MEDICAL .SCHOOL LIISMAIIO' ELEMENTS OF Clinical Bacteriology FOR PHYSICIANS AND STUDENTS BY DR. ERNST .LEVY Professor in the University of Strasburg i. E. AND DR. FELIX KLEMPERER Private Decent in the University of Strasburg i. E. SECOND ENLARGED AND REVISED EDITION AUTHORIZED TRANSLATION BY AUGUSTUS A. ESHNER, M.D. Professor of Clinical Medicine in the Philadelphia Polyclinic ; Physician to the Philadelphia Hospital ; etc. PHILADELPHIA W. B. SAUNDERS 925 WALNUT Street Copyright, 1900, by w. b. Saunders. Press of •W%B.*|Al»IDEKS,VP^II^.t)4iPblM TRANSLATOR'S NOTE. Works on bacteriology, on pathology, and on clinical diagnosis are not wanting in any language, but we know of only one on clinical bacteriology, and as a consideration of the subject from this aspect must appeal to a large body of the profession, it was thought a service might be ren- dered by a translation of this admirable publication, whose authors are well and favorably known for their original work in both clinical medicine and bacteriology. The general practitioner can scarcely be expected to be a trained and practised bacteriologist, but he must have a working familiarity with the subject of bacteriology in order that he may possess clear conceptions as to the etiology of disease and the nature of the resultant morbid processes, leading to a rational application of measures and methods of prophylaxis and treatment. To these ends it is hoped the present publication will contribute. The work of translation is coupled with especial and personal pleasure from the fact that both junior author and translator participated simultaneously in the work in bacteriology in the Hygienic Institute at Berlin in the spring of 1890. It has been thought advantageous to add illustrations to the English version. A. A. E. Philadelphia, February, igoo. 4^5 Digitized by the Internet Archive in 2007 with funding from IVIicrosoft Corporation http://www.archive.org/details/elementsofclinicOOIevyrich PREFACE TO THE SECOND EDITION. Scientific activity in the domain of clinical bacteriology has been not less pronounced than fruitful during the period that has elapsed since the appearance of the first edition of this book. By including the numerous results of recent investigation the size of the present volume has been considerably increased. Chapters have been added on Plague and Botulism, and those on Immunity, Diph- theria, Typhoid Fever, Actinomycosis, Examination of Air and of Water, and others, have been radically revised ; and in all other sections numerous changes and additions have been made. We believe the work adapted to the present position of bacteriologic knowledge, and hope that this edition may have the same friendly reception accorded the first edition. The Authors. CONTENTS PART L , PAGE I. Morphology and Biology of Bacteria 17 II. Infection 33 III. Immunity, Immunization, and Cure 49 IV. Methods of Culture and of Examination 74 PART n. INFLAMMATION AND SUPPURATION. Morphology of the Causative Agents of Inflammation I15 The Pathogenic Properties of the Causative Agents of Inflammation with Relation to Animals 123 The Occurrence of the Causative Agents of Inflammation and Suppura- tion in Healthy Persons and Outside the Body 126 The Occurrence of the Causative Agents of Inflammation in Disease . . 127 Cutaneous Suppuration 127 Erysipelas 129 Phlebitis and Lymphangitis 132 Inflammations of the Nose and Throat 133 Angina 134 Otitis Media 135 Meningitis ' . 136 Bronchitis 138 Pleuritis 138 Pneumonia 142 Endocarditis 148 Pericarditis 150 Myocarditis 15 1 Peritonitis 151 Cholecystitis and Cholangitis .' 154 Abscess of the Liver 155 Nephritis -157 Perinephritis 158 Pyelonephritis 159 13 14 CONTENTS. PAGE Inflammatory Diseases of the Eye i6o Pyemia and Septicemia l6i Puerperal Fever 163 Osteomyelitis 164 Pyocyaneous General Infection 165 PARTm. SPECIFIC DISEASES OF BACTERIAL ORIGIN. Typhoid Fever 166 Asiatic Cholera 181 Cholera Nostras and Summer Diarrhea 198 Plague 200 Diphtheria 207 Tetanus 230 Botulism 245 Tuberculosis 251 Fowl-tuberculosis 275 Pseudo- tuberculosis 276 Leprosy 278 Influenza 281 Anthrax 287 Glanders 300 Malignant Edema 305 Proteus-infection 308 Gonorrhea 311 Syphilis 314 Hydrophobia (Lyssa ; Rabies) 321 Smallpox (Variola) . ' "325 Acute Exanthemata 328 Whooping-cough (Pertussis) 330 Articular Rheumatism 331 Relapsing Fever 332 PART IV. I. MYCOSES (INFECTIONS WITH FILAMENTOUS AND BUDDING FUNGI). Morphology and Biology of the Filamentous and the Budding Fungi . 336 Pathogenic Activity of Filamentous and Budding Fungi for Animals . . 339 Diseases in Human Beings Induced by Filamentous and Budding Fungi 341 Actinomycosis 354 Pathogenic Streptothrices 362 Pathogenic Yeasts 363 CONTENTS. 15 PAGE II. INFECTIONS WITH TliE LOWEST FORMS OF ANIMAL LIFE. Dysentery (Amebic Enteritis) and Tropical Abscess of the Liver . . . 364 Malaria 372 Leydenia Gemmipara Schaudinn 387 APPENDIX. I. BACTERIOLOGIC EXAMINATION OF SOIL, AIR, AND WATER. Soif 389 Air ... 