iediccL E 101 •» RGSCOPY . .. \ *-* - r ,-^s ¥ /rx /^^ "X ] IDLDOif ARCIi -L'VRD flfeebical JEpitome Series. MICROSCOPY, BACTERIOLOGY, AND HUMAN PARASITOLOGY. A MANUAL FOR STUDENTS AND PRACTITIONERS. BY P. E. ^ARCHINARD, A. M., M. D., Bacteriologist Louisiana State Board of Health and City Board of Health, New Orleans. SECOND EDITION, REVISED AND ENLARGED. ILLUSTRATED WITH ONE HUNDRED ENGRAVINGS AND SIX PLATES. LEA & FEBIGER, PHILADELPHIA AND NEW YORK. Entered according to Act of Congress, in the year 1912, by LEA & FEBIGER, In the Office of the Librarian of Congress. All rights reserved. 2_ PREFACE TO SECOND EDITION. IN reviewing this work the author has made such additions as were necessary to bring it to date as a concise presentation of Bacteriology, as it should be understood by all students and practitioners of medicine. He has broadened its scope by including information on a few protozoa nearly akin to bac- teria. This will make the book more useful to advanced students, as well as to general practitioners seeking to enlarge their knowledge of the microscopical world by attending spe- cial post-graduate courses. The author acknowledges with thanks the very valuable services rendered in the revision by Dr. W. H. Seeman, Pro- fessor of Clinical Microscopy and Tropical Medicine in the Post-Graduate Department of the Tulane University of Louisiana. P. E. A. NEW ORLEANS, 1912. CONTENTS. INTRODUCTION. PAGES The Refraction of Light and the Microscope 17-22 THE REFRACTION OF LIGHT : The Two Fundamental Laws of Refraction ; The Principles of Refraction by Lenses . . . 17-19 THE MICROSCOPE: The Simple Microscope; The Compound Microscope ; The Lenses and Lens-systems of the Micro- scope; The Care of the Microscope . . ..... 19-27 CHAPTER I. The Fundamental Principles . . 28-42 THE HISTORY OF BACTERIOLOGY ............. 28 THE CLASSIFICATION OF COHN FOR BACTERIA 28 THE DEFINITION OF BACTERIA 29 THE MORPHOLOGICAL CLASSIFICATION OF BACTERIA : The Coccus; The Bacillus; The Spirillum 30 THE SIZE OF BACTERIA 33 THE REPRODUCTION OF BACTERIA: Fission; Spdrulation . . 33-34 THE MOTILITY OF BACTERIA 36 THE RELATION OF OXYGEN TO BACTERIAL LIFE 37 THE RELATION OF DEAD AND LIVING ORGANIC MATTER TO BACTERIA 37 THE ESSENTIAL CONDITIONS OF BACTERIAL GROWTH : Heat ; Moisture ; Decomposable Organic Material ; Special Chemi- cal Reaction of the Culture-medium 37-39 THE INERT AND INHIBITTVE CONDITIONS OF BACTERIAL LIFE THE VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA . 39-42 8 CONTENTS, CHAPTER II. PAGES The Examination and the Staining of Bacteria ...... 42-57 THE EXAMINATION OP BACTERIA : The Hanging-Drop Prepa- ration 42-43 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 43-57 CHAPTER III. The Process, Media, and Utensils of the Cultivation of Bacteria 57-71 THE PROCESS OF THE CULTIVATION OF BACTERIA 57 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 57-65 THE UTENSILS OF THE CULTIVATION OF BACTERIA .... 65-71 CHAPTER IV. The Inoculation of Culture-Media with Bacteria 71-78 THE METHOD OF INOCULATING FLUID MEDIA ..... 71 THE METHODS OF INOCULATING SOLID MEDIA 71-75 THE CULTIVATION OF ANAEROBIC BACTERIA: The Incubator and the Thermostat 75-78 CHAPTER V. Sterilization, Disinfection, and Antisepsis ...... 79-87 THE METHODS OF STERILIZATION 79 THE METHODS OF DISINFECTION 84-86 THE METHODS OF ANTISEPSIS: The Common Disinfectants . 86-87 CHAPTER VI. The Inoculation of Animals and their Study 88-94 THE INOCULATION OF ANIMALS : The Various Methods of In- oculation of Animals 88-91 THE OBSERVATION OF THE INOCULATED ANIMAL : The Roux- Nocard Method of Culture and Observation . . 92-94 CONTENTS. 9 CHAPTER VII. PAGES Infection and Immunity 94-103 INFECTION : The Theories of Infection ; The Avenues and Factors of Infection 94-97 IMMUNITY AND ITS VARIETIES: The Methods of Producing Immunity ; The Antitoxic and Antimicrobic Blood- serums ; The Theories of Immunity 97-103 CHAPTER VIII. Antibodies and Opsonins 103-108 ANTIBODIES 103-106 OPSONINS 106-108 CHAPTER IX. The Pathogenic Bacteria 108-117 THE PYOGENIC MICROCOCCI AND ALLIED BACILLI .... 108-109 THE INDIVIDUAL FEATURES OF THE PYOGENIC BACTERIA : Staphylococcus Pyogenes Aureus; Staphylococcus Pyo- genes Albus; Staphylococcus Citreus; Streptococcus Pyogenes ; Micrococcus Tetragenus 109-113 GONORRHCEA : Micrococcus Gonorrhoese (Gonococcus) ; Micro- coccus Catarrhalis ; Bacillus Pyocyaneus ; Pneumococcus or Pneumobacillus ; Bacillus Coli Communis ; Bacillus Typhosus; Bacillus Tuberculosis 113-117 CHAPTER X. The Other Pathogenic Micrococci and Allied Bacilli— Mi- crococcus Pneumoniae, Epidemic Cerebrospinal Men- ingitis, and Malta Fever ... 117-125 PNEUMONIA: Micrococcus Pneumonise Croupos* (Diplococ- cus Pneumonise ; Micrococcus Pasteuri ; Micrococcus of Sputum Septicaemia) ; Streptococcus Mucosus ; Pneumo- coccus of Friedlaender (Bacillus Pneumoniae of Fluegge ; Bacillus Mucosus Capsulatus) ; I'acillus of Rhinoscleroma 117-122 EPIDEMIC C'EREBROSPINAL MENINGITIS: Diplococcos Intra- ct'lluhiris Meningitidis 122-124 MAI/PA OK MEDITERRANEAN FEVER: Micrococcus Melitensis 124-12") 10 CONTENTS. CHAPTEK XL Tuberculosis 126-132 BACILLUS TUBERCULOSIS 126-132 CHAPTER XII. Leprosy and Syphilis 133-136 LEPROSY : Bacillus Leprse 133-135 SYPHILIS: Bacillus Smegmatis (formerly called the Bacillus of Syphilis) ; Streptococcus of Syphilis 135-136 CHAPTER XIII. Glanders (Farcy) 137-141 BACILLUS MALLEI 137-141 CHAPTER XIV. Anthrax 141-146 BACILLUS ANTHRACIS - 141-146 CHAPTER XV. Diphtheria and Pseudodiphtheria 146-159 DIPHTHERIA : Bacillus Diphtherise 146-154 PSEUDODIPHTHERIA : Bacillus Pseudodiphtherise ; The Anti- toxin Treatment of Diphtheria ; Bacillus Xerosis . . . 154-159 CHAPTER XVI. Tetanus, Malignant (Edema, and Symptomatic Anthrax 159-173 TETANUS: Bacillus Tetani ; Anaphylaxis 159-166 MALIGNANT (EDEMA : The Bacillus of Malignant (Edema . 166-167 SYMPTOMATIC ANTHRAX : Bacillus Anthracis Symptomatici ; Bacillus Aerogenes Capsulatus ; Bacillus Botulinus . . . 168-173 CHAPTER XVII. Typhoid Fever 173-183 BACILLUS TYPHOSUS 173-176 DIFFERENTIATION OF BACILLUS TYPHOSUS FROM ALLIED GROUPS 176-179 THE BLOOD-SERUM DIAGNOSIS OF TYPHOID FEVER .... 179-182 VACCINATION AGAINST TYPHOID FEVER 181-183 CONTENTS. 11 CHAPTEK XVIII. PAGES Bacillus Coli Communis 184-187 BACILLUS PROTEUS VULGARIS 186-187 CHAPTER XIX. Asiatic Cholera - 187-193 SPIRILLUM CHOLERA ASIATICS (COMMA BACILLUS) . . . 187-193 CHAPTEE XX. Influenza 193-196 BACILLUS OF INFLUENZA 193-195 PERTUSSIS BACILLUS 195-196 CHAPTEK XXL Bubonic Plague 196-199 BACILLUS PESTIS ... 196-199 CHAPTEK XXII. Dysentery, Hog Cholera, and Chicken Cholera 199-205 DYSENTERY: Bacillus Dysenterise 199-201 HOG CHOLERA : Bacillus sui Pestifer 201-202 CHICKEN CHOLERA : Bacillus Choleras Gallinarum 202-203 MORAX-AXENFELD BACILLUS 203-204 BACILLUS OF DUCREY 204 CHAPTER XXIII. Spirilla 205-213 RELAPSING FEVER : Spirillum Obermeieri 206 • SpiROCH-aETA PALLIDA : Treponema Pallida 207-211 YAWS 211 VINCENT'S ANGINA 211-212 SPIROCH^ETA GALLINARUM 212 CHAPTER XXIV. The Pathogenic Micro-organisms Other than Bacteria . . 213-234 ACTINOMYCOSIS, MALARIA, AND AMO3BTC COLITIS : StreptO- thrix 213-214 ACTINOMYCOSIS: Streptothrix Actinomyces (Ray Fungus); Other Pathogenic Streptoth rices ........... 214-216 12 CONTENTS. PAGES YEASTS . . . • 217 MOULDS 217-219 MALARIA : Plasmodium Malarise 220-229 PROTOZOA FOUND IN BLOOD : Filaria Sanguinis Hominis ; Trypanosomes ; Leishmania Donovan! ; Distomum Hsema- tobium . 229-231 AMCEBIC COLITIS: Amoeba Coli 232-234 CHAPTER XXV. Intestinal Parasites 235-246 ASCARIS. 235-236 OXYURIS 236-237 T^ENIA 237-241 TRICHINA 241-242 HOOK-WORM ..." 242-246 CHAPTEE XXVI. The Unknown Causes of Other Infectious Diseases - 246-249 BABIES 246-248 YELLOW FEVER 248-249 SCARLET FEVER 249 CHAPTER XXVII. Bacteriological Examinations of Water, Air, and Soil . 249-257 BACTERIOLOGICAL INVESTIGATION OF WATER' 249-255 BACTERIOLOGICAL EXAMINATION OF THE AIR 255-256 BACTERIOLOGICAL EXAMINATION OF THE SOIL 256-257 MICROSCOPY AND BACTERIOLOGY. INTRODUCTION. THE REFRACTION OF LIGHT AND THE MICROSCOPE. THE REFRACTION OF LIGHT. Definition. — Refraction is the property possessed by trans- parent media of altering the rays of light which pass through them. It is to this property possessed by lenses, the trans- parent media of microscopes, that these instruments owe their magnifying power. The Two Fundamental Laws of Refraction. I. When a ray of light passes from a denser to a rarer medium, it is refracted away from a line drawn perpendicu- larly to the plane which divides the media ; and vice versa, when the light passes from a rarer to a denser medium, it is refracted toward that perpendicular. II. The sines of the angles of incidence and refraction — that is, of the angles which the ray makes with the perpendicular before and after its refraction — bear to one another a constant ratio for each substance, which is known as its index of refraction. The Principles of Refraction by Lenses. Microscope lenses are chiefly convex ; those of other forms are used to make certain modifications in the rays passing through the convex lens, and so render their performance more exact. 2— M. B. 17 18 INTRODUCTION. Focus of a Lens. — For a lens to give a perfect image of an object, all the rays of light coming from that object and pass- ing through the lens must meet at the same point on the other side of the lens. This point is known as the focal point or focus of the lens. The Spherical Aberration of Lenses. Definition. — It is difficult, however, so to construct a con- vex lens that all the rays of light that pass through it shall come to the same focus. As a rule, the rays which traverse its peripheral or marginal portion come to a shorter focus than those which pass through its more central portion. Correction. — Distortion of the image is thus caused, and is known as spherical aberration. Theoretically, spherical aber- ration might be corrected by making the curvature of the periphery of the lens less than that of its more central por- tion ; but the difficulties in the mechanical construction of such a lens would be very great, and opticians have found it more practical to correct this defect by coupling with a con- vex lens a concave one of less curvature, but which is subject to exactly the opposite error of refraction. Doublets. — These combinations of convex and concave lenses, or doublets, act as a single convex lens. Triplets. — Sometimes two convex and one concave lens are used in combination, and are called triplets. The Chromatic Aberration of Lenses. Definition. — When light traverses a convex lens, the differ- ent colors which compose it do not all come to the same focus — that is, the colors are of unequal refrangibility ; and the image then is seen chiefly in that color which chances to be in focus. This color-distortion is especially noticeable with the marginal rays, and is known as chromatic aberration. Correction. — Though exclusion of the marginal rays can, as with spherical aberration, partly correct this defect, yet this is not sufficient, and chromatic aberration is remedied best by constructing the convex lenses of the combination THE MICROSCOPE. 19 mentioned above of crown glass, and the concave lenses of flint glass, as those two kinds of glass have opposite proper- ties with regard to refrangibility. THE MICROSCOPE. Microscopes are of two kinds : simple and compound : The Simple Microscope. The ordinary hand magnifying-glass and the dissecting microscope are examples of simple microscopes. Their magnifying power depends upon one lens or several lenses acting as one double convex lens. To obtain a clear, enlarged image of the object, the latter must be in its princi- pal focus, and the shorter the focus of the lens the greater its magnifying power. The focal length, or focus, of the lens depends on the degree of curvature of the lens. In expressing the magnifying power of lenses, the size of an object as seen by the unaided eye at ten inches distance is taken as unity. A lens having a magnifying power of ten diameters, or linears, is one which enlarges the object ten times in each linear direction. The Compound Microscope. The compound, or ordinary, microscope consists of the stand and lenses. The stand comprises the following parts : 1 . Base or foot ; 2. Pillars, or upright, which may be jointed or not ; 3. Arm connecting the pillars with the — 4. Body, containing the — 5. Draw-tube ; moved up and down rapidly or slowly by means of the — 6. Coarse adjustment — used to bring the object into view ; 7. Fine adjustment — used only when the object is already in view, to bring out more clearly its details. 20 INTRODUCTION. 8. Stage — the flat part on which is laid . the object to be examined, and which is perforated by a central hole to allow illumination of the object from below. The stage may be circular or square, stationary or movable, and mechanical. Underneath the Stage are Found the Parts: 9. Flat mirror for low-power, and when Abbe condenser is used. 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, \ inch. In continental Europe the numerator of the fraction is often omitted, the ^ objective being called 3, and the fy 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 LEXS. 23 All recent microscopes have the equivalent focal lengths of the objectives marked in the metric system, which is prefer- able. Thus the ^ inch will be represented by the 16 m.m., the J inch by the 4 m.m., the T^ inch by the 1.9 m.m. Th'e 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 24 INTRODUCTION. 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 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 THE TYPES OF OCULAR LENS. 25 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. 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 26 INTRODUCTION. 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 described in the preceding paragraph, and if this is not sufficient, pass a little water over the surface and wipe dry. If the soiling is due to balsam or other resinous sub- stance, clean gently with benzole or xylol. The objective system of lenses is held together by a shellac, soluble in alco- hol, and all instrument-makers caution against the use of alcohol on any part of the microscope. The back surface of the objective need never get dirty ; but when it does, insert- ing a soft rag into the objective and gently turning it around is sufficient to cleanse it. Never screw apart the different lenses of the objective, as it takes an expert optician *o 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 ^ mch 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. A dark field illuminator, or condenser for dark ground illum- ination, consists of a condenser in which the central portion is darkened, and the rays of light, being admitted from the circumference, are reflected to the centre by means of a mir- rored edge. This arrangement produces a lateral illumina- tion of the object against a black or grayish background. To use this special condenser the ordinary Abbe" condenser must be removed and the dark field illuminator substituted. A THE TYPES OF OCULAR LENS. 27 drop of oil of cedar must be placed on top of the condenser, and the slide, which must be as thin as possible, placed on it. The cover-glass used should also be very thin. Either a 4 m.m. or an oil immersion objective may be used. For this form of illumination an intense light is needed, either direct sunlight or some form of improved electric lamp being best. 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 ? 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 ? 28 THE FUNDAMENTAL PRINCIPLES. 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. Describe a condenser for dark field illumination. CHAPTER I. THE FUNDAMENTAL PEINCIPLES. THE HISTORY OF BACTERIOLOGY. WHEN in the latter part of the seventeenth century Anthony von Leuwenhoek, by means of his magnifying- glasses, first discovered organisms in decaying vegetable infusions, he may be said to have laid the very first stone in the foundation of what later on was to be the Science of Bacteriology. It was very long after this, however, before sufficient facts were collected to place this science upon a firm basis, and it remained for a genius like the immortal Pasteur and the eminent talents of the equally great Koch to build up the superstructure of bacteriology so as to have it accepted by all as the true basis of scientific medicine. When first observed, these microorganisms were supposed 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 THE DEFINITION OF "BACTERIA." 29 no chlorophyl, and are unable, like other vegetables, to obtain their nourishment from the carbon dioxide and nitrogen of the atmosphere, on the contrary, like animals, requires higher carbohydrate and nitrogenous substances, which they decom- pose into their primitive elements for their subsistence. A few of them possess some plant coloring-matter, and some seem able to thrive in a simple saline solution from which ab- solutely 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- 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. Closely allied to the Bacteria are the Protozoa (singular, Protozoon). They are also unicellular in form, but on account of metabolic considerations are classed in the animal kingdom. The differences between some of them and bacteria are, in the light of our present knowledge, academic, and a work on bacteriology may well take in its scope the consideration of some protozoa. 30 THE FUNDAMENTAL PRINCIPLES. 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 sarcinas ; 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. III. The Spirillum. The spirilla or curved or spiral bacteria develop either singly or in pairs or in long twisted or corkscrew filaments. MORPHOLOGICAL CLASSIFICATION OF BACTERIA. ;U 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 inn in divisions are permanent — that is, micrococci always develop into micrococci, bacilli into bacilli, and spirilla into spirilla. FIG. 2. e d e a Staphylococci b. Streptococci, c. Diplococci. d. Tetrads, e. Sarcinae. (Abbott.) FIG. 3. Diplococcus of pneumonia, with surrounding capsule. (Park.) Occasionally under peculiar conditions what are known as involution-forms are produced, forms which may scarcely be 32 THE FUNDAMENTAL PRINCIPLES. FIG. 4. a. Bacilli in pairs. 5. Single bacilli, c and d. Bacilli in threads, e and /. Bacilli of variable morphology. (Abbott.) FIG. a and'rf. Spirilla in short segments and longer threads— the so-called comma forms and spirals. &. The forms known as spirochaeta. c. The thick spirals some- times known as vibrios. (Abbott.) FIG. "'•if a. Spirillum of Asiatic cholera (comma bacillus) ; normal appearance in fresh cult- ures, b. Involution-forms of this organism as seen in old cultures. (Abbott.) recognized as those belonging to the original bacteria. These points are shown by Figs. 2, 3, 4, 5, 6. THE REPRODUCTION OF BACTERIA. 33 THE SIZE OF BACTERIA. Bacteria require to be seen and studied by the highest powers of the microscope ; they vary in size from 0.2 to 30 mikrons. (A mikron isy^Q-o millimeter; about ^Wo incn-) The micro- cocci have a diameter of from 0.2 to 1 mikron or more. Bacilli and spirilla vary in length from 2 to 30 mikrons or more ; in breadth, from 1 to 4 mikrons. The average length of pathogenic bacilli is 3 mikrons. THE REPRODUCTION OF BACTERIA. As mentioned in the foregoing paragraphs, bacteria multi- ply by fission. I. Fission. In the case of cocci, the round or oval cells show a little indentation beginning in the membrane at two, four, or eight points of its periphery, according as the division is to occur in two, four, or eight parts ; this indentation increases until the original cell is divided into hemispheres, quadrants, or octants, as the case may be. These parts remain attached to one another until complete spheric cocci are formed from each part, and they then separate or not according to the nature of the bacteria. In some forms of diplococci, as the gonococci, complete spheres are never formed, the cells remaining attached to each other in pairs as hemispheres. In the case of nearly all bacilli and spirilla the cells increase to nearly double their original size before division, and the division always takes place in the direction of the length of the bacterium. The daughter cell remains attached for a while to its parent cell after fission is complete ; occasionally this attachment persists for a long time, so that large filaments consisting of a number of bacteria are formed. II. Sporulation. 1. The Endospore. Division by fission is the usual mode of the reproduction of bacteria, but at times, depending upon various circum- 3— M. B. 34 THE FUNDAMENTAL PRINCIPLES. stances to be mentioned later, what are known as spores are formed by a number of bacilli and spirilla. These spores, which are perhaps the equivalents of seeds for the higher plants, are formed in this way : in the body of the bacillus, generally at its centre, occasionally at one of its poles, a number of dark highly refractile granules accumulate, and are soon changed into an oval, glistening, highly refractile body which is surrounded by a membrane of the same com- position as that of the cell itself, but thicker and more resistant. One spore only is formed in each cell. Some- times the spore-formation causes no change in the shape of the rod. At other times there is a bulging of the centre of the body of the bacillus, where the spore is located, with a general tapering to the two ends, giving to the bacillus the shape of a spindle. This is called a clostridium. Again, when the spore is formed at one of the poles, there is some- times a bulging of that part, giving to the bacillus the appearance of a nail or drum-stick, whence the name of drum- mer-bacillus for the cell. Fig. 7 shows these forms well. FIG. 7. a. Bacillus subtilis with spores, b. Bacillus anthrads with spores, c. Clostridiumform with spores, d. Bacillus of tetanus with endospores. (Abbott.) Soon after the formation of the spore under proper favor- able conditions the rest of the body of the cell disintegrates and breaks down, and the oval spore is liberated. The spores are characterized, on account of their thick membrane, by resistance to external influences which would be fatal to the bacilli themselves, such, for instance, as extremes of heat or cold, desiccation, and the action of chemicals; also resisting staining by the penetration into THE REPRODUCTION OF BACTERIA. 35 their body of certain dyes which have great affinity for the protoplasm of ordinary bacteria, so that a special method must be adopted for their staining, as will be described later. Spores therefore preserve the species, when these would be destroyed if dependent solely on the bacilli for their preser- vation. 2. The Arthrospore. The foregoing spore-formation, known as endospores, is the usual mode of spore-formation found in bacteria, and is lim- ited to the rod and spirilla forms ; but another form of spore, called arthrospore, is mentioned by some as occurring occasionally in the round or cocci forms. This consists in a special jointed projection forming from the outside of the cells, and capable later of developing into the original cell. This form of spore-formation is generally doubted at the present time. Spores are incapable of producing other spores, and can, only when placed in suitable conditions, develop into the type of bacilli which gave them birth. When this occurs the spore begins to elongate, loses its glistening appearance, and finally its membrane ruptures at one end or in the centre and gives exit to a fully developed bacillus. Spores may be distinguished from rounded bacteria by means of their brighter, more glistening appearance, their power of resisting stains, and the fact that in suitable media they develop into bacilli. Significance of Sporulation. — Whether the original idea that spores form, in bacteria capable of producing them, only when the latter are submitted to external noxious influences, and for the purpose of perpetuating the species, or whether, as more recently maintained, sporulation is the result of the highest expression of the complete development of bacteria, is not at present fully determined, though the dictum of authorities inclines to the latter view, and the spore-formation of some of the best-known and studied bacteria, as anthrax, seems to lend color to this theorv. 36 THE FUNDAMENTAL PRINCIPLES. THE MOTILITY OF BACTERIA. Motility, or the power of transporting themselves from place to place, is possessed by a number of bacteria. This is effected by filamentous or hair-like processes arising from the body of the bacteria, and called flagella. It must not be confounded with the peculiar whirling or dancing move- ment so often seen under the microscope, even in inorganic particles, and called the Brownian movements. Motility, except in two instances, has so far been observed only in bacilli and spirilla. Its rapidity depends on the particular bacterium, its mode of cultivation, the age of the culture, and other similar factors. Some bacteria after repeated arti- ficial cultivation seem to lose their motility, which, however, may be restored fully by passing through an animal. FIG. 8. a. Spiral forms with a flagellum at only one end. 6. Bacillus of typhoid fever with flagella given off from all sides, c. Large spirals from stagnant water with wisps of flagella at their ends (Spirillum undula). (Abbott.) The flagella are hair-like processes consisting of the same material as the bacterial cell-membrane, and are so minute as scarcely to be visible under the highest power of the micro- scope unless they are stained by special processes, as will be described in the chapter on staining. Whenever there is only a single flagellum at one of the poles of the bacillus, this is said to be monotrocha ; whenever there is a single flagellum at each pole of the bacterium, it is said to be amphitrocha ; whenever there is a cluster of flagella at one pole, the bac- terium is said to be lophotrocha ; and finally, when a varying number of flagella seem to arise from different portions of the body of the bacterium, it is said to be peritrocha. These features are shown in Fig. 8. ESSENTIAL CONDITIONS OF BACTERIAL GROWTH. 37 THE RELATION OF OXYGEN TO BACTERIAL LIFE. Bacteria are divided into aerobic and anaerobic according as they require oxygen or not for their development. Some thriving best in the presence of oxygen are able to develop, however, without the presence of this gas ; these are called facultative anaerobic. Others thriving best without oxygen but able to develop in the presence of this gas are called facultative aerobic. THE RELATION OF DEAD AND LIVING ORGANIC MATTER TO BACTERIA. Bacteria are also divided into saprophytes and parasites, according as they require for their development merely the presence of decomposable organic matter or the body of a living higher organism, as host, on which they live. Should they also be able to live outside their host, they are called facultative parasites. The disease-producing germs are always parasitic. THE ESSENTIAL CONDITIONS OF BACTERIAL GROWTH. For the proper development of bacteria the following are required : heat, moisture, and the presence of some decom- posable organic matter, with special chemical reaction of the culture-medium. I. Heat. A temperature between 10° and 40° C. is required for the development of adult bacteria, but the degree varies greatly according to the different species. Some of the parasites thrive best at a temperature in the neighborhood of that of the human body, about 36° or 37° 0. ; others, the saprophytes, seem to grow best at the ordinary room temperature, between 20° and 30° C. ; some few again seem to be able to grow and multiply at a temperature near the freezing-point ; and not long ago a number have boon shown to develop abun- dantly at a temperature above 70° C. As a general rule, however, a temperature of 0° C. and one above 60° C. are fatal 38 THE FUNDAMENTAL PRINCIPLES. within a few minutes for the ordinary non- spore-bearing bacteria. Spores are able to resist for a long time the effects of cold and excessive heat. Some have developed after having been im- mersed for a long time in liquid air (temperature, — 200° C.j, and also after exposure to dry heat of above 150° F. for sixty minutes and moist heat for thirty or forty minutes. II. Moisture. A certain amount of moisture is absolutely indispensable for the growth of bacteria, desiccation being fatal within a few minutes for nearly all the fully formed bacteria. Spores, however, are capable of developing after being kept dry for an indefinite period. III. Decomposable Organic Material. A certain amount of decomposable organic matter is indis- pensable for the development of the bacteria. This they decompose into simple elements, and are so able to obtain the nitrogen and carbon necessary for their sustenance. At the same time they set free carbon dioxide and nitrogen from the remainder, and so provide for the nourishment of the higher vegetables, which must have these substances free in order to support life. And in so doing bacteria render a service of incalculable value, as without- it life in the animal kingdom would soon be extinct. Different bacteria require a greater or lesser proportion of the proteid substances for their nutrition ; and according as substances contain these in a more or less favorable condition for absorption they are said to be more or less good culture- soils or media. Some few species seem to be able to live in saline solution and in other media where there is no appreciable amount of organic matter, but in these cases they probably obtain their nutrition from the decomposition of slight traces of ammonia and the carbon dioxide of the air contained in the water. Again, some bacteria seem to have the power of decomposing VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA. 39 ammonia and building up nitrite and nitrate compounds ; these are known as the nitrifying bacteria, and as a class are im- portant, and are being carefully studied. IV. Special Chemical Reaction of the Culture-medium. Bacteria are likewise profoundly influenced in their growth by the reaction of the medium in which they grow, most bac- teria requiring a neutral or faintly alkaline medium, some few a faintly acid one. This characteristic is found an element of danger for bacterial life : for the bacteria which require alkaline surroundings have, as a rule, the property of secret- ing acids, so that after a while they render the medium unfit for their own further growth long before the pabulum is exhausted. THE INERT AND INHIBITIVE CONDITIONS OF BACTERIAL LIFE. 1. Diffuse daylight seems to have little or no influence on bacterial growth, but most bacteria and their spores are killed after a more or less prolonged exposure to the direct rays of the sun, a fact of great importance in practical hygiene. 2. Electric currents and the X-rays seem to have but little influence on bacterial growth. 3. Compressed air seems to retard the growth of bacteria. 4. A number of chemical substances either kill off bacteria or arrest their growth — as will be spoken of more fully under the head of Antiseptics and Disinfectants. THE VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA. These are manifold and various, and occasionally attempts at classification have been based on some of these special functions. 1. Fermentation. — The alcoholic and acetic acid fermentation are the work of the yeast fungi ; but the butyric and lactic acid fermentation in milk are caused by a number of bacteria, 40 THE FUNDAMENTAL PRINCIPLES. the most prominent of which are those that bear the respec- tive names. This class of bacteria are called the zymogenic. 2. Putrefaction is caused by a variety of bacteria called saprogenic. 