Gift of D.W. Bennett, M.D, eo Ube flfeebfcal Epitome Series, MICROSCOPY AND BACTERIOLOGY. A MANUAL FOR STUDENTS AND PRACTITIONERS, BY P. E.^.RCHINARD, 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. 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 BY 3TT & THOMSON, PHILAOA, PRESS OF WM. J. DORNAN, PHILADA. 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 list of questions appended to each chapter will be useful to students in quizzing and reviewing. P. E. A. NEW ORLEANS, 1903. 3 EDITOR'S PREFACE. IN arranging for the editorship of The Medical Epitome Series the publishers established a few simple conditions, namely, that the Series as a whole should embrace the entire realm of medicine ; that the individual volumes should au- thoritatively cover their respective subjects in all essentials ; and that the maximum amount of information, in letter- press and engravings, should be given for a minimum price. It was the belief of publishers and editor alike that brief works of high character would render valuable service not only to students, but also to practitioners who might wish to refresh or supplement their knowledge to date. To the authors the editor extends his heartiest thanks for their excellent work. They have fully justified his choice in inviting them to undertake a kind of literary task which is always difficult namely, the combination of brevity, clear- ness, and comprehensiveness. The authors have shown a consistent interest in the work and an earnest endeavor to cooperate with the editor throughout the undertaking. Co- operation of this kind ought to result in useful books, in brief manuals as contradistinguished from mere compends. The editor desires at this opportunity to express his appre- 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 Kefraction ; The Principles of Refraction by Lenses . . . 17-19 THE MICEOSCOPE: The Simple Microscope; The Compound Microscope; The Lenses and Lens-systems of the Micro- scope; The Care of the Microscope . . . . 19-27 CHAPTEE 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 OF 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 CHAPTEE 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 Theories 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 VHI. 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 GONORRHCEA: Micrococcus Gonorrhoea (Gonococcus) ; Bacil- lus Pyocyaneus; Bacillus Pyogenes Fcetidus; Pneumo- coccus or Pneumobacillus ; Bacillus Coli Communis , Bacillus Typhosus; Bacillus Tuberculosis 104-107 CHAPTER IX. The Other Pathogenic Micrococci and Allied Bacilli Micrococcus Pneumonia, Epidemic Cerebrospinal Men- ingitis, and Malta Fever 108-115 PNEUMONIA: Micrococcus Pneumonise Crouposae (Diplococ- cus Pneumonias ; Micrococcus Pasteuri ; Micrococcus of Sputum Septicaemia) ; Pneumococcus of Friedlaender (Bacillus Pneumonise of Fluegge) 108-112 EPIDEMIC CEREBROSPINAL MENINGITIS: Diplococcus Intra- cellularis Meningitidis 112-113 MALTA OR MEDITERRANEAN FEVER: Micrococcus Melitensis 113-115 CHAPTER X. Tuberculosis 115-120 BACILLUS TUBERCULOSIS . 115-120 10 CONTENTS. CHAPTEE XL PAGES Leprosy and Syphilis .... 120-123 LEPROSY: Bacillus Leprae 120-122 SYPHILIS : Bacillus of Syphilis ; Streptococcus of Syphilis . 122-123 CHAPTER XII. Glanders (Farcy) 12^128 BACILLUS MALLEI 124-128 CHAPTEE XIIL Anthrax 128-133 BACILLUS ANTHRACIS 128-133 CHAPTEE XIV. Diphtheria and Pseudodiphtheria 133-145 DIPHTHERIA: Bacillus Diphtherias 133-140 PSEUDODIPHTHERIA: Bacillus Pseudodiphtheriae ; The Anti- toxin Treatment of Diphtheria 140-145 CHAPTEE XV. Tetanus, Malignant (Edema, and Symptomatic Anthrax 145-156 MALIGNANT (EDEMA: The Bacillus of Malignant (Edema . 152-153 SYMPTOMATIC ANTHRAX: Bacillus Anthracis Symptomatici . 153-156 CHAPTEE 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 CHAPTEE XVII. Bacillus Coli Communis 165-168 CHAPTEE XVHL 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 180-182 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 1 8 1NTR OD UCTlONi Focus of a Lens. Foi 1 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 direct 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 its 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 crown 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, T}- 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 Microscope : A, ocular or eye-piece ; B, objective system ; c, stage ; D, iris diaphragm (its opening may be diminished or increased by means of a small lever) ; E, mirror or reflector; F, coarse adjustment; G, fine adjustment; H, substage con- denser (Abbe's) ; i, nose-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 J 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 INTE OD UCTION. 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 OCULAE 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. 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 lenses 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 apochromatic 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. in the latter part of the seventeenth century Anthony von Leuwenhoek, by means of his magnify ing- 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. Cohn 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 defined as fol- lows : Unicellular vegetables of low organization, devoid of chlorophyl (plant color ing -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 sarcinae ; 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. 00 o <*> , * d e a e a. Staphylococci b. Streptococci, c. Diplococci. d. Tetrads, e. Sarcinse. (Abbott.) FIG. 3. Diplococcus of pneumonia, with surrounding capsule. (Park.) MORPHOLOGICAL CLASSIFICATION OF BACTERIA. 31 FIG. 4. \ v A few drops of it are placea 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 agilis, Bacillus typhosusj Bacillus subtilis, bacillus of malignant oedema, 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 j resit , 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-fuchsiri, 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. j ^ foJ^ ^'iM^rwvi Qu^JU^* *' ,. 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. Kuhne's method. Ziehl-Neelsen's method. THE CULTIVATION OF BACTERIA. 55 CHAPTER III. THE PROCESS, MEDIA, AND UTENSILS OF THE CULTI- VATION OF BACTERIA. THE PROCESS OF 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. 1. 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 milk 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 be 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 Roux. 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. pjCL^DK^X \ Q 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 septicsemic 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 of 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 V, 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 \j, able to destroy bacterial life or to neutralize their toxins. *>T 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 effect 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 septicaemic ? When is it toxjemic ? 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 by 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 pyogcnes aureus, albus, and citreus ; the Staphylococcus cereus albus, the Staphylococcus cereus aureus (Passet) ; the Staphylococcus cereus flavus (Passet). 2. The Micrococcus pyogenes tennis (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 Baeiliux />//o";/" //.""*. iyphosrs, and tuber- culosis are sometimes the cause of pus-prod'uctioir, 'as -pure 100 THE PATHOGENIC BACTERIA. cultures of these organisms have been found in some cases of abscesses during the respective infections. Nearly all pyogenic 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 pyogenic staphylococci, also FIG. 47. 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 ;c"r^;in fee^lar, groups or masses resem- bling a bunch of 1 grapes, hence its name. Sometimes it is seen INDIVIDUAL FEATURES OF PYOGENIG 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 b. 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, b. 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 septicaemia and of many surgically common infections (Fig. 48 and Plate I.). FIG. 48. 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. Streptococcus 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, semitransparent 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 BACTERIA. VIII. Micrococcus Tetragenus. The Micrococcus tetragenus 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. GONORRHCEA. IX. Micrococcus Gonorrhoeas (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 QONOREHCEA. 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. I ..< ' *^m. s $m 7; 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 (J-ohm 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- organisniSj 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 ite GONORRHCEA. 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 citreusf Describe the streptococcus pyogenes. Where is it found? Of how many elements are its chains formed? What is the effect of intravenous, intni- peritoneal, and subcutaneous inoculations? Where were the MicrococcMS cereus albus and flavus found, and by whom? What are the characteristics of the Micrococcus tetragenns 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 MICROCOCCL CHAPTER IX. THE OTHER PATHOGENIC MICROCOCCI AND ALLIED BACILLI MICROCOCCUS PNEUMONIA, EPIDEMIC CEREBROSPINAL MENINGITIS, AND MALTA FEVER. PNEUMONIA. I. Micrococcus Pneumonias Crouposae (Diplococcus Pneumoniae ; Micrococcus Pasteuri ; Micrococcus of Sputum Septicaemia). History. The Micrococcus pneumonice crouposce was discov- ered in September, 1880, by Sternberg, 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, 6. 