BIOLOGY LIBRARY G flDebfcal Epitome Series. MICROSCOPY AND BACTERIOLOGY. A MANUAL FOR STUDENTS AND PRACTITIONERS, BY P. E. ARCHINARD, A. M., M. D., Demonstrator of Microscopy and Bacteriology, Tulane University of Louisiana, Medical Department. SERIES EDITED BY V. C. PEDERSEN, A. M., M. D., Instructor in Surgery and Assistant Anaesthetist at the New York Polyclinic Medical School and Hospital; Deputy Genito- Urinary Surgeon to the Out-Patient Department of the New York Hospital; Physician-in- Charge, St. Chrysostom's Dispensary; Anaesthetist to the Roosevelt Hospital (First Surgical Division). ILLUSTRATED WITH SEVENTY-FOUR ENGRAVINGS. LEA BROTHERS & CO., PHILADELPHIA AND NEW YORK BIOLOGY LIBRARY THfc LIBRARY OF CONGRESS, Two Copies Received JUL 24 1903 Ctpyrifht Entry * 1 COPY XXo. N«. fc f Maiu Lib. Agric, Entered according to Act of Congress, in the year 1903, by LEA BROTHERS & CO., In the Office of the Librarian of Congress. All rights reserved. ELECTROTYPED BV WESTCOTT & THOMSON. PHILADA, PRESS OF WM. J. DORNAN, PHILAOA. AUTHOR'S PREFACE. THE scope of an Epitome of Bacteriology and Microscopy obviously affords little or ^no opportunity for original work, nor indeed would it be desirable to do more than represent the actual status of these cognate sciences. Standard text- books have accordingly been consulted freely. The merit of an Epitome consists in affording a concise and clear presentation of essentials, command of which enables the student to build a sound superstructure of knowledge. The practitioner may also use such ' a. volume to post himself on the main facts/of' Bacteriology and Microscopy, and the technique. The Ifst of questions appended to each chapter will be useful to students in quizzing and reviewing. P. E. A. NEW ORLEANS, 1903. 3 268464 6 EDITOR'S PREFACE. elation of their helpfulness in the matter of producing the proper character of work. In order to render the volumes suitable for quizzing, and yet preserve the continuity of the text unbroken by the interpolation of questions throughout the subject-matter, which has heretofore been the design in books of this type, all questions have been placed at the end of each chapter. This new arrangement, it is hoped, will be convenient alike to students and practitioners. VICTOR C. PEDERSEN. NEW YORK, 1903. CONTENTS. INTRODUCTION. PAGES The Refraction of Light and the Microscope 17-27 THE REFRACTION OF LIGHT : The Two Fundamental Laws of Refraction ; The Principles of Refraction by Lenses . . . 17-19 THE MICROSCOPE: The Simple Microscope; The Compound Microscope ; The Lenses and Lens-systems of the Micro- scope; The Care of the Microscope 19-27 CHAPTER I. The Fundamental Principles 27-41 THE HISTORY OF BACTERIOLOGY 27-28 THE CLASSIFICATION OF COHN FOR BACTERIA , 28 THE DEFINITION OF BACTERIA . 28-29 THE MORPHOLOGICAL CLASSIFICATION OF BACTERIA: The Coccus ; The Bacillus ; The Spirillum 29-31 THE SIZE OF BACTERIA . 31 THE REPRODUCTION OF BACTERIA: Fission; Sporulation . . 32-34 THE MOTILITY OF BACTERIA 35 THE RELATION OF OXYGEN TO BACTERIAL LIFE 36 THE RELATION OF DEAD AND LIVING ORGANIC MATTER TO BACTERIA 36 THE ESSENTIAL CONDITIONS OF BACTERIAL GROWTH : Heat ; Moisture ; Decomposable Organic Material ; Special Chemi- cal Reaction of the Culture-medium . . 36-38 THE INERT AND INHIBITIVE CONDITIONS OF BACTERIAL LIFE 38 THE VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA . 38-41 8 CONTENTS. CHAPTER II. PAGES The Examination and the Staining of Bacteria 41-54 THE EXAMINATION OF BACTERIA : The Hanging-Drop Prepa- ration 41-42 THE STAINING OP BACTERIA: The General Mode of Proced- ure ; The Most Commonly Used Stains ; The Application of the Dyes ; The Special Methods of Staining ; The Staining of Capsules; The Staining of Spores; The Staining of Flagella ; The Staining of Bacteria in Tissues 42-54 CHAPTER III. The Process, Media, and Utensils of the Cultivation of Bacteria 55-68 THE PROCESS OF THE CULTIVATION OF BACTERIA 55 THE MEDIA OF THE CULTIVATION OF BACTERIA: The Most Commonly Used Liquid Culture-Media ; The Most Com- monly Used Solid Culture-Media; The Most Commonly Used Special Culture-Media - 55-62 THE UTENSILS OF THE CULTIVATION OF BACTERIA .... 62-68 CHAPTER IV. The Inoculation of Culture-Media with Bacteria 68-75 THE METHOD OF INOCULATING FLUID MEDIA ....... 68 THE METHODS OF INOCULATING SOLID MEDIA 68-72 THE CULTIVATION OF ANAEROBIC BACTERIA: The Incubator and the Thermostat 72-75 CHAPTER V. Sterilization, Disinfection, and Antisepsis 76-84 THE METHODS OF STERILIZATION 81 THE METHODS OF DISINFECTION 81-83 THE METHODS OF ANTISEPSIS: The Common Disinfectants . 83-84 CHAPTER VI. The Inoculation of Animals and their Study 85-91 THE INOCULATION OF ANIMALS : The Various Methods of In- oculation of Animals 85-88 THE OBSERVATION OF THE INOCULATED ANIMAL : The Roux- Nocard Method of Culture and Observation 89-91 CONTENTS. 9 CHAPTER VII. PAGES Infection and Immunity 91-99 INFECTION: The Theoiies of Infection; The Avenues and Factors of Infection 91-94 IMMUNITY AND ITS VARIETIES : The Methods of Producing Immunity ; The Antitoxic and Antimicrobic Blood- Serums; The Thories of Immunity 94-99 CHAPTER VIII. The Pathogenic Bacteria 99-107 THE PYOGENIC MICROCOCCI AND ALLIED BACILLI .... 99-100 THE INDIVIDUAL FEATURES OF THE PYOGENIC BACTERIA: Staphylococcus Pyogenes Aureus: Staphylococcus Pyo- genes Albus ; Staphylococcus Citreus; Streptococcus Pyogenes ; The Micrococcus Cereus Albus ; The Micrococ- cus Cereus Flavus ; The Micrococcus Pyogenes Tenuis ; Micrococcus Tetragenus 100-104 GONORRHOEA : Micrococcus Gonorrhoeas (Gonococcus) ; Bacil- lus Pyocyaneus ; Bacillus Pyogenes Foetidus ; Pneumo- coccus or Pneumobacillus ; Bacillus Coli Communis, Bacillus Typhosus ; Bacillus Tuberculosis 104-107 CHAPTER IX. The Other Pathogenic Micrococci and Allied Bacilli— Micrococcus Pneumonias, Epidemic Cerebrospinal Men- ingitis, and Malta Fever , • 108-115 PNEUMONIA: Micrococcus Pneumoniae Crouposae (Diplococ- cus Pneumonia?; Micrococcus Pasteuri ; Micrococcus of Sputum Septicaemia) ; Pneumococcus of Friedlaender (Bacillus Pneumoniae of Fluegge) 108-112 EPIDEMIC CEREBROSPINAL MENINGITIS: Diplococcus Intra- cellnlaris Meningitidis 112-113 MALTA OR MEDITERRANEAN FEVER: Micrococcus Melitensis 113-115 CHAPTER X. Tuberculosis 115-120 BACILLUS TUBERCULOSIS • • 115-120 10 CONTENTS. CHAPTER XL PAG PS Leprosy and Syphilis 120-123 LEPROSY : Bacillus Leprae 120-1 22 SYPHILIS: Bacillus of Syphilis ; Streptococcus of Syphilis . 122-123 CHAPTER XII. Glanders (Farcy) . . . , 124-128 BACILLUS MALLEI 124-128 CHAPTER XIII. Anthrax . 128-133 BACILLUS ANTHRACIS 128-133 CHAPTER XIV. Diphtheria and Pseudodiphtheria 133-145 DIPHTHERIA: Bacillus Diphtherias 133-140 PSEUDODIPHTHERIA: Bacillus Pseudodiphtheriae ; The Anti- toxin Treatment of Diphtheria 140-145 CHAPTER XV. Tetanus, Malignant (Edema, and Symptomatic Anthrax 145-156 MALIGNANT (EDEMA: The Bacillus of Malignant (Edema . 152-153 SYMPTOMATIC ANTHRAX : Bacillus Anthracis Symptomatic! . 153-156 CHAPTER XVI. Typhoid Fever 156-165 BACILLUS TYPHOSUS • . 156-159 DIFFERENTIATION OF BACILLUS TYPHOSUS FROM ALLIED GROUPS 159-162 THE BLOOD-SERUM DIAGNOSIS OF TYPHOID FEVER . . . 162-164 VACCINATION AGAINST TYPHOID FEVER 164-165 CHAPTER XVII. Bacillus Coli Communis 165-168 CHAPTER XVIII. Asiatic Cholera 168-174 SPIRILLUM CHOLERA ASIATICS (COMMA BACILLUS) . . . 168-174 CONTENTS. 11 CHAPTER XIX. PAGES Influenza 174-176 BACILLUS OF INFLUENZA 174-176 CHAPTER XX. Bubonic Plague 176-179 BACILLUS PESTIS 176-179 CHAPTER XXI. Relapsing Fever 179-180 SPIRILLUM OBERMEIERI 179-180 CHAPTER XXII. Dysentry, Hog Cholera, and Chicken Cholera 180-185 DYSENTRY : Bacillus Dysentericae 180-1 82 HOG CHOLERA: Bacillus sui Pestifer 182-183 CHICKEN CHOLERA : Bacillus Cholerse Gallinarum 183-185 CHAPTER XXIII. The Pathogenic Micro-organisms other than Bacteria . . 185-196 ACTINOMYCOSIS, MALARIA, AND AMCEBIC COLITIS : StreptO- thrix 185-186 ACTINOMYCOSIS: Streptothrix Actinomyces (Ray Fungus); Other Pathogenic Streptothrices 186-188 MALARIA: Plasmodium Malarise 189-193 AMCEBIC COLITIS : Amoeba Coli 194-196 CHAPTER XXIV. Bacteriological Examinations of Water, Air, and Soil . 196-204 THE BACTERIOLOGICAL INVESTIGATION OF WATER .... 196-202 BACTERIOLOGICAL EXAMINATION OF THE AIR 202-203 THE BACTERIOLOGICAL EXAMINATION OF THE SOIL . . . 203-204 MICROSCOPY AND BACTERIOLOGY. INTRODUCTION. THE REFRACTION OF LIGHT AND THE MICROSCOPE. THE REFRACTION OF LIGHT. Definition. — Refraction is the property possessed by trans- parent media of altering the rays of light which pass through them. It is to this property possessed by lenses, the trans- parent media of microscopes, that these instruments owe their magnifying power. The Two Fundamental Laws of Refraction. I. When a ray of light passes from a denser to a rarer medium, it is refracted away from a line drawn perpendicu- larly to the plane which divides the media ; and vice versa, when the light passes from a rarer to a denser medium, it is refracted toward that perpendicular. II. The sines of the angles of incidence and refraction — that is, of the angles which the ray makes with the perpendicular before and after its refraction — bear to one another a constant ratio for each substance, which is known as its index of refraction. The Principles of Refraction by Lenses. Microscope lenses are chiefly convex ; those of other forms are used to make certain modifications in the rays passing through the convex lens, and so render their performance more exact. 2— M. B. 17 18 INTRODUCTION. Focus of a Lens. — For a lens to give a perfect image of an object, all the rays of light coming from that object and pass- ing through the lens must meet at the same point on the other side of the lens. This point is known as the focal point or focus of the lens. The Spherical Aberration of Lenses. Definition. — It is difficult, however, so to construct a con- vex lens that all the rays of light that pass through it shall come to the same focus. As a rule, the rays which traverse its peripheral or marginal portion come to a shorter focus than those which pass through its more central portion. Correction. — Distortion of the image is thus caused, and is known as spherical aberration. Theoretically, spherical aber- ration might be corrected by making the curvature of the periphery of the lens less than that of its more central por- tion ; but the difficulties in the mechanical construction of such a lens would be very great, and opticians have found it more practical to correct this defect by coupling with a con- vex lens a concave one of less curvature, but which is subject to exactly the opposite error of refraction. Doublets. — These combinations of convex and concave lenses, or doublets, act as a single convex lens. Triplets. — Sometimes two convex and one concave lens are used in combination, and are called triplets. The Chromatic Aberration of Lenses. Definition. — When light traverses a convex lens, the differ- ent colors which compose it do not all come to the same focus — that is, the colors are of unequal refrangibility ; and the image then is seen chiefly in that color which chances to be in focus. This color-distortion is especially noticeable with the marginal rays, and is known as chromatic aberration. Correction. — Though exclusion of the marginal rays can, as with spherical aberration, partly correct this defect, yet this is not sufficient, and chromatic aberration is remedied best by constructing the convex lenses of the combination THE MICROSCOPE. 19 mentioned above of crown glass, and the concave lenses of flint glass, as those two kinds of glass have opposite proper- ties with regard to refrangibility. THE MICROSCOPE. Microscopes are of two kinds : simple and compound : The Simple Microscope. The ordinary hand magnifying-glass and the dissecting microscope are examples of simple microscopes. Their magnifying power depends upon one lens or several lenses acting as one double convex lens. To obtain a clear, enlarged image of the object, the latter must be in its princi- pal focus, and the shorter the focus of the lens the greater its magnifying power. The focal length, or focus, of the lens depends on the degree of curvature of the lens. In expressing the magnifying power of lenses, the size of an object as seen by the unaided eye at ten inches distance is taken as unity. A lens having a magnifying power of ten diameters, or linears, is one which enlarges the object ten times in each linear direction. The Compound Microscope. The compound, or ordinary, microscope consists of the stand and lenses. The stand comprises the following parts : 1 . Base or foot ; 2. Pillars, or upright, which may be jointed or not ; 3. Arm connecting the pillars with the — 4. Body, containing the — 5. Draw-tube ; moved up and down rapidly or slowly by means of the — 6. Coarse adjustment — used to bring the object into view ; 7. Fine adjustment — used only when the object is already in view, to bring out more clearly its details. 20 INTRODUCTION. 8. Stage — the flat part on which is laid the object to be examined, and which is perforated by a central hole to allow illumination of the object from below. The stage may be circular or square, stationary or movable, and mechanical. Underneath the Stage are Found the Parts: 9. Flat mirror for low-power, and 10. Concave mirror for high-power objectives. The mirror is so arranged as to allow motion in all direc- tions. For ordinary histological purposes it is usually fixed perpendicularly to the stage, and gives direct light ; occasion- ally it is placed in an oblique direction, giving oblique light. 11. Diaphragm. — Immediately below the stage and about two inches above the mirror, though freely movable up and down, is found the diaphragm or stop : used to prevent the peripheral or diffuse rays of light from the mirror from reaching the object, and to allow only the more central and di ect rays to illuminate the same. The holes in the dia- phragm are of different sizes, the smaller ones being used with the higher power and the larger ones with the lower power class of work. 12. Condenser. — For very high powers, especially such as are used in bacteriology, besides the foregoing parts, there are on the substage the condenser, which is a lens, or system of lenses, used to concentrate still further the light from the mirror on the object. The condenser most commonly in use is known by the name of its introducer as the Abbe. The condenser should be exactly central, and, as a rule, it should be brought almost into contact with the object on the stage. 13. Iris Diaphragm. — Immediately below the condenser, instead of the ordinary diaphragm, what is known as an iris diaphragm is used (so called from Jts peculiar variability, like the iris of the eye). 14. Nose-piece or Revolver. — At the bottom of the tube a mechanical piece, which enables one to attach two or three objectives to the microscope at the same time, is known as the nose-piece or revolver. THE MICROSCOPE. 21 15. Lenses. — These are so important that a detailed descrip- tion of them is necessary. The Lenses and Lens-systems of the Microscope. Lenses. — The lenses of an ordinary microscope are of two kinds : those attached to the end of the tube nearer the object, and known by the name of the objective lens, or objective system of lenses, and. those fitting the end of the tube into which the observer looks, known as the eye-piece or ocular lens. The Objective Lens or System of Lenses. The objective is the principal lens or system of lenses of the microscope. It is that which gives the greatest part of the magnifying power to the instrument. As ordinarily arranged, it is composed of a number of lenses connected together in various ways, and known as combinations or systems. The combination nearest the object is called the front combination, or front lens, and that nearest the ocular the back combination, or back lens. There may be one or more intermediate systems between these. Each combination, or system, consists of a concave lens of flint glass and a con- vex lens of cro\vn glass ; the whole combination acts as a double convex lens. The purposes of having lenses of vari- ous shapes and materials is to correct what is known as chromatic (colored) and spherical aberration or distortion (see Fig. 1). Designation of the Objective. — Objectives are designated, as a rule, by their equivalent focal lengths. This length is usu- ally given in inches or fractions thereof — for instance, 1 inch, J inch, ^ inch. In continental Europe the numerator of the fraction is often omitted, the ^ objective being called 3, and the | inch being called 7. These numbers indicate that the objective produces a real image of the same size as is pro- duced by a simple convex lens whose principal focal distance would be that indicated by the number. And as " the rela- tive size of object and image vary directly as their distance 22 INTRODUCTION. FIG. 1. — G denser (Abbe's) ; I, no'se-'piece, from the centre of the lens," the less the equivalent focal distance of the objective, the greater is its magnifying power. THE TYPES OF OBJECTIVE LENS. 23 An objective of ^ inch, or No. 3, therefore, magnifies less than one of ^ inch, or No. 7. The working distance of the microscope — that is, the dis- tance between the objective and the object — is always less than the equivalent focal distance of .the objective. THE TYPES OF OBJECTIVE LENS. 1. Dry and Immersion Objectives. In the dry objectives, nothing intervenes between the objec- tive and the object to be examined except air : all low-power objectives are dry. In the immersion objectives, some liquid, such as water, glycerin, or oil (homogeneous immersion objectives), must be placed upon the cover-glass over the object and make contact between the cover-glass and the objective. Such lenses are known, respectively, as water-, glycerin-, and oil-immersion lenses. In homogeneous immersion objectives the oil has the same refracting index as the front lens of the objective. 2. Non-achromatic objectives are objectives in which the color-distortion is not corrected, and the image produced is bordered by a colored fringe ; they also show spherical dis- tortion. 3. Achromatic objectives are those in which the color- aberration is corrected. 4. Aplanatic objectives are those in which the spherical aberration is corrected. All better classes of objectives are both achromatic and aplanatic. 5. Apochromatic objectives are objectives in which .rays of three spectral colors combine at one focus instead of rays of two colors, as in the ordinary achromatic. They are highly achromatic objectives. 6. Adjustable objectives are objectives in which the distance between the front and back combinations may be regulated by means of a milled-head screw. This is useful in dry or water-immersion objectives to correct the dispersion of light caused by different thicknesses of the cover-glasses. The angular aperture of an objective is the angle formed 24 INTRODUCTION. between the most diverging rays issuing from the axial point of an object that may enter and take part in the formation of an image. By axial point is meant a point situated in the extended optical axis of the microscope. The optical axis of the microscope is a line drawn from the eye through the middle of the tube to the centre of the objective. Relation of Size and Working Distance of the Lens. — The larger the lens and the less its working distance, the greater the angle of aperture. For dry objectives the greater the angular aperture, the better the definition of the objective. Numerical aperture is the capacity of an optical instrument for receiving rays from the object and transmitting them to the image, and the numerical aperture of a microscopic objec- tive is, therefore, determined by the ratio between its focal length and the diameter of the emergent pencil at the point of its emergence — that is, the utilized diameter of a single- lens objective or of the back lens of a compound objective. It is the ratio of the diameter of the .emergent pencil to the focal length of the lens, or, in other words, it is the index of refraction of the medium in front of the objective multiplied by the sine of half the aperture. The Ocular Lens or Eye-piece. The eye-piece, or ocular, is the lens, or combination of lenses, placed in the tube at the point of observation. It acts as a simple microscope and serves to magnify the image of the object. The ocular consists of two lenses, one -situated nearer the eye, known as the eye-lens, and the other known as the field-lens. The ocular is said to be positive when the image is formed beyond it ; and nega- tive when it is formed within it, between the field-lens and eye-lens. In the positive ocular the two lenses act together as a simple microscope and magnify the image. In the negative ocular the field-lens acts with the objective in making clearer the image, and with the eye-lens in help- ing to correct some of the aberrations. The eye-lens also magnifies the image. THE TYPES OF OCULAR LENS. 25 THE TYPES OF OCULAR LENS. 1. Compensating oculars, which correct the chromatic aber- ration of the ray outside of the axis. 2. Projecting oculars, used with the projecting microscope or for microphotography. 3. Spectroscopic oculars. These three types, among many, are the most important and most frequently employed. The designation of oculars is by %their magnifying power and equivalent focal distance, and also by numbers, the smaller number designating the lower power, and vice versa.w The field of the microscope is the lighted portion which is seen when one looks through the microscope with the instru- ment in focus. The eye-point is the distance from the instrument at which the eye may look through with the least strain. The Care of the Microscope. Keep the instrument cleaned, and see that all mechanical parts move smoothly and evenly. Keep the mirror, con- denser, and diaphragm central — that is, in the optical axis. Bring the object into view with the coarse adjustment, and define the details in it by means of the fine adjustment. See that no dirt or dust of any kind covers the lenses. Should the field be blurred or dim, after proper focusing and light- ing, the fault is either with the lenses or the cover-glass is soiled. Tests for the Sources of Dimness in the Object. — By revolv- ing the ocular with the eye in position, the dimness, when due to the ocular, will also move. By moving gently the object with the hands, the dimness will move if due to dirt on the cover-glass. Should the blurring be stationary in both the above tests, it is due to soiling of the objective. To cleanse the lenses of the ocular, blow on both surfaces of each lens and wipe dry with a fine silk handkerchief, old soft linen rag, or, better, rice-paper. To cleanse the objective, wipe, put the lens into the instrument and test it as de- 26 INTRODUCTION. scribed in the preceding paragraph, and if this is not suffi- cient, pass a little water or absolute alcohol over the surface and wipe dry. If the soiling is due to balsam or other resin- ous substance, clean gently with benzole or xylol. The back surface of the objective need never get dirty ; but when it does, inserting a soft rag into the objective and gently turn- ing it around is sufficient to* cleanse it. Never screw apart the different lemes of the objective, as it takes an expert optician to put them into proper position. Always see that the cover- glass is clean and dry on its upper surface. Never bring the front lens of the objective into direct contact with the object or 'cover-glass. For bacteriological work, it is indispensable to have a microscope supplied with an Abbe condenser and an oil-im- mersion objective of -fa inch focus. Different objectives according to their construction require different tube-lengths of the microscope to magnify at their fullest power and give their best definition. Manufacturers generally supply full information as to the proper tube-length for each instrument. QUESTIONS. What is refraction ? Give the two laws of refraction. What is the type of the microscope lenses ? What is the focus of a lens? What is meant by spherical aberration ? How is this corrected ? What are doublets and triplets? What is meant by refrangibility ? How is this corrected in microscope lenses? How many kinds of microscope are there? What is a simple microscope ? How is the magnifying power of lenses expressed? What is meant by a compound microscope ? Give the different parts of a compound microscope ? What is meant by direct light? What purpose does a diaphragm serve? What is a condenser? What is meant by an iris diaphragm ? What is a nose-piece ? How are the lenses of an ordinary microscope called ? What is an objective ? How many lenses or combinations of lenses does an ordinary objective contain ? How is the magnifying power of objectives designated? THE FUNDAMENTAL PRINCIPLES. 27 What is meant by the focal distance of an objective? The working dis- tance? What is the difference between the dry and immersion objectives? What is a homogeneous immersion objective? What is meant by a non-achromatic objective? What is meant by an achromatic objective? An aplanatic objective ? What is an apochroinatic objective? What is meant by an adjustable objective? What is the angular aperture of an objective? What is meant by the actual point of an objective? What is the optical axis of a microscope ? What relation does the size of the lenses have to its angular aperture? What is the numerical aperture of an objective ? What is the ocular of a microscope ? Of how many lenses does it consist? What is the difference between the positive and the negative ocular. What is a compensating ocular? A projecting? How are oculars designated ? What is the field of a microscope ? What is the eye-point? What care should be given to a microscope? Describe the tests for determining the cause of an obscure image. Describe the methods of cleansing the lenses of the microscope. CHAPTER I. THE FUNDAMENTAL PRINCIPLES. THE HISTORY OF BACTERIOLOGY. WHEN in the latter part of the seventeenth century Anthony von Leuwenhoek, by means of his magnifying- glasses, first discovered organisms in decaying vegetable infusions, he may be said to have laid the very first stone in the foundation of what later on was to be the Science of Bacteriology. It was very long after this, however, before sufficient facts were collected to place this science upon a firm basis, and it remained for a genius like the immortal Pasteur and the eminent talents of the equally great Koch to build up the superstructure of bacteriology so as to have it accepted by all as the true basis of scientific medicine. When first observed, these microorganisms were supposed 28 THE FUNDAMENTAL PRINCIPLES. to be animalcules, and were accepted as such until the middle of the nineteenth century, when F. Colin classed them as belonging to the vegetable kingdom, and listed them among the fungi, making of them the third variety of fungi, the schizomycetes or cleft fungi ; the other two being the saccharo- mycetes or sprouting fungi (the yeast plant), and the hyphomy- cetes or mucorini (the moulds). THE CLASSIFICATION OF COHN FOR BACTERIA. This, as just given, is accepted to-day by all authori- ties, though it is open to criticism. Although it is true that the great majority of these organisms like the fungi possess no chlorophyl, and are unable, like other vegetables, to obtain their nourishment from the carbon dioxide and nitrogen of the atmosphere, but, on the contrary, like animals, require higher carbohydrate and nitrogenous substances, which they decompose into their primitive elements for their subsistence. A few of them, however, possess some plant coloring-matter, and some seem able to thrive in a simple saline solution from which absolutely no nitrogen is to be obtained. THE DEFINITION OF " BACTERIA." The proper name therefore for these organisms, and the one generally adopted, is bacteria, which is the plural of the Latin substantive bacterium. They may be denned as fol- lows : Unicellular vegetables of low organization, devoid of chlorophyl (plant coloring-matter}, and multiplying by fission. The bacteria cells consist of a cell-membrane and protoplasm, which latter is sometimes clear and sometimes granular, but with no nuclei. The cell-membrane is a firm, tough envelope, very much like cellulose, which occasionally in some bacteria becomes viscid and gelatinous in its outer layers, forming a sort of bright halo around the bacteria, called a capsule. This gelatinous matter occasionally serves to bind two or more bacteria together, and gives to them quite a characteristic grouping which helps to distinguish them from others. In some instances the membranous envelope interferes consid- MORPHOLOGICAL CLASSIFICATION OF BACTERIA. 