392 Water 396 THE BACTERIA PRINCIPALLY FOUND IN SOIL, AIR, AND WATER. I. Bacilli 401 II. Micrococci 405 III. Vibrios 407 II. DISINFECTION. Disinfecting Agents 411 Disinfection of the Hands 418 Disinfection of Mucous Membranes 419 Disinfection of Instruments and Dressings 419 Disinfection of Feces and Cesspools 419 Disinfection of Bath-water, Wash-water, etc 420 Disinfection of Urine 421 Disinfection of Sputum 421 Disinfection of Body-clothing and Bed-linen 422 Disinfection of Beds and Clothing 423 Disinfection of Articles of Food 423 Disinfection of the Sick-room 423 Disinfection of Ships, Vehicles, Railway-cars, etc 425 Index 427 I \ )p 0 . 425 Clinical Bacteriology, PART I L MORPHOLOGY AND BIOLOGY OF BACEB3UA. The minute forms of life constituting in largest part the exciting agents of the infectious diseases occupy the lowest level in the vegetable kingdom. They are designated bac- teria or fission-fungi. The latter term is, however, not well chosen. The botanic conception of fungi includes those lower forms of vegetable life that are without the coloring- matter of leaves (chlorophyl) or similar coloring-matters (chromophyls). There are not a few bacteria, however, that are capable of generating such coloring-matters, and, thus, also of decomposing carbon dioxid and its combi- nations, and utilizing the products of this activity. There is, thus, no justification for the term fission-fungi, and it is, therefore, preferable to designate the minute forms of life under consideration as bacteria. There is as yet no strictly scientific, natural classification of bacteria. For the present the adoption of an artificial system, based upon such physical features as the size, the shape, and the arrangement of the bacteria, must suffice (F. Cohn). (See Figs, i, 2, 3.) Three main divisions are recognized: (i) Spheric bacteria (cocci, micrococci, coc- caceae) ; these are spheric cells. (2) Rod-shaped bacteria (bacilli, bacteriaceae) ; these are rod-like, cylindric cells. (3) Spiral bacteria (vibrios, spirilla, spirillaceae) ; these are twisted in both the horizontal and the vertical direc- tion, and are, thus, comparable to the windings of a screw or of a corkscrew. 2 17 18 CLINICAL BACTERIOLOGY. The individual bacterial cells exhibit at times a homo- geneous translucent, at other times a granular protoplas- mic, structure that, on the whole, possesses the properties of all other protoplasmic structure, but that is not, as in the cells of the higher orders of plants it is, differentiable 0|?i \^ i^ fj /< lO 11 Fig. I.— Various forms of bacteria: i and 2, Round and oval micrococci ; 3, diplo- cocci ; 4, tetracocci, or tetrads; 5, streptococci; 6, bacilli; 7, bacilli in chains, the lower showing spore-formation ; 8, bacilli showing spores, forming drumsticks and Clostridia ; 9 and 10, spirilla; 11, spirochetse (McFarland). Fig. 2. — Diagram illustrating the morphology of the spirilla: a, ^, c, Spirilla; d, y rotated, and METHODS OF CULTURE AND OF EXAMINATION. 101 the inner space is cut off completely from the outer air. (Fig. 32.) Novy's apparatus may also be connected with an air-pump, and be exhausted of air ; or, finally, it may be freed of oxygen with the aid of an alkaline solution of pyrogallic acid. (See method of Buchner, below.) Aiiaerobic pure cultures in test-tubes may be prepared in the following manner : 1. In a High Layer. — Stab-cultures are made in tubes that are filled with nutrient material to a higher level than usual, and that are boiled again shortly before being used. In the lower portions of the culture, free from oxygen, to which the needle has penetrated, development takes place, whereas the upper portions of the medium containing oxygen remain sterile. The anaerobic bacteria thrive more vigorously when reducing substances are added to the nutrient media — e. g., two per cent, of grape-sugar or from 0. 3 to o. 5 per cent, of formic acid. 2. In an Atmosphere of Hydrogen. — The test-tubes are closed with sterilized rubber stoppers doubly perforated, through which two glass tubes bent at a right angle lead into the interior. (Fig. 28.) Through the longer tube, which reaches almost to the nutrient medium and is closed with a cotton stop- per, hydrogen is passed, while through the shorter tube atmospheric air is expelled. As soon as gas in pure state escapes through the shorter arm, both tubes are sealed by heat, or closed by means of rub- ber tubes. Inoculation must, of course, have been effected before the introduction of the hydrogen. J. With Complete Exclusion of Air. — A test-tube with a long-drawn-out tenuous neck is filled with nutritive material and inoculated in the usual manner. The neck is then connected with an air-pump, and when the air has been completely ex- hausted, the tube is sealed by heat. 4. According to the Method of Buchner. — The culture-tubes, closed by means of a loosely introduced cotton stopper, are in- troduced into a large, hermetically sealed tube, whose floor is covered with alkaline solution of pyrogallic acid ( i gram Fig- 33' — Buchner's method of making anaerobic cultures. 