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 Diplococcus 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 MICROCOCCL 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 Micrococcus 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 Pneumonise 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 pneumonias 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 pneumonias is fatal to mice and 112 THE PATHOGENIC M1CROCOCCI. 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 vertebra?, 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 test-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 MICROCOCCL 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 pneumonise ; 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. V Tuberculous Sputum Stained by Gabbett's Method. Tubercle Bacilli seen as Red Rods; all else is Stained Blue. (Abbott.) TUBERCULOSIS. 115 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 f 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. Nocard, 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 flagella. 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 whicli 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. It 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- tical ly, 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 the'se 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 peculiai 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 E ? Why has the tubercle bacillus been thought to be a streptothrix? CHAPTER XI. LEPROSY AND SYPHILIS. LEPROSY. Bacillus Leprae. 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 reproduei^gthe 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 anilin 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 leprse 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, arid 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 conclusion. 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. T/ie 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 hacillus 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-seruin, 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 anthracis, 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 disease. 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 AXTHHAC1S. 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 ANTHRAC1S. 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 oedema 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 antJtracis 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 Erlenmeyer 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 tne 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 ; 6. 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 j 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 diphtherice is aerobic, but can grow in the presence of oxygen, and is therefore a facultative anaerobic ; it is non-motile, has no nagella, 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 two 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 a 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 (liji/itheriw, 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 befree 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 osdema 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, PSEUDODIPHTHERIA. 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- V 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 WO 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- therias 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 urticarial 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 diphtherix discovered ? How does it answer the postulates of Koch with regard to pathogenic bacteria ? Where is the Bacillus diphtherise 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 Loefller's blood-serum for the culture of the Bacillus diphtherise prepared ? Describe the growth of this bacillus on Loefller'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 diphtherise 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 1884, and cultivated by Kitasato in 1889. 10 M. B. 146 TETANUS, MALIGNANT (EDEMA, 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 c. in the intestinal secretions of animals. Morphology. The bacillus of tetanus as obtained in cult- ures is seen in one of two forms, either in the vegetative form or as a spore -bearing bacterium. Its vegetative form is a short rod with round ends, occur- ring singly or in pairs, or sometimes forming long filaments. FIG. 60. Bacillus tetani : A, vegetative stage ; B, spore-stage, showing pin-shapes. (Abbott.) Its spore-bearing form is quite characteristic, resembling a pin ; this is due to the fact that the spore is formed at one end of the bacillus, and as the bacillus bulges at that portion the typical appearance of a pin is given to the bacillus (Fig. '60). _ The Bacillus tetani stains with all the anilin dyes, and also by Gram's method. Biologic Characters. The Bacillus tetani is purely an- aerobic, not developing at all in the presence of oxygen. It grows in all culture-media at a temperature as low as 18 or TETANUS. 147 FIG. 61. 20 C., but best at 37 C. It does not grow at a tempera- ture below 14 C. Cultures must be kept in a hydrogen atmosphere, as the presence of the oxy- gen of the air prevents their growth. On the surface of gelatin the cultures resemble very much those of the Ba- cillus subtilis, but liquefy the medium more slowly (Fig. 61). In gelatin stab-cultures it grows in the depth of the medium, and the col- onies have very much the appearance of a fir tree. Its growth is very slow in this medium, but the addition of from 1 to 2 per cent, of glucose to the gelatin increases materially the rapidity of the growth. The growth on agar is very much like that on gelatin, but it causes no liquefaction of the medium. In bouillon it grows at 37 C., in the depth of the tube, with the production of gases. It does not cause coagula- tion of milk, and produces no acids in its cultures. All cultures of it are noted for their characteristic disagreeable odor. To obtain pure cultures of the Bacillus tetani, a number of methods have been resorted to ; that recommended by Kit- asato is as follows : The pus or secretion of the wound in a case of tetanus, or some garden or stable soil containing the sporing form of Bacillus tetani, is plated on agar, or streak cultures are made on this me- dium witli the secretions of the wound or with the contaminated soil. These agar plates or tubes arc kept at the fraenkei and p'feiffer.) temperature of the incubator for three or four days, so as to allow the growth of all bacteria contained therein. At Colonies of the tetanus bacillus four days old.made by distributing the organ- is'ms through a tube nearly filled with glucose-gelatin. Cultivation in an atmos- phere of hydrogen. (From 148 TETANUS, MALIGNANT (EDEMA, ETC. the end of that time cover-glass preparations are made from the colonies along the streak or on the surface of the agar plates. If some of the characteristic pin-shaped Ba- cillus tetani are found, the cultures are treated as follows : They are exposed from three-fourths to one hour to the temperature of 80 C. ; in this way all the fully formed bacteria and the greater part of the spores are killed, the vegetative form of the Bacillus tetani included, but spores of this bacillus remain alive. Then from these cultures fresh gelatin, bouillon, or agar tubes are inoculated, and the same grown at the temperature of the room or incubator in an atmosphere of hydrogen. If the original substance experi- mented with contained the Bacillus tetani, characteristic cult- ures will be seen in this medium in a few days, and may subsequently be transplanted. Motility and Thermal Death-Points. The Bacillus tetani in the spore-bearing variety is non-motile ; the vegetative form is quite motile, though no flagella have been discovered. A temperature of 58 C. will destroy the non-spore-bearing variety in a half-hour ; 60 C. will kill them in five minutes ; and 65 C. instantaneously. Spores, however, are able to resist a temperature of 80 C. for two hours, but are killed by a temperature of 100 C. in from four to five min- utes. When dried, the spores are capable of retaining their vitality for months and years. Carbolic acid (5 per cent.) will not kill them in less than ten hours ; but if 0.5 per cent, hydrochloric acid be added to the carbolic acid solution, spores will be destroyed in two hours. Bichloride of mer- cury (1 in 1000) will destroy them in three hours. Bi- chloride (1 in 1000) to which 6.5 per cent, hydrochloric acid has been added will kill them in thirty minutes. Tetanin. The Bacillus tetani secretes a powerful poison, known as tetanin, which diffuses in the cultures and is not retained in the cell-body. The symptoms of tetanus are due to the action of this toxin, and not to the influence of the bacteria themselves. Tetanus is strictly a toxsemic disease. This is proved by the fact that inoculations with cultures of bacilli in which the TETANUS. 