29 erably with the staining of the protoplasm of the bacteria cells, so that special methods of staining have to be adopted for these. Again, those bacteria which are generally found surrounded by a capsule, when grown in artificial media seem to lose the power of developing capsules. THE MORPHOLOGICAL CLASSIFICATION OF BACTERIA. Bacteria are divided into three varieties : (1) the rounded form or coccus (plural cocci) ; (2) the rod-shaped form or bacillus (plural bacilli) ; and (3) the curved or spiral form, spirillum (plural spirilla). I. The Coccus. Varieties. — The cocci, which are not always round, but very often oval in form, are further distinguished according as they appear : singly and of large size, as megacocci; of small size, as micrococci; double — that is, two of the cells adhering together, as diplococci ; in chains — that is, a number of cells adhering together in single file — as streptococci; in groups very like a bunch of grapes, as staphylococci ; in groups of four, as tetrads or merismopedia ; in groups of eight arranged in cubes, as sarcinsB ; in irregular masses united by an intercellular substance and imbedded in a tough gelati- nous matrix, as ascococci. II. The Bacillus. Morphology. — The bacilli or rod-shaped (desmo-) bacteria are distinguished by the fact that their two longest sides are parallel to each other ; the two short sides being at times straight, at others concave, and at others again, convex. Varieties. — They are said to be (1) slender when their breadth is to their length as 1 to 4 or more, and (2) thick when it is as 1 to 2. They develop singly or in pairs or in long threads or filaments, being attached together always by their narrow ends. 30 THE FUNDAMENTAL PRINCIPLES. III. The Spirillum. The spirilla or curved or spiral bacteria develop either singly or in pairs or in long twisted or corkscrew filaments. The Variations in Development of Each Species. Though under varied conditions of growth the form of any one species may vary considerably, yet these three main divisions under similar conditions are permanent — that is, micrococci always develop into micrococci, bacilli into bacilli, and spirilla into spirilla. FIG. 2. °o Q_ o&o a. Staphylococci d . e 6. Streptococci, c. Diplococci. d. Tetrads, e. Sarcinse. (Abbott.) FIG. 3. Diplococcus of pneumonia, with surrounding capsule. (Park.) MORPHOLOGICAL CLASSIFICATION OF BACTERIA. 31 FIG. 4. ••». •*'\' \ -->— '* I x •-. »»... •>— \ d e / a. Bacilli in pairs, b. Single bacilli, c and d. Bacilli in threads, e and /. Bacilli of variable morphology. (Abbott.) FIG. 5. c d a and d. Spirilla in short segments and longer threads— the so-called comma forms and spirals, b. The forms known as spirochseta. c. The thick spirals some- times known as vibrios. (Abbott.) FIG. 6. fl, a. Spirillum of Asiatic cholera (comma bacillus) ; normal appearance in fresh culf ures. 6. Involution-forms of this organism as seen in old cultures. (Abbott.) Occasionally under peculiar conditions what are known as involution-forms are produced, forms which may scarcely be recognized as those belonging to the original bacteria. These points are shown by Figs. 2, 3, 4, 5, 6. 32 THE FUNDAMENTAL PRINCIPLES. THE SIZE OF BACTERIA. Bacteria require to be seen and studied by the highest powers of the microscope ; they vary in size from 0.2 to 30 mikrons. (A mikron is yoVo millimeter ; about -^^-^ inch.) The micro- cocci have a diameter of from 0.2 to 1 mikron or more. Bacilli and spirilla vary in length from 2 to 30 mikrons or more ; in breadth, from 1 to 4 mikrons. The average length of pathogenic bacilli is 3 mikrons. THE EEPRODUCTION OF BACTERIA. As mentioned in the foregoing paragraphs, bacteria multi- ply by fission. I. Fission. In the case of cocci, the round or oval cells show a little indentation beginning in the membrane at two, four, or eight points of its periphery, according as the division is to occur in two, four, or eight parts ; this indentation increases until the original cell is divided into hemispheres, quadrants, or octants, as the case may be. These parts remain attached to one another until complete spheric cocci are formed from each part, and they then separate or not according to the nature of the bacteria. In some forms of diplococci, as the gonococci, complete spheres are never formed, the cells remaining attached to each other in pairs as hemispheres. In the case of nearly all bacilli and spirilla the cells increase to nearly double their original size before division, and the division always takes place in the direction of the length of the bacterium. The daughter cell remains attached for a while to its parent cell after fission is complete ; occasionally this attachment persists for a long time, so that large filaments consisting of a number of bacteria are formed. II. Sporulation. 1. The Endospore. Division by fission is the usual mode of the reproduction of bacteria, but at times, depending upon various circum- THE REPRODUCTION OF BACTERIA. 33 stances to be mentioned later, what are known as spores are formed by a number of bacilli and spirilla. These spores, which are perhaps the equivalents of seeds for the higher plants, are formed in this way : in the body of the bacillus, generally at its centre, occasionally at one of its poles, a number of dark highly refractile granules accumulate, and are soon changed into an oval, glistening, highly refractile body which is surrounded by a membrane of the same com- position as that of the cell itself, but thicker and more resistant. One spore only is formed in each cell. Some- times the spore-formation causes no change in the shape of the rod. At other times there is a bulging of the centre of the body of the bacillus, where the spore is located, with a general tapering to the two ends, giving to the bacillus the shape of a spindle. This is called a clostridium. Again, when the spore is formed at one of the poles, there is some- times a bulging of that part, giving to the bacillus the appearance of a nail or drum-stick, whence the name of drum- mer-bacillus for the cell. Fig. 7 shows these forms well. FIG. 7. Q a. Bacillus siibtilis with spores, b. Bacillus anthracis with spores, c. Clostridiumform with spores, rf. Bacillus of tetanus with endospores. (Abbott.) Soon after the formation of the spore the rest of the body of the cell disintegrates and breaks down, and the oval spore is liberated. The spores are characterized, on account of their thick membrane, by resistance to external influences which would be fatal to the bacilli themselves, such, for instance, as extremes, of heat or cold, desiccation, and the action of chemi- cals ; also, to a great extent, staining by the penetration into 3— M. B. 34 THE FUNDAMENTAL PRINCIPLES. their body of certain dyes which have great affinity for the protoplasm of ordinary bacteria, so that a special method must be adopted for their staining, as will be described later. Spores therefore preserve the species, when these would be destroyed if dependent solely on the bacilli for their preser- vation. 2. The Arthrospore. The foregoing spore-formation, known as endospores, is the usual mode of spore-formation found in bacteria, and is lim- ited to the rod and spirilla forms ; but another form of spore, called arthrospore, is mentioned by some as occurring occasionally in the round or cocci forms. This consists in a* special jointed projection forming from the outside of the cells, and capable later of developing into the original cell. This form of spore-formation is generally doubted at the present time. Spores are incapable of producing other spores, and can, only when placed in suitable conditions, develop into the type of bacilli which gave them birth. When this occurs the spore begins to elongate, loses its glistening appearance, and finally its membrane ruptures at one end or in the centre and gives exit to a fully developed bacillus. Spores may be distinguished from rounded bacteria by means of their brighter, more glistening appearance, their power of resisting stains, and the fact that in suitable media they develop into bacilli. Significance of Sporulation. — Whether the original idea that spores form, in bacteria capable of producing them, only when the latter are submitted to external noxious influences, and for the purpose of perpetuating the species, or whether, as more recently maintained, sporulation is the result of the highest expression of the complete development of bacteria, is not at present fully determined, though the dictum of authorities inclines to the latter view, and the spore-form a ti on of some of the best-known and studied bacteria, as anthrax, seems to lend color to this theory. THE MOTILITY OF BACTERIA. 35 THE MOTILITY OF BACTERIA. Motility, or the power of transporting themselves from place to place, is possessed by a number of bacteria. This is effected by filamentous or hair-like processes arising from the body of the bacteria, and called flagella. It must not be confounded with the peculiar whirling or dancing move- ment so often seen under the microscope, even in inorganic particles, and called the Brownian movements. Motility, except in two instances, has so far been observed only in bacilli and spirilla. Its rapidity depends on the particular bacterium, its mode of cultivation, the age of the culture, and other similar factors. Some bacteria after repeated arti- ficial cultivation seem to lose their motility, which, however, may be restored fully by passing through an animal. FIG. 8. a. Spiral forms with a flagellum at only one end. 6. Bacillus of typhoid fever with flagella given off from all sides, c. Large spirals from stagnant water with wisps of flagella at their ends (Spirillum undula). (Abbott.) The flagella are hair-like processes consisting of the same material as the bacterial cell-membrane, and are so minute as scarcely to be visible under the highest power of the micro- scope unless they are stained by special processes, as will be described in the chapter on staining. Whenever there is only a single flagellum at one of the poles of the bacillus, this is said to be monotrocha ; whenever there is a single flagellum at each pole of the bacterium, it is said to be amphitrocha ; whenever there is a cluster of flagella at one pole, the bac- terium is said to be lophotrocha ; and finally, when a varying number of flagella seem to arise from different portions of the body of the bacterium, it is said to be peritrocha. These features are shown in Fig. 8. 36 THE FUNDAMENTAL PRINCIPLES. THE RELATION OF OXYGEN TO BACTERIAL LIFE. Bacteria are divided into aerobic and anaerobic according as they require oxygen or not for their development. Some thriving best in the presence of oxygen are able to develop, however, without the presence of this gas ; these are called facultative anaerobic. Others thriving best without oxygen but able to develop in the presence of this gas are called facultative aerobic. THE RELATION OF DEAD AND LIVING ORGANIC MATTER TO BACTERIA. Bacteria are also divided into saprophytes and parasites, according as they require for their development merely the presence of decomposable organic matter or the body of a living higher organism, as host, on which they live. Should they also be able to live outside their host, they are called facultative parasites. The disease-producing germs are always parasitic. THE ESSENTIAL CONDITIONS OF BACTERIAL GROWTH. For the proper development of bacteria the following are required : heat, moisture, and the presence of some decom- posable organic matter, with special chemical reaction of the culture-medium. I. Heat. A temperature between 10° and 40° C. is required for the development of adult bacteria, but the degree varies greatly according to the different species. Some of the parasites thrive best at a temperature in the neighborhood of that of the human body, about 36° or 37° O. ; others, the saprophytes, seem to grow best at the ordinary room temperature, between 20° and 30° C. ; some few again seem to be able to grow and multiply at a temperature near the freezing-point ; and not long ago a number have been shown to develop abun- dantly at a temperature above 70° C. As a general rule, however, a temperature of 0° C. and one above 60° C. are fatal ESSENTIAL CONDITIONS OF BACTERIAL GROWTH. 37 within a few minutes for the ordinary non-spore-bearing bacteria. Spores are able to resist for a long time the effects of cold and excessive heat. Some have developed after having been im- mersed for a long time in liquid air (temperature, — 200° C.j, and also after exposure to dry heat of above 150° F. for sixty minutes and moist heat for thirty or forty minutes. II. Moisture. A certain amount of moisture is absolutely indispensable for the growth of bacteria, desiccation being fatal within a few minutes for nearly all the fully formed bacteria. Spores, however, are capable of developing after being kept dry for an indefinite period. III. Decomposable Organic Material. A certain amount of decomposable organic matter is indis- pensable for the development of the bacteria. This they decompose into simple elements, and are so able to obtain the nitrogen and carbon necessary for their sustenance. At the same time they set free carbon dioxide and nitrogen from the remainder, and so provide for the nourishment of the higher vegetables, which must have these substances free in order to support life. And in so doing bacteria render a service of incalculable value, as without it life in the animal kingdom would soon be extinct. Different bacteria require a greater or lesser proportion of the proteid substances for their nutrition ; and according as substances contain these in a more or less favorable condition for absorption they are said to be more or less good culture- soils or media. Some few species seem to be able to live in saline solution and in other media where there is no appreciable amount of organic matter, but in these cases they probably obtain their nutrition from the decomposition of slight traces of ammonia and the carbon dioxide of the air contained in the water. Again, some bacteria seem to have the power of decomposing 38 THE FUNDAMENTAL PRINCIPLES. ammonia and building up nitrite and nitrate compounds ; these are known as the nitrifying bacteria, and as a class are im- portant, and are being carefully studied. IV. Special Chemical Reaction of the Culture -medium. Bacteria are likewise profoundly influenced in their growth by the reaction of the medium in which they grow, most bac- teria requiring a neutral or faintly alkaline medium, some few a faintly acid one. This characteristic is found an element of danger for bacterial life : for the bacteria which require alkaline surroundings have, as a rule, the property of secret- ing acids, so that after a while they render the medium unfit for their own further growth long before the pabulum is exhausted. THE INERT AND INHIBITIVE CONDITIONS OF BACTERIAL LIFE. 1. Diffuse daylight seems to have little or no influence on bacterial growth, but most bacteria and their spores are killed after a more or less prolonged exposure to the direct rays of the sun, a fact of great importance in practical hygiene. 2. Electric currents and the X-rays seem to have but little influence on bacterial growth. 3. Compressed air seems to retard the growth of bacteria. 4. A number of chemical substances either kill off bacteria or arrest their growth — as will be spoken of more fully under the head of Antiseptics and Disinfectants. THE VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA. These are manifold and various, and occasionally attempts at classification have been based on some of these special functions. 1. Fermentation. — The alcoholic and acetic acid fermentation are the work of the yeast fungi ; but the butyric and lactic acid fermentation in milk are caused by a number of bacteria, VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA. 39 the most prominent of which are those that bear the respec- tive names. This class of bacteria are called the zymogenic. 2. Putrefaction is caused by a variety of bacteria called saprogenic. 3. Pigments and colors are produced by a number of bacteria called chromogenic. Sometimes the pigment is secreted by the cells and diffused in the surrounding media, the bacteria remaining uncolored. At other times the pigment is limited to the cell-protoplasm and membrane, the surrounding medium remaining uncolored. As a rule, these pigments are produced only in an atmosphere of oxygen. 4. Some bacteria, called photogenic, phosphorescent, or fluorescent, have the property of emitting and producing light. 5. Bacteria secrete poisonous substances which are some- times highly deadly, and which from their character are classed as ptomaines and toxalbumins. The ptomaines are crystallizable basic substances, closely allied to the vegetable alkaloids. The toxalbumins are non-crystallizable substances, similar to albumin or protein. It is by the poisonous effects of these toxins that most bacteria affect the human body. 6. Some bacteria liquefy gelatin, and this fact is made use of in differentiating the different species. This liquefaction has been demonstrated to be caused by a soluble peptonizing ferment secreted by the bacteria cells, and after filtration of a bouillon culture of the liquefying bacterium the filtrate free from bacteria possesses the same power of liquefying gelatin. 7. Some bacteria produce acids and other alkalies — as may be demonstrated by their action. 8. Various gases, such as carbon dioxide, marsh gas, hydro- gen sulphide, etc., are the products in some instances of bacterial growth. 9. A number of bacteria produce odors without the apparent production of gases. 10. A few produce aromatics, and are used extensively in the arts. 11. Again, a certain class peptonize milk by means of 40 THE FUNDAMENTAL PRINCIPLES. enzymes which they secrete. Some cause a reduction of the nitrites. 12. Others oxidize nitrogen into nitrites, and even nitrates. 13. Finally, some bacteria cause disease in man and animals. These, called pathogenic, are of the greatest interest to physicians, and it is the discovery of this pathogenic prop- erty which in the last twenty years has given such an impetus to the study of bacteriology. QUESTIONS. Who first discovered microorganisms iu decaying vegetable infusions ? To what kingdom were they thought to belong at first? What classification is generally accepted at present ? Name the three varieties of fungi. What criticism may be made of this classification ? Give a proper definition of bacteria. Of what does the bacteria cell consist ? What causes some bacteria to have a capsule ? Are the capsules generally retained by bacteria when grown artificially ? What causes some bacteria to be more difficult of staining than" others? Into what three varieties or species are bacteria divided ? What are megacocci ? Micrococci ? Diplococci ? Merismopedia ? Sarcinsc ? Streptococci ? Staphylococci ? Ascococci ? What is the shape of bacilli ? When are they said to be slender? When thick ? How do they develop ? What are spirilla ? Can one species develop into another species? What are the limits of size of bacteria ? What is a mikron ? What is the average length of the pathogenic bacteria? How do bacteria multiply? Describe this process in the case of cocci, bacilli, and spirilla? What are spores, and how are they formed ? How do spores compare with bacteria in their power of resisting injurious external influences and of staining ? To what is this due? What is meant by a clostridium ? What is meant by a drummer-bacillus ? What is the name of the ordinary spores? What is meant by arthrospores ? What is the theory of spore-formation ? What is meant by motility, and what species have this power? What are flagella? What names are given to bacteria according to the position and number of their flagella? What is meant by aerobic bacteria? Anaerobic? Facultative aerobic? Facultative anaerobic? What are saprophytes? Parasites? What conditions are required for the proper development of bacteria? What is the most favorable temperature for bacterial growth ? What is the effect of cold? Of excessive heat? How do spores differ from bacteria in their reaction to heat and cold ? EXAMINATION AND STAINING OF BACTERIA. 41 What is the effect of moisture on bacterial life ? Of desiccation ? What pabulum is necessary for the life of bacteria ? How do you explain the life of bacteria in saline solution and in media with no appreciable amount of organic matter? What is meant by culture-soils or media ? What other conditions influence bacterial growth ? What is the effect of sun-light on bacteria ? Of electric currents ? X-rays ? Of compressed air? What are the manifold functions of bacteria ? Enumerate and explain the same. What is the chemical difference between a ptomaine and a toxalbumin? What are zymogenic ? Chromogenic? Photogenic? Pathogenic bacteria? CHAPTER II. THE EXAMINATION AND THE STAINING OF BACTERIA. THE EXAMINATION OF BACTERIA. FOE the purpose of examining bacteria the highest power of the microscope is necessary, although many are seen with an ordinary dry ^ or ^ objective. Ordinarily, however, the ^ oil-immersion objective is indispensable for the proper study of microorganisms. Bacteria are examined either alive and in their natural con- dition, or dried on microscope slides or cover-glasses as a thin film, and stained. 1. For the purpose of examining bacteria in their natural condition, it is only necessary, when the bacteria are in liquid medium, to put a droplet of the liquid on a slide, cover lightly with a thin cover-glass, put upon the stage of the microscope, and bring into focus the y1^ inch oil-immersion objective, being careful to close almost completely the iris diaphragm underneath the substage condenser. When the bacteria are in solid medium, a minute particle of the culture is taken up on a sterilized platinum needle and stirred up in a small drop of sterilized water on a slide, and a cover-glass applied as above. In these ways the form, shape, mode of grouping, and motility 42 EXAMINATION AND STAINING OF BACTERIA. of the bacteria may be very well seen and carefully in- vestigated. The Hanging-drop Preparation. When it is necessary to keep them under observation for some time, however, or when it is desired to study their development, multiplication, or sporulation, what is known as the hanging-drop preparation or culture is resorted to. This consists in placing a small drop of the liquid contain- ing the bacteria on a thin cover-glass and placing upon this cover-glass a slide with a concavity in its centre, known as the hanging-drop slide, after having carefully lubricated the edges of the surface around the concavity with vaselin, so that the slide will adhere to the cover-glass when it is pressed down on it. In this way a hermetically sealed, transparent, moist chamber is obtained, which may be kept under observa- tion on the stage of a microscope almost indefinitely. FIG. 9. Longitudinal section of hollo\y ground glass slide for observing bacteria in hanging drops. (Abbott.) 2. The foregoing are very simple and useful methods for rapid examinations of bacteria, and have many applications, but they are far from satisfactory in all cases, as they fail to bring out full details of bacterial structure. For this purpose recourse must be had to the staining methods introduced by Koch, and perfected by Weigert, Loeffler, and many others. In this method bacteria are examined dead. THE STAINING OF BACTERIA. I. The General Mode of Procedure. One or several droplets of the suspected liquid are spread thinly and evenly on the surface of a slide or thin cover-glass, THE STAINING OF BACTERIA. 43 or, in case of a solid, a small particle is diluted with sterile water and spread in the same way on the slide or cover-glass. This is allowed to dry in the air, protected from dust, or else is held in a suitable forceps high up over the flame of an alcohol lamp or Bunsen gas-burner until dry, thus forming a thin film on the surface of the glass. After this step has been carefully taken the slide or cover-glass is taken up with a pair of forceps and passed several times through the flame, film-side up, for one-half to one second at each pass, three times in the case of a cover-glass and eight or ten passes in the case of a slide ; which is for the purpose of setting the prepa- ration, or coagulating the albuminoids and fixing them to the glass so that they will not be easily washed away in the sub- sequent procedures. After allowing the preparation to cool well, it is ready for the staining reagents. II. The Most Commonly Used Stains. The basic anilin dyes, such as fuchsin, methylene-blue, gentian- violet or methyl- violet, and Bismarck-brown, are most commonly employed. They are made into saturated alcoholic solutions, to be kept in stock, and are freely diluted with water whenever required for use. III. The Application of the Dyes. Nearly all the known bacteria, with the exceptions to be mentioned later, are readily stained by the watery solutions of any of the basic anilin dyes. The film on the slide or cover-glass, prepared as just described, is covered by a few drops of the stain, or the cover glass, film-side down, is floated in a watch-glass full of the staining solution ; at the end of from one-half to two or three minutes the staining fluid is poured off, the slide or cover-glass washed rapidly in water and then allowed to air-dry ; after which, in the case of cover-glass preparations, they are inverted upon a drop of Canada balsam on a slide and examined with the oil-immer- sion lens ; or, when slides have been prepared, after wash- 44 EXAMINATION AND STAINING OF BACTERIA. ing and drying a drop of cedar oil is put over the prepa- ration, and the same is examined with the oil-immersion objective without the use of a cover-glass. Air-bubbles are often caught between the balsam and the cover-glass. Gentle uniform pressure begun at the centre of the cover-glass and progressively applied toward its periphery will ordinarily remove them. If this should fail, heat care- fully applied until the balsam is quite soft will aid in the riddance of the others. IV. The Special Methods of Staining. As stated above, this method may be applied for the stain- ing of nearly all bacteria. Some, however, are not so easily stained, and special methods must be resorted to to increase the penetrating power of the dye. The most commonly used will be here described. 1. Loeffler's Method. In this method, instead of using the ordinary watery solu- tion of an anilin dye, Loeffler's alkaline solution of methylene- blue is used. This is prepared as follows : Concentrated alcoholic solution of methylene-blue, 30 parts; Caustic potash solution (1 : 10,000), 100 " . Mix well and filter. This method stains well all the ordinary bacteria, but is specially useful for the staining of the bacillus of diphtheria. 2. Koch-Ehrlich's Method. Anilin water is prepared by adding a few drops of anilin oil, drop by drop, to distilled water in a test-tube, shaking well after the addition of each drop and until the liquid assumes a milky appearance, after which it is filtered through moistened filter-paper until the filtrate is absolutely clear. To 100 parts of this clear filtrate of anilin water, 10 parts THE STAINING OF BACTERIA. 45 of absolute alcohol and 10 parts of an alcoholic solution of fuchsin, methylene-blue, or gentian- violet, are added, and the whole thoroughly mixed and filtered. The preparation is better when made fresh in small quantity at each time it is needed, as it decomposes in a few days. In Koch-Ehrlich's method this anilin water (violet, blue, or red) is used instead of the simple watery solution of the dye. It possesses much more penetrating power, and this again may be increased by gently heating the slide or cover- glass, over a Bunsen burner, while it is being stained. It is applicable whenever it is desirable so to fix the color in the bacteria that one may by means of decolorizing agents remove the color from the surrounding objects and tissue and fix it solely on the bacteria themselves. Some bacteria so retain the color by this method that it serves to distinguish them from others which they very much resemble, but which do not possess the same persistent retention of the stain. The usual decolorizing agent employed is either ordinary alcohol or diluted sulphuric or hydrochloric acid (1 : 4). 