3. Pigments and colors are produced by a number of bacteria called chromogenic. Sometimes the pigment is secreted by the cells and diffused in the surrounding media, the bacteria remaining uncolored. At other times the pigment is limited to the cell-protoplasm and membrane, the surrounding medium remaining uncolored. As a rule, these pigments are produced only in an atmosphere of oxygen. 4. Some bacteria, called photogenic, phosphorescent, or fluorescent, have the property of emitting and producing light. 5. Bacteria secrete poisonous substances which are some- times highly deadly, and which from their character are classed as ptomaines and toxalbumins. The ptomaines are crystallizable basic substances, closely allied to the vegetable alkaloids. The toxalbumins are non-crystallizable substances, similar to albumin or protein. It is by the poisonous effects of these toxins that most bacteria affect the human body. 6. Some bacteria liquefy gelatin, and this fact is made use of in differentiating the different species. This liquefaction has been demonstrated to be caused by a soluble peptonizing ferment secreted by the bacteria cells, and after filtration of a bouillon culture of the liquefying bacterium the filtrate free from bacteria possesses the same power of liquefying gelatin. 7. Some bacteria produce acids and other alkalies — as may be demonstrated by their action in certain fluid media. 8. Various gases, such as carbon dioxide, marsh gas, hydro- gen sulphide, etc., are the products in some instances of bacterial growth. 9. A number of bacteria produce odors without the apparent production of gases. 10. A few produce aromatics, and are used extensively in the arts. 11. Again, a certain class peptonize milk by means of VITAL MANIFESTATIONS OR FUNCTIONS OF BACTERIA. 41 enzymes which they secrete. Some cause a reduction of the nitrites. 12. Others oxidize nitrogen into nitrites, and even nitrates. 13. Finally, some bacteria cause disease in man and animals. These, called pathogenic, are of the greatest interest to physicians, and it is the discovery of this pathogenic prop- erty which in the last twenty years has given such an impetus to the study of bacteriology. QUESTIONS. Who first discovered microorganisms in decaying vegetable infusions? To what kingdom were they thought to belong at first? What classification is generally accepted at present? Name the three varieties of fungi. What criticism may be made of this classification ? Give a proper definition of bacteria. What is a protozoon (plural protozoa) ? Of what does the bacteria cell consist ? What causes some bacteria to have a capsule ? Are the capsules generally retained by bacteria when grown artificially ? What causes some bacteria to be more difficult of staining than others? Into what three varieties or species are bacteria divided ? What are megacocci ? Micrococci ? Diplococci ? Merismopedia ? Sarcinse ? Streptococci ? Staphylococci ? Ascococci ? What is the shape of bacilli? When are they said to be slender? When thick ? How do they develop ? What are spirilla ? Can one species develop into another species ? What are the limits of size of bacteria ? What is a mikron ? What is the average length of the pathogenic bacteria ? How do bacteria multiply ? Describe this process in the case of cocci, bacilli, and spirilla ? What are spores, and how are they formed ? How do spores compare with bacteria in their power of resisting injurious external influences and of staining ? To what is this due ? What is meant by a clostridium ? What is meant by a drummer-bacillus? What is the name of the ordinary spores? What is meant by arthrospores ? What is the theory of spore-formation ? What is meant by motility, and what species have this power ? What are flagella ? What names are given to bacteria according to the position and number of their flagella? What is meant by aerobic bacteria? Anaerobic? Facultative aerobic? Facultative anaerobic? What are saprophytes? Parasites? What conditions are required for the proper development of bacteria? What is the. most favorable temperature for bacterial growth ? What is the, elfect of cold ? Of excessive heat ? How do spores differ from bacteria in their reaction to heat and cold ? 42 EXAMINATION AND STAINING OF BACTERIA. What is the effect of moisture on bacterial life ? Of desiccation ? What pabulum is necessary for the life of bacteria ? How do you explain the life of bacteria in saline solution and in media with no appreciable amount of organic matter ? What is meant by culture-soils or media ? What other conditions influence bacterial growth ? What is the effect of sun-light on bacteria ? Of electric currents ? X-rays ? Of compressed air? What are the manifold functions of bacteria ? Enumerate and explain the same. What is the chemical difference between a ptomaine and a toxalbumin ? What are zymogenic? Chromogenic? Photogenic? Pathogenic bacteria? CHAPTER II. THE EXAMINATION AND THE STAINING OF BACTEKIA. THE EXAMINATION OF BACTERIA. FOR the purpose of examining bacteria the highest power of the microscope is necessary, although many are seen with an ordinary dry J or ^ objective. Ordinarily, however, the 11f oil-immersion objective is indispensable for the proper study of microorganisms. Bacteria are examined either alive and in their natural con- dition, or dried on microscope slides or cover-glasses as a thin film, and stained. 1. For the purpose of examining bacteria in their natural condition, it is only necessary, when the bacteria are in liquid medium, to put a droplet of the liquid on a slide, cover lightly with a thin cover-glass, put upon the stage of the microscope, and bring into focus the y1^ inch oil-immersion objective, being careful to close almost completely the iris diaphragm underneath the substage condenser. When the bacteria are in solid medium, a minute particle of the culture is taken up on a sterilized platinum needle and stirred up in a small drop of sterilized water on a slide, and a cover-glass applied as above. In these ways the form, shape, mode of grouping, and motility THE STAINING OF UACTERIA. 1:> of the bacteria may be very well seen and carefully in- vestigated. The Hanging-drop Preparation. When it is necessary to keep them under observation for some time, however, or when it is desired to study their development, multiplication, or sporulation, what is known as the hanging-drop preparation or culture is resorted to. This consists in placing a small drop of the liquid contain- ing the bacteria on a thin cover-glass and placing upon this cover-glass a slide with a concavity in its centre, known as the hanging-drop slide, after having carefully lubricated the edges of the surface around the concavity with vaselin, so that the slide will adhere to the cover-glass when it is pressed down on it. In this way a hermetically sealed, transparent, moist chamber is obtained, which may be kept under observa- tion on the stage of a microscope almost indefinitely. FIG. 9. Longitudinal section of hollow-prronml glass slide for observing bacteria in hanging drops. (Abbott.) 2. The foregoing are very simple and useful methods for rapid examinations of bacteria, and have many applications, but they are far from satisfactory in all cases, as they fail to bring out full details of bacterial structure. For this purpose recourse must be had to the staining methods introduced by Koch, and perfected by Weigert, Loeffler, and many others. In this method bacteria are examined dead. THE STAINING OF BACTERIA. I. The General Mode of Procedure. One or several droplets of the suspected liquid are spread thinly and cren/i/ on the surface of a slide or thin cover-glass, 44 EXAMINATION AND STAINING OF BACTERIA. or, in case of a solid, a small particle is diluted with sterile water and spread in the same way on the slide or cover-glass. This is allowed to dry in the air, protected from dust, or else is held in a suitable forceps high up over the flame of an alcohol lamp or Bunsen gas-burner until dry, thus forming a thin film on the surface of the glass. After this step has been carefully taken the slide or cover-glass is taken up with a pair of forceps and passed several times through the flame, film-side up, for one-half to one second at each pass, three times in the case of a cover-glass and eight or ten passes in the case of a slide; which is for the purpose of setting the prepa- ration, or coagulating the albuminoids and fixing them to the glass so that they will not be easily washed away in the sub- sequent procedures. After allowing the preparation to cool well, it is ready for the staining reagents. II. The Most Commonly Used Stains. The basic anilin dyes, such as fuchsin, methylene-blue, gentian-violet or methyl-violet, and Bismarck-brown, are most commonly employed. They are made into saturated alcoholic solutions, to be kept in stock, and are freely diluted with water whenever required for use. III. The Application of the Dyes. Nearly all the known bacteria, with the exceptions to be mentioned later, are readily stained by the watery solutions of any of the basic anilin dyes. The film on the slide or cover-glass, prepared as just described, is covered by a few drops of the stain, or the cover glass, film-side down, is floated in a watch-glass full of the staining solution ; at the end of from one-half to two or three minutes the staining fluid is poured oif, the slide or cover-glass washed rapidly in water and then allowed to air-dry; after which, in the case of cover-glass preparations, they are inverted upon a drop of Canada balsam on a slide and examined with the oil-immer- sion lens ; or, when slides have been prepared, after wash- THE STAINING OF BACTERIA. 45 ing and drying a drop of cedar oil is put over the prepa- ration, and the same is examined with the oil-immersion objective without the use of a cover-glass. Air-bubbles are often caught between the balsam and the cover-glass. Gentle uniform pressure begun at the centre of the cover-glass and progressively applied toward its periphery will ordinarily remove them. If this should fail, heat care- fully applied until the balsam is quite soft will aid in the riddance of the others. IV. The Special Methods of Staining. As stated above, this method may be applied for the stain- ing of nearly all bacteria. Some, however, are not so easily stained, and special methods must be resorted to to increase the penetrating power of the dye. The most commonly used will be here described. 1. Loeffler's Method. In this method, instead of using the ordinary watery solu- tion of an anilin dye, Loeffler's alkaline solution of methylene- blue is used. This is prepared as follows : Concentrated alcoholic solution of methylene-blue, 30 parts; Caustic potash solution (1:10,000), 100 « . Mix well and filter. This method stains well all the ordinary bacteria, but is specially useful for the staining of the bacillus of diphtheria. 2. Koch-Ehrlich's Method. Anilin water is prepared by adding a few drops of anilin oil, drop by drop, to distilled water in a test-tube, shaking well after the addition of each drop and until the liquid assumes a milky appearance, after which it is filtered through moistened filter-paper until the filtrate is absolutely clear. To 100 parts of this clear filtrate of anilin water, 10 parts 46 EXAMINATION AND STAINING OF BACTERIA. of absolute alcohol and 10 parts of an alcoholic solution of fuchsin, methylene-blue, or gentian-violet, are added, and the whole thoroughly mixed and filtered. The preparation is better when made fresh in small quantity at each time it is needed, as it decomposes in a few days. In Koch-Ehrlich's method this anilin water (violet, blue, or red) is used instead of the simple watery solution of the dye. It possesses much more penetrating power, and this again may be increased by gently heating the slide or cover- glass, over a Bunsen burner, while it is being stained. It is applicable whenever it is desirable so to fix the color in the bacteria that one may by means of decolorizing agents remove the color from the surrounding objects and tissue and fix it solely on the bacteria themselves. Some bacteria so retain the color by this method that it serves to distinguish them from others which they very much resemble, but which do not possess the same persistent retention of the stain. The usual decolorizing agent employed is either ordinary alcohol or diluted sulphuric or hydrochloric acid (1 : 4). 3. Gram's Method. Treat the object to be colored with anilin-water gentian- violet for about three minutes, after which immerse in Gram's fluid. This consists of: Iodine, 1 part; Iodide of potassium, 2 parts ; Water, 300 " . Maintain this immersion for five minutes, then pass the preparation through alcohol and rinse in water. If the object is still of a violet color, treat it again with Gram's fluid and alcohol until no violet is visible to the naked eye. This method is applicable to a number of bacteria, and serves as a mode of differentiation between some of them which could not otherwise be distinguished under the microscope. It serves also to color the capsule of bacteria, and, slightly modified, is useful for the stain of bacteria in tissue. A contrast-color should be given to the uncolored parts. THE STAINING OF BACTERIA. 47 4. Ziehl's Carbol-fuchsin Method. Make a solution of carbol-fuchsin as follows : Fuchsin, 1 part; Crystallized carbolic acid, 5 parts ; Alcohol, 10 " j Water, 100 " . Immerse the glass in or cover same with the carbol-fuchsin solution, heat gently over the flame of a Bunsen burner, gradu- ally bringing to a point just below boiling; repeat this two or three times ; after which immerse in nitric acid solution (1 part of acid to 3 parts of water) until the color is scarcely visible to the naked eye. To ascertain this, wash off the acid from the film with water. If color is still faintly visible, remove it by dipping into alcohol ; wash in water, dry, mount in Canada balsam, and examine. A contrast-color may be given to the rest of the specimen by employing methylene-blue. This is a useful method in coloring cover-glass preparations for tubercle bacilli or for the Bacillus leprae. 5. Gabbett's Method. This is a modification of Ziehl's method, and is perhaps the best method, on account of its simplicity and rapidity, for the staining of the tubercle bacillus in secretions. It is as fol- lows : Prepare a slide or cover-glass film as indicated, im- merse in Ziehl's carbol-fuchsin solution for ten minutes, remove to Gabbett's sulphuric acid methylene-blue solution for three to five minutes, rinse in water, dry, mount, examine. The tubercle bacilli are colored red and the other bacteria and cell- nuclei are colored blue. Gabbett's solution consists of : Methylene-blue, 1 to 2 parts; Sulphuric acid, 25 " ; Water, 75 " . 48 EXAMINATION AND STAINING OF BACTERIA. Besides the foregoing means, which will stain the bacteria in films, bacteriologists adopt special methods for the staining of bacteria in ^tissues, and for the staining of spores, flagella, and the capsules of bacteria. V. The Staining of Capsules. 1. Welch's Glacial Acetic Acid Method. Prepare the cover-glass in the usual way. Cover the film with glacial acetic acid, pour off the acid immediately, do not wash in water, cover the film with anilin- water gentian- violet solution for three or four minutes, wash in a 0.5 to 2 per cent, solution of sodium chloride, dry, mount, and examine. The acetic acid coagulates the mucin of the capsules and renders the same distinctly visible. 2. Johne's Method. A cover-glass film prepared in the usual way is covered with a solution of gentian-violet and heated until steam rises. The stain is then washed off in water, and the cover-glass put into a 2 per cent, acetic acid solution for from ten to fifteen seconds. It is again washed in water, dried, and mounted in balsam. VI. The Staining of Spores. Though with the ordinary method of staining, spores in bacteria may be recognized by their highly refractive appear- ance and by the fact that they have not taken the color, they may be stained themselves, however, by special methods. The First Method (Abbott's). A cover-glass preparation is covered with Loeffler's alkaline methylene-blue solution and held by its edge with forceps over the Bunsen burner flame until the fluid begins to boil. It is then removed from the flame, and after a few seconds heated again. This step is repeated a number of times for one or two minutes, after which it is washed in water, and decolor- THE STAINING OF BACTERIA. 49 ized, until all blue coloring visible to the naked eye has dis- appeared, in the following solution : Alcohol (80 per cent.), 98 parts ; Nitric acid, 2 " . The cover-glass is then dipped for a few seconds into the fol- lowing solution : Saturated alcoholic solution of eosin, 10 parts ; Water, 90 '" . After this it is again rinsed in water, dried, and mounted. The Second Method. Float a cover-glass preparation film-side down in a watch- glass full of Koch-Ehrlich's fuchsin solution. Take the watch- glass by its edge with a pair of forceps and hold same over a low Bunsen flame until the staining fluid begins to boil. Remove from the burner, and after a few minutes repeat this process five or six times. After cooling, the cover-glass, without washing in water, is transferred to a second watch- glass containing a decolorizing solution as follows : Absolute alcohol, 100 parts ; Hydrochloric acid, 3 " Place the cover-glass, film-side up, at the bottom of this watch-glass and let it remain for one or two minutes. Remove, wash in water, stain with methylene-blue solution for one or two minutes, wash rapidly in water, dry, and mount. By this method the spores will be stained red and the body of the bacteria cells blue. The Third Method. Cover-glasses are prepared in the usual way. After fix- ing, the preparation is immersed in chloroform for two min- utes, washed in water, placed for one or two minutes in a 5 4— M. B. 50 EXAMINATION AND STAINING OF BACTERIA. per cent, solution of chromic acid, again washed in water, and stained in hot Ziehl's carbol-fuchsin solution for five minutes. The staining fluid is poured off and, without washing in water, the preparation is decolorized in 5 per cent, sulphuric acid. After this it is again washed in water, and finally stained for two or three minutes in the watery methylene-blue solution. The spores will be stained red, the body of the cells blue. The action of the chloroform is to dissolve the fatty crys- tals that may be in the preparation. The chromic acid acts on the membrane of the spores and permits the entrance of the stain. The Fourth Method (Fiocca's). Ten to 20 parts of an alkaline solution of an anilin color are added to 20 c.c. of a 10 per cent, ammonia solution in a watch-glass ; then heat is applied until steam commences to be given off. The cover-glass stays in this hot solution from three to five minutes ; it is then taken out and washed in a 20 per cent, solution of nitric or sulphuric acid to decolorize it, then washed again, when a contrast-color may be given. It must be remembered that there is considerable difference in the behavior of spores of different bacteria to the staining methods described; some stain very readily and others with considerable difficulty. Practice will be the best guide as to which method is best to employ and to what extent it should be carried out. VII. The Staining of Flagella. The hair-like processes of the bacteria which serve for their locomotion, the flagella, can not, on account of their fineness, be seen in any stained specimen, nor can they be stained by any of the ordinary methods just described for staining bacteria. In order to make them visible, it is neces- sary to use special stains in which the action of mordants plays an essential part. 1. Loeffler's Method. This is the most common method, and is as follows : Clean very carefully a thin cover-glass, and spread very thinly and THE STAINING OF BACTERIA. 51 evenly upon it as few as possible of the bacteria to be exam- ined. This is done by diluting with sterilized water a number of times the culture containing them. The cover-glass is dried and fixed in the ordinary way. The following solution, known as a mordant, is then applied : Tannic acid (20 per cent, solution in water, filtered), 10 parts; Cold saturated solution of ferrous sulphate, filtered, 5 " ; Saturated alcoholic solution of fuchsin, 1 part. A few drops of it are placed on the film, and the cover-glass taken up with a pair of forceps and held over the flame of a Bunsen burner until the solution begins to steam, but not allowing the boiling-point to be reached. It is next washed rapidly in water, and then in absolute alcohol. The bacteria are to be stained in anilin- water fuchsin solution in the ordi- nary way. Practice has shown, however, that different bacteria behave differently when exposed to this staining, and Locffler 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 agUis, Bacillus typhosus, Bacillus subtilisj bacillus of malignant oedema, Bacillus anthracis symptomatic •/. 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 52 EXAMINATION AND STAINING OF BACTERIA. to the mordant, and those which produce alkalies require the addition of an acid. 2. Bunge's Method. Bunge's method, a modification of Loeffler's method, is as follows : Prepare a saturated solution of tannin in water, and also a 5 per cent, solution of sesquichloride of iron in distilled water ; to 3 parts of the tannin solution add 1 part of the iron solution. To 10 parts of such a mixture add 1 part of concentrated watery solution of fuchsin. This mordant should never be used fresh, but only after it has been exposed to the air for several days. The cover-glass, thoroughly cleaned, is covered over by this mordant for five minutes, after which it is slightly warmed. It is then washed, dried, and stained faintly with a little carbol-fuchsin. 3. Pitfield's Method. The following solution is used as a mordant : Tannic acid (10 per cent, solution, filtered), 10 parts; Corrosive sublimate (saturated aque- ous solution), 5 " ; Alum (saturated aqueous solution), 5 " ; Carbol-fuchsin, 5 " Let this stand, and pour off the clear fluid. This mordant will keep for one or two weeks. The staining fluid is prepared as follows : Alum (saturated aqueous solution), 10 parts; Gentian-violet (saturated alcoholic solution), 2 " . This stain is to be prepared fresh every second or third day. The modus operandi is as follows : On an absolutely clean cover-glass make a thin film as already described. Treat the film with the mordant applied cold for twenty-four hours, or THE STAINING OF BACTERIA. 53 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, Bowhill's, 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 54 EXAMINATION AND STAINING OF BACTERIA. 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 either oil of turpen- tine or thyme for two minutes, then in xylol, and mounted in xylol balsam. THE STAINING OF BACTERIA. 55 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. 6. Revised Directions for Making and Using the Wright Blood-stain. To a 0.5 per cent, aqueous solution of sodium bicarbonate add methylene-blue (B. X. or "medicinally pure") in the proportion of 1 gm. of the dye to each 100 c.c. of the solu- tion. Heat the mixture in a steam sterilizer at 100° C. for one full hour, counting the time after the sterilizer has be- come thoroughly heated. The mixture is to be contained in a flask or flasks of such size and shape that it forms a layer not more than 6 cm. deep. After heating, allow the mixture to cool, placing the flask in cold water if desired, and then filter it to remove the precipitate which has formed in it. It should, when cold, have a deep purple-red color when viewed in a thin layer by transmitted yellowish artificial light. It does not show this color while it is warm. To each 100 c.c. of the filtered mixture add 500 c.c. of a 0.1 per cent, aqueous solution of "yellowish, water-soluble " eosin, and mix thoroughly. Collect the abundant precipitate which immediately appears on the filter. When the pre- cipitate is dry, dissolve it in methylic alcohol (Merck's "reagent") in the proportion of 0.1 gm. to 60 c.c. of the alcohol. In order to facilitate solution, the precipitate is to be rubbed up with the alcohol in a porcelain dish or mortar with a spatula or pestle. This alcoholic solution of the pre- cipitate is the staining fluid. It should be kept in a well- 56 EXAMINATION AND STAINING OF BACTERIA. stoppered bottle because of the volatility of the alcohol. If it becomes too concentrated by evaporation and thus stains too deeply, or forms a precipitate on the blood-smear, the addition of a suitable quantity of rnethylic alcohol will quickly correct such faults. It does not undergo any spon- taneous change other than of concentration by evaporation, according to my personal experience. A most important fault encountered in the working of some samples of this fluid is that it fails to stain the red blood-corpuscles a yellow or orange color, but stains them a blue color, which cannot be readily removed by washing with water. This fault I have recently discovered to be due to a peculiarity of the eosin employed. It can be eliminated by using a proper " yellowish, water-soluble " eosin.1 Application of the Staining Fluid to Blood-films. — 1 . Cover the film with a noted quantity of the staining fluid by means of a medicine-dropper. 2. After one minute add to the staining fluid on the film the same quantity of distilled water by means of a medicine- dropper, and allow the mixture to remain for two or three minutes, according to the intensity of the staining desired. A longer period of staining may produce a precipitate. Eosin- ophilic granules are best brought out by a short period of staining. The quantity of the diluted fluid on the preparation should not be so large that some of it runs off. 3. Wash the preparation in water for thirty seconds, or until the thinner portions of the film become yellow or pink in color. 4. Dry and mount in balsam. Films more than a few hours old do not stain as well as fresh ones. 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. 1 Such an eosin I have obtained from K. L. Emerson, No. 739 Boylston ,»St., Boston. THE CULTIVATION OF BACTERIA. 57 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. Pi tfi eld's method. How would you stain bacteria in tissue? Give Gram's method. Weigert's method. Kuhne's method. Ziehl-Neelseri's method. Give directions for making Wright's blood-stain. Give directions for using same. 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 FOR THE CULTIVATION OF BACTERIA. All substances that contain carbon and nitrogen compounds in assimilable form associated with water may be used as culture- 58 THE CULTIVATION OF BACTERIA. 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 necessary to sterilize the same before using, and for this pur- pose Avhat 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 MEDIA FOR THE CULTIVATION OF BACTERIA. 59 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 : 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 60 THE CULTIVATION OF BACTERIA. chopped up very fine and covered with 1 litre of sterilized water, and put into an ice-box for twenty-four hours, after which the aqueous extract is obtained by nitration 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 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. II. The Most Commonly Used Solid Culture -Media. 1. Gelatin. Nutrient gelatin is prepared as follows : A meat-infusion, as described for bouillon, is prepared and put into a large Bohemian flask of 2 liters capacity ; to this meat-infusion are added for each litre : Chloride of sodium, 5 grams ; Peptone,. 10 " ; Best quality gelatin 100 " . The flask, loosely closed with a plug of absorbent cotton, is cooked over a water-bath until all the gelatin is dissolved. This will take from an hour and a half to two hours. After this the mixture, which will be found to be decidedly acid, is neutralized by means of a solution of sodium carbonate1 THK MEDIA FOR THE CULTIVATION OF BACTERIA. 61 added drop by drop until the mixture is faintly alkaline, as in the bouillon. The flask is again put over the water-bath for an hour, after which the mixture i-s filtered hot through absorbent cotton and sterilized in the steam sterilizer for twenty minutes each day on three consecutive days. Great care must be exercised to introduce the medium into the sterilizer only when steam is actively being generated, and not to allow it to cool in the sterilizer. Advantages. — Gelatin is a most excellent medium, and remains solid at room temperature (22° or 24° C.), but is readily liquefied when exposed to a higher temperature. Not only does it serve as an excellent medium for the culti- vation of nearly all bacteria that grow out of the incubator temperature (36° to 37° C.), but it offers by the plate-method one of the best culture-soils for isolating bacteria. As some of the bacteria liquefy gelatin and others do not, it is useful also in the differentiation of these species. On account of its clearness, its easy preparation, and the other advantages mentioned above, it is one of the best culture- soils available. The disadvantages lie chiefly in the fact that it liquefies at a much lower temperature than that of the incubator, and can not therefore be used for the cultivation of some patho- genic bacteria which grow only at blood temperature. 2. Agar. Nutrient agar is prepared much in the same manner as gelatin, except that 20 or 30 grams of agar are added, in- stead of the gelatin, to the meat-infusion, and the process of cooking must be much more prolonged (four to five hours), as the agar is much slower in dissolving. It is necessary also sometimes, after dissolving the agar and neutralizing the mixture, to add the white of one or two eggs in order to clarify the solution before filtration. This is done by re- moving the flask from the fire and allowing it to cool to a temperature below 70° C. After clarification the mixture is again cooked for one and one-half to two hours and filtered through absorbent cotton two or three times. This filtration 62 THE CULTIVATION OF BACTERIA. is a much more difficult process than with gelatin, and must be carried on in the steam sterilizer. After filtration the agar is distributed among test-tubes for use, and is sterilized by the same procedure as nutrient gelatin. Nutrient agar has a melting-point much higher than that of gelatin, about 42° C., so that it may be used for the culti- vation of those bacteria which grow only at or best at the temperature of the human body. It possesses all the advan- tages of gelatin, but is not so clear and transparent, and is not liquefied by the secretions of any known bacteria. It is useful also for the isolation of bacteria by means of plate cultures. The disadvantages of agar, when compared with gelatin, lie chiefly in the greater difficulty of its preparation, and especially of its filtration. 3. The glycerin-agar culture is obtained by adding 5 per cent, of neutral glycerin to nutrient agar before sterilization. This makes a very favorable medium for the cultivation of the tubercle bacillus and of some other pathogenic bacteria. 4. A mixture of agar and gelatin in bouillon is sometimes used so as to obtain the advantages of the two substances com- bined. This mixture is prepared from bouillon in. the usual way by dissolving in the bouillon 0.75 per cent, of agar and 5 per cent, of gelatin in the manner outlined in the foregoing paragraphs. There are a number of special filtering apparatus constructed for the purpose of facilitating the filtration of agar. None in the author's estimation has any advantages over filtration through absorbent cotton in the steam sterilizer. 