149 toxins have been destroyed produce no symptoms whatever. These toxins are destroyed by a temperature of 60 to 65 C., by prolonged exposure to diffuse daylight, or by exposure for one hour to direct sunlight, and cultures containing spores so exposed are innocuous to animals. The author has succeeded in several instances in obtaining the tetanus bacillus by the following cultivation-method : After thoroughly heating a bouillon tube or a liquid gela- tin tube so as to expel as much as possible all the oxygen, the culture is allowed to cool to a temperature a little below 80 C. The suspected material is then inoculated deep into the tube, and the surface of the medium is covered by a layer of 1 to 2 c.c. of paraffin oil, a cotton plug inserted, and a rubber cap applied over the tube. In this way he has obtained cultures with great facility. In one case, notably, cultures were made from the surface of a nail, that caused a wound which produced tetanus in an adult. In another case a piece of diphtheritic membrane wrapped in a piece of gauze and kept on the hearth over night, was handed to him from a diphtheria patient for examination. The next day to his surprise besides the diphtheria bacilli a few bacilli resembling the tetanus bacilli were found. A piece of this membrane was inoculated into bouillon prepared in the fore- going manner, and he obtained after three or four days a pure culture of the tetanus bacillus which proved fatal to guinea- pigs. Pathogenesis. The animals susceptible to the Bacillus tetani are man, horses, guinea-pigs, rabbits, and mice. Dogs are little susceptible, and birds scarcely at all. Amphibians can not be infected. The inoculation of animals is made by means of a liquid culture injected subcutaneously or by means of some of the contaminated material introduced into a deep pocket in the subcutaneous tissue. The period of incubation is more or less prolonged, varying from a few days to occasion- ally two or three weeks. During this time the bacilli seem to be generating their poison. After this has been accomplished, the toxic effects are very marked and rapidly fatal, the symp- toms showing first in the parts nearest to the point of inocu- 150 TETANUS, MALIGNANT (EDEMA, ETC. lation. These symptoms consist in spasms of the muscular system, and generally end in death. The blood and the urine of inoculated animals is toxic to other susceptible animals. At the autopsy, apart from the slight inflammatory changes at the point of inoculation, with occasionally the discovery of a few bacilli at that point, no changes are observed in the organs, excepting an intense congestion of the nervous system. Bacilli deprived of toxins injected into animals are taken up by the phagocytes. Preparation of the tetanus toxin is very easy. A bouillon culture of the Bacillus tetani is grown in an atmosphere of hydrogen at a temperature of 37 C. for from two to four weeks. At the end of that time the culture is filtered through a porcelain filter, and the filtrate is found to contain the tetanin, which is best kept in the dark, and preserved by the addition of 0.5 per cent, of phenol. The power of this toxin, called tetanin, is very great, 5-0 oliTo-o c ' c * ^eing suffi- cient to kill a 15-gram mouse in three to four days. Occa- sionally this toxicity is very much increased, and Burger and Cohn have succeeded in obtaining tetanin, which in doses of c - c ' was fetal to mice. This is b far the most FoTTooT o powerful poison known; taken in this proportion it would mean that about ^ milligram would be fatal to man. Com- pare this with atropine, the fatal dose of which is about 130 milligrams, and anhydrous prussic acid, the fatal dose of which is 54 milligrams, and a fair idea of its toxicity will be obtained. Tetanin acts on animals only when introduced into the cir- culation; given by the mouth it possesses no poisonous prop- erties. The blood of animals dead or affected with tetanus is poi- sonous to other animals in the same way as cultures of the bacillus itself. But it is possible to inoculate animals with doses small enough to produce no fatal effects ; and animals so inoculated are protected from future infection, and their blood and fluid secretions will serve to protect other animals when injected in doses less than the fatal dose. The dis- TETANUS. 151 covery of this fact by Behring and Kitasato has been the open- ing wedge to serum therapy. Tetanus antitoxin, like diphtheria antitoxin, is produced by inoculating large animals, like the horse, with minute doses of the toxin, diluted at first with Gram's iodine solution, and artificially establishing in the horse an immunity against the poison. The dose of the toxin is gradually increased, and injected every few days into the animals until immense doses (600 to 700 c.c.) may be injected at one time without pro- ducing any marked symptoms. When immunity has thus been secured, blood is taken from the animal and its serum tested, when it is found to have decided powers of neutralizing the toxin. Tetanus antitoxin is useful chiefly as a preventative against tetanus, and in veterinary medicine has been found of great value. When applied to the human subject, however, the results have not been so satisfactory, for it is used then only as a therapeutic agent. At the time of its employment the symptoms of tetanus have generally shown themselves, and these are exceedingly rapid and violent in their effects, and commonly fatal. A number of observers have derived very decided benefit from its use, however, especially by injecting it into the ventricles of the brain, where it may act by directly and locally combating the poisonous action of the tetanin present. Tetanus antitoxin is measured somewhat differently than is diphtheria antitoxin. Its strength is expressed as follows : 1 in 1,000,000 or I in 10,000,000. This means that 1 c.c. of the antitoxin is capable of protecting from infection 1,000,000 or 10,000,000 grams of guinea-pig. In some cases an antitoxin of 800,000,000-gram power has been obtained. This antitoxin, however, does not retain its power very long, and deteriorates quickly in the fluid form. It is gener- ally made into a powder, which may be dissolved into a neutral saline solution for use. 152 TETANUS, MALIGNANT (EDEMA, ETC. MALIGNANT (EDEMA. The Bacillus of Malignant (Edema. History. Malignant oedema is caused by a very malignant bacillus, discovered by Pasteur, studied by Koch and Kitt, and found in the soil of gardens and in the dust of streets, which, when inoculated into animals, rapidly produces the disease. Morphology. Bods from 3 to 5 mikrons in length and 1.10 mikron in thickness. They occur singly or in pairs in cult- ures, rarely forming threads. The ends are square in appo- sition when two bacilli come together, but rounded when the bacilli are single or at the free ends of united bacilli. This bacillus stains with all the ordinary methods of stain- ing, but does not stain by Gram's method. It forms spores, situated at or near the centre of the ba- cillus, causing a swelling of the bacterium. (Plate III.) Biologic Characters. The bacillus of malignant cedema is an obligate anaerobic, and does not grow at all in the presence of oxygen. It grows in all culture-media in hydrogen gas, liquefies gelatin, and rapidly liquefies blood-serum. In gelatin and bouillon it grows at the bottom of the tube, and in the liquid gelatin the colonies are in the form of spheres, which are scarcely discernible at first, but which, on account of the fermentation developed by the bacilli causing clouding of the medium, become more and more apparent. On agar plates in a hydrogen atmosphere it grows as whit- ish bodies, which under the magnifying glass are seen to consist of branching and interlacing lines radiating irregu- larly from the centre to the periphery. The colonies grow at ordinary temperature, but best at 37 C. Pathogenesis. Men, horses, calves, dogs, sheep, chickens, pigeons, rabbits, guinea-pigs, are all susceptible to the disease. Inoculation of animals is performed subcutaneously by in- troducing a small particle of the suspected material or culture into a deep pocket. The symptoms developed in animals are a rapid and extensive oedema, with bloody effusions at the PLATE III Bacillus CEdematis Maligni. (Abbott.) A. (Edema-fluid, from site of inoculation of guinea-pig, showing long and short threads. B. Spore-formation, from culture. SYMPTOMATIC ANTHRAX. 153 point of inoculation, involving also the muscular tissues. The internal organs show little change, excepting the spleen, which is enlarged. The bacilli are rarely found in the blood of the heart when the autopsy is performed immediately after death, but they are found in limited numbers in the internal viscera. If the autopsy is delayed, however, the whole body of the animal becomes infected with the bacillus. This bacillus is grown, like the tetanus and other anaerobics, in atmospheres of hydrogen only. SYMPTOMATIC ANTHRAX. Bacillus Anthracis Symptomatici. History. Ferrer and Bellinger discovered a bacillus in the disease of animals known as black leg, quarter evil, or quarter ill, which is also found in humid soils in certain localities during the summer months, especially when those places have been contaminated with discharges from infected animals. Morphology. The description of this microorganism given by Kitasato is as follows : Actively motile rods, 3 to 5 mikrons in length, and from 0.5 to 0.6 mikron in thickness, occurring singly, occasionally in pairs, never forming filaments (Fig. 62). It stains by all the anilin colors and by Gram's method. It forms spores, which are situated at or near one of the poles, giving a swollen appearance to the bacillus. Biologic Characters. In the vegetative type it is actively motile, but loses its motion in the spore-bearing form. It can not be cultivated in an atmosphere of oxygen. It is purely anaerobic, and does not grow in an atmosphere of car- bonic acid gas. It grows best when glucose (1.5 to 2 per cent.) or glyc- erin (4 to 5 per cent.) is added to the culture-medium. It grows in all media. It liquefies gelatin. It grows best at the temperature of the incubator, 37 C., but does not grow at a temperature below 14 C. In deep-seated punct- ures of gelatin or agar it grows in three or four days, and produces during its growth gas bubbles. The colonies appear 154 TETANUS, MALIGNANT (EDEMA, ETC. as globules which cause liquefaction of the gelatin and coalesce into irregular tabulated liquid areas. The dried spores retain their vitality for months. They resist a tem- perature of 80 C. for one hour, but five minutes' exposure at 100 G. is sufficient to destroy them. Carbolic acid (5 per cent.) is not effective as a disinfectant in less than ten hours. The vegetative form, however, is killed in from three FIG. 62. Bacillus of symptomatic anthrax: A, vegetative stage gelatin culture; B, spore-forms agar-agar culture. (Abbott.) to five minutes. Bichloride of mercury (1 : 1000) will kill the spores in two hours. Pathogenesis. Cattle, sheep, goats, guinea-pigs, and mice are susceptible animals. Horses, asses, and rats show only slight local swelling, but no general infection. Dogs, cats, rabbits, chickens, pigeons, and hogs are immune. Inocula- tions are generally made deep into the subcutaneous tissue either with pure cultures of the microorganisms or from bits of tissue of a suspected animal. The symptoms are a rise of temperature, followed by painful swelling at the point of inoculation. Death takes place in from one to two days. QUESTIONS. 