3. Gram's Method. Treat the object to be colored with anilin-water gentian- violet for about three minutes, after which immerse in Gram's fluid. This consists of: Iodine, 1 part; Iodide of potassium, 2 parts ; Water, 300 " . Maintain this immersion for five minutes, then pass the preparation through alcohol and rinse in water. If the object is still of a violet color, treat it again with Gram's fluid and alcohol until no violet is visible to the naked eye. This method is applicable to a number of bacteria, and serves as a mode of differentiation between some of them which could not otherwise be distinguished under the microscope. It serves also to color the capsule of bacteria, and, slightly modified, is useful for the stain of bacteria in tissue. A contrast-color should be given to the uncolored parts. 46 EXAMINATION AND STAINING OF BACTERIA. 4. Ziehl's Carbol-fuchsin Method. Make a solution of carbol-fuchsin as follows : Fuchsin, 1 part; Crystallized carbolic acid, 5 parts ; Alcohol, 10 " ; Water, 100 " . Immerse the glass in or cover same with the carbol-fuchsin solution, heat gently over the flame of a Bunsen burner, gradu- ally bringing to a point just below boiling; repeat this two or three times ; after which immerse in nitric acid solution (1 part of acid to 3 parts of water) until the color is scarcely visible to the naked eye. To ascertain this, wash off the acid from the film with water. If color is still faintly visible, remove it by dipping into alcohol ; wash in water, dry, mount in Canada balsam, and examine. A contrast-color may be given to the rest of the specimen by employing methylene-blue . This is a useful method in coloring cover-glass preparations for tubercle bacilli or for the Bacillus leprse. 5. Gabbett's Method. This is a modification of Ziehl's method, and is perhaps the best method, on account of its simplicity and rapidity, for the staining of the tubercle bacillus in secretions. It is as fol- lows : Prepare a slide or cover-glass film as indicated, im- merse in Ziehl's carbol-fuchsin solution for ten minutes, remove to Gabbett's sulphuric acid methylene-blue solution for three to five minutes, rinse in water, dry, mount, examine. The tubercle bacilli are colored red and the other bacteria and cell- nuclei are colored blue. Gabbett's solution consists of : Methylene-blue, 1 to 2 parts ; Sulphuric acid, 25 " ; Water, 75 " . THE STAINING OF BACTERIA. 47 Besides the foregoing means, which will stain the bacteria in films, bacteriologists adopt special methods for the staining of bacteria in tissues, and for the staining of spores, flagella, and the capsules of bacteria. V. The Staining of Capsules. 1. Welch's Glacial Acetic Acid Method. Prepare the cover-glass in the usual way. Cover the film with glacial acetic acid, pour off the acid immediately, do not wash in water, cover the film with anilin-water gentian-violet solution for three or four minutes, wash in a 0.5 to 2 percent, solution of sodium chloride, dry, mount, and examine. The acetic acid coagulates the mucin of the capsules and renders the same distinctly visible. 2. Johne's Method. A cover-glass film prepared in the usual way is covered with a solution of gentian- violet and heated until steam rises. The stain is then washed off in water, and the cover-glass put into a 2 per cent, acetic acid solution for from ten to fifteen seconds. It is again washed in water, dried, and mounted in balsam. VI. The Staining of Spores. Though with the ordinary method of staining, spores in bacteria may be recognized by their highly refractive appear- ance and by the fact that they have not taken the color, they may be stained themselves, however, by special methods. The First Method (Abbott's). A cover-glass preparation is covered with Loeffler's alkaline methylene-blue solution and held by its edge with forceps over the Bunsen burner flame until the fluid begins to boil. It is then removed from the flame, and after a few seconds heated again. This step is repeated a number of times for one or two minutes, after which it is washed in water, and decolor- 48 EXAMINATION AND THE STAINING OF BACTERIA. ized, until all blue coloring visible to the naked eye has dis- appeared, in the following solution : Alcohol (80 per cent.), 98 parts ; Nitric acid, 2 " . The cover-glass is then dipped for a few seconds into the fol- lowing solution : Saturated alcoholic solution of eosin, 10 parts ; Water, 90 " . After this it is again rinsed in water, dried, and mounted. The Second Method. Float a cover-glass preparation film-side down in a watch- glass full of Koch-Ehrlich's fuchsin solution. Take the watch- glass by its edge with a pair of forceps and hold same over a low Bunsen flame until the staining fluid begins to boil. Remove from the burner, and after a few minutes repeat this process five or six times. After cooling, the cover-glass, without washing in water, is transferred to a second watch- glass containing a decolorizing solution as follows : Absolute alcohol, 100 parts ; Hydrochloric acid, 3 " . Place the cover-glass, film-side up, at the bottom of this watch-glass and let it remain for one or two minutes. Remove, wash in water, stain with methylene-blue solution for one or two minutes, wash rapidly in water, dry, and mount. By this method the spores will be stained red and the body of the bacteria cells blue. The Third Method. Cover-glasses are prepared in the usual way. After fix- ing, the preparation is immersed in chloroform for two min- utes, washed in water, placed for one or two minutes in a 5 THE STAINING OF BACTERIA. 49 per cent, solution of chromic acid, again washed in water, and stained in hot Ziehl's earbol-fuchsin solution for five minutes. The staining fluid is poured off and, without washing in water, the preparation is decolorized in 5 per cent, sulphuric acid. After this it is again washed in water, and finally stained for two or three minutes in the watery methylene-blue solution. The spores will be stained red, the body of the cells blue. The action of the chloroform is to dissolve the fatty crys- tals that may be in the preparation. The chromic acid acts on the membrane of the spores and permits the entrance of the stain. The Fourth Method (Fiocca's). Ten to 20 parts of an alkaline solution of an anilin color are added to 20 c.c. of a 10 per cent, ammonia solution in a watch-glass ; then heat is applied until steam commences to be given off. The cover-glass stays in this hot solution from three to five minutes ; it is then taken out and washed in a 20 per cent, solution of nitric or sulphuric acid to decolorize it, then washed again, when a contrast-color may be given. It must be remembered that there is considerable difference in the behavior of spores of different bacteria to the staining methods described; some stain very readily and others with considerable difficulty. Practice will be the best guide as to which method is best to employ and to what extent it should be carried out. VII. The Staining of Flagella. The hair-like processes of the bacteria which serve for their locomotion, the flagellaj can not, on account of their fineness, be seen in any stained specimen, nor can they be stained by any of the ordinary methods just described for staining bacteria. In order to make them visible, it is neces- sary to use special stains in which the action of mordants plays an essential part. 1. Loeffler's Method. This is the most common method, and is as follows: Clean very carefully a thin cover-glass, and spread very thinly and 4— M. B. 50 EXAMINATION AND STAINING OF BACTERIA. evenly upon it as few as possible of the bacteria to be exam- ined. This is done by diluting with sterilized watei* a number of times the culture containing them. The cover-glass is dried and fixed in the ordinary way. The following solution, known as a mordant, is then applied : Tannic acid (20 per cent, solution in water, filtered), 10 parts; Cold saturated solution of ferrous sulphate, filtered, 5 " ; Saturated alcoholic solution of fuchsin, 1 part. A few drops of it are placed on the film, and the cover-glass taken up with a pair of forceps and held over the flame of a Bunsen burner until the solution begins to steam, but not allowing the boiling-point to be reached. It is next washed rapidly in water, and then in absolute alcohol. The bacteria are to be stained in anilin- water fuchsin solution in the ordi- nary way. Practice has shown, however, that different bacteria behave differently when exposed to this staining, and Loeffler himself has modified it to meet these requirements. Having found that the addition of an alkali favors the staining of flagella in some of the bacteria, he has added to his stain 1 per cent, of sodium hydrate. In other cases, having found that an acid helps to bring out the flagella, he has added to his stain a solution of sulphuric acid in water of such strength that 1 c.c. will neutralize 1 c.c. of the sodium hydrate solution. The following bacteria require an acid solution added to the stain : Bacillus pyocyaneus, the spirillum of Asiatic cholera, Spirillum rubrum, Spirillum concentricum, Spirillum Metch- nikowi. The following bacteria require an alkaline addition to the staining solution : Bacillus mesentericus, Micrococcus agitts, Bacillus typhosus, Bacillus subtilis, bacillus of malignant redema, Bacillus anthracis symptomatici. In a general way one may say that bacteria that produce acid in the media in which they grow require the addition of an alkali THE STAINING OF BACTERIA. 51 to the mordant, and those which produce alkalies require the addition of an acid. 2. Bunge's Method. Bunge's method, a modification of Loeffler's method, is as follows : Prepare a saturated solution of tannin in water, and also a 5 per cent, solution of sesquichloride of iron in distilled water ; to 3 parts of the tannin solution add 1 part of the iron solution. To 10 parts of such a mixture add 1 part of concentrated watery solution of fuchsin. This mordant should never be used fresh, but only after it has been exposed to the air for several days. The cover-glass, thoroughly cleaned, is covered over by this mordant for five minutes, after which it is slightly warmed. It is then washed, dried, and stained faintly with a little carbol-fuchsin. 3. Pitfield's Method. The following solution is used as a mordant : Tannic acid (10 per cent, solution, filtered), 10 parts; Corrosive sublimate (saturated aque- ous solution), 5 " ; Alum (saturated aqueous solution), 5 " ; Carbol-fuchsin, 5 " . Let this stand, and pour off the clear fluid. This mordant will keep for one or two weeks. The staining fluid is prepared as follows : Alum (saturated aqueous solution), 10 parts; Gentian-violet (saturated alcoholic solution), 2 " . This stain is to be prepared fresh every second or third day. The modus operandi is as follows : On an absolutely clean cover-glass make a thin film as already described. Treat the film with the mordant applied cold for twenty-four hours, or 52 EXAMINATION AND STAINING OF BACTERIA. with the mordant applied steaming, but not boiling, for three minutes ; wash off thoroughly in water and dry ; treat with the stain in the same manner as with the mordant ; wash in water, dry, mount, and examine with an oil-immersion lens. Other methods, such as Van Ermengem's, BowhilTs, etc., are highly recommended, but in the author's hands have given no better result than the foregoing three methods. VIII. The Staining of Bacteria in Tissues. 1. The Staining of Sections. Sections should be cut in the ordinary way in paraffin or celloidin. The sections are first put into water for a few minutes, then transferred to watch-glasses containing watery solutions of any basic anilin dye, and allowed to remain from five to ten minutes ; they are next removed, rinsed in water, decolorized in a 0.1 solution of acetic acid for a few seconds, again washed in water, then for a few minutes in absolute alcohol, and placed in cedar oil or xylol. They are allowed to remain in xylol from one-half to one minute. They, are finally spread thinly on a spatula and brought to the slides, where the excess of fluid is taken up with blotting-paper ; after which a drop of xylol balsam is placed on the sections, which are covered by thin clean cover-glasses, when they are ready for examination. 2. Gram's Method for Staining Bacteria in Tissue. This is practically the same as the method for cover-glass preparations. The section is stained in anilin-water gentian-violet (Koch- Ehrlich) diluted with one-third its volume of water. The section remains in this for about ten minutes at the tem- perature of the incubator. From this it is taken out and washed alternately in Gram's iodine solution and alcohol until all the naked-eye color has been extracted. It is then put into a watery solution .of eosin or Bismarck-brown for one minute, again washed in alcohol a few seconds, and then put THE STAINING OF BACTERIA. 53 for one-quarter minute in absolute alcohol. After this it is transferred to xylol for one-half minute, then lifted to a slide, mounted in Canada balsam, and examined. 3. Weigert's Modification of Gram's Method. Stain sections in the Koch-Ehrlich anilin-water gentian-violet solution for five or ten minutes ; wash them afterward in water or physiological salt solution. Transfer to slide and remove excess of fluid with blotting-paper. Treat with the iodine solution of Gram for three minutes. Take up the ex- cess of solution with blotting-paper. Cover the section with anilin oil, wash out the oil with xylol, and mount in xylol balsam. The anilin oil in this case acts as a decolorizing agent, and should be removed carefully, otherwise the speci- men will not keep. 4. Kuehne's Carbolic Methylene-Blue Method. Put the sections into the following solution for one-half hour : Methylene-blue in substance, 1^ part ; Absolute alcohol, 10 parts. Rub thoroughly in a mortar, and when the blue is completely dissolved add 100 parts of a 5 per cent, carbolic acid solu- tion. This solution should be made fresh when needed. The sections are stained for fifteen minutes in this solution and then washed in water until free from it. They are next transferred to a 2 per cent, hydrochloric acid solution, then to a solution of carbonate of lithium (of the strength of 6 to 8 drops of a concentrated watery solution of the salt to 10 drops of water), and from this they are again washed in water and in absolute alcohol containing sufficient meth- ylene-blue in substance to give it a blue color, then for a few minutes in anilin oil to which a little methylene-blue in sub- stance has been added, and they are then rinsed out in pure anilin oil ; from this they are placed in oil of turpentine or thymol for two minutes, then in xylol, and mounted in xylol balsam. 54 EXAMINATION AND STAINING OF BACTERIA. The advantages of this method are that it is generally ap- plicable. Bacteria are not robbed of their color, and the tissue is sufficiently decolorized to render the bacteria visible and to admit of a contrast-stain. 5. Ziehl-Neelsen's Method. The sections are warmed in a solution of carbol-fuchsin for one hour at a temperature of about 45° to 50° C., decolorized for a few seconds in a 5 per cent, sulphuric acid solution, then put into 70 per cent, alcohol, then in absolute alcohol for a few seconds to dehydrate, then in xylol to clear, and mounted on a slide in xylol balsam. QUESTIONS. What powers of the microscope are necessary for the examination of bacteria ? How are bacteria examined alive ? How is a hanging-drop preparation made ? What is the usual method of staining bacteria? What are the most usual stains used for bacteria? What is Loeffler's method ? Describe the Koch-Ehrlich method. What are the usual decolorizing agents used ? What is Gram's method ? Describe Ziehl's carbol-fuchsin method. Describe Gabbett's method. Give Welch's method of staining the capsule of bacteria. Give Johne's method. Give Abbott's method of staining spores. Moeller's method. Fiocca's method. Give Loeffler's method of staining flagella. Which bacteria require the addition of acid to the mordant in order to stain their flagella? Which require the addition of alkalies? Describe Bunge's method of staining flagella. Pitfield's method. How would you stain bacteria in tissue? Give Gram's method. Weigert's method. Kuhue's method. Ziehl-Neelsen's method. THE CULTIVATION OF BACTERIA. 55 CHAPTER III. THE PKOCESS, MEDIA, AND UTENSILS OF THE CULTI- VATION OF BACTERIA. THE PROCESS OP THE CULTIVATION OF BACTERIA. As mentioned before, bacteria can not be separated from one another by form and appearance under the microscope only. Indeed, in a number of instances, and even with the highest power of the microscope, some very inoffensive bac- teria resemble very much and can not be differentiated from some that are highly pathogenic. Especially is this the case with the group of cocci. In all such cases it is necessary to study the properties and mode of growth, and for this purpose the bacteriologist must use and prepare suitable soils, which are known by the name of culture -media. These culture-media must themselves be absolutely free from all live bacteria — that is, sterile ; or, if they naturally contain bacteria, or if bacteria have been introduced during their preparation, these must be destroyed, or, in bacteriological language, the media must be sterilized. THE MEDIA OF THE CULTIVATION OF BACTERIA. All substances that contain carbon and nitrogen compounds in assimilable form associated with water may be used as culture- soils for bacteria. The culture-media used ordinarily are either natural or artificial. They may be liquid or solid ; or, again, they may be solid at the temperature used, and liquefied at a temperature not high enough to destroy bacterial life. I. The Most Commonly Used Liquid Culture-Media. l. Milk. Milk, as contained in the udders of the cow, is an excel- lent culture-medium, and is generally sterile. In its col- lection, however, it usually becomes contaminated — that is, bacteria are introduced into the milk : so much so that it is 56 THE CULTIVATION OF BACTERIA. necessary to sterilize the same before using, and for this pur- pose what is known as the discontinuous or fractional steril- ization by steam is resorted to. Mode of Preparing Sterilized Milk. — Sterilized test- tubes from 5 to 7 inches in length, and about from 1 to 1J inches in diameter, are filled to one-third their capacity with raw milk. The test-tubes are plugged tightly with ordinary cotton-batting, and are submitted to live steam in the steam sterilizer, at 100° C., for twenty minutes each time, on three consecutive days. Before sterilization tincture of blue litmus may be added to the milk, and in this way the generation of acids by bacteria may easily be ascertained. Milk prepared in the foregoing manner offers an excellent culture-soil for nearly all forms of bacteria ; it serves also for differentiating between certain species accordingly as these have the property of coagulating the casein in the rnilk rapidly, slowly, or not at all. 2. Animal Blood-Serum. Animal blood-serum obtained from a slaughter-house is an exceedingly useful culture-medium. Its mode of preparation is as follows : In large cylindrical jars the fresh fluid blood is collected and allowed to remain untouched for a half-hour or an hour. After this, with a clean sterilized glass rod the coagulum that begins to form is detached from the sides of the vessel. The vessel then, well covered and. protected from dust, is put into an ice-box, and at the end of twenty-four hours the clot, consisting of fibrin and of blood-corpuscles, is firm and sinks to the bottom of the vessel, leaving a clear serum above and around it. This clear serum may IDC siphoned or pipetted out and distributed among sterilized test-tubes, which, after being plugged with absorbent cotton-batting, are sterilized in Koch's serum steril- izer by the low temperature process to be described later. Loeffler's modification of this method is generally used in all municipal laboratories for the cultivation and diagnosis of the bacillus of diphtheria : THE MEDIA OF THE CULTIVATION OF BACTERIA. 57 Beef or mutton-blood is collected in the usual way, and to 8 parts of the clear serum 1 part of glucose-bouillon is added. This mixture distributed among test-tubes is sterilized and hardened in a slanting position in a steam sterilizer at a tem- perature between 80° and 90° C., for an hour each day dur- ing a whole week. 3. The serum of ascitic fluid and (4) the fluid of hydrocele are sometimes used for the cultivation of bacteria, and are prepared in the same manner as ordinary blood-serum. 5. Urine. Urine may also be used for the cultivation of bacteria. For this purpose it is obtained by means of a sterilized cathe- ter directly from the bladder, where it is generally sterile. It is safest, however, to sterilize it by steam for one hour before use. 6. Pasteur's Solution. Filtered water, 100 parts; Cane-sugar, 10 " ; Ammonium tartrate, 1 part. With the addition of 1 part of the ashes of yeast this was formerly extensively used as a culture-medium, but is now seldom used. 7. Bouillon. Bouillon is the most frequently used of all the fluid media. It is prepared as follows: 1 pound of fresh lean beef is chopped up very fine and covered with 1 liter of sterilized water, and put into an ice-box for twenty-four hours, after which the aqueous extract is obtained by filtration through muslin by pressure, sufficient water being added if necessary to make up the original liter. To this filtrate 10 grams of peptone and 5 grams of sodium chloride are added, and the whole is cooked on a water-bath or in an enamelled iron kettle for a half-hour, after which sufficient of a saturated solution of sodium carbonate is added drop by drop to give the mixture a slight alkaline reaction. This, after cooking 58 THE CULTIVATION OF BACTERIA. for fifteen or twenty minutes, is filtered through absorbent cotton several times into test-tubes and sterilized by steam for twenty minutes on three successive days. The addition of 5 per cent, neutral glycerin to this bouillon makes an excellent liquid culture-medium for tubercle bacilli, and is highly recommended by Rotix. Any standard beef-extract, such as Liebig's, Armour's, Wyeth's, etc., may be used in making this bouillon, instead of the meat itself, 5 grams of the extract being added to 1 liter of water, the rest of the process being the same. II. The Most Commonly Used Solid Culture-Media. 1. Gelatin. Nutrient gelatin is prepared as follows : A meat-infusion, as described for bouillon, is prepared and put into a large Bohemian flask of 2 liters capacity ; to this meat-infusion are added : Chloride of sodium, 5 grams ; Peptone, 10 " ; Best quality gelatin 100 " . The flask, loosely closed with a plug of absorbent cotton, is cooked over a water-bath until all the gelatin is dissolved. This will take from an hour and a half to two hours. After this the mixture, which will be found to be decidedly acid, is neutralized by means of a solution of sodium carbonate added drop by drop until the mixture is faintly alkaline, as in the bouillon. The flask is again put over the water-bath for an hour, after which the mixture is filtered hot through absorbent cotton and sterilized in the steam sterilizer for twenty minutes each day on three consecutive days. Great care must be exercised to introduce the medium into the sterilizer only when steam is actively being generated, and not to allow it to cool in the sterilizer. Advantages. — Gelatin is a most excellent medium, and remains solid at room temperature (22° or 24° C.), but is readily liquefied when exposed to a higher temperature. THE MEDIA OF THE CULTIVATION OF BACTERIA. 59 Not only does it serve as an excellent medium for the culti- vation of nearly all bacteria that grow out of the incubator temperature (36° to 37° C.), but it offers by the plate -method one of the best culture-soils for isolating bacteria. As some of the bacteria liquefy gelatin and others do not, it is useful also in the differentiation of these species. On account of its clearness, its easy preparation, and the other advantages mentioned above, it is one of the best culture- soils available. The disadvantages lie chiefly in the fact that it liquefies at a much lower temperature than that of the incubator, and can not therefore be used for the cultivation of some patho- genic bacteria which grow only at blood temperature. 2. Agar. Nutrient agar is prepared much in the same manner as gelatin, except that 20 or 30 grams of 2 or 3 per cent, agar are added, instead of the gelatin, to the meat-infusion, and the process of cooking must be much more prolonged (four to five hours), as the agar is much slower in dissolving. It is necessary also sometimes, after dissolving the agar and neutralizing the mixture, to add the white of one or two eggs in order to clarify the solution before filtration. This is done by removing the flask from the fire and allowing it to cool to a temperature below 70° C. After clarification the mixture is again cooked for one and one-half to two hours and filtered through absorbent cotton two or three times. This filtration is a much more difficult process than with gelatin, and must be carried on in the steam sterilizer. After filtration the agar is distributed among test-tubes for use, and is sterilized by the same procedure as nutrient gelatin. Nutrient agar has a melting-point much higher than that of gelatin, about 42° C., so that it may be used for the culti- vation of those bacteria which grow only at or best at the temperature of the human body. It possesses all the advan- tages of gelatin, but is not so clear and transparent, and is not liquefied by the secretions of any known bacteria. It is useful also for the isolation of bacteria by means of plate cultures. 60 THE CULTIVATION OF BACTERIA. The disadvantages of agar, when compared with gelatin, lie chiefly in the greater difficulty of its preparation, and especially of its nitration. 3. The glycerin-agar culture is obtained by adding 5 per cent, of neutral glycerin to nutrient agar before sterilization. This makes a very favorable medium for the cultivation of the tubercle bacillus and of some other pathogenic bacteria. 4. A mixture of agar and gelatin in bouillon is sometimes used so as to obtain the advantages of the two substances com- bined. This mixture is prepared from bouillon in the usual way by dissolving in the bouillon 0.75 per cent, of agar and 5 per cent, of gelatin in the manner outlined in the foregoing paragraphs. There are a number of special filtering apparatus constructed for the purpose of facilitating the nitration of agar. None in the author's estimation has any advantages over filtration through absorbent cotton in the steam sterilizer. 5. Potato Culture. Koch called attention to the great value of potato cultures for differentiating species of bacteria. The mode of preparing potatoes is as follows : A sound potato, of good size, with an intact epidermis, is chosen, and thoroughly washed and scrubbed with a brush to remove all dirt ; after which, with a sharp-pointed knife, all the eyes and discolored parts are cut out of the potato. It is then washed again in water and put into a solution of 1 : 500 bichloride of mercury for an hour, after which it is cooked in the steam sterilizer for forty minutes on each of two suc- cessive days. Just before inoculation the potato is split into halves by means of a sterilized knife and allowed to fall into a moist chamber cut surface up. The moist chamber consists of a double glass dish, the upper one of which is larger than the lower. This chamber should be thoroughly rinsed with a 1 : 500 solution of bichloride of mercury before use. Some precautions are necessary to prevent contamination of the potato from external germs. The hand that cuts the THE MEDIA OF THE CULTIVATION OF BACTERIA. 