102 CLINICAL BACTERIOLOGY. of pyrogallic acid, lo cu. cm. of i per cent, potassium hydroxid). (Fig. 33.) Pyrogallic acid has the peculiarity of taking up oxygen, and in this way the space in which the cultures are exposed is quickly freed of oxygen. Raw eggs also may be employed for anaerobic culture. One extremity of the ^^^ is thoroughly cleansed with mercuric chlorid and sterile water, a puncture is made with a needle sterilized in the flame and still hot, and with the platinum needle a portion of the pure culture is introduced into the interior of the ^^'g. The small opening made is then closed with hot sealing-wax. MICROSCOPIC EXAMINATION AND STAINING OF BACTERIA. To examine bacteria in the living state in the hanging drop, a small drop is removed from a fluid culture by means of the platinum loop sterilized in the flame, and it is then placed in the center of a cover-slip carefully cleansed with alcohol. (.Fig. 34.) The cavity of a slide that has been 0 Fig. 34. — The " hanging drop" seen from above and in profile (Mallory and Wright). excavated at its center is surrounded with vaselin, and the slide is pressed face down upon the cover-slip, so that the drop is suspended exactly in the center of the cavity. If it be desired to prepare a hanging drop from a solid culture, a drop of sterile water or of bouillon is first placed upon the cover-slip, and to this is added a minimal amount of the bacteria to be examined. It is useful in some cases to add to the hanging drop a small amount of a dilute staining solution : e. g., 2l carbol-fuchsin solution (p. 104) diluted four or five times. The small amount of coloring- matter added does not influence the vital activity of the bacteria. These are stained faintly, but the motile varieties METHODS OF CULTURE AND OF EXAMINATION. 103 continue in active movement, and in many of them, as a result of the action of the stain, even the flagella become visible. In making examinations in hanging drop it is advisable first to find the border of the drop with a low power of the microscope, and then to scrutinize it with higher powers. After having observed sufficiently the size and the shape of the bacteria in a thin layer, and while comparatively quiet, the central portions of the drop are brought into the field. Instead of a simple hanging drop, a culture may be made in hanging drop, and the growth of the individual bac- terial cell, the formation of spores and their germination, etc., can be observed directly under the microscope. As a matter of course the cover-slip must previously have been sterilized by being passed through the flame. After the slip has cooled, a drop of sterile bouillon or gelatin is placed at its center and is inoculated. The observation may then be continued, perhaps with the aid of a warm stage, or of a special small incubator in which the entire microscope is introduced. In order to decide with certainty whether the movement observed in certain bacteria is dependent upon their own motility, upon molecular movement, or upon that due to currents, it is at times necessary to employ liquid gelatin as. a diluting fluid instead of sterile water. The degree of temperature necessary for liquefaction is readily obtained by using the warm stage. All such observations are, of course, made through a narrowed diaphragm. Examination of Stained Preparations. — For staining purposes basic aniline dyes are employed that possess the property of staining nuclei and bacteria. The most impor- tant of those in use are gentian -violet, methyl -violet, fuch- sin, methylene-blue, vesuvin, and malachite-green. These stains, with the exception of the last two, are kept in readi- ness in concentrated alcoholic solution, and the two ex- cepted, vesuvin and malachite-green, in about one per cent, watery solution. From the alcoholic stock-solutions the usual watery staining solutions are prepared by dilution with from ten to twenty times the amount of distilled water. A still simpler means consists in permitting a few drops of the stock -solution to pass through a filter into a watch-glass containing distilled water. By the addition of certain sub- stances that, to a certain extent, play the part of mordants, 104 CLINICAL BACTERIOLOGY. the staining power of these substances may be increased considerably. Among substances to be mentioned in this connection are : 1. Potassium hydroxid (employed by Loffler in combina- tion with methylene^blue) : 30 cu. cm. of concentrated alcoholic solution of methylene-blue. ICX) cu. cm. of o.oi per cent, solution of potassium hydroxid (i : 10,000). This so-called Loffler's solution stains well, and may be preserved for an almost indefinite time. 2. Aniline water : Four or 5 cu. cm. of aniline oil, with 100 cu. cm. of dis- tilled water (i part of aniline oil to about 20 parts of water), are vigorously shaken and passed through a moist- ened filter. To the clear filtered aniline water (100 cu. cm.) are added 1 1 cu. cm. of a concentrated alcoholic solution of fuchsin or of gentian-viplet or methyl-violet. It is more convenient to filter the aniline water in a watch-glass and to add the solution of fuchsin or gentian-violet