155 The autopsy reveals an extensive swelling of the subcutane- ous tissues with emphysema. The oedematous fluid is blood- stained, and the muscles are dark and prominent. The lymphatic glands are involved. The internal organs show little change. In the fluid of the oedema the bacilli are found in large numbers, lying singly. Early autopsy reveals no bacteria in the blood, only a few in the internal organs. Late autopsy shows a considerable quantity of organisms that have invaded the whole body. The bacilli in the body arc- found to contain spores. This serves as a differentiation, in addition to other points, between it and the Bacillus anthraeis. Immunity. One attack of the disease if not fatal affords protection against future attacks. The opposite of this hap- pens with malignant oedema, one attack of which seems to predispose to other attacks. QUESTIONS. When, where, and by whom was the Bacillus tetani discovered and culti- vated? How many forms of the Bacillus tetani are there, and how are these dis- tinguished ? What is the characteristic appearance of the spore-bearing form ? In what atmosphere does it grow best, and why? What is the temperature-limit of its growth ? How does it grow in gelatin ? In agar? In bouillon? In milk? What is Kitasato's method of obtaining pure cultures of this bacillus? In what form is it motile ? What agents and chemicals are the spores capable of resisting, and to what extent? What is tetanin? Describe a method of growing anaerobic bacilli with the use of paraffin oil. What animals are susceptible to the infection of tetanus? Describe the autopsy of an animal inoculated with Bacillus tetani. Where and how are inoculations in animals made? What symptoms are produced by tetanin injection? What type of infection is tetanus ? How is tetanus toxin prepared? What is the degree of toxieity of tetanin? How is the antitoxin of tetanus prepared? How is it used and for what purpose? Why is it of more use in veterinary than in human medicine? How is the strength of tetanus antitoxin expressed? By whom was discovered the bacillus of malignant oedema? Where is it found ? What is its appearance ? 156 TYPHOID FEVER. How does it stain ? How does it behave in the presence of oxygen? How does it grorv on different media ? What animals are susceptible ? How are inoculations performed ? What symptoms a e produced by inoculation? By whom was tho bacillus of symptomatic anthrax discovered? Where is it found ? What diseases of animals are produced by it? Give the description of microorganisms containing spores and vegetative forms. How does it stain ? What effect does the addition of glucose to media have upon the growth of this organism ? How does it grow in different media ? What are the effects of temperature on its growth ? How is it fatal to animals ? What animals are susceptible? How is it differentiated from other bacilli ? What is the effect of a non-fatal attack of this disease? How does symptomatic anthrax compare with malignant oedema? CHAPTER XYI. TYPHOID FEVER. Bacillus Typhosus. History. The presence of a microorganism in cases of typhoid fever was discovered by Eberth, in 1880; it was named the Bacillus typhosus ; but until isolated and described by Gaffky, in 1884, it was not fully recognized. It is found after death in the blood, spleen, liver, intestines, Peyer's patches, and mesenteric ganglia, and during life in the blood, especially when the same is taken from the spleen by means of a hypodermatic syringe, in the rose patches, in the urine and feces, and outside the human body, occasionally in water and soil contaminated with dejecta of typhoid patients, and often in milk, which is due probably to the cleansing of the utensils in which the milk is collected with water con- taminated with the bacilli (Figs. 63 and 64). Morphology. The Bacillus typhosus appears as a rod with BACILLUS TYPHOSUS. 157 rounded extremities, from 2 to 4 mikrons in length, and 0.6 to 0.8 mikron in breadth. At times it appears as short ovals ; at others the bacilli are joined together, forming long threads. It stains with all the anilin dyes, but not quite so readily as other bacteria. It does not stain by Gram's method. In stained preparations clear spaces are observed in the body of the cells. This has given rise to the belief that the bacteria contain spores. There are, however, no spores, for those clear spaces do not stain by any of the spore-staining processes, and bacteria in which they are found are less resistant to external FIG. 63. FIG. 64. /', Wtof Bacillus typhosus, from culture twenty-four hours old, on agar- agar. (Abbott.) Bacillus typhosus, showing flagella stained by Loeffler's method. (Abbott.) influences than others. This bacillus has numerous fine, hair- like flagella, which are not to be seen in unstained prepara- tions or preparations stained by the ordinary methods, but it requires the flagella-stain of Loeffler to bring them out. Biologic Characters. The Bacillus typhosus is aerobic, but grows also without the presence of oxygen ; it is therefore facultative anaerobic. It is non-spore-bearing, and is actively motile, the motions at times being very rapid. It grows in nearly all the artificial media, even at the room temperature, but best at a temperature of 37 C. Its growth at 20 C. is rather slow, but quite rapid at the temperature of the body. On gelatin plates its colonies appear as small, yellowish, punctiform bodies, becoming in a short time round and 158 TYPHOID FEVER. irregularly notched, resembling droplets of oil. In gelatin stab-cultures they appear as small thick disks, finely dentated, of a pearl-like color. They do not liquefy gelatin. On agar plates they appear as round, irregular, shiny colo- nies of a blue or grayish-white color, and develop very abun- dantly. In agar stab-cultures the growth is chiefly on the surface, and in the depth of the medium there is scarcely any appreciable development. On lactose-litmus agar colonies are pale blue. On potato the growth is exceedingly variable, and not characteristic, as formerly believed. Sometimes it is scarcely appreciable, at other times it forms a film like a thin veil of the same color as the potato itself. Again, at times the growth is somewhat luxuriant and of a whitish color. It does not coagulate milk. It does not cause fermentation in glucose-, lactose-, or sac- charose-bouillon. It does not produce indol in such quantity as is detected by the ordinary tests. Vitality. It is killed by an exposure of ten minutes to 60 C., and in much shorter time by exposure to higher tem- peratures. In the dried conditions it may be preserved for months. Agglutination. Persons who have suffered from an attack of typhoid fever or animals which have been inoculated with cultures of this bacillus have generated in their blood-serum a substance called agglutinin. This agglutinin has the prop- erty when mixed with cultures of the Bacillus typhosus of suddenly arresting the motion of the bacilli and of causing their clumping or agglutination, which is quite characteristic, and is made use of for the diagnosis of typhoid fever, as will be described later. Pathogenesis. None of the lower animals, as far as has been ascertained, is naturally susceptible to contract or develop typhoid fever. Indeed, the typical lesions of the disease as found in man have rarely been induced in the lower animals by inoculations with the typhoid bacillus. Intraperitoneal, subcutaneous, and intravascular inoculations, in rabbits, guinea-pigs, and mice, will produce marked infec- tion even in those animals, in the form of general septicaemia, DIFFERENTIATION OF THE BACILLUS TYPHOSUS. 