61 potato should be thoroughly sterilized in a 1 : 1000 bichloride of mercury bath. It is better also after washing the potato and before submitting it to the bichloride bath to wrap it in some thin tissue paper, and to keep it in this paper until ready for inoculation. Fio^ll. Preparation of a Potato for Test-tube Culture. — By means of a cork-borer (Fig. 10) a cylinder is cut from a sound potato. This cylinder is cut obliquely FIG. 10. Nest of cork-borers, used to cut potatoes for test-tube cultures. into two pieces, and each placed into a large test- tube (Fig. 11), in which it is sterilized and cooked on three successive days for a half-hour in a steam sterilizer. 6. Potato-paste is sometimes used for cultivation. For this purpose a potato is boiled, peeled, and mashed with a little sterilized water, placed in a suitable glass dish, and sterilized for one-half hour on three successive days in a steam sterilizer. 7. Bread-paste is a useful medium for the growth of moulds, and is made in the same way as potato- Potatube.test paste. III. The Most Commonly Used Special Culture-Media. In making final distinctions between the different species of bacteria the following special media are occasionally used : 1. The Peptone Solution. Dry peptone, 1 part; Sodium chloride, J " ; Distilled water, 100 parts. This is filtered, decanted into test-tubes, and sterilized in the steam sterilizer. 62 THE CULTIVATION OF BACTERIA. This preparation is chiefly used for determining whether the bacteria secrete indol or not. It is necessary therefore to see that the peptone preparation used be chemically pure, also that the solution be free from the presence of carbohydrates. 2. Glucose-, lactose-, and saccharose-bouillon. These are made by adding to the bouillon after filtration and before steriliza- tion from 1 to 2 per cent, of the desired kind of sugar. THE UTENSILS OF THE CULTIVATION OF BACTERIA. For making and keeping cultures the following instruments, glassware, and utensils are required : 1 . Perfectly clean glass tubes, 5 to. 7 inches long and J to FIG. 12. FIG. 13. Glass test-tube. Erlenmeyer flask. 1J inches in diameter (Fig. 12). with ordinary cotton-batting. 2. Erlenmeyer flasks (Fig. 13). These should be plugged THE UTENSILS OF THE CULTIVATION OF BACTERIA. 63 3. Cylindrical brushes with reed handle, and wire-handled brush of Lentz & Sons, for the purpose of cleaning test-tubes (Fig. 14). FIG. 14. Brushes for cleaning test-tubes : a, with reed handle ; 6, with wire handle. 4. Bohemian glass flasks of 1 and 2 liters capacity. 5. Platinum needles, straight and looped, mounted in glass rods (Fig. 15). FIG. 15. (a) Looped and (6) straight platinum wires in glass handles. 6. Plates of ordinary glass about 4 by 5 inches, and Russia iron boxes to hold them during sterilization (Fig. 16). FIG. 16. Russia iron box for holding plates, etc., during sterilization in dry heat. (Abbott.) 7. Glass benches for supporting plates (Fig. 17). 64 THE CULTIVATION OF BACTERIA. FIG. 17. Glass benches for supporting plates. 8. Graduated measuring cylinders of 100 and 1000 c.c. capacity (Fig. 18). 9. Graduated pipettes of 1 c.c. capacity divided into tenths, and 10 c.c. divided into c.c. (Fig. 19). FIG. 18. FIG. 19. FIG. 20. FIG. 21. Measuring cylinder. Graduated pipette. Sternberg bulb. 10. Sternberg bulbs (Fig. 20). 11. Bulb-pipettes (Fig. 21). 12. Petri's double dishes (Fig. .22). FIG. 22. Bulb-pipette. Petri's double dish, now generally used instead of plates. (Abbott.) THE UTENSILS OF THE CULTIVATION OF BACTERIA. 65 FIG. 23. FIG. 24. FIG. 25. Moist chamber with a knob on the Wooden filter-stand. Iron stand with upper dish. rings. FIG. 26. Iron tripod with water-bath. FIG. 27. Wire baskets. 5— M B. 66 THE CULTIVATION OF BACTERIA. Pinchcock. 13. Moist chambers for potato and plate cultures (Fig. 23). 14. Glass funnels of different sizes, 1, 4, and 8 ounces. 15. Wooden filter-stands (Fig. 24). FIG. 30. FIG. 29. Fermentation-tube on left side; ordin- ary tube on right side. Anatomical jar for collecting blood. 16. Iron stands with rings and clamps (Fig. 25). 17. Iron tripods with water-baths (Fig. 26). 18. Test-tube racks of any standard design. QUESTIONS. 67 19. Square and round iron wire baskets for sterilizing test- tubes (Fig. 27). 20. Perforated tin buckets for sterilizing potatoes. 21. Pinchcocks (Fig. 28) for holding test-tubes. 22. Fermentation-tubes (Fig. 29). FIG. 31. Wolf huegel's ruled plate for counting colonies. 23. A 2- and a 4-liter anatomical jar, with tightly fitting cover, for the collection of blood-serum (Fig. 30). 24. Wolf huegel's ruled plates for counting colonies (Fig. 31). 25. Bunsen burners of different sizes. QUESTIONS. What is meant by a culture-medium ? What is meant by the sterilization of culture-media ? How is milk prepared as a culture-medium ? How is milk sterilized when used for a medium ? Why is tincture of litmus added to milk medium ? How does milk help in differentiating between different species of bacteria ? What is the process of making blood-serum ? How is blood-serum sterilized? What is Loeffler's modification of the blood-serum method ? What other animal fluids are occasionally used as culture-media? What does Pasteur's solution consist of? How would you prepare bouillon for cultures ? What are the principal solid cultures ? How would you prepare a nutrient gelatin? How is nutrient gelatin sterilized ? What are the advantages of using gelatin as a culture ? What are its dis- advantages? How would you prepare nutrient agar? What is difficult in the prepara- tion of it? How would you sterilize it? What are its advantages and dis- advantages when compared to gelatin ? How is agar-glycerin made ? How is agar-gelatin made ? What is the best way to filtrate agar ? 68 INOCULATION OF CULTURE-MEDIA WITH BACTERIA. Give Koch's method of preparing potatoes for a culture? What are the precautions necessary for protecting the potato from external germs ? How would you prepare a potato test-tube culture ? How would you prepare Dunham's solution? How are glucose-, lactose-, and saccharose-bouillon prepared ? CHAPTER IV. THE INOCULATION OF CULTURE-MEDIA WITH BACTERIA. THE METHOD OF INOCULATING FLUID MEDIA. FOR the purpose of cultivating bacteria in liquid media it is only necessary to introduce the smallest possible particle containing the bacteria, into the media, by means of a steril- ized platinum needle (Fig. 15). For this procedure the culture-tube is held slightly inclined between the thumb and fingers of the left hand, its cotton plug removed by a twist- ing motion and placed between the backs of the third and fourth fingers of this hand, being careful not to touch that por- tion of the cotton which fits inside of the tube. The inoculating needle is rapidly introduced into the liquid and gently stirred around, after which the cotton plug is replaced, and the plat- inum needle sterilized by heating to redness in the flame. It is good practice to accustom one's self never to take up a platinum needle, whether it is known to be inoculated or unin- oculated, without first sterilizing it by heating it to redness in the flame , and allowing it to cool for a few seconds before taking up with it the inoculating material. The bacteria might other- wise be destroyed by the heat. Again, after use the needle should always be sterilized in the same manner before putting it down. THE METHODS OF INOCULATING SOLID MEDIA. 1. For the inoculation of potatoes and other solid media not hereafter mentioned, it is only necessary to streak the surface of the medium with a platinum needle or other instru- ment which has been dipped into or which contains a small THE METHODS OF INOCULATING SOLID MEDIA. 69 fragment of the contaminating material, care having been taken beforehand to sterilize thoroughly the needle or instrument. 2. Gelatin culture-tubes are inoculated in one of three ways : a. Stab culture, made by puncturing the centre of the solidified gelatin mass with a platinum needle previously charged with the bacteria. b. Slant cultures, made by gently passing over the surface of the medium the inoculating needle; for this purpose the gelatin is made to solidify with the tube in an inclined posi- tion, so as to give a larger surface for inoculation. c. Plate cultures, made by inoculating the gelatin mass, which has been previously liquefied by submitting it to a temperature of 30° C., with a platinum needle or loop as for liquid cultures, and pouring the liquid mass rapidly and evenly on a sterilized glass plate, allowing it to solidify well protected from dust. The plate method, introduced by Koch, is of great value for the separation and isolation of bacteria. In this manner each bacterium introduced into the liquefied gelatin is fixed by the hardening of the gelatin and develops as a separate colony, the number of colonies being, as a rule, equal to the number of bacteria originally introduced. Each colony grows in its own peculiar way, because each species has a definite way of growing in gelatin. This method therefore not only serves for the separation of the bacteria themselves, but also enables us to recognize one species from another. Indeed, the classical method of Koch for the separation and isolation of bacteria, though modified in some particulars, has not been essentially changed since its introduction. It is as follows : Three sterilized gelatin culture-tubes about a third full and containing about 10 c.c. of the medium are liquefied by being submitted to a temperature of 30° C. Tube I. is inoculated with one or two loopfuls of the platinum needle from the contaminated substance, its cotton plug replaced, and the contents well shaken. After sterilizing the platinum needle one or two loopfuls from tube No. I. are introduced into tube 70 INOCULATION OF CULTURE-MEDIA WITH BACTERIA No. II. ; both tubes are again plugged, and tube No. II. is in turn well shaken. The platinum needle is again sterilized, and finally one or two loopfuls from tube No. II. are intro- duced into Tube No. III., and the contents of the latter well stirred. The three tubes are kept on a water-bath at a temperature of between 25° and 30° C., so as to keep the mass liquid. Meanwhile three sterilized glass plates are arranged on three cooling-stages, as depicted in Fig. 32. The gelatin from each FIG. 32. Levelling-tripod with glass cooling-chamber for plates. of the tubes I., II., III. is slowly and evenly poured over the surface of the plates, correspondingly designated as No. I., II., III., and allowed to solidify. It is necessary during the pouring and solidification of the gelatin on the plates that they be carefully protected by a cover against the dust and bacteria from without. After solidification is perfect the plates are transferred into culture-dishes placed on glass benches (Fig. 17), and properly labelled. To harden the gelatin more rapidly^ ice or iced water is generally kept in the lower dish of the cooling-stage. To insure THE METHODS OF INOCULATING SOLID MEDIA. 71 evenness of surface in the gelatin on the glass plates a levelling- tripod is used, as seen in Fig. 32. This tripod is easily set by means of a levelling glass. Results. — In this manner each of the separate bacteria con- tained in the media will develop as separate colonies on the gelatin plates. Plate IM made from tube L, will contain a large number of bacteria ; plate II. will contain the bacteria in much smaller number; and plate III. will contain only a few colonies, well separated ; and in this way the characteristic growth of the separate colonies and their action on gelatin may be carefully followed out and studied. By this means also the observer may under a magnifying-glass with a fine sterile platinum needle pick out the individual colonies and inoculate fresh tubes, and so obtain pure cultures of any liv- ing organism. 3. Plate cultures of agar are made in the same way, but require more care in their preparation, as agar does not melt at a temperature below 42° C., and solidifies again at a tem- perature of 39° or 40° C., so that after inoculation in the liquid state the tubes must be kept in a water-bath between 40° and 42° C. until they are poured into the plates. The agar plates, however, may be incubated at blood tempera- ture (37° C.), and the growth of bacteria noted. In recent years small double dishes, known as Petri's culture- dishes (Fig. 22), have been introduced to take the place of the plates. For the inoculation, liquefied gelatin or agar is poured into the lower dish, and this is quickly covered by the upper dish. For this method no levelling apparatus and cooling-stage are required. For counting the colonies in the plates the apparatus of Wolfhuegel (Fig. 31) has been adopted. This consists of regularly lined glass plates, divided into squares and arranged as seen in the figure. By placing the culture-plate under the lined plate it is easy to ascertain the number of colonies contained in each of, for example, ten squares, and by a simple process of multiplication the number of colonies in the whole plate. 4. Instead of pouring out the liquid gelatin from the tubes 72 INOCULATION OF CULTURE-MEDIA WITH BACTERIA. into plates after inoculation, Esmarch rolls these tubes in a vertical position until the gelatin is completely solid. This is hastened by rolling the tubes as shown in Fig. 33. By this excellent method there is less likelihood of contamination than by the plate method. 5. Gelatin and especially agar plates are occasionally made in a different way. The liquid medium is poured on glass plates and allowed to solidify before inoculation. When well hardened the surface is streaked with the inoculating material FIG. 33. Demonstrating Booker's method of rolling Esmarch tubes on a block of ice. on a platinum needle. In this way the colonies grow along the streak much more superficially than they do in the ordi- nary plate method. 6. Agar and blood-serum slant cultures are made in the same manner as similar cultures on gelatin. They have one advantage over the gelatin culture by reason of being able to withstand the temperature of the incubator, 37° C. THE CULTIVATION OF ANAEROBIC BACTERIA. Exclusion of oxygen is absolutely necessary. For this pur- pose a number of methods have been suggested and used, some of which require special and elaborate apparatus. All THE CULTIVATION OF ANAEROBIC BACTERIA. 73 the foregoing methods of inoculation and cultivation are avail- able only for the aerobic bacteria. 1. Cultivation of Tetanus Bacillus. — The following method FIG. 34. FIG. 35. Jar for anaerobic cultures. FIG. 36. Small incubator. Mercurial thermo-regulator. has been found very useful for the cultivation of this strictly anaerobic bacillus, aud answers all purposes in the author's opinion. 74 INOCULATION OF CULTURE-MEDIA WITH BACTERIA. A culture-tube three-fourths full of the medium is heated to the boiling-point and allowed to cool to a temperature in the neighborhood of 40° C.; then a platinum needle charged FIG. 37. Incubator used in bacteriological work : a represents the incubator set up and containing a cage of tubes and a Petri dish; 6, represents a vertical section of the incubator and displays the water-chamber, inner chamber, walls, vents, ther- mometer, valve, etc. with the material for inoculation is dipped down to the bot- tom of the tube. With fluid media, immediately thereafter a layer of paraffin oil is poured on the surface of the liquid QUESTIONS. 75 before introduction of the cotton plug. With solid media, the fluid is allowed to solidify after inoculation, and after solidification the paraffin oil is poured on the surface of the medium. This effectually shuts out the oxygen, and as a rule allows a luxuriant growth of anaerobic bacteria. 2. Special apparatus, with oxygen replaced by hydrogen, is also used for the cultivation of anaerobic bacteria. The Incubator and The Thermostat. For the purpose of growing bacteria it is often necessary or desirable to obtain a constant temperature. The ordinary body-temperature, 37° C., is the most favorable for the growth of the pathogenic bacteria. Apparatus especially constructed for maintaining a constant temperature are known as incuba- tors or thermostats. They are generally made of double- walled metal, and contain water, which by means of a gas-jet at the bottom of the apparatus may be kept at a constant temperature (Fig. 37). For regulating the gas-supply and to maintain the constant temperature, instruments known as thermo-regulators are used. A number of these are highly complicated, but for ordinary work is recommended Reichert's or Dunham's mer- curial thermo-regulator (Fig. 35). QUESTIONS. How do you inoculate a fluid culture-medium ? How do you inoculate potato cultures? In how many ways may gelatin tubes be inoculated ? What is meant by a stab-culture? By a slant culture? How are plate cultures made? Describe the method of making plate cultures according to Koch. How are agar plate cultures made ? How are colonies on plates counted? Describe the method of making cultures in Petri dishes? How are Esmarch's culture-tubes made? Give a good method for the cultivation of anaerobic bacteria. What is an incubator? What is a thermo-regulator ? 76 STERILIZATION, DISINFECTION, AND ANTISEPSIS. CHAPTER V. STERILIZATION, DISINFECTION, AND ANTISEPSIS! Definitions. — The freeing of substances from the live bacteria they may contain or that may have collected on their surfaces is called sterilization. This is accomplished either by means of heat or the use of chemicals. Erroneously sometimes the term sterilization is used to indicate the destruction of bacteria by the application of heat, the term disinfection being used then for their destruction by chemical agents. To disinfect a substance is to destroy in it or on it all the harmful or infectious bacteria, without necessarily killing all the living bacteria. THE METHODS OF STERILIZATION. I. Substances chemically sterilized are unfit for bacterial culture, except in very rare instances when the chemical agents are very volatile. II. In laboratory work, therefore, where the aim is the cultivation of bacteria, heat is the only method of sterilization used. This is applied either in the form of dry heat or moist heat (steam). 1. All substances which may be passed through the Bunsen flame and heated red-hot are usually sterilized in this manner. 2. Other implements, such as instruments and glassware, which would be injured by the direct flame, but withstand con- siderable heat, are sterilized by dry heat in an oven at a temperature of 160° to 180° C. for an hour. 3. For culture-media, one resorts to sterilization by steam, except in rare instances, when filtration under pressure through unglazed porcelain is considered sufficient. Experience has taught that steam at a temperature of 100° C. will kill all known bacteria and their spores within an hour, and Pasteur has demonstrated that steam under a pressure of two or three atmospheres, at about 130° C., will THE METHODS OF STERILIZATION. 77 destroy all known bacteria and their spores within fifteen or twenty minutes. The sterilization of culture-media, then, is usually done by the means of steam, with or without pressure. 4. In some instances, when exposure to steam for one hour would be prejudicial to the medium, what is known as dis- continuous or fractional sterilization is resorted to. The medium is steamed during three consecutive days for twenty minutes each time ; during the interval it is kept in favorable FIG. 38. FIG. 39. Laboratory hot-air sterilizer. Rose-burner. conditions for the development of bacteria. In this manner the first heat destroys all the fully formed bacteria that may exist. The favorable temperature in the interval between the first and second heatings allows all the spores contained in the medium which may not have been destroyed in the first heating to develop into fully formed germs, which are destroyed by the second application of steam on the ensuing day. The application of heat on the third day is to make 78 STERILIZATION, DISINFECTION, AND ANTISEPSIS. sure of the destruction of all spores which may have resisted the two previous applications. It has been demonstrated that this sterilization is very effective and complete, and substances which have undergone it may be kept indefinitely bacteria- free thereafter. 5. Another method of fractional sterilization is occasionally used for certain media which cannot withstand the tempera- ture of boiling water without deteriorating. It consists in FIG. 40. Steam sterilizer, pattern of Koch. (Abbott.) submitting the medium to a temperature of 68° to 70° C. for from two to three hours during seven consecutive days. This method is necessarily very imperfect, and though successful in those cases in which no spores have to be destroyed and where none of the bacteria contained in the medium are of the pyrogenic variety, it cannot but have a very limited application, and media so sterilized should not be used for THE METHODS OF STERILIZATION. 79 culture purposes, except after they have been submitted for several days to the temperature of the human body in the thermostat and remained sterile during this control-test. 6. For generating dry heat for the sterilization of glass- ware, implements, and instruments used in making cnltures, the hot-air oven and rose-burner are most usually employed, and a temperature usually of 180° Co maintained in the oven for one and one-half hours (Figs. 38 and 39). FIG. 41. Arnold steam sterilizer. (Abbott.) The oven consists of a double- walled metallic box, with a double-walled front door, with a copper bottom, the sides and door encased in asbestos boards. The heat is applied by means of a rose gas-burner (Fig. 39) to the bottom of the box. The objects to be sterilized are put upon perforated metallic shelves in the box. In the top of the apparatus are two per- forated openings for the insertion of the thermometers. In this portion the oven is also provided with a perforated slid- ing window to allow escape of the overheated air. 80 STERILIZATION, DISINFECTION, AND ANTISEPSIS. 7. For applying moist heat, bacteriologists commonly use a. Koch's apparatus (Fig. 40). b. Arnold's apparatus (Fig. 41). 8. For the application of steam under pressure preference is felt for the autoclave of Chamberlain or of Wiesnegg (Fig. 42). 9. For discontinuous fractional sterilization at a low tem- FIQ. 42. A B Autoclave, pattern of Wiesnegg: A, external appearance; B, section. (Abbott.) perature the apparatus in more general use is the blood-serum sterilizer of Koch (Fig. 43). 10. Filtration under pressure through unglazed porcelain, the pores of which are too small to allow bacteria to go through, will render certain liquid substances sterile or bacteria-free. This method is made use of in the case of certain pathogenic bacteria which secrete soluble poisons which we desire to separate from the bacteria themselves. The Chamberlain THE METHODS OF DISINFECTION. 81 filter of unglazed Sevre porcelain is the only one to be recom- mended for this purpose. FIG. 43. Chamber for sterilizing and solidifying blood-serum. (Koch.) THE METHODS OF DISINFECTION. I. Disinfection or destruction of infectious bacteria may with certainty be accomplished by heat, and indeed in the labora- tory when the total destruction of the bacteria is desired with the material containing them heat is the most effective measure. In other cases, however, when the destruction of the bac- teria themselves and the preservation of the contaminated sub- stance are desired, recourse must be had to such measures as will destroy the bacteria alone. Substances which are able to destroy bacteria or their spores are known as germicides or disinfectants, and those substances which retard bacterial growth are called antiseptics. II. In the use of chemicals for disinfecting, one should always bear in mind that their mode of action is not a cata- lytic one, but that they owe their virtue to the power of 6— M. B. 82 STERILIZATION, DISINFECTION, AND ANTISEPSIS. forming definite chemical compounds with the bacteria cells, which are thus rendered innocuous. They must, therefore, come into direct contact with the bacteria themselves, and in the combination so formed they and the bacteria change their chemical properties. In the choice of chemical disinfectants, one must be guided by the species of bacteria to be destroyed, by the number of these bacteria, by the nature of the media containing the germs, by the substances to be disinfected, and by the quan- tity of material to be disinfected. All chemicals so used must be of a definite strength, and must be made to act for a specified time, the quantity and strength of the disinfec- tant and the time varying with the nature of the chemical and the species of bacteria to be destroyed. To test the germicidal power of substances, the following is a convenient method : To young bouillon cultures of the bacteria to be acted upon sufficient of the chemical to be tested is added to make the proper dilution, and at given intervals of time a few droplets of this mixture are inocu- lated on sterile agar and gelatin tubes and the result carefully noted — for instance, supposing it is intended to test the germi- cidal power of carbolic acid toward some pus organism. To 9.90 c.c. of a bouillon culture of a pus germ in a test-tube 0.10 c.c. of carbolic acid is added, making the mixture a 1 per cent, carbolic acid dilution. The tube is well shaken, and at the end of one minute a few droplets of the mixture are taken on a sterile platinum needle and inoculated on a fresh tube of gelatin or agar, another fresh agar tube is inoculated with the mixture in the same manner after two minutes, another again after five minutes, and a fourth in fifteen minutes, a fifth in a half hour, a sixth in an hour, and a seventh in two hours. The growth in the inoculated tubes would indicate the action of 1 per cent, carbolic acid upon the given pus germ in the specified time. The same process is repeated, using a 2 per cent, dilution of the car- bolic acid, and next a 3 per cent., a 4 per cent., and a 5 per cent, dilution. Higher than 5 per cent, solutions of carbolic acid are not possible with any but water too hot for this work. THE METHODS OF ANTISEPSIS. 83 In other substances a 10 percent, dilution would be the last step of the test. Thus are obtained results which are fixed and definite, stating positively the strength of disinfection used and the time of its application. Cautions. — In conducting these experiments one must remember always that the growth of bacteria on the solid tubes may be considerably retarded by the action of the dis- infectants, and one .must not accept the result as conclusive unless those cultures have been kept under observation for a considerable length of time. Again it is proper, whenever practicable, to conduct the experiments at the temperature of the human body, 37° C., as experience has demonstrated that at this and higher temperature the disinfectant power of chemicals is increased. THE METHODS OF ANTISEPSIS. Substances that retard the growth of bacteria without, however, destroying them are called antiseptics. It is clear that all disinfectants when used in a more diluted form, or when allowed to act for a shorter space of time than is re- quired for them to show their germicidal power, act as anti- septics. I. The Common Disinfectants. Carbolic acid, strength 3 to 5 per cent., efficient in one hour. Bichloride of mercury, solution 1 : 1000 or 1 : 500, acts from within a few minutes to a half-hour. Chlorinated lime, containing free chlorine, is an efficient germicide in an hour's time in the strength of from 5 to 10 per cent. Boiling water, to which 2 or 3 per cent, sodium carbonate is added, is an efficient germicide in an hour. Sulphur dioxide gas, when used dry, has little or no disin- fectant power, and bacteria have been able to withstand an atmosphere containing from 10 to 12 per cent, of this gas for several hours. In the presence of moisture, however, it 84 STERILIZATION, DISINFECTION, AND ANTISEPSIS. forms sulphurous acid, and is then an efficient germicide in as low a percentage as 4 or 5 per cent. Formalin, in 2 to 5 per cent, strength, or as formaldehyde gas, is a powerful germicide. A number of other substances have been recommended, and have been used as germicides, but the above are of more general and practical value. II. Enumeration of the common antiseptics would be too lengthy to be stated in a book of this kind. In conclusion, one should bear in mind that chemical agents must act directly on the bacteria cells themselves — that is, they must penetrate ; and that they are the more efficient the more quickly they form chemical compounds with those cells — or, as usually expressed, the more penetrating power they possess — and anything that interferes with this chemical com- bination interferes with the action of the disinfectant. QUESTIONS. What is meant by sterilization ? By disinfection ? At what temperature and how long does it take dry heat to sterilize? How long does it take live steam to sterilize? How long does it take steam under pressure to sterilize? How are implements in the bacteriological laboratory sterilized? How are culture-media sterilized ? What is discontinuous or fractional sterilization by steam, and how is it accomplished ? How is sterilization at a low temperature effected? When is it used? What are its disadvantages? What forms of apparatus are generally used to generate steam for sterili- zation ? What are the only filters which can remove bacteria from liquid media? What are germicides or disinfectants? What are antiseptics? How would you test the value of a germicide ? What chemicals are generally used as disinfectants? State the value of carbolic acid as a disinfectant ; of bichloride of mercury ; of the chlorinated lime ; of sodium carbonate ; of sulphur dioxide, and of formaldehyde. THE INOCULATION OF ANIMALS AND THEIR STUDY. 