacilli 3 days ture. 650 diameters. old 100 X. (Frankel and Pfeiffer.) in 48 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. The growth never becomes more prominent ; the potato must have a neutral or acid reaction. Milk. — The bacteria grow very well in milk, producing a slightly acid reaction, but no coagulation. PATHOGENIC BACTERIA. 117 Carbolized Gelatine. — Gelatine which has added to it ^ per cent, carbolic acid will allow the typhoid bacillus to develop, other similar bacilli being destroyed. Glucose Gelatine. — In glucose gelatine there is no gas-produc- tion. Indol is likewise not generated by the typhoid bacillus, whereas it is by the colon bacillus. On Eisner's potato-gelatine the colon bacillus and the typhoid bacillus grow readily. The medium of Hiss is of great assistance in isolating the germ. Fig. 61. The Widal agglutination reaction (Slater and Spitta). The Gruber- Widal blood-serum test, or, as otherwise known, the agglutination-phenomenon (Fig. 61), has the following history: About 1889, Charrin and Roger observed in the serum of im- munized animals that the B. pyocyaneus arranged itself in little clumps. Other investigators reported the same thing for other bacteria, and Metschnikoff added that motility was destroyed. In 1895, Bordet showed that the serum of cholera-immunized animals, when mixed with bouillon cultures of cholera spirilla, affected their motility and caused them to form masses, or "Klumpen," as the Germans call it- R. Pfeiffer, in the same year, showed that the introduction of immune serum at the same time with virulent cholera spirilla 118 ESSENTIALS OF BACTERIOLOGY. into the peritoneum of guinea-pigs, prevented infection from taking place, and the spirilla were transformed into granular masses. He likewise showed this reaction to be specific, the serum of cholera-immune animals acting only on cholera vibrio; and hence he suggested using the serum as a means of diagnosis for the cholera vibrio and typhoid bacillus. Gru- ber about the same time made some studies upon the use of this serum property in differentiating bacteria, but it was con- sidered as yet a property connected in some way with immu- nity. In 1896, Widal and Griinbaum, working separately, developed what is now spoken of as the " Widal serum-test," or "Widal reaction." 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 micro- scope. Within fifteen minutes to an hour the motility of the bacilli will cease, and they will have arranged themselves into clusters, as if stuck or glued together. If this reaction occurs within an hour, and with the proper dilution of the serum, the case is one of typhoid. Widal first used the serum of the blood ; this has been modified so that even a drop of dried blood is suf- ficient. 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 1 : 5. One drop of this is then mixed with one drop of a bouillon culture of typhoid bacilli of about 24 hours' growth, and examined under the microscope in the hanging drop. Weaker dilutions of the serum have been rec- ommended (1 : 50), and this should be used in cases of doubt So far, about 95 per cent, of the cases examined, and which clin« ically were considered typhoid fever, have given a positive reac- tion. It is not often present until the fifth day of the fever, and disappears usually within a year, though in some individuals it has been found ten years after an attack of the disease. PATHOGENIC BACTERIA. 119 The agglutinating properties have been found in nearly all the secretions of the body — tears, urine, milk, pleuritic effusions, serous fluid from blisters, etc. There is no relation between the reaction and the bactericidal power of the serum ; the agglutination is not a destruction. 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 mobility. The test must occur within a certain limit of time to be of value, since agglutination is liable to appear of itself with non- typhoid sera after a period of an hour. As a clinical test of the disease it has considerable value, although operative at a time when other symptoms have devel- oped sufficiently to determine the diagnosis. Staining. — Colored with the ordinary aniline dyes, when they are warmed; since they are easily decolorized, acids should be avoided. Gram's method is not applicable. Tissue sections stained as follows : — Alkaline methylin-blue . .1 hour. Alcohol 5 seconds. Aniline oil 5 minutes. Turpentine oil 1 minute. Xylol and Canada bals. Such a specimen should first be examined with low power, to focus little colored masses, then examined with immersion lens ; these masses will be found composed of bacilli. Similar Bacteria. The Neapolitanus bacillus of Emmerich, or fxces bacillus of Brieger, resembles the typhoid bacillus in many ways, the colonies being the same and its structure similar. But the growth on potato is very different; a thick, yellow, pasty layer is formed thereon. 120 ESSENTIALS OF BACTERIOLOGY. The colon bacillus not only resembles the typhoid germ in form, but also in some of the pathologic processes produced. For points of resemblance and difference, see Bacillus coli com- munis. In Water. Bacilli have been found which also resemble ty- phoid bacilli, and one must be very careful not to make any positive statement. Examination of Water for Typhoid Bacilli. — When a water is supposed to contain typhoid bacilli, 500 c.cm. of the same is mixed with 20 gtt. of |-per cent, carbolic acid, which destroys many of the saprophytes. Plates are then made as described under Water Analysis. Those colonies which then form and have a tendency to liquefy, are touched on second day with permanganate of potassium, and when so colored, destroyed with bichloride of mercury. Those that now develop are transferred by inoculation to fresh plates. At the end of eight days they are examined under microscope ; every colony not possessing motile bacilli is dis- carded. The motile bacilli are tested with Gram's method of staining ; those that do not take the stain are alone retained. Cultures are made from these upon potatoes, and, if the char- acteristic growth occurs, then only can they be called typhoid bacilli with any certainty. Pathogenesis. — Lower animals have npt yet been given enteric fever, though their death has been caused by injection of the bacilli into the veins of the ear. In man it has been found in the urine, blood, sputum, 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 sub- sided. It is found only in this disease, and regularly. Way of Infection. — The bacilli in the dejecta of the diseased person find their way into drinking water, milk, or dirty clothes, and so into the alimentary tract of a person predisposed to the disease. They enter the blood through the lymphatics, and so become lodged in various organs. They are quite resistant, liv- ing for some time in the soil and water, and are not affected as PATHOGENIC BACTERIA. 121 other organisms by carbolic acid. An epidemic has been traced to the eating of oysters taken from contaminated water. Persistence in Water. — Franckland kept bacilli alive in water, sterilized by heat, 75 days ; in filtered water at 19° C, 5 days ; at 6° C, 12 days. In ordinary water they are likely to be de- stroyed in a few days by the overgrowth of other bacteria. Products. — Brieger found a ptomaine in the cultures which he named typhotoxin with the formula C9HnN02. It has no specific action. A toxalbumen 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. Paracolon or paratyphoid bacilli are members of the colon group recently described by Widal, Gwyn, Schottmuller, and others. They are of importance, since they produce fevers clinically resembling a mild form of typhoid, and which are rarely fatal. They may be the sole cause of the disease, and probably 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 coagu- lated. On potato a slight visible growth occurs ; indol is usually not formed. Typhoid sera do not agglutinate paracolon bacilli, and vice versa; also different paracolon infections may not agglutinate each other. The Bacillus enteritidis of Gartner is a related form. Bacillus psittacosis is an allied form occurring in parrots, and producing hemorrhagic septicemia in them and experi- ment animals. The disease is readily communicated to man from the affected birds, and causes, after ten days' incubation, a disease, the chief symptoms of which are fever, delirium, vomiting, diarrhoea, and albuminuria, about a third of the cases ending fatally. The organism is agglutinated by strong dilutions of typhoid serum, but the clumping is incomplete and the bacillus differs further from the typhoid bacillus in its growth on potato and in the nature of the infection produced. 122 ESSENTIALS OF BACTERIOLOGY. Bacillus Coli Communis. (Escherich.) Found in human feces, intestinal canal of most animals, in pus and water. Form. — Short rods with very slow movement, often associated in little masses resembling the typhoid germ, flagellated, does not form spores. Fig. 62. Bacillus coli communis, from an agar-agar culture; X 1°00 (Itzerott and Niemann). Properties.— Does not liquefy gelatine, causes fermentation in saccharine solutions in the absence of oxygen, produces acid fermentation in milk. Growth.— On potato a thick, moist, yellow-colored growth. Very soon after inoculation on gelatine a growth similar to typhoid. It can also develop in carbolized gelatine, and with- stands a temperature of 45° C. without its growth being de- stroyed. Pathogenesis.— Inoculated into rabbits or guinea-pigs, death follows in from one to three days, the symptoms being those of diarrhoea 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 germs. With the blood of immunized animals a serum reaction PATHOGENIC BACTERIA. 123 similar to that of typhoid fever may be obtained with cultures of colon bacilli. The colon bacillus is held responsible for most of the complications of typhoid fever, such as peritonitis, cholangitis, etc., by many writers. Staining. — Ordinary stains; do not take Gram. Site.— The bacillus has been found very constant in acute peritonitis and in cholera nostras. Its presence in water would indicate fecal contamination, as it is normally present in the intestine. Points of Resemblance between Bacillus Typhi and Bacillus Coli Communis. — 1. Microscopic appearance; 2. Agar and gelatine cultures ; 3. Sometimes growth on potato the same ; 4. Stain- ing peculiarities ; 5. Resistance to carbolic acid. Points of Difference : Colon Bacillus. Less motile, Gelatine colonies develop more rapidly, Produces gas on dextrose or lactose media, Coagulates milk, Produces indol, Growth on potato visible, Changes neutral red to yellow. Typhoid Bacillus. Actively motile, Develop more slowly, Does not, Does not, Does not, Invisible, Does not reduce neutral red. Differences are also noted in the growth on special media, such as those of Hiss and Eisner. Varieties. — By some bacteriologists the following bacilli are all considered forms of the colon bacillus : B. lactis aerogenes of Escherich, B. cavicida of Brieger, B. neapolitanus of Emmerich, B. enteritidis of Gartner, and, together with some other allied organisms, they are spoken of as the " colon group." 124 ESSENTIALS OF BACTERIOLOGY CHAPTER III. PATHOGENIC BACTERIA — CONTINUED. Spirillum Cholerse. (Koch.) Comma bacillus of cholera. Origin. — Koch, as a member of the German expedition sent to India, in 1883, to study cholera, found this micro-organism in the intestinal contents of cholera Fig. 63. patients, and by further experiments identified it with the disease. Form.— The microbe as seen ordi- narily appears as a short, arc-like body, about half the size of a tubercle bacillus, but when seen in large 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 Comma bacillus, pure cul- is broad, and possesses a flagellum at ture. 600 diameters. one or more rarely both ends. Properties. — They are very motile ; liquefy gelatine. They are easily affected by heat and dryness. Spores have not been found, though some (Hiippe) claim arthrospores, but these bodies represent only degenerative changes. Growth. — Develops at ordinary temperatures on all nutrient media that have an alkaline or neutral reaction. They are facultative anaerobic. Colonies, gelatine. — After 24 hours, small white points which gradually come to the surface, the gelatine being slowly lique- fied, a funnel-shaped cavity formed holding the colony in its narrow part, at the bottom, and on the fifth day all the gelatine 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. 9W PATHOGENIC BACTERIA 125 Stab Culture.— After 30 hours a growth can be distinguished along the needle track, and on the surface a little cavity has been formed, filled up by a bubble of air, and this liquefaction 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 collec- FlGJ- 64- tions at the bottom of the fluid gelatine. In eight weeks the bacilli have perished. Agar. — Stroke cultures. A shiny white layer lasts many months. Potato. — A yellow honey- like transparent layer, if the potato is kept at ani- mal heat. Bouillon. — A wrinkled scum is soon formed in bouillon. They live wel] and grow in sterilized mill* and sterilized water, re^ maining virulent in the latter for many months. In ordinary water, the bacteria present are destructive to the comma bacillus, and they die in a few days. Dunham's Peptone Solution. — Useful for the development of nitrites and the indol reaction. WidaVs serum test, as used in typhoid, is applicable in the diagnosis of cholera, using cholera cultures in place of the typhoid. Staining. — They are colored well with watery aniline solu- tions. The flagella can be well seen by staining according to the flagella stain. Pathogenesis. — Experiment animals are not subject to cholera Asiatica, but by overcoming two obstacles Koch has produced choleraic symptoms in guinea-pigs. Nieati and Rietsch pre- vented peristalsis and avoided the acidity of the stomach juices Cholera colonies after 30 hours 100 X . (Frankel and Pfeiffer.) 126 ESSENTIALS OF BACTERIOLOGY. by direct injection into the duodenum, after tying the gall-duct. Koch alkalinizes the gastric juice with 5 c.cm. of 5 per cent, sol. of sodii carbonas, and then injecting 2 grams of opium tinc- ture for every 300 grams of weight into the peritoneal cavity paralyzes peristalsis. The cholera culture then introduced through a stomach-tube, the animals die in forty-eight hours, presenting the same symptoms in the appearance of the intes- tines as in cholera patients, the serous effusion containing great numbers of spirilla. 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 drink- ing water. Soiled clothes to fingers, fingers to the mouth, etc. ; torpid catarrhal affection of the digestive tract predisposing. The microbe is not found in the blood or any organ other than the intestines, the tissue of the small intestines. It is also found in the vomit and the intestinal contents. Fig. 65. Comma bacillus in mucus, from a case of Asiatic cholera. Products. — " Cholera red." When chemically pure nitric or sulphuric acid is added to nutrient peptone cultures of the PATHOGENIC BACTERIA. 127 cholera bacillus a rose-red color is produced. This will not take place with other bacilli unless nitrous acid is present. The cholera bacillus forms nitrites from the nitrates present in the media, and also indol. The mineral acid splits the nitrites, setting free nitrous acid, which, with the indol, forms the red reaction. This pigment has been isolated and extracted and called " cholera red." A ptomaine, identical with cadaverin, and sev- eral other alkaloids have been obtained from the cultures. A toxalbumen and a toxicpeptone have lately been isolated, but no special actions ascribed to them. 