159 in which the bacilli have been recovered in the general cir- culation and in the internal organs. The feeding of animals with articles contaminated with typhoid fever germs has in some instances, when the animal's vitality was very much lowered, produced infection, and sometimes lesions in the intestines and mesenteric ganglia very much resembling those found in human beings. Differentiation of Bacillus Typhosus from Allied Groups. I. General Features. In many respects the Bacillus typho- sus resembles very much the Bacillus coli communis, both from a morphological point of view as well as in its cultural peculiarities. The differentiation between the two is some- times quite difficult, and it is necessary to cultivate the bacilli in all the known artificial media to come to a conclusion about their identity. The points of differentiation are the following : The Bacillus coli communis is generally thicker and much less motile than the typhoid bacillus. The coli communis grows much more rapidly in all media. The flagella of the typhoid bacillus are more numerous. The Bacillus typhosus does not coagulate milk, and the coli communis does. Its growth on litmus-agar remains blue, that of the coli com- munis becomes red from the production of acids. The Ba- cillus typhosus produces no indol, as ascertained by the ordinary Dunham's test, but the coli communis produces indol very rapidly. The Bacillus typhosus does not produce fermenta- tion in lactose or glucose media, whereas the coli communis produces fermentation and fermentative gases. On potato the growth of Bacillus typhosus is almost invisible, while that of Bacillus coli communis is abundant, creamy, and of a dark- brown color. The serum of the blood from typhoid fever cases agglutinates cultures of Bacillus typhosus. It has no action on Bacillus coli communis II. Widal's and Chantemesse's Differentiation. Two tubes of agar or gelatin to which 2 per cent, of lactose-sugar has 160 TYPHOID FEVER. been added are allowed to melt and a sufficient quantity of neutral litmus tincture is added to them to give a deep- violet color. The tubes are sterilized and are inoculated, one with the Bacillus typhosus, and the other with the Ba- cillus coli communis. If agar tubes are used, they- are placed in the incubator at 37 C. When the colonies grow, those of the Bacillus typhosus retain the blue color, while the colonies of the Bacillus coli communis become of a bright-red color, and at the bottom of the tube can be seen bubbles of gas. III. Eisner's Method of Differentiation. This consists in employing an acid mixture of gelatin, potato juice, and potas- sium iodide, which contains neither peptone nor sodium chlo- ride. It is used to separate not only the coli communis, but also the ordinary saprophytes from the Bacillus typhosus. The saprophytes do not develop at all in this medium, and the colonies of coli communis and Bacillus typhosus show marked differences in their behavior on plates made of this mixture, and are easily separated. At the end of twenty- four hours tubes of this mixture inoculated with the sus- pected material will contain a large number of coli communis colonies, which have the same appearance as cultures of this bacillus on ordinary agar plates, whereas there will scarcely be visible development of colonies of the Bacillus typhosus. After forty-eight hours the Bacillus typhosus will appear as small, pale, almost transparent colonies, easily distinguished from the dark granular colonies of the coli bacillus. Accord- ing to Abbott, Eisner's medium is thus prepared : "Grate 1 kilogram of pealed potato and allow this to stand over night in a refrigerator ; then press out all juice, using an ordinary meat-press for the purpose ; filter this fresh juice cold to remove as much of the starch-granules as possible. If this is not done, the starch when heated swells to such an extent as to render filtration almost impracticable. Boil the filtrate and again filter. Test the filtrate for acidity by titrating 10 c.c. with a decinormal solution of sodium hy- droxide, the indicator used being 6 drops of the ordinary 0.5 per cent, solution of phenolphtalein in 50 per cent, alcohol. The acidity of the juice should be such as to re- DIFFERENTIATION OF THE BACILLUS TYPHOSUS. 161 quire 3 c.c. of a decinormal sodium hydroxide solution to neutralize 10 c.c. of the juice. If the acidity is found to be greater than this, which is usually the case, dilute with water until the proper degree is reached. If less than this, the juice may be concentrated by evaporation. It is desirable that this acidity should be due to the acids normally present in the potato, and that it should not be artificially obtained by the addition of other acids. Now add 10 per cent, of gelatin (with no peptone and no sodium chloride present), dissolve by boiling, and again test the acidity, using 10 c.c. of the mixture and phenolphtalein as before. Deduct 3 c.c. (the acidity of the potato juice that is to be maintained) from the number of c.c. of the decinormal sodium hydroxide solu- tion requisite to neutralize the 10 c.c. of the gelatin mixture, and from the resulting figure calculate the amount of normal solution of sodium hydroxide needed for the entire volume, and add it. Boil, clarify with an egg, and filter through paper in the usual manner. To the filtrate add potassium iodide in the proportion of 1 per cent., decant into tubes, and sterilize." IV. Stodard's and Hiss' Differentiation. By this method use is made of the great motility of the Bacillus typhosus to differentiate it from the coli communis. It is valuable at times. Success in this procedure depends on the important fact that in a semifluid mixture the Bacillus typhosus, on account of its great motility, will diffuse much more rap- idly from the point of inoculation to nearly all parts of the medium, whereas the coli communis, having only a sluggish or no motion at all, develops only at the place of immediate inoculation. For detailed accounts of these methods the reader is referred to larger treatises on bacteriology. Sources of Pure Cultures. From the spleen of typhoid fever cases pure cultures of the bacillus may be readily ob- tained, in early autopsies, and during life ; blood extracted by means of a hypodermatic syringe from this organ will almost always show the bacillus. Indiscriminate punctures of the spleen during life, however, are not to be recom- mended, as this procedure is not free from danger. 11 M. B. 162 TYPHOID FEVER. The Bacillus typkosus has occasionally been obtained froni abscesses in the subcutaneous tissue and internal organs in pure cultures in some cases of typhoid fever, showing that this bacillus is at times the cause of suppuration. Artificial Susceptibility. Animals resisting the effects of inoculation with the Bacillus typhosus can be made suscepti- ble by the simultaneous introduction of other saprophytes which seem to overcome their immunity. The Blood-Serum Diagnosis of Typhoid Fever. The diagnosis of typhoid fever by the blood-serum method is to-day generally employed. As mentioned before, this is based on the principle discovered by Pfeiffer, that the blood of persons suffering with typhoid fever, or who may recently have had the disease, when mixed with young cultures of Eberth's bacillus, has the property of arresting the active motion of the bacilli, and causing their agglutination or clumping. This power resides in the serum, and is due to a substance called agglutinin. Widal inaugurated the blood-test for typhoid fever, and sug- gests that to 1 c.c. of bouillon culture, not more than twenty- four hours old, and grown at a temperature of 35 C., 0.10 c.c. of the serum to be. tested be added. The serum may be obtained either by allowing the drawn blood to coagulate, or by means of a small blister. In the space of from five to ten minutes all motion of the bacilli is arrested, and these come together, forming peculiar clumps. This clumping may be seen both in the hanging drop, and even by the naked eye in culture-tubes. Ordinarily the hanging-drop method is adopted, as it requires much less serum, and is therefore less injurious and vexatious to the patient, Wyatt Johnston's Dried Blood Method. This observer has demonstrated that the same reaction may be obtained by the use of dried blood instead of fresh serum, and that even after the blood has been dried for several days or weeks it still retains its agglutinating power. The procedure in detail is as follows : THE BLOOD-SERUM DIAGNOSIS OF TYPHOID FEVER. 163 A drop of the blood to be tested is obtained from the finger or lobe of the ear and allowed to dry on a clean slide. With a platinum wire a few loopfuls of sterile water are mixed with the dried blood and the same is diluted until about of the same color as normal blood. One loopful of this blood mixt- ure is added to 40 or 50 loopfuls of a bouillon culture of the Bacillus typhosus twenty hours old, on a cover-glass, and a hanging drop made in the usual way. In the course of a half- to one hour, if the blood comes from a case of typhoid fever of sufficient duration, not less than six or seven days, cessation of motion and clumping of the bacteria in the culture drop will have been completely effected. In the experience of the author in 'the Municipal Labora- tory of New Orleans with more than 6000 cases, this test FIG. 65. Outfit used by the Municipal Laboratory of New Orleans for the collection of blood for the typhoid fever test. has given satisfactory results. The plan, which is a modifica- tion of the New York Board of Health method, is as follows : At the diphtheria depots blood slides are left with blank forms giving directions (Fig. 65). Directions for Preparing Specimen of Blood. Clean thor- oughly the tip of the finger or lobe of the ear, and prick with a clean needle deep enough to cause several drops of blood to exude ; two or three drops are then placed on the slide of the outfit. Let the blood dry, then place the slide in holder, fill out the blank form, and return to depot where obtained. On the following day a report of the result of examination will be mailed or telephoned to the attending physician. 