85 CHAPTER VI. THE INOCULATION OF ANIMALS AND THEIR STUDY. THE INOCULATION OF ANIMALS. ITS purposes are to differentiate between bacteria. In order to study their virulence it is often necessary to test their action on animals. In the laboratory the smaller animals, such as mice, rats, guinea-pigs, and rabbits, are chiefly used. Technic. — The inoculation is made in a number of ways, depending on the species of the bacteria, the nature of its toxin, and the rapidity of action desired. The Various Methods of Inoculation of Animals. 1. Sometimes, though rarely, the inoculation is made by rubbing a solid or liquid culture over the abraded epidermis, very much in the same manner as vaccine is introduced into the human subject. 2. Subcutaneous inoculation — that is, into the connective tissue under the skin — is an important method. For this purpose the hair is shaved from part of the back or abdomen, the skin well washed and disinfected as well as it may be, with a 5 per cent, carbolic acid solution. Then the skin is seized with a pair of sterilized forceps, and with a sterilized scalpel a small nick is made into it, after which a small sterilized pair of scissors is introduced in the areolar tissue and a pocket made for some little distance into this tissue. Into this pocket the inoculating material or bacterial culture (especially when a solid culture has been employed) is intro- duced, by means of a sterilized forceps or a platinum loop, care being taken to avoid touching the edges of the wound with the instrument. If a liquid culture is being used, particularly in appreciable quantity, it may be introduced subcutaneously by means of a hypodermatic syringe and needle well sterilized beforehand. 86 THE INOCULATION OF ANIMALS AND THEIR STUDY. Guinea-pigs and rabbits are usually inoculated in the abdom- inal wall ; rats or mice in the loose tissue at the root of the tail. Animal Holders. — Various instruments have been devised FIG. 44. The Voges holder for guinea-pigs. "(Abbott.) for keeping animals quiet during this operation, the most use- ful of which are : Voges' guinea-pig-holder (Fig. 44) ; Kitasato's mouse-holders (Fig. 45) ; and the basket mouse-holder (Fig. 46). 3. Intravenous injection or inoculation into the circulation THE INOCULATION OF ANIMALS. 87 consists in injecting directly into the veins of the animal in the direction of the circulation, the material to be inoculated. Necessarily the material used must be a liquid, and the injec- tion must be done slowly and with precaution. Intravenous injection is used especially in rabbits. The most convenient point of injection is into the vein of the ear known as the posterior auricular vein, which is easily penetrated from the dorsal surface of the ear, where it lies superficial and FIG. 45. Kitasato's mouse-holder. (Abbott.) Mouse-holder, with mouse in proper position. (Abbott.) imbedded firmly in the areolar tissue. For the pur- pose of making these injections an ordinary hypodermatic syringe with the needle used for morphine injections in man is employed. All that is required is that both syringe and needle be sterilized. The mode of procedure is as follows : The rabbit is firmly held by an assistant. The ear chosen is taken between the thumb and forefinger of the left hand and after washing and sterilizing the skin as thoroughly as possible, the vein on the posterior edge of the ear is sought 88 THE INOCULATION OF ANIMALS AND THEIR STUDY. for. If it is invisible, pressure at the root of the ear will make it prominent. From the dorsal surface the hypodermatic needle is in- serted at its distal extremity and the material slowly injected. Care should be taken to have the needle penetrate the vein, and the first few drops of injected liquid will show whether it does so or not ; for if the needle is outside the vein a bulla will immediately be found at the site of injection. A little practice renders this method very easy. 4. Inoculation into the lymphatics is done best with a hypo- dermatic syringe and the injection is made into the testicles. 5. Intraperitoneal inoculation requires much care and the same antiseptic precautions as when opening the peritoneum for a laparotomy. The skin, cleanly shaven and disinfected as thoroughly as possible, is opened in the linea alba midway between the sternum and pubis, through an incision, from an inch and a half to two inches long and penetrating the fascia. The edges of the wound being held apart, the connective tissue and muscles are separated with a pair of sterilized, blunt-pointed scissors. If a liquid inoculating material is used, it may now be introduced with a sterile hypodermatic needle into the peritoneal cavity, avoiding as much as possi- ble the wounding of the intestines, which is not difficult. If the material employed is solid, the peritoneum is opened with scissors and the solid particle introduced into the cavity by means of a sterile needle or forceps. The wound is care- fully sutured and closed by a layer of collodion. 6. Intrapleural inoculation is performed much more rarely on account of the danger of wounding the lungs, and when used the same precaution must be taken as for the intra- peritoneal method. 7. Inoculation into the anterior chamber of the eye is per- formed occasionally to study in the living animal the changes produced locally by bacteria. With a sharp-pointed bistoury an incision is made in the cornea, at its sclerotic attachment near the inner canthus, and the material introduced and applied directly upon the iris, by a sterilized needle or by means of a small forceps. THE OBSERVATION OF THE INOCULATED ANIMAL. 89 THE OBSERVATION OF THE INOCULATED ANIMAL. 1 . After inoculation animals should be observed carefully and all changes in their condition noted. Their temperature should be taken several times a day, their body weight recorded every day under the same conditions ; their behavior as to food noticed ; the state of their fur and any sign of paralysis or of convulsions carefully observed. 2. After death, the autopsy should be performed as soon as practicable. For that purpose the animal should be laid upon its back on a board, its four legs stretched widely apart and attached to the sides of the board by strings, or nailed, and the nose should also be carefully nailed down. By means of a 5 per cent, carbolic acid solution the skin of the body from the chin to the pubis should be carefully sterilized and all hair shaved off. The place of inoculation is now to be carefully examined and described. Examination of the Abdominal and Pelvic Contents. — After this an incision is made in the skin only, and that carefully dissected away from the subcutaneous tissue and hooked back so as to prevent its contaminating the underlying tissues. After this by means of a metallic spatula, heated to redness, the muscular tissue is singed all along the line of the next incision, along the linea alba from the pubis to the sternum, along the arches of the rib and obliquely from the sterno- clavicular junctions to the tip of the last two ribs. With a pair of sterilized blunt-pointed scissors the peritoneal cavity is opened. All changes within it are carefully noted, cultures made from all exudates, or inflammatory products apparent, bouillon, agar, and blood-serum tubes being used for that pur- pose, and cover-glasses being also prepared for the microscope. Then, by means of the same heated spatula, the surfaces of the different organs are thoroughly singed, and with a spear-head, thick sterilized platinum wire, the organ is penetrated and cultures made from the small pieces of organs or blood adhering to the wire ; bouillon, agar, and blood-serum being used, and cover-glasses being also prepared. From all cultures so obtained plate cultures should be made, 90 THE INOCULATION OF ANIMALS AND THEIR STUDY. and the several bacteria therein isolated, and pure cultures made. After complete examination of the abdominal and pelvic organs, by means of a thick pair of scissors the ribs are cut off along the singed lines, the sternum turned up, and exam- ination and cultures of the thoracic organs made in the same way as for the abdominal organs. 3. Alter the complete autopsy the animal should be inciner- ated, and if this is not practicable, it should be kept bathed at least for two hours in a 5 per cent, carbolic acid solution, and finally boxed in quicklime before burial. Cultures from the human body at autopsies should be made as described for lower animals. 4. Cultures from secretions of living animals and man should be made immediately upon their passage and must be always collected in sterilized vessels. The examinations and the cult- ures should be prepared forthwith, using agar, bouillon, and serum as media. The Roux-Nocard Method of Culture and Observation. History. — Recently observations made by Roux and Nocard for the growth of microorganisms in culture in the live animal have shown that a number of minute bacteria are found associated with certain diseases, notably the pleuro- pneumonia of cattle. These microorganisms require a much higher power of the microscope than that generally in use to bring them into view, a magnification of 2000 at least being necessary. Technic. — Small collodion flasks are made thoroughly sterile, filled with blood from the suspected animal, and then closed with sterile collodion. These tubes or flasks are after- ward introduced into the abdominal cavity of live rabbits and guinea-pigs, and allowed to remain for a few days, after which they are taken out and examined, when the small motile microorganisms as mentioned above will be discovered. Importance. — This method of observation and cultivation in the live animal body seems to open a large field for the INFECTION AND IMMUNITY. 91 future investigation of such diseases as scarlet fever, measles, smallpox, rabies, etc., which, though unquestionably of microbic origin, have so far failed to reveal any specific germ for their causation. QUESTIONS. Why are animals inoculated ? What different methods are used for animal inoculation in the laboratory ? Describe the subcutaneous method. The intravenous method. The intra- lymphatic method. The intraperitoneal method. What instruments are used to inoculate liquid cultures into the veins of animals ? What should be observed in inoculated animals ? How should an autopsy be made in the case of an animal dead after inoculation ? What precautions are necessary in making cultures from tissues and organs in dead animals to prevent contamination from outside ? How should secretions of animals and men be collected for bacterial exam- ination ? What form of culture have Eoux and Nocard proposed for cultivation of bacteria ? CHAPTER VII. INFECTION AND IMMUNITY. INFECTION. BACTEEIA which produce diseases in animals and man are known as the pathogenic bacteria, and the process by which disease is produced is called infection. The mode of communication of these infections to man or to animals is not fully demonstrated. The following explana- tions are the more plausible. The Theories of Infection. 1. The rapid multiplication of bacteria in the blood and organs of infected animals is supposed to interfere with their bodily functions, and so cause disease and death. This is the so-called mechanical theory of infection, and finds support in such diseases as anthrax, when in fatal cases every capillary 92 INFECTION AND IMMUNITY. and organ of the animal is teeming with microorganisms, and in so-called septicaemic diseases where the microorgan- isms may be found in greater or lesser number in the blood and organs. II. The bacteria secrete or contain in their cell-bodies poisonous substances (toxins) which act deleteriously on the animal economy through its own molecules. This, the chemi- cal theory, is the accepted one of to-day, and finds its ready explanation in nearly all infectious diseases, especially in those which, like diphtheria and tetanus, may be superin- duced by inoculations of cultures from which the bacteria have been eliminated by filtration. The so-called toxaemic diseases are so produced. The Avenues and Factors of Infection. A. Infection of the animal body is effected by one of three ways : I. Through the respiratory tract. II. Through the digestive tract. III. Through the wounded or unwounded surface of the skin or mucous membrane. B. Conditions and Factors. — These are various and play an important part in infection. Some of them have reference (1) to the infecting material, or chiefly (2) to the animal ex- perimented upon. To the first class belong the species of bacteria, the quantity of infected material introduced, the cultural conditions of the bacteria, the presence or absence of the so-called mixed infection in which more than one species is taking part, the method of its introduction, and, in some cases, the time elapsed since the infection occurred. The conditions which depend upon the animal are the follow- ing : the amount of natural resistance to the bacterial poison, the condition of health of the animal. It must be remembered that some species of bacteria are much more injurious than others either on account of the rapidity with which they are able to develop in the human or animal economy, or on account of the large quantity of INFECTION. 93 toxins which they generate, or on account of the highly poi- sonous property of these toxins. 1. The quantity of bacteria used plays an important part because there is a more or less marked natural resistance in the animal body to the action of bacteria or their poisons. When these are introduced in small quantity only, they fail to produce any effect, and it requires a certain definite amount i)f bacteria to produce disease in the animal body. This amount varies with the species of the bacteria. 2. The condition of the bacterial culture when introduced into the animal body is an important factor in the subsequent course of the infection, for bacteria under different conditions secrete toxins which are more or less injurious, and the same bacteria grown under the same conditions are able at different times to produce toxins of more or less virulence. When the condition of growth or the environment of the bacteria varies, their cultural aspects and the amount of toxins they are able to produce vary also. So much so is this the case that bacteria are grown under peculiarly disadvantageous surroundings — high temperature, or the addition of a small proportion of antiseptics to their cultural fluid so as to pro- duce bacteria of less virulence — in other words, to attenuate them. Methods of Attenuation. — Bacteria from young liquid cult- ures are known to be more virulent than those from older cultures. Again, cultures are made through the body of resisting animals so as to diminish the virulence of the cult- ures. Or, again, the cultures are passed through artificial media for a number of generations to diminish their virulence. The converse of this happens also, and bacteria grown or passed through the bodies of susceptible animals acquire more and more virulence. 3. The method of introduction of the bacteria contributes considerably to the degree of infection from the fact that nearly all bacteria have certain affinities for different tissues of the body where they exert their most baneful influence, and the nearer akin to those tissues is the place of the intro- duction in the body the more rapidly and energetically is the 94 INFECTION AND IMMUNITY. bacterial influence felt. Again, the different secretions of the body have more or less germicidal effect, so that bacteria, as a rule, are more potent in their effect when introduced directly into the circulation. 4. The association of bacteria among themselves has occa- sionally the power of increasing the toxic effects of the inocu- lated germs, sometimes the two germs acting simultaneously on the animal body and producing what is known as " double," "mixed," or " associated " infection. At other times, some of the germs, though not pathogenic, are able to destroy the resistance of the body to the action of other toxic germs, as, for instance, the injection of tetanus bacillus with some ordinary saprophyte is capable of producing symptoms when the introduction of the tetanus germs alone would utterly fail. Occasionally a beneficial association of germs may be ob- served, the presence of the secretion of some bacteria being prejudicial to the growth of other bacteria or neutralizing their toxins. 5. The condition of the human or animal body as to per- fect health, as has already been remarked, offers more or less resistance to the bacterial poison. When, however, the gen- eral health is below par this resistance is diminished and the animal is much more susceptible to the action of the germs. 6. The time elapsed since the infection is often of great moment. In some cases germs will lurk in an organ for a long time, after which, through circumstances very little understood, they will suddenly and violently begin to cause symptoms and often death. Diseases of the appendix and gall-bladder in man are among the more familiar examples of this phenomenon. IMMUNITY AND ITS VARIETIES. Resistance to the action of pathogenic bacteria is called immunity, and is either natural or acquired. I. Natural immunity is present in all such cases where, for instance, some species of animals can not be affected by cer- IMMUNITY AND ITS VARIETIES. 95 tain bacteria or their toxins, which are injurious to other species, 'or, as occasionally happens, when some individuals in a susceptible species are refractive. II. Acquired immunity is manifested when a susceptible animal is protected from the further noxious influences of bacteria either from the fact of having suffered an attack of the disease caused by the bacteria, or when it has been made artificially insusceptible. Examples of Natural Immunity. — Rats can not be success- fully inoculated with the anthrax bacillus, though other rodents are very susceptible. Again, pigeons are not sus- ceptible but are immune to the anthrax bacillus. The expla- nation of this natural immunity is not easily given. It is supposed in some cases to be due to the mode of living of the immune animal, or to some condition of its secretions, or to some substances found in its blood and tissues which are able to destroy bacterial life or to neutralize their toxins. These substances are called alexins. Examples of Acquired Immunity. — This may be due, as just mentioned, to a previous attack of disease, and when due to this it lasts in the majority of instances during the life of the animal. In other cases acquired immunity can be arti- ficially induced in animals, and according to the methods used for its production is said to be active or passive. 1. Active acquired immunity is produced by the action of living germs or their toxins introduced into the animal. 2. Passive acquired immunity is obtained by a direct trans- ference of an immunizing substance from an immune animal to a susceptible one. Active immunity takes some time to develop, but, as a rule, lasts longer than passive immunity, which is immediately established. The Methods of Producing Immunity. I. Inoculation, or the introduction of small quantities of live bacteria, so as to produce a mild attack of the disease. This method is dangerous from the fact that it is hard to ascertain how small a quantity of bacteria may be introduced without 96 INFECTION AND IMMUNITY. its being prejudicial to life, and from the danger of spread- ing the infection. II. Vaccination, or the introduction of attenuated bacteria, which attenuation is obtained either by submitting the bacteria to a higher degree of heat during their cultivation or by add- ing a small proportion of an antiseptic to their culture-media, or by using bacteria which have grown for a long period of time in artificial media, or by using bacteria which have grown in the bodies of natural immunes. III. Intoxication, or the introduction of the toxins of the bacteria in small broken but frequently repeated doses, or in cases where the toxic eifect of bacteria is due to substances contained in the cell-body itself by the injection of the dead bacilli. This is the method used for the production of the diphtheria and tetanus antitoxins. IV. Antitoxins, or the introduction of bacterial products of any one of the first three processes into other animals, these substances, known by the name of antitoxins, being able to confer immunity to susceptible animals. V. By the inoculation of an emulsion of tissues, consisting in the introduction into the animal of the emulsion of certain tissues which are known to be the tissues susceptible to the action of the bacterial poison. VI. By introducing into the animal inert particles, such as carmine, mixed with the bacteria. Forced immunization of animals consists in introducing gradually and in increasing doses bacterial toxins in sufficient quantity to produce a reaction, but in quantity too small to produce deleterious effects. In this way it has been found that animals immunized produce substances in their tissue- fluids which when inoculated in susceptible animals serve to protect them against the deleterious action of those bacteria or their toxins. The Antitoxic and Antimicrobic Blood-Serums. The blood-serum of animals used for the purpose of pro- tecting others is said to be antitoxic, when it has been obtained IMMUNITY AND ITS VARIETIES. 97 by the action of the toxins of the bacteria on the animal ; and to be antimicrobic, when it has been obtained by means of the action of virulent or attenuated cultures on those animals. Uses. — Antitoxic serum is employed chiefly in the toxic diseases, such as diphtheria, tetanus, etc., and antimicrobic serum is used particularly in the invasive diseases, such as plague, typhoid fever, cholera, etc. Theories in Explanation. — A number have been suggested. Some believe that the antitoxin is a chemically changed toxin ; others claim that it is a sort of enzyme produced by the toxin ; others again state that it is the product of the cytic activity developed by the toxin; again others consider that it acts as a sort of combining ferment in the same manner as those fer- ments which favor coagulation of the fibrin in the blood. The Theories of Immunity. How these substances act so as to produce immunity in ani- mals is a subject that has occupied investigators considerably in recent years. I. The abstraction theory (Pasteur's) is to-day only of his- torical interest. It was believed to be due to the fact that the pabulum necessary for the life of the specific bacteria had been consumed, and that these bacteria could no longer live in the animal. II. The retention theory (Chauveau's), in which it was sup- posed that microorganisms left in the system certain substances which were antagonistic to their further growth, is still worthy to-day of some consideration. III. The theory of phagocytosis (Metchnikoff's), by which immunity was supposed to be due to the action of the white blood-corpuscles, which have the power of absorbing and destroying bacteria, is not tenable to-day in its original entirety. That the leucocytes play a certain part in the immunizing process cannot be denied, but the phagocytic property is more probably due to the fact that the animal is immune than the cause of the immunization. 7— M. B. 98 INFECTION AND IMMUNITY. Immunity is, in general terms, certainly produced by certain secretions formed in the animal's body, and secreted by it to protect itself from the attack of the invading bacteria, and dis- tributed in all the tissues, but found especially in the serum of the blood. IV. The chain-theory (Ehrlich's) claims that this immuniz- ing substance is developed on account of the fact that the poi- sonous substances introduced by the microbes or the secretions of the microbes in the animal body combine with certain elements of the tissue and destroy them, subtracting them from other elements with which they were naturally in com- bination ; this stimulates the natural resistance of the tissues and causes an increased production of the substances attacked by the bacteria in such a way that an overproduction results, and this makes the animal more resistant to the further intro- duction of the poison. This is certainly the most plausible explanation of immunity offered to this day. A passing remark, however, may only be offered on this subject, and those who are interested must be directed to con- sult larger works, in which these views are explained at length. QUESTIONS. What is infection ? How are bacteria called which produce disease in animals ? How is the action of pathogenic bacteria on the animal body explained ? When is a disease said to be septicsemic ? When is it toxseniic ? Name the three modes by which the animal body may be infected. What conditions favor infection ? What conditions in the infecting material increase its power? What conditions in the animal increase the rapidity of infection ? What part does the quantity of bacteria introduced in the inoculation play in the infection? What is meant by attenuation ? What conditions of the cultures make the bacteria more virulent? What is the effect of passing for a number of generations pathogenic bac- teria through artificial media? What part does the mode of introduction of the bacteria in the animal body play in infection? What is meant by double infection ? What is immunity ? What is meant by natural immunity ? What is meant by acquired immunity ? Give some examples of natural immunity? What produces acquired immunity in animals? THE PATHOGENIC BACTERIA. 99 What are active and passive immunity ? What artificial methods are used to produce immunity? What is meant by inoculation? Vaccination ? Intoxication ? How does tissue suspension produce immunity ? What influence does the injection of inert particles have upon immunity? What is meant hy forced immunity ? What is meant by an antitoxic serum ? By an antimicrobic serum ? What classes of disease are protected against by antitoxic serum? What by antimicrobic serum ? How is this anti-action explained? What is the theory of abstraction ? What is meant by the retention theory ? What is the theory of Metchnikoff ? What is Ehrlich's chain-theory? CHAPTER VIII. THE PATHOGENIC BACTERIA. THE PYOGENIC MICROCOCCI AND ALLIED BACILLI. THE most commonly found bacteria in pus are cocci (pyo- cocci). A few are bacilli. The list includes: 1. The Staphylococcus pyogenes aureus, albus, and citreus ; the Staphylococcus cereus albus 9 the Staphylococcus cereus aureus (Passet) ; the Staphylococcus cereus flavus (Passet). 2. The Micrococcus pyogenes tenuis (Rosenbach). The Micrococcus tetragenus is sometimes found associated with the foregoing two varieties in abscesses or in pus cavities, and are also able to produce abscesses at the place of injec- tion in animals. 