5. Potato Culture. Koch called attention to the great value of potato cultures for differentiating species of bacteria. The mode of preparing potatoes is as follows : A sound potato, of good size, with an intact epidermis, is chosen, and thoroughly washed and scrubbed with a brush to remove all dirt ; after which, with a sharp-pointed knife, all the eyes and discolored parts are cut out of the potato. It is then TllK MEDIA FOR THE (HJLTIVAT/OX <>!<' nAfTKHfA. <)•'> washed again in water and put into a solution of 1 : 500 bichloride of mercury for an hour, after which it is cooked in the steam sterilizer for forty minutes on each of two suc- cessive days. Just before inoculation the potato is split into halves by means of a sterilized knife and allowed to fall into a moist chamber cut surface up. The moist chamber consists of a double glass dish, the upper one of which is larger than the lower. This chamber should be thoroughly rinsed with a 1 : 500 solution of bichloride of mercury before use. Some precautions are necessary to prevent contamination of the potato from external germs. The hand that cuts the potato should be thoroughly sterilized in a 1 : 1000 bichloride of mercury bath. It is better also after washing the potato and before submitting it to the bichloride bath to wrap it in some thin tissue paper, and to keep it in this paper until ready for inoculation. Preparation of a Potato for Test-tube Culture. — By means of a cork-borer (Fig. 10) a cylinder is cut from a sound potato. This cylinder is cut obliquely FIG. 10. FIG. 11. Nest of cork-borers, used to cut potatoes for test-tube cultures. into two pieces, and each placed into a large test- tube (Fig. 11), in which it is sterilized and cooked on three successive days for a half-hour in a steam sterilizer. 6. Potato-paste is sometimes used for cultivation. For this purpose a potato is boiled, peeled, and mashed with a little sterilized water, placed in a suitable glass dish, and sterilized for one-half hour on three successive days in a steam sterilizer. 7. Bread-paste is a useful medium for the growth of moulds, and is made in the same way as potato- paste. Potato in test- tube. 64 THE CULTIVATION OF EACTfiRIA. III. The Most Commonly Used Special Culture -Media. In making final distinctions between the different species of bacteria the following special media are occasionally used : 1. The Peptone Solution. Dry peptone, 1 part; Sodium chloride, J " ; Distilled water, 100 parts. This is filtered, decanted into test-tubes, and sterilized in the steam sterilizer. This preparation is chiefly used for determining whether the bacteria secrete indol or not. It is necessary, therefore, to see that the peptone preparation used be chemically pure, also that the solution be free from the presence of carbohydrates. 2. Glucose-, lactose-, and saccharose-bouillon. These are made by adding to the bouillon after filtration and before ster- ilization from 1 to 2 per cent, of the desired kind of sugar. 3. Conradi's bile medium is prepared as follows : Take of fresh ox-bile, 900 c.c. ; Glycerin, 100 c.c. ; Peptone, 20 grams. Mix, divide off into suitable quantities, and sterilize by frac- tional sterilization. 4. Jackson's bile medium is used for isolation of B. coli and B. typhosus. These bacilli, especially the latter, outgrow all other bacteria in this medium. This medium consists of undiluted ox-bile, containing 1 per cent, peptone and 1 per cent, lactose. Fermentation tubes are filled with this medium and sterilized by fractional sterilization. 5. Dorsett's egg medium is especially used for cultivating Bacilli tuberculosis. It is made as follows : 1. The yolks and whites of a number of eggs are thor- oughly mixed in a wide-mouthed flask. 2. For every four eggs add 25 c.c. distilled water. Strain through cloth. 3. Place 10 c.c. in tubes, and slant at 73° C. for five hours on two days. THE UTENSILS OF THE CULTIVATION OF BACTERIA. 65 4. On third day, five hours at 76° C. 5. Finish sterilization by fifteen minutes in steam ster- ilizer at 100° C. Before inoculation add 2 or 3 drops of distilled water to each tube. In preparing this medium careful asepsis must be observed. THE UTENSILS OF THE CULTIVATION OF BACTERIA. For making and keeping cultures the following instruments, glassware, and utensils are required : 1. Perfectly clean glass tubes, 5 to 7 inches long and J to FIG. 12. FIG. 13. Glass test-tube. Erleniiieyer ilask. 1J inches in diameter (Fig. 12). These should be plugged with ordinary cotton -batting. 2. Erlenmeyer flasks (Fig. 13). 5— M. B. 66 THE CULTIVATION OF BACTERIA. 3. Cylindrical brushes with reed handle, and wire-handled brush of Lentz & Sons, for the purpose of cleaning test-tubes (Fig. 14). FIG. 14. Brushes for cleaning test-tubes : a, with reed handle ; 6, with wire handle. 4. Bohemian glass flasks of 1 and 2 liters capacity. 5. Platinum needles, straight and looped, mounted in glass rods (Fig. 15). FIG. 15. (a) Looped and (ft) straight platinum wires in glass handles. 6. Plates of ordinary glass about 4 by 5 inches, and Russia iron boxes to hold them during sterilization (Fig. 16). FIG. 16. Russia iron box for holding plates, etc., during sterilization in dry heat. (Abbott.) 7. Glass benches for supporting plates (Fig, 17). THE UTENSILS OF THE CULTIVATION OF BACTERIA. 67 KIG. 17. Glass benches for supporting plates. 8. Graduated measuring cylinders of 100 and 1000 c.c. capacity (Fig. 18). 9. Graduated pipettes of 1 c.c. capacity divided into tenths, and 10 c.c. divided into c.c. (Fig. 19). FIG. 18. FIG. 19. FIG. 20. FIG. 21. Measuring cylinder. Graduated pipette. Sternberg bulb. Bulb-pipette. 10. Sternberg bulbs (Fig. 20). 11. Bulb-pipettes (Fig. 21). 12. Petri's double dishes (Fig. 22). FIG. 22. Petri's double dish, now generally used instead of plates. (Abbott.) 68 THE CULTIVATION OF BACTERIA. FJG. 23. FIG. 24. FIG. 25. Moist chamber with a knob on the Wooden filter-stand. Iron stand with upper dish. rings. FIG. 26. Iron tripod with water-bath. FIG. 27. Wire baskets. THE UTENSILS OF THE CULTIVATION OF BACTERIA. 69 FIG. 28. Pinchcock. 13. Moist chambers for potato and plate cultures (Fig. 23). 14. Glass funnels of different sizes, 1, 4, and 8 ounces. 15. Wooden filter-stands (Fig. 24). FIG. 30. FIG. 29. Fermentation-tube on left side; ordin- ary tube on right side. Anatomical jar for collecting blood. 16. Iron stands with rings and clamps (Fig. 25). 17. Iron tripods with water-baths (Fig. 26). 18. Test-tube racks of any standard design. 70 THE CULTIVATION OF BACTERIA. 19. Square and round iron wire baskets for sterilizing test- tubes (Fig. 27). 20. Perforated tin buckets for sterilizing potatoes. 21. Pinchcocks (Fig. 28) for holding test-tubes. 22. Fermentation-tubes (Fig. 29). FIG. 31. Wolf huegel's ruled plate for counting colonies. 23. A 2- and a 4-liter anatomical jar, with tightly fitting cover, for the collection of blood-serum (Fig. 30). 24. Wolf huegel's ruled plates for counting colonies (Fig. 31). 25. Bunsen burners of different sizes. QUESTIONS. What is meant by a culture-medium ? What is meant by the sterilization of culture-media ? How is milk prepared as a culture-medium ? How is milk sterilized when used for a medium ? Why is tincture of litmus added to milk medium ? How does milk help in differentiating between different species of bacteria ? What is the process of making blood-serum ? How is blood-serum sterilized ? What is Loeffler's modification of. the blood -serum method ? What other animal fluids are occasionally used as culture-media? What does Pasteur's solution consist of? How would you prepare bouillon for cultures ? What are the principal solid cultures ? How would you prepare a nutrient gelatin ? How is nutrient gelatin sterilized ? What are the advantages of using gelatin as a culture ? What are its dis- advantages? How would you prepare nutrient agar? What is difficult in the prepara- tion of it? How would you sterilize it? What are its advantages and dis- advantages when compared to gelatin ? How is agar-glycerin made ? What is the best way to filtrate agar? Give Koch's method of preparing potatoes for a culture? What are tin- precautions necessary for protecting the potato from external germs ? INOCULATION OF CULTURE-MEDIA WITH BACTERIA. 71 How would you prepare a potato test-tube culture ? How would you prepare Dunham's solution? How are glucose-, lactose-, and saccharose-bouillon prepared ? How is Conradi's bile mixture prepared? Jackson's bile mixture? Dor- sett's egg mixture for cultivating tubercle bacilli? CHAPTER IV. THE INOCULATION OF CULTURE-MEDIA WITH BACTERIA. THE METHOD OF INOCULATING FLUID MEDIA. FOR the purpose of cultivating bacteria in liquid media it is only necessary to introduce the smallest possible particle containing the bacteria, into the media, by means of a steril- ized platinum needle (Fig. 15). For this procedure the culture-tube is held slightly inclined between the thumb and fingers of the left hand, its cotton plug removed by a twist- ing motion and placed between the backs of the third and fourth fingers of this hand, being careful not to touch that por- tion of the cotton which fits inside of the tube. The inoculating needle is rapidly introduced into the liquid and gently stirred around, after which the cotton plug is replaced, and the plat- inum needle sterilized by heating to redness in the flame. It is good practice to accustom one's self never to take up a platinum needle, whether it is known to be inoculated or unin- oculated, without first sterilizing it by heating it to redness in the flame, and allowing it to cool for a few seconds before taking up with it the inoculating material. The bacteria might other- wise be destroyed by the heat. Again, after use the needle should always be sterilized in the same manner before putting it down. THE METHODS OF INOCULATING SOLID MEDIA. 1. For the inoculation of potatoes and other solid media not hereafter mentioned, it is only necessary to streak the surface of the medium with a platinum needle or other instru- ment which has been dipped into or which contains a small 72 INOCULATION OF CULTURE-MEDIA WITH BACTERIA. fragment of the contaminating material, care having been taken beforehand to sterilize thoroughly the needle or instrument. 2. Gelatin culture-tubes are inoculated in one of three ways : a. Stab culture, made by puncturing the centre of the solidified gelatin mass with a platinum needle previously charged with the bacteria. 6. Slant cultures, made by gently passing over the surface of the medium the inoculating needle ; for this purpose the gelatin is made to solidify with the tube in an inclined posi- tion, so as to give a larger surface for inoculation. c. Plate cultures, made by inoculating the gelatin mass, which has been previously liquefied by submitting it to a temperature of 30° C., with a platinum needle or loop as for liquid cultures, and pouring the liquid mass rapidly and evenly on a sterilized glass plate, allowing it to solidify well protected from dust. The plate method, introduced by Koch, is of great value for the separation and isolation of bacteria. In this manner each bacterium introduced into the liquefied gelatin is fixed by the hardening of the gelatin and develops as a separate colony, the number of colonies being, as a rule, equal to the number of bacteria originally introduced. Each colony grows in its own peculiar way, because each species has a definite way of growing in gelatin. This method therefore not only serves for the separation of the bacteria themselves, but also enables us to recognize one species from another. Indeed, the classical method of Koch for the separation and isolation of bacteria, though modified in some particulars, has not been essentially changed since its introduction. It is as follows : Three sterilized gelatin culture-tubes about a third full and containing about 10 c.c. of the medium are liquefied by being submitted to a temperature of 30° C. Tube I. is inoculated with one or two loopfuls of the platinum needle from the contaminated substance, its cotton plug replaced, and the contents well shaken. After sterilizing the platinum needle one or two loopfuls from tube No. I. are introduced into tube THE METHODS OF INOCULATING SOLID MEDIA. 73 No. II. ; both tubes are again plugged, and tube No. II. is in turn well shaken. The platinum needle is again sterilized, and finally one or two loopfuls from tube No. II. are intro- duced into Tube No. III., and the contents of the latter well stirred. The three tubes are kept on a water-bath at a temperature of between 25° and 30° C., so as to keep the mass liquid. Meanwhile three sterilized glass plates are arranged on three cooling-stages, as depicted in Fig. 32. The gelatin from each FIG. 32. Levelling-tripod with glass cooling-chamber for plates. of the tubes I., II., III. is slowly and evenly poured over the surface of the plates, correspondingly designated as No. I., II., III., and allowed to solidify. It is necessary during the pouring and solidification of the gelatin on the plates that they be carefully protected by a cover against the dust and bacteria from without. After solidification is perfect the plates are transferred into culture-dishes placed on glass benches (Fig. 17), and properly labelled. To harden the gelatin more rapidly, ice or iced water is generally kept in the lower dish of the cooling-stage. To insure 74 INOCULATION OF CULTURE-MEDIA WITH BACTERIA. evenness of surface in the gelatin on the glass plates a levelling- tripod is used, as seen in Fig. 32. This tripod is easily set by means of a levelling glass. Results. — In this manner each of the separate bacteria con- tained in the media will develop as separate colonies on the gelatin plates. Plate L, made from tube L, will contain a large number of bacteria ; plate II. will contain the bacteria in much smaller number ; and plate III. will contain only a few colonies, well separated ; and in this way the characteristic growth of the separate colonies and their action on gelatin may be carefully followed out and studied. By this means also the observer may under a magnify ing-glass with a fine sterile platinum needle pick out the individual colonies and inoculate fresh tubes, and so obtain pure cultures of any liv- ing organism. 3. Plate cultures of agar are made in the same way, but require more care in their preparation, as agar does not melt at a temperature below 42° C., and solidifies again at a tem- perature of 39° or 40° C., so that after inoculation in the liquid state the tubes must be kept in a water-bath between 40° and 42° C. until they are poured into the plates. The agar plates, however, may be incubated at blood tempera- ture (37° C.)? and the growth of bacteria noted. In recent years small double dishes, known as Petri's culture- dishes (Fig. 22), have been introduced to take the place of the plates. For the inoculation, liquefied gelatin or agar is poured into the lower dish, and this is quickly covered by the upper dish. For this method no levelling apparatus and cooling-stage are required. For counting the colonies in the plates the apparatus of Wolfhuegel (Fig. 31) has been adopted. This consists of regularly lined glass plates, divided into squares and arranged as seen in the figure. By placing the culture-plate under the lined plate it is easy to ascertain the number of colonies contained in each of, for example, ten squares, and by a simple process of multiplication the number of colonies in the whole plate. 4. Instead of pouring out the liquid gelatin from the tubes THE CULTIVATION OF ANAEROBIC BACTERIA. 75 into plates after inoculation, Esmarch rolls these tubes in a vertical position until the gelatin is completely solid. This is hastened by rolling the tubes as shown in Fig. 33. By this excellent method there is less likelihood of contamination than by the plate method. 5. Gelatin and especially agar plates are occasionally made in a different way. The liquid medium is poured on glass plates and allowed to solidify before inoculation. When well hardened the surface is streaked with the inoculating material FIG. 33. Demonstrating Booker's method of rolling Esmarch tubes on a block of ice. on a platinum needle. In this way the colonies grow along the streak much more superficially than they do in the ordi- nary plate method. 6. Agar and blood-serum slant cultures are made in the same manner as similar cultures on gelatin. They have one advantage over the gelatin culture by reason of being able to withstand the temperature of the incubator, 37° C. THE CULTIVATION OF ANAEROBIC BACTERIA. Exclusion of oxygen is absolutely necessary. For this pur- pose a number of methods have been suggested and used, some of which require special and elaborate apparatus. All 76 INOCULATION OF CULTURE-MEDIA WITH BACTERIA. the foregoing methods of inoculation and cultivation are avail- able only for the aerobic bacteria. 1. Cultivation of Tetanus Bacillus. — The following method FIG. 34. FIG. 35. Jar for anaerobic cultures. FIG. 36. Small incubator. Mercurial thermo- regulator. has been found very useful for the cultivation of this strictly anaerobic bacillus, aud answers all purposes in the author's opinion. THE CULTIVATION OF ANAEROBIC BACTERIA. 77 A culture-tube three-fourths full of the medium is heated to the boiling-point and allowed to cool to a temperature in the neighborhood of 40° C.; then a platinum needle charged FIG. 37. Incubator used in bacteriological work : a represents the incubator set up and containing a cage of tubes and a Petri dish ; 6, represents a vertical section of the incubator and displays the water-chamber, inner chamber, walls, vents, ther- mometer, valve, etc. with the material for inoculation is dipped down to the bot- tom of the tube. With fluid media, immediately thereafter a layer of paraffin oil is poured on the surface of the liquid 78 INOCULATION OF CULTUEE-MEDIA WITH BACTERIA. before introduction of the cotton plug. With solid media, the fluid is allowed to solidify after inoculation, and after solidification the paraffin oil is poured on the surface of the medium. This effectually shuts out the oxygen, and as a rule allows a luxuriant growth of anaerobic bacteria. 2. Special apparatus, with oxygen replaced by hydrogen, is also used for the cultivation of anaerobic bacteria. The Incubator and The Thermostat. For the purpose of growing bacteria it is often necessary or desirable to obtain a constant temperature. The ordinary body-temperature, 37° C., is the most favorable for the growth of the pathogenic bacteria. Apparatus especially constructed for maintaining a constant temperature are known as incuba- tors or thermostats. They are generally made of double- walled metal, and contain water, which by means of a gas-jet at the bottom of the apparatus may be kept at a constant temperature (Fig. '37). For regulating the gas-supply and to maintain the constant temperature, instruments known as thermo-regulators are used. A number of these are highly complicated, but for ordinary work is recommended Reichert's or Dunham's mer- curial thermo-regulator (Fig. 35). QUESTIONS. How do you inoculate a fluid culture-medium ? How do you inoculate potato cultures? In how many ways may gelatin tubes be inoculated? What is meant by a stab-culture? By a slant culture? How are plate cultures made ? Describe the method of making plate cultures according to Koch. How are agar plate cultures made? How are colonies on plates counted? Describe the method of making cultures in Petri dishes? How are Esmarch's culture-tubes made? Give a good method for the cultivation of anaerobic bacteria. What is an incubator? What is a thermo-regulator? STKRILIZATfOX, DISINFECTION, AND ANTISEPSIS, 79 CHAPTER V. STERILIZATION, DISINFECTION, AND ANTISEPSIS^ Definitions. — The freeing of substances from the live bacteria they may contain or that may have collected on their surfaces is called sterilization. This is accomplished either by means of heat or the use of chemicals. Erroneously sometimes the term sterilization is used to indicate the destruction of bacteria by the application of heat, the term disinfection being used then for their destruction by chemical agents. To disinfect a substance is to destroy in it or on it all the harmful or infectious bacteria, without necessarily killing all the living bacteria. THE METHODS OF STERILIZATION. I. Substances chemically sterilized are unfit for bacterial culture, except in very rare instances when the chemical agents are very volatile. II. In laboratory work, therefore, where the aim is the cultivation of bacteria, heat is the only method of sterilization used. This is applied either in the form of dry heat or moist heat (steam). 1. All substances which may be passed through the Bunsen flame and heated red-hot are usually sterilized in this manner. 2. Other implements, such as instruments and glassware, which would be injured by the direct flame, but withstand con- siderable heat, are sterilized by dry heat in an oven at a temperature of 160° to 180° C. for an hour. 3. For culture-media, one resorts to sterilization by steam, except in rare instances, when filtration tinder pressure through unglazed porcelain is considered sufficient. Experience has taught that steam at a temperature of 100° C. will kill all known bacteria and their spores within an hour, and Pasteur has demonstrated that steam under a pressure of two or three atmospheres, at about 130° C., will 80 STERILIZATION, DISINFECTION, AND ANTISEPSIS. destroy all known bacteria and their spores within fifteen or twenty minutes. The sterilization of culture-media,, then, is usually done by the means of steam, with or without pressure. 4. In some instances, when exposure to steam for one hour would be prejudicial to the medium, what is known as dis- continuous or fractional sterilization is resorted to. The medium is steamed during three consecutive days for twenty minutes each time ; during the interval it is kept in favorable FIG. 38. FIG. 39. Laboratory hot-air sterilizer. Rose-burner. conditions for the development of bacteria. In this manner the first heat destroys all the fully formed bacteria that may exist. The favorable temperature in the interval between the first and second heatings allows all the spores contained in the medium which may not have been destroyed in the first heating to develop into fully formed germs, which are destroyed by the second application of steam on the ensuing day. The application of heat on the third day is to make THE METHODS OF STERILIZATION. 81 sure of the destruction of all spores which may have resisted the two previous applications. It has been demonstrated that this sterilization is very effective and complete, and substances which have undergone it may be kept indefinitely bacteria- free thereafter. 5. Another method of fractional sterilization is occasionally used for certain media which cannot withstand the tempera- ture of boiling water without deteriorating. It consists in FIG. 40. Steam sterilizer, pattern of Koch. (Abbott.) submitting the medium to a temperature of 68° to 70° C. for from two to three hours during seven consecutive days. This method is necessarily yery imperfect, and though successful in those cases in which no spores have to be destroyed and where none of the bacteria contained in the medium are of the pyogenic variety, it cannot but have a very limited application, and media so sterilized should not be used for 6— M. B. 82 STERILIZATION, DISINFECTION, AND ANTISEPSIS. culture purposes, except after they have been submitted for several days to the temperature of the human body in the thermostat and remained sterile during this control-test. 6. For generating dry heat for the sterilization of glass- ware, implements, and instruments used in making cultures, the hot-air oven and rose-burner are most usually employed, and a temperature usually of 180° C0 maintained in the oven for one and one-half hours (Figs. 38 and 39). FIG. 41. Arnold steam sterilizer. (Abbott.) The oven consists of a double- walled metallic box, with a double- walled front door, with a copper bottom, the sides and door encased in asbestos boards. The heat is applied by means of a rose gas-burner (Fig. 39) to the bottom of the box. The objects to be sterilized are put upon perforated metallic shelves in the box. In the top of the apparatus are two per- forated openings for the insertion of the thermometers. In this portion the oven is also provided with a perforated slid- ing window to allow escape of the overheated air. THE METHODS OF STERILIZATION. 83 7. For applying moist heat, bacteriologists commonly use a. Koch's apparatus (Fig. 40). b. Arnold's apparatus (Fig. 41). 8. For the application of steam under pressure preference is felt for the autoclave of Chamberlain or of Wiesnegg (Fig. 42). 9. For discontinuous fractional sterilization at a low tem- FIG. 42. Vk. A B Autoclave, pattern of Wiesnegg: A, external appearance; B, section. (Abbott.) perature the apparatus in more general use is the blood-serum sterilizer of Koch (Fig. 43). 10. Filtration under pressure through unglazed porcelain, the pores of which are too small to allow bacteria to go through, will render certain liquid substances sterile or bacteria-free. This method is made use of in the case of certain pathogenic bacteria which secrete soluble poisons which we desire to separate from the bacteria themselves. The Chamberlain 84 STERILIZATION, DISINFECTION, AND ANTISEPSIS. filter of unglazed Sevre porcelain is the only one to be recom- mended for this purpose. FIG. 43. Chamber for sterilizing and solidifying blood-serum. (Koch.) THE METHODS OF DISINFECTION. I. Disinfection or destruction of infectious bacteria may with certainty be accomplished by heat, and indeed in the labora- tory when the total destruction of the bacteria is desired with the material containing them heat is the most effective measure. In other cases, however, when the destruction of the bac- teria themselves and the preservation of the contaminated sub- stance are desired, recourse must be had to such measures as will destroy the bacteria alone. Substances which are able to destroy bacteria or their spores are known as germicides or disinfectants, and those substances which retard bacterial growth are called antiseptics. II. In the use of chemicals for disinfecting, one should always bear in mind that their mode of action is not a cata-, lytic one, but that they owe their virtue to the power of THE METHODS OF DISINFECTION. 85 forming definite chemical compounds with the bacteria cells, which are thus rendered innocuous. They must, therefore, come into direct contact with the bacteria themselves, and in the combination so formed they and the bacteria change their chemical properties. In the choice of chemical disinfectants, one must be guided by the species of bacteria to be destroyed, by the number of these bacteria, by the nature of the media containing the germs, by the substances to be disinfected, and by the quan- tity of material to be disinfected. All chemicals so used must be of a definite strength, and must be made to act for a specified time, the quantity and strength of the disinfec- tant and the time varying with the nature of the chemical and the species of bacteria to be destroyed. To test the germicidal power of substances, the following is a convenient method : To young bouillon cultures of the bacteria to be acted upon sufficient of the chemical to be tested is added to make the proper dilution, and at given intervals of time a few droplets of this mixture are inocu- lated on sterile agar and gelatin tubes and the result carefully noted — for instance, supposing it is intended to test the germi- cidal power of carbolic acid toward some pus organism. To 9.90 c.c. of a bouillon culture of a pus germ in a test-tube 0.10 c.c. of carbolic acid is added, making the mixture a 1 per cent, carbolic acid dilution. The tube is well shaken, and at the end of one minute a few droplets of the mixture are taken on a sterile platinum needle and inoculated on a fresh tube of gelatin or agar, another fresh agar tube is inoculated with the mixture in the same manner after two minutes, another again after five minutes, and a fourth in fifteen minutes, a fifth in a half hour, a sixth in an hour, and a seventh in two hours. The growth in the inoculated tubes would indicate the action of 1 per cent, carbolic acid upon the given pus germ in the specified time. The same process is repeated, using a 2 per cent, dilution of the car- bolic acid, and next a 3 per cent., a 4 per cent., and a 5 per cent, dilution. Higher than 5 per cent, solutions of carbolic acid are not possible with any but water too hot for this work. 86 STERILIZATION, DISINFECTION, AND ANTISEPSIS. In other substances a 10 percent, dilution would be the last step of the test. Thus are obtained results which are fixed and definite, stating positively the strength of disinfection used and the time of its application. Cautions. — In conducting these experiments one must remember always that the growth of bacteria on the solid tubes may be considerably retarded by the action of the dis- infectants, and one must not accept the result as conclusive unless those cultures have been kept under observation for a considerable length of time. Again it is proper, whenever practicable, to conduct the experiments at the temperature of the human body, 37° C., as experience has demonstrated that at this and higher temperature the disinfectant power of chemicals is increased. THE METHODS OF ANTISEPSIS. Substances that retard the growth of bacteria without, however, destroying them are called antiseptics. It is clear that all disinfectants when used in a more diluted form, or when allowed to act for a shorter space of time than is re- quired for them to show their germicidal power, act as anti- septics. I. The Common Disinfectants. Carbolic acid, strength 3 to 5 per cent., efficient in one hour. Bichloride of mercury, solution 1 : 1000 or 1 : 500, acts from within a few minutes to a half-hour. Chlorinated lime, containing free chlorine, is an efficient germicide in an hour's time in the strength of from 5 to 10 per cent. Boiling water, to which 2 or 3 per cent, sodium carbonate is added, is an efficient germicide in an hour. Sulphur dioxide gas, when used dry, has little or no disin- fectant power, and bacteria have been able to withstand an atmosphere containing from 10 to 12 per cent, of this gas for several hours. In the presence of moisture, however, it QUESTIONS. 87 forms sulphurous acid, and is then an efficient germicide in as low a percentage as 4 or 5 per cent. Formalin, in 2 to 5 per cent, strength, or as formaldehyde gas, is a powerful germicide. A number of other substances have been recommended, arid have been used as germicides, but the above are of more general and practical value. II. Enumeration of the common antiseptics would be too lengthy to be stated in a book of this kind. In conclusion, one should bear in mind that chemical agents must act directly on the bacteria cells themselves — that is, they must penetrate ; and that they are the more efficient the more quickly they form chemical compounds with those cells — or, as usually expressed, the more penetrating power they possess — and anything that interferes with this chemical com- bination interferes with the action of the disinfectant. QUESTIONS. What is meant by sterilization ? By disinfection ? At what temperature and how long does it take dry heat to sterilize? How long does it take live steam to sterilize ? How long does it take steam under pressure to sterilize? How are implements in the bacteriological laboratory sterilized? How are culture-media sterilized? What is discontinuous or fractional sterilization by steam, and how is it accomplished ? How is sterilization at a low temperature effected? When is it used? What are its disadvantages? What forms of apparatus are generally used to generate steam for sterili- zation ? What are the only filters which can remove bacteria from liquid media? What are germicides or disinfectants? What are antiseptics? How would you test the value of a germicide ? What chemicals are generally used as disinfectants? State the value of carbolic acid as a disinfectant ; of bichloride of mercury ; of the chlorinated lime ; of sodium carbonate ; of sulphur dioxide, and of formaldehyde. 