164 TYPHOID FEVER. The blood only of fever patients is to be used. Should the report be negatived and the case be suspicious, the physician in attendance is requested to send another specimen, and in every case to notify the bacteriologist as to whether the labo- ratory diagnosis is finally in harmony with the clinical diag- nosis or at variance with it. Sources of Error. One, which must be remembered, is due in some cases to the persistence of the reaction for a number of years after a typhoid attack : so that a reaction may appear in health or in affections other than typhoid fever, if the patient has previously suffered from the disease. In cases in which the reaction is marked, it may apparently be positively stated that the patient has, or has had, typhoid fever within a few years. Diagnostic Values. If the reaction is present, but not well marked, only probable diagnosis may be made. If the reac- tion is absent in a patient sick seven days, the diagnosis of typhoid fever may be excluded. The experiment has not been tried long enough and not in a sufficient number of cases to permit a positive statement as to the earliest date of the appearance of the reaction in typhoid fever. Vaccination Against Typhoid Fever. Wright and Semple have recently practised the vaccination of human beings against typhoid fever, and extensive ob- servations have been made in India and South Africa in the British Army. For this purpose a typhoid vaccine consisting of a bouillon emulsion made from a slant agar culture of the Bacillus typhosus twenty-four hours old is used. The cult- ure is killed by heating it for five minutes at a temperature of 60 C. From a half to a quarter of the whole culture is used for one vaccination, and the culture must be of such a strength that a fourth of it is capable of killing a 300- to 400-gram guinea-pig, when the same is injected into it, without killing the bacilli. The results obtained by these vaccinations have been en- couraging and seem to open up a promising field for the serum-therapy of typhoid fever. BACILLUS COLI COMMUNIS. 165 Antityphoid Serum. Bokenham has recently succeeded in immunizing a horse by using a filtered bouillon culture of the typhoid bacillus, and he claims that the horse's serum has immunizing power when injected into guinea-pigs. QUESTIONS. What name is usually given to the microorganism causing typhoid fever? By whom and when was it discovered ? Where is it found in the human body? Where is it occasionally found outside of the human body? Describe the Bacillus typhosus. What are its staining peculiarities? How do you stain the flagella of the Bacillus typhosus ? Why do you say that it contains no spores? How does it behave in the presence of oxygen ? Is it motile ? At what temperature does it grow best ? What is its growth on gelatin? On agar? On lactose-litmus-agar ? On potato? In Dunham's solution? In the fermentation-tube ? Does it liquefy gelatin ? What is the thermal death-point of the Bacillus typhosus ? What is agglutinin ? How are animals inoculated with the Bacillus typhosus f What are the points of difference between the Bacillus typhosus and the Bacillus coli communis f Give the Widal-Chantemesse method of distinguishing between colonies of typhoid and coli communis? Give Eisner's method of separating the Bacillus typhosus from the Bacillus coli communis and water bacteria. Give Abbott's mode of preparing Eisner's medium. On what are Stodard's and Hiss' methods based? In what organ may the bacillus be obtained in pure cultures? How may the resistance of animals to typhoid inoculation be overcome? On what does the serum-test of typhoid fever depend ? How may serum be obtained for this test ? Describe the methods pursued with dried blood in municipal laboratories. CHAPTER XVII. Bacillus Coli Gommunis. History. It was discovered by Escherich, 1885, and is found in health as a constant inhabitant of the intestinal tract chiefly in the large intestine and also in the excretions from 166 BACILLUS COLI COMMUNIS. that tract. In pathological conditions it is met with, in asso- ciation with other bacteria, a. In acute enteritis, cholera morbus, in certain forms of dysentery ; it is easily demon- strable in large numbers, and has been thought by some to be the cause of those diseases, but this is not so. Its pres- ence in the healthy individual in nearly all cases is sufficient to show the falsity of this position, b. It has also been found in cases of peritonitis, endocarditis, and in suppurating inflammation of the liver and the kidney. At autopsies it occurs in various organs and in nearly all conditions. Asso- ciated with specific microorganisms it has also been proved to exist in the blood of patients in articulo mortis. Outside the human body it has been discovered in water and soil con- taminated with fecal matter. Etiologic Relations. For a long time this bacillus was looked upon as a harmless saprophyte ; latterly experiments have established the fact that it is often the cause of inflam- matory conditions in the body, and that in a number of other instances it is pathogenic from the fact that it lowers the vitality of the body and enables other germs to act delete- riously. Morphology. This bacillus is polymorphous and very closely resembles the typhoid bacillus in shape. It is a rod with rounded extremities, in very young cultures appearing almost oval with a bright centre. Later on the bacilli coa- lesce and appear as long threads. They possess flagella ; not so numerous, however, as the Bacillus typhosus. These fla- gella may be stained by the Loeffler method. It has no spores and stains by all the ordinary anilin dyes, but not by the Gram method. Biologic Characters. It is aerobic and facultative anaerobic. It is motile at times, and at other times appears to be motion- less. Its motility is always of the sluggish kind. Cultures which when young contain organisms with decided motion, have on being kept for some time shown that the bacilli have lost their motility. It grows on all the artificial culture- media and at the temperature between 10 and 40 C. Its growth, though retarded at the temperature above 40 C., BACILLUS COLI COMMUNIS. 167 is not altogether stopped until 45 C. is reached. Exposure to a temperature of 65 C. for five minutes destroys the bac- teria. Exposure to cold has no effect on the bacteria, and in some instances the author has been able to cultivate bacteria which had been exposed to the temperature of liquefied air for several minutes. In bouillon the bacillus grows very rapidly and renders the bouillon cloudy ; pellicles are formed on the surface of the medium, and there is also a thick deposit at the bottom of the tube. A strong fecal odor can be detected. On gelatin plates the colonies appear as small, spherical, blue-gray points, somewhat dentated at the margin. With a magnifying glass the colonies are brownish, lozenge-shaped or irregularly round, coarsely granular. In gelatin stab- cultures along the track of the needle are seen a series of small spherical colonies in rows and separated from each other. On the surface of the tube the growth is of a dirty gray color. It does not liquefy gelatin. On agar-agar the growth has nothing characteristic. On agar to which 2 per cent, glucose has been added bubbles may be seen along the line of growth, due to the gases of fermen- tation. On lactose-litmus-agar the colonies develop very rapidly and are of a pinkish color. On potato it grows rap- idly in the beginning, being of a bright-yellow color which later becomes brown. The growth in serum is similar to that on agar. It produces indol in peptone solution and coagulates milk very rapidly. It ferments lactose- and glucose-bouillon. Pathogenesis. Bouillon cultures of this bacillus injected intravenously or into the peritoneal cavity of a rabbit cause death in less than twenty-four hours. On autopsy intense hyperaBmia of the peritoneum, ecchymotic 'spots of the intes- tines, swelling of Peyer's patches, and enlargement of the spleen are found. Subcutaneous inoculations are followed by abscesses formed at the point of inoculation and by internal conditions similar to those produced by intravascular injec- tions. Injected into the pleural cavity it gives rise in twenty- four hours to a purulent pleurisy accompanied by a large effusion in the cavity and the formation of false membrane. 168 ASIATIC CHOLERA. QUESTIONS. When and by whom was the Bacillus coli communis discovered ? Where is it found in the body in health ? In pathological conditions? What pathological conditions are found to be due to the presence of this microorganism? Where is it found outside the human body ? Describe the Bacillus coll communis. How do its flagella compare with those of the typhoid bacillus ? How is it stained ? How does it behave in reference to oxygen ? What is peculiar about its motility ? How does it grow on artificial media and at what temperature? What is its thermal death -point? What is the effect of cold ? How does it grow in bouillon ? On gelatin? On lactose-litmus-agar ? On potato? In milk? In Donovan's peptone solution ? What is the effect of intraperitoneal and intravascular inoculations in animals ? What lesions are found at the autopsy? What lesions are produced by subcutaneous inoculation ? By intrapleural inoculation ? CHAPTEK XVIII. ASIATIC CHOLERA. Spirillum Gholerae Asiatic ae (Comma Bacillus). History. In the Cholera Congress at Berlin,, 1884, Koch made the announcement that he had been able to isolate from the intestinal dejecta of cholera patients a microorganism which he believed to be the cause of the disease. His experi- ments were carried out in a number of cholera-infested places and on a large number of patients. His conclusions, though very much questioned at the time, are to-day accepted by all, and his Spirillum cholerce Asiaticce, more commonly known as the comma bacillus, is recognized as the etiological factor in Asiatic cholera. Morphology. This microorganism belongs to the class of spirilla called by some authorities vibrios. It is found in the secretions of cholera patients and in cult- SPIRILLUM CHOLERA ASIATICS. 