3. The Streptococcus pyogenes is found associated with the staphylococci in purulent accumulations, and is sometimes itself responsible for pus-production in the body. 4. The gonococcus is the cause of specific suppuration of the urethra and often elsewhere in the body. 5. The pneumococcus is often found in abscesses which occur in the course of the disease in pneumonic patients. 6. 7, and 8. The Bacillus pyocyaneus, typhosus, and tuber- culosis are sometimes the cause of pus-production, as pure 100 THE PATHOGENIC BACTERIA. cultures of these organisms have been found in some cases of abscesses during the respective infections. Nearly all pyogenie organisms are facultative anaerobics. THE INDIVIDUAL FEATURES OF THE PYOGENIC BACTERIA. I. Staphylococcus Pyogenes Aureus. The Staphylococcus pyogenes aureus, by far the most fre- quent pus organism, is found a. in health on the surface of the skin, also of the mucous membranes in the digestive tube, and upper part of the respiratory tract, and b. in pathological conditions in pus irrespective of its localization, either alone or in association with the other pyogenie staphylococci, also FIG. 47. r* A. /?/'* //, / Twr/t//j/'/M - y f Preparation from pus, showing pus-cells, A, and staphylococci, C. (Abbott.) in the blood in cases of general infection, and a number of cases of extensive suppurating lesions, abscesses, suppu- rating tumors, furuncles, etc.; and c. outside of the human body in the air, in dust, and occasionally in water. Morphology. — The Staphylococcus pyogenes aureus is a small rounded cell having a diameter of 0.9 to 1.2 mikrons, found either singly or in irregular groups or masses resem- bling a bunch of grapes, hence its name. Sometimes it is seen INDIVIDUAL FEATURES OF PYOGENIC BACTERIA. 101 in pairs, as a diplococcus. Its appearance in pus as well as in culture-media is the same in general as is seen in Fig. 47. Principal Biologic Characters. — The Staphylococcus pyogenes aureus is a facultative anaerobic. It clouds bouillon in twenty- four hours at 37° C., and shows from the second day a yel- lowish precipitate, which gradually increases in color and at the bottom of the tube appears of a golden yellow. It lique- fies gelatin. Stab-cultures on this media at 20° C. on the second or third day have the appearance of a funnel, at the bottom of which is an orange-yellow deposit. At the end of three days the gelatin in the tube is completely liquefied. On gelatin plates colonies of a dark-yellowish color are observed with a centre of more or less intense orange color. On agar-agar the colonies appear small, regularly spherical, and of an orange-yellow. Plates made from this medium have the same characteristics as on gelatin, being more or less pigmented yellow. It does not liquefy agar. The cult- ures on blood-serum have the same characteristics as on agar. The Staphylococcus pyogenes aureus stains with all the anilin dyes, and also by Gram's method. Pathogenesis. — When inoculated into the blood of an animal, the Staphylococcus aureus rapidly causes a fatal septicaemia. Rabbits and guinea-pigs die, as a rule, in twenty-four to forty-eight hours after inoculation, and the organisms may be found generally disseminated in the blood-capillaries of the organs, and are also found in the blood taken from the heart- Inoculations into the peritoneal cavity cause a purulent peritonitis of a virulent character, generally ending in death of the animal. Injected under the skin, this organism pro- duces localized abscesses. II. Staphylococcus Pyogenes Albus. The Staphylococcus pyogenes albus, like its companion the aureus, exists as a saprophyte : a. on the surface of the skin in man, and 6. in association with the aureus in abscesses and superficial phlegmons. Although clinicians are in the habit of considering it as an 102 THE PATHOGENIC BACTERIA. achromogenic variety of the preceding, it is, however, some- what less pathogenic. Its morphological characters are the same as those of the aureus, with the exception that it does not form pigment and its colonies are of a milk-white color. III. Staphylococcus Citreus. The Staphylococcus pyogenes citreus is of identical mor- phology with the two preceding varieties, with the exceptions that its growth is of a lemon-yellow color and that it liquefies gelatin more slowly. It is found in association with the Sta- phylococcus aureus and albus in pus of acute abscesses, espe- cially in the liver. IV. Streptococcus Pyogenes. The Streptococcus pyogenes is found : a. in the lymphatics of the skin in patients suffering from erysipelas, 6. in pus, c. in the false membranes in cases of diphtheria, d. in surgi- cal and e. obstetrical complications of erysipelas, and /. as a frequent causative agent of puerperal septica3mia and of many surgically common infections (Fig. 48 and Plate I.). FIG. 48. K ** &* r • ,»j f* Streptococcus pyogenes. (Abbott.) Morphology. — The streptococcus is a micrococcus varying in size from 1 to 4 mikrons in diameter, spherical in shape and arranged as a chain of variable length. When grown in liquid media, this chain consists of from 30 to 40 elements, but in solid media a chain usually consists of from 7 to 10 cocci. In young cultures the diameters of all the cocci of the PLATE I. Streptoeoeous Pyogenes in Pus. (Abbott.) INDIVIDUAL FEATURES OF PYOGENIC BACTERIA. 103 chain are equal ; in older cultures they vary very much even in the same chain. Biologic Characters. — The streptococcus is aerobic and fac- ultative anaerobic. At 37° C. it clouds bouillon in twenty- four hours, and this becomes again clear at the end of three or four days, when small spherical bodies may be seen at the bottom of the tube. The bouillon becomes acid. On gelatin it forms small spherical opaque colonies about the size of a pin-head, which cease to increase after the third or fourth day. It does not liquefy gelatin. On agar-agar also it forms spherical colonies of the size of a pin-head, semi transparent and of a grayish- white appearance, shaped somewhat like a bead. It does not develop on potato. The streptococcus does not live longer than three weeks in cultures. It stains by the Gram method, and also by the other anilin dyes. Pathogenesis. — Intravenous inoculations in animals produce variable effects. The germ usually kills the animal, causing a rapid general septicaemia ; at other times the animal reacts only slightly. Subcutaneously it causes erysipelas and the formation of abscesses. All laboratory animals are susceptible to infection by means of the streptococcus pyogenes. V. The Micrococcus Cereus Albus. VI. The Micrococcus Cereus Flavus. These were found in pus by Passet associated with other organisms. Their pathogenesis has not been fully established. They differ from the other groups of cocci just described by the shiny, waxy appearance of their growth. VII. The Micrococcus Pyogenes Tenuis. This was found in pus by Rosenbach, is very irregular in size and somewhat larger than the Staphylococcus albus. On agar-agar its biology presents a thin opaque streak along the line of inoculation, resembling a thin layer of varnish. Its pathogenic properties have never been fully determined. 104 THE PATHOGENIC BACTERL4. VIII. Micrococcus Tetragenus. The Mierococcm tetrayenus was obtained by Koch a. from cavities of tuberculous lungs, b. in the sputum of phthisical patients in the last stages of the disease, c. in the pus of buccal and d. ocular abscesses. It has been found by Morinier e. in the normal saliva and /. even in the saliva of newborn babes. Morphology. — A micrococcus with a diameter of about 1 mikron, formed in groups of 8 (tetrads) and enveloped by a transparent gelatinous substance. Principal Biologic Properties. — It is a facultative anaerobic. On agar it forms thick granular spherical colonies of a white or grayish color. It does not liquefy gelatin. It stains with all the anilin dyes and readily by Gram's method. Pathogenesis. — When inoculated into guinea-pigs subcutane- ously, the animals die rapidly and abscesses are formed at the point of inoculation. The micrococcus at the autopsy may be found in all the organs and in the blood taken from the heart. GONORRHOEA. IX. Micrococcus Gonorrhceae (Gonococcus). Discovered by Neisser in 1879, the gonococcus causes the specific suppuration of gonorrhoea. Pathogenesis. — This micrococcus, or diplococcus, as it is generally called, has a special affinity for the urethral mucous membrane, finding lodgement in the epithelial cells lining this canal. It sometimes causes inflammation with or with- out suppuration in other parts of the human body, such as the conjunctiva, appendages of the uterus, in the peritoneum and articulations. Cutaneous and muscular abscesses have occasionally been found to be caused by the gonococcus. Morphology. — -These micrococci are usually found united in pairs presenting the appearance of grains of coffee, the two opposing sides being generally flattened or concave. In stained preparations the flattened surfaces are separated by an unstained interspace. The gonococci are found free in OONOREHCEA. 105 the pus, but more often as small masses in the pus or epithe- lial cells. This serves partly to distinguish them from other pus cocci (Fig. 49). Principal Biologic Characters. — It is aerobic, but is very difficult to cultivate outside the human body. A number of investigators have succeeded in cultivating it on human blood- serum obtained from the placenta of a recently delivered woman ; others have been successful with ascitic fluid and with the fluid of hydrocele. The cultures grow at a tempera- ture of between 30° and 35° C. Finger has succeeded in cultivating it in sterile acid urine with 0.5 per cent, of peptone. FIG. 49. Pus of gonorrhoea, showing diplococci in the bodies of the pus-cells. (Abbott.) The gonococcus will not grow on gelatin, agar-agar, potato, or in bouillon. It stains with the basic anilin dyes, especially with gentian- violet. It does not stain by the Gram method. This is a valuable point to differentiate it from the pus cocci, which all stain by the Gram method. Pathogenesis. — Toure succeeded in causing urethritis in dogs by injecting into their urethras cultures in acid media. Finger and Gohm have caused acute urethritis, which rapidly disap- peared, by intra-articular injections of cultures into dogs and rabbits. Pus containing the gonococci when inoculated into 106 THE PATHOGENIC BACTERIA. man have reproduced the disease in many instances. Pus cultures of the gonococci have also given positive results in many cases : Wertheim, 5 times in 5 cases ; Bockardt, 6 times in 10 cases; Finger, 3 times in 14 cases. Subcutaneous injections of the culture produce considerable tumefaction and redness at the point of inoculation, but no abscess-formation. X. Bacillus Pyocyaneus. The Bacillus pyocyaneus is found frequently in suppurating wounds, especially in burns. It colors the pus green and the dressings a bluish-green, without showing any color-influence on the local condition of the wound. Pathogenesis. — It exists in pus associated with other micro- organisms, and is considered an inoffensive saprophyte in most cases. It may, however, under certain conditions become pathogenic. Morphology. — It is a delicate rod with rounded or pointed ends. Biologic Characters. — It is aerobic and grows readily on all artificial media and imparts to them a bright-green color. It liquefies gelatin and stains readily with all anilin dyes. XI. Bacillus Pyogenes Fcetidus. This organism was first obtained by Passet from suppurat- ing surfaces in the vicinity of the lower bowel. Morphology. — It is a short bacillus with rounded ends, usually found in pairs or in short chains. Biology. — It is an aerobic, motile, and grows on all media. Stains with all the anilin dyes. The cultures are noted on account of the disagreeable putrefactive odor which they emit. It derives its name from this feature. XII. Pneumococcus or Pneumobacillus. Friedlaender discovered this organism. It is sometimes found : a. in pus associated with other organisms and b. in cases of pneumonia as the sole factor of the disease and its GONORRHfEA. 107 secondary abscesses. The pus produced by it is thick, and creamy white in color. Pathogenesis. — It frequently causes suppuration in the serous membranes — pleura, peritoneum, pericardium, and lungs. It has also on some occasions caused suppuration in the viscera and in the subcutaneous and deep cellular tissue. XIII. Bacillus Coli Communis. XIV. Bacillus Typhosus. XV. Bacillus Tuberculosis. These three organisms are sometimes found associated with pus-formation, and have been thought to be occasionally the chief suppurative agents. The discussion of this subject, however, will be properly taken up under the head of the description of these bacilli. QUESTIONS. What are the pyococci ? Describe the Staphylococcus pyogenes aureus. How does it act on bouillon, on gelatin, on agar? Where is this organism found in the human body ? Where outside of the human body ? What is the effect on animals of intravenous injections of this organism? What of subcutaneous inoculation ? In what respect does the Staphylococcus pyogenes albus differ from the aureus? The Staphylococcus pyogenes citreus? Describe the streptococcus pyogenes. Where is it found ? Of how many elements are its chains formed? • What is the effect of intravenous, intra- peritoneal, and subcutaneous inoculations? Where were the Micrococcus cereus albus and flavus found, and by whom? What are the characteristics of the Micrococcus tetragenus f What is the gonococcus? Where is it found? How is it recognized under the microscope ? What media are best suited for its growth? How is it differentiated from other pus cocci ? What other bacteria cause suppuration or are found in pure cultures in abscesses. 108 THE PATHOGENIC MICROCOCCI. CHAPTER IX. THE OTHER PATHOGENIC MICROCOCCI AND ALLIED BACILLI— MICROCOCCUS PNEUMONIA, EPIDEMIC CEREBROSPINAL MENINGITIS, AND MALTA FEVER. PNEUMONIA. I. Micrococcus Pneumonias Crouposae (Diplococcus Pneumonias ; Micrococcus Pasteuri ; Micrococcus of Sputum Septicaemia). History. — The Micrococcus pneumonice crouposce was discov- ered in September, 1880, by Steinberg, in the blood of rabbits which he had inoculated subcutaneously with his own saliva ; also by Pasteur, in December, 1880, in the saliva of a child who had died of pneumonia in a Paris hospital. This was confirmed and studied by Fraenkel, Weichselbaum, and others. It is found: a. in the saliva of about 50 per cent, of healthy individuals, b. in the rusty sputum of pneumonic patients and in the fibrinous exudation of 75 per cent, of the cases of pneumonia, c. in a large number of cases of meningitis com- plicating pneumonia or associated with pneumonia, d. occa- sionally where no pneumonia exists, e. also in abscesses. Morphology. — Micrococcus pneumonice is a small oval coccus appearing alone or united in pairs, occasionally forming chains with four or five elements resembling streptococci. In the animal body it is generally oval and double, as a diplococcus, surrounded by a capsule (Fig. 50). In solid media it grows as a micrococcus, a diplococcus, or as a chain like the streptococcus with scarcely more than four or five elements. In liquid media the cells are more nearly round, and the chains contain sometimes as many as eight or ten elements (Fig. 51). It stains by the anilin dyes, and also by Gram's method. Biologic Characters. — The Micrococcus pneumonice is aerobic and facultative anaerobic. Like most cocci it is non-motile, PNEUMONIA. FIG. 50. 109 Piplococcus of pneumonia from blood, with surrounding capsule. (Park.) and therefore has no flagella. It grows on all culture-media, very little at a temperature below 24° C., best at a tempera- ture of 37° C. At a temperature above 42° C. all growth FIG. 51. Pneumococcus from bouillon culture, resembling streptococcus (Park.) ceases. It is killed in a few minutes by exposure to a tem- perature of 52° C. If grown at 42° C. for twenty- four hours, 110 THE PATHOGENIC MICROCOCCI. its culture becomes very much attenuated, practically losing its virulence. In bouillon it grows rapidly, and in twenty-four hours causes a distinct cloudiness of the medium. At the end of forty-eight hours its growth ceases, and in four or five days the bouillon becomes clear again, the bacillary growth being deposited at the bottom of the tube. In 15 per cent, gelatin at 24° C. its growth is slow. The gelatin is not liquefied. On blood-serum at the temperature of 37° C. it grows as clear, almost transparent spots. Its growth on agar is very much like that on blood-serum. It does not grow on potato. It causes coagulation of milk. Immunization. — The inoculation of animals with attenuated cultures grown at 42° C. for twenty-four hours seems to protect the animal from the after-infection of virulent cultures. An infusion made of the tissues of immunized animals seems to have a protective influence when injected simultaneously or shortly before virulent cultures in susceptible animals. Pathogenesis. — Mice and rabbits are very susceptible to the action of the Microeoccus pneumonice, guinea-pigs much less so. When injected subcutaneously into mice and rabbits, it produces a general septicaemia, with considerable swelling at the place of injection and the formation of a fibrinous mem- brane. The spleen is enlarged, and the bacteria may be found in all the internal organs and in the blood, but no specific pneumonia is developed. When intrathoracic injections are made in the lung substance, it produces a marked lobar pneumonia with considerable fibrinous exudate, and also symptoms of general infection. Injected in the dog intra- thoracically, it may produce marked croupous pneumonia, the animal generally recovering in two or three weeks after presenting all the different stages of the disease. II. Pneumococcus of Friedlaender (Bacillus Pneumoniae of Fluegge). The organism was discovered and described by Friedlaender in 1883, and believed by him to belong to the class of cocci, PNEUMONIA. Ill but recognized afterward as a bacillus. It is found : a. in a number of cases of pneumonia in the fibrinous exudate, 6. in the blood, and c. in the sputum. Morphology. — Short rods, with rounded ends, united in pairs, sometimes in fours, having a decided capsule when taken directly from the blood of the animal. When grown on artificial media the capsule disappears. Occasionally the capsule surrounds each individual cell, at other times it is around the cells, united in pairs or fours. This capsule may be distinctly brought out by the special method of staining capsules mentioned in the chapter on staining. The 'Bacillus pneumonice stains well with all anilin dyes, but does not stain well by Gram's method — a diagnostic point differentiating it from the Micrococcus pneumonice. Biologic Characters. — It is aerobic and facultative anaerobic, non-motile, and has no flagella. It grows in all the media at a temperature of between 16° and 20° C., but grows best at the temperature of the blood, 37° C. Growth ceases at a temperature exceeding 46° C. Its growth in cultures is exceedingly long lived, so that after a year or longer it has grown upon transplantation into a suitable culture. Its growth in bouillon is cloudy. It does not liquefy gela- tin. Stab-cultures in gelatin have quite a characteristic appearance, growing in the form of a nail. The head of the nail is at the point where the inoculating needle enters the gelatin, the path of the needle through the gelatin marking the body of the nail. The head of the nail is a white mass of shiny appearance ; the body is opaque and made up of white spherical colonies. It produces bubbles of gas in gela- tin. On gelatin plates colonies appear in twenty-four hours as small white spheres which increase rapidly in size, and in a short time on the surface of the plate large masses are formed. Its growth on agar is much like that on gelatin. On blood-serum the growth is abundant, viscid, and grayish white in color. On potato it grows rapidly and abundantly, and is yellowish white in appearance. Pathogenesis. — The Bacillus pneumonice, is fatal to mice and 112 THE PATHOGENIC MICEOCOCCI. guinea-pigs. Dogs and rabbits are immune. Intrapleural injections in susceptible animals result in a decided pleuritic effusion with formation of fibrinous membranes, intense con- gestion of the lungs on the injected side, great enlargement of the spleen, and general involvement of the blood (septi- caemia) and internal organs ; the bacillus being found every- where. EPIDEMIC CEREBROSPINAL MENINGITIS. III. Diplococcus Intracellularis Meningitidis. This organism was discovered by Weichselbaum, in 1887, in pus-cells (polymorphonuclear leucocytes) of the cerebro- spinal exudate of cases of epidemic cerebrospinal meningitis. Morphology. — The micrococcus occurs in bunches or in chains of three or four elements, the elements in the chain showing marked variation in size. Stains with all the anilin dyes and is decolorized by Gram's method. It shows marked variation of the different elements in their power of taking color ; some elements being deeply stained, others scarcely at all. The organism has a low vitality ; exposure in the dry state for twenty-four hours to direct sunlight at the body temperature, 37° C., is sufficient to kill it. At the room temperature it is killed in seventy-two hours when dried. To obtain cultures from man, of this bacillus, what is known as lumbar puncture of the spine must be made. The patient is placed on the left side very much in the same position as is used for intraspinal cocainization, the skin of the patient and hands of the operator are thoroughly sterilized, and an ordi- nary antitoxin-serum needle is introduced into the spinal canal, between the second and third lumbar vertebrae, the skin being pierced a little to the right of the spinous process. The needle is driven in for 4 cm. in a child, and 7 to 8 cm. in an adult, until the spinal canal is reached, when the spinal fluid is allowed to drop into a clean sterilized ttfst-tube. From 5 to 15 c.c. of fluid are generally taken for examination. Cover-glasses are prepared and a number of cultures are made. This puncture seems to be followed by no ill effect. MALTA OR MEDITERRANEAN FEVER. 113 Biologic Characters. — This coccus is aerobic and is a faculta- tive saprophyte, non-motile, has no flagella, and grows on all culture-media, but rather irregularly, thriving best on ordinary or Loeffler's blood-serum. In inoculating cultures from the exudate of patients, a large quantity of exudate must be used and a number of tubes inoculated, as otherwise no growth may be obtained. It seems to grow best when the exudate taken comes from a recent, acute case. It does not cloud bouillon, but causes a scanty deposit on the side and at the bottom of the fluid. On glycerin-agar and blood-serum it grows as transparent, shiny colonies. It does not liquefy gelatin nor does it grow on potato. It grows only at the temperature of the body, 37° C., in two or three days. Cultures of this bacillus live only for five or six days, so that it is necessary to transplant them every third or fourth day. Pathogenesis. — It can not be inoculated into animals by the ordinary methods used, but intrameningeal injections, either spinal or under the cerebral dura, produce a characteristic meningitis and fibrinous exudate, the bacteria invading at times the lungs, but never being found in the blood. MALTA OR MEDITERRANEAN FEVER. IV. Micrococcus Melitensis. This organism was demonstrated by Surgeon-Major Bruce, of the British Army, as the cause of what is known as Malta or Mediterranean fever. Morphology. — Round or oval cocci 0.5 mikron in diameter, occurring solitary or in pairs, in cultures occasionally form- ing chains, and staining by the usual anilin dyes but not by Gram's method. The micrococcus is non-motile, but Gordon claims to have demonstrated the presence of from one to four flagella. Biologic Characters. — It is aerobic. It grows very scantily on gelatin at 22° C. only at the end of several weeks, and does not liquefy the gelatin. It grows best in agar, stab 8— M. B. 114 THE PATHOGENIC MICROCOCCI. cultures showing growth only at the end of several days. The colonies appear as pearly-white spots scattered around the points of puncture, and as minute round white colonies along the course of the needle-track, which increases in size, and after some weeks a rosette-shaped growth is seen upon the surface. Along the line of puncture the growth assumes a yellowish-brown color. At 35° C. the colonies become visible only at the end of seven days ; at 37° C. they are seen in three or four days. It does not grow on potato. Pathogenesis. — This micrococcus is not pathogenic for mice, guinea-pigs, or rabbits, but subcutaneous injections in mon- keys have induced fever, the animal dying in from thirteen to twenty-one days. At the autopsy the spleen is found enlarged and contains the micrococcus. In man the micrococcus is found in the enlarged spleen in great numbers. Agglutination. — Recent cultures of Micrococcus melitensis are agglutinated by the blood-serum of patients suffering from Malta fever, and occasionally with some this reaction is manifested a year after recovery. This agglutinating effect has been obtained in a dilution as high as 1 in 1000. QUESTIONS. Give the several names of the Micrococcus pneumonix ; by whom and how was it discovered? Where is it found ? What is its morphology? How does it stain ? How does it behave with regard to oxygen ? Does it possess flagella? Is it motile ? In what media and at what temperature does it grow? What is its thermal death-point? How does it grow in bouillon, gelatin, agar, blood-serum? What protects animals from inoculations with virulent cultures? What animals are susceptible? What are the effects of subcutaneous and intrathoracic injection of animals? What is the synonym of the pneumococcus? Is it a coccus ? By whom was it discovered ? Where is it found ? Give its morphology. Its staining properties. Give its principal bio- PLATE II. "•:. ' ^ > '. •;- '/A/ */:• >• U ^ t ^ Tuberculous Sputum Stained by Gabbett's Method. Tubercle Bacilli seen as Red Rods; all else is Stained Blue. (Abbott.) TUBERCUL OSIS. 1 1 5 logical characters. How does it grow in bouillon, in gelatin, on agar, on blood-serum, on potato? What animals are susceptible? Describe the effects of subcutaneous or intrathoracic inoculations. How is it differentiated from the preceding germ? Where is the Diplococcus intracellularis meningitidis found ? By whom was it discovered ? Give its morphology, its staining properties, its principal biologic charac- ters? How is lumbar puncture performed ? What animals are susceptible? How and where should the inoculation be performed ? Who discovered the Micrococcus melitensis ? Where was it found ? State its morphology, staining, its biologic characters. What animals are susceptible ? In what dilution does the blood of cases of Malta fever agglutinate cult- ures of this micrococcus? CHAPTER X. TUBERCULOSIS. Bacillus Tuberculosis. History. — That tuberculosis, the scourge of the human race, was caused by a microorganism, had long been sus- pected there is no doubt, but it was not until Koch's dis- covery of the bacillus tuberculosis in 1882 that this was at all proved. (Plate II.) Morphology. — The Bacillus tuberculosis is a strict parasite. It is aerobic and grows at the temperature of the human body. It is a slender rod from 1.5 to 3.5 mikrons in length, and from 0.2 to 0.5 mikron in breadth, occurring singly or in pairs united by their narrow extremities. It is found in all tuberculous growths and secretions, but especially in the sputum of tuberculous patients, where its presence is the best confirmatory evidence of the existence of the disease. Biologic Characters. — It grows with difficulty on any of the artificial media. Koch succeeded in growing it on blood- serum. It does not grow in gelatin. It thrives best on 8 per cent, glycerin-agar or, in the mixture of Roux and 116 TUBERCULOSIS. Noeard, 8 per cent, glycerin-bouillon. In this bouillon, kept at a temperature of 37° C., at the end of from twelve to fourteen days it forms a small pellicle on the surface. In slant cultures of glycerin-agar and blood-serum it grows over the surface of the medium as a dried-up, scaly-looking mass. According to some authorities, it is a spore-bearing bacterium ; others fail to find the existence of spores in it. It is non-motile, though occasionally slight movements have been detected in it. It appears to have no fiagella. It is usually killed by exposure to 70° C., but in the dried state may be preserved alive for a considerable time even at a tem- perature approaching 100° C. Staining. — It is difficult to stain by the usual staining methods, and requires the use of special staining technic. Koch's method of staining it consists in adding liquor potassse to the alkaline anilin dyes. Ehrlich's modification of Koch's method, which consists in preparing anilin water and adding this to the solution of an anilin dye, is perhaps the best method of bringing out the tubercle bacillus. The mode of procedure for the staining of bacilli in secre- tions, especially in sputum, has been described in the chapter on staining, as the Koch-Ehrlich method, or the Ziehl carbol- fuchsin method, or, better still, as Gabbett's modification of Ziehl's method. In tissue the bacillus is stained best by an application of either method, which will also be found described in the chap- ter on staining. When so stained, the bacillus shows a number of unstained places in the cell-body, somewhat resembling spores. They have given rise to the opinion that the bacilli are spore-form- ing, but the fact that when the usual method for staining spores is applied these spots remain unstained seems to prove that they are not spores, but are due possibly to some degen- eration in the protoplasm of the bacillus. Nature and Occurrence. — As mentioned, the tubercle bacillus is a strict parasite, and is found only in tuberculous tissues and in the secretions from tuberculous patients, especially the BACILLUS TUBERCULOSIS. 