88 THE INOCULATION OF ANIMALS AND THEIR STUDY. CHAPTER VI. THE INOCULATION OF ANIMALS AND THEIR STUDY. THE INOCULATION OF ANIMALS. ITS purposes are to differentiate between bacteria. In order to study their virulence it is often necessary to test their action on animals. In the laboratory the smaller animals, such as mice, rats, guinea-pigs, and rabbits, are chiefly used. Technic. — The inoculation is made in a number of ways, depending on the species of the bacteria, the nature of its toxin, and the rapidity of action desired. The Various Methods of Inoculation of Animals. 1. Sometimes, though rarely, the inoculation is made by rubbing a solid or liquid culture over the abraded epidermis, very much in the same manner as vaccine is introduced into the human subject. 2. Subcutaneous inoculation — that is, into the connective tissue under the skin — is an important method. For this purpose the hair is shaved from part of the back or abdomen, the skin well washed and disinfected as well as it may be, with a 5 per cent, carbolic acid solution. Then the skin is seized with a pair of sterilized forceps, and with a sterilized scalpel a small nick is made into it, after which a small sterilized pair of scissors is -introduced in the areolar tissue and a pocket made for some little distance into this tissue. Into this pocket the inoculating material or bacterial culture (especially when a solid culture has been employed) is intro- duced, by means of a sterilized forceps or a platinum loop, care being taken to avoid touching the edges of the wound with the instrument. If a liquid culture is being used, particularly in appreciable quantity, it may be introduced subcutaneously by means of a hypodermatic syringe and needle well sterilized beforehand. THE INOCULATION OF ANIMALS. 89 Guinea-pigs and rabbits are usually inoculated in the abdom- inal wall ; rats or mice in the loose tissue at the root of the tail. Animal Holders. — Various instruments have been devised Fid. 44. The Voges holder for guinea-pigs. (Abbott.) for keeping animals quiet during this operation, the most use- ful of which are : Voges' guinea-pig-holder (Fig. 44) ; Kitasato's mouse-holders (Fig. 45) ; and the basket mouse-holder (Fig. 46). 3. Intravenous injection or inoculation into the circulation 90 THE INOCULATION OF ANIMALS AND THEIR STUDY. consists in injecting directly into the veins of the animal in the direction of the circulation, the material to be inoculated. Necessarily the material used must be a liquid, and the injec- tion must be done slowly and witli precaution. Intravenous injection is used especially in rabbits. The most convenient point of injection is into the vein of the ear known as the posterior auricular vein, which is easily penetrated from the dorsal surface of the ear, where it lies superficial and FIG. 45. Kitasato's mouse-holder. (Abbott.) Mouse-holder, with mouse in proper position. (Abbott.) imbedded firmly in the areolar tissue. For the pur- pose of making these injections an ordinary hypodermatic syringe with the needle used for injections in man is employed. All that is required is that both syringe and needle be ster- ilized. The mode of procedure is as follows : The rabbit is firmly held by an assistant. The ear chosen is taken between the thumb and forefinger of the left hand, and after washing and sterilizing the skin as thoroughly as possible, the vein on the posterior edge of the ear is sought THE INOCULATION OF ANIMALS. 91 for. If it is invisible, pressure at the root "of the ear will make it prominent. From the dorsal surface the hypodermatic needle is in- serted at its distal extremity and the material slowly injected. Care should be taken to have the needle penetrate the vein, and the first few drops of injected liquid will show whether it does so or not ; for if the needle is outside the vein a bulla will immediately be found at the site of injection. A little practice renders this method very easy. 4. Inoculation into the lymphatics is done best with a hypo- dermatic syringe and the injection is made into the testicles. 5. Intraperitoneal inoculation requires much care and the same antiseptic precautions as when opening the peritoneum for a laparotomy. The skin, cleanly shaven and disinfected as thoroughly as possible, is opened in the linea alba midway between the sternum and pubis, through an incision, from an inch and a half to two inches long and penetrating the fascia. The edges of the wound being held apart, the connective tissue and muscles are separated with a pair of sterilized, blunt-pointed scissors. If a liquid inoculating material is used, it may now be introduced with a sterile hypodermatic needle into the peritoneal cavity, avoiding as much as possi- ble the wounding of the intestines, which is not difficult. If the material employed is solid, the peritoneum is opened with scissors and the solid particle introduced into the cavity by means of a sterile needle or forceps. The wound is care- fully sutured and closed by a layer of collodion. 6. Intrapleural inoculation is performed much more rarely on account of the danger of wounding the lungs, and when used the same precaution must be taken as for the intra- peritoneal method. 7. Inoculation into the anterior chamber of the eye is per- formed occasionally to study in the living animal the changes produced locally by bacteria. With a sharp-pointed bistoury an incision is made in the cornea, at its sclerotic attachment near the inner canthus, and the material introduced and applied directly upon the iris, by a sterilized needle or by means of a small forceps. 92 THE INOCULATION OF ANIMALS AND THEIR STUDY. THE OBSERVATION OF THE INOCULATED ANIMAL. 1. After inoculation animals should be observed carefully and all changes in their condition noted. Their temperature should be taken several times a day, their body weight recorded every day under the same conditions ; their behavior as to food noticed ; the state of their fur and any sign of paralysis or of convulsions carefully observed. 2. After death, the autopsy should be performed as soon as practicable. For that purpose the animal should be laid upon its back on a board, its four legs stretched widely apart and attached to the sides of the board by strings, or nailed, and the nose should also be carefully nailed down. By means of a 5 per cent, carbolic acid solution the skin of the body from the chin to the pubis should be carefully sterilized and all hair shaved off. The place of inoculation is now to be carefully examined and described. Examination of the Abdominal and Pelvic Contents. — After this an incision is made in the skin only, and that carefully dissected away from the subcutaneous tissue and hooked back so as to prevent its contaminating the underlying tissues. After this by means of a metallic spatula, heated to redness, the muscular tissue is singed all along the line of the next incision, along the linea alba from the pubis to the sternum, along the arches of the rib and obliquely from the sterno- clavicular junctions to the tip of the last two ribs. With a pair of sterilized blunt-pointed scissors the peritoneal cavity is opened. All changes within it are carefully noted, cultures made from all exudates, or inflammatory products apparent, bouillon, agar, and blood-serum tubes being used for that pur- pose, and cover-glasses being also prepared for the microscope. Then, by means of the same heated spatula, the surfaces of the different organs are thoroughly singed, and with a spear-head, thick sterilized platinum wire, the organ is penetrated and cultures made from the small pieces of organs or blood adhering to the wire ; bouillon, agar, and blood-serum being used, and cover-glasses being also prepared. From all cultures so obtained plate cultures should be made, and the several bacteria therein isolated, and pure cultures made. THE OBSERVATION OF THE INOCULATED ANIMAL. 93 After complete exumiiuition of the abdominal and pelvic organs, by means of a thick pair of scissors the ribs are cut off along the singed lines, the sternum turned up, and exam- ination and cultures of the thoracic organs made in the same way as for the abdominal organs. 3. Alter the complete autopsy the animal should be inciner- ated, and if this is not practicable, it should be kept bathed at least for two hours in a 5 per cent, carbolic acid solution, and finally boxed in quicklime before burial. Cultures from the human body at autopsies should be made as described for lower animals. 4. Cultures from secretions of living animals and man should be made immediately upon their passage and must be always collected in sterilized vessels. The examinations and the cult- ures should be prepared forthwith, using agar, bouillon, and serum as media. 5. It is wise, in collecting spinal exudates or urine, to col- lect some of the material directly into sterilized centrifuge tubes, so that sediments for immediate examination or culture making may be quickly obtained without contamination. The Roux-Nocard Method of Culture and Observation. History. — Observations made by Roux and Nocard for the growth of microorganisms in culture in the live animal have shown that a number of minute bacteria are found associated with certain diseases, notably the pleuropneumonia of cattle. These microorganisms require a much higher power of the microscope than that generally in use to bring them into view, a magnification of 2000 at least being necessary. Technic. — Small collodion flasks are made thoroughly sterile, filled with blood from the suspected animal, and then closed with sterile collodion. These tubes or flasks are after- ward introduced into the abdominal cavity of live rabbits and guinea-pigs, and allowed to remain for a few days, after which they are taken out and examined, when the small motile microorganisms as mentioned above will be discovered. Importance. — This method of observation and cultivation in the live animal body seems to open a large field for the 94 INFECTION AND IMMUNITY. future investigation of such diseases as scarlet fever, measles, smallpox, rabies, etc., which, though unquestionably of microbic origin, have so far failed to reveal any specific germ for their causation. QUESTIONS. Why are animals inoculated ? What different methods are used for animal inoculation in the laboratory ? Describe the subcutaneous method. The intravenous method. The intra- lymphatic method. The intraperitoneal method. What instruments are used to inoculate liquid cultures into the veins of animals ? What should be observed in inoculated animals? How should an autopsy be made in the case of an animal dead after inoculation ? What precautions are necessary in making cultures from tissues and organs in dead animals to prevent contamination from outside ? How should secretions of animals and men be collected for bacterial exam- ination ? What form of culture have Eoux and Nocard proposed for cultivation of bacteria ? CHAPTER VII. INFECTION AND IMMUNITY. INFECTION. BACTEEIA which produce diseases in animals and man are known as the pathogenic bacteria, and the process by which disease is produced is called infection. The mode of communication of these infections to man or to animals is not fully demonstrated. The following explana- tions are the more plausible. The Theories of Infection. 1. The rapid multiplication of bacteria in the blood and organs of infected animals is supposed to interfere with their bodily functions, and so cause disease and death. This is the so-called mechanical theory of infection, and finds support in such diseases as anthrax, when in fatal cases every capillary INFECTION. 95 and origin of the animal is teeming with microorganisms, and in so-called sopticaemic 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 96 INFECTION AND IMMUNITY. toxins which they generate, or on account of the highly poi- sonous property of these toxins. Certain bacteria and their toxins have been proved to exercise a selective " affinity " for certain tissues ; e. ,. •» £?/.• *'V ?m '• ,>// Pus of gonorrhoea, showing diplococci in the bodies of the pus-cells. (Abbott.) 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. The gonococcus will not grow on gelatin, agar-agar, potato, PLATE II Tuberculous Sputum Stained by Gabbett's Method. Tubercle Bacilli seen as Red Rods; all else is Stained Blue. (Abbott.) GONORR1KEA. 115 or in bouillon. The most commonly used medium for the growth of the gonoeocci is that of Wertheim. It consists of three parts of meat infusion agar mixed with one part of imcoagulated ascitic fluid, hydrocele fluid, or blood serum. The agar is melted and mixed with the fluid or serum at about blood temperature. Blood smeared over agar as for the B. influenzas makes a good medium. 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 G-ohin have caused acute urethritis, which rapidly disap- peared, by intra-articular injections of cultures into dogs and rabbits. Pus containing the gonoeocci when inoculated into man have reproduced the disease in many instances. Pus cultures of the gonoeocci 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 in- jections of the culture produce considerable tumefaction and redness at the point of inoculation, but no abscess-formation. VII. Micrococcus Catarrhalis. The Micrococcus catarrhaiw is a commonly found organ- ism, occurring in inflammations of the upper respiratory tract. Morphology and Staining. — It is identical with the Micro- coccus gonorrhoeas in its shape and staining qualities. Biology. — It can be differentiated by the fact that M. catar- rhalis grows readily on all culture-media. From the inenin- gococcus it can be differentiated by its growth at room tem- perature (below 25° C. and even below 20° G), and by its white colonies. Micrococcus catarrhalis does not ferment any sugars. Micrococcus meningitidis ferments dextrose and maltose. Micrococcus gonorrhoeas ferments dextrose alone. 116 THE PATHOGENIC BACTERIA. VIII. Bacillus Pyocyaneus. The Bacillus pyocyaneus is found frequently in suppurating wounds, especially in burns. It colors the pus green and the dressings a bluish-green, without showing any color-influence on the local condition of the wound. Pathogenesis. — It exists in pus associated with other micro- organisms, and is considered an inoffensive saprophyte in most cases. It may, however, under certain conditions, be- come pathogenic. Morphology. — It is a delicate rod with rounded or pointed ends, actively motile and flagellated at one end. 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. IX. Pneumococcus or Pneumobacillus. Friedlaender discovered this organism. It is sometimes found: a, in pus associated with other organisms, and />, in cases of pneumonia as the sole factor of the disease and its secondary abscesses. The pus produced by it is thick and creamy white in color. Pathogenesis. — It frequently causes suppuration in the ser- ous membranes — pleura, peritoneum, pericardium, and lungs. It has also on some occasions caused suppuration in the vis- cera and in the subcutaneous and deep cellular tissue. X. Bacillus Coli Communis. XI. Bacillus Typhosus. XII. 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. THE PATHOGENIC MICROCOCCL 117 QUESTIONS. What are the pyococci ? Describe the Staphylococcus pyogenes aureus. How does it act on bouillon, on gelatin, on agar ? Where is this organism found in the human body ? Where outside of the human body ? What is the effect on animals of intravenous injections of this organism? What of subcutaneous inoculation ? In what respect does the Staphylococcus pyogenes albus differ from the aureus? The Staphylococcus pyogenes citreus? Describe the streptococcus pyogenes. Where is it found ? Of how many elements are its chains formed ? What is the effect of intravenous, intra- peritoneal, and subcutaneous inoculations ? What are the characteristics of the Micrococcus tetragenus f What is the gonococcus? Where is it found ? How is it recognized under the microscope ? What is Wertheim's method of growing gonococcus ? What is the Micrococcus cafcarrhalis? Where is it found ? How is it dif- ferentiated from the gonococcus ? How is it differentiated from the Micro- coccus meningitidis ? 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 ? CHAPTER X. THE OTHER PATHOGENIC MICROCOCCI AND ALLIED BACILLI— MICROCOCCUS PNEUMONIA, EPIDEMIC CEREBROSPINAL MENINGITIS, AND MALTA FEVER. PNEUMONIA. I. Micrococcus Pneumoniae Crouposae (Diplococcus Pneumonia ; 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. 118 THE PATHOGENIC MICROCOCCL FIG. 50. Piplococcus of pneumonia from blood, with surrounding capsule. (Park.) It is found : a. in the saliva of about 50 per cent, of healthy individuals, b. in the rusty sputum of pneumonic patients and in the fibrinous exudation of 75 per cent, of the cases of pneumonia, c. in a large number of cases of meningitis com- FIG. 51. Pneumococcus from bouillon culture, resembling streptococcus (Park.) plicating pneumonia or associated with pneumonia, d. occa- sionally where no pneumonia exists, e. also in abscesses. PNEUMONIA. 119 Morphology. — Jttcrocoecus pneumonia? 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 Hficrococeus pneumonice is aerobic and facultative anaerobic. Like most cocci it is non-motile, 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 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, its culture becomes very much attenuated, practically losing its virulence. In sputum it lives a long time. Twenty per cent, alcohol is the best disinfectant (Wadsworth). 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. Hiss has devised a medium for the differentiation of streptococci and pneumococci. It con- sists of beef serum, 1 part, distilled water, 2 parts, to which is added 1 per cent, inulin (Inulin C. P.) and enough litmus to render the medium a clear transparent blue. Pneumococci ferment the inulin with acid production, rendering the litmus red, ; ;in b^ng 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 70 oo^ ooo c-c- was fatal ^0 mice. This is by far the most 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. 165 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 1 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 noutral saline solution for use. 166 TETANUS, MALIGNANT (EDEMA, ETC. Anaphylaxis. Anaphylaxis (dud, against ; Aazt7, protection) is a term which embraces a certain series of untoward phenomena ob- served when an otherwise benign proteid material has been absorbed by an animal previously rendered hypersusceptible to that proteid. In the earlier experiments with antitoxin for diphtheria and tetanus, such untoward accidents were some- times noted and their occurrence laid at the door of hyper- susceptibility to the toxines. It is now known that they* were due to the proteid contained in the horse-serum. A great deal of work has been done in an endeavor to ferret out the causes of and laws governing anaphylaxis. So far the results have been contradictory, and no reliable information for prac- tical guidance has been furnished. The subject of anaphylaxis is mentioned here on account of the prejudice that has arisen in certain quarters against antitoxin administration through fear of anaphy lactic results, when it is known that a previous dose of antitoxic serum has been given. In the light of our present knowledge of the efficacy of antitoxins against diphtheria and tetanus, the latter in a pro- phylactic way especially, we do not believe it is justifiable in refraining from their free use on the grounds of "anaphylaxi- phobia." MALIGNANT (EDEMA. The Bacillus of Malignant (Edema. History. — Malignant O3dema 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. — Rods from 3 to 8 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. TETANUS. 167 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 oedema is motile and flagellated and 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 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 ike, tetanus and other anaerobics, in atmospheres of hydrogen only. One attack produces immunity against a second attack. 168 TETANUS, MALIGNANT (EDEMA, ETC. 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). FIG. 62. Bacillus of symptomatic anthrax: A, vegetative stage— gelatin culture; B, spore-forms — agar-agar culture. (Abbott.) It stains by all the anilin colors. It forms spores, which are situated at or near one of the poles, giving a swollen appear- ance to the bacillus. Biologic Characters. — In the vegetative type it is actively motile, but loses its motion in the spore-bearing form. It SYMPTOMATIC ANTHRAX. 169 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 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° C. 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 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. The autopsy reveals an extensive swelling of the subcutane- ous tissues with emphysema. The cedematous 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 are found to contain spores. This serves as a differentiation, 170 TETANUS, MALIGNANT (EDEMA, ETC. in addition to other points, between it and the Bacillus anthracis. Immunity. — One attack of the disease, if not fatal, affords protection against future attacks. Active immunization has been practised on animals with extracts of infected meats. The first vaccine used consists of about 0.01 gm. of dried extract previously heated to 100° C. for five or six hours. This is emulsified with 0.85 per cent, saline solution. In about ten days a similar amount of the second vaccine is given. In this instance the extract has been heated to 90° C. only. These vaccines should be in- jected under strict aseptic precautions near the end of the tail of the animal. Bacillus Aerogenes Capsulatus. History. — This bacillus was discovered by Welch and de- scribed by Welch and Nuttall in 1892. Morphology and Staining. — It resembles slightly the anthrax bacillus. It is very variable in size, however, varying in length from 3—6 //., sometimes being nearly as broad as long, especially in artificial cultures. Generally they occur singly, but occasion- ally in chains. Long chains are found in the blood, but never, in artificial media. This is considered by Welch a great point of differentiation from the Bacillus anthracis. The bacillus of Welch is non-flag 'ellated, non-motile, and constantly capsulated. It stains by all the ordinary anilin stains and by Gram's stain. Sometimes involution forms on artificial media give up the stain. Biology. — It is an obligatory anaerobe, growing well on all media, producing in deep stab culture characteristic gas-bub- bles. Pathogenesis. — It is slightly pathogenic for rabbits, highly so for guinea-pigs. In man, in infected compound fractures and in puerperal infections, it has been found. Gangrene foudroyante (emphysematotis gangrene) seems to be due to infection by Welch's bacillus. It has been found in some instances in the apparently normal intestine. SYMPTOMATIC ANTHRAX. 171 Bacillus Botulinus. History. — Yon Ermengen in 1896 discovered the Bacillus botulinus (botulinus, a sausage) as the cause of an outbreak of meat poisoning. He recovered the bacilli from fatal cases of sickness produced by eating infected ham, finding the bacilli in the stomachs and spleens. He also recovered similar bacilli from the remains of the infected ham. Morphology and Staining. — This bacillus is from 4 to 6 //. in length and from 0.9 to 1.2 /j. in width. It has rounded ends and occurs either singly or in small chains. It is flagellated and slightly motile. In certain cultures spores are formed, especially at low temperatures (20° C.). Biologic Characters. — Bacillus botulinus is an obligate anae- robe, growing best at a temperature below 37.5° C. and above 20° C., the optimum temperature being 25° C. It grows on all culture-media, provided the reaction be neutral or slightly alkaline. Gas is formed in the cultures. Milk is not coagu- lated. It grows best on gelatine, which is liquefied. In the liquefied zone surrounding the colonies the constant motion of the growth may be noticed, and this is regarded by von Ermengen as characteristic. In dextrose bouillon cloudiness and gas are formed. Pathogenesis. — The injection of meat, usually ham or sau- sage or canned meat, infected with B. botulinus, gives rise to symptoms after an incubation of about thirty-six hours.- The symptoms are those of a profound intoxication, and most se- vere in character ; strangely, vomiting and purging are rare. Headache, chilliness without fever, trembling, and giddiness are followed in severe cases by dilated pupils, loss of voice, inability to swallow, and loss ot consciousness. In fatal cases dyspnoea and general paralysis are the precursors of death. Laboratory animals are very susceptible, guinea-pigs, cats, mice, and monkeys especially so. Rabbits are more resistant, and birds even more so. The bacilli may be recovered from the spleen after death. Lesions are those of a parenchymatous degeneration of the organs, with general hypereinia and widely distributed minute hemorrhages. 172 TETANUS, MALIGNANT (EDEMA, ETC. Toxins and antitoxins have been demonstrated. It is be- lieved that there is very little, if any, toxin production within the human host. The train of disastrous symptoms following the swallowing of infected meat are supposed to be due to toxins formed in the meat before it was ingested. 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 toxicity 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 ? How does it stain ? How does it behave in the presence of oxygen? How does it grow on different media ? What animals are susceptible ? How are inoculations performed ? What symptoms are produced by inoculation? By whom was the 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? TYPHOID FEVER. 173 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 ? What is anaphylaxis? How is active immunization practised on animals with extract of infected meats ? What is Bacillus aerogenes capsulatus ? The Bacillus botulinus ? CHAPTER XVII. 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 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 174 TYPHOID FEVER. 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. // Bacillus fyphosus, from culture Bacillus typhosus, showing flagella twenty-four hours old, on agar- stained by Loeffler's method. (Abbott.) agar. (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, punctiforrn bodies, becoming in a short time round and 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. PLATE IV Bacillus of Influenza in Sputum. (Abbott.) BACILLUS TYPHOSUS. 175 On lactose -litmus agar colonies arc 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, although it causes acid formation in glu- cose, but not in lactose or saccharose media. For isolating Bacillus typhosus from blood, Conradi's bile medium is to be recommended. For isolation from water, urine, or fasces Jackson's medium gives better results (see section on Culture- media for these media). It does not produce indol in such quantity as is detected by the ordinary tests. Vitalityv — 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. Precipitins and lysins have also been demonstrated. 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, in which the bacilli have been recovered in the general cir- culation and in the internal organs. The feeding of aiamuls 176 TYPHOID FEVER. 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. The usual sources of infection are water or milk ; in a lesser degree vegetables and fruit con- taminated with human dejecta. Flies, especially the common house-fly, have been found to be potent carriers of these bacteria. 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 PLATE V Bacillus of Bubonic Plague. (Abbott.) A. In pus from suppurating bubo. B. The bacillus very much enlarged to show peculiar polar staining. nirFERENTfATTON OF THE J3ACILLUS TYPHOSUS. Ill II. Widal's and Chantemesse's Differentiation. — Two tubes of agar or gelatin to which 2 per cent, of lactose-sugar has 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. Jf 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 12— M. B. 178 TYPHOID FEVER. 0.5 per cent, solution of phenolphthalein in 50 per cent, alcohol. The acidity oi the juice should be such as to re- 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 wafer 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. V. The Bacillus fsecalis alkaligenes, described by Petruschky in 1896, which is very nearly identical in appearance and culture qualities, does not produce acids in media containing dextrin, levulose, maltose, or mannit, and B. typhosus, on the contrary, does produce acid in these media. DIFFERENTIATION OF THE BACILLUS TYPHOSUS. 179 VI. The para-typhoid group of bacilli, which are generally classed as intermediate between B. coli and the B. typhosus, can be distinguished from B. typhosus on account of the power of the para-typhoid group to ferment dextrose. They (the para-typhoid group) are unable, however, to coagulate milk, nor do they produce indol in peptone solutions. This differ- entiates them from the B. coli, which possesses these prop- erties. 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. The Bacillus typhbsus has occasionally been obtained from 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. Cultures may be obtained from the blood of the finger, ear, or, prefera- bly, a superficial vein early in the disease by using the Conradi medium and plating the growth at the end of twenty-four hours. From the faeces, usually after the second week of the disease, and for an indefinite period (in typhoid carriers) the Bacillus typhosus may be recovered. Numerous workers have reported the isolation from the rose spots and some even from the sputum of typhoid-fever cases. 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 180 TYPHOID FEVER. 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 : 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. AVith 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 12,000 cases, this test Till-: BLOOD-SERUM DIAGNOSIS OF TYPHOID FEVER. 181 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). FIG. 65. Outfit used by the Municipal Laboratory of New Orleans for the collection of blood for the typhoid fever test. 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. The blood only of fever patients is to be used. Should the report be negative 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. 182 TYPHOID FEVER. 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 agglutination reaction is capable of manifestation macroscopically, even with killed bacteria, and many preparations and methods of their use are in vogue. Their advantage, of course, lies in their stability and lack of dangerousness, but with the availability of free board-of-health laboratories, whence returns may be received in twenty-four to forty-eight hours, it would seem that even in the remotest communities the promiscuous use by general practitioners of these various " diagnostica " would mean the needless sacrifice of accuracy to haste. 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. Similar injections of vaccines have been practised by the Germans in East Africa. The United States government has given its approval to this method. The recruits in nearly all the armies of the world receive the preventive inoculation. Nurses and physicians exposed to infection take advantage of the method. The usual dose of the first injection is 500,000,000 dead B. typhosus. At the end of the first and second weeks injections of double this amount are given. As the favorable reports of the active immunity produced from all over the world cover several hundred thousand cases, this method of preventive inocula- QUESTIONS 183 tion against typhoid fever may be considered of certain value. During the immunization no evil effects of any great conse- quence are usually experienced. 