169 ures as a short curved rod, from 0.8 to 2 mikrons in length, by 0.3 to 0.4 niikron in breadth. Sometimes two of these rods are united together by either end, with the convex sur- face looking different ways, appearing then as the Roman letter S ; at other times a number of the rods are united together forming a long spirillum. These latter forms are especially seen in older cultures. In young cultures the rods are generally single or lying together and parallel to each other. This peculiar mode of grouping serves in the recogni- tion of this bacterium. The Spirillum cholerce Asiatics stains with all the anilin dyes, but rather poorly. It seems to have a more active FIG. 66. Spirillum of Asiatic cholera. Impression Involution-forms of the spirillum cover-slip from a colony thirty-four hours of Asiatic cholera, as seen in old old. (Abbott.) cultures. (Abbott.) affinity for the fuchsin dye. It does not stain by the Gram method. Young cultures take the stain much more readily than older cultures, and in these what is known as involution- forms long, thready filaments of different thickness are often found. The spirillum contains no spores, but has a single flagellum at each end (Figs. 66 and 67). Biologic Characters. The comma bacillus is strictly aerobic, and though it grows in an atmosphere in which the oxygen is diminished, it can not grow in the absence of this gas. This fact is the cause of its surface growth in fluid media. It is an artificially motile spirillum, especially when lately obtained from cholera cases or in young cultures. It grows 170 ASIATIC CHOLERA. in all artificial media, provided these are neutral or slightly alkaline. Its growth on gelatin plates and stab-cultures is quite characteristic. At the end of a few hours on gelatin plates the colony appears as a light whitish point, which grows very rapidly, liquefying slightly the gelatin around it. This FIG. 68. b c d Stab-culture of the spirillum of Asiatic cholera in gelatin, at 18 to 20 C. : a, after twenty-four hours ; 6, after forty -eight hours ; c, after seventy-two hours d ' after ninety-six hours. (Abbott.) liquefaction of the gelatin seems to be accompanied by evaporation of the liquid, so that the colony sinks into the depth of the space left in the gelatin by the liquefaction, and the whole surface of the plate seems to be punched out. In gelatin stab-cultures the surface growth shows liquefaction of the gelatin around the colony, and this liquefaction gradually SPIRILLUM CHOLERA ASIATICS. 171 enlarges and extends along the track of the inoculating needle, being broader at the surface and forming a short funnel, which from the evaporation of the liquefied gelatin at the top gives it a characteristic appearance (Fig. 68). Its growth on agar resembles very much the growth on gelatin, but the medium is not liquefied. Milk is coagulated by the formation of acids in the medium. In peptone- bouillon the medium is clouded, and a pellicle forms on the surface. Vitality. Its growth is very rapid, and advances best at a temperature between 35 and 37 C., but continues at a tem- perature as low as 17 C. Its growth is stopped at a tem- perature of 16 C., and the bacterium is destroyed in five minutes by an exposure to 65 C. Freezing does not destroy it. Dryness destroys it very rapidly, but in the moist state it may be kept frequently for several days and sometimes for several months. Rapidity of Growth. When associated with other bacteria in cultures it grows at first much more readily than any of the known bacteria, having a tendency to form a surface growth. At the end of eighteen to twenty hours, however, it is outstripped in its growth by the other bacteria, and in twenty-four to forty-eight hours its growth ceases altogether, and in a few days scarcely any spirillum may be found in the cultures. This is not due to the fact that it is destroyed by its association with the other bacteria, but more because the pabulum necessary for its growth is consumed. The rapidity of the growth of this bacterium and the fact of its growing on the surface of liquids are a great help in its isolation from cholera dejecta, which when diluted with a large amount of peptone-bouillon shows in a few hours a peculiar surface growth, which consists almost of a pure culture of cholera spirilla. Pathogenesis. None of the domestic animals contracts the disease naturally. But their immunity seems to be due to the fact that the bacteria that they ingest at the time that they are exposed are destroyed by the acidity of the gastric juice. 172 ASIATIC CHOLERA. Artificial Susceptibility. A number of ingenious devices have been resorted to to render animals susceptible to inocula- tion of the comma bacillus. The method of Koch is ingenious and very successful. It consists in neutralizing the acidity of the gastric juice in a guinea-pig by the inoculation of 10 c.c. of a 5 per cent, solution of carbonate of sodium. This is introduced into the stomach by means of a soft catheter. A few minutes after- ward 10 c.c. of young bouillon cultures of the cholera spiril- lum are introduced also into the stomach through the same catheter, and immediately an intraperitoneal injection of 1 c.c. of laudanum is made into the animal, for the purpose of retard- ing peristaltic action. The animal for an hour or so remains in a stupefied condition from the action of the opiate, but it soon revives. It shows, however, a complete loss of appetite, and at the end of twenty-four hours begins to show signs of paralysis of the hind extremities, with coldness of the sur- face. This paralysis gradually increases until in forty-eight hours the animal dies, showing pathologically some lesions resembling those found in man in cases of cholera i. e., a large amount of white serous exudate in the intestinal canal, with intense congestion of the intestines. Pure colonies of the spirillum may be obtained from these secretions. Intraperitoneal injections in animals are followed by death in two or three hours. The symptoms are those of a rapid and intense peritonitis. Immunity. When the injections into animals are made in quantities too small to produce death the animal is protected for a time from subsequent fatal doses, and its serum has been found useful to protect animals of the same species against inoculations with fatal doses of the bacteria. The blood-serum of these immunized animals, as well as that of cholera patients, has been found in a dilution of 1 to 50 to possess the power of agglutination when mixed with young bouillon cultures of the bacteria. This may be used as a diagnostic test of the disease. The organism is seldom or never found in the general cir- culation nor in the internal organs of cholera cases. QUESTIONS. 173 Diagnosis. For this purpose the rate of the growth on gelatin plates and the rapidity of the indol-formation in Dunham's solution are made use of. The experiments are carried on as follows : The small flocculent masses found in the discharges of choleraic patients are taken and mixed with a large quantity of diluted peptone-bouillon, or preferably with Dunham's solution of peptone, and put into the incubator for three or four hours. At the end of that time a few drops from the surface of the liquid are taken and inoculated on gelatin plates, when characteristic colonies are developed in a few hours. Cover-glass preparations are also made, and if rods with a morphological appearance of cholera bacteria are found, agar plates are also made in this way. Melted agar is poured into Petri dishes, and these put into the incubator for a few hours in order to allow the evaporation water to collect on the surface of the agar ; this water is poured off and the dishes inoculated by streaking the surface with the suspected material. In a very short time characteristic colonies develop along the line of the streak. The cholera bacteria are of very rapid growth, but possess little or no resisting power, being destroyed by the physical measures just mentioned, and also in a very short time by the use of weak disinfectants. Vaccinations against cholera have been performed on an ex- tensive scale in cholera-infected countries. Haffkine's method of injecting attenuated or small doses of virulent cultures of the cholera spirillum as a means of protection against an attack of cholera seems to have rendered considerable service in protecting persons exposed to the disease ; and experiments made by Ferran, in Spain, with attenuated cultures seem to have given encouraging results at the time of the cholera visitation. QUESTIONS. When and by whom was the Spirillum choleras Asiaticse (comma bacillus) discovered ? Describe the spirillum. What is the peculiar arrangement of the bacteria in cultures and secre- tions? 