117 sputum. It is also found in substances that have been con- taminated with those secretions, and occasionally are wafted in the air in this manner. Pathogenesis. — The tubercle bacillus is pathogenic for man and for nearly all the lower animals, especially the herbivora, though the carnivora and birds are alike susceptible to it, and traces of the disease have even been found in cold-blooded animals. It may infect the whole animal economy, giving rise to local manifestations in the shape of nodules which contain the bacillus. The usual mode of infection of animals is through the re- spiratory tract, but sometimes through the gastro-intestinal tract. Infection may occasionally be produced by the intro- duction of the bacilli through abrasions of the skin, as in the case of dissectors or pathologists, when it gives rise to local- ized tuberculous nodules on the hands, which at any time may become the source of infection of the general organism. The usual mode of inoculation of animals is either by intra- peritoneal inoculation, when it gives rise to a general tubercu- losis involving especially the glands of the abdomen and the lungs, or by subcutaneous inoculation, when a small quantity of the culture or a small bit of the suspected substance is used. The usual contaminating substance for man is the secretion of tuberculous patients, which may be deposited on utensils used by others, or which through carelessness may have dried in the room, thus contaminating the dust of the apartment, which, wafted through the air, is brought into contact with the mucous membrane of the respiratory organs of susceptible individuals. In this way the air of hospital wards of con- sumptives and the various articles of furniture in rooms inhabited by consumptives have been proved to be infec- tious. The drinking of contaminated milk and the eating of meat from tuberculous animals are believed in some instances to have spread the disease. This, however, is not thoroughly proved, and recently the eminent Koch has asserted that this mode of contamination is exceedingly rare, and is an equation 118 TUBERCULOSIS. which in the treatment and the prevention of tuberculosis may be altogether neglected. ft has been assumed that human, avian, and bovine tuber- culosis are identical. In a remarkable paper on tuberculosis by Koch, read before the Tuberculosis Congress in 1901, at Berlin, he denies this identity, and shows by a number of experiments that cattle can not be inoculated with the secre- tion of tuberculous patients, and that man is not affected by eating meat from contaminated oxen. As regards the transmission of tuberculosis, the part played by heredity is almost nil. It has failed of demonstration that foetuses or young children from intensely tuberculous mothers have in their secretions or tissues the tubercle bacil- lus ; and reasoning by analogy, as in Bang's method, the sepa- ration of newborn calves from their tuberculous mothers has completely succeeded in eliminating tuberculous diseases from these calves, it must be assumed that like precautions would produce identical results in man. The tubercle bacilli secrete a poisonous material, which is chiefly contained in the bacterial cells themselves, and is known by the name of tuberculin. This tuberculin is believed to be a preventative against tubercular diseases ; and in 1890 Koch proclaimed that by means of injections of this substance he had succeeded in curing tuberculosis. This promise has not been fully realized, but Koch's discovery has given us valu- able information, and has demonstrated that by injection of this tuberculin healthy animals may be recognized and so separated from tuberculous ones long before the disease could be diagnosed in the latter by physical signs ; for the former are not affected by small doses of tuberculin, whereas animals that have the least tuberculous taint will show decided reac- tion when injected with tuberculin. This procedure is used extensively in all civilized countries nowadays for the diag- nosis of tuberculosis in cattle and other animals. The original tuberculin of Koch is prepared from an extract of glycerin-bouillon of virulent bacteria, in which the bacteria themselves are quickly killed by exposure to a higher tem- perature, and filtered away by a Chamberlain filter. 0.025 BACILLUS TUBERCULOSIS. 119 c.c. of such an extract will in tuberculous animals develop marked reactionary symptoms, whereas when used in healthy animals it gives rise to no reaction. This tuberculin has a beneficial action in man, especially an action on local tuberculous diseases, such as lupus, tuberculous joints, etc. It is dangerous, however, when used therapeu- tically, because it shows a tendency to stimulate the develop- ment of dormant tuberculosis. Recently different forms of tuberculin have been prepared by Koch, known as tuberculin A, O, and R. Tuberculin A. — This is prepared by extracting the bacilli with decinormal salt solution, and acts very much like ordi- nary tuberculin, being even more severe in effect. Tuberculin 0. — This is prepared by pounding the dried tubercle bacilli and extracting with distilled water, the emulsion being then passed through the centrifuge. The residue after centrifugation is dried and again pounded and extracted with water, and these processes repeated until no solid residue is left. The whitish liquids from all these operations are mixed, and the result is tuberculin R. Tuberculin O is identical in effect to tuberculin A and has an immunizing effect. Tuberculin R gives rise to little reaction, but has a decided immunizing effect. The fluid in tuberculin R is made so that 1 c.c. corresponds to 10 milligrams of solid matter, and must be diluted with sterile salt solution to bring it to the required strength. In applying the same therapeutically the dose of tuberculin R for an adult is ^-^ to 1 milligram. It must be used hypo- dermatically, and should be administered every other day. The dose should not give rise to a temperature exceeding 1 degree C. This produces very satisfactory results in the treatment of lupus, but so far in tuberculous diseases of the lung its effects have not come up to expectation. Recently, the tubercle bacillus, on account of its peculiar growth in some cases in which it seems to present projecting proc- esses or branches, has been thought by some to belong to the 120 LEPROSY AND SYPHILIS. higher bacteria, being probably a streptothrix, closely related to the actinomyces. QUESTIONS. When and by whom was the Bacillus tuberculosis discovered ? How does the bacillus behave in the presence of oxygen ? What is the size of the tubercle bacillus? In what tissues and secretions of tuberculous animals is it usually found ? How is it best artificially grown ? What temperature is most favorable fur its growth ? How high a temperature does it resist? Give two methods of staining the tubercle bacillus in cultures or in the se- cretions of animals. Give the mode of staining the bacteria in tissue? What has given rise to the idea of spores in the bacillus? What animals besides man are the most susceptible to tuberculous diseases? What two forms of infection follow inoculation of this bacillus? What is the usual mode of infection in man ? Mention some cases of localized tuberculosis in man. How are animals in- oculated to produce the disease ? What are the usual infecting agents in man ? What part does tuberculous milk or tuberculous meat play in the dissem- ination of tuberculosis? What was the subject of Koch's paper at the Congress of Tuberculosis, in 1901? What part does heredity play in the transmission of tuberculosis? What is tuberculin ? What diagnostic purpose does tuberculin serve ? How is tuberculin prepared ? What is meant by Tuberculin A, O, and R? Why has the tubercle bacillus been thought to be a streptothrix ? CHAPTER XI. LEPROSY AND SYPHILIS. LEPROSY. Bacillus Leprse. History. — The specific cause of leprosy is a bacillus known as the Bacillus leprce, discovered by Hansen, and confirmed by Neisser, in 1879. The bacillus is found a. in the tissues of leprous patients, and b. in the secretions, with the exception of the urine. It has never been found in the blood. Morphology. — The bacilli are small straight rods with pointed ends, sometimes curved, measuring from 5 to 6 mi- LEPROSY. 121 krons in length, non-motile, resembling very much the tubercle bacillus, but are more uniform in length and not so frequently bent. When stained, their protoplasm shows unstained spaces similar to those of the tubercle bacillus, which are regarded by some as spores. Biology. — Bordoni-Uffreduzzi claims to have cultivated the bacillus through a number of generations in glycerinized gela- tin. Byron (Researches Loomis Laboratory, 1892) made a pure culture of the bacillus on agar. From the secretions and scrapings obtained from an ulcer of the nares in a leper the author found upon examination a great many bacilli lying in cells, some cells containing as many as 3 or 4 bunches, and was able to procure a pure culture on Loeffler's blood-serum and glycerin-agar. The growth upon the serum very much resembled a twisted band of yellowish-gray color, and developed very rapidly at 37° C. Cultures in bouillon and potato did not develop. The Bacillus leprce stains very readily with the anilin dyes, and also by Gram's method. It very greatly resembles the tubercle bacillus in retaining its color when subsequently treated with strong solutions of mineral acids. An interesting point about the staining of the Bacillus leprce which will permit differentiation from the Bacillus tuber- culosis is that the lepra bacillus is rapidly stained by the Gram method, while the tubercle bacillus stains with great difficulty by it, and must remain at least twenty-four hours in the color dish before taking the stain. Baumgarten's differentiation between these two bacteria is to subject cover-glass preparations which have been smeared with scrapings from leprous nodules or ulcers for five minutes in the Ehrlich solution, and afterward to decolorize with solu- tion of nitric acid in alcohol, 1 part of acid to 10 parts of alcohol. The bacillus of Hansen will be stained, while the tubercle bacillus will not. A number of investigators have by inoculation with fresh extirpated leprous tissue succeeded in reproducing the disease in the lower animals. Tedeschi inoculated a monkey under the dura mater, and death resulted in six days. Many lepra 122 LEPROSY AND SYPHILIS. bacilli were found in the spleen and spinal cord at the autopsy. Nature of Leprosy. — Besnier, with many others, contends that leprosy is a bacterial disease exclusively limited to man, and that the microorganisms will reproduce themselves in man alone, and not in animals. Dyer, from observation of leprosy in fifty cases in Louis- iana, concludes positively that the direct cause of the disease is the lepra bacillus. The indirect cause is contagion. The disease therefore is not hereditary. A very useful method of diagnosis for physicians who wish to make a speedy and positive proof of leprosy, and have no microtome or laboratory facilities, is to remove a bit of skin or scraping near a tubercle or nodule and place the same in a mortar with some saline solution and triturate until a homo- geneous solution results, adding from time to time enough saline solution to prevent drying. A small quantity of this emulsion is transferred to a clean cover-glass, air-dried, and fixed over a flame, stained with the Ziehl carbol-fuchsin for five minutes, then washed in water, counterstained, and de- colorized with Gabbett's solution of methylene-blue and sul- phuric acid for two minutes, washed again in water, dried, and mounted in Canada balsam. The bacilli will appear red, while the rest of the tissue will be stained blue. SYPHILIS. Bacillus of Syphilis. History. — In 1884-1885 Lustgarten described a bacillus which he had discovered in the primary sore and secondary manifestations of syphilis. Rarely could this bacillus be found in the tertiary stages of the disease. In size and shape the bacillus very closely resembles that of tuberculosis, but differs from it especially in its cultural peculiarities and also in its staining properties with the anilin dyes. For instance, the bacillus could not be cultivated on any of the artificial media, not even on those on which the Bacillus tuberculosis could be made to grow ; and in staining QUESTIONS. 123 the Bacillus syphilidis it showed considerable difficulty in tak- ing up the auilin colors, yet when stained according to Ehr- lich's or ZiehPs method it very quickly parted with its colors when washed in mineral acids, especially sulphuric acid, con- trary to what happens in the case of the Bacillus tuberculosis. When decolorized, however, by means of alcohol the Bacillus syphilidis retained the dye for a considerable time. For the staining of sections the following method is recom- mended : Place the section in a cold solution of anilin-water gentian-violet for from twelve to twenty-four hours at the room temperature, or for two hours at a temperature of 40° C. Wash a few minutes in absolute alcohol, then put the section for some seconds into 1.5 per cent, solution of per- manganate of potassium, pass rapidly (for one or two seconds) into sulphuric acid solution, wash thoroughly in water, and mount on xylol balsam. When stained by this method the Bacillus syphilidis shows considerable resemblance to the Bacillus tuberculosis, being of similar size and showing similar refractive spots in the body of the cell. As mentioned above, this bacillus has not been successfully cultivated artificially, and inoculations of animals have also been barren of results. Streptococcus of Syphilis. Vanniessen, by collecting blood of syphilitic subjects, and allowing same to coagulate in sterilized tubes, has been able from the serum of this blood to cultivate a streptococcus which he believes to be the etiological factor in syphilis. His experiments, however, have failed of confirmation by others. QUESTIONS. When and by whom was the Bacillus leprx found ? Where is it found ? Describe the Bacillus leprse. Does it contain spores? How is it grown artificially ? How does it stain ? How do you differentiate Bacillus leprse from the Bacillus tuberculosis by staining? What animals are susceptible to the infection? Give a ready method for the diagnosis of a leprous ulcer or nodule, and give a diagnosis of leprosy in a suspected case. Describe the Bacillus syphilidis of Lustgarten ? Where is it found? How does it stain ? How decolorized ? How does it stain in tissue ? How does it grow in artificial culture-media? Differentiate between Bacillus syphilidis and Bacillus tuberculosis. 124 LEPROSY AND SYPHILIS. CHAPTER XII. GLANDERS (FARCY). Bacillus Mallei. GLANDERS is a disease of the horse and ass tribe, charac- terized by the formation of nodules in the mucous membrane of the mouth and respiratory passages. These nodules, very prone to ulcerate, give rise to profuse suppuration, and very soon afterward the lymphatic glands of the neck begin to enlarge. These glands soften early and discharge a very virulent pus. Secondarily the lungs become infected, the infectious material forming small nodules very much resem- bling tubercles in appearance. History. — In 1882, Loeffler discovered in the discharges and tissues of animals affected with this disease a specific micro- organism which he called Bacillus mallei. Morphology. — Glanders bacillus is a bacillus with rounded or pointed ends, occurring generally singly, occasionally in pairs, seldom or never forming threads. The bacillus is non- motile, and therefore possesses no flagella. Spores. — Some observers claim to have discovered spores in the glanders bacillus, but, reasoning by analogy, those shiny particles described as spores are really not spores ; they are the same as the shiny particles discovered in stained prepara- tions of the Bacillus tuberculosis, and they cannot be stained by the usual methods of spore-staining, nor do the bacteria containing same resist conditions which are usually resisted by other spore-bearing bacteria. The observation of Loeffler, however, that this microorganism is able to grow after being kept in the dry state for a long time, makes it appear as if some form of permanent spore existed. Biology. — The Bacillus mallei grows readily on all ordinary media at a temperature between 25° and 38° C. Its growth is very slow, and on this account its isolation and cultivation GLANDERS. 125 by the usual plate-methods are rather difficult. Upon nutrient agar it appears as a moist opaque layer. On gelatin its growth is much less voluminous than on agar. It does not liquefy the gelatin. In blood-serum the growth is opaque, moist, of a bright-yellow color ; the serum is not liquefied. On potato at 37° C. its growth is rapid, moist, and of an amber-yellow color, which becomes darker with age and finally becomes of a reddish-brown. It causes clouding of bouillon, with a tenacious, ropy sediment. In litmus milk it produces acidity FIG. 52. Bacillus of glanders (Bacillus mallei), from culture. (Abbott.) in four or five days, as seen by the change of color from blue to red. It also causes coagulation of the milk. Bacillus mallei is very susceptible to the effect of high tem- perature. At 40° C. it will grow for twenty or more days. It will not grow at 43° C., and if exposed to that temperature for forty-eight hours it is destroyed. It is killed by a tem- perature of 50° C. in five hours, and does not survive more than five minutes at a temperature of 55° C. It is aerobic and facultative anaerobic. 126 LEPROSY AND SYPHILIS. It stains readily with all the anilin dyes, but presents in its body conspicuous irregularity of staining, showing places stained very deeply and others that have scarcely any dye at all. It is difficult to stain in tissues from the fact that, though readily stained, the bacillus parts very quickly with its color- ing-matter in the presence of a decolorizing agent, and even in the alcohol used to dehydrate the tissue. A number of methods for staining sections of tissue for the bacillus of glanders have been suggested. The following is the best : Transfer the sections from alcohol to distilled water, put the sections upon a slide and absorb the water with blotting- paper, stain for a half-hour with a few drops of a 10 per cent, solution of carbol-fuchsin in water, remove the superfluous stain with blotting-paper, wash the sections three times in a 0.3 per cent, acetic acid solution, not allowing the acid to act more than ten seconds each time, and remove the acid by care- fully washing with distilled water. Absorb all water with blotting-paper, and heat moderately over the flame so as to drive off the remaining water. Clear in xylol and mount in xylol balsam. In properly stained tissues the bacilli will be found more numerous in the centre of the nodule, becoming fewer as the periphery is approached. The animals susceptible to infection by glanders, besides horses and asses, are guinea-pigs, cats, and field-mice. The rabbit is very little so ; dogs and sheep still less so. Man is susceptible, and not seldom the infection terminates fatally. House-mice, rats, cattle, and hogs are insusceptible. For inoculation experiments the guinea.-pig is made use of. The experiment is generally performed by subcutaneous inocu- lation of the culture or a small piece of the nodule from the diseased animal. The most prominent symptom in the animal is the enlargement of the spleen, with formation of nodules in that organ and in the liver. From these nodules the glanders bacillus may be obtained in pure culture. The animals live from six to eight weeks. The specific character of the inflammation of the mucous membrane of the nostrils QUESTIONS. 127 is almost always present. The joints become swollen and the testicles enormously distended ; the internal organs — lungs, kidney, spleen, and liver — are the seats of the nodular deposits, from which bacilli may be obtained in pure cultures. Diagnosis. — The method of Strauss for the recognition of the disease is of great importance clinically. With it in a short time a diagnosis may be arrived at, while by the ordi- nary methods of inoculation it would take weeks to come to a certain cdnclusion. Its details are these : Into the perito- neal cavity of a male guinea-pig a bit of the suspected tissue is introduced. If the case be one of glanders, in about thirty hours the testicles begin to swell and the skin covering them becomes red and shining, and there is evidence of ab- scess-formation. TJie tumefaction of the testicle is a true diagnostic sign. Mallein, the toxic principle secreted by the bacillus, has been isolated from old glycerin-bouillon cultures of the Bacillus mallei. For this purpose the cultures are steamed in a sterilizer for several hours and then filtrated through a Chamberlain por- celain filter and evaporated to one-tenth of their volume. This mallein is used as a diagnostic test for glanders in animals, very much as tuberculin is for tuberculosis. It produces when injected in very small quantity a rise of a degree and a half C. if the animal be at all infected with the disease, but in healthy animals injection is followed by no febrile reaction. Some observers have asserted that the injection of this mallein into susceptible animals will protect them from the disease ; other observers assert that the blood -serum of nat- urally immune animals is curative when injected into infected animals. But these points are not fully determined. QUESTIONS. What are the synonyms for the bacillus of glanders? What are the symptoms of glanders in the horse ? By whom and when was this bacillus discovered ? Describe the bacillus. Does it contain spores? Give reasons for and against its spore-formation. 128 ANTHRAX. What are its cultural peculiarities, if any, on agar, on gelatin, on potato, on blood-serum, in bouillon, in litmus milk? Give a method of staining the glanders bacillus in tissue. Give the method of inoculation of a guinea-pig, and the prominent symptoms. How long does the animal live ? Give Strauss' method of inoculation for diagnosis. What is mallein ? How is it obtained ? What are its uses ? Does it pro- tect from glanders ? CHAPTER XIII. ANTHRAX. Bacillus Anthracis. History. — The Bacillus anthracix, discovered and described by Davaine, in 1868, is the first bacillus that was demon- strated to be pathogenic to man and animals. It is found in the blood and tissues of animals which have died of this disease, which is known as splenic fever, and charbon. It produces in these animals a genuine septicaemia, the capillaries all over the body teeming with the microorganisms. No bacteria have more than the Bacillus anthracis helped to establish the three postulates of Koch used in testing the pathogenicity of bacteria. These postulates are as follows : I. For a microorganism to be considered the cause of a dis- ease, it must at all times be found in the organs, blood, or secretions of an animal dead or affected with the dist^ase. II. It must be possible to isolate this organism and obtain it in pure cultures from the same sources. It may also be grown for several generations in artificial culture -media. III. Inoculation of these pure cultures into susceptible animals must give rise to the same symptoms and changes found in the animal originally affected, and the same bacteria must be found in their blood, tissues, or secretions. Morphology. — The anthrax bacillus is a rod bacterium measuring from 2 to 3 mikrons when found in the blood and BACILLUS ANTHRACIS. 129 tissues of animals ; from 20 to 25 mikrons when obtained from cultures; and of a uniform thickness of 1.25 mikrons. The ends of the rod seem a little thicker than the rest of the body, and under a low power look square, but with a higher power they are seen to be concave (Fig. 53). FIG. 53. & Bacillus anthracis, highly magnified to show swellings and concavities at extremities of the single cells. (Abbott.) It is found singly or in pairs in the blood and tissues of diseased animals, but when cultivated in bouillon or in the hanging drop it forms long threads which may or may not contain spores. It is stained by all the alkaline anilin dyes, the spores FIG. 54. Threads of Bacillus anthracis containing spores. X about 1200. (Abbott.) remaining uncolored ; but the latter are easily stained by any of the special methods for staining spores described in the chapter on staining. Biologic Characters. — The Bacillus anthracis is anaerobic, but can grow without the presence of oxygen. When grown 9— M. B. 130 ANTHRAX. with free access of oxygen in artificial culture-media it forms long filaments or threads, which are formed by the union of a number of bacilli. In the presence of free oxygen elliptical bright spots, one to each segment of the thread, are observed; these are the spores. This bacillus grows at all temperatures between 12° and 45° C., but it does not form spores at a temperature below 18° or above 42° C. Its maximum of growth is at 37.5° C. (Fig. 54). In the blood and tissues of animals it does not sporulate. The bacterium is non-motile and has no flagella. In bouillon it grows very rapidly, forming twisted thread- like masses, resembling cotton, in the mass of the bouillon FIG. 55. Colony of Bacillus anthracis on agar-agar. (Abbott.) and at the bottom of the tube, but it does not cloud the medium. On agar its growth is quite characteristic, forming colonies which look like irregularly twisted knots of thread resem- bling cotton-wool ; this peculiar growth has been given the name of the head of Medusa (Fig. 55). On gelatin its growth is very like that on agar, but it liquefies the medium. On potato it grows rapidly as a dull white, thread-like mass. Resistance to Thermal Changes. — It does not grow at a temperature below 12° C. or above 45° C. It may, however, when containing spores, be kept for almost an indefinite period even when dried and exposed to a high temperature, and be BACILLUS ANTHRACIS. 