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. Antityphoid Serum. — Bokenham succeeded in immunizing a horse by using a filtered bouillon culture of the typhoid bacil- lus, and he claimed 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 ami when wTas 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? What are the points of difference between the Bacillus typhosus and the Bacillus coli communis ? 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 comminiis 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 overcom6? 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. 184 BACILLUS COLI COM MUNIS. CHAPTER XVIII. Bacillus Coli Communis. 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 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. 6. 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. BACILLUS COLI COMMUNIS. 185 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 sho\vn 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., 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 b,e 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. A type of B. coli, styled by Dunham Bacillus coli communior, has been isolated. It is supposed to bo a distinct variety. The only present known difference from B. coli communis is 186 BACILLUS COL1 COMMUNIS. that B. coli communior produces gas from saccharose and B. coli com munis does not. Pathogenesis. — Bouillon cultures of Bacillus coli com munis injected intravenously or into the peritoneal cavity of a rabbit cause death in less than twenty-four hours. On autopsy in- tense hypersemia of the peritoneum, ecchymotic spots of the intestines, swelling of Payer'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. Agglutinins, precipitins, and lysins against B. coli have been demonstrated in immune sera. Bacillus Proteus Vulgaris. History. — Described by Hauser in 1885. Its distribution is wide in nature, and it is of interest chiefly on account of the fact that it is a frequent source of contamination in cultures. Morphology and Staining. — In appearance B. proteus re- sembles the members of the typho-colon group, but varies more in size. It stains by all the ordinary stains and not by Gram's method. Biologic Characters. — It forms no spores, and grows best in an atmosphere at about room temperature. The optimum temperature for its growth is 25° C. It grows readily on all culture-media. Blood-serum is usually liquefied. The growth on gelatine, however, is characteristic, the colonies being irreg- ular (protean) and the gelatin liquefied. Milk is rendered acid and coagulated at first with subsequent liquefaction. On potato a dirty yellowish growth results. Pathogenesis. — As the bacilli of this group have always boon regarded as pure saprophytes, flourishing where putre- faction existed, very little pathogen icity has been ascribed to them. Some cases of meat poison have, however, been traced to members of this group. The Bacillus liquefaciens septicus of urine, which is a factor in the production of a cystitis, has been described by Krogius and no doubt belongs to this group. ASIATIC CHOLERA. 187 QUESTIONS. When and by whom was the Bacillus coli communis discovered ? When* 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 lint-illus coli communis. I l<>\v 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? Onlactose-litmus-agar? On potato? In milk? In Donovan's peptone solution ? What is the effect of intraperitoueal and intravascular inoculations in animals? What lesions are found at the autopsy ? What lesions are produced by subcutaneous inoculation ? By intrapleural inoculation ? What is the Bacillus proteus vulgaris? Its morphology? Biologic char- acter ? Pathogenesis ? CHAPTER XIX. ASIATIC CHOLERA. Spirillum Cholerse Asiaticae (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 SptriUum cholcrcc 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- 188 ASIATIC CHOLERA. ures as a short curved rod, from 0.8 to 2 mikrons in length, by 0.3 to 0.4 mikron 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 Asiaticce stains with all the anilin dyes, but rather poorly. It seems to have a more active FIG. 66. **A 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 only a flagellum at one 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 cannot 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 SPIRILLUM CHOLERA ASIATICM 189 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. Stab-culture of the spirillum of Asiatic cholera in gelatin, at 18° to 20° C. : a, after twenty-four hours; b, after forty-eight hours; e, 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 190 ASIATIC CHOLERA. 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. SPIRILLUM CHOLERA AS1ATICM 191 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 wrhite 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. — One attack protects against being subsequently infected. 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. 192 ASIATIC CHOLERA. 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. This growth is accom- panied by abundant indol production. Upon addition to such a culture of sulphuric acid the well-known cholera-red reaction takes place. Other 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 condensation 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. Several closely allied spirilla have been described by Metchnikoff, Finkler and Prior, and others, but they do PLATE VI FIG. I J Spirocheta Paliicla. Smear from Hard Chancre, Giemsa's stain. X 1OOO. FIG 2 J | M 0, Spirocheta Refringens. Smear from Chancroid. X 10OO. INFLUENZA. 19:1 not give the " cholera red " reaction, with the exception of the first named. The spirillum of Metchnikoff is very poisonous to pigeons, which are not susceptible to the cholera vibrio. QUESTIONS. When and by whom was the Spirillum cholerse Asiatics (comma bacillus) discovered ? Describe the spirillum. What is the peculiar arrangement of the bacteria in cultures and secre- tions ? 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 dryness ? 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 intraperitoueal 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? OHAPTEli XX. 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- is— M. B. 194 INFLUENZA. 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.) 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. Pigeon blood seems even better. 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 BACILLUS OF INFLUENZA. 195 lungs for a considerable length of time; especially is this^the case with tuberculous patients, in whose sputum is very often found the Bacillns influenzce. By inoculating animals in the brain, the nervous phenomena of this disease have been easily reproduced. One attack of influenza does not seem to give immunity against another. Closely resembling the Bacillus influenza, but unlike it, in growing without the presence of haemoglobin in artificial media, is the Koch- Weeks bacillus. This bacillus is found in a certain form of conjunctivitis of an acute and apparently epidemic nature. These bacilli are somewhat longer and thinner than B. influenza, and, unlike the latter, can be grown on serum-agar. There is another bacillus found in rabbits causing pleuro-pneumonia in them, described by Beck as motile and growing in all media. Pertussis Bacillus. History. — From a case of whooping-cough Bordet and Gengou in 1900 isolated a bacillus, but it was a few years later before cultures were successful. Morphology. — The Bordet-Gengou bacillus is a small, ovoid bacillus found scattered among the pus-cells early in cases of whooping-cough. Sometimes they occur in pairs, end to end, and on account of their shortness are sometimes taken for cocci. Staining. — This bacillus may be stained with Loeffler's blue or dilute carbol-fuchsin. By Gram's method it is not stained. Cultivation was successful on a special medium composed of potato, glycerin, agar, and defibrinated rabbit's blood. The optimum temperature for growth is 37.5° C. Pathogenesis. — Animal inoculation has been negative. The almost constant presence of these bacilli in the sputum of early cases of whooping-cough is the only argument for its etiological significance. It is readily differentiated from B. influenza, which it re- sembles by its inability to grow readily on haemoglobin media. 196 BUBONIC PLAGUE. 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 ? 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 ? Describe the pertussis bacillus and its peculiarities. CHAPTER XXI. 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 BACILLUS PESTIS. 197 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- 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 does not cloud bouillon. Cultures in this medium show a number of flocculi in the tube and a deposit at the bottom. It does not cause fermentation, and it gives no indol reaction. It coagulates milk. Vitality. — The Bacillus pest-is is very susceptible to the action of disinfectants, 1 per cent, carbolic acid being suf- ficient to kill it in one hour. Pathogenesis. — Man, mice, rats, guinea-pigs, rabbits, cats, hogs, horses, chickens, and sparrows are very susceptible to the disease. Squirrels are proved to be of considerable danger as carriers. Pigeons, dogs, amphibia, and bovines appear to enjoy immunity. During an epidemic of bubonic plague sus- ceptible animals seem to contract the disease naturally. For experimentation subcutaneous inoculation with liquid cultures of the bacillus is generally resorted to. The changes produced are : Swelling of oedematous character at the point of inoculation, and involvement of the lymphatic glands; death resulting in from twenty-four to forty-eight hours. At the autopsy the local swelling is found to be due to an oadern- atous condition of the part, the bloody fluid containing a large number of bacilli. The neighboring lymphatic glands are also greatly inflamed, and some of them are found sup- purating. In their substance the pest bacilli are also found in great number. There also occurs a purulent exudate in the peritoneal and pleural cavities. The internal organs, liver, lungs, adrenal bodies, and spleen are very much affected. Three forms of the disease are recognized in man : the bubonic or ganglionic, the septicaemic, and the pneumonic form, the most frequent of these being the bubonic, and the most fatal the pneumonic. 198 BUBONIC PLAGUE. Infection generally takes place through an abrasion of the skin, but the disease may be caused by inhalation of the pest bacilli. The usual form of the disease presents the following symp- toms : a sudden rise of temperature accompanied by great prostration and delirium, and the occurrence of lymphatic swellings (buboes) affecting chiefly the glands corresponding to the inoculated portion. These become very much enlarged, and have a tendency to soften and suppurate. In severe cases death occurs in forty-eight hours ; in others, the dura- tion of the disease is somewhat longer. The prognosis is more favorable, the longer the duration of the disease. Char- acteristic bacilli are found in the lymphatic glands, and also occasionally in the blood. Immunity. — Persons who have recovered from an attack of bubonic plague or animals that have survived inoculations are found to be immune for a certain period of time. This immunity is due to a substance developed in the serum of those animals, which may also when inoculated into suscepti- ble animals protect them from infection with bubonic plague. Artificial immunity may also be conferred by injecting cult- ures of the dead bacilli. Agglutination. — The serum of immune animals possesses also an agglutinating action when mixed with bouillon cult- ures of the Bacillus pestis, very much the same as the agglu- tinative action of typhoid or cholera serum. Precipitins are also found. Serotherapeutics. — Yersin claims to have developed a serum in the horse which is not only protective, but also curative, when injected into the human being. His experiments, carried on in a number of cases, seem to indicate this serum to have some decided beneficial effect when administered early in the disease, the proportion of deaths in cases so treated being scarcely 8 per cent., whereas the mortality in non- inoculated cases is as high as 80 per cent. To obtain the blood-serum from horses, he immunizes them with the dead bouillon cultures of the Bacillus pestis. Haffkine has practised on an extensive scale protective in- DYSENTERY, NOG AND CHICKEN CHOLERA. 199 oculations against bubonic plague by injections of dead cult- ures. This immunity seems to last for several weeks. Recently, observations have demonstrated that Yersin r-iMMim has absolutely no protective or curative properties. Ilaffkine's protective inoculations are still held in favor, however. QUESTIONS. By whom and when was the Bacillus pestis discovered? Describe this bacillus. Its mode of staining. Its principal biologic char- acters. What temperature suits its growth best? What is the effect of high tem- perature? How does it grow on gelatin ? On agar ? On bouillon ? What is its behavior with regard to fermentation and to indol pro- duction ? How does it affect milk? How is it affected by disinfectants? What animals are susceptible to this disease? How are inoculations performed? Describe the symptoms and lesions caused by inoculation. What three forms of bubonic plague are recognized in man? Which is tiie most frequent? Which the most fatal? How does infection take place? What are the symptoms of the disease, and what the lesions in man ? How is immunity conferred in this disease? Does the serum in cases of plague contain agglutinating power? How does Yersin manufacture his protective serum? What does he claim for it ? How does Haffkine practise his protective inoculations? CHAPTER XXII. DYSENTERY, HOG CHOLERA, AND CHICKEN CHOLERA DYSENTERY. Bacillus Dysenteriae. History. — This bacillus was first found in the intestinal con- tents and in the visceral walls and mesenteric glands in cases of acute epidemics of dysentery by Shiga, in Japan in 1898, and this observation was confirmed afterward by Flexner in a study of dysentery of the Philippine Islands. It seems to belong to the typho-colon group. 200 DYSENTERY, HOG AND CHICKEN CHOLERA. Morphology. — The Bacillus dysenteries is of medium size, with round ends, containing no spores. The flagellate char- acter and motility of the B. dysenterise have been denied by many observers. It stains by the ordinary anilin dyes, but not by Gram's method. Biologic Characters. — It is aerobic, but may be grown with- out oxygen. It grows at the ordinary room temperature, but best at the temperature of the human body, and does not liquefy gelatin. Its growth on agar is not characteristic, slightly resembling the typhoid growth, and on gelatin the growth is pearl col- ored, somewhat like the typhoid, but later becomes moist. On potato it sometimes has also an invisible growth ; at other times its growth is rather voluminous and grayish brown in color. It clouds bouillon without forming a pellicle on the surface. It does not liquefy blood-serum. Litmus milk at the end of three days becomes of a pale lilac color, but the milk is not coagulated. In six or seven days the medium becomes dark blue. It produces no indol. By means of fermentation tests Hiss has divided the dysentery 'bacilli into four groups : Group I. ferments only dextrose. Group II. ferments dex- trose and mannit. Group III. ferments dextrose, mannit, and saccharose. Group IV. ferments dextrose, mannit, mal- tose, saccharose, and dextrin. None of these groups liquefy gelatin. None are capable of forming acid from lactose and none produce gas in carbohydrate media. Agglutination. — The serum of affected animals has an agglutinating power on young cultures of the bacillus. By agglutination methods group differentiation has been proved. It is pathogenic for laboratory animals. When injected intraperitoneally into animals it produces a purulent peri- tonitis, with involvement of the mesenteric glands and swollen spleen, and the liver is covered with an exudate. The intes- tinal glands and Peyer's patches show signs of inflammation. The bacillus may be recovered from the exudate, and also in limited quantity from the organs. Subcutaneous injections show swelling and oedema at the point of inoculation, with HOG CHOLERA. 201 involvement of the lymphatic glands, and are also followed by effusion in the serous cavities. By alkalinizing the secretions of the stomach, animals have been infected by feeding with the bacillus, and in those ani- mals lesions very much resembling the disease in man have been reproduced, and pure cultures of the bacillus obtained from the secretions of the intestines. Both toxins and endotoxins have been demonstrated in cultures of B. dysenteric, and antitoxins have been demon- strated in immune animals. That the poison is in the cell-body of the Bacillus dysen- teries, and is not a secretion of the cells, is demonstrated by the fact that by heating cultures to a temperature above 60° C., which kills the bacilli, does not seem to have any effect on the activity of the poison. Protective inoculations in animals have been performed with positive results, and the serum of immunized animals has been found to possess the power of agglutination, and to be in some degree both protective and curative. HOG CHOLERA. Bacillus Sui Pestifer. History. — In the dejecta of hogs affected with cholera Salmon and Smith have succeeded in isolating a bacillus which they found to be the specific cause of this disease. Morphology. — It is a short, thick rod, 1.20 to 1.50 mikrons in length, and 0.6 to 0.7 mikron in breadth, actively motile, containing flagella, stains by all the anilin dyes, but not by Gram's method. Biologic Characters. — It is aerobic and grows in all the cult- ure-media. Its growth on gelatin is visible in from twenty- four to forty-eight hours ; the colonies appear irregularly round and the gelatin is not liquefied. On agar-agar the colonies are translucent and rather circumscribed. Upon potato the colonies are yellow. Bouillon is clouded and a thin surface growth may be observed. In milk it does not generate acids and does not coagulate it. 202 DYSENTERY, HOG AND CHICKEN CHOLERA. It produces gas copiously, but no indol. Vitality. — It withstands drying for a long time. Its ther- mal death-point is 54° C. Pathogenesis. — It is intensely pathogenic for every labor- atory animal, death being preceded always by a rise of temperature, and postmortem lesions affecting chiefly the liver and kidneys are seen. Sometimes the lesions are found in the intestines and Peyer's glands also. The bacillus is found in all the organs. Artificially swine are inoculat ^ with dif- ficulty. Immunity in animals has been produced by Salmon and Smith by injections of gradually increasing doses of cultures of this bacillus. De Schweinitz has isolated from cultures of the bacteria pure toxic substances with which he has been able to produce immunity. By subcutaneous inoculations with these toxins in cows he was able to develop in their blood-serum an antitoxic substance capable of protecting ani- mals from the disease. The serum of infected animals has a remarkable agglutinating power; with a dilution of 1 to 10,000, agglutination can be obtained in an hour. CHICKEN CHOLERA. Bacillus Choleras Gallinarum. History. — This bacillus was observed by Perroncito, in 1878, and described by Pasteur. The cause of the disease known in fowls as chicken cholera is due to a short, broad bacillus, with rounded ends, occurring singly or united to form filaments. This bacillus stains in a peculiar way with the anilin dyes, its two poles being markedly stained, whereas the centre of the bacillus is scarcely stained at all, giving it very much the appearance of a micrococcus, which it was at first believed to be by Pasteur. It does not stain by Gram's method. Biology. — It produces no spores and is non-motile. It is easily killed by heat and drying. It grows on all ordinary culture-media. On gelatin in two days the cultures appear to the naked eye as small white points ; under the microscope CHICKEN CHOLERA. 203 the colonies are granular and concentric. It does not liquefy gelatin. In stab-cultures its growth on this media resembles that of a nail with a flat head, the head of the nail being closer to the surface of the medium than the point. Its growth in agar and bouillon offers nothing character- istic. The bacillus is strictly aerobic. Pathogenesis. — Chickens, geese, pigeons, sparrows, mice, and rabbits are susceptible animals. Guinea-pigs are im- mune. By inoculation the disease produced in susceptible animals is that of a general septicaemia, the bacillus being found in the blood and all the internal organs. By feeding the contaminated material to animals the lesions are limited to the intestines, having the appearance of true cholera. Protective inoculations have been performed by using atten- uated cultures, the attenuation being arrived at, as recom- mended by Pasteur, by using cultures two or three months old. This bacillus has been used extensively in Australia for the destruction of rabbits. It is said that Avith two gallons of a bouillon culture as many as 2000 rabbits may be destroyed. This bacillus has been described by different authors under a number of names : as the bacillus of rabbit septicaemia, by Koch ; the bacillus of swine plague, by Loeffler ; Bacilli cuni- cucilidi, by Fluegge, and others. MORAX-AXENFELD BACILLUS. History. — In some cases of subacute conjunctivitis Morax, in' 1896, found a diplobacillus which he described as the etio- logical factor. Axenfeld, shortly after, corroborated his findings. Morphology and Staining. — This bacillus is about 2 //. long, ovoid in shape, and generally placed end to end in pairs. It is stained readily by the usual aniline dyes and not by Gram's method. Biologic Characters. — It grows on all media containing blood-serum and of alkaline reaction. Loeffler's blood-serum mixture is liquefied. 204 DYSENTERY, HOG AND CHICKEN CHOLERA. Pathogenesis. — In man a subacute conjunctivitis is produced by this bacillus. Animal inoculation has been negative. BACILLUS OF DUCREY. History. — Ducrey, in 1889, in the discharges from chan- croids and their concomitant buboes was able to demonstrate these bacilli constantly. Morphology and Staining. — This bacillus is extremely small, scarcely half a micron in width. In length it measures from one to two micra. It occurs in short chains, or in parallel rows, or in irregular groups. In pus these bacilli are usually found in pus-cells. They are stained readily but irregularly by the ordinary aniline dyes, the poles staining more deeply. By Gram's method they are decolorized. Biologic Character. — B. Ducrey is non-motile, non-flagel- lated, and non-spore bearing. It is artificially grown with great difficulty. The best growths have been obtained on agar containing human blood. Growths on coagulated blood, agar, and rabbit's blood, and even serum agar have been suc- cessful. The pus obtained directly from the bubo with a sterile needle is placed immediately on the culture-medium. The material obtained by scraping from the deeper portions of the chancroid may be used. Small transparent gray firm colonies usually appear after forty-eight hours. Pathogenesis. — Successful inoculation has been practised on man by Besan9on and others. Animal inoculation has been negative. QUESTIONS. Dysentery. When and by whom was the Bacillus dysentericse discovered and described ? What is its morphology ? How does it stain ? Give its principal biologic characters. How does it grow on agar? On potato ? On bouillon ? In litmus milk ? Does it produce indol? Does its serum have an agglutinating power ? What is the effect of an intraperitoneal injection in animals? Of a sub- cutaneous injection? SPIRILLA. 205 How are animals affected by feeding of the bacilla ? Where is the toxin of the Bacillus dysentericse contained? May animals be immunized against dysentery by inoculation with Bacil- lus dysentericse ? What claims are made for the serum of inoculated animals? Hog Cholera. By whom was the bacillus of hog cholera discovered ? Give its morphology. Its staining. Its principal biologic characters. How does it grow on gelatin and agar ? On potato ? In bouillon ? In milk? Does it produce gases? What is its thermal death-point? How does it affect laboratory animals? Has immunity been produced by injections of cultures of this bacillus ? Has a toxin been isolated from the bacillus of hog cholera ? What are the properties of the serum of immunized animals ? Describe the Morax-Axenfeld bacillus. Chicken Cholera. By whom was the Bacillus choleras gallinarum discovered, and by whom described ? Describe this bacillus. How does it stain ? Give its principal biologic characters. What is the appearance of the cultures on gelatin to the naked eye ? How do they look when seen under the microscope? What animals are subject to infection ? Which animals are immune? What sort of infection is produced in animals by the inoculation of this bacillus ? What is effected by feeding animals with material contaminated with this bacillus ? How are protective inoculations performed? To what extent is this bacillus pathogenic to rabbits? Under what different names has this bacillus been described by various authors ? CHAPTER XXIII. SPIRILLA. ALTHOUGH spirillse and spirochsetae are divisions of the spirillacese and belong to the bacteria, there are several organisms which are labelled spirochsetse and spirillae about whose vegetable nature there is a considerable doubt. The difficulty attending their classification is increased by the fail- 206 SPIRILLA. lire of the many attempts to grow many of these organisms. Schaudinn, working principally with the " Spirochseta " pal- lida, seems inclined to classify them as protozoa. RELAPSING FEVER. Spirillum Obermeieri. History. — As early as 1873 Obermeier discovered in the blood of patients suffering from relapsing fever a long, spirillum-like microorganism, measuring from 20 to 30 mikrons, having the power of active motion. His observations have since been confirmed by a number of other investigators. This spirillum, which has not been cultivated artificially, is found in the blood and spleen, but never in the secretions of patients affected with relapsing fever. Morphology and Biology. — It stains readily by all the anilin dyes, but does not stain by Gram's method. It is actively motile and contains no spores. In the blood it is found in two forms: (1) during the pyrexia as long twisted filaments ; (2) after the crisis of the fever is reached it is seen in the leucocytes as short degenerated curved rods. Pathogenesis. — This spirillum is not pathogenic to the lower animals, with the exception of the monkey, rat, or mouse. Blood taken from patients during the paroxysm when inoc- ulated in other individuals may give rise to relapsing fever. One attack of the disease seems to confer immunity from future attacks in a greater or less degree. Agglutinins have been demonstrated in immune sera. Dulton and Todd have demonstrated that many of the native African fevers are due to spirochaetee which they con- sider identical with those of Obermeier, although the former are somewhat smaller and the clinical symptoms produced by them milder. They have shown that the African disease is transmitted by a tick (the Ornithodorus moubata). SPIROCHJETA PALLIDA. 207 SPIROCILETA PALLIDA. Treponema Pallida. History. — Schaudinn and Hoffman in 1905 discovered in primary and secondary lesions of syphilis spirochaetse differ- ing considerably from the previously known ones. He called them Spirochceta pallida (Plate VI, Fig. 1). On account of his belief in their animal nature he has since renamed them Treponema pallida. Morphology and Staining. — The Treponema pallida is dis- tinguished by its extreme thinness and the acuteness of its curves or spirals, which are almost angular and usually aver- age ten in number. In fresh preparations, seen best with the aid of a dark field illumination, they are observed to be very active, their motion being mostly of a rotary character, though occasionally they may be seen to bend on themselves. The Treponema pal/Ida measures from 4-10 p. in length, and rarely exceeds 0.5 p in width. The Treponema pallida are best demonstrated in fresh prep- arations. The aid of a condenser for dark field illumination is almost a necessity, although sometimes under favorable condi- tions they may be demonstrated with an ordinary condenser with the diaphragm closed as for urine work. The spirochsetse are very refractile and show as glistening, undulating threads. For this method of examination, as well as for making smears, it is necessary to obtain the material from the deeper layers of tissue, using a curette if necessary. The admixture of blood- corpuscles should be avoided, as the serum is best. For mak- ing stained preparations all the ordinary blood stains are useful, Giem.sa's method being probably the best. The Treponema pal- lida does not stain with the ordinary anilin dyes. If a drop of India-ink and a drop of syphilitic material be mixed and a smear made and allowed to dry, the Treponema pallida will show unstained on a contrasted black background. For begin- ners with untrained eyes this is an uncertain method, and must not be recommended to any one except for its quickness. Schaudinn described a larger, coarser, more highly refractik spiral which he afterward found in company with the Trepo- 208 SPIRILLA. nema pallida. He called it the Spirochaeta refringens (Plate VI, Fig. 2). Its curves are fewer and more rounded, and in stained preparations it stains more deeply than Treponema pallida. Schaudinn regards its finding as accidental and unimportant. For staining in tissues Levaditi's method is most used, as follows : 1. Cut fresh tissue in cubes 2-4 m.m. square. 2. Harden in solution formalin (10 per cent.) for twenty- four hours. 3. Wash in distilled water. 4. 96 per cent, ethyl alcohol for twenty-four hours to de- hydrate. 5. Wash in distilled water. 6. Place in 3 per cent, nitrate of silver solution for three to five days at a temperature of 37.5° C. and in the dark. 7. Give the tissue a short rinsing in distilled water. 8. Place for twenty-four hours in fresh solution of pyrogal- lic acid, 2 per cent., formalin, 5 per cent., in distilled water. 9. Wash in distilled water. 10. Dehydrate in ethyl alcohol of graded strength, from 25 per cent, to absolute. 11. Imbed in paraffin and make thin sections. 