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 material (200 c. cm. of drinking-water) to about 10 c. cm. of bouillon, and place the mixture for twenty-four hours in an incubator, which will cause rapid reproduction, and then the organisms can be readily discovered. HafFkine has obtained a great reduction in mortality in cholera regions by the use of anti-cholera vaccines as pro- tective and curative measures. Cholera Immunity of Pfeiffer. — Intraperitoneal, subcutaneous, and intravenous injections of living or dead cholera bacteria cause a disease in animals similar to the cold stage of cholera. Death is the result of toxemia. If the animal lives, the blood has protective properties of a specific nature; it has bacteri- cidal properties against cholera vibrio, and by the injection of this serum into non-immune animals it renders them immune. The blood-serum of convalescents and cholera-vaccinated indi- viduals contains the same bactericidal substances. Bacteria Similar to the Spirillum of Cholera. Finkler-Prior Vibrio, or Spirillum Finkleri. Origin. — Found in the intestinal contents of a patient suffer- ing from cholera Asiatica in 1884, by Finkler and Prior, who thought it identical with the spirillum of cholera; it differs from it, however, in many ways, and has been found in healthy persons. Form. — Somewhat thicker than the cholera vibrio: but forms the long spirilla less often. Has flagella. 128 ESSENTIALS OF BACTERIOLOGY. Liquefies gelatine in a short It Fig. 67. Properties. — It is very motile time. Growth. — It grows quickly at ordinary room temperature. is facultative aerobic. Colonies on Gelatine Plates. — Round, finely granular colonfes, which in twenty-four hours are ten times as large as the cholera colonies, and in forty-eight hours the whole plate is liquefied, it being then impossible to distinguish any separate colonies. The microscopic appearances in no way resemble the cholera colony. Stab Cultures. — The gelatine is lique- fied from above downwards, like a stock- ing in appearance, and in three days is completely liquid. Potato. — At ordinary temperature a thick gray layer covering the whole sur- face. Water. — It soon perishes in water. Staining. — Ordinary aniline dyes. Pathogenesis. — For man it has no spe- Fig. 66. Spirillum Finkleri. 700 diameters. (Fl !ugge.) Stab Culture. (Finkler- Prior.) cific action. If it is injected into Guinea pigs, prepared as described under the cholera bacillus, they die, the intestines having a foul odor, and the bacilli then found in great numbers. Spirillum Tyrogenum. (Deneke.) Origin.— In 1885 Deneke found in old cheese a spirillum very similar in appearance to the cholera spirillum. Form. — The same as the cholera vibrio. w I CC 6 D 1 5 D o w 1 O 1 ^ S W a a o tf s D ,§ H H S J 2 — D 1 E- O & < d w I - w g D f H ° 03 & a 2 E PATHOGENIC BACTERIA. 129 Properties. — Very motile, liquefy gelatine. Growth. — They grow quicker than the cholera, and slower than the Finkler; they are also facultative aerobic. Colonies. — At first resemble cholera colonies ; have, however, a yellow-green iridescence, and are more irregular; also grow more rapidly. Stab Cultures. — A thick line along the needle-track and yellow colonies forming at the bottom, on the surface a bubble of air similar to the cholera. The gelatine is liquid in two weeks. Potato. — At brood-heat a thin yellow membrane, but not always constant. Staining, as cholera bacillus. Pathogenesis. — When injected into animals prepared as for the cholera bacillus, a certain number die. Vibrio Metschnikovi. (Gamaleia.) Origin. — In the intestines of fowls suffering from a gastro- enteritis, common in Kussia. Gamaleia found a spirillum which bears so close a resemblance to the cholera bacillus, both in form and growth, that it cannot be distinguished by these character- istics alone. Form. — As cholera bacillus. Growth. — Two kinds are found on the gelatine plate— one that is identical in appearance with the cholera colony, the other more liquefying, resembling the Finkler spirillum. If now a second plate be inoculated from either one of these forms, both kinds again are found grown, so that it is not a mixture of two bacilli. Stab Culture. — Similar to the cholera growth, a trifle faster in growing. Staining, as cholera. Pathogenesis.— To differentiate it from cholera, these bacilli, when injected into animals, prove very fatal, and no especial precautions need be taken to make the animal susceptible. In the pigeon, guinea-pig, and chicken it produces a hemorrhagic oedema, and a septicaemia which has been called " Vibrion septicaemia." The blood and organs contain the spirilla in great numbers. Products.— The nitrites are formed just as in cholera bacillus, and the red reaction given when mineral acids added to gelatine cultures. Certain products also which, when injected, give 130 ESSENTIALS OP BACTERIOLOGY. immunity. The cultures are first heated for one half hour at 100° C, which destroys the germs, and then this sterilized pro- duct injected. (5 c.cm. of a five days' old sterilized culture.) In a couple of weeks 1 to 2 c.cm. of the infected blood can be injected without causing any fatal result. A great many more spirilla resembling the spirillum of chol- era have been isolated from drinking-waters in the past few years, and some bacteriologists are inclined to consider them as varieties of the true cholera spirillum which require only certain conditions to make them pathogenic. Among these, besides those already described, are Spirillum Berolinesis, S. Dunbar, S. Danubicus, S. Wernicke, S. BonhofF, S. Weibeli, S.« Schuylkiliensis, S. Milleri, S. Aquatilis. The last two are non- pathogenic for experiment animals. Bacteria of Pneumonia. Two forms of bacteria have been found in this disease, and thought at different times to be the cause of the same. Neither one of them is constant in pneumonia ; and since many other pathological processes have shown them they can hardly be set down as the sole cause of 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 microbe was not constant, in fact was rare. A. Frankel obtained in the majority of cases of pneumonia a microbe that he had described in 1884 under the name of sputum-septicaemia micrococcus. Weichselbaum called it " Diplococcus pneumonias," and be- lieved it to be the real cause of pneumonia. It has been found in many other serous inflammations, and also in the mouths of healthy persons. 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. Streptococcus pyogenes and staphylococcus pyogenes aureus have been found in some cases. PATHOGENIC BACTERIA 131 Pneumo-bacillus (Pneumococcus). (Friedlander.) Origin. — In the lung of a croupous-pneumonia person, by Friedlander, in 1882. Fig. 68. Bacillus pneumoniae of Friedlander, from the expectoration of a pneumonia patient ; X 1000 (Frankel and Pfeitterj. Form.— Small, almost oval-shaped rods, nearly as wide as they are long ; often in pairs, they were at first believed to be cocci. In bouillon cultures the rod-form becomes more visible. In tissues each bacillus is surrounded by a faint capsule ; but not around those developed in artificial cultures. Spores have not been found. Properties.— They are immobile ; do not liquefy gelatine. A gas is produced in gelatine cultures. Growth.— Grows rapidly on all media at ordinary temperature ; is facultative aerobic. Colonies.— On gelatine plates. Small white round colonies, reaching the surface in the course of three or four days ; appear- ing then as little buttons, with a porcelain-like shimmer, the edges smooth. 132 ESSENTIALS OF BACTERIOLOGY. Pig. 69. Stab Culture. — A growth along the needle-track, but on the surface a button-like projection, which gives to the growth the appearance of a nail driven into the gelatine, its head resting on the surface ; therefore such cultures are called "Nail cultures.'''' See Fig. 69. Old cultures are colored brown, and contain bubbles of gas. Potato. — A yellow, moist layer in a few days at brood-heat. Gas bubbles develop. Staining.— The ordinary aniline stains. The sections do not take Gram's method ; are therefore not suited for double staining. Capsule.— Stained as follows :— Cover glasses. 1. Acetic acid, two minutes. 2. Allow acetic acid to dry by blowing air upon it through a glass tube. 3. Saturated, aniline water. Gent, violet, ten seconds. 4. Rinse in water. Mount in Canada balsam. For Sections. Bacillus of Pneumo- f cone, ale. gent, violet, 50.0 nia. stab Culture. 1. Stain in warm ] aqua, 100.0 LNaii culture.) I acetic acid, 10. M. for 24 hours. 2. Rinse in one per cent, acetic acid. 3. Alcohol to dehydrate. Mount in balsam. The capsule will be found stained a light blue, the bacillus a deep blue. (See also the capsule stain of Hiss, p. 35.) Pathogenesis. — Animals are not affected unless the culture is injected intrapleura. Pneumobacillus of Frankel. (A. Frankel and Weichselbaum.) Synonyms. — Pneumococcus ; Diplococcus of Pneumonia ; Mi- crococcus of sputum septicaemia ; Micrococcus Pasteuri ; Diplo- coccus lanceolatus. Origin. — A. Frankel found it in the sputum of pneumonic PATHOGENIC BACTERIA. 133 patients, thinking it at first to be the micrococcus of sputum septicemia ; later he believed it to be the cause of pneumonia. Form. — Oval cocci they were at first called, but they are now known to be rod-shaped, being somewhat longer than broad ; varying, however, much in size and shape. Usually found in pairs, sometimes in filaments of three and four elements. In the material from the body a capsule surrounds each rod. In the artificial cultures this is not found. Fig. 70. Bacillus of pneumonia in blood of rabbit 1000 X. (Frankel and Pfeifler.) Properties. — They are without self-movement; do not liquefy gelatine. There are no spores. Growth.— Grow only at high temperature, 35° C. ; are facul- tative anaerobic. The culture media must be slightly alkaline ; the growth is slow. Colonies on Gelatine Plates. — Since the temperature must be somewhat elevated, the gelatine media need to be thicker than usual (15 per cent, gelatine), in order to keep it solid, and a temperature of 24° C. used. Little round white colonies, some- what granular in the centre, growing very slowly. Stab Cultures. — Along the needle-track small separate white granules, one above the other, like a string of beads. Stroke Culture. — On agar, transparent, almost invisible little drops resembling dew moisture. 134 ESSENTIALS OF BACTERIOLOGY. Bouillon. — They grow better here than in the other media, remaining alive a longer period of time. Staining.— Takes Gram's method and the other aniline stains very readily. The capsule stained the same way as that of the Friedlander bacillus. Pathogenesis. — Rabbits and guinea-pigs, if subcutaneously in- jected, die in the course of a couple of days with septicaemia. (0.1 com. of a fresh bouillon culture suffices.) Autopsy shows greatly enlarged spleen and myriads of bacilli in the blood and viscera, the lungs not especially affected. If injected per trachea, a pneumonia occurs. In man in 90 per cent, of croupous pneumonia they are found and usually only during the existence of the rusty sputum, i. e., the first stage. Fig. 71. 1& ** Micrococcus tetragenus in sputum (tubercle bacillus also). They have also been found in pleuritis, peritonitis, pericarditis, meningitis, and endocarditis. They stand in some intimate re- lation with all infectious inflammations of the body. Their presence in healthy mouth secretion does not speak against this, it requiring some slight injury to allow this ever-present germ to develop into disease. Anti-toxin of Pneumonia. (Klemperer.) The injection of very diluted cultures of the virulent bacilli in- travenously has produced an immunity in rabbits and guinea- pigs. The serum of such artificially immune animals when filtered PATHOGENIC BACTERIA. 135 through a Chamberland filter and injected into a rabbit suffer- ing with pneumonia, cured the same; or when injected into a susceptible animal produced in it immunity very quickly. This principle is ascribed to an anti-toxin formed in the tissues by the diluted proteids, and this anti-toxin neutralizes the toxicity of the strong virus. Bacillus of Rhinoscleroma. (Frisch. 1882.) It was found in the tissue of a rhinoscleroma, but resembles the Friedlander bacillus in nearly every respect, and as the disease rhinoscleroma was not reproduced by the inoculation of the bacillus in animals, it can be considered identical. The growth, cultures, and pro- perties are the same as the pneumobacillus of Friedlander. Diplococcus Intracellularis Meningitidis. Weichselbaum claims to have found a special diplococcus in epidemic cerebro- spinal meningitis, which differs in a few respects from the pneu- mococcus of Frankel : growth most abundant on blood-serum — round, white, shining colonies in twenty-four hours. It does not take Gram's stain ; does not affect animals when injected eubcutaneously. Inoculated into the meninges of the dog and goat, a meningitis has been produced, and when found in the exudate of the meninges lies in the protoplasm and nuclei of the leucocytes. The organism has many points in common with the gonococcus, but differs from it in the ease of cultivation. Micrococcus Tetragenus. (Koch. Gaffky). Origin. — Koch found this microbe in the cavity of a tuber- culous 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. 71. In artificial culture, sometimes found in pairs. A capsule of light gelat- inous consistence surrounds each tetrad. Properties. — They are immobile ; do not liquefy gelatine. Growth. — They grow well on all nutrient media at ordinary and brood temperatures ; are facultative aerobic. They grow slowly. Colonies in gelatine 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. 136 ESSENTIALS OF BACTERIOLOGY. Fig. 72. v\ 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. 72. Potato. — A thick slimy layer which can be loosened in long shreds. Staining.— Colored with the ordinary ani- line stains. Gram's method also applicable. Pathogenesis.— White mice and guinea-pigs die in a few days of septicaemia when injected with the tetragenus cultures, and the micro- coccus is then found in large numbers in the blood and viscera. Field mice are immune. In the cavities of tubercular 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. Capsule Bacillus. (Pfeiffer.) Origin. — Stringy exudate and blood of a dead guinea-pig. Form. — Thick little rods, sometimes in long threads. Large oval capsules in the stained preparations. Properties. — Immotile, not liquefying, an odorless gas in gelatine cultures. Growth. — At ordinary temperatures, rap- idly; facultative anaerobin. Gelatine Plates. — Oval points, and like a por- celain button on the surface. Stab Cultures. — Like the pneumonia bacillus of Friedlander. Potatoes. — Abundant growth, yellow color and moist, coming off in strings. Staining. — Hot fuchsin colors the capsule intensely ; carefully decolorizing with acetic acid, the capsules are red or light violet around the deeply-tinged bacillus. Gram's method not applic- able. Pathogenesis. — Subcutaneously injected in mice, they die in 48 hours. Rabbits die when a large quantity is injected into Stab Culture. Micrococcus tetra- genus. PATHOGENIC BACTERIA. 