or methyl- violet until a metallic, opalescent pellicle appears upon the surface. These solutions of Ehrlich stain well, but they possess the disadvantage that they decompose rapidly, and they must, therefore, be freshly prepared each time that they are used. J. Carbolic acid: One gram of fuchsin is dissolved in 10 cu. cm. of 96 per cent, alcohol, and then 90 cu. cm. of 5 per cent, carbolic acid are added. This solution of Ziehl does not possess quite so intense a staining power as the aniline-water solutions, but it has over these the not inconsiderable advantage of greater durability. In an analogous manner carbolic-acid gentian-violet, and carbolic-acid methylene-blue solutions are prepared, both of which possess excellent staining qualities, and can be preserved for a long time. Preparation and Staining of Cover-glass Specimens. — In making stained preparations the suspected material is spread in as thin a layer as possible upon a cover-glass thoroughly cleansed with alcohol and dried, a small drop being taken, by means of the platinum loop, directly from liquid cultures not more than one or two days old, or a small amount of the bacterial mass from solid cultures being rubbed up in a drop of sterilized water (or tap-water, which METHODS OF CULTURE AND OF EXAMINATION. 105 may be considered sufficiently sterile for this purpose) upon the cover-glass. The preparation is permitted to dry in the air, and then, in order to fix it, it is passed three times, with moderate rapidity, through the flame of a spirit-lamp or of a Bunsen burner. In order to obtain satisfactory preparations from cultures it is useful, before staining, to immerse the cover-glasses for from one-half to one minute in from a one to a four per cent, solution of acetic acid. By this means the preparation is cleared, while the bacteria in no wise suffer. The acetic acid is blown off by means of a glass tube, or the cover-slip is simply dried carefully be- tween filter-paper. By means of a pipet or of a small filter (it is usually well to filter the staining solutions before they are used) several drops of the staining solution are placed upon the smeared surface of the cover-slip (conveniently grasped with Cornet's forceps) ; the stain is permitted to exert its influence for a short time, and is then washed off Fig- 35- — Stewart's cover-glass forceps. in water. If the staining solution has been placed in a watch-glass, the cover-slip is carefully placed upon the sur- face of the fluid so that it floats thereon with its smeared surface down. As to the length of time that the cover- glasses should be exposed to the influence of the staining solution, no definite statement can be made. This will de- pend upon the thickness of the layer, the concentration of the stain, and the tingibility of the bacteria. In general, from half a minute to a minute is required for staining. . The time of exposure to the action of the staining fluids may be materially shortened by application of heat. For this pur- pose the cover-glass is held with the forceps directly over the flame until the vapor of steam is given off from the staining solution. The stained specimen is thoroughly washed in water, then carefully dried between filter-paper, and mounted in xylol-Canada balsam. If blood-preparations are to be examined for bacteria, it is likewise well to treat the smeared cover-glasses according to the acetic-acid 106 CLINICAL BACTERIOLOGY. method, as by this means the blood coloring-matter and a portion of the plasma are removed. The specimens prepared in the manner described show the size and the shape of the bacteria, but fail to disclose their mutual positions in the colonies. It this information be desired, so-called contact-preparations or impress-prepa- rations must be made. A carefully cleaned cover-slip is lightly applied or impressed upon an isolated bacterial colony of a gelatin-plate or an agar-plate, and immediately removed. After it has been dried, this preparation is further treated exactly in the manner described. Staining of Sections of Tissue. — Bits of tissue hard- ened according to the usual methods and embedded in cel- loidin are cut with the aid of the microtome. The sections are next placed in distilled water, and from this are trans- ferred to one of the staining solutions mentioned. In this they are permitted to remain in general from two to fifteen minutes. The excess of coloring-matter is removed by means of dilute acetic acid (i : looo), and the sections are rinsed in water for the removal of the acid, dehydrated with absolute alcohol, cleared with xylol, and mounted upon a slide in xylol-Canada balsam. It is advisable first to spread the sections upon slides, and to undertake the staining, the decolorization, the dehydration, and the clearing upon these. On the whole, this mode of procedure agrees with that originally described by Weigert. If the tissue has been embedded in paraffin, this must be extracted with xylol before staining, and the xylol be then removed with alcohol. The methods of staining thus far described have