12. Mount and examine. By this method the treponema show as blackened, untrans- parent spirals. Pathogenesis. — The treponemata are pathogenic for all races and ages of man. They have been found in all stages of syph- ilis— in the chancre, lymph-nodes, and even in gummata. In cases of congenital syphilis treponemata have been demon- strated in all the foetal organs. Animal Inoculation. — After many failures by injection of syphilitic material, successful inoculations have been made by many investigators by the implantation of pieces of fresh syphilitic material under the skin of the eyelid in the genital mucous membrane. In these cases successful inoculations were followed by definite syphilitic lesions, and Treponema pallida recovered from the primary sore and the enlarged lymphatics. Immunity. — The fact that one attack o£ syphilis confers SPIROCHuETA PALLID A. 209 immunity against a second has been a long known clinical doctrine. Diagnosis. — Wassermann Reaction. — The finding of the tre- ponenia in the lesions constitutes the best means of diagnosis at our command. In the absence of this proof recourse has been had of late years to a reaction based on the principles enunciated by Bordet and Gengou, elaborated by Wassermann, and altered and simplified by Noguchi and others. We have learned that when infection has taken place or when certain substances are injected, that antibodies or amboceptors specific for the cause of the infection or for these substances are pro- duced in excess. We have also learned that in the presence of complement or alexin union will take place between ambo- ceptors and their specific poison or antigen. The complement is necessary in the union and is utilized in its consummation. Given then a known antigen (made of syphilitic material) and a known definite complement, we can tell, by the addition of an unknown serum and proper incubation, whether the un- known serum contains the specific syphilitic amboceptors. If it does, the union will take place between the amboceptors and the antigen, and the complement will be consumed in bringing this union about. The complement will, therefore, not be available for future use. If the specific amboceptors are not present in the unknown serum, no union is possible between the complement and antigen. Complement, there- fore, will still be free and available for further use. The positiveness or negativeness of the above reaction is demon- strated by making use of a hsemolytic reaction in which com- plement, of course, is needed. If the reaction has been pos- itive, no complement will be available and no haBmolysis will take place. If negative, the complement will enter the hsemolytic combination of complement hsemolysin corpuscles, and haBmolysis will take place. The technique, as given by Noguchi, is briefly as follows : Seven small test-tubes are used, and contain the following: Tube 1. — 1 drop patient's serum + complement 4- antigen. Tube 2. — 1 drop patient's serum + complement (no anti- gen). 14— M. B. 210 SPIRILLA. Tube 3. — 1 drop known syphilitic serum + complement + antigen. Tube 4- — 1 drop known syphilitic serum + complement (no antigen). Tube 5. — :1 drop known normal serum -f complement 4 antigen. Tube 6. — 1 drop known normal serum + complement (no antigen). Tube 7. — Complement alone. To each tube 1 c.c. of 1 per cent, emulsion of human cor- puscles is added, and the mixture shaken and placed in an incubator at blood heat for one hour. At the end of one hour two units of amboceptor are added to each tube, and incubation again resorted to for one hour. In tube 3 certainly, and in tube 1 if the patient's serum is syphilitic, no haemolysis will take place, for the complement will be bound, as explained before. If patient's serum is not syphilitic, haemolysis will take place in tube 1 . By drying amboceptors and antigen in measured quantities on blotting- or filter-paper, Noguchi has further simplified the technique, as well as furnishing staple reagents. The substances needed for the above tests are prepared as follows : 1. Patient's serum. 2. Fresh guinea-pig serum, obtained by bleeding a guinea- pig into a sterile Petri dish. For making the test 0.1 c.c. of a mixture of 1 c.c. of serum to 1.5 c.c. of normal salt solution is used. 3. Antigen. — This consists of the acetone insoluble residue of syphilitic organs which have been previously macerated and extracted with alcohol, evaporated, dissolved in ether, and pre- cipitated by acetone. This residue is dissolved in ether and mixed by shaking with salt solution until the ether has evap- orated. About 0.2 grani of the sticky residue is used to the 100 c.c. of salt solution. In its finished state it is opalescent green without sediment. 4. Human Corpuseles. — (This is the essential of Noguchi's modification, as the patient's own corpuscles may be used in- SPIROCHJETA PALLIDA. 211 stead of the sheep's corpuscles, as in the Wassermann test.) These are washed in salt solution and used in a 1 per cent, emulsion in salt solution, 1 c.c. being used. 5. Aniboceptor against human blood-corpuscles. This is obtained by the repeated injections of washed human blood- corpuscles into rabbits, the serum being tested by titration against human corpuscles. By reversing the above test unknown antigen may be determined. Yaws. Castellan! in 1905 described a spirochsete closely resembling Treponema pallida, which he found in cases of yaws. The question of their identity or non-identity is still sub judice. Vincent's Angina. History. — Vincent in 1896 described an inflammatory dis- ease of the mouth, pharynx, and especially of the tonsils, whose lesions resembled in appearance those of diphtheria, the course of the disease, however, being much milder. Morphology. — In the above-mentioned disease Vincent found and described bacteria of two kinds, a fusiform or spindle- shaped bacillus and a spirillum not unlike that of relapsing fever in appearance. Some bacteriologists claim that these differently shaped organisms are of a like nature, merely rep- resenting different developmental stages of the same organism. Others claim, however, that they represent distinct varieties of organisms living in symbiosis. In length the fusiform bacilli measure from 4—10 /JE, the spirilla from 6—12 //. The spirilla have shallower, more rounded, and fewer curves than the Treponema pallida. I have found a 1 : 10 carbol-fuchsin solution the best stain. Ordinary blood stains and even Loeffler's blue may be used ; Giemsa's blood stain gives splendid results. An irregular intensity of the stain is a characteristic of these bacteria. Pathogenesis. — As mentioned above, these organisms are found in inflammatory conditions of the mouth and pharynx, 212 SPIRILLA, either alone or associated with streptococci or other bacteria. Their actual pathogenicity or saprophytism has never been determined. Animal inoculation has had negative results. In some cases of noma Weaver and TunniclifF have found spirilla and fusiform bacteria in great quantities. I have also, in a case of noma which proved fatal, had some of the secre- tion scraped from the lesions submitted for examination, and found spirilla and fusiform bacilli accompanied by streptococci and staphylococci, all in great numbers. Spirochaeta Gallinarum. History. — Described as the etiological factor in an infectious disease of chickens and fowl by Marchoux and Salimenbeni in 1903. Morphology and Staining. — In shape it is almost the counter- part of the Obermeier spirillum, and stains readily with dilute carbol-fuchsin or any of the blood stains. Pathogenesis. — Chickens, geese, ducks, and pigeons are very susceptible. The disease is ordinarily spread among the fowl by a species of tick or by swallowing infected matter. Sub- cutaneous injection of infected blood produces the disease, which is characterized by fever, diarrhrea, and often ends fatally. The organism may be recovered from the blood and organs. Immunity has been induced by the injection of attenuated immune serum. It is generally believed that the Spirochazta anserince, de- scribed by Saccharoff in 1891, is identical with the above organism. QUESTIONS. What microorganism is the cause of relapsing fever? By whom and when was it discovered ? Describe it. How does it stain? May it be cultivated artificially ? Where is it found in cases of relapsing fever? Is it motile? Does it contain spores ? PATHOGENIC MICROORGANISMS. 213 In what two forms is it found in the blood ? For what animals is it pathogenic? May blood of relapsing fever give rise to other cases of the disease by inoculation? Does one attack of relapsing fever confer immunity upon the subject ? What is the Spirochseta or Treponema pallida ? Its morphology and staining? What is the Spirochseta refringeus? How distinguished from the pallida? What is Levaditi's method for staining the Spirocbseta pallida in tissues ? Pathogenesis of spirochaeta pallida ? Describe the Wassermann test in syphilis. Noguchi's modification. Describe the spirochsete of yaws. What is Vincent's angina ? Describe the Spirochseta gallinarum. What is Spirochseta anserhm>? CHAPTER XXIV. THE PATHOGENIC MICROORGANISMS OTHER THAN BACTERIA. ACTINOMYCOSIS, MALARIA, AND AMOEBIC COLITIS. Streptothrix. THE streptothrix group, which has not as yet been clearly defined, presents a number of varieties which have been found pathogenic. This group of microorganisms resembles the bacteria, yet differ from them in a number of important respects, and are associated, on the other hand, with the moulds. They resemble the moulds in so far that they develop from spore-like bodies into dichotomously branching threads, which grow into colonies having more or less the appearance of true myeelia. Under favorable conditions some of the threads become fruit hypha3 and break up into a number of spore-like bodies. These sporiform bodies differ from bac- terial spores in the fact that they are stained by the ordinary method of staining. They resemble the bacteria in the fact 214 PATHOGENIC MICROORGANISMS. that they occur as threads which may under careful cultiva- tion become divided into short segments. They do not have a double wall like the moulds, and are not filled with fluid containing granules, and the segments of the filaments have no distinct partition separating one from the other. The most thoroughly studied streptothrices are : the Strep- tothrix actinomyces, or ray fungus, the Streptothrix madurce, and the Streptothrix Eppingeri, all of which have been found associated with important pathological lesions and are be- lieved to be the cause of special diseases. The bacteria of tuberculosis and diphtheria are believed by some to belong to the class of streptothrices, because at times they show a tendency to form branched segments. This view, however, is not generally accepted. It is interesting to note that the lesions caused by the Streptothrix have very much the appearance of tuberculous lesions, being almost indistinguishable from tuberculosis except for the absence of the Bacillus tuberculosis. In diseases attributable to these fungi, microscopical exam- inations of the tissues reveal the streptothrices in the tissues, and these have been cultivated artificially, and by inocula- tions into animals have reproduced lesions identical with those of the original disease. The most important of the diseases caused by one of the streptothrices, and the only one which will be studied in this volume, is : ACTINOMYCOSIS. Streptothrix Actinomyces (Ray Fungus). History. — It was described first by Bollinger, in 1877, and found in the disease of cattle known as big-jaw or wooden- tongue, and contained in the tissues and exudates. In man the disease first described by Israel, in 1885, seems to be iden- tical with this cattle disease. Morphology. — In pus from the affected parts are small yel- lowish granular masses from 2 to 5 millimeters in diameter. Under the microscope these granules are seen to consist of a number of threads which radiate from a centre to a bulbous, ACTINOMYCOSIS. 215 club-like termination, the whole mass having very much the appearance of a rosette. Sometimes the free ends of the thread are only slightly or not at all swollen (Fig. 69). The threads which compose the centre of the mass are from 0.3 to 0.5 mikron in width. The clubs are from 0.6 to 0.8 mikron in width. These mycelia stain by all the ordinary anilin colors, and also by Gram's method, though by these methods their fine structure is not always brought out. The best stain for the fungus is Mallory's stain, which is as fol- lows : Stain the secretions on the slide with gentian-violet, dehy- drate, and clear with anilin oil to which a little basic fuchsin FIG. 69. Actinomycosis fungus in pus. Fresh, unstained preparation. Magnified about 500 diameters. (Abbott.) has boon added, and mount in xylol balsam. In this way the cocci in the centre and the threads of the mycelium are stained blue ; the club-like extremities are stained red. Biologic Characters. — The Streptotfirix actinomycex grows in all the ordinary artificial media, but from the fact that all the mycelia seen under the microscope are not living, it is often necessary to make several cultures before obtaining a satisfactory one. It grows both with and without oxygen, either at the room temperature or at the temperature of the incubator, and is not. resistant to hont, being destroyed by a temperature of 75° C. in five minutes. 216 PATHOGENIC MICROORGANISMS. Its growth on blood-serum and agar is as isolated colonies on the surface of these media. The colonies are yellowish red and covered with a sort of fluffy down. After a few weeks the colonies run together and form a thick wrinkled mass which sinks into the media. On gelatin the growth causes slow liquefaction. On potato the colony is yellowish red and limited in extent, has a viscid, membranous appearance, and is slow in progress. In bouillon it causes no clouding, but develops on the surface of the medium as a distinct granular growth forming a membranous film, which afterward sinks to the bottom of the tube. Pathogenesis. — Cattle are the most frequently affected animals, though the disease has been seen in swine, dogs, and horses. The common location of the disease is in the jaw. It is not transmissible from animal to man. Inoculations of pure cultures are negative, though some observers have suc- ceeded by intravascular inoculations in producing tumors from which the fungus was obtained in pure cultures. The disease is sometimes quite prevalent among animals, and appears to result from the ingestion of vegetable products which contain the streptothrix. In the earliest stages the parasites give rise to tumors resembling tuberculous growths, and as these reach a larger size there is proliferation of the surrounding connective tissue. The tumors are then very hard, resembling osteosar- comata. Suppuration finally takes place, due to the action of the fungus itself, or more probably to secondary infection of the tumor by pus-producing organisms. The Other Pathogenic Streptothrices. The streptothrix of madura foot, described by Wright, in the Journal of Experimental Medicine, 1898. and the Strepto- thrix farcinicus, discovered by Nocard in 1888, in a disease of cattle resembling farcy in horses ; the Streptothrix Eppin- geri, discovered by Eppinger in a case of acute abscess of the brain ; and the Streptothrix pseudotuberculosa, described by Flexner in 1897, are of interest, and the reader is referred to larger works on Bacteriology for their description. THE YEASTS -THE-MOULDS. 217 THE YEASTS. The yeasts, on account of their multiplication by budding, form a distinct class among fungi. They are widely sepa- rated in their morphology from the schizomycetes or cleft fungi (bacteria), while between them and the hyphomycetes or mucorini (the moulds) there are occasional features of simi- larity. This is especially shown in the occasional formation in certain yeasts of hyphse and mycelia. The yeasts ordi- narily consist of cells averaging 15 /;. in length and 10 n in width, and consisting of a cell membrane and protoplasm con- taining vacuoles and highly refractile globules and nuclear granules. Multiplication takes place by the extrusion of the cell membrane of the mother-cell to form the daughter-cell (budding). For a time the maternal protoplasm continues to enter the daughter-cell freely and nourish it until the grad- ual narrowing of the connecting isthmus between the two is cut off, as the daughter-cell reaches maturity. Yeasts are more of interest in the trades, but the reports at different times by Busse, Zinnser, and others of human infec- tion by the Saccharomyces hominis have naturally drawn the attention of medical men more strongly to the subject. THE MOULDS. The hyphomycetes or moulds constitute the third division of the fungi, as suggested by Cohn (p. 28). They occur ex- tensively in nature and constitute a frequent source of con- tamination in cultures, on which they appear as a fluffy white light layer, having the appearance of adherent cotton or wool. This layer, if examined, will be found to consist of a number of interlacing threads or mycelia, from which larger branches, called hyphcBj extend to terminate in bulbous extremities called sporangia. These latter contain the small rounded spores, which escape when the sporangia burst at maturity to develop into new mycelia, if the environment is suitable. This method constitutes the asexual type of reproduction, and is common both to the higher class of moulds, the iiiycomycetes, and to 21 8 PA THOGENIC MICROORGANISMS. the phycomycetes. The latter, besides being distinguished by their lack of partitions between the mycelia, have the power of sexual reproduction. This is accomplished by neighboring hyphce throwing out lateral branches called gametophores, which meet, with free intermingling of their protoplasm. Soon an independent cell is formed at their junction, from which an ordinary branch is thrown out, gradually forming at the tip an asexual sporangium. The moulds are responsible for various skin diseases which will be briefly mentioned. Thrush is caused by the o'idium albicans (oi'dium, a form of fungus between the yeasts and moulds, possessing some of the characteristics of both). Langenbeck, in 1839, described the oidium albicans, which consist of budding cells resembling tho.se of yeasts. They are 10 to 20 v in diameter and sometimes produce mycelia. They grow on all culture-media and stain with all ordinary aniline dyes. Some types liquefy gelatin. Pathogenesis. — In cases of malnutrition, especially with un- clean surroundings, o'idium albicans is able to cause an in- flammation of the tongue and mouth. This inflammation is attended by the formation of pearly white slightly elevated translucent spots, which tend to spread quickly and may coalesce. Ringworm. — Tinea circinata, Trichophyton tonsurans. History. — The credit of the discovery of these moulds as the cause of ringworm belongs to Gruby, who, in 1849, de- scribed these tricophyta. Morphology and Staining. — They consist of mycelia in great numbers with enclosed spores. Staining is easy, but not needed. For examination a hair-bulb should be plucked out and examined in a solution of caustic potash. Biologic Character. — Tricophyton tonsurans grows on all ordinary culture-media." Gelatin is liquefied. Pathogenesis. — In man ringworm of the scalp and body are produced by contact with infected individuals or the use of infected articles. Domestic animals may become infected, and constitute sources of infection. The disease consists THE MOULDS. 219 essentially in circular scaly formations surrounding the hairs, and eventually causing the latter to fall out. Favus. — Tinea favosa, caused by the Achorion Schonleinii, is a contagious skin disease, occurring usually on the scalp and characterized by cup-shaped yellow crusts perforated by hair. The " favus cup," or scutulum, is characteristic. Morphology. — The Achorion Schonleinii consists of slender raycelial threads which may contain spores. The threads are broader and the joints more numerous than in ringworm. The spores are highly refractile and measure 20 to 40 P. in length. For examination staining is unnecessary, though easy. A piece of the scutulum, or crust, or the hair-bulb is examined in a drop of liquor potassae. Biologic Character. — It grows readily on all culture-media, best on acid agar at 37.5° C., yellowish discs being formed. Material from the centre of the " favus cup " or the ground- up cup may be used to plant in liquid agar, plates being then poured (Plant). Tinea versicolor (chromophytosisy pityriasis versicolor) is caused by the microsporon furfur. History. — Microsporon furfur as the cause of this skin dis- ease was discovered by Eichstedt in 1846. Morphology. — It resembles the tricophyton, but the spores have a tendency to aggregate in masses, and the hyphse are generally bent at right angles. It can be demonstrated by putting some of the fine scales in liquor potassse and examin- ing or may be stained with the ordinary dyes. Biologic Character. — It grows especially well on potato, developing in four or five days white or yellowish-white colonies. Pathogenesis. — In man it causes an infection of the skin, of the trunk principally, characterized by macular yellowish furfuraceous patches. 220 PATHOGENIC MICROORGANISMS. MALARIA. Flasmodium Malariae. History. — In 1880 Laveran discovered in the blood of cases of intermittent fever a microorganism which he believed to be the cause of this disease. This microorganism belongs to the animal kingdom of the family of the protozoa, and has received a number of names. The one generally applied to-day is that of Plasmodium malariae. Later investigations have shown that this protozoa has two cycles of existence. One cycle is reproduced in man, and the other cycle in the body of some insects of the mosquito tribe, the Anopheles claviger or Anopheles maculapennis. In the red blood-corpuscles of man the parasites go through an undetermined number of life-cycles, and then pass into the middle intestine of certain species of mosquitoes, in which they go through the various phases of a new life- cycle, ending in the salivary glands, and from these when the mosquito bites a human being, in order to obtain nourish- ment, the parasite passes again into man. The phase of life which is completed in man is the cause of malarial fever. In the younger stages the parasites in this life-cycle appear as very small amoaboid bodies, which have a more or less rapid motion, and which are found in the red blood-corpuscles, nourishing themselves with the substance of these corpuscles and converting their haemoglobin into a black pigment known as melanin. They increase in size, cease their motion, and by a process of fission multiply ; the daughter cells resulting from this fission become free in the plasma and invade other blood-corpuscles, in which they go through the same life-cycle. The two principal symptoms of malaria, ansemia and inter- mittent fever, are related to this life-cycle, the anaemia being due to the destruction of a large number of the red blood- corpuscles by the parasites, and the fever is manifested when the parasite is undergoing multiplication. In their growth the parasites of malaria have been shown MALARIA. 221 to belong to different varieties, differing in their appearance, their mode of division, and the length of time which they take to go through their whole life-cycle. Three varieties are at present recognized : the tertian, which goes through its life-cycle in man in forty-eight hours ; the quartan, whose life-cycle is three days ; and the aestivo-autumnal, whose life- cycle is indefinite and irregular. These different species have constant characteristics, and are not transformed from one into another, though Laveran claims that they are modifications of one and the same species, and are interchangeable. Recent researches have demonstrated that each variety of malaiial parasite is the cause of a particular kind of malarial fever, and by a micro- scopic examination of the blood of a patient one may authori- tatively state the kind of malaria with which that patient is affected. In addition to the life-cycle which begins and ends in the human subject, there is another one which only begins in man. For instance, some of the parasite bodies increase in size ; they do not divide, but getting free in the blood-plasma they are found as bodies of characteristic shape, larger than the red blood-corpuscles. These bodies circulate in the blood for a number of days, not giving rise to any phenomena ; and if they remain sterile, they degenerate and disappear. Upon exam- ination it is found that some of those bodies throw off flagella which move with great rapidity among the red blood-cor- puscles ; others do not present this phenomenon. In the sestivo-autumnal parasites, on account of their appearance these bodies have been called crescent bodies. Some persons regard these forms as degenerated forms ; but it has been definitely ascertained that those bodies which degenerate and disappear, when they remain in man are capable in the intes- tines of certain species of mosquitoes of starting a second life-cycle which may be described as follows : When a mos- quito of the Anopheles variety bites a person in whose blood these crescent bodies are present, or their analogous form in the other species of parasites, some of them are taken up with the blood and lodge in the mid-intestine of the mos- 222 PATHOGENIC MICROORGANISMS. qu-ito. Certain of these crescent-forms give out flagella which are motile filaments containing chromatin, and these loose filaments penetrate and fecundate other crescent-forms. And the fecundated bodies are after this able to penetrate the epithelium of the intestines and travel between the muscular fibres of the intestines. There is therefore a differentiation of sex between the crescent bodies : those becoming flagellated are the male elements, the microgametocytes ; and the others, which do not become flagellated, are macrogametes, the fe- male. These macrogametes after fecundation develop between the muscles of the intestines of the mosquito, becoming sur- rounded with a capsule and acquiring the characteristics of typical sporozoa. After a while its nucleus divides into a number of smaller ones, which in their turn become the nucleus of the dividing cell itself, the sporozoite. These sporozoites are set free by rupture of the capsule of the sporozoa, and are scattered throughout the body of the mos- quito, some finding lodgement in the tubules of the salivary glands, and when the insect again stings man, sporozoites are inoculated into him together with the irritating secretion of the gland. This cycle lasts in the mosquito from eight to ten days, and varies with the species of the parasite. It is likely that this represents the whole life of the malarial parasite, and it has been demonstrated that the infected mosquito does not trans- mit the malarial parasite to its larva, the two life-cycles in man and the mosquito being sufficient to explain the known practical facts. The three varieties of the malarial parasites found in the human blood differ as to size, distribution of their pigments, in the number of daughter cells produced from one parasite, and the length of time required for the completion of their life-cycle. One form, the tertian (Figs. 70 and 71), requires forty-eight hours ; another, the quartan (Figs. 72 and 73), re- quires seventy-two hours ; and a third form, the sestivo -autumnal (Figs. 74 and 75), has an indefinite life-cycle. Occasionally there are seen what are known as the double -tertian and double-quartan forms of malarial fever, in which the fever is MALARIA. 223 produced by several generations of one of those two forms of parasites going through their life-cycles beginning at differ- ent times and on successive days, so that the fever has the appearance of a quotidian (Fig. 76) form of fever. The three different varieties of parasites will be best understood by re- ferring to Figs. 70 to 76. FIG. 70. Tertian malarial plasmodium: 1, Hyaline form; 2, pigmented ring , , pigmented forms; 7, segmenting forms; 8, flagellate form (microgametocyte) ; 9, non-flatrellate form (macrosametc) : 10. segmenting form after destruc" form; 3-6, juKiutuiiuu minis, /, segmciiiuJK lurms ; o, imgeimie lunu (.imcrogumetocyte) ; 9, non-flagellate form (macrogamete) ; 10, segmenting form after destruction of red corpuscles. Examination of the Blood of Man for Diagnostic Purposes.— The following two methods always serre best : (1) the fresh blood examination and (2) the examination of stained speci- mens. Whenever practicable, fresh blood examination offers the easiest and best method for diagnosis. The technic is as follows : Thoroughly clean cover-glasses and slides, being careful to remove all greasy matter. Cleanse also the skin of the lobe of the ear or tip of a finger, make an incision with a sharp-pointed knife, wipe off the first **"• -;> >'> • 17 FIG. 71. — Tertian malarial plasmodium : 1-4, Ring forms of tertian parasite ; 5 ring form (conjugation form of Eyving) ; 6-10, pigmented organisms ; 11-14, nearly full-grown forms, showing diffusion of the chromatin; 15-17, segmenting forms within red corpuscle; 18, segmenting forms after destruction of red corpuscle ; 19, flagellum (microgamete) ; 20, sporozoite. L^ FIG. 72. — Quartan malarial plasmodium: 1, Hyaline forms; 2-5, pigmented forms ; 6, 7, segmenting forms ; 8, segmenting form's after the destruction of red corpuscle ; 9, flagellate form (microgametocyte) ; 10, non-flagellate form (macro- gamete). 224 '&'. 4 /•*• •«*' 7 FIG. 73. — Quartan malarial plasmodium : 1-4, Ring forms of quartan parasites; 5-9, pigmented parasites ; 10-12, segmenting forms of quartan parasite ; 13, segment- ing stage after destruction of red corpuscle. 'J a •/;' 13 r FIG. 74.— Tertian sestivo-autumnal malarial plasmodium : 1, 4, Hyaline ring form ; 2, 3, 7, pigmented ring form ; 5, 6, pigmented forms ; 8, young intracorpuscular cres- cent; 9, segmenting forms ; 10, flagellate form (microgametocyte) ; 11-14, crescentic forms. 15— M. B. 225 r*«* 17 IH ^ '**'*/ A'- 19 ' * V* ' 2" 24 31 .irt " 34 "3r, , 37 FIG. 75.— Tertian sestivo-autumnal jilasmodia : 1, 3, 4, r>, 6, 7, 8, 9, 10, 15, King forms of tertian testivo-autumnal plasmodium ; "2, Intracellular form; 11, 13, 14, 16, 17, pigmented ring forms ; 12, red corpuscle, showing infection with two " ring forms " ; 18, 19, pigmented forms, just prior to segmentation ; 20, 21, 23, 24, round and ovoid forms developed from crescents ; 22, macrogamete ; 25-36, crescentic forms of sestivo-autumnal plasmodium (tertian) ; 29, ovoid form ; 37, segmenting form : 38, sporozoites ; a, segmenting form of quotidian sestivo-autumnal plasmodium. 226 MA LARIA. 227 exuding drop, touch the top of the next drop with a clean cover-glass held with forceps, being careful to avoid touching the skin, and taking the drop when small, so that the corpus- cles will be spread out in a uniform layer, not in rouleaux, when the cover-glass is laid on the slide ; press the cover- glass on to the slide gently ; if the cover-glass and slide are clean, the blood will spread in an even thin layer; examine with a y^ oil immersion. Prep- arations made in this way will show, if examined immediately, the amoeboid plasmodium in- side of the red blood-cells, being especially recognizable by the movements or the contained pigment. It is possible some- times to keep such preparations for several hours. When examination of fresh blood is not practicable, or when it is desired to preserve the preparation, resort to the procedure of staining must be had. This is best accomplished by the methylene-blue and eosin methods, either applied to- gether, or each dye being used separately, as follows : A drop of blood is taken as just described, spread evenly upon a thin cover-glass, and allowed to air-dry ; the film is then set by immersing the cover-glass for twenty to thirty minutes in a mixture of equai parts of absolute alcohol and ether ; after drying, the mixed stain (methylene-blue and eosin) is applied over the surface of the. film and allowed to remain for five minutes ; it is then poured off, the preparation washed thoroughly in distilled Quotidian sestivo-autumnal mala- rial plasmodium : 1-4, Hyaline ring forms ; some cells show infection with more than one organism; 5-7, pig- mented forms ; in 6 one hyaline form ; 8, segmenting forms ; segmentation complete within infected red blood- corpuscle ; 9, flagellate form (microga- metocyte); 10, 11, 13, 15, crescentic forms ; 12, ovoid form ; 14, nou-flagel- late forms (macrogamete). 