137 the circulation. The blood and juices have a peculiar stringy fibrinous consistence. Bacillus of Influenza. (Pfeiffer, 1892.) A small bacillus about one-half the size of the bacillus of mouse septicaemia, and arranged in chain-form. It develops Fig. 73. » . • * <« Bacillus influenzae, from a gelatin culture; X 1000 (Itzerott and Niemann). upon blood-serum agar. It is aerobic. Without movement; does not take the Gram stain. It is best stained with diluted carbol-fuchsin, the contrast-stain being Loffler's methylene- blue. Upon glycerine-agar, over which a drop of blood has been spread, in an incubator at the end of twenty-four hours a very delicate growth occurs, which resembles condensed moisture. It is found in the sputum and in the bronchial nasal secretions and blood of influenza patients, but cannot as yet be said to be the cause of influenza. Micro-Organisms of Suppuration. The suppuration of wounds is due to the presence of germs. The knowledge of this fact is the basis of the antiseptic treatment in surgery ; for when the microbes can be destroyed or their entrance prevented, the wounds are made clean and kept without suppurating. Vari- ous forms of bacteria have been found in septic processes, and 138 ESSENTIALS OF BACTERIOLOGY-. the formation of pus cannot be ascribed to any particular one alone ; some, more common than others, are found in nearly all forms of suppuration ; others give rise to special types. Wounds are often irritated by for- FlG# 74, eign bodies and chemicals, and a dis- charge occurs in them even when every aseptic and antiseptic precau- tion has been taken ; but such a dis- charge is free from bacteria, and no more like pus than a benign growth is like a malignant one. Fig. 75. Streptococcus pyogenes: cult- ure upon agar-agar two days old (Frankel and Pfeiffer). Streptococcus pyogenes (Jakob). Streptococcus Pyogenes. (Rosenbach.) Streptococcus erysipe- latis. (Fehleisen.) Origin. — Fehleisen discovered this microbe in the lymphatics 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. Form. — Small cocci singly and in chain-like groups. Spores PATHOGENIC BACTERIA. 139 have not been found, though it is supposed because of their permanency that spores are present. Properties. — They are immotile, do not liquefy gelatine. Growth. — They grow slowly, usually on the surface, and best at higher temperatures. Colonies. — In three days a very small grayish speck, which hardly ever becomes much larger than a pin-head ; under micro- scope, looking yellowish, finely granular, the edges quite defined. Stub Cultures. — Along the needle-track little separated colonies like strings of beads, which after a time become one solid white string. Stroke Culture. — Little drops, never coalescing, having a bluish tint. 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. 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. In man, inoculations have been made to produce an effect upon carcinomatous growths. Erysipelas was always produced thereby. When it occurs upon the valves of the heart, endo- carditis results. Puerperal fever is caused by the microbe in- fecting the endometrium, the Streptococcus puerperalis of Frankel being the same germ. In scarlatina, variola, yellow fever, cerebro-spinal meningitis, and many similar diseases, the microbe has been an almost con- stant attendant. It is often associated with the diphtheria bacillus in true diphtheria, and is the cause of many of the diphtheritic affections of the throat in which the diphtheria bacillus is absent. An antistreptococcic serum has been used as a curative agent in puerperal fever, scarlatina, and other diseases sup- posed to be due to this germ. A mixture of a culture of Pyogenes and Prodigiosus has been 140 ESSENTIALS OF BACTERIOLOGY. used as an injection, with apparent benefit, in inoperable cases of sarcoma. Staphylococcus Pyogenes Aureus. (Rosenbach.) Fig. 76. Staphylococcus pyogenes albus (Jakob). Origin. — Found commonly in pus (80 per cent, of all suppura- tions), in air, water, and earth ; also in sputum of healthy persons. Form. — Micrococci in clusters like bunched grapes, hence the name staphylo, which means grape. They never form chains. Spores have not been found, though the cocci are very resistant. Properties. — Without movement ; liquefying gelatine. It gives rise to an orange-yellow pigment in the various cultures. Growth. — It grows moderately fast at ordinary temperature, and can live without air, a facultative serobin and anterobin. Colonies on Gelatine. — On second day small dots on the surface, containing in their centre an orange-yellow spot. The gelatine all around the colony is liquefied ; the size is never much greater than that attained the second day. Colonies on Agar. — The pigment remains 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 yel- low granular mass, the gelatine being all liquid. Stroke Culture on Agar. — The pigment diffused over the sur- face where the growth is, in moist masses. PATHOGENIC BACTERIA. 141 Fig. Potato. — A thin white layer which gradually becomes yellow and gives out a doughy smell. 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 phleg- monous condition arises, the capillaries be- come plugged with masses of cocci, infarct occur in kidney and liver, and metastatic ab- scesses form in viscera and joints. Garre\ by rubbing the culture on his forearm, caused carbuncles to appear. Several varieties of the pyogenic staphylo- cocci are recognized according to their color- producing properties and slight variations of growth. Of these, the staphylococcus pyog- enes aureus is the most virulent, and is con- sidered 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 floor of houses. Staphylococcus pyogenes albus differs from the preceding only in the absence of Stab culture. Micro- pigment and in its slight virulence. Welch coccus Py°genes ° aureus. describes a variety constantly found both on the skin and in its deeper layers, which he calls the staphylo- coccus epidermidis albus. Micrococcus Pyogenes Citreus. (Passett.) This liquefies gel- atine 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 pyo- genes 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. 142 ESSENTIALS OF BACTERIOLOGY. 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 varnished over. Bacillus Pyocyaneus. (Gessard.) Synonyms. — Bacterium seruginosum, bacillus fluorescens. (Schroter.) The bacillus of bluish-green pus. Origin. — Found in 1882 in the green pus in pyocyeemia. Form. — Small slender rods with rounded ends, easily mistaken for cocci. Often in groups of four and six, without spores. Properties. — Very motile ; liquefy gelatine rapidly ; a peculiar sweetish odor is produced in the cultures, and a blue pigment. Fig. 78. *«*N*«- Bacillus pyocyaneus, from an agar-agar culture ; X 1000 (Itzerott and Niemann). Growth. — Develops readily at ordinary temperature, growing quickly and mostly on the" surface ; it is aerobic. Colonies on gela- tine plate, in two or three days a greenish iridescence appears over the whole plate, the colonies having a funnel-shaped lique- faction, and appearing under low power when still young, as yellowish green, the periphery being granulated. Stab Cultures. — Mainly in upper strata, the liquefaction funnel- shaped, the growth gradually settling at the bottom, a rich green PATHOGENIC BACTERIA. 143 shimmer forming on the surface, and the gelatine having a deep fluorescence. Potato. — The potato is soaked with the pigment, a deep fold of green occurring on the surface. Staining. — With ordinary aniline dyes. Pathogenesis. — When animals are injected with fresh cultures in the peritoneal cavities or cellular tissues, a rapidly spreading oedema with general suppuration develops. The bacilli are found in the viscera and blood. If a small quantity is injected, a local suppuration occurs, and if the animal does not die it then can withstand large quanti- ties. 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 com- pound. The bacillus has no especial action on the wound, and is found sometimes in perspiration of healthy persons. Bacillus Pyocyaneus. |3. (Ernst.) A bacillus found in gray- ish pus-colored bandages. The only especial difference between this and the above is the formation of brownish-yellow pigment instead of pyocyanin. The form and appearance of cultures otherwise the same. Micrococcus Gonorrhoeae. Gonococcus. (Neisser.) In 1879 Neisser demonstrated the presence of this germ in the secretion of specific urethritis. Fi®. 79. 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. :■%. •v»- ' Immotile. Culture. — On gelatine-agar or potato they do not grow, and only upon human-blood Gonococci in gon- serum have they given any semblance of a nne^methyi' *toi£ growth. The temperature must be between (650 diameters.) Four to twelve such pairs are often found together. r 144 ESSENTIALS OF BACTERIOLOGY. 83° and 37° C, and the growth occurs very slowly and sparsely. Method of Cultivation (Wertheim).— Gonorrheal pus is mixed in a test-tube with liquid human blood serum of 40° C. temper- ature, and two dilutions are made with blood of the same tem- perature. An equal quantity of 2 per cent, agar solution is now poured into each tube, and three glass dishes are covered at once with this mixture. After being in the brood oven for twenty-four hours colonies can be discovered. Fig. 80. Gonococcus in urethral pus; X 1000 (Frankel and Pfeiffer). In three days a very thin, almost invisible, moist 3rellowish growth, seemingly composed of little drops. Under low power small processes are seen shooting out from the smooth border. It requires to be then transferred to fresh media, as it quickly perishes. Cultivation has also occurred on acid gelatine, gelatine con- taining acid urine, in acid urine itself, and albuminous urine with agar. Staining. — Colored easily with all ordinary aniline stains. PATHOGENIC BACTERIA. 145 Gram's method is not applicable, this being one of its main diagnostic features. 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 methylin blue (alcoholic solution) for 15 seconds, and rinsed in water. The gonococci are dark blue, the protoplasm of the cell pink, and the nucleus a light blue, the gonococci lying in the proto- plasm next to the nucleus. Other bacteria are similar to the gonococci in form ; they are distinguished from the gonococcus, in that they are colored with Gram's method, whereas the micrococcus of gonorrhoea is not. The points on which the diagnosis is to be made are the char- acteristic biscuit shape, the intracellular position of the organ- ism, and its failure to stain with Gram. Pathogenesis. — The attempts to infect the experiment ani- mals with gonorrhoea have so far been without success. In man, upon a healthy urethra, a specific urethritis was produced with even the 20th generation of the culture. Gonorrhoeal ophthalmia contains the cocci in great numbers, and endocarditis and gon- orrhceal rheumatism are said to be caused by the cocci. The microbes have been found long after the acute attack, when only a very slight oozing remained, and the same were very virulent. The specific inflammations of the generative organs of the female are due to this microbe, having gained entrance through the vagina, extending its influence. It is found chiefly in the superficial layers of the mucous membrane. A temperature of 40° C. for 12 hours destroys the gonococci. Gonotoxin.—A toxin has been isolated which causes fever, loss of weight, and finally death. The urethra is not immunized by repeated injections. In man the toxin causes painful indura- tions lasting several days. Similar Microbes found in the Urethra and Vagina. Micrococcus Citreus Conglomerata. (Bumra.) Similar to 10 116 ESSENTIALS OP BACTERIOLOGY. the gonococci in form, they are, however, easily cultivated, and form yellow colonies which dissolve the gelatine and grow rap- idly; the surface of the gelatine is at first moist and shiny, but later on wrinkled. Colored with Gram's method, and have no spe- cial pathological action. Found in the air and gonorrhoeal pus. Diplococcus Albicans Amplus. (Bumm. ) In vaginal secretion. The diplococci are much larger than the gonococci, but similar in form. They are also cultivated upon gelatine plates, grayish- white colonies, which slowly liquefy gelatine. They grow mode- rately rapid. Stained with Gram's method, and have no pathogenic action. Diplococcus Albicans Tardissimus. (Bumm.) Origin. — In urethral pus. Form, like gonococci. Properties, immotile ; do not liquefy gelatine. Growth, very slow at ordi- nary temperature, but more rapid at brood-heat. The colonies are small white points, which under low power appear brown and opaque. Agar Stroke Culture. — Grayish-white growth, which after two months is like a skin upon the surface. Staining. — Takes Gram's method. Pathogenesis. — None known. Micrococcus Subflavus. (Bumm.) Origin.— In lochial discharges, in vagina and urethra of healthy persons. Form. — As gonococci. Properties. — Not motile ; liquefy gelatine slowly ; a yellow- brownish pigment. Growth. — Grows slowly on all media, forming on gelatine, after two weeks, a moist yellowish surface growth. Potato. — Small half-moon-shaped colonies which, after three weeks, become light-brown in color, and covering the surface as a skin. Staining. — Colored ivith Gram. Pathogenesis. — Not acting upon the mucous membrane, but when injected in cellular connective tissue, an abscess results which contains myriads of diplococci. The gonococcus is distinguished from all these similar micro- cocci by being found usually within the cell protoplasm. PATHOGENIC BACTERIA. 147 Secondly.— Not stained with Gram's method. Thirdly. — Refusing to grow readily upon gelatine. All the similar bacteria being easily cultivated. 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 verdict until we have tested the micro-organism as above. When the germ so tested is found, the process can be called specific without a doubt. Fig. 81. / * :^v Bacillus of Tetanus with spores. Bacillus of Tetanus. (Nicolaier-Kitasato.) Oi*igin.— Nicolaier found this bacillus in the pus of a wound in one who had died of tetanus, describing it in 1884. Kitasato has since then been able to isolate and cultivate this germ. (1889.) Form.— A very delicate, slender rod, somewhat longer than the bacillus of mouse septicaemia, which is the smallest bacillus. When the spores form, a small swelling occurs at the end where the spore lies, giving it a drum-stick shape. 148 ESSENTIALS OF BACTERIOLOGY. Properties.- JSot very motile, though distinctly so ; liquefies gelatine slowly. The cultures give rise to a foul-smelling gas. Growth.— Develops very slowly, best at brood-heat (36° to 38° Fig. 82. Fig. 83. tf Racillus tetani : culture four days old in glucose-gelatine (Frankel and Pfeiffer). Six days' culture of bacillus of tetanus in gelatine (deep stab). (Frankel and Pfeiffer.) C), and only when all oxygen is excluded, an obligatory ancero- bin. In an atmosphere of carbon dioxide gas it cannot grow, but in hydrogen it nourishes. PATHOGENIC BACTERIA. 149 Colonies on gelatine plates in an atmosphere of hydrogen. Small colonies. After four days a thick centre and radiating wreath-like periphery, like the colonies of bacillus subtilis. Hujh Stab-Culture. — (The gelatine having 2 per cent, glucose added and filling the tube.) Along the lower portion of the needle- track, a thorny-like growth, little needle-like points shooting out from a straight line. The whole tube becomes clouded as the gelatine liquefies, and then the growth settles at the bottom of the tube. Agar. — At brood-heat, on agar, the growth is quite rapid, and at the end of forty-eight hours gas bubbles have formed and the growth nearly reached the surface. Bouillon. — Adding glucose to the bouillon gives a medium in which an abundant growth occurs. Cultivation from Spores. — Kitasato, by exposing a portion of sus- pected material to a temperature of 80° C. for one hour, killed off all the spores save those of tetanus, which were then cultivated. 