the property of staining all bacteria in the same way. There is, however, a method that stains some of the bacteria specifically — that is, Gram's method. Gram's Method. — The prepared cover-slips from a fresh culture from twenty-four to forty-eight hours' old are placed in aniline-water gentian -violet solution (cover-glasses for one or two minutes, sections directly out of alcohol for ten or fifteen minutes), and then for half a minute, or for two and a half minutes or three minutes, in an iodin potassium- iodid solution, consisting of iodin i.o, potassium iodid 2.0, water 300.0. Decolorization is next effected in alcohol, and is continued as long as any color is given off. The prepara- tion, which finally appears colorless (light gray), is then METHODS OF CULTURE »l«PjQF-SgCAMINATl€«S. Jj07 X^/n;-- .-.,.. •'"^ rinsed in water, dried, and niounterM4,^0att^"d^ j45ali Stained in this way certain bacteria assumed Dltiislf^lack color, while others yield up the stain completely on decol- orization. Gram's method, which is of especial importance in the identification of bacteria, is most difficult of applica- tion. The slightest variations in its technic may render the results doubtful. It is well, therefore, to make a control- stain in every instance, examining simultaneously, in addition to the bacteria under investigation, another variety of bacteria that with certainty either stains (staphylococci) or decolorizes (bacterium coli). Giinther's Modification of Gram's Method. — After exposure to the action of the iodin potassium-iodid solution, the specimen is placed for half a minute in alcohol, then for a short time (ten seconds) in three per cent, hydrochloric- acid alcohol, and, finally, for complete decolorization, in pure alcohol. Before mounting, the sections are cleared in oil of cloves or in xylol. Weigert's Modification of Gram's Method. — After treatment with aniline-water gentian -violet solution, the specimen is rinsed in water, and the section is spread upon a slide, iodin potassium-iodid is added, and then removed with bibulous paper. Next follow differentiation, dehydra- tion, and clearing with aniline oil and xylol, and mounting in Canada balsam. Double Staining. — To make a contrast between the bacteria and the surrounding tissue, it is advisable, after staining by Gram's method, to stain the sections further with a dilute watery solution of vesuvin, safranin, or car- mine, and to rinse in alcohol. In this way, however, the bacteria under some circumstances lose a portion of their stain. For this reason it is preferable to apply the contrast- stain before using Gram's method of staining the bacteria. To this end the sections are washed in water, stained in picrocarmine solution, again washed in water and placed in alcohol, after which the staining, according to the method of Gram, can be undertaken at once or at any convenient time. The staining of the tissues is in no way interfered with by Gram's method. Not all bacteria remain stained after application of Gram's method : a very consider- able number can not be demonstrated by this means. Particulars in this regard will be found in the special section. 108 CLINICAL BACTERIOLOGY. Staining of Capsules. — Some bacteria are character- ized by the formation of capsules in the blood and in other animal substances (anthrax-bacilli, pneumococci, ba- cillus of Friedlander, etc.). Occasionally, the capsules appear also in cultures, and their demonstration can be made in the following method of Johne : The preparations are stained in a warmed two per cent, solution of gentian- violet, are rinsed in water, decolorized for from ten to twenty seconds in two per cent, acetic acid, and are washed and mounted in water (not Canada balsam, as this causes the capsules to shrink). Staining of Spores. — Spores are stained with extraor- dinary difficulty, because of the dense and impenetrable membrane by which they are surrounded. The usual methods of staining are not sufficient for the staining of spores. This may be effected by exposure of cover-glass specimens prepared in the usual way with spore-containing bacteria for an hour in hot carbol-fuchsin solution or aniline- water fuchsin-solution, which is from time to time brought to the boiling-point. By addition of solution undue con- centration is avoided. The specimen is taken directly from the stain, and is placed in ten per cent, hydrochloric acid or in three per cent, hydrochloric-acid alcohol, in which it is washed for about a minute. By this means everything is decolorized with the exception of the spores, which re- tain their stain. It is here, also, useful to make a contrast- stain, by exposing the specimen briefly to the action of a watery solution of methylene-blue or of malachite -green. The bacilli then appear blue or green respectively, and the spores, on the contrary, a bright red. If it be desired to stain the spores without reference to the bodies of the bacteria, the specimens may, according to a suggestion of Buchner, be placed for half a minute in concentrated sulphuric acid. By this means the vegetative forms lose their power of staining, whereas the spores, after thorough rinsing in water, are readily susceptible to the action of the carbolfuchsin„ Good results are obtained with the aid of the apparently complicated, but quite reliable, method of staining spores proposed by Moller. The cover-glass, dried in air, is placed for two or three minutes in absolute alcohol, is then rinsed in water, and kept for two minutes in chloroform. After again rinsing in water it is exposed for one or two minutes METHODS OF CULTURE AND OF EXAMINATION. 