228 PATHOGENIC MICROORGANISMS. water three or four times, then dried and mounted in balsam, and examined. In this preparation the red cells will be stained •pinky the leucocytes pale blue and their nuclei dark blue; the parasites will be stained very dark blue and their pigment- granules remain unstained. The mixture of methylene-blue and eosin is prepared as follows : Saturated aqueous solution of methylene-blue, 1 part ; Alcoholic solution of eosin (1 per cent.), 2 parts. Do not filter. Giemsa's, Leishman's, and Wright's are best routine stains. Loeffler's methylene-blue, and Ehrlich's hsematoxylin and eosin, give also at times very excellent preparations. The varieties of plasmodia may be distinguished from each other. Differentiation between the Tertian and Quartan Parasites : 1st. The tertian parasite completes its life-cycle in two days ; that of the quartan requires three days. 2d. The tertian parasite has a tendency to discolor the blood-cells and to enlarge them. The quartan does not dis- color so much, and never enlarges its containing red cell ; on the contrary, the cell generally appears smaller. 3d. The quartan parasite has better-defined and clearer outlines and coarser pigment-granules than the tertian. 4th. In fission the quartan parasite divides into six to twelve daughter cells, which are larger and contain each a refractive granule in its centre. The tertian parasite divides into fifteen to twenty daughter cells, smaller, and with no central granule. Inoculation. — Blood infected with either kind of parasite when inoculated into healthy individuals has in a number of cases produced that variety of fever specific for the particu- lar parasite, and this special parasite has been found in the blood of the inoculated individual. PROTOZOA FOUND IN BLOOD. 229 FIG. 77. PROTOZOA FOUND IN BLOOD. Among the protozoa occasionally found in the blood are the following : Filaria Sanguinis Hominis. Morphology. — The filaria when fully grown are slender and hair-like, measuring 3-6 inches long. They live in the lymphat- ics and tissues exclusively, and dis- charge from there their embryos, which circulate then in the blood. An embryo measures about forty times the diameter of a red blood- corpuscle in length and equals its diameter in width. The embryo of F. bancrofti (Fig. 77) (F. nocturna) is found free in peripheral blood only at night, that of the F. diurna only in the daytime, while that of F. perstans is found with equal fre- quency during the night and day. Pathogenesis. — Chyluria, lymph- scrotum, and elephantiasis are caused by the F. bancrofti and the latter sometimes by F. diurna. Craw- craw, a parasitic disease, is caused by F. perstans. Infection is sup- posed to be carried in water in which are deposited the ova by mosquitoes, in which they develop. An. Trypanosomes. Trypanosomiasis. — Many diseases in man and animals are due to inva- sion by trypanosomes. Several vari- eties of these have been discovered and described. In general they are characterized by a flagellum and undulating mem- brane, a centrosome, nucleus, and nucleolus. The species of Ex. Nerv. Larva of Filaria bancrofti in the blood ofn.au, in Egypt : Nerv,, nervous system; ex., excretory; «//., anus. X 514. (Looss.) 230 PATHOGENIC MICROORGANISMS. greatest importance is the Trypanosoma gambiensis, the etio- logical factor in sleeping sickness. It is a little shorter and thicker than T. lewisi a'nd T. brucei. It measures from 20-25 fj. long and 2-3 //. in width. In life it has a slow undu- lating movement. Its flagellum extends from the centrosome through the body, curving around the nucleus and extending beyond the nucleolus at the anterior end. The nucleus is oval and central. The Trypanosoma gambiense is conveyed to man by the bite of the tse-tse fly (Glossina palpalis). The trypan- osomes are stained by the Wright or other blood-stains. Leishmania Donovani. In certain forms of fever of irregular character and sup- posed to be of malarial origin, Leishman and Donovan, work- ing independently, found certain protozoal organisms which they considered the etiological factors. Morphology and Staining. — The Leish man-Donovan bodies are small elliptical or circular bodies 2-3 /j. in diameter, and contain chromatin. This is arranged in a small rod-shaped portion and a larger surrounding circular portion. The chro- matin is usually arranged in the short axis of the ellipse. To demonstrate this organism, it is generally necessary to obtain blood by puncture of the liver or spleen. The organisms are usually found either singly or in bunches free in the blood of these organs. For staining, Wright's stain may be used. The Leishman stain gives splendid results, as well as any of the Romanowsky modifications. By Wright's stain the chro- matin is colored dark blue, the rest of the organism is stained a paler blue. Distomum Hsematobium, first described by Bilharz, causes a disease found in Africa and Arabia, characterized by anaemia, emaciation, and haemor- rhages from the bladder and rectum. The adult male worm measures about 12 m.m. long and 1 m.m. broad. The female is much longer and thinner, and in copulation is received into a groove in the body of the male. The ova are discharged into the blood and lodge in the capillaries of the rectum and bladder and elsewhere. They are discharged at times in the PROTOZOA FOUND IN BLOOD. 231 urine and faeces. The ova are oval and terminate at one end in a process or spine. In some ova this process is directly placed at the centre of one pole, the tenninal spined ova ; in others it is placed a little to the side, the lateral spined ova. The former are supposed to be more frequently found in the bladder, the latter in the rectum. Very often in examining an ovum in a drop of urine or faeces, the contained myracidium or embryo may be seen moving about in the containing shell, which resembles a melon seed in shape. If watched long enough and under favorable conditions, it may be seen to leave its shell and swim free in the fluid by the aid of its cilia. FIG. 79. Fio. 78. Distoma hepaticum (Leuckart). Distoma lanceolatum (X8) and eggs (von Jaksch). Another fluke of common finding in the Philippines, and hence of great interest to us, is the Distomum westermanii. It causes epidemic haemoptysis. Diagnosis. — The diagnosis is made by examining the spu- tum, where the very characteristic eggs are found. They are slightly smaller than those of bilharzia. and have a characteris- tic hood or top to them. Cases of haemoptysis in Chinese residing on the Pacific coast of this country, and believed to be tubercular, have been found to be due to the Paragonum westermanii. Other flukes, causing diarrhoea, anaemia, emaciation, and sometimes death in animals, are the Fasciola hepatica (Fig. 78) in ruminants, the Distomum lanceolatum, (Fig. 79) in sheep and cattle, and the Distomum felineum in cats. 232 PATHOGENIC MICROORGANISMS. AMCEBIC COLITIS. Amoeba Coli. History. — In certain forms of chronic dysentery accom- panied with ulcerations of the lower bowel, and which very often give rise to suppuration in internal organs, especially the liver, an animal parasite, the Amoeba coli, has been accu- rately described since 1875 by Losch, of St. Petersburg. Losch's observations have been confirmed since by a number of other observers. According to Schaudinn, the amoebae coli of Losch are really two distinct organisms, one of which is sometimes found in the intestines of healthy people, giving rise to no symptoms, and the other giving rise to the amoebic dysentery as found in the tropics. He calls the harmless amoeba Entamoeba coli, and the dys- entery amoeba the Entamoeba Jiistolytica, and gives the method of differentiating between the two as follows : In the dysenteric form the external membrane, or ectoplasm, is more plainly de- veloped and refractile and hyaline than the Entamoeba coli. In the Entamoeba coli the hyaline pseudopodial plasma, in contrast to the remaining plasma, is much less refractile than the Entamoeba histolytica, in which there is always a plainly developed ectoplasm existing as a plasma zone, and possessing a stronger refraction than the endoplasm. The nucleus of Entamoeba histolytica is very difficult to see in fresh speci- mens. Its position is excentric, its shape is changeable. Morphology. — The amoeba is a protozoon, and consists of protoplasm which exhibits under different conditions various forms. In the quiescent condition it is spherical in shape, and may be recognized from the other cellular elements by its greater refraction of light and by its pale-green color. Its size is from 10 to 25 mikrons. The body consists of two parts : an inner part, or endoplasm, which is generally gran- ular and of dark color ; and an outer part, or ectoplasm, which is pale and white. These two parts may be best made out in QUESTIONS. 233 the motile amoeba. A nucleus more or less central is also easily made out. The Amoeba coll is stained easily by any of the nticleolar stains, especially by methylene-blue. In its body may often be seen foreign bodies, especially red blood-cells, but it rarely contains leucocytes or fat. The motion of the amoeba is caused by the mechanism of pseudopodia, which are blunt homogeneous processes, the pro- trusion of a portion of the ectoplasm. Motion is sometimes gradual and deliberate, at other times rapid, and is modified by variations in temperature. Biology. — Nothing is known as to the functions of nutri- tion, respiration, and reproduction of the amoeba. It is found occasionally in health in the secretions of the lower bowel, and in cases of dysentery may disappear partially or com- pletely from the stools during convalescence. They are also frequently found in the pus of hepatic abscesses. Examination of the feces, especially the slimy portion of dysenteric stools, and the pus in cases of suspected amoebic infection, should be made in fresh specimens, when the live amoebae may be easily recognized. Dried preparations are made as for bacterial examination, and colored with aqueous solution of methylene-blue. QUESTIONS. Streptothrix. What is a streptothrix ? How does it resemble moulds? How does it dif- fer from moulds? How does it resemble bacteria? Which are the best known of the streptothrices ? Why are the Bacillus tuberculosis and Bacillus diphtheria; believed to belong to the class of the streptothrices ? What is the appearance of the lesion caused by streptothrices? Describe the Streptothrix adinomyces,nr ray fungus. How is it best stained? Give Mallory's staining method. Give the principal biologic characters of the Streptothrix actinomyces, How does it grow on blood-serum and agar ? On gelatin ? On potato ? In bouillon? What animals are susceptible to streptothrix infection ? Is the disease transmissible from animal to animal or from animal to man? How are ani- mals infected ? Describe the lesions produced by streptothrix infection. What causes the suppuration? 234 PATHOGENIC MICROORGANISMS. What other pathogenic streptothrices have been described and studied ? What are the yeasts ? How do they differ from the moulds ? What are the moulds? Describe them. What diseases are the moulds responsible for? What is a ringworm ? Its morphology and staining properties ? Biolog- ical character? What causes favus? Give the morphology and biological character of Achorion Schonleinii. What is tsenia versicolor? What fungus causes it? What its staining prop- erties and biological character? What disease does it cause? Malaria. What is the Plasmodium malarise f How many life-cycles has it? Where are those phases of life completed ? Describe the life-cycle of the plasmodium in man. What are the two principal symptoms of malaria, and what relation have thev to the development of the parasite? What differentiates the varieties of the malarial parasites from each other ? What three varieties are at present recognized ? What forms of fever are caused by these different varieties? What life-cycle only begins in the body of man ? Describe the flagellated and non-flagellated bodies? What are the crescent bodies? How do those crescent bodies or their homologues develop in the body of the mosquito? What are the microgametocytes? What are the macrogametes ? Describe the development of the fecundated macrogametes in the mos- quito ? What are the sporozoites, and how do they infect man ? What is the duration of the life-cycle parasite in the mosquito? Does the mosquito transmit the malarial parasite to its larva? What is meant by the double-tertian and double-quartan forms of mala- rial fever ? To what is the quotodian form of fever due? Give the technic for the examination of fresh malarial blood for diag- nostic purposes. How are stained specimens prepared for examination? What is the differentiation between the quartan and tertian parasite in the blood ? Is the blood of malarial patients infectious ? Amoeba Coli. Where is the Amoeba coli found? To what kingdom does it belong? Describe it. What is the endoplasm ? What is the ectoplasm ? How is Amceba coli stained ? What may be seen in the body of the Amceba coli? What causes the motion of the amoeba ? How is examination for the Amoeba coli practised ? How do the Amceba coli and Amoeba histolytica differ? ASCARIS. 235 CHAPTER XXV. INTESTINAL PARASITES. IN connection with the examination of amoeba?, it is well to be on the lookout for the commoner form of intestinal para- sites which may be found. ASCARIS. Chief among them in frequency is the common round worm, or Ascaris lumbricoides (Figs. 80 and 81). It is a cylindrical worm, pointed at both ends, and of a color ranging from yellowish to red. Near the head is a constriction or neck. The male is shorter than the female, and curves at the posterior extremity. An adult worm possesses transverse markings, and four longitudinal bands ex- tend from one extremity to FK4. 81. FIG. 80. Ascaris lumbricoides (von Jaksch): a, Worm, half natural size ; b, head, slightly magnified; c, egg (eye-piece I, objective 8 A, Reichert). Egg of the common ascaris (Ascaris hvmbricoides) of man as found in faeces. Seen with super- ficial focus. Greatly enlarged. (Stiles.) the other. The female measures from 10-30 c.m. in length. Its ova, which are numerous and which may be demonstrated 236 INTESTINAL PARASITES. by placing a drop of faeces on a slide and placing a cover-slip over same and examining with an ordinary eye-piece and 16 m.m. objective, measures 60-70 by 35-50 //.. They are oval in shape and have a very thick albuminous envelope, which has a rough fringed appearance. Usually, on account of pigment absorbed in the intestines, the ova, especially the envelopes, are colored from yellow to reddish brown. Infec- tion may take place through infected food or by means of dirty hands. Sometimes children constantly reinfect themselves by scratching about the anus, FIG. 82. and thus infecting their hands, etc. This worm may be present in any part of the alimentary tract. OXYURIS. Next in frequency among the intestinal parasites is the se(dy thread, or pin worm, the Oxyuris vermicularis (von Jaksch): a, Head ; Embryo of the common pin worm b, male; c, female; d, eggs. (Oxyuris vermicularis) of man, in the eggshell, as found in fresh faeces (Leuckart). Oxyuris vermicularis (Figs. 82 and 83). This worm usually inhabits the rectum and lower parts of the colon. The male measures from 3-5 m.m. in length, .16-.6 m.m. in breadth; the female is about three times as long. The ova are not seen free in the faeces unless decomposition of the worm has taken place. By crushing a mature female beneath a cover-glass, a large number of ova may be demonstrated. They are pale and oval, measuring from 40-50 /. long. The endoplasm is TJENIA. 287 fine and granular and the outer envelope thin and smooth. There is nearly always a flattening on the side of one of the poles, which breaks the symmetry of the ovoid and is a dis- tinguishing characteristic of the ova. Infection takes place as with the ascaris. The Trichocephalus dispar, or Trichocephalus trichyuris, the very common whip worm, is considered to be of no great pathological significance. Its ova are very often found in the faeces, and resemble waiters with handles at each end. The ova are about the length of the oxytiris, but somewhat nar- rower. They are brown in color, with a double contoured thick envelope and the protoplasm is coarser. The "knobs77 on the ends mark them effectively from confusion with other ova. T-ENIA. Of the tienia, or tapeworms, there are three principal vari- eties. Common to all is the intervascular system, intercom- municating between segments (proglottides), and the her- FIG. 84. Tsenia saginata: Head; proglottis ; egg (Reichert's eye-piece III, objective IV). (von Jaksch.) maphroditic character of the latter. The ova are very rarely found in the intestines. They may be found in a mature link, usually any one after the 450 from the head, by teasing 238 INTESTINAL PARASITES. the link on a clean slide and examining with the | (16 in.m.) objective. The Tsenia saginata (Fig. 84), or beef tapeworm, which is the prevailing one in the United States, is distinguished prin- cipally by its head, which is 2 m.m. long and nearly square in shape. Its head is not provided with hooks. The scolex (head) of the Tsenia solium (Figs. 85 and 86), or FIG. 85. Head of Trcnia solium ; X 45. (Lcuckart.) pork tapeworm, is about the size of a common pin, and is sur- rounded by a projecting rostellum which has on it a double row of booklets. Both species of worm are provided with FIG. 86. Ova of Tsenia solium (Leuckart): a, With yolk ; b, without yolk, as in mature seg- ment. The hard brown shell is indicated. sucking disks, or suckers, four in number, by which they attach themselves to the intestines and through which they nourish. The T. saginata is longer than the T. soliu-m, the former measuring, when fully grown, 12-20 feet, the latter 6—12 feet in length. Larger and longer than either is the fish tapeworm of Europe, the Bothriocephalus latus (Figs. 88 and TJZNIA. 239 89), which measures as much as 30 feet in length. In all of these varieties below the head is a thin constricted neck, which develops gradually into broad, flattened, connected segments, or proglottideS) each of which contains male and female sexual organs. The links or segments of the T. saginata are larger and not nearly as equilateral as those of the T. solium, which measure, when mature, about 1 c.m. long and 8 m.m. wide, the links of the T. saginata being more the shape of a key- stone. The head of the T. saginata is provided only with suckers, while the T. solium has, besides the four suckers, an FIG. 87. Tsenia cucumerina: Head; proglottis ; magnified (von Jakscb). armature of hooklets. The uterus of the T. saginata has many lateral branches (15-30), while the T. solium' has fewer (10- 20). The uterus of the Bothriocephalm latus is dark and small and located in the centre of the segment, which at maturity is nearly square. The head of the bothriocephalus is characterized by two grooves in its side, which are probably suckers. The head is oblong in shape. It takes about three to three and a half months for the seg- ments of the T. saginata to appear in the faeces after infection 240 INTESTINAL PARASITES. FIG. 88. has taken place. Boiling of suspected meat will destroy the infection. Some cases of infection by the smaller tapeworm, or Tsenia nana, have been reported. This is the dog tapeworm, and its length is about J inch. These worms have a life-cycle in different animals. The T. saginata in cattle, the T. solium in hogs, the bothriocephalus in fish, and the T. nana in dogs. When swallowed by these animals the ova reach the stomach, where the shells are dis- solved and the larvae freed. These emigrate to different parts of the body — muscles, liver, brain, etc.— where they become encysted, and are then called Cysticerci. These cysticerci, macroscopically, can be seen as small grayish-white, hard cysts. Meat infected with them is called "measly." When incom- pletely cooked and swallowed by man, this sort of meat constitutes the chief source of infection. The FIG. 89. Bothriocephalus latus. Bothriocephalus latus : Head. protecting envelope of the cysticercus is dissolved in the 241 stomach juices, and the contained scolex, or head, is then free to attach itself to the mucous membrane and to grow into maturity. The ova of T. solium are round, with a striated double-contoured envelope. They measure from 40-50 /j. in diameter. The ova of the T. saginata are larger and coarser. They are both of a brown color and dense appearance. The ova of bothriocephalus are somewhat larger and more oval ; the envelope is thinner. A form of tsenia, fortunately very rare in the United States, but more common in countries in which the association with dogs is common, especially in such countries as Iceland, where the transportation problem is settled by the employment of dogs, and where, on account of the cold, very often dogs and men are huddled together, the Tsenia echinococcus very often causes serious results. It is a parasite of the intestinal canal of dogs principally. The adult worm consists of a head and three segments. It measures in length 4 to 5 m.rn. The head has four sucking disks and a double row of booklets. The eggs are numerous, and when swrallowed the larvaB are liber- ated and burrow into the liver, spleen, muscles, and other parts of the body. Here they develop into cysts with clear contents (hydatids). These cysts increase in size and develop- ment by a system of interior projection or budding which is continuous, daughter and granddaughter cells being formed. From the inner granular lining of these daughter and grand- daughter cells broad capsules are developed. From the lining of these latter, scolices with sucking disks are formed. These, when ingested by a dog or man, may develop into tsenia. Sometimes these cysts rupture. The fluid contained in them is non-albuminous in character, an important point when aspi- ration has been done for diagnosis, and may contain booklets. In echinococcus cyst of the kidney booklets may appear in the urine. The sputum may show booklets in echinococcus cyst of the lung. TRICHINA. A species of worm which causes considerable illness and fatality, less so now than formerly, is the Trichina spiralis. 16-M. B. 242 INTESTINAL PARASITES. FIG. 90. Nerv. CEs. The larvae of this worm, ingested in infected pork which has been insufficiently cooked, are freed from their capsular encyst- inent in the stomach, and then proceed to the intestine, where they mature in about a week. If not passed out in the faeces, the female worm, which is about 3 or 4 m.m. in length (about three times the length of the male), will burrow into the lympli- spaces, where she deposits her embryos in enormous numbers. These are carried in the blood-stream, and ultimately find lodgment in the voluntary muscles, where they become encapsulated. They are coiled up, either singly or in small groups, in the capsules, which are oval in shape. These latter sometimes become calcified, but in spite of this the larvae may live for a long time. The larvae measure from J to 1 m.m. in length. The parasite may be discovered in the stools early in the disease. In infected meat the larvae are seen as. white specks, which by means of a microscope can be readily examined. Sections may be made, or a small piece of the "specked" meat crushed between a slide and cover-glass. The coil-shaped organism is typical. Staining is unnecessary. HOOKWORM (Figs. 90-95). By far the most important intestinal parasite to us in this country is the Necator americanus, a member of the strongyloides group, sometimes improp- , o%, , , ° . ,r 7, , -i i erly called the Ankylostoma duodenale. rj^ ig & ^^ rQund ^^ ^^ bling in macroscopic appearance a The female adult measures from 10-12 Gen. faeces -. Nerv., Nervous sys- tem; (Es., oesophagus ; int.. intestines; gen., genital pri- medium; em., anus. > 228. small coarse thread. m.m. in length; the male, from 8—10 m.m. The cephalic end HOOKWORM. 243 of the worm terminates in a buccal bursse, which contains the oval papilhe above, the chitinous capsule and ventral lancet ventral] y, the lateral lan- cet to the side, and, most FIG. 94. important, the single con- ical tooth dorsal! y. This _ ^^^MFM&i^-j™t. FIG. 93. FIG. 91. FIG. 92. FIG. 91.— New World male hookworm. Natural size (Stiles). FIG. 92.— New World female hookworm. Natural size (Stiles). FIG. 93.— The same, enlarged to show the position of the anus (a) and the vulva (r) : in, month (Stiles). I-'IG. '.»!.— Dorsal view of anterior end of New World hookworm: b. c., Buccal cav- ity; c. v., cervical papillae: d. in. (., dorsal median tooth, projecting prominently into the Imreal cavity: . in length. They are distinguished by their oval shape and the paleness of their protoplasm, and by the fact that there is nearly always a clean space between the ectosarc and endosarc. The ova of the Old AVorld hookworm are larger than those of the necator. In case of a negative examination, small bits of the faeces should be mixed thoroughly with saline or calcium chloride solution and centrifuged. This washing and centrifuging should be repeated two or three times and the washed sediment examined as before. Very often ova which are few in number will be detected in this way. QUESTIONS. What are the most common intestinal parasites which may be found in man ? Describe the Ascaris lumbricoides. Its ova. How may infection take' place ? ' Describe the Oxyuris vermicularis. Its ova. What is the distinguishing characteristic of its ova? How may infection take place? Describe the Trichocephalns dispar. Its ova. How may these be recog- nized? What are the three principal varieties of Taenia? Describe the Tjenia saginata. Its ova. Describe the Tsenia sol mm. Its ova. Describe the Bothrioccphalus. Its ova. Describe the Taenia nana. Its ova. In what different animals do these worms have a life-cycle? What are Cystercerci ? Describe the Tamia echinococcus. Describe the Trichina spiralis. Describe the Necator americauus, or New World hookworm. 246 UNKNOWN CA USES OF OTHER INFECTIOUS DISEASES. How is it to be distinguished from the Old World hookworm, or Aukylos- toma duodenale ? Describe its mode of entrance in the human body. How does it affect the human body? What disease is caused by it? Describe its ova. Where are they found? CHAPTER XXVI. THE UNKNOWN CAUSES OF OTHER INFECTIOUS DISEASES. IN rabies, yellow fever, scarlatina, and measles the organisms whose etiological claims have been advanced are many and varied. So far the claims of none are sufficiently advanced or supported to demand treatment in this book. The finding of the so-called " Negri" bodies and the method of immuniza- tion introduced by the immortal Pasteur will be referred to in connection with Rabies. The intermediary function of the Steyo- myia mosquito in yellow fever is well known, and a knowledge of the more distinctive features of this mosquito is desirable. BABIES. Rabies is an intensely fatal microbic disease, with an incu- bation period, in ordinary cases, of from two weeks to two months. In some cases incubation periods of six months have been reported. Negri, in 1903, described certain bodies which he observed constantly present in cases of rabies. He found them in all parts of the brain, but particularly in the " hippo- campus major" and " cerebellum. " These constitute, accord- ing to a preponderance of authorities, a valuable means of diagnosis in doubtful cases. They may be demonstrated in section, stained with eosin and methylene-blue. They stain pink, and may be recognized by their oval shape and average length of about 1 5 /*. Fairly good preparations may be obtained, generally by crushing a small piece of infected brain sub- stance beneath the cover-glass and then staining with one of RABIES. 247 the rapid blood-stains (Giemsa's or Wright's). The Negri bodies will show blue on a darker blue field. Immunity. — Pasteur succeeded in working out a method by which active immunization could be accomplished. He found that he could obtain a virus of nearly constant and fixed vir- ulence by passing it through rabbits, and that this virulence could be diminished by drying over caustic potash. He found out that ordinary fresh " standard" virus — virus fixe — would prove fatal to rabbits in six or seven days, and that this vir- ulence was decreased more and more as drying progressed, until, after fourteen days' drying, it was completely lost. He also demonstrated that by beginning wit^ a very weak virus and injecting virus of gradually increasing strength, it is possible to produce immunity. Briefly, the technique is as follows : " Street virus" or virus obtained from " mad dogs," is passed through rabbits until the incubation period is reduced to one week. The virus is then potent enough to work with. Rabbits are inoculated with an emulsion of the medulla ob- tained from animals dead from rabies. This inoculation is done, after removing a small piece of bone, intracranially. The inoculation is best made at a point in the angle between the sagittal and coronal sutures. As soon as the animal has died of rabies (six to seven days) autopsy is aseptically per- formed and the cord removed and hung by a sterile thread in a bottle or cylinder containing at the bottom some pieces of Potassium Hydrate C. P. A small piece of the infected me- dulla is now emulsified with salt solution by means of a sterile glass rod, and a fresh rabbit or two injected as before. This is done in order to keep the supply up. The bottle or cylinder containing the injected cord is put away in a dark place at a temperature of 25° C., to be used after suitable drying. The treatment in cases of rabies infection is begun as soon as possible after infection. The injections are given subcutaueously, beneath the skin of the abdomen usually. The dose for injection is prepared by emulsifying about 1 c.m. of the dried cord in about 3 c.c. of 0.85 per cent, salt solution. About two-thirds of this amount is injected, care being taken to avoid injecting coarse sediment. The dosage, 248 UNKNO WN CA USES OF OTHER INFECTIO US DISEASES. it will be readily seen, is only approximate. The treatment is begun with an emulsion of the cords of the 14th and 13th days of drying. On the second day the treatment is made up of the cords of the 12th and llth days of drying; on the third day, of the 10th and 9th ; on the fourth, of the 8th and 7th ; and on the fifth, of the cord which has been drying six days. Thus the virulence of the injection is increased until the third-day cord is reached, when we must revert back to the fifth, and then fourth, both for two days, finally winding up the treatment with the third-day cord. The above method, as practised in the Pasteur Institute, is used for ordinary cases, and consumes^about fifteen days. In severe cases two separate treatments are given on each of the first three days, the sixth- day cord being reached in the second injection of the third day, and the treatment, which in other particulars is the same, is continued for eighteen to twenty -one days. The depth of a bite from a mad dog is supposed to influence the severity of its effects. Bites about the face, head, and upper extremities are considered more dangerous than those of the lower extremities. YELLOW FEVER. While much has been said and written about yellow fever, nothing is definitely known about its cause except that the Stegomyia calopus orfasciata acts as a carrier of the infection. It is a delicate, dark gray mosquito, with lyre-shaped mark- ings on its thorax. It has white spotting and striping on its abdomen, but none on its wings. Its third pair of legs are marked with bands. The blood of a yellow fever patient is capable of transmitting the disease only during the first three days of the disease. During this time the American Commission in Cuba showed as little as 0.1 c.c. of infected serum was capable of reproducing infection if injected directly into a non-immune. For infection to take place by the bite of a mosquito, it is necessary that the mosquito should have sucked the blood of a yellow fever patient during the first four or five days, and that at least twelve days shall have elapsed between that time and the time of biting a non-immune. As only the female stegomyia is capable of trans- BACTERIOLOGICAL INVESTIGATION OF WATER. 