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 tetanus, the tetanic condition starting from the point of infection. At the autopsy nothing characteristic or abnormal is found, and the bacilli have disappeared, except near the point of entrance. This fact is explained as follows : Several toxic products have been obtained from the cultures, and they are produced in the body, and give rise to the morbid symptoms. These have been isolated, and when injected singly cause some of the tetanic symptoms. The virus enters the circulation, but does not remain in the tissues. The spores are very resistant to heat, drying, and chemicals. Four toxins (among them tetanin, tetanotoxin, and spasmo- toxin) have been found. The blood and the urine contain the toxin and are fatal to animals. Immunity. — Kitasato, by inoculation of sterilized cultures, has caused immunity to the effects of virulent bacilli. An anti-toxin obtained by Tizzoni and Cattani from the serum of animals made immune by sterilized cultures has been used 150 ESSENTIALS OF BACTERIOLOGY. with curative effects in several cases of tetanus in man. It is a globulin, but differs from the anthrax anti-toxin, and it is found exclusively in the serum. By precipitation with alcohol and drying in vacuo the anti-toxin 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 puncture has also been recommended. Anti-toxin is more beneficial in chronic cases than in acute. Habitat. — The bacillus is present in garden earth, in manure ; and it has been isolated even from mortar. The earth of special districts seems to contain the bacilli in greater quantities. Fig. 84. Bacillus of malignant oedema, from the body-juice of a guinea-pig inoculated with garden earth ; X 1000 (Frankel and Pfeiffer). Bacillus (Edematis Maligni. (Koch, 1881.) Vibrion Septique. (Pasteur, 1875.) Origin. — In garden earth, found lately also in man, in severe wounds when gangrene with oedema had developed. Identical with the bacillus found in Pasteur's septicaemia. Form. — Rods somewhat smaller than the anthrax bacilli, the PATHOGENIC BACTERIA. 151 ends rounded very sharply. Long threads are formed. Very large spores which cause the rods to become spindle-shaped. Fig. 85. Fig. 86. ESfr-wl Cultures in agar of malignant (Edema, after 24 hours, at 37° C. (Fr&nkel and Pfeiffer.) Bacillus of malignant oedema growing in glucose-gelatine (Frankel and Pfeiffer). Properties. — Very motile ; liquefy gelatine ; do not produce any foul gaseous products in the body. 152 ESSENTIALS OF BACTERIOLOGY. Growth. — Grows rapidly, but only when the air is excluded, and best at brood or body heat. Roll Cultures. — (After Esmarch's method.) Small, round colo- nies with fluid contents, under low power, a mass of motile threads in the centre, and at the edges a wreath-like border. High Stab- Culture. — With glucose gelatine, the growth at first seen in the bottom of the tube, with a general liquefaction of the gelatine, gases develop and a somewhat unpleasant odor. Agar. — The gases develop more strongly in this medium, and the odor is more prominent. Guinea-Tig Bouillon. — In an atmosphere of hydrogen cloud- ing of the entire culture medium without any flocculent pre- cipitate until third day. Staining.— Are stained with the ordinary dyes, but Gram's method is not applicable. Pathogenesis. — When experiment animals, mice or guinea- pigs, are injected with a pure culture under the skin they die in 8 to 15 hours, and the following picture presents itself at the autopsy : In guinea-pigs from the point of infection, spreading over a large area, an oedema of the subcutaneous tissues and muscles, which are saturated with a clear red serous exudate free from smell, containing great quantities of bacilli. The spleen is enlarged, especially in mice. The bacilli are not found in the viscera, but are present in great numbers on the surface, i. e., in the serous coverings of the different orgfcns ; 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 gan- grene. 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 bloody serum of animals dead with the disease. Spirillum of Relapsing Fever. (Obermeier.) Syn. — Spirochseta Obermeieri. Origin. — Found in the blood of recurrent fever patients, described in 1873. PATHOGENIC BACTERIA. 153 Form.— Long, wavy threads (16 to 40 fx long), a true spiril- lum ; flagella are present. Properties. — Very motile. Has not been cultivated. Staining. — Ordinary aniline stains. Bismark brown best for tissue sections. Pathogenesis. —Found in the organs and blood of recurrent fever. Man and monkeys inoculated with blood from one suf- fering from this disease become attacked with the fever, and in their blood the spirillum is again found. It is found in the blood, only in the relapses (during the fever). After the attack the spirilla gather in the spleen and gradually die there. It has been found in the brain, spleen, liver, and kidneys. In the secretions it has not been discovered. ©°©« Fio. 87. .© <&Pod& ° Spirochseta Obermeieti in the blood (von Jaksch). Bacillus of Soft Chancre. (Ducrey-Unna.) 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. Cultivation. — Unsuccessful. Staining.— With borax, methylen-blue, decolorized with weak acetic acid. Pathogenesis. — Probably a mixed infection occurs in most chancroids, especially if buboes result. The bacillus of Ducrey 154 ESSENTIALS OF BACTERIOLOGY. is not found in unopened buboes, though often contaminating the ulcerated ones. Bacillus Icteroides. (Sanarelli, 1897.) Fig. 88. Considered the cause of yellow fever by Sanarelli, but Stern- berg and Novy regard this as not determined. It is not identical, as was once supposed, with the Bacillus X of Stern- berg, which is a variety of the Bacillus Coli. Origin. — In the tissues and blood of yellow-fever patients. Form. — A small bacillus with rounded ends, often arranged in pairs, sometimes in threads, with lateral flagella. Properties. — Motile, readily stained, decolorized by Gram's method. Does not liquefy gelatine -nor produce glucose fermen- tation. Aerobic. No acid reaction in milk. Growth. — Gelatine plates ; white kidney -shaped colonies, with a central darker portion or nucleus. Agar. — Colonies look like drops of paraffin, with margins raised above the surface. Kept alternately at 22° C. and 37° C, the colonies take on a characteristic appearance, as if an im- pression had been made in soft wax. PATHOGENIC BACTERIA. 155 Potato. — Creamy pale growth, turning brown in a week. Pathogenesis. — Dogs and rabbits, when inoculated with pure cultures, are affected with symptoms exactly similar to those seen in yellow-fever patients— a hemorrhagic gastro-enteritis, steatosis of the liver, and albuminuria. Two theories exist as to the etiology of yellow fever : (a) That it is due to the Bacillus icteroides. The results of Sanarelli have not been universally accepted, (b) That the causative agent is so small as to be microscopically invisible, and that it is transmitted from man to man through the bite of a mos- quito, the Stegomyia fasciata, which acts as intermediate host. Individuals who have allowed themselves to be bitten by in- fected mosquitoes have contracted the disease, while others exposed to infection in all ways but this have remained well. Bacillus of Bubonic Plague. (Yersin and Kitasato, 1894. ) 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, especially among those living under unsanitary conditions. Nearly at the same time Yersin and Kitasato, working inde- pendently, discovered in the bubonic swellings and blood 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 secretions of affected individuals. Form. — Short, thick rods with an indistinct capsule, rounded ends. Growing in chains in fluid media. Properties. — Immotile. Stains readily. No spores. Culti- vated best in oxygen, but is facultative anaerobic. Stains stronger at the ends, producing bipolar appearance. Gelatine not liquefied. Easily destroyed by sunlight and drying. Growth.— Best at 37° C. Gelatine. — At 22° C, in 24 hours white, point-like colonies on the plates, with broad and flat surface, turning gray and then brown. Stab. — Snow-white, spreading out on the surface to the edge, and fluorescent. 156 ESSENTIALS OF BACTERIOLOGY. Bouillon. — Granular precipitate, with clear fluid above. Agar 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. No gas formation in glucose media. Staining readily with all basic dyes. Pathogenesis. — After subcutaneous injection in rats death fol- lows in 40 to 60 hours, with symptoms of severe toxaemia and convulsions. The point of infection shows a local oedema and inflammation of the lymphatics. All the organs congested and surrounded by a bloody exudate. The characteristic bacilli in all the tissues and secretions. Nearly all the domestic animals Fig. 89. I ,1s Ij ** »? '■ * \ * '*. * ' <# V'~svx (Billings.) { Frog Plague. (E berth.) Here the bacteria are motile. They grow on potatoes and are similar to the typhoid bacillus in gelatine. They form small embolic processes in the capillaries. They cause only a local disturbance in rabbits when subcutnneously injected. An acid fermentation is produced in milk. ,. . . f Hog Cholera. (Salmon.) 1 Swedish Swine Plague. (Lelander.) The bacteria of this third division are very motile. The hog- cholera bacilli lie in the spleen and other organs in small masses like the typhoid bacillus. Rabbits die in four to eight days without any local disturb- ance. The growth on potato is strong. The Swedish swine-plague bacillus occupies a position be- tween that of Hog Cholera and Bacillus Coli Communis. The various swine-plague bacilli are but little active in fowls, differing thus widely from the chicken cholera bacillus. Bacillus of Erysipelas of Swine. (Loffler, Schiitz.) Schweine- roilaufbacillus (German). Bouget du pore (French). 168 ESSENTIALS OF BACTERIOLOGY. Origin. — Found in the spleen of an erysipelatous swine by Loffler in 1885. Form.— One of the smallest forms of bacilli known ; very thin, seldom longer than 1 p, looking at first like little needle-like crystals. Spores have not been found. Properties. — They are motile ; do not liquefy gelatine. Growth in culture at ordinary temperature, very slowly, and the less oxygen the better the growth. Gelatine Plate. — On third day little silver-gray specks, seen best with a dark background, coalescing after awhile, pro- ducing a clouding of the entire plate. Stab Cultures. — In a few days a very light, silvery -like clouding, which gradually involves the entire gelatine ; 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 tor- pidity develops with diarrhoea 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 exhaustion in 24 to 48 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 all the viscera. One attack, if withstood, protects against succeeding ones. Immunity. — Has also been attained b}T injecting vaccines of two separate strengths. Bacillus Murisepticus. (Koch.) Mouse septicaemia. 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 cul- tures exactly similar to those of swine erysipelas. BACTERIA PATHOGENIC FOR ANIMALS. 169 The pathological 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 umai depis." Form. — Yery small cocci seldom in chains. Properties, immotile ; liquefying gelatine. Growth.— Growth occurs best between 20° and 37° C, is very rapid, and irrespective of oxygen. Plates of Gelatine.— White round colonies, some on the surface 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 mammary gland of sheep, a u mal de pis" is produced which causes the death of the animal in 24 to 48 hours. The breast is found oedematous, 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. Bacillus Alvei. (Cheshire and Cheyne.) Bacillus melittoph- tharus. (Cohn.) Origin.— In foul-brood of bees. Form. — Slender rods, with round and conical-pointed ends ; very large oval spores, the rod becoming spindle-shaped when they appear. Properties. — Motile, liquefying gelatine rapidly. Grotvth.— Grows best between 20° C. and 37° C, very slowly ; aerobic. Gelatine Plates.— Small grooves are slowly formed, which unite 170 ESSENTIALS OF BACTERIOLOGY. so as to form a circle or pear-shaped growth, from which linear grooves again start. Stab Culture.— Grows first on surface, then gradually along the needle-track, long processes shooting out from the same, clouding the gelatine. Later, air-buhbles form like the cholera culture, and in two weeks the whole gelatine liquefied. Staining. —Do not take aniline dyes very well. Gram's method is, however, applicable. Pathogenesis. — If a pure culture is spread over the honey- comb containing bee larvae, or if bees are fed upon infected material, foul-brood disease will occur. Mice, if injected, die in a few hours. (Edema around the point of infection, and many bacilli contained in the cedematous fluid, otherwise no changes. Micrococcus Amylovorus (Burrill.) Origin.— In the disease called "Blight," which affects pear- trees and other plants. Form.— Small oval cells, never in chains, more the form of a bacillus. Pathogenesis. — Introduced into small incisions in the bark of pear-trees the trees perished from the " blight." The starch of the plant cell was converted into carbon dioxide, hydrogen, and butyric acid. Bacterium Termo. (Cohn.) This was a name given to a form of micro-organism found in decomposing albuminous material, and was supposed to be one specific germ. Hauser, in 1885, found three different distinct microbes which he grouped under the common name of Proteus, which have the putrefying properties ascribed to B. Termo. Proteus Vulgaris. 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 gelatine ; forms hydrogen sulphide gas ; causes putrefaction in meat. Growth. —Growth very rapid, best at 24° C, is facultative aerobic. Gelatine Plates. — Yellowish-brown, irregular colonies, with prolongations in every direction, forming all sorts of figures ; an BACTERIA PATHOGENIC FOR ANIMALS. 171 impression preparation shows these spider-leg processes to con- sist of bacilli in regular order. Stab Culture. — The gelatine soon liquid, a gray layer on the surface, but the chief part of the culture in small crumbs at the bottom. Pathogenesis.— Rabbits and guinea-pigs injected subcutane- ously die quickly, a form of toxaemia, hemorrhagic condition of lungs and intestines present. When neurin is injected previ- ously the animals do not die. This ptomaine is supposed to be generated by the proteus vulgaris. Proteus Mirabilis. (Hauser.) Differs from P. vulgaris in that the gelatine is less rapidly liquefied. Found also in putrid material. Proteus Zenkeri. (Hauser.) Does not liquefy gelatine ; otherwise similar to the other two. We have now considered some of the characteristics of the more important bacteria. The scope of this work does not allow a more extended study than we have made, which, as we are aware, has been very superficial. The larger works must be referred to, if a deeper interest is taken in the subject. APPENDIX YEASTS AND MOULDS. In works on bacteria, these true fungi, yeasts and moulds, are usually considered. They are so closely related to bacteria, and so often contaminate the culture media, and are so similar in many respects, that a description is almost a necessity. But there are several thousand varieties, and we cannot attempt to describe even all of the more important ones. It will answer our purpose to detail a few of the more common kinds, and give the principal features of the different orders. ' Saccharomycetes or Yeasts increase through budding; the spores are attached to the mother cell like a tuber on a potato. Yeasts are the cause of alcoholic fermentation in the saccha- roses. A description of the most common ones will suffice. Saccharomyces Cerevisise. (Torula Cerevisice.) This is the ordinary beer yeast. Form. — Bound and oval cells ; a thin membrane inclosing a granular mass, in which usually can be seen three or four irre- gular-shaped spores. When these become full grown they pass through the cell wall and form a daughter cell. Sometimes long chains are produced by the attached daughter cells. Growth.