131 subsequently grown when brought in a suitable medium. The spores resist a freezing temperature, and even the tem- perature of liquid air, for almost an indefinite time. They are killed by dry heat at a temperature of 140° C. only after three hours' exposure, and at 150° C. only after one hour's exposure. By moist heat at the temperature of 100° C. they are killed in from three to four minutes. They resist the action of 5 per cent, carbolic acid for five minutes. Its non-sporing forms are killed by a temperature of 54° C. Pathogenesis. — Cattle, sheep, horses, mice, guinea-pigs, and rabbits are all susceptible to the action of the bacilli. Am- phibia, dogs, white rats, and birds are not susceptible. Sus- ceptible animals may be infected in one of four ways : through the abrasions of the skin and mucous surfaces, through the respiratory tract, through the alimentary tract, or by subcu- taneous inoculation, as generally practised in the laboratory. When the bacillus is inoculated subcutaneously into animals, the animal shows little or no inflammation at the point of inoculation, but marked oadema of the subcutaneous tissue at a distance from the inoculating point, with small points of blood extravasation in this tissue. To the naked eye there is very little change in the internal organs except in the spleen, which is enlarged, darker, and soft. Bacilli may be found everywhere in the capillaries, in organs and blood, but espe- cially in the vessels of the lungs, the liver, and in the glom- eruli of the kidneys. Death takes place in from one to three days according to the size of the animal and the dose given. The most susceptible animal is the mouse, next comes the guinea-pig, and then the rabbit, and so uniformly is the resistance of these animals shown to the action of inocula- tions with anthrax that the virulence of attenuated cultures used for protective inoculations are tested on those animals. Immunization. — Pasteur has demonstrated that attenuated cultures of the Bacillus anthracis when injected into susceptible animals are capable of protecting the same against the action of the virulent bacillus, subsequently inoculated, and against an attack of the disease itself. His inoculation or vaccination consists in using cultures that have been attenuated by means 132 ANTHRAX. of heat. For that purpose the bacteria are cultivated in large Erlenrneyer flasks at a temperature of between 42° and 43° C., for a period of time varying from ten to thirty days, when they do not form spores. The pathogenic power of these cultures is tested every few days on guinea-pigs and rabbits, and when a small dose of the culture will kill a mouse and a guinea-pig, but fails to kill a rat, it is called vaccine No. 2. In a few days more this same culture will fail to kill a guinea-pig, but will still kill a mouse ; it is then vaccine No. 1. In veterinary practice large animals, as sheep, cattle, and horses, are inoculated with aseptic precautions with 3 c.c. of vaccine No. 1. Then they show little or no reaction. In ten days or two weeks more they are inoculated with vaccine No. 2, when they again show some little reaction ; and a few days after this second vaccination they are able to withstand an inoculation of virulent cultures of the bacilli. This mode of vaccination has been of inestimable value by making it possible to stop the ravages of epidemics of anthrax. It is practised extensively in countries like France, Germany, and Russia, where the disease is very prevalent among sheep and cattle. In the Southern States the author has had occasion to use it extensively during the last few years, with decided benefit. The manner of infection among animals with the bacilli has not been fully demonstrated. It seems to occur in the ma- jority of cases from the soil, possibly from the fact that animals which have died of the disease have been buried too near the surface. It is therefore advisable that animals dead from anthrax be buried at a depth not less than six feet from the surface, as the soil at that depth is 15° C. even in sum- mer. Consequently the bacilli developed in the dead bodies so buried, both on account of the low temperature of the soil and of the deprivation of oxygen, will not form spores and are not likely therefore to survive for any length of time. DIPHTHERIA AND PSEUDODIPHTHERIA. 133 QUESTIONS. Where and by whom was the Bacillus anthracis first discovered ? What are the three postulates of Koch ? Describe the anthrax bacillus? How does it stain ? How does it appear in the blood of animals? How in culture-media ? When does it form spores ? How does it grow on gelatin ? How on agar ? How on potato? At what temperature does it grow ? When does it cease to form spores ? Are spores found in the animal body? How resistant are the spores ? In what four ways are animals injected? How are animals inoculated ? Describe the lesions found in animals after subcutaneous inoculation ? How are cultures attenuated to prepare the anthrax vaccine? What is vaccine No. 1 ? Vaccine No. 2? How is protecting vaccination practised ? CHAPTER XIV. DIPHTHERIA AND PSEUDODIPHTHERIA. DIPHTHERIA. Bacillus Diphtherias. History. — The infectious nature of diphtheria had been sus- pected for a long time when Klebs in 1883, and later Loeffler in 1884, discovered and accurately described in the false membranes of diphtheritic patients the presence of a micro- organism which bears their combined name — Klebs-Loeffler. Indeed, no infectious disease has been better studied from its etiological and therapeutical standpoints than diphtheria, and it conforms absolutely to the postulates of Koch before men- tioned : that is, it is found in animals sick with the disease, it may be cultivated artificially, and pure cultures inoculated into susceptible animals produce the disease. The disease is not produced by any other germs, and besides injection of its toxins produces in animals substances which are of immu- nizing value when injected into susceptible animals. 134 DIPHTHERIA AND PSEUDODIPHTHERIA. The Bacillus diphtheria? is found a. in false membranes of diphtheritic origin ; b. occasionally in the mouth and nose of healthy individuals ; and c. in the dust of rooms inhabited by diphtheritic patients, or on articles of clothing or furniture which, though they may not have come into direct contact with the patients, yet have been in the. same room with them. Morphology. — The Klebs-Loeffler bacillus is a short rod, from 2 to 6 mikrons in length, and from 0.2 to 0.8 mikron in breadth, being found longer in certain cultures than in others, and when grown for several generations in artificial media. The rods occur singly or in pairs, or in irregu- lar groups ; they may be straight or sometimes slightly curved. Occasionally one or both of the extremities are thicker than the rest of the body of the cell ; at other times the centre of the cell bulges and the end of the cell tapers (Figs. 56, 57, 58). FIG. 56. FIG. 57. One of very characteristic forms of diphtheria bacilli from blood-serum cultures, showing clubbed ends and ir- regular stain. X 1100. Stain, meth- ylene-blue. (Park.) Extremely long form of diphtheria bacillus. This culture has grown on artificial media for four years and pro- duces strong toxin. X 1100. (Park.) Bacillus diphtherice stains with all of the anilin dyes and by Gram's method, but better with Loeffler's alkaline meth- ylene-blue solution. For the purpose of differentiation the Neisser special stain is often used. The bacilli cells do not stain uniformly ; they contain large DIPHTHERIA. 135 granules, occasionally situated at one or both extremities or in its central portion, which stain much more deeply than the rest of the cells, and which make of a stained diphtheria prepara- tion quite a characteristic picture under the microscope. Neisser's Differential Method. — Some forms of false diph- theria bacilli which can not be separated from diphtheria FIG. 58. Diphtheria bacilli characteristic in shape but showing even staining. In appear- ance similar to the xerosis bacillus. X 1100. Stain, methylene-blue. bacilli by their mode of growth or by their appearance under the microscope, but which are not toxic, must be differen- tiated from the toxin-producing bacilli ; and Neisser has suggested the following method, which is used in a number of municipal laboratories. It consists of two solutions, as follows : Solution No. 1. Alcohol (96 per cent.), 20 parts ; Methylene-blue, 1 part ; Distilled water, 950 parts ; Glacial acetic acid, 50 parts ; Solution No. 2. Bismarck-brown, 1 part ; Hot distilled water, 500 parts. 136 DIPHTHERIA AND PSEUDODIPHTHERIA. Put a cover-glass prepared in the usual way for two or three seconds into No. 1 ; then pass into No. 2 and let it remain there for three to five seconds ; wash, air-dry, mount in balsam. The body of the bacteria will be stained brown, and the usually darkly stained granules with the Loeffler method will be stained blue. If the bacilli under examina- tion are true diphtheria bacilli, the majority of them will show the blue granule. If the bacilli are pseudodiphtheritic bacilli, scarcely any or few will show a blue stain in their interior. Biologic Characters. — The Bacillus diphtherias is aerobic, but can grow in the presence of oxygen, and is therefore a facultative anaerobic ; it is non-motile, has no flagella, does not form spores, and does not liquefy gelatin. Its thermal death-point is 58° C. It grows at ordinary room temperature, but slowly. Its maximum of growth is between 37° and 38° C. It is easily killed by disinfectants. Exposure to direct sunlight destroys the bacilli in a few days. In albuminous fluid and in the dark it may live, even when dried, for months. It grows on all artificial culture- media, but best in blood-serum prepared after the formula of Loeffler, a modification of which, employed in many munic- ipal laboratories, is as follows : Blood-serum from sheep or calves, 3 parts ; Peptone-bouillon containing 1 per cent. of glucose, 1 part. Mix, distribute among test-tubes, sterilize, and harden by ex- posing in a slanting position in a steam sterilizer at 97° C. for twro hours. On this mixture at 37° C. after twelve hours the colonies are round, grayish-white, about the size of a pin-head ; later they become larger, elevated, and yellowish, with the centre more opaque than the periphery. At the end of a few days the colonies have a diameter of from 3 to 5 milli- meters. In bouillon at 37° C. the cultures present small clots deposited on the side and at the bottom of the tube. Some DIPHTHERIA. 137 of the culture floats on the surface of the liquid, forming a thin whitish pellicle. The bouillon, which is at first cloudy, becomes in a few days clear, and remains so. The sugars con- tained in the bouillon are fermented, and it is due to their fermentation that this medium has at first a tendency to be acid ; but subsequently, when the fermentation is complete, become decidedly more alkaline. On gelatin the colonies develop very slowly. They appear white, round, irregu- larly notched, and somewhat granular, never attaining a large size. On agar the growth presents the same characteristics as on blood-serum ; but on the surface of agar plates the colonies are quite characteristic, having a dark elevated centre and flat periphery, with a radiated appearance and indented edges. On potato the growth is invisible at first ; and at the end of several days a thin whitish veil seems to cover the portion of the potato which has been inoculated. In milk it grows at a temperature as low as 20° C., without any appre- ciable change of the medium. Pathogenesis. — Diphtheria, along with tetanus, should be classified among the toxic diseases. As a matter of fact, the symptoms met with in cases of diphtheria are due to the effects of the toxins secreted by the bacilli; very few, if any, of the microorganisms are ever found in the blood or deep- seated organs in cases of this disease ; and filtered cultures from which the bacilli have been completely eliminated, when inocu- lated into animals give rise to symptoms identical with those induced by inoculation of the virulent bacilli themselves. Roux and Yersin, by the filtration of cultures through un- glazed porcelain, have been able to separate from the bacilli ii toxalbumin which, when injected under the skin of rabbits and guinea-pigs, produces the blood-poisoning, renal and nervous symptoms met with in pure diphtheria. Welch and Abbott have repeated these experiments, and having estab- lished the same facts have come to the same conclusion as to the action of this toxalbumin. Subcutaneous inoculations of the diphtheria bacilli will pro- duce death in guinea-pigs in about thirty-six hours. The fol- lowing lesions are found at the autopsy : General oedema at 138 DIPHTHERIA AND PSEUDODIPHTHERIA. the point of inoculation, with the formation of a false mem- brane. Marked congestion of the adrenal bodies, serous or serosanguinolent effusions in the pleural cavities, and swollen spleen. A few of the bacilli may be found at the point of inoculation and in the fluid of the oedema. In the blood and internal organs no bacilli can be found, showing that the symptoms are purely toxic. Roux and Yersin have also been able to produce the false membrane giving rise to the disease, by the inoculation of rabbits and guinea-pigs into the mucous surfaces or into the skin, and they have reproduced in animals the characteristic diphtheria paralysis. This paralysis, best seen in the rabbit, usually begins in the posterior extremities and gradually ex- tends over the whole body, death being caused by paralysis of the heart and respiratory organs. Different cultures of diphtheria bacilli, though emanating from equally virulent cases of diphtheria, and grown under the same conditions, show at times a great variation in toxicity. The explanation of this has not been as yet satisfactorily given. But this fact we should remember when testing the efficacy of antitoxins in neutralizing the toxins of diphtheria. Diphtheria Diagnosis. — Clinically it is not always easy to differentiate diphtheria in its early stages from other affections of the throat and nose which are characterized by the pres- ence of exudates. In view of the recent therapeutical advances in diphtheria, it is important that a very early diagnosis be made. For this purpose, accepting the almost unanimous opinions of experts, that diphtheria is due to the presence of diphtheria bacilli in the membranous exudate, boards of health, cities, and hospitals have established a diphtheria service for the purpose of facilitating the early recognition of the disease. In order to carry out this method, a central laboratory with all facilities is established, and in cities a number of supply-depots are located within reach of the practising physician, where the material in complete outfits necessary to make cultures from the throats of suspected cases of diphtheria may be procured. These outfits consist of a blood- serum culture-tube (Fig. 59) made after the formula of Loeffler, DIPHTHERIA. 139 and a swab or applicator kept in a well-sterilized test-tube. This swab is a small iron rod roughened on one of its ends, and on which a little absorbent cotton is twisted. The test- tube containing the swab is plugged with absorbent cotton and then thoroughly sterilized by dry heat for one hour at 150° C. The blood-serum and swab are neatly packed together in a small pasteboard or wooden box, together with a blank form giving instructions as to how to make the cultures. The cultures from the throat are made as follows : The patient is put in the best possible light, and if he is a child is held firmly by an assistant, the mouth is opened, the FIG. 59. Culture-box used in municipal laboratories to prepare cultures from throats of diphtheria suspects. tongue depressed by means of a spoon or other instrument, the swab taken out of its containing tube and gently rubbed over the false membrane or exudate in the throat, if any, or if no false membrane be present, over the surface of the pillar of the fauces, after which, without laying down the swab, the serum-tube is taken, the plug of cotton removed, and the surface of the swab which has been in contact with the throat of the patient is gently and freely rubbed over the surface of the blood-serum, being careful not to break into it, 140 DIPHTHERIA AND PSEUDOD1PHTHERIA. and certain to rub all sides of the swab upon the serum. After which the swab is returned to its tube, both tubes plugged, and the whole outfit with the blank form filled in is returned to the laboratory. On receiving the tube at the laboratory it is incubated at a temperature of 37° C. for twelve hours, at the end of which time it is ready for exami- nation. If the case is one of diphtheria, the typical diph- theria growth is found on the surface of the culture. This consists of grayish or yellowish-white glistening spots, and a cover-glass preparation made of these shows in typical cases the Klebs-Loeffler bacillus, as short, thick rods, with rounded edges, irregular in shape, showing a decided staining in some parts of their body, deficient in color in other parts, and characterized chiefly by the variety of form of the different bacteria forming the culture. In exceptional cases it is possible to find colonies as early as five or six hours after incubation. Indeed, for cases outside of the city limits, in the munic- ipal laboratory in New Orleans, it has been possible to make examinations of the swabs themselves by making cover-glass preparations from the same even two or three days after they were prepared, and in a great majority of the cases come to a positive or negative conclusion, verified later clinically and also bacteriologically, by cultures made from these same swabs. It is essential for these examinations that the cultures from the throats of suspected cases be made before antiseptics have been applied to the throat, or, if that is not possible, the cult- ures should be made at an interval of at least two or three hours after such applications, as otherwise the antiseptics may have acted on the bacilli on the surface of the membrane and destroyed them or greatly inhibited their growth. PSEUDODIPHTHERIA. Bacillus Pseudodiphtherise. Another source of error in the application of this method comes from the pseudodiphtheria bacilli which are found in PSEUDODIPHTHERIA . 141 cultures, and which greatly resemble the virulent Bacillus diphtherice, but have no pathogenic power. These pseudobacilli are of two kinds: I. It is not possible to separate the first kind from the true diphtheria bacilli either by morphology or cultural properties. When injected into the lower animals they are non-virulent, because they secrete no toxin. II. The second kind, in the opinion of the author, are very improperly so-called, for they are not diphtheria bacilli, and can with little difficulty be differentiated from true diph- theria bacilli by their appearance, mode of staining, and their cultural properties. Differential Diagnosis. — The method of staining suggested by Neisser, as mentioned in the beginning of the chapter, is applicable especially to the recognition of the second form of pseudobacilli. For the recognition of the non-toxin-producing form, ex- periment on animals is the only means of differentiating. What appear to be true diphtheria bacilli have been found in the throat and mouth in about 1 per cent, of a number of healthy persons examined, but generally in individuals who have come into contact with diphtheria patients, or when diphtheria was prevalent in the community at the time of the examination. Those persons are always a source of danger to others, and they no doubt are in a great measure responsible for the spread of the disease. The experiments of Roux and Yersin have shown that the various cultures of diphtheria bacilli have different potency in the production of toxins, and that occasionally bacilli grown under conditions, the same as much as possible, may at different times produce more or less toxins, and of a greater or lesser virulence. These facts bacteriologists are in no position to explain, and the toxicity of a diphtheria culture may only be determined by experimentation on animals. The Antitoxin Treatment of Diphtheria. The discovery made by Roux, that the diphtheria bacilli secrete a toxin which, when injected into susceptible ani- 142 DIPHTHERIA AND PSEUDODIPHTHERIA. mals, produces all the symptoms of true diphtheria, was soon followed by the discovery of Behring, which showed that the blood-serum of animals injected with the bacilli of diph- theria contains a substance which when inoculated into sus- ceptible animals is able to immunize them from lethal doses of the bacilli. These substances, called antitoxins, are obtained from ani- mals having little or no susceptibility to the disease, and they have been used extensively both in the prevention and cure of diphtheria since 1894. These antitoxins as exhibited therapeutically are obtained from the blood-serum of horses, as first suggested by Roux, and are prepared as follows : Immunization. — A good-sized horse, which has been demon- strated to be free from tuberculosis and glanders, by the injecting of tuberculin and mallein, and free from all rheumatic and chronic disease, is gradually immunized to the diphtheritic poison by being injected with very small doses of the virulent toxins from a diphtheria bouillon culture filtrated through porcelain. The initial dose consists of 0.10 c.c. mixed with an equal quantity of Gram's iodine solution ; this should produce little or no constitutional disturbance, and very little if any local effect. Four or five days after this first injection a second injection, consisting of pure toxin 0.10 c.c., is used, and every four or five days thereafter injections are re- peated in progressively larger doses until the animal is able to withstand doses of from 400 to 500 c.c. of toxin. During those injections the animal may show decided local effects, such as swelling and oedema at the point of inocula- tion, but no very marked constitutional disturbances. During the progress of this immunization, at intervals, by punctur- ing of the jugular vein with a sterilized trocar, some blood is withdrawn from the animal and its serum tested as to its antitoxic value, and when the same is found sufficient the toxin injections are repeated at longer intervals to maintain the antitoxic property of the animal's serum, and the next process is begun. Standardization. — A large quantity of blood, 4 or 5 liters, PSEUDODIPHTHER1A. 143 is extracted from the immunized horse at one time, collected in well-sterilized vessels, and allowed to clot in an ice-chest for two or three days, after which the clear serum is pipetted off and stored in sterilized flasks, the antiseptic strength of the serum being properly labelled on each flask. This anti- toxin power, called units, is estimated as follows : Ten times a fatal dose of a toxin, that is known to kill a 250-gram guinea-pig within three days, is mixed with differ- ent quantities of the serum to be tested, say, 0.10, 0.01, 0.001 c.c., and these mixtures injected into different guinea- pigs, Nos. 1, 2, and 3, respectively. Should guinea-pig No. 1 survive the mixed injection, and guinea-pigs Nos. 2 and 3 die. the antitoxin is said to contain 10 times 10 units in 1 c.c. ; that is, it is an antitoxin of 100-unit power. Should guinea- pigs 1 and 2 survive, the antitoxin is one which in 1 c.c. has protecting powers amounting to 10 multiplied by 20, or 200 antitoxin units. Should guinea-pig No 3 also survive this injection, then the serum used is equivalent to 10 times 100, or 1000 antitoxin units per c.c. No serum should be accepted for use in the treatment of diph- theria unless its immunizing or antitoxic power is equivalent to at least U200 units per c.c.; a serum, used as a protective only may be accepted with 100 units antitoxic power per c.c. In order to test the antitoxin and for the purpose of im- munizing animals, it is necessary to produce toxins of a standard virulence. This, as has been seen, is not always a task of easy performance. The standard of toxins accepted in all laboratories and establishments in which antitoxin is manufactured is a toxin of which 0.10 c.c. is able to kill a 250- or 300- gram guinea-pig within three days, and no toxins should be used excepting such as have this power. It is best manufactured by growing virulent cultures of Bacillus diph- theria? in large Erlenmeyer flasks, with free access of air and at a temperature of 37° C. The height of the toxicity of the culture is reached in about eight to ten days, when the culture should be removed from the incubator and filtered through a Chamberlain porcelain filter, tested on guinea-pigs, and if found of the required strength put away in sterile 144 DIPHTHERIA AND PSEUDO DIPHTHERIA. bottles. Unless it shows that 0.10 c.c. when injected into a guinea-pig of 250 grams causes death of the animal within three days, it should not be accepted. The German government adopted this as a standard strength for toxins, and no antitoxin is put on the market unless its value has been tested by means of its power of neutralizing so many units of this standard toxin. Value of the Antitoxin Treatment of Diphtheria. — It has now been used eight years; and has been of inestimable impor- tance. As a therapeutic agent given within the first three days of the disease, it has reduced the mortality of diphtheria more than one-half. When used after the third day it is of less value, but still shows decidedly good effects. When used as a preventative in persons exposed to the danger of contagion with this disease, it gives protection for several weeks. Dose of Antitoxic Serum. — As a prophylactic from 200 to 500 units should be used, according to the age. For the purpose of treatment not less than 2000 to 3000 units should be injected at one time, and that as early as possible in the course of the disease ; and this dose should be repeated in twenty-four hours unless decided beneficial effects are noticed. The experience of the author, based on the examination of several thousand cases of diphtheria treated by serum in New Orleans, has shown that, with the exception of an occa- sional urticaria! rash, no untoward effect follows this treat- ment. The explanation of this eruption has not been given, but it is very probably due to some other elements contained in the blood-serum of the horse, and appears to be much more common following the use of serum taken from some horses than from that of others ; it appears to have no rela- tion to the antitoxic power of the serum. QUESTIONS. When and by whom was the Bacillus fliphtherise discovered ? How does it answer the postulates of Koch with regard to pathogenic bacteria ? Where is the Bacillus diphtheria found ? Describe the appearance of the Klebs-Loeffler bacillus. TETANUS, MALIGNANT (EDEMA, ETC. 145 Describe the staining of this bacteria. What characterizes cultures of the diphtheria bacillus? How are false or pseudobacilli differentiated from true diphtheria bacilli? Describe the Neisser method of staining the diphtheria bacilli. How does it behave in the presence of oxygen ? Is it motile ? Has it flagella ? Does it contain spores? At what temperature does it grow ? What is its thermal death-point ? How does it behave in the presence of disinfectants? How is it affected by direct sunlight ? How does it behave in the albuminous fluid? How in the dark? How is Loeffler's blood-serum for the culture of the Bacillus diphtherise prepared ? Describe the growth of this bacillus on Loeffler's medium, in bouillon, on gelatin, on agar, on potato, in milk? Why is diphtheria a toxic disease? How is the toxin of diphtheria obtained? Give the effects of inoculation of diphtheria bacilli on guinea-pigs. What is the effect of the inoculation of those bacteria on mucous surfaces of animals? How do the different cultures of Bacillus diphtheriss vary as to their virulence ? Give the boards' of health measures for diagnosing diphtheria by means of cultures. How is the inoculation of cultures made in those cases? What are the sources of error in this form of examination? What two forms of pseudobacilli are found ? How are they recognized from true virulent bacilli? How is the toxin prepared? How is it gauged ? What is constant diphtheria toxin ? What is the result of the antitoxin treatment of diphtheria ? What is the result of its prophylactic use? What dose should be given as a prophylactic? What dose should be given in the treatment of diphtheria cases? CHAPTER XV. TETANUS, MALIGNANT (EDEMA, AND SYMPTOMATIC ANTHRAX. TETANUS. Bacillus Tetani. History. — Bacillus tetani was discovered by Nicolaier in 1 884, and cultivated by Kitasato in 1889. 10— M. B. 146 TETANUS, MALIGNANT CEDEMA, ETC. It is found a. in wounds in cases of tetanus, b. as a sapro- phyte in the soil, especially manured soil of gardens and stables, and vdL UNIVERSITY OF CALIFORNIA LIBRARY