109 to the action of five per cent, chromic acid, is again rinsed in water, and is stained for two minutes in steaming con- centrated carbol-fuchsin solution. The preparation is then treated for a short time with five per cent, sulphuric acid (being passed through once or at most twice), is thoroughly rinsed in water, is counterstained quite deeply with methylene-blue or malachite-green, is again rinsed, and is dried and mounted in Canada balsam. The spores appear deep red, the bodies of the bacteria blue or green. Staining of Flagella (Loffler). — In staining flagella a mordant is first used : Twenty grams of tannic acid are dissolved in 80 cu. cm. of hot water, and 50 cu. cm. of a saturated watery solution of ferrous sulphate that has stood in the cold for twenty-four hours with an excess of ferric sulphate are added, and finally 10 cu. cm. of a concentrated alcoholic solution of fuchsin. This fuchsin-solution may be advantageously permitted to stand exposed to air for several weeks ; it stains the better the older it is. The specimens themselves must be prepared with cover- slips most carefully cleansed, and in such a way that an extremely thin layer is spread upon the cover-slip with little rubbing — so much of the material as will adhere to the tip of a platinum needle must yet be largely diluted. Quite young agar-cultures, from fourteen to eighteen hours old, and at the most twenty-four hours, are employed, as the flagella can not be stained in older cultures. After the preparations have been thoroughly dried in the air, the mor- dant is dropped upon them, and its action is permitted to continue for a minute. It is then entirely washed off, the cover-glass is dried, and upon it is filtered aniline-water gentian-violet or methyl-violet or fuchsin-solution. Heat is now applied carefully by means of a small flame, until vapors of steam arise. Then, after a minute, the prepara- tion is washed in water, is dried, and is mounted. This modification by Giinther of Lofifier's method of staining flagella yields quite satisfactory results. By another method the preparations, dried in air, are exposed for from six to twelve hours to the action of a two per cent, solution of tannic acid and one-half per cent, hydrochloric acid, are rinsed in water, immersed for an hour in a watery solution of iodin, again washed in water, and then stained for half an hour in aniline-water gentian-violet. The specimen should then be mounted, not in Canada balsam, but in solution of iodin. no CLINICAL BACTERIOLOGY. DETERMINATION OF THE PATHOGENICITY (OR THE SPE- CIFICITY) OF BACTERIA BY ANIMAL EXPERIMENTATION. The possession of pure cultures renders it possible to undertake experiments upon animals, and thus to deter- mine the changes that are induced in the animal organism by a given variety of bacteria. In order to consider a bacterium as the cause of an in- fective disease, in order to declare it specific for this disease, it must comply with three conditions formulated by Koch : In the first place, it must be present in all cases of the given disease ; in the second place, it must occur only in this dis- ease ; and, in the third place, it must be capable of induc- ing essentially the same disease in experiments on animals. Animal experimentation, therefore, plays a most important role in bacteriology. In order to mfect animals with bacteria various ways are open. The natural portals of infection may be em- ployed, and, besides, new ones may be artificially estab- lished through which the microbes are introduced into the organism. (a) Cutaneous Inoculation. — Quite superficial wounds of the skin are made in animals (as in vaccination), and these are smeared, by means of the platinum wire, with a small amount of pure culture. In guinea-pigs and in mice, in- stead of cutaneous inoculation, an incision is made with scissors through the margin of the ear, and the injured places are rubbed with the inoculating material. (6) Subcutaneous Inoculation. — By means of a scalpel or an inoculating needle, a pocket is formed in the subcuta- neous connective tissue, into which the bacterial material is introduced ; or the bacteria suspended in water or in bouillon are introduced beneath the skin by means of a hypodermic syringe. (c) Intravenous Injection. — By means of a hypodermic syringe the infecting fluid is injected directly into a vein that either lies quite superficially (as the marginal vein of the rabbit) or is exposed by dissection. Entrance of air into the vein may cause