— They can be cultivated as bacteria in bouillon, but they grow best in beer. There are several varieties of beer yeast, each one giving a characteristic taste to the beer. Brewers, by paying special attention to the nutrient media, cultivate yeasts which give to their beers individual flavors. Mixed yeast gives rise to a poor quality of beer. Saccharomyces Rosaceus. S. Niger and S. Albicans. These yeasts are found in the air ; and instead of producing alcoholic (173) 174 APPENDIX. fermentation they give rise to a pigment in the culture media. They grow upon gelatine which they do not liquefy. Saccharomyces Mycoderma. This yeast forms a mould-like growth, a skin, on the surface of fermented liquids, but does not cause any fermentation itself. It forms the common u mould" on wine, preserves, and " sour krout." Pathogenic Yeasts. In recent years a number of workers have interested themselves in experiments with yeasts in their relation to disease; and under the name of Blastomycetes, San- felice has grouped yeasts that produce tumors resembling epi- theliomata ; and he has tried to prove that the so-called animal parasites found in malignant growths, and variously known as coccidia and sporozoa, are yeasts. The whole subject is still under discussion. Oidium. A form which seems to be the bridge between the yeast and the moulds is the oidium. Sometimes it resembles the yeasts, sometimes the moulds, and often both forms are found in the same culture. Several are pathogenic for man. Oidium Lactis. Origin. — In sour milk and butter. Form.— The branches or hyphens break up into short rod-like spores. No sporangium, as in moulds. Growth.— In milk it appears as a white mould. Artificially cultured on gelatine plates, or milk gelatine plates, it forms satin-like, star-shaped colonies, which slowly liquefy. Under microscope the form of the fungus is well seen. Agar Stroke Culture.— The little stars, very nicely seen at first ; then the culture becomes covered with them, causing a smeared layer to appear over the whole surface, with a sour odor. Properties. — The milk is not changed in any special way. It is not pathogenic for man or animals. It is found when the milk begins to sour. Oidium Albicans. {Soor.) Thrush Fungus. Origin. — Mucous membrane of the mouth, especially of infants. Form. — Taken from the surface of the culture, a form like yeasts ; but in the deeper layers, mycelia with hyphens occur. Growth. — Not liquefying; snow-white colonies on gelatine plates. YEASTS AND MOULDS. 17o Stab Culture.— Radiating yellow or white processes spring from the line made by the needle, those near the surface having oval ends. Potatoes.— The yeast form, develops as thick white colonies. Bread Mash. — Snow-white veil over the surface. Pathogenesis. — In man the parasitic thrush, or "white mouth, n is caused by this fungus. In the white patches the spores and filaments of this microbe can be found. Rabbits receiving an intravenous injection perish in twenty-four to forty-eight hours, the viscera being filled with mycelia. True Moulds. Fliigge has made five distinct divisions of moulds. It will, however, serve our purpose to classify those to be described under three headings : Penicillium, Mucor, and Aspergillus. Penicillium Glaucum. Origin.— The most widely distributed of all moulds, found wherever moulds can exist. Form. — From the mycelium, hypha? spring which divide into basidia (branches), from which tiny filaments arise (sterigmata ), arranged like a brush or tuft. On each sterigma a little bead or conidium forms, which is the spore. In this particular fungus the spores in mass appear green. Growth. — It develops only at ordinary temperatures, forming thick grayish-green moulds on bread-mash. At first these ap- pear white, but as soon as the spores form, the green predomi- nates. Gelatine is liquefied by it. Mucor Mucedo. Next to the penicillium glaucum, this is the most common mould. Found in horse dung, in nuts, and apples, in bread and potatoes as a white mould. Form. — The mycelium sends out several branches, on one of which a pointed stem is formed which enlarges to form a globu- 'ar head, a spore-bulb, or Sporangium. The spore-bulb is par- ilioned off into cells in which large oval spores lie. When the spores are ripe a cap forms around the bulb, the walls break down and the wind scatters the spores, leaving the cap or M columella"11 behind. Growth. — Takes place at higher temperatures on acid media. It is not Pathogenic. 176 APPENDIX Achorion Schbnleinii. Trichophyton Tonsurans. Microsporon Furfur. These three forms are similar to each other in nearly every particular and resemble in some respects the oidium lactis, in other ways the mucors. The first one, Achorion Schbnleinii, was discovered by Schonlein in 1839, in Favus, and is now known as the direct cause of this skin disease. Fig. 91. Achorion Schbnleinii (after Kaposi). Origin, — Found in the scaly crusts of favus. Form. — Similar to oidium lactis. Growth. — Is very sparse. On gelatine round white masses inclosed by a zone of liquefied gelatine. In milk it is destroyed. Pathogenesis. — Causes favus in man. Trichophyton Tonsurans. Found, in 1854, by Bazin, in Tinea. Form. — Similar to the achorion or favus fungus. Growth. — Somewhat more rapid than the favus, and the gela- tine quickly liquefied, Old cultures are of an orange-yellow color. Colonies have a star-shaped form. Pathogenesis. — Herpes tonsurans and the various tineee are produced by this fungus. Microsporon Furfur. Found in tinea versicolor, almost iden- YEASTS AND MOULDS. 177 tical with the above, forms dry yellow spots, usually on the chest in persons suffering from wasting diseases. Aspergillus Glaucus. Origin. — In saccharine fruits. Form. — The hypha has formed upon its further end a bulb, from which pear-shaped sterigmata arise and bear upon their ends the conidia or spores. Growth.— Best upon fruit juices. Non-pathogen fc. The mould is green. Aspergillus flams has the tufts and spores of a yellow color. A. Fumigatus. Is pathogenic for rabbits when injected into them. At the autopsy their viscera are found filled with the mould. Examination of Yeasts and Moulds. Yeasts and moulds are best examined in the unstained condition. A small portion of the colony rubbed up with a mixture of alcohol and a few drops of liquor ammonia ; of this, a little is brought upon the glass- slide covered with a drop of glycerine and the cover-glass pressed upon it. If the preparation is to be saved, the cover-glass is secured by ringing around the edges. Yeasts take methylin-blue stain very well. Cladothrices and Streptothrices. The streptothrix and cladothrix groups are classed with the higher bacteria, but their exact status is still undetermined. They may be consid- ered as representing the transition from the bacteria to the lower fungi. Streptothrix, or Cladothrix Actinomyces (ray fungus). Actinomycosis is a disease caused in man and cattle by this organism, which is commonly found in grain, particularly barley. It is probable that several varieties of the parasite can produce the characteristic lesions. It has been discovered in all countries and in various organs of the body, although its place of election is about the lower jaw, where it tends to form hard ulcerating abscesses, affecting other organs secondarily. Form. — In the granular masses of an abscess cylindrical fila- ments are matted together, and radiating outward from this zone are club-shaped branches, as the petals of an aster. In the center of the granule are numerous cocci-like bodies, and some 12 178 APPENDIX. of the ovoid or club-shaped hyphae lie detached from the clusters. Through cultivation it was found that the ovules give rise to filaments, and they then form the ovules again. Cultivation. — At 38° C. on glycerine-agar in a period of one to two weeks, pointed scales about the size of a millet-seed, center dry and prominent, margins hyaline, composed only of filaments, short and long, massed together, but no clubbed forms. By some the clubs are considered the spore organs ; by others they are thought to be encapsulated or thickened filaments. Pathogenesis. — When a portion of the growth obtained in eggs was injected into the abdominal cavity of a rabbit, actinomy- cotic processes developed upon the peritoneum. It usually gains access to the living body through a wound in the gum or some caries of the teeth. A new growth is formed, ulceration being first set up. The new tissue, composed of round cells, then undergoes soft- ening, purulent collections form and the normal structure is destroyed. The usual seat is in the maxillary bones, but the fungus has been found in the lungs, tonsils, intestines, and various other organs in man and cattle. Examination. — Well seen in the unstained condition. From the pus or scraping a small portion is taken and squeezed upon the glass slide ; if calcareous matter is present, a drop of nitric acid will dissolve the same. Glycerine will preserve the preparation. Staining.— Cover-glass specimens stained best with Gram's method. Tissue sections should be stained as follows : — Ziehl's carbol-fuchsin, ten minutes. Kinse in water. Cone, alcohol. sol. of picric acid, five minutes. Rinse in water. Alcohol, 50 per cent., fifteen minutes. Alcohol absolute, clove oil, balsam. The rays stained red, the tissue yellow. Streptothrix Madurae. (Vincent.) Origin. — Found in the disease known as Madura foot, or Mycetoma, an ulceration affecting the feet, especially of indi- YEASTS AND MOULDS 179 viduals living in the tropics. Two varieties, the pale and the black, have been described. Form. — Branched filaments resembling the actinomyces strep- tothrix in the mycelia. Spores are seen. Cultivation. — In liquid media containing vegetable infusions growth occurs best. Temper- ature of 37° C. most suited. Fig. 92. The colonies near the surface become colored red. Agar. — Glazed colonies, at first colorless, then rose-col- ored, about the size of a pea, with the central part umbili- cated and pale. Gradually the rose color fades. Acid Potato. — A slow and meager growth. Pathogenesis. — Only local re- action has been caused by in- oculation in animals. In man the disease usually follows a slight injury and attacks the leg or foot, slowly forming a nodu- lar growth, which in the course of months or a year begins to soften and ulcerate, and with the sero-pus are discharged numerous little granules, some black, some pink, containing mycelia. The limb becomes much deformed, the tissue ■WP< • M Streptothrix Madura; in a secti eased tissue(Vincent) dis- vascularized, and the degenerated area. filled with the strep- tothrix filaments.^ Staining. — The organism itself stained with ordinary stains. Gram's method for the tissue. Streptothrix Farcinica. (Nocard.) Bovine Farcy, Farcin du Boeuf. 180 APPENDIX. Origin. — A disease affecting cattle and giving rise to tubercle- like lesions in the lungs, liver, and spleen. Common in France. Form. — Small interwoven mass of threads arranged in tufts found in the centers of the tubercles. Culture. — At body-temperature in various media. Bouillon. — Colorless masses irregular in size and shape. Agar and Gelatine. — Small, rounded, opaque colonies, thicker at the periphery. Potato. — Rapid growth of pale yellow dry scales, consisting of many spores. Pathogenesis. — Pure cultures introduced into the peritoneum of guinea-pigs give rise in 9 to 20 days to tubercle-like lesions. Subcutaneous injections cause abscesses with secondary in- volvement of the lymphatics, ending in recovery. Dogs, horses, and rabbits are immune. Staining. — Wright's double stain for tissues; also Gram's. Examination of Air, Soil, and Water. Air. — Many germs are constantly found in the atmosphere about us. Bacteria unaided do not rise into the air and fly about ; they usually become mixed with small particles of dirt or dust and are moved with the wind. The more dust the more bacteria, and therefore the air in summer contains a greater number than the air in winter, and all the other differences can be attributed to the greater or less quantity of dust and wind. Methods of Examination. The simplest method is to ex- pose a glass or dish covered with gelatine in a dust-laden atmosphere or in the place to be examined. In the course of 24 to 48 hours colonies will be seen formed wherever a germ has fallen. But this method will not give any accurate results in regard to the number of bacteria in a given space ; for such a purpose somewhat more complicated methods are needed, so that a certain amount of air can come in contact with the culture media at a certain regulated rate of speed. Hesse's Method. This is the most useful of the various methods in vogue. A glass cylinder, 70 centimetres long and 3.5 centimetres in diameter, is covered at one end, by two rubber caps, the inner AIR, SOIL, AND WATER. 181 one having a hole in its centre 10 millimetres in diameter ; and at the end B a rubber cork fits in the cylinder; through this cork a glass tube 10 mm. in diameter passes, which is plugged at both ends with cotton. The cylinder and fittings are first washed in alcohol and sublimate and then placed for one hour in the steam chamber. Removing the cork of the cylinder, 50 cubic centimetres of sterile gelatine in a fluid condition are introduced and rolled out on the sides of the tube, after the manner of Esmarch, leaving a somewhat thicker coating along the under side of the Fig. 93. cylinder. The aeroscope, as the cylinder and its fittings are called, is placed upon an ordinary photographer's tripod and the glass tube, which passes through the rubber cork, connected with an aspirator, the cotton having first been removed from its 82 APPENDIX. Fig. 94. outer end. The aspirator consists of two ordinary wash-bottles connected with each other by a rubber tube, 0. They are at- tached to the tripod with a small hook one above the other, the upper one half filled with water and slightly tilted. When the apparatus is wanted, the outer rubber cap at the end A of the aeroscope is removed, the air can then pass through the small hole in the other cap, and the germs fall upon the gelatine in the tube, the cotton in the small glass tube at the other end preventing the germs from getting out. The aspirator is set in use by tilting the upper bottle so that the water flows into the lower, this creates suction and draws the air through the aeroscope. The amount entering estimated by the capacity of the wash-bottle. The rate at which it enters depending upon the rate of the flow of water, which can be regulated. Hesse advises for rooms and closed spaces 1 to 5 litres, at the rate of 2 minutes a litre, and for open spaces, 10 to 20 litres at 4 minutes a litre. Plate cultures can be made from the colonies which de- velop in 8 to 10 days in the cylinder. Petri's Method. The air pumped or sucked through sand filters, and the sand then mixed with gelatine. Sand is sterilized by heating to redness, and while still warm placed in test tubes which are Sand filter then plugged. (Sand which has been passed a ter e n. through a sieve with meshes 0.25 millimetre wide is the kind required.) A glass tube 9 centimetres long is pro- vided with two portions of sand each 3 cm. long and £ cm. apart, little plates of brass gauze keeping the portions in position. The tube and its contents now sterilized in hot air oven at 150° C, the ends having first been plugged with cotton. One end of the tube is then fitted with a rubber cork through which passes a glass tube, which is connected with an aspirator (a hand-pump with a known capacity). AIR, SOIL, AND WATER. 