174 INFLUENZA. How does the comma bacillus stain ? Does it contain spores? Has it flagella ? How does it behave in the presence of oxygen ? Is it motile? What condition of the media is necessary for its growth ? How does it grow on gelatin plates ? In stab- cultures? On agar? In pep- tone-bouillon ? At what temperature does it grow? What is its thermal death-point ? What is the effect of cold ? Of dryuess ? How long may it be kept in a moist state ? How does it grow when associated with other bacteria? What peculiarities of its growth are made use of in those cases to isolate it? What is the cause of the natural immunity of domestic animals to cholera ? How has Koch succeeded in inoculating the lower animals through the stomach ? What effect has inoculation of animals with cultures of the comma bacillus ? What is the effect of intraperitoneal inoculation ? How are animals made immune against cholera inoculation ? What is the effect of the blood-serum of immunized animals on other animals? Where are the organisms found in cholera patients or at the autopsy in a cholera case ? How is the cholera bacillus isolated from cholera dejecta? How much resisting power has the comma bacillus? What is Haffkine's method of protection against cholera? CHAPTEK XIX. INFLUENZA. Bacillus of Influenza. History. In 1892 Pfeiffer and Cannon independently iso- lated from the bronchial and nasal secretions of cases of influenza, and from the blood in some cases, a small micro- organism which they believed, with apparent correctness, to be the cause of the disease. Morphology. The bacillus so isolated may be described as follows : a small, thick rod, occurring singly or in pairs, stained with difficulty by the ordinary anilin dyes, but fairly well with a diluted Ziehl solution or Loeffler's methylene- blue ; not stained by Gram's method. The body of the rod stains less well than the ends. It has no flagella and contains no spores. (Plate IV.) PLATE IV. .*\ *w v V Bacillus of Influenza in Sputum. (Abbott.) QUESTIONS. 175 Biologic Characters. The bacillus of influenza is strictly aerobic, not growing at all without oxygen. It is non-motile, and grows at a temperature between 26 and 43 C. It grows but rather poorly in all media that may be sub- mitted to this temperature, unless the surface of the media be smeared over with fresh sterilized blood, when the growth is quite luxuriant. On glycerin-agar or in blood-serum tubes on which fresh rabbit's blood has been smeared, it grows as transparent watery colonies, resembling very much dew- drops. The colonies have no tendency to coalesce. In bouillon to which a little fresh blood has been added it grows luxuriantly. It does not cause clouding of the medium, but its colonies are seen as little flakes adhering to the sides of the tube and forming a deposit at the bottom. Vitality. The bacillus of influenza is destroyed in two or three hours by drying. It has very little resisting power, and in water lives scarcely twenty-four hours. In pneu- monia occurring during the course of this disease the bacilli are often found in the body of the leucocytes. Pathogenesis. Outside of the human race none of the lower animals seems to be susceptible to the disease, excepting perhaps the monkey, and by inoculation it is difficult to produce any symptom in laboratory animals. In man, how- ever, the bacillus is constantly found in the bronchial and nasal secretions, also in the pneumonic patches so often found in the course of this disease. At autopsies it has been found also in the spleen and occasionally in the blood. Some per- sons have a natural power of retaining live bacilli in the lungs for a considerable length of time; especially is this the case with tuberculous patients, in whose sputum is very often found the Bacillus influenzas. By inoculating animals in the brain, the nervous phenomena of this disease have been easily reproduced. QUESTIONS. By whom and when was the bacillus of influenza discovered ? Describe this bacillus and its staining peculiarities? Does it possess flagella ? Spores ? What are the principal biologic characters of the bacillus of influenza ? 176 BUBONIC PLAGUE. At what temperature does it grow ? In what artificial media does it grow ? What must be added to this media to facilitate its growth ? What is the appearance of the colonies, in glycerin ? On agar ? In blood- serum ? How does it grow in bouillon ? What is the resisting power of this bacillus ? What animals are susceptible ? Where is the bacillus found in animals? What is peculiar about the retention of this bacillus by some persons? How may this explain the spread of the disease ? CHAPTER XX. BUBONIC PLAGUE. Bacillus Pestis. History. Under various names and from the remotest times epidemics of bubonic plague have appeared in the old world, causing an immense fatality. Yersin and Kitasato, in 1894, working independently, have both discovered the pathogenic germ of this disease in the suppurating buboes, blood, internal organs, and excretions of persons affected, and called it the Bacillus pestis. Morphology. This bacillus is a short, oval, thick rod, occurring singly or in pairs, or sometimes by the union of a number forming long filaments or threads. Staining with all the anilin dyes, but not by Gram's method. In stained preparations the centre of the bacilli cell stains less well than the ends of the rod, giving it quite a characteristic appearance. (Plate V.) This bacillus has no flagella. Biologic Characters. The Bacillus pestis is a non-motile aerobic. It grows at all temperatures, but best between 36 and 39 C. It is killed by a temperature of 80 C. after an exposure of a half-hour, and in five minutes by an exposure to 100 C. in the steam sterilizer. It grows in all the arti- PLATE V. Bacillus of Bubonic Plague (Abbott.) A. In pus from suppurating bubo. B. The bacillus very much enlarged to show peculiar polar staining. BACILLUS PESTIS. 177 ficial media. On gelatin, after twenty-four or thirty-six hours the colonies appear as small, sharply defined, round, white masses which do not liquefy the medium. Its growth in agar in the incubator is a little more rapid than in gelatin. It .~> mode of preparing sterilized, 50 Monotrocha, 35 Mordant, 50 NATURAL immunity, 94 Neisser's gonococcus, 104 Nicolaier, tetanus bacillus of, 145 Numerical aperture, 24 ABERMEIER'S spirillum, 179 V Objective, angular aperture of an, 23 to cleanse, 25 designation of, 21 Ocular, to cleanse the lenses of, 25 lens, 24 types of, 25 (Edema, malignant (see Malignant cedema), 152 Optical axis, 24 Oxygen, relation of, to bacterial life, 36 PARASITES, 36 Passet's bacillus pyogenes foe- tidus. 10<> Passive immunity, 95 14 Bact. Pasteur, bacteriological researches of, 27 Pasteur's abstraction theory, 97 Pathogenic bacteria, 99 Pelvic contents, examination of, 89 Pepton solution, 61 Peritrocha. 35 Pfeiffer's bacillus, 174 Phagocytosis theory, 97 Pitfield's flagella stain, 51 Plasmodium malariae, 189 Plate cultures of agar, 71 Pneumobacillus (Friedlaender's), 106 p:ithogenesis, 107 Pneumococcus, 99 Friedlaender's, 110 biologic characters of, 111 discovery of, 110 morphology of, 111 pathogenesis of, 111 Pneumonia, 108 Potato as culture-media, 60 preparation of, for test-tube culture, 61 Pseudodiphtheria, 140 differential diagnosis of, 141 Ptomaines, 39 Putrefaction, causes of, 39 RAY fungus, 186 Refraction, law of, 17 Reichert's thermo-regulator, 75 Relapsing fever, 179 Retention, theory of, 97 Roux-Nocard method of culture, 90 history of, 90 importance of, 90 technic of, 90 QACCHAROMYCETES, sprouting k3 fungi, 28 Saprophytes, 36 Schizomycetes, cleft fungi, 28 Sedgwick-Tucker method for examin- ing air, 203 Septicaemia sputum, 108 Serotherapeutics, 178 Scrum, agglutinating action of, 178 antityphoid, 165 Shiga's bacillus, 180 Soil examination for bacteria, 203 Spirillum, 30 cholera Asiatics, 168 Obermeieri, 179 biology of, 179 210 INDEX. Spirillum Obermeieri, history of, 179 morphology of, 179 pathogenesis of, 180 Spleen, typhoid bacilli in, 161 Spores, staining of, 47 (Abbott) first method, 47 second method, 48 third method, 48 (Fiocca) fourth method, 49 Sporozoite, 191 Sporulation, 32 significance of, 34 Sputum septicaemia, 108 Staining, 33 methods, 42, 116 Bowhill's method, 52 Bunge's method, 51 Ehrlich's modification of Koch's method, 44, 116 Gabbett's modification of Ziehl's method, 46, 116 Koch's method, 116 Pitfield's method, 51 Van Ermengem's method, 52 Ziehl's carbol-fuchsin method, 46, 116 Stains, 43 Staphylococcus cereus albus, 99 aureus (Passet), 99 flavus, (Passet), 99 pyogenes albus, 101 aureus, 100 features of, 100 morphology of, 100 citreus, 102 Sterilization, definition of, 76 fractional, 77 methods of, 76 et seq. Streptococcus of syphilis, 123 Streptothrix, 185 actinomyces of, 186 Eppingeri, 186 et seq. madurse, 186 pseudotuberculosa, 188 resemblance of, to bacteria, 185 to moulds, 185 Subcutaneous inoculation, 85 Sulphur dioxide as antiseptic, 83 Syphilis, 122 history of, 122 TETANIN, 148 Tetanus, 145 antitoxin, 151 bacillus, cultivation of, 73 history of, 145 toxin, 150 Tissues, staining of bacteria in, 52 Gram's method, 52 Kuehne's carbolic methylene- blue method, 53 Weigert's method (modification of Gram), 53 Ziehl-Neelsen's method, 54 Toxalbumins, 39 Tuberculin A, O, and E, 119 diagnosis of tuberculosis by, 118 Koch's, 118 Tuberculosis, 115 history of, 115 transmission of, 118 Typhoid fever, 156 vaccination against, 164 VAN NEISSEN'S streptococcus, 123 Voges' guinea-pig holder, 86 WATER, bacillus coli communis in, 202 bacteria in, 196 cholera bacilli in, 201 counting of colonies in, 198 et seq. examination of, 197 pathogenic germs in, 201 typhoid bacilli in, 201 Weigert's modification of Gram's method, 53 Wiesnegg's autoclave, 80 EESIN plague, 176 7IEHL-NEELSEN'S method, 54 L DATE DUE SLIP UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL LIBRARY THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW . ^ * lw-10,'33 HOOL LIBRARY