immediate death through air- embolism. It is therefore necessary to exclude carefully all air-bubbles from the system, and, further, to compress the vein with a small cotton pad, and close the wound immediately after removal of the needle. METHODS OF CULTURE AND OF EXAMINATION. Ill (d) Inoculation of the Anterior Chamber of the Eye. — With a cataract-needle a small incision is made at the junction of the cornea with the sclerotic, the aqueous humor is per- mitted to escape, and the infecting material is introduced. The wound closes and heals rapidly. {e) Inoculation of the Cavities of the Body. ^-The needle of the syringe is introduced into the selected cavity (pleural or peritoneal), and the suspension of bacteria is injected. In intraperitoneal inoculation the needle of the syringe, after thorough cleansing of the abdominal wall, is introduced subcutaneously in a horizontal direction ; then the syringe is elevated, and the needle pushed on until the disappearance .of resistance indicates that the extremity is free in the abdominal cavity. . (/") Subdural Inoculation. — A trephine-opening is made to one side of the sagittal suture in order to avoid injury to the Longitudinal sinus, and, with the aid of a curved needle, the fluid is introduced beneath the dura. (^g) Inocidation by Inhalation. — The bacterial mass is minutely subdivided by means of a spray, which is intro- duced through a tube into a closed space in which the experimental animals are placed, (Ji) Inoculation through tJie G astro-intestinal Tract. — The food of the animals is saturated with the bacterial fluid, or this is introduced into the stomach by means of a tube, the jaws of the animal being held apart with the aid of a hollow wooden gag, through which an elastic so-called Nelaton catheter is passed carefully into the stomach. For special purposes (introduction of bacteria into the liver or into the portal vein or into a loop of intestine) celi- otomy becomes necessary. After especially careful sterili- zation of instruments, hands, and field of operation, the cutaneous incision is made, the muscles are divided layer by layer, and, finally, the peritoneum is divided upon a grooved director. At the conclusion of the inoculation the wound is closed by interrupted peritoneal, muscular, and cutaneous sutures, and it is covered with iodoform-coUodion. All of these various inoculations must naturally be car- ried out with the most rigorous cleanliness. The skin at the point of inoculation must be shaved and washed with soap, solution of mercuric chlorid, alcohol, and ether. All instruments employed in the inoculation are sterilized by boiling in a one per cent, soda-solution. The disinfection 112 CLINICAL BACTERIOLOGY. of the ordinary hypodermic syringe is more difficult, and for this reason quite a number of steriHzable syringes (Fig. 36) have been devised (Roux, Koch, Lewin). The ordinary hypodermic syringe is conveniently and safely disinfected by filling it with, and permitting it to remain for from twelve to twenty-four hours in, a five per cent, solution of carbolic acid, and then removing the carbolic acid by repeated rinsing in sterilized water. In deal- ing with especially infectious and conspicuously resistant bacteria (anthrax and tetanus), however, the syringe of Roux is employed, and it is boiled for ten minutes in a one per cent, solution of soda. In inoculating the anterior chamber of the eye the greatest care must be taken to secure disinfection of the conjunctival sac. This is cleansed Fig. 36. — I, Roux's bacteriologic syringe ; 2, Koch's syringe ; 3, Meyer's bacteriologic syringe. with a I : 3000 solution of mercuric chlorid, which is removed by irrigation with sterilized water. Anesthesia is effected by means of boiled cocain-solution. The inoculated animals are carefully observed, their tem- perature is taken at regular intervals, the evacuations and the occurrence of convulsions, etc., are noted, and, in short, all manifestations of disease are looked for. Should an in- oculated animal die, the autopsy is to be conducted with every precaution. It is usually made as soon as possible after death, in order to avoid the occurrence of putrefaction. The animal, placed upon its back, is stretched upon a board, with somewhat raised borders. The whole surface of the animal is moistened with a solution of mercuric chlorid, in order to prevent the generation of dust. The METHODS OF CULTURE AND OF EXAMINATION. 