183 If a hundred litres of air pass through the tube in fifteen min- utes the germs should all be arrested in the first sand filter. And when the filters are removed and thoroughly mixed with gelatine, each filter for itself, there should be no colonies de- veloped from the second filter, i. c, the one nearest the aspirator. Sedgwick-Tucker Method. A special form of tube is used, called an aerobioscope. It consists of a neck 2.5 cm. in length, Fig. 95. Sedgwick-Tucker aerobioscope. an expanded portion 15 cm. long, and a long narrow tube of 15 cm. After sterilization the tube is partly filled with granulated sugar, which is the filtering material. By means of a vacuum gauge and an air-pump, or ordinary aspirating bottles, the vol- ume of air passing through the apparatus can be determined. After the air has been passed through, the sugar is gently shaken from the narrow tube into the expanded portion, and 20 c. c. of liquefied gelatine is poured in. The sugar dissolves, and the mixture is then rolled on the inner side of the glass as an Esmarch tube. This part of the apparatus is divided into squares to make the counting of colonies easy. The aerobio- scope is very highly recommended. Varieties Found in Air. The only pathogenic bacteria found with any constancy are the staphylococcus aureus and citreus; but any bacterium can be, through accident, lifted into the atmo- sphere, and in certain places may be always found — the bacillus tuberculosis, for example, in rooms where many consumptives are living. Non-Pathogenic. The micrococci predominate. Sarcinse, yeasts, and moulds constantly contaminate cultures. In the ordinary habitations the average number of germs to the litre of air does not exceed five. Around water-closets, where one would imagine a great num- ber to exist, owing to the undisturbed condition of the air, but few will be found. 184 APPENDIX. Examination of Water. The bacteriological examination of water is to-day of as much importance as the chemical analy- sis, and must go hand in hand with it. At the start we must say that a water containing thousands of germs to the cubic centimeter is far less dangerous than one containing but two germs, if one of these two be a typhoid ba- cillus. It is not the number that proves dangerous, it is the kind. If a natural water contains more than 500 germs to the cubic centimeter, it were well to examine its source. Bacteriology performs the greatest service in testing the devices which are intended to render water fit for drinking. As a diagnostic aid the examination is of but little use. An epidemic of typhoid fever occurs, the water is suspected, an ex- amination is undertaken ; but the days of incubation and the days passed before the water is analyzed have given the typhoid germs, if any had been present, ample time to disappear, since in water that contains other bacteria they live a very short time only. Again, the water tested one day may be entirely free and the next day contain a great number, and before the typhoid germ can be proven to be present in that particular water, the epidemic may be past. Purity of Waters. The purest water we have is the natural spring water— water that has slowly filtered its way through various layers of gravel and sand and comes finally clear and sparkling from the ground. It is without germs ; but let such a water stand walled up in cisterns or wells, it becomes as surface water, open to all sorts of impurities, and the bacterial nature of it changes every moment. Artesian or Driven Well. The driven well will secure to a cer- tain extent a pure water. It is the only form of well or cistern that will insure this, since the water does not become stagnant in it ; but it may connect with an outhouse, the soil being very loose, allowing the products of germs of refuse water to find their way into the well. If a chemical examination shows increased amounts of chloride of sodium, a contamination can be mooted. Filtered Water. Dangerous as surface water is, the greater quantity used, is such : the inhabitants of larger towns and cities AIR, SOIL, AND WATER. 185 using chiefly the rivers and other large waters which course near them for drinking purposes. A purification or filtration can in a certain measure render these waters harmless. Filtration is often carried on on a large scale in the water- works of cities and towns. Bacteriological examination is here of great service to deter- mine if a water, which has been filtered and may have a very clear appearance, and give no harmful chemical reaction, yet be entirely free, or nearly so, from germs ; in other words, if the filter is a germ filter or not. Charcoal Sponge and Asbestos, the materials formerly in use, are objectionable because germs readily develop on them and clog them, so that they require frequent renewal. In very large filters, sand and gravel give the best results ; the number of germs in a cubic centimetre is reduced to forty or fifty and kept at that number. This is a very pure water for a city water, though, as we stated before, not a safe one, for among those forty germs very dangerous ones may be found. It is then necessary for the users to refilter the water before drinking it, through a material which will not allow any germs to pass. Pasteur-Chamberland Filter. This very perfect filter, which is now in almost universal use, consists of a piece of polished porcelain in the form of a cylinder closed at one end and pointed at the other. It is placed in another cylinder of glass or rubber and the pointed portion connected with a bottle containing the water, or directly with faucet of the water-pipe. The water courses through the porcelain very slowly and comes out entirely free from germs ; pipe-clay, bisque, infusorial earth, and kaolin are also perfect filters. The only disadvantage is the long time it takes for the water to pass through. Pressure is used to accelerate the passage in the form of an aspirator or air-pump. The force of the hydrant water is also sufficient to produce a steady, small stream. These porcelain cylinders can easily be sterilized and the pores washed out. All the cylinders or bougies are not germ proof, so that they must be tested, and most of them must be cleaned every fourth day, or they will allow germs to pass through. 186 APPENDIX. Boiling as a means of purifying. When such a filter cannot be obtained, the only alternative is to boil all the water to be used for drinking ; and this should especially be done in times of typhoid and cholera epidemics. Methods of Examination. Since the germs rapidly multiply in stagnant water, an examination must not be delayed longer than an hour after the water has been collected. Every pre- caution must be taken in the way of cleanliness to prevent con- tamination ; sterilized flasks, pipettes, and plugs must be at hand, and the gelatine tubes best inoculated on the spot. If this cannot be done, the sample should be packed in ice until it arrives at the laboratory. The sample is placed in a steri- lized glass flask, and the flask then closed with a sterile cotton plug. A sterilized pipette is then dipped into the flask and 1 c.c. of the water withdrawn and added to a tube of gelatine, the gelatine being in a fluid condition. To a second tube, £ c.c. is added. The tubes are then shaken so as to thor- oughly mix the water with the gelatine, and then poured upon wide glass plates, one plate for each tube ; the plates are then placed in the moist chamber, and in two or three days examined. A temperature of 18° to 20° C. is best. Many water- bacteria are hindered by higher degrees of heat. If the germs are equally divided, there should be one-half the number on one plate that there is on the other; thus the ? c.c. serves as control. Water that is very rich in germs requires dilution with ster- ilized water 50 to 100 times. Fewer colonies will be found on agar than on gelatine even at the same temperature. To count the colonies which develop upon the plates, a spe- cial apparatus has been designed, known as Wolfhiigel's Apparatus. A glass plate divided into squares, each a centimeter large, and some of these subdivided. This plate is placed above the gelatine plate with the colonies, and the number in several quadrants taken, a lens being used to see the smaller ones. The petri saucers can be used instead of plates, and an appa- ratus on the Wolf hiigel plan can be obtained to count the colo- nies. It is best to count all the colonies on the plate or dish. AIR, SOIL, AND WATER. 187 Agar and bouillon are used in qualitative analyses. A large quantity of the water is taken (about 100 c. cm.) and mixed with 25 c.c. of bouillon ; the mixture is then placed in an in- cubator. The ordinary water-bacteria do not bear the higher temperatures very well, and therefore pathogenic organisms— as cholera, for instance — will be found almost in pure cultures. The growth of intestinal bacteria is also favored by glucose bouillon (2 per cent.), and fermentation ensues. If a fermenta tion-tube (Smith's) is used, the gas collects at one end, and the bacteria can be further cultivated and studied. Varieties Found. The usual kinds found are non-patho- genic, but, as is well known, typhoid and cholera are princi- pally spread through drinking water, and many other germs may find their way into the water. Some of the common varieties give rise to fluorescence, or produce pigment. Eisenberg gives 100 different varieties as ordinarily found. Other intestinal diseases also are supposed to be water borne, and the presence of large numbers of the Bacillus coli communis is strongly suggestive of sewage contamination. Ice supplies require the same supervision as water supplies, for many bac- teria, like the typhoid bacillus, retain their vitality for weeks after freezing. The Examination of the Soil. The upper layers of the soil contain a great many bacteria, but because of the difficulty in analyzing the same, the results are neither accurate nor con- stant. The principal trouble lies in the mixing of the earth with the nutrient medium ; little particles of ground will cling to the walls of the tube, or be imbedded in the gelatine, and may contain within them myriads of bacteria. As with water, the soil must be examined immediately or very soon after it is collected, the bacteria rapidly multiplying in it. When the deeper layers are to be examined, some precautions must be taken to avoid contamination with the other portions of the soil. One method, very laborious and not often practical, is to dig a hole near the spot to be examined and take the earth from the sides of this excavation. Frankel's Borer. Frankel has devised a small apparatus in the form of a borer, which contains near its lower end a small 188 APPENDIX. cavity, which can be closed up by turning the handle, or opened by turning in the opposite direction. It is introduced with the cavity closed, and when it is at the desired depth, the handle is turned, the earth enters the cavity, the handle again turned, incloses it completely, and the borer is then withdrawn. The earth can then be mixed with the gelatine in a tube, and this gelatine then rolled on the walls of the tube after the man- ner of Esmarch, or it can be poured upon a glass plate, and the colonies developed so. Another method is to wash the earth with sterilized water, and the water then mixed with the gelatine, as many of the germs are taken up b}r the water. The roll-cultures of Esmarch give the best results, many of the varieties usually found being anaerobic. Animals inoculated with the soil around Berlin die almost always of malignant oedema, and with that of some other towns invariably of tetanus. Many of the germs found are nitrogen formers and play a great role in the economy of the soil. Nitrifying organisms are found in the superficial layers of the earth. Organic matters found in sewage and in the faecal evac- uations of animals form the basis for their activity, whereby nitrates, ammonias, and nitric acid result. The nitrogen neces- sary for the growing plant is thus produced. The nitro-monas of Winogradsky belongs to this group. The Bacteria of Milk and Other Foods. Milk as secreted is sterile, but at every step in its passage from the cow to the con- sumer it is liable to contamination. Even the lower portion of the teat is a source of infection, owing to the presence of stagnated milk from the former milking, and, as consumed, milk usually contains thousands to millions of bacteria to the cubic centimetre. Sterilization or Pasteurization and supervision of the dairies should always be carried out on milk used for infant feeding. 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 not infrequent so-called ptomaine poisoning observed after the consumption of ice-cream, sausage, canned meats, etc., is the result of the action of bacteria or their products EXAMINATION OF THE HUMAN BODY. ] go, BACTERIOLOGIC EXAMINATION OF THE ORGANS AND CAVITIES OF THE HUMAN BODY. The body, on account of its constant contact with the sur- rounding air, is necessarily exposed to infection, and we would be likely to find on the skin and in the oral, anal, and nasal cavities the varieties of micro-organisms 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 micro-organisms met with on the skin are non-pathogenic, although underneath the nails and in the hair, pus-forming micro-organisms often occur, producing sometimes serious abscesses. In the sweat-glands and the sebaceous glands various organ- isms have been found. The Staphylococcus epidermidis albus of Welch is present normally. In foul-smelling perspiration of the feet Rosenbach found Saprogenes No. II., which is pathogenic for rabbits. Micrococcus cereus albns and flavus, Diplococcus liquefa- ciens albus and flavus, Staphylococcus pyogenes aureus, and Streptococcus pyogenes are found underneath the nails. In eczema, Diplococcus albicans tardus, D. citreus liquefa- ciens, D. flavus liquefaciens, and Ascobacillus citreus. In colored sweat, Micrococcus hsematodes, Bacillus pyocya- neus. 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. 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 xero- sis, are special germs found on the conjunctiva; the other va- rieties of air- and water-organisms, and those usually present on the skin, are also found. 190 APPENDIX. The Mouth. The mouth is a favorite seat for the development of bacteria. The alkaline saliva, the particles of food left in the teeth, the decayed teeth themselves, all furnish suitable soil for their growth. Quite a number of germs have been isolated and their prop- erties 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, Micrococcus gingivae pyogenes, Bacillus dentalis viridans, B. pulpae pyogenes, Microccocus of sputum-septicaemia, and M. salivarus septicus are a few of the germs cultivated by Miller and Biondi from the mouth. 