249 milling the disease, and she is known to live for long periods after sucking the blood, it is claimed that this viability consti- tutes the xole reason why only this species is infectious. Other species of Culicidce deposit their eggs and die usually before twelve days have elapsed since their time of feeding, too short a time for the development of the infection. SCARLET FEVER. Various etiological factors have been proposed as responsible for scarlet fever. Mallory's intracellular "bodies" have been unsubstantiated. It has been assumed by some writers that streptococci, possibly Streptococci pyogenes, are responsible. About this subject, however, nothing has been brought for- ward the claims of which justify consideration now. QUESTIONS. What is rabies? What is its period of incubation ? Give Pasteur's treatment in suspected or positive cases of rabies. CHAPTER XXVII. BACTERIOLOGICAL EXAMINATIONS OF WATER, AIR, AND SOIL. THE BACTERIOLOGICAL INVESTIGATION OF WATER. BOTH qualitative and quantitative bacteriological examina- tions of water are often resorted to in order to test the adapta- bility of this substance for human and animal consumption. By the quantitative test the number of bacteria present in tlio water is ascertained without any reference to their patho- genic character, and when this number exceeds 500 bacteria per c.c. the water is condemned. This is evidently not a very fair test, but it is not without value when it is consid- ered that in a number of instances the virulence of some pathogenic microorganisms is increased when they are inocu- lated together with saprophytic germs ; and again, that the 250 EXAMINATIONS OF WATER, AIR, AND SOIL. introduction of non-pathogenic bacteria occasionally so dimin- ishes the animal resistance that animals resistant to inocula- tions by some pathogenic bacteria are subsequently rendered susceptible. This quantitative analysis is especially useful in cases in which the mean quantity of bacteria in a given body of water is already known, and as a matter of comparison to ascertain whether any new source of contamination has been introduced. The examination is made as follows : A sample of the water is collected in clean sterilized bottles or tubes. If the water is from a pump, well, or from a cistern, it should be allowed to run for a few minutes before the sample is taken. If the water is from a spring, river, or any collection of water, the sample for examination should be taken a foot or two below the surface of the water. Agar and gelatin plates should be immediately inoculated with the water. When this is not practicable the plates should be made at as early a time as possible, the samples meanwhile being kept on ice, near the freezing-point, to prevent the further development of bacteria. For the purpose of collecting water for examination, glass bulbs after the pattern of Sternberg (Fig. 96) are very use- ful. These consist of a sphere blown on the end of a glass FIG. 96. Glass bulb for collecting samples of water. (Abbott.) tube, the stem at the other end terminating in a capillary tube. After thoroughly cleaning these bulbs, a negative vacuum is established therein by introducing in each tube a few drops of water, allowing same to boil over a gas-flame, and when the water has completely vaporized into steam the capillary end of the bulb is brought into the flame and the apparatus sealed. When it is desired to collect a sample of BACTERIOLOGICAL INVESTIGATION OF WATER. 251 water, the capillary end of the bulb is broken under the water and the vacuum in the tube causes a suction of the fluid into the bulb until same is about three-fourths full. When this is accomplished, the bulb should again be sealed and packed in ice. When making gelatin and agar plates a known quantity of water should always be used to each plate, say '0.01, 0.05, 0.10, or 1 c.c., the quantity varying according to the amount of suspected contamination of the water. The plates are made as already described in the chapter on the making of plates, only plate No. 1, however, being made. After twenty-four or forty-eight hours the colonies on the plate are counted ; and as it is assumed that each colony is grown from a single bacterium, the number of colonies on the plate represents the number of bacteria originally in the sample. Two sets of plates should always be made, one on gelatin, which is kept at the room temperature, and another on agar for the incubator. When the number of colonies on the plates is very large, plates should be made with still greater dilution, so as to obtain plates with only a moderate number of bac- teria, in order to facilitate the count. Absolutely sterile water should always be used to make the dilution. In expressing results, the number of bacteria in 1 c.c. of the water is men- tioned. For counting the colonies on the plates the counter of Wolfhuegel (Fig. 31) is used, which consists of a wooden framework, at the bottom of which may be put a glass plate, white or black, in order to form a background for the plate containing the colonies. Over this background the plate of which the colonies are to be counted is placed, and over this a transparent glass plate ruled in square centimeters, and held in position just above the colonies, but without touching thorn. It is easy in this way to count the colonies in each square centimeter, and so make out the total number of col- onies on the plate. When this is found too tedious, a number of squares at different portions of the plate may be counted, an average established, and that average multiplied by the number of squares will give approximately the number of colonies on the plate. By multiplying the number of col- 252 EXAMINATIONS OF WATER, AIR, AND SOIL. onies on the plate according to the degree of dilution of the water, it is easy to arrive at tha number of bacteria in a cubic centimeter of the water. .Thus if the average number of col- onies per square is 15, and there are 100 squares on the plate, and the amount of water used in making the plate was 0.10 c.c., then 15 X 100 X 10 equals 15,000, which represents the number of bacteria present in 1 c.c. of the water. Petri dishes may be used instead of plates, and for the counting of colonies on the same special means have been or ^ 6 Fakes' apparatus for counting colonies (reduced one-third). devised. That of Fakes' apparatus (Fig. 97) is a cheap and convenient one. It consists of a sheet of paper on which is printed a black disc ruled with white lines. The Petri dish is placed centrally upon this paper, and the colonies between the white lines are counted, the whole circle being divided into sixteen equal segments, as seen in the figure. BACTERIOLOGICAL INVESTIGATION OF WATER. 253 For counting colonies, a small hand lens, such as is repre- sented in Fig. 98, is often necessary. FIG. 98. Lens for counting colonies. Instead of plates and Petri dishes, Esmarch tubes may be used as follows : A definite quantity of the water is added to melted gelatin in a test-tube and the same rolled as described previously. A special counter for Esmarch tubes, provided with a mag- nifying glass, is used in counting the colonies (Fig. 99). FIG. 99. Esmarch's apparatus for counting colonies in rolled tubes. (Abbott.) As previously mentioned, the value of this quantitative examination of the bacteria of water is not very great, because, 254 EXAMINATIONS OF WATER, AIR, AND SOIL. though of great utility, it does not give definite information as to the poisonous organisms of the water. For this pur- pose a qualitative examination for pathogenic germs is of much more value. This is not, however, as easily per- formed, and in the great majority of cases gives negative results. The bacteria most often sought in this way are the bacillus of cholera and the bacillus of typhoid fever, both of which are short-lived in water, and have only rarely been demon- strated, partly perhaps on account of their low vitality in water, and partly also because they are looked for at a date considerably later than that at which they were originally contained in the water. From the very nature of these bacteria it is easy to understand why they should be short- lived. The presence in the water of a number of ordinary saprophytes interferes with the growth of pathogenic bacteria, and either destroys them or consumes the pabulum neces- sary for their growth. Sedimentation of the water, which is constantly taking place, carries along with insoluble inorganic matter the bacteria to the bottom ; and in running water the oxygen of the air and direct sunlight act as efficient germicides. In making qualitative examinations of water for typhoid and cholera germs, what has been said in the special chapters on those germs should be borne in mind. It is advan- tageous, for instance, to mix the water with three times its volume of sterile bouillon, and to incubate this mixture before making the plates. For cholera, after six hours in the incubator the plates may be made, taking for this purpose the fluid from the upper portion of the mixture, as this germ grows rapidly, and chiefly on the surface of the fluid. For typhoid fever the incubation should be continued for two or three days before making the plates. By this incu- bation the ordinary saprophytes are retarded in growth, as they have less tendency to thrive at the incubator tempera- ture, whereas the reverse is the case for pathogenic germs, which grow much more rapidly at 37° C., and are thus in BACTERIOLOGICAL EXAMINATION OF THE AIR. 255 relatively larger numbers. Two sets of plates should be made as described in the quantitative test, one to be kept at the room temperature and the other to be incubated, and after these have grown for twenty-four to forty-eight hours the colonies should be examined under a low power of the micro- scope, and the colonies picked up with a platinum needle and planted in fresh agar and gelatin tubes, and these plated again until pure cultures are obtained. Eisner's method, described in the chapter on typhoid fever, is a very good method for the separation of water bacteria from typhoid bacteria, and the reader is referred to that chapter for its description. The addition of antiseptics, in small amounts, helps in the recognition of pathogenic germs, such as typhoid, in water, as these antiseptics interfere more materially with the growth of saprophytes than they do with that of the pathogenic germs. The reader is again reminded that the isolation of typhoid fever germs from water is difficult. It is easier to examine water for the Bacillus coli communis, which when present shows contamination by animal or human excreta, and makes the water unfit for consumption. By inoculating glucose- or lactose-bouillon in fermentation- tubes, the presence of this bacillus is easily made out on ac- count of the rapid development of gases it produces ; and plates made from the bouillon in the closed arm of the fermentation-tube will yield in such cases almost pure cult- ures of this bacillus. BACTERIOLOGICAL EXAMINATION OF THE AIR. A number of methods have been suggested for this pur- pose ; some consist in exposing plates of nutrient media, such as gelatin and agar, in the air, the bacteria falling on these plates and developing ; or, again, causing by slow aspiration a measured volume of air to pass through substances, such as sterilized sand or granulated sugar, afterward making agar and gelatin plate cultures with them. Quiet air contains but few bacteria, but air in motion, as in blowing wind, carries 256 EXAMINATIONS OF WATER, AIR, AND SOIL. solid substances, such as dirt, etc., loaded with bacteria. These may travel in suspension in the air for a considerable distance. The Sedgwick-Tucker method is perhaps the best procedure for the examination of air. For this purpose an apparatus known as the aerobioscope (Fig. 100) is required. In this apparatus a certain amount of sterile dry granulated sugar is introduced into the narrow part of the tube at d ; and at a a small roll of fine brass wire-gauze is inserted in order to provide a stop for the filtering material which is placed over it, as, for example, the sugar ; then by means of an air-pump a certain defi- nite quantity of air is sucked through the aerobioscope, after which the apparatus is closed with sterile cotton at FIG. 100. The Sedgwick-Tucker aerobioscope. (Abbott.) b and at c, and by gentle tapping, the contaminated sugar is forced into the larger portion of the tube at e; when this is accomplished 20 c.c. of liquefied sterile gelatin are poured in the larger part of the tube, the sugar dissolved in this gelatin, and an ordinary Esmarch tube made. The colonies may be counted as in an Esmarch tube, and pure cultures of the isolated colonies may be made on other plates. THE BACTERIOLOGICAL EXAMINATION OF THE SOIL. In the study of the soil for microorganisms special instru- ments for collecting the soil at different depths have been in- vented. C. Fraenkel's apparatus is perhaps the most useful. Small fragments of the soil to be examined should be dis- solved in liquid gelatin or agar, plates made, and the colonies counted as for the examination of water. The objection to this method, however, lies in the fact that QUESTIONS. 257 the solid particles of earth interfere considerably with the counting of the colonies, and to obviate this it is necessary at times to dissolve the soil in a certain quantity of sterile water, and to make plate cultures from this water. It should always be remembered, however, in making examinations of the soil, that a number of the bacteria found in it are anaerobics, and should be cultivated as such. Soil taken near the surface is always rich in bacteria, and the further down the investigator proceeds the smaller is the number of bacteria found, until at a distance of a meter and a half from the surface all bacteria have disappeared. In examinations of excavations made in the city of New Orleans some three years ago the author had occasion to verify the foregoing fact fully ; cultures made from mud at different depths showed a constant diminution of micro- organisms, until at a depth of between five and six feet no bacteria could be obtained. QUESTIONS. What sort of bacteriological examinations of water are made ? What is meant by a quantitative test ? Why is this riot a^ fair test? How is a quantitative analysis of water made ? What instrument is useful for that purpose ? How should water be collected ? What dilutions should be used? How is this dilution made ? How are the colonies on plates counted? Describe Wolfhuegel's apparatus for counting colonies on plates? How are Petri dishes used ? Describe Fakes' apparatus for counting colonies in Petri dishes. How are Esmarch tubes made? For what bacteria is the qualitative analysis of water made ? What are the difficulties in making qualitative analysis? How is examination of water for the cholera germ made? For typhoid fever germ? Describe Eisner's method of examining water for typhoid fever bacilli. What influence has the addition of antiseptics to water for bacterial exam- ination? How is water to be examined for tin- presence of Bacillus coli communisf What is the significance of the Hacilhtx co/i communis in water? Describe a method for the bacteriological examination of air. Describe Sedgwick-Tucker's method. How is an examination of the. soil made V What bacteria are found in the soil ? At what depth from the surface is the soil free from bacteria? 17 M. I1,. INDEX. A. AB-BE condenser, 20 Abdominal contents, examination of, 92 Abstraction theory, 101 Achorion Schonleinii, 219 Acquired immunity, 98 Actinomyces, 214 biologic characters of, 215 Mallory's stain of, 215 morphology of, 214 staining of, 215 Actinomycosis, 214 history of, 214 pathogenesis 'of , 216 Active immunity, 98 Aerobic bacteria, 37 Agar, filtration of, 62 media, 61 Agglutination of M. melitensis, 125 serum, 200 Agglutinin, 103, 180 Aggressins, 108 Air, 255 bacteriological examination of, 255 Sedg wick-Tucker method, 256 Alexins, 108 Amoeba coli, 232 biology of, 233 morphology of, 232 motion of, 233 staining of, 233 Amoebic colitis, 232 history of, 232 Amphitrocha, 36 Anaerobic bacteria, 37 cultivation of, 75 Anaphylaxis, 166 Angina, Vincent's, 211 Animals, inoculation of, 88 observation of, 92 Ankylostoma duodenale, 242 Anterior chamber of eye, inocula- tion into, 91 Anthrax, 141 bacillus of, 141 biology of, 143 morphology of, 142 pathogenesis of, 144 resistance of, to thermal changes, 144 staining of, 142 immunization of, 145 Antibodies, 103 Antigen, 106 Antimicrobic blood-serums, 100 Antisepsis, methods of, 86 Antiseptics, 84, 86 Antiserum, 106 Antitoxic blood-serums, 100 Antityphoid scrum, 182 Arnold steam sterilizer, 82 Arthrpspore, 35 Ascaris, 235 lumbricoides, 235 Attenuation, methods of, 96 Autopsy on animals, 92 Axial point, 24 B. BACILLEN emulsion, 131 Bacillus, 29 aerogenes capsulatus, 170 biology of, 170 history of, 170 morphology of, 170 259 260 INDEX. Bacillus aerogenes capsulatus, pathogen esis of, 170 staining of, 170 anthracis, 141 symptomatici, 168 biology of, 168 history of 168 immunity from, 170 morphology of, 168 pathogenesis of, 169 spores of, 168 staining of, 168 botulinus, 171 biology of, 171 history of, 171 morphology of, 171 pathogenesis of, 171 staining of, 171 cholerae gallinarum, 202 biology of, 202 inoculation of, 203 pathogenesis of, 203 staining of, 202 coli communis, 116, 184 biology of, 185 etiologic relations of, 184 history of, 184 morphology of, 184 _/ . pathogenesis of, 186 cuniculicidi (Fluegge), 203 diphtherisc, 146 of Ducrey, 204 biology of, 204 history of, 204 morphology of, 204 pathogenesis of, 204 staining of, 204 dysenteriae, 199 biology of, 200 history of, 199 inoculation of, 201 morphology of, 200 faecalis alkaligenes, 178 leprse, 133 biology of, 133 history of, 133 inoculation of, 134 morphology of, 133 staining of, 134 mallei, 137 biology of, 137 Bacillus mallei, inoculation of, 139 morphology of, 137 spores in, 137 staining in, 137 of Morax-Axenfeld, 203 biology of, 203 history of, 203 morphology of, 203 pathogenesis of, 204 staining of, 203 mucosus capsulatus, 120. See Pneumococcus. of pertussis, 195 pestis, 196 biology of, 196 immunity from, 198 morphology of, 196 pathogenesis of, 197 vitality of, 197 pneumonise (Fluegge). See Pneumococcus. proteus vulgaris, 186 biology of, 186 history of, 186 morphology of, 186 pathogenesis of, 186 staining of, 186 pseudodiphtherise, 154 staining of. 154 varieties of, 154 pyocyaneus, 109, 116 biology of, 116 morphology of, 116 pathogenesis of, 116 of rabbit septicaemia (Koch), 203 of rhinoscleroma, 122 smegmatis, 135 staining of, 135 sui pestifer, 201 biology of, 201 history of, 201 immunity from, 201 morphology of, 201 pathogenesis of, 202 vitality of, 202* of swine plague (Loeffler), 203 of syphilis, 235 tetani, 159 biology of, 160 cultivation of, 76 morphology of, 160 INDEX. 261 Bacillus tetani, motility of, 162 pathogenesis of, 163 spores of, 160 staining of, 160 thermal death-point of, 162 tuberculosis, 106, 116, 126 Koch's discovery of, 126 morphology of, 126 nature of, 127 occurrence of, 127 pathogenesis of, 127 staining of, 127 transmission of, 128 typhosus, 106, 116, 173 artificial susceptibility of, 179 biology of, 174 comparison of, with Bacilli coli, 176 cultures of, 175, 179 differentiation of, from allied groups, 176 history of, 173 inoculation with, 175 morphology of, 173 serum diagnosis of, 179 staining of, 173 vitality of, 175 varieties of, 30 xerosis, 158 Bacteria, anaerobic, 37 cultivation of, 57 definition of, 29 destruction of, 89 examination of, 42 isolation of (Koch's), 72, 73 morphology of, 30 motility of, 36 reproduction of, 33 size of, 33 staining of, 43 varieties of, 30 Bacterial growth, chemical reaction of culture-medium and, 38 decomposable organic material and, 38 essential conditions of, 37 heat and, 37 moisture and, 38 life, 39 inert conditions of, 39 inhibitive Conditions of, 39 Bacteriolysin, 103 Bacterium, 29 Bichloride of mercury as antiseptic, 86 Blood in typhoid fever, preparing specimen of, 180 Blood-serum as culture-media, 58, 75 Blood-stain, Wright's, 55 Boiling water as an antiseptic, 86 Bordet-Gengou phenomenon, 106 Bothriocephalus latus, 238 Bouillon. See Culture-media. Bread-paste, 63 Brownian movements, 36 Bubonic plague, 196 0. CALMETTE'S ophthalmic reaction, 131 Cannon's influenza bacillus, 193 Capsules, staining of, 48 Johne's method, 48 Welch's method, 48 Carbolic acid as an antiseptic, 86 Cerebrospinal meningitis, epidemic, 122 Chamberlain's filter, 83 Chauveau's retention theory, 101 Chemical agents, 87 Chemicals, choice of, 85 use of, for disinfecting, 84 Chicken cholera, 202 Chlorinated lime as an antiseptic, 86 Cholera spirillum, 187 artificial susceptibility to, 191 diagnosis of, 193 diagnostic test of, 191 growth of, 190 history of, 187 immunity against, 191 morphology of, 187 pathogenesis of, 190 staining of, 188 vaccination against, 192 vitality of, 190 Chromophytosis, 219 Clostridium, 33 Coccus, varieties of. 30 Cohn, classification of, for bacteria, 28 262 INDEX. Colonies, counting of, 74 Comma bacillus. See Cholera spi- rillum. Condenser, dark field, 26 Conradi's bile medium, 64 Control test, 82 Cultivation of anaerobic bacteria,75 of bacteria, 57 utensils used, 65-70 of tetanus bacillus, 76 Culture-media, agar, 61 blood-serum, 58 bouillon, 59 bread-paste, 63 Conradi's bile, 64 Dorsett's egg, 64 Eisner's, 177 gelatin, 60 glucose-bouillon, 64 glycerin-agar, 62 Jackson's bile, 64 lactose-bouillon, 64 milk, 52 Pasteur's solution, 59 peptone solution, 64 potato, 62 potato-paste, 63 saccharose-bouillon, 64 urine, 59 Cultures, agar slant, 75 blood-serum, 75 diphtheria bacillus, 153 from secretions, 93 human body, 93 thoracic organs, 93 Cysticerci, 240 D. DARK field condenser, 26 illuminator, 26 Deny's bouillon filtrate, 131 Dimness, tests for sources of, in the object, 25 Diphtheria, 146 antitoxins, 155 preparation of, 155 standardization of, 156 bacillus, 146 biology of, 149 cultures of, 153 Diphtheria bacillus, distribution of, 147 morphology of, 147 pathogenesis of, 150 powers of resistance of, 149 staining of, 148 Neisser's method, 148 diagnosis of, 152 treatment of, antitoxin, 155 Diplococcus intracellularis meningi- tidis, 122 biology of, 123 cultures of, from man, 122 discovery of, 122 morphology of, 122 pathogenesis of, 123 Disinfection, methods of, 84 Distomum hematobium, 230 diagnosis of, 231 westermanii, 231 Dorsett's egg medium, 64 Drummer-bacillus, 34 Dyes, application of, 44 E. EBERT'S bacillus, 173 Ehrlich's chain theory, 101 Eisner's medium, 177 Entamceba coli and histolytica. See Amoeba coli. Epidemic cerebrospinal meningitis. 122 Examination of air, 255 of fa3ces in dysentery, 233 of soil, 256 of water, 249 F. FARCY, 137 Favus, 219 Fermentation, 39 alcoholic and acetic acid, 39 butyric and lactic acid, 39 Filaria sanguinis hominis, 229 morphology of, 229 pathogenesis of, 229 Fission, 33 Flagella, 36 INDEX. 263 Flagella, staining of, 50 Bunge's method, 52 Loeffler's method, 50 Pitfield's method, 52 Formalin as an antiseptic, 87 G. GASES, 40 Gelatin media, 60 Germicidal power, test for, 85 Germicides, 84. See also Antisep- tics. Glanders, 137 Glycerin-agar media, 62 Gonococcus, 109, 113 Gonorrhoea, 113 Gram's method, 53 Gruber-Widal reaction, 104 H. H^MOLYSINS, 104 demonstration of, 105 Hanging-drop, 43 Heat, sterilization by, 82, 83 Hog-cholera, 201 Hookworm, 242 disease, 242 Hyphomyoetes (mucorini), 28 I. ILLUMINATOR, dark field, 26 Immunity, 87 acquired, 98 active, 98 methods of, 99 natural, 98 passive, 100 racial, 98 theories of, 101 Incubator, 78 Infection, associated, U7 avenues of, 95 bacteria in, quantity of, 96 chemical theory of, 95 definition of, 94 factors of, 95 mechanical theory, 94 Influenza, 193 Influenza, bacillus of, 193 biology of, 194 history of, 193 morphology of, 194 pathogenesis of, 194 vitality of, 194 Inoculation of animals, 88 of fluid media, 71 of gelatin culture-tubes, 72 methods, 88 of solid media, 71 Intestinal parasites, 235 Intraperitoneal inoculation, 91 Intrapleural inoculation, 91 Intravenous injection, 89, 90 J. JACKSON'S bile medium, 64 K. KITASATO'S mouse-holder, 90 Koch's bacillus of rabbit septicemia, 203 sterilizer, 81 tubercle bacillus, 126 tuberculin, 130 A, O, and R, 130 Kuehne's carbolic methylene-blue method, 54 LAVERAN'S malarial parasite, 220 Leishmania Donovani, 230 morphology of, 230 staining of, 230 Lens, focus of, 18 type of objective, 23 Lenses, chromatic aberration of, 18 objective of, 21 ocular of, 21 spherical aberration of, 18 Leprosy, 133 diagnosis of, 134 nature of, 134 Light, direct, 20 oblique, 20 producing bacteria, 40 refraction of, 17 264 INDEX. Loeffler's blood-serum, 59 glanders bacillus, 124 Losch, amoeba of, 232 Lustgarten's bacillus, 135 Lymphatics, inoculation into, 91 Lysins, 104 M. MACROGAMETES, 222 Malaria, cycles of, 220 in man, 220 in mosquito, 220 Malarial fever, 220 examination of blood in, 223 mosquitoes in, 220 symptoms of, 220 parasite, 220 characteristics of, 221 differentiation of, 228 inoculation of, 228 staining of, 127 varieties of, 221 Malignant oedema, bacillus of, 166 biology of, 167 morphology of, 166 pathogenesis of, 167 spores of, 167 Mallein, 140 Mallory's stain for ray fungus, 215 Malta fever, 164 Melanin, 220 Meningitis, epidemic cerebrospinal, 122 Metchnikoff's phagocytosis theory, 101 Methods, special, of staining, 45 . Gabbett's, 47 Gram's, 46 Koch-Ehrlich's, 45 Loeffler's, 45 Ziehl's carbol-fuchsin, 47 Micrococcus catarrhalis, 115 biology of, 115 morphology of, 115 staining of, 115 gonprrhoeae, 113 biology of, 114 morphology of, 114 pathogenesis of, 113 staining of, 115 Micrococcus melitensis, 124 biology of, 124 morphology of, 124 pathogenesis of, 124 pasteuri, 117 pneumonia crcuposae, 117 biology of, 119 history of, 117 immunization of, 120 intrathoracic injection of, 120 morphology of, 119 subcutaneous injection of, 120 pyogenes tenuis (Rosenbach), 109 tetragenus, 109, 113 morphology of, 113 pathogenesis of, 113 properties of, 113 Microgametocytes, 191 Microscope, care of, 25 compound, 19 dark field illuminator of, 26 lenses of, 17 simple, 19 working distance of, 23 Microsporon furfur, 219 Milk as culture-medium, 58 mode of preparing sterilized, 58 Monotrocha, 36 Morax-Axenfeld bacillus, 203 Moro's ointment, 132 Mordant, 51 Moulds, 217 N. NATURAL immunity, 98 Necator americanus, 242 Negri bodies, 246 Neisser's gonococcus, 113 Nicolaier, tetanus bacillus of, 159 Noma, 212 Numerical aperture, 24 0. OBERMEIER'S spirillum, 206 Objective, angular aperture of an, 23 > cleansing of, 25 designation of, 21 INDEX. 265 Ocular, cleansing of lenses of, 25 lens, 24 types of, 25 (Edema, malignant. See Malignant oedema. Oidium albicans, 218 Ophthalmic reaction, Calmette's, 131 Opsonic index, 107 ( )psonins, 106 Optical axis, 24 Oxygen, relation of, to bacterial life, Oxyuris, 236 vermicularis, 236 Ozaena, 122 P. PARASITES, 37 intestinal, 235 Paratyphoid bacilli, 179 Pasteur's abstraction theory, 101 treatment in rabies, 247 Pathogenic bacteria, 108 Pelvic contents, examination of, 92 Peptone solution, 64 Peritrocha, 36 Pertussis bacillus, 195 history of, 195 morphology of, 195 pathogenesis of, 195 staining of, 195 Pfeiffer's bacillus, 193 phenomenon, 104 Phagocytosis theory, 101 Pit field's flagella stain, 52 Pityriasis versicolor, 219 JPlasmodium malariae, 220 Plate cultures of agar, 74 Pneumobacillus (Friedlaender's) , 116 .Pneumococcus, 109 Friedlaender's, 120 biology of, 121 discovery of, 120 . morphology of, 121 pathogenesis of, 121 Pneumonia, 117 Potato as culture-media, 62 preparation of, for test-tube cul- ture, 63 Precipitin, 103 demonstration of, 104 Protozoa, 29, 220, 229 Pseudodiphtheria, 154 differential diagnosis of, 154 Ptomaines, 40 Putrefaction, causes of, 40 R. RABIES, 246 immunity to, 247 . Negri bodies in, 246 Pasteur treatment in, 247 Racial immunity, 98 Ray fungus, 214 Refraction, law of, 17 Reichert's thermo-regulator, 78 Relapsing fever, 206 Retention, theory of, 101 Rhinoscleroma, bacillus of, 122 Ringworm, 218 Roux-Nocard method of culture, 93 history of, 93 importance of, 93 technic of, 93 SACCHAROMYCES hominis, 217 Saccharomycetes, sprouting fungi, 28 Saphrophytes, 37 Scarlet fever, 249 Schizomycetes, cleft fungi, 28 Sedgwick-Tucker method for ex- amining air, 256 Serotherapeutics, 198 Serum, agglutinating action of, IDS antityphoid, 183 Shiga's bacillus, 199 Side-chain theory, Ehrlich's, 101 Soil, examination of, for bacteria, 256 Spirillum, 30, 205 cholera Asiaticae, 187 Obermeieri, 206 biology of, 206 history of, 206 morphology of, 206 patriogenesis of, 206 266 INDEX. Spirochaeta anserinse, 212 gallinarum, 212 history of, 212 morphology of, 212 pathogenesis of, 212 staining of, 212 pallida, 207 animal inoculation of, 208 diagnosis of, 209 history of, 207 immunity to, 208 morphology of, 207 pathogenesis of, 208 staining of, 207 Wassermann reaction and, 209 refrigerens, 208 Spleen, typhoid bacilli in, 179 Spores, staining of, 48 (Abbott) first method, 48 second method, 49 third method, 49 (Fiocca) fourth method, 50 Sporozoite, 222 Sporulation, 33 significance of, 35 Sputum septicaemia, 117 Staining, 34 methods, 43, 127 BowhilFs, 53 Bunge's, 52 Ehrlich's modification of Koch's, 45, 127 Gabbett's modification of Ziehl's, 47, 127 Koch's, 127 Pitfield's, 52 Van Ermengem's, 53 Wright's, 55 Ziehl's carbol-fuchsin, 47, 127 Stains, 44 Staphylococcus cereus albus, 108 aureus (Passet), 108 flavus (Passet), 108 pyogenes albus, 111 aureus, 109 features of, 109 morphology of, 109 citreus, 111 Stegomyia calopus, 248 Sterilization, definition of, 79 fractional, 80 Sterilization, methods of, 79 Streptococcus mucosus, 120 of syphilis, 136 Streptothrix, 213 Eppingeri, 214 madurae, 214 pseudotuberculosa, 216 resemblance of, to bacteria, 213 to moulds, 213 Subcutaneous inoculation, 88 Sulphur dioxide as antiseptic, 86 Syphilis, 135 spirochaeta pallida and, 207 Wassermann reaction in, 209 T. T^NIA, 237 echinococcus, 241 nana, 240 saginata, 238 solium, 238 Tapeworm. See Taenia. Tetanin, 162 Tetanus, 157 antitoxin, 165 bacillus, cultivation of, 76 history of, 159 toxin, 164 Thrush, 218 Tinea circinata, 218 favosa, 219 versicolor, 219 Tissues, staining of bacteria in, 53 Gram's method, 53 Kuehne's carbolic methyl- ene-blue method, 54 Weigert's method (modifica- tion of Gram), 54 Ziehl-Neelson's method, 55 Toxalbumins, 40 Treponema pallida, 207. See Spi-- rochaeta pallida. Trichina, 241 spiralis, 241 Trichocephalus dispar, 237 trichyuris, 237 Trichophyton tonsurans, 218 Trypanosoma gambiensis, 230 Trypanosomes, 229 Trypanosomiasis, 229 INDEX. 267 Tuberculin A, O, and R, 130 diagnosis of tuberculosis by, 129 Koch's, 129 Tuberculosis, 126 history of, 126 Moro's ointment in, 132 ophthalmic reaction of Calmette in, 131 transmission of, 129 Typhoid fever, 173 transmission of, by house fly. 176 vaccination against, 182 UNCINARIASIS, 245 V. VAN NIESSEN'S streptococcus, 136 Vincent's angina, 211 history of, 211 morphology of, 211 pathogenesis of, 211 Voges' guinea-pig holder, 87 Von Pirquet's skin reaction, 131 W. WASSERMANN reaction, 106, 209 Water, bacillus colicommunis in, 255 bacteria in, 249 cholera bacilli in, 254 counting of colonies in, 251 examination of, 249 pathogenic germs in, 254 typhoid bacilli in, 254 Weigert's modification of Gram's method, 54 Wiesnegg's autoclave, 83 Wright's blood-stain, 55 method for opsonic index, 107 Y. YAWS, 211 Yeasts, 217 Yellow fever, 248 transmission of, 248 stegomyia in, 248 Yersin's plague serum, 199 Z. ZIEHL-NEELSEN'S method, 55 Date Due FF8 28 19S1 ...MicroscopyL bacteri- 1912 otogy, and human par- -1932- FEB o LIBRARY COLLEGE OF DENTISTRY UNIVERSITY OF CALIFORNIA