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 patients is always saturated with tubercle bacilli. Ear. In the middle ear of new-born infants no pathogenic organisms were found, but quite a number of non-pathogenic ones. In affections of the ear the pneumo-bacillus and the Staphylococcus pyogenes are most frequent. 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, B. striatus albus et flavus, B. capsulatus mucosus, and Vibrio nasalis are some of the organisms described 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 dimin- ished in quantity or absent altogether, the conditions for the EXAMINATION OF THE HUMAN BODY. 191 growth of bacteria are more favorable. The alimentary canal of the newly born infant is sterile, but in a few hours micro- organisms begin to appear. Some gastric bacteria normally present are Sarcina ventric- uli, Bacterium lactis aerogenes, Bacillus subtilis, B. amylo- bacter, B. megaterium. The intestinal organisms are more numerous, and the mu- cous lining of the intestines and the secretions there present are favorable to germ-growth. Bacillus geniculates, 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 microbic activity, but experiments with animals have recently shown that life and digestion can proceed in a perfectly sterile condi- tion. Food and air sterilized will not develop bacteria in the faeces. In the faeces 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, B. butyricus, B. putrificus coli, B. lactis aerogenes, B. coli commune, B. 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 albi- cans 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, and it is considered identical with the so-called syphilis bacillus of Lustgarten. In urethral pus a number of diplococci other than the gono- cocci have been isolated. 192 APPENDIX. 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. A description of uro-bacteria will be found on page 91. Micro-organisms of the Blood. Many of the bacteria de- scribed in the body of this book are found in the blood of the animal they infect ; thus, anthrax bacilli are always found in the blood, whereas tubercle bacilli seldom, if ever, enter this secretion. When animals are subcutaneously injected with pneumo- cocci they are found in large quantities in the blood. The dis- eases of a hemorrhagic nature affecting fowls and swine usually show the presence of bacteria in the vascular system. Bacteria may be recovered from the blood in all forms of septic infection, such as general sepsis, malignant endocarditis, and puerperal sepsis. Method of Examination. — A drop of blood can be spread on a cover-glass and stained with the ordinary dyes, as sputum, pus, or serum ; 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 (1 to 5 per cent.). The haemoglobin is thereby extracted, and the corpuscles appear then only as faint out- lines. Instead of " fixing " by heat, Canon employs alcohol for five minutes, especially in staining for influenza bacilli, which have been detected in the blood. This method, however, requires the presence of enormous numbers of bacteria in order to succeed, and the plan com- monly employed consists in making " blood cultures." As large a quantity of blood as possible — never less than 10 c.c. — is taken from a superficial vein, the median basilic, for ex- ample, 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 trans- ferred to culture tubes, which are then studied in the custom- arv manner. PLATE IV. -' < h ! y i . ' \> H I o BACILLI OF SYMPTOMATIC ANTHRAX, WITH SPORES iooo* (Frankel and Pfeiffer.) DIPHTHERIA BACILLUS PURE CULTURE ioooX- (Frankel and Pfeiffer.) PLATE V. PFEIFFER'S CAPSULE BACILLUS IN BLOOD ioooX- 'Frankel and Pfeiffer.) YEAST-CELUS 500 X- (FrSnkel and Pfeiffer.) PLATE VI. PENICILLIUM GLAUCUM 500 X. (Frankel and Pfeitter.) ASPERGILLUS FUMIGATUS 500 X- (Frankel and Pfeiffer.) INDEX Abbe's condenser, 27 Achorion Schonleinii, 176 Actinomyces, 177 Actinomycosis, 177 Aerobin, 24 .Kstivo-autumnal form of malarial parasite, 161 Agar-agar, 54 bouillon, 54 glycerine, 55 Agglutination reaction for tubercle bacillus, 106 Agglutinins, 7G Air, examination of, 180 Alexin, 75 Amboceptor, 75 Amoeba dysenteriae, 163 Anaerobin, 24 Anilin dyes, 30 oil, 31 oil water, 31 Animals for experiment, 76 tuberculosis in, 105 Anopheles, 159 Anthrax, 94 Anthraxin, 97 Antitoxin of diphtheria, 113 of pneumonia, 134 of tetanus, 149 Antituberculous serum, 106 Arnold's sterilizer, 45 Arthrospores, 22 Asexual cycle in man, 160 Aspergillus fumigatus, 177 glaucus, 177 Autoclave of Chamberland, 46 Bacillus acidilactici, 84 aerogenes capsulatus, 158 Bacillus, alvei, 169 amylobacter, 85 anthracis, 94 avicidis, 165 Boas-Oppler, 93 butyricus, 85 capsule, 136 coeruleus, 88 coli communis, 122 comma, 124 erythrosporus, 88 fluorescens, 142 fluorescens liquefaciens, 88 geniculatus, 93 icteroides, 154 indicus, 81 Klebs-Loffler, 110 lactis cyanogenus, 86 erythrogenes, 86 lepra, 107 mallei, 108 megaterium, 82 melittophtharus, 169 mesentericus vulgatus, 81 Milzbrand, 94 murisepticus, 168 mycoides, 82 cedematis maligni, 150 of American swine plague, 167 of anthrax, 94 of bluish-green pus, 142 of bubonic plague, 155 of cattle plague, 167 of chicken cholera, 165 of diphtheria, 110 pseudo-, 114 of dysentery, 157 of French swine plague, 167 of glanders, 108 237 238 INDEX. Bacillus of hog cholera, 167 of influenza, 137 of malignant oedema, 150 of mouse septicaemia, 1 68 of ox plague, 167 of rabbit septicaemia, 167 of soft chancre, 153 of steer plague, 167 of Swedish swine plague, 167 of swine erysipelas, 167 of symptomatic anthrax, 164 of syphilis, 108 of tetanus, 147 of typhoid fever, 115 paracolon, 121 paratyphoid, 121 phosphorescens gelidus, 89 indicus, 89 indigenus, 89 pneumo-, 131 potato, 81 prodigiosus, 80 pseudo-diphtheria, 114 psittacosis, 121 pyocyaneus, 142 P-, 143 ramosus, 82 smegma, 108 spinosus, 84 subtilis, 83 tuberculosis, 97 products of, 106 violaceus, 87 Bacteria, 17 action in causing disease, 70 asporogenic, 22 effect on body, 71 fluorescent, 88 higher, 17 in air, 183 in milk, 84 in urine, 91 in water, 87, 184 infective, 71 life of, 23 lower, 17 non-pathogenic, 80, 183 of food, 188 of hemorrhagic septicaemia, 166 Bacteria of milk, 188 of pneumonia, 130 origin of, 23 pathogenic, 25, 70, 94, 183 persistence in water, 121 phosphorescent, 89 pyogenic, 71 similar to cholera, 127 staining of, 30 structure of, 18 suppurative, 71 tables of, 194-235 toxic, 70 unstained, 27 vital actions of, 24 Bactericie du charbon, 94 Bacteriolysis, 75 Bacterium acidi lactici, 84 aeruginosum, 142 Balticum, 90 Fischeri, 90 Pflugeri, 90 syncyanum, 86 termo-, 170 ureae, 91 zopfi, 83 Beef extract, 48 Beggiatoa alba, 90 Behavior of bacteria to Gram' stain, 41 Benches for glass plates, 63 Biedert's method. 103 Black-leg, 164 Blight, 170 Blood cultures, 192 Blood-coagulum, 58 Blood-serum as media, 55 Boas-Oppler bacillus, 93 Botkin's method, 69 Bouillon, 48 gelatine, 52 guinea-pig, 59 preparation of, 48 Bovine tuberculosis, 105 Bowhill's orcein stain, 40 Bread mash, 51 Brood-oven, 56 Brownian movements, 19 Buchner's method, 69 INDEX. 239 CArsuLE stain of Hiss, 35 of Welch, 35 Carbol-thionin solution, 34 Cattle plague, 167 Cell contents, 18 wall, 18 Celloidin sacs, 79 Cellular theory, 74 Charbon symptomatique, 164 Charcoal niter, 185 Chemotaxis, 74 Cholera, 124 Pfeitfer's immunity, 127 Cladothrices, 177 Cladothrix actinomyces, 177 dichotoma, 90 Clostridium, 21 - butyricum, 85 Complement, 75 Cotton plugs, 47 Cover-glass preparations, 35 Crenothrix, 90 Kulmiana, 90 Cultivation, 42 artificial, 42 methods of, 42 of anaerobins, 67 Cultures, appearances of, 64 egg, 59 glass-plate, 62 glass-slide, 60 potato, 49 rolled, 64 test-tube, 60 Cytase, 75 Dkcolorants, 32 Diphtheria, 110 Diplococcus albicans ampins, 146 tardissimus, 146 lanceolatus, 132 of meningitis, 135 of pneumonia, 130 Disinfectants, 42 Drying specimens, 35 Dunham's rosalic acid solution, 59 peptone solution, 125 Dysentery, 157 Effect of age, 26 Ehrlich's side-chain theory, 75 Eisner's medium, 59 Endospores, 21 Enteric fever, 115 Esmarch's method, 67 • tubes, 64 Examination of human body, 189 Experiments on animals, 76 Favus, 176 Ferments, 25, 71 Filters, 185 hot-water, 53 Pasteur-Chamberland, 185 sand, 182 Fishing, 66 Fission, 20 Fission-fungi, 17 Flagella, 19 staining of, 41 Fluorescence, 26 P'ood, bacteria of, 188 Foul-brood, 169 Franker s borer, 187 method for anaerobins, 68 stain for tubercle bacillus, 101 Frog plague, 167 Fuchsin, carbol-, 31, 33 Fungi, 173 Fungus, rav, 177 thrush, 174 Gabbett's stain, 34, 101 Gametes, 160 Gas formation, 26 Gelatine, 52 bouillon, 52 carbolized, 117 paste, 37 plates, 170 Gelatinous membrane, 18 Germination, 22 Glucose broth, 49 Glycerin broth, 49 Gonococcus, 143 Gonorrhoea, 143 Gonotoxin, 145 240 INDEX Gram's stain, 33 Gruber-Widal blood-serum test, 117 HjEMAMCEBA malarise, 159 vivax, 159 Hsemomenas prcecox, 160 , Hanging-drop, 29 Haptins, 75 Haptophores, 75 Heat as disinfectant, 43 dry, 43 moist, 44 Hemorrhagic septicaemia, 166 Herpes tonsurans, 176 Hesse's method for air, 180 for anaerobins, 67 Hiss' capsule stain, 35 typhoid medium, 59 Hog cholera, 167 Homogeneous lens, 26 Hot-air oven, 43 Hot- water filter, 53 Hiippe's method, 68 Immersion lens, 26 Immune body, 75 Immunity, 72 theories of, 74 Incubators, 56 Infection, 70 conditions necessary to produce, 70 > Inoculation of animals, 76 Iodin, 32 Iris blender, 27 Iron box for plates, 62 Japanese method, 55 Jenner's stain, 162 Klatsch preparations, 66 Koch's rules, 79 stain, 33 steam-chest, 44 Kiihne's method, 41 stain, 33 Leprosy, 107 Leptothrix buccalis, 90 Liborius's method for anaerobins, 67 Locomotion, 19 Loftier' s alkaline stain, 33 blood serum, 111 mordant, 33 Lysins, 75 Macrocytases, 75 Macrogamete, 161 Macrophages, 74 Malarial parasite, 159 Malignant oedema, 150 Mallein, 110 Marchoux's thionin stain, 162 Marmorek's serum, 106 Material from animals, 79 Media, nutrient, 48 solid, 49 transparent, 52 Merozoites, 160 Metchnikoff ' s theory, 74 Microbe en huit, 165 Micrococci similar to gonococcus, 145 Micrococcus amylovorus, 170 cereus albus, 141 flavus, 141 cholera gallinarum, 165 citreus conglomeratus, 145 indicus, 81 melitensis, 159 of gonorrhoea, 143 of mal de pis, 169 of sputum septicaemia, 132 Pasteuri, 132 pyogenes aureus, 141 citreus, 141 tenuis, 142 subflavus, 146 tetragenus, 135 ureae, 91 Microcytases, 75 Microgametes, 160 Microgametocytes, 160 Micro-organisms of suppuration, 137 Microphages, 74 Microscope, 26 NDEX. 241 Microsporon furfur, 176 Milzbrand, 94 Moist chamber, 50 Mordants, 31 Mosquito, sexual cycle in, 160 Moulds, 175 examination of, 177 Mouse septicaemia, 168 Movements, vibratory, 19 Mucor mucedo, 175 Mycoprotein, 18 Nail culture, 132 Neisser's stain, 34 Nicolle's solution, 34 Nitrification, 25 Nitromonas, 188 Nivellier apparatus, 62 Nutrient media, 48 Odors in cultures, 25 Oi'dium, 174 albicans, 174 lactis, 174 Oil immersion, 26 Orcein stain, 40 Oxidation, 25 Parasites, 23 malarial, 159 Park's method, 69 Pasteur filter, 185 Pathogenic yeasts, 174 Penicillium glaucum, 175 Peptone solution, 58 Dunham's, 125 Petri's sand Altera, 182 saucer, 63, 186 Pfeiffer's immunity from cholera, 127 Phagocytic theory, 74 Phosphorescence, 25 Pigmentation, 25 Platinum needles, 28 Pneumo-bacillus, 131, 132 Potato cubes, 51 cultures, 49 inoculation of, 51 in test-tubes, 51 16 Potato mash, 51 Precipitins, 76 Products of tubercle bacilli, 106 Proteins, 25 Proteus, 170 mirabilis, 171 vulgaris, 170 Zenkeri, 171 Protozoa, pathogenic, 159 Pseudo-diphtheria bacilli, 114 Ptomaines, 25, 71 Putrefaction, 25 Pyocyanin, 143 Pyrosoma bigeminum, 163 Quartan forms of malarial para- site, 161 Rabbit septicaemia, 167 Rauschbrand, 165 Ray fungus, 177 Receptors, 75 Reduction, 25 Relapsing fever, 152 Removing excess of stain, 36 Reproduction, 20 Rosalie acid solution, 59 Rotz, 108 Rouget du pore, 167 Roux's stain, 34 test-tube, 51 Saccharomyces albicans, 173 cerevisiae, 173 mycoderma, 174 niger, 173 rosaceus, 173 Sapremia, 71 Saprophytes, 23 Sarcina, 92 alba, 93 aurantica, 93 flava, 93 lutea, 92 rosea, 93 ventriculi, 93 Schizomycetes, 17 Sehizophyceae, 17 Schizophyta, 17 242 INDEX. Schultz's method, 49 Schweinerotlauf, 167 Sedge wick-Tucker method, 183 Septicemia, 71 Serum, antituberculous, 106 test, 125 Serum-agar, 111 Sexual cycle in mosquito, 160 Slides, concave, 29 Small-pox, 163 Soil, examination of, 187 Solutions, composite, 31 formulae of, 32 saturated, 32 stock, 31, 32 weak, 31, 32 Soor, 174 Spasmotoxin, 149 Specimens, cover-glass, 35 cutting of, 37 drying of, 35 Klatsch, 66 Spirillum, 18 cholera?, 124 concentricum, 92 Finkleri, 127 of relapsing fever, 152 rubrum, 92 tyrogenum, 128 Spirochete Obermeieri, 152 Spores, arthro-, 21, 22 contents of, 21 endo-, 21, 22 formation of, 21, 22 requisites for, 22 resistance of, 22 staining of, 40 Sporidium vaccinale, 163 Sputum, hardened, 104 Stain, alkaline, 31, 33 aniline-water, 32 chloroform methyl-blue, 87 Gabbett's, 34 Gram's, 33 Hiss', 35 Koch's, 33 Kiihne's, 33 Loffler's, 33 Neisser's, 34 Stain, Roux's, 34 Unna's, 33 Welch's, 35 Ziehl-Neelsen, 33 Staining, Ernst's method of, 41 general method of, 35 Gram's method of, 39 Jenner's method of, 162 Kiihne's method of, 41, 109 Loffler's method of, 109 Marchoux's method of, 162 of capsule of bacillus of pneu- monia, 132 of flagella, 41 of malarial parasite, 162 of milk, 87 of spores, 40 of sporogenic bodies, 41 of tissue sections, 37, 39 rapid method of, for bacillis tu- berculosis, 101 slow method of, for bacillus tu- berculosis, 102 solutions, 31 special methods of, 39 Weigert's method of, 41 Wright's method of, 162 Staphylococcus, 18 pyogenes albus, 141 aureus, 140 epidermidis albus, 141 Sterilization, 42 fractional, 47 Streptococcus, 18 erysipelatis, 138 puerperalis, 139 pyogenes, 138 Streptothrix, 177 farcinica, 179 Madura, 178 Suppuration, 137 Swine erysipelas, 167 plague, 167 Tertian form of malarial para- site, 161 Test-tubes, 47 Tetanin, 119 Tetanotoxin, 149 INDEX 243 Tetanus, 147 Thermo-regulator, 58 Thread reaction, 76 Thrush, 174 Tinea, 176 Tissue preparations, 37 Toxalbumins, 25, 71 Toxins, 25, 71 nature of, 71 Toxophores, 75 Trichophyton tonsurans, 176 Trypanosomes, 163 Tuberculin, 106 Tuberculin E, 106 Tuberculocidin, 106 Tuberculosis, 97 in animals, 105 Typhoid fever, 115 medium of Hiss, 59 Typhotoxin, 121 Urine media, 59 Vaccinia, 163 Vibrio, Finkler-Prior, 127 Metschnikovi, 129 Vibrion septique, 150 Water, bacteria in, 87, 127, 180 examination of, 184 persistence of bacilli in, 121 Weigert's method of staining, 41 Welch's capsule stain, 35 Widal's serum test, 125 Wild plague, 167 Wire cages, 47 Wolf hiigel's apparatus, 186 Wright's chromatin stain, 162 method, 69 Yeasts, 173 examination of, 177 pathogenic, 174 Yellow fever, etiology, 155 Ziehl's solution, 33 Zoogloea, 18 SAUNDERS' BOOKS Nervous and Mental Diseases, Children, Hygiene, Nursing', and Medical Jurisprudence W. 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In this new edition the book has been thoroughly revised, and upward of two thousand new terms have been added, thus bringing the book absolutely up to date. The book con- tains hundreds of terms not to be found in any other dictionary, over ioo original tables, and many handsome illustrations. PERSONAL OPINIONS Howard A. Kelly, M. D., Professor of Gynecology, Johns Hopkins University, Baltimore. " Dr. Dorland's dictionary is admirable. It is so well gotten up and of such conve- nient size. No errors have been found in my use of it." Roswell Park, M. D., Professor of Principles and Practice of Surgery and of Clinical Surgery, University of Buffalo. " I must acknowledge my astonishment at seeing how much he has condensed within relatively small space. I find nothing to criticize, very much to commend, and was inter- ested in finding some of the new words which are not in other recent dictionaries." PERSONAL HYGIENE. Galbraith's Four Epochs qf Woman's Life Second Revised Edition — Recently Issued The Four Epochs of Woman's Life : A Study in Hygiene. By Anna M. Galbraith, M. D., Fellow of the New York Acad- emy of Medicine, etc. With an Introductory Note by John H. Musser, M. D., Professor of Clinical Medicine, University of Pennsylvania. 121110 volume of 247 pages. Cloth, $1.50 net. In this instructive work are stated, in a modest, pleasing, and conclusive manner, those truths of which every woman should have a thorough knowl- edge. Written, as it is, for the laity, the subject is discussed in language readily grasped even by those most unfamiliar with medical subjects. Birmingham Medical Review, England " 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 whole- Pyle's Personal Hygiene A Manual of Personal Hygiene : Proper Living upon a Physiologic Basis. By Eminent Specialists. Edited by Walter L. Pyle, A. M., M. D., Assistant Surgeon to Wills Eye Hospital, Philadelphia. Octavo volume of 441 pages, fully illustrated. Cloth, $1.50 net. NEW (2d) EDITION— RECENTLY ISSUED In this new second edition there have been added new chapters on Home Gymnastics and Domestic Hygiene, besides an Appendix of Emergency Pro- cedures. Boston Medical and Surgical Journal "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." SAUNDERS' BOOKS ON Draper's Legal Medicine A Text-Book of Legal Medicine. By Frank Winthrop Draper, A. M., M. I)., Professor of Legal Medicine in Harvard University, Boston. Handsome octavo of 573 pages, illustrated. Cloth, $4-oo net. RECENTLY ISSUED The author of this work has had twenty -six years' experience as Medical Examiner for the city of Boston, his investigations comprising nearly eight thousand deaths under a suspicion of violence. Hon. Olin Bryan, LL. B. Professor of Medical Jurisprudence, Baltimore Medical College " It is comprehensive, thorough, and must, of a necessity, prove a splendid acquisition to the libraries of those who are interested in medical jurisprudence." Jakob arid FisherV Nervous System and its Diseases Atlas and Epitome of the Nervous System and its Diseases. By Professor Dr. Chr. Jakob, of Erlangen. From the Second Revised German Edition. Edited, with additions, by Edward D. Fisher, M. D., Professor of Diseases of the Nervous System, University and Bellevue Hospital Medical College, New York. With 83 plates and copious text. Cloth, $3.50 net. In Saunders' Hand- Atlas Series. The matter is divided into Anatomy, Pathology, and Description of Dis- eases of the Nervous System. The plates illustrate these divisions most completely ; especially is this so in regard to pathology. The exact site and character of the lesion are portrayed in such a way that they cannot fail to impress themselves on the memory of the reader. Philadelphia Medical Journal ** We know of no one work of anything like equal size which covers this important and complicated field with the clearness and scientific fidelity of this hand-atlas." DISEASES OF CHILDREN. g American Text-Book of Diseases of Children American Text-Book of Diseases of Children. Edited by Louis Starr, M. I)., Consulting Pediatrist to the Maternity Hospital, etc. ; assisted by Thompson S. Westcott, M. D., Attending Physician to the Dispensary for Diseases of Children, Hospital of trie University of Pennsylvania. Handsome octavo, 1244 pages, profusely illustrated. Cloth, $7.00 net; Sheep or Half Morocco, $8.00 net. SECOND REVISED EDITION To keep up witli the rapid advances in the field of pediatrics, the whole subject-matter embraced in the first edition has been carefully revised, new articles added, some original papers amended, and a number entirely rewrit- ten and brought up to date. British Medical Journal " May be recommended as a thoroughly trustworthy and satisfactory guide to the subject of the diseases of children." Paul's Fever Nursing' Nursing in the Acute Infectious Fevers. By George P. Paul, M. D. , Assistant Visiting Physician to the Samaritan Hos- pital, Troy, N. Y. 121110 of 200 pages. Cloth, $1.00 net. JUST ISSUED Dr. Paul has written his book especially for the trained nurse, so that all extraneous matter has been studiously avoided. Great stress has been laid upon care and management in each disease, as this relates directly to the duties of the nurse. The work discusses fever in general, then each acute infectious fever separately, and finally those practical procedures necessary to the proper management of the fevers described. io SAUNDERS' BOOKS ON Friedenwald & Ruhrah's Dietetics for Nurses Dietetics for Nurses. By Julius Friedenwald, M. D., Clinical Professor of Diseases of the Stomach, College of Physi- cians and Surgeons, Baltimore; and John Ruhrah, M. D., Clinical Professor of Diseases of Children, College of Physicians and Surgeons, Baltimore. i2moof 363 pages. Cloth, $1.50 net. JUST ISSUED This work has been prepared to meet the needs of the nurse, both in the training school and after graduation. Rectal alimentation and the feeding of operative cases are fully described. Edinburg Medical Journal. " It appears to us to contain all the practical side of dietetics, of handy size and devoid of padding." Lewis' Anatomy and Physiology for Nurses Anatomy and Physiology for Nurses. By LERoy Lewi^ M. D., Surgeon to and Lecturer on Anatomy and Physiology for Nurses at the Lewis Hospital, Bay City, Michigan. i2mo of 317 pages, with 146 illustrations. Cloth, $1.75 net. JUST ISSUED The author has based the plan and scope of the work on the methods he has employed in teaching the subjects, and has made the text unusually simple and clear. The object was so to deal with anatomy and physiology that the student might easily grasp the primary principles, at the same time laying a broad foundation for a wider study. NURSING. 1 1 De Lee's Obstetrics for Nurses Obstetrics for Nurses. By Joseph B. De Lee, M. D., Pro- fessor of Obstetrics in the Northwestern University Medical School, Chicago ; Lecturer in the Nurses' Training Schools of Mercy, Wesley, Provident, Cook County, and Chicago Lying-in Hos- pitals. 121110 of 460 pages, fully illustrated. Cloth, $2.50 net. JUST ISSUED— NEW 2nd (EDITION The illustrations in Dr. De Lee's work are nearly all original, and repre- sent photographs taken from actual scenes. The text is the result of the author's eight years' experience in lecturing to nurses. J. Clifton Edgar, M. D., Professor of Obstetrics and Clinical Midwifery, Cornell University, Neiv York. " It is far and away the best that has come to my notice, and I shall take great pleasure in recommending it to my nurses, and students as well." Davis' Nursing Obstetric and Gynecologic Nursing. By Edward P. Davis, A. M., M. D., Professor of Obstetrics, Jefferson Medical College, Phila. i2mo, 400 pages, illustrated. Buckram, $1.75 net. RECENTLY ISSUED— SECOND REVISED EDITION The Lancet, London " Not only nurses, but even newly qualified medical men, would learn a great deal by a perusal of this book. It is written in a clear and pleasant style, and is a work we can recommend." Beck's Reference Handbook for Nurses A Reference Handbook for Nurses. By Amanda K. Beck, Chicago. 121110 of 150 pages. Flexible morocco, $1.25 net. RECENTLY ISSUED This little book contains information upon every question that comes to a nurse in her daily work, and embraces all the information that she requires to carry out any directions given by the physician. Boston Medical and Surgical Journal " Must be regarded as extremely useful, not only for nurses, but for physicians." SAUNDERS' BOOKS ON Hof matin and Peterson's Legal Medicine Atlas of Legal Medicine. By Dr. E. von Hofmann, of Vienna. Edited by Frederick Peterson, M. 1)., Clinical Profes- sor 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. In Saunders'' Hand-Atlas Series. By reason of the wealth of illustrations and the fidelity of the colored plates, the book supplements all the text-books on the subject. Moreover, it furnishes to every physician, student, and lawyer a veritable treasure-house of information. The Practitioner, London " The illustrations appear to be the best that have ever been published in connection with this department of medicine, and they cannot fail to be useful alike to the medical jurist and to the student of forensic medicine." Chapman's Medical Jurisprudence Medical Jurisprudence, Insanity, and Toxicology. By Henry C. Chapman, M. D., Professor of Institutes of Medicine and Medical Jurisprudence in Jefferson Medical College, Phila- delphia. Handsome 1 21110 of 329 pages, fully illustrated. Cloth, $1.75 net. RECENTLY ISSUED— THIRD REVISED EDITION, ENLARGED This third edition has been thoroughly revised and greatly enlarged, so as tc bring it absolutely in accord with the very latest advances in this important branch of medical science. Medical Record, New York "The manual is essentially practical, and is a useful guide for the general practitioner, besides possessing literary merit." IfUSSING. 13 Golebiewski and Bailey V 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 Saunders' Hand- Atlas Series. This work contains a full and scientific treatment of the subject of accident injury ; the functional disability caused thereby ; the medicolegal questions involved, and the amount of indemnity justified in given cases. The Medical Record, New York " This volume is upon an important and only recently systematized subject, which is growing in extent all the time. The pictorial part of the book is very satisfactory." 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, Superintendent of the Training School for Nurses at the Carney Hospital, South Boston, Mass. 1 2mo, 300 pages. 31.50 net. JUST ISSUED— NEW (3rd) 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." 14 SAUNDERS' BOOKS ON StoneyV Nursing Practical Points in Nursing : for Nurses in Private Practice. By Emily M. A. Stoney, Superintendent of the Training School for Nurses at the Carney Hospital, South Boston, Mass. 466 pages, fully illustrated. Cloth, $1.75 net. THIRD REVISED EDITION— RECENTLY ISSUED 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. The Lancet, London '* A very complete exposition of practical nursing in its various branches, including obstetric and gynecologic nursing. The instructions given are full of useful detail." Stoney's Technic for Nurses Bacteriology and Surgical Technic for Nurses. By Emily M. A. Stoney, Superintendent of the Training School> Carney Hospital, South Boston. Revised by Frederic R. Grif- fith, M. D., Surgeon, N. Y. i2mo, 278 pages, illus. #1.50 net. RECENTLY ISSUED— NEW (2d) EDITION Spratling on Epilepsy Epilepsy and Its Treatment. By William P. Spratling, M. D. , Medical Superintendent of the Craig Colony for Epilep- tics, Sonyea, New York. Octavo of 522 pages, fully illustrated. Cloth, $4.00 net. CHILDREN AND HYGIENE. 15 Griffiths Care of the Baby The Care of the Baby. By J. P. Crozer Griffith, M. D., Clinical Professor of Diseases of Children, University of Penn- sylvania. i2mo, 436 pages. Illustrated. Cloth, $1.50 net. RECENTLY ISSUED— THIRD EDITION, REVISED 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." Crothers' Morphinism Morphinism and Narcomania from Opium, Cocain, Ether, Chloral, Chloroform, and other Narcotic Drugs ; also the Etiol- ogy, Treatment, and Medicolegal Relations. By T. D. Croth- ers, M. D., Superintendent of Walnut Lodge Hospital, Hartford, Conn. Handsome i2mo of 351 pages. Cloth, $2.00 net. The Lancet, London " An excellent account of the various causes, symptoms, and stages of morphinism, the discussion being throughout illuminated by an abundance of facts of clinical, psychological, and social interest." Abbott's Transmissible Diseases The Hygiene of Transmissible Diseases: Their Causa- tion, Modes of Dissemination, and Methods of Prevention. By A. C. Abbott, M. D., Professor of Hygiene and Bacteriology, University of Pennsylvania. Octavo, 35 1 pages, with numerous illustrations. Cloth, $2.50 net. SECOND REVISED EDITION The Lancet, London " We heartily commend the book as a concise and trustworthy guide in the subject with which it deals, and we sincerely congratulate Professor Abbott." 1 6 SAUNDERS* BOOKS ON NUI&ING. American Pocket Dictionary 4th Ed.— Recently issued American Pocket Medical Dictionary. Edited by W. A. Newman Dorland, M. D., Assistant Obstetrician to the Hospital of the University of Pennsylvania. Containing the pronunciation and defi- nition of the principal words used in medicine and kindred sciences, with 64 extensive tables. Handsomely bound in flexible leather, with gold edges, $ I. 00 net; with patent thumb index, #1.25 net. Morrow's Immediate Care of Injured Just Ready 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 350 pages, with 250 illustrations. 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 fcFOR 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. Grafctrom's Mechano-Therapy JSSS^zSSL 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. 121110, 200 pages, illustrated. Cloth, $1.25 net. Sh^w on Nervous Diseases and Insanity Recently Issued — 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. 121110 of 204 pages, illustrated. Cloth, $1.00 net. In Saunders" 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. l2mo volume of 259 pages. Cloth, #1.00 net. /// Saunders* Question -Com fend Series. Saunders' Compends SAUNDERS' Question Compends, arranged in question- and-answer form, are the latest, most complete, and best illustrated series of compends ever issued. They are now recognized as the standard authorities in medical literature with students and practitioners in every city of the United States and Canada. Since the first appearance of these in- valuable student-helps there have been sold over 265,000 copies. The entire series has been kept thoroughly revised and enlarged when necessary, many of them being in their fifth and sixth editions. A COMPLETE LIST OF VOLUMES Cloth, $1.00 net per copy, unless otherwise noted 1. ESSENTIALS OF PHYSIOLOGY. 2d edition. By Sidney P. BUDGETT, M.D. 2. ESSENTIALS OF SURGERY. 7th ed. 90 illustrations. By Edward Martin, M.D. 3. ESSENTIALS OF ANATOMY. 7* ed. 151 illustrations. By C. B. Nancrede, M.D. 4. ESSENTIALS OF MEDICAL CHEMISTRY. 6th ed. By Law- rence Wolff, M.D. Revised by A. Ferree Witmer, Ph.D. 5. ESSENTIALS OF OBSTETRICS. 6th ed. 75 illustrations. By W. Easterly Ashton, M.D. 6. ESSENTIALS OF PATHOLOGY AND MORBID ANATOMY. By H. Harlow Brooks, M.D. A new work. Preparing. 7. ESSENTIALS OF MATERIA MEDICA, THERAPEUTICS, AND PRESCRIPTION-WRITING. 7th ed. By Henry Morris, M.D. Revised by W. A. Bastedo, Ph.G., M.D. 8,9. ESSENTIALS OF PRACTICE OF MEDICINE. By W. R. Williams, M.D. (Double number, $1.75 net.) (Continued on Opposite Page) Saunders' Compends 10. ESSENTIALS OF GYNECOLOGY. 6th ed. With 57 illustrations. By Edwin B. Cragin, M.D. Revised by Frank S. Mathews, M.D. 1 1. ESSENTIALS OF DISEASES OF THE SKIN. 6th edition. 61 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. A new work, fully illus- trated. Just Ready. 14. ESSENTIALS OF DISEASES OF THE EYE. trated. By Edward Jackson, M.D. 15. ESSENTIALS OF DISEASES OF CHILDREN. Wm. M. Powell, M.D. 17. ESSENTIALS OF DIAGNOSIS. 2d ed. M.D., and A. A. Eshner, M.D. 19. ESSENTIALS OF NOSE AND TH By E. B. Gleason, M.D. 20. ESSENTIALS OF BACTERI and 6 plates. By M. V Vogel, M.D 21. ESSENTIALS OF 4th ed. 53 ill by Smith F 24. ESSENTIA! illus- trate 25. ESSr Orations. By 55nQ(j 925 Walnut St., Phila.