113 abdominal wall is thoroughly cleansed with the same solu- tion, and then, with instruments sterilized in the flame, it is divided and dissected back on either side sufficiently for the flaps to be fastened to the board with small upholstery- tacks. After renewed irrigation a fold of peritoneum is raised, the abdominal cavity opened with a freshly sterilized knife, and the peritoneum thrown back on either side as far as possible. Pieces of all the organs (liver, spleen, kidney, possibly testicle) are removed, and placed in sterilized glass jars for further investigation. From the tissue-fluids of the organs and from the blood cover-slip preparations are made, and at the same time cultures are prepared and poured into plates. As a matter of course, the macroscopic appearance of the organ also is observed, and every patho- logic alteration is carefully noted. If necessary, portions of the various organs are hardened and cut with the micro- tome, in order that the distribution of the bacteria in the tissues can be studied subsequently in stained sections. In opening the thoracic cavity the xiphoid process is raised with sterilized forceps, and the ribs on the left side, and then those on the right, are divided with scissors, and, finally, the manubrium sterni above is divided. The heart is now exposed, and is opened with a sterilized and cooled knife ; and cultures, plates, and streak-preparations are made from the heart's blood. At the conclusion .of the autopsy the instruments and the dissecting table are thoroughly disinfected, and the animal cadaver is incin- erated. Special circumstances justify at times the holding of the autopsy at a later period, or subsequent investigation of individual organs. Thus, typhoid-bacilli are more easily cultivated from the spleen of a patient dead of typhoid fever, if that organ has been kept for some time, than if it is examined fresh. Also, in the blood of rabbits destroyed by pneumonia the bacteria can be more readily demon- strated about twelve hours after death than immediately after. Under these circumstances an increase of the bac- teria in the cadaver must obviously take place. However valuable animal experimentation often proves, it is, nevertheless, not capable, in every instance, of securing the desired information. It fails frequently, and especially in connection with those diseases that occur as infections exclusively in man — as for instance, cholera, typhoid fever, 8 114 CLINICAL BACTERIOLOGY. etc. ; but even in these cases experiments on animals are not useless, for they demonstrate the toxic activity of the bacteria in question, and they lead, above all, to a knowledge of the anti-bodies formed in the blood-serum. Also, the other of Koch's postulates — that a microbe, to be considered as the cause of a disease, must be present only in association therewith — is not fulfilled in certain diseases. Those infections that are due to the bacteria giving rise to inflammation and suppuration are sometimes caused by the one and sometimes by the other of these germs. The clinical picture of these diseases depends less upon the species of infecting bacteria than upon the site at which the infection is localized. This is true of otitis media, of men- ingitis, of empyema, etc. ; at least, we are as yet not in a position with regard to these diseases to set up different clinical pictures in accordance with the various bacterial findings. A streptococcus-meningitis is clinically not sharply differentiated from a pneumococcus-meningitis or from a meningitis caused by staphylococci, etc. The con- ception of the specificity of bacteria, which is . otherwise fully applicable to the etiology of infectious diseases, must be abandoned with relation to these common excitants of inflammation. PART II INFLAMMATION AND SUPPURATION* Almost all bacteria exhibit under some circumstances in- flammatory and suppurative properties (phlogogenic or pyo- genic activity). Inflammation and suppuration may also be induced by chemic substances — as, for instance, ammonia, oil of turpentine, etc.; but, above all, by bacterial metabolic products (ptomains, proteids, etc.) when these are cm- ployed apart from the bacteria. For practical purposes, however, this purely chemic mode of origin is of no note- worthy importance, and in almost every suppurative and inflammatory process microorganisms are the responsible agents. The bacteria that are found in the large majority of cases in inflammatory and suppurative areas, and that are known as the common (not specific) causes of inflam- mation and suppuration, are as follows : 1. The so-called pyogenic cocci (staphylococci, strep- tococci, pneumococci, etc.). 2. The bacterium coli commune, and the entire group of related bacteria. 3. The far less common pneumonia-bacillus of Fried- lander. 4. The bacillus pyocyaneus. MORPHOLCXJY OF THE CAUSATIVE AGENTS OF INFLAMMATION. Staphylococcus Pyogenes Aureus (Fig. 37). — This ap- pears in the form of spherical nonmotile cells (micrococci), from 0.7 to 1.2 fi in diameter, generally arranged like bunches of grapes ; hence the designation staphylococci. They stain readily with all basic aniline dyes, and also according to Gram's method. The temperature-minimum 115 116 CLINICAL BACTERIOLOGY. is +6° C. (42.8° F.), the temperature -maximum +44° C. (111.2° F.), the temperature-optimum from +34° 0.(93.2° F.) to -{-38° C. (100.4° F-) • thus, the temperature of the body. On gelatin-plates, with low powers of the micro- scope, they form at first round, coarsely granular colonies, with sharply limited borders, and of whitish-gray color ; later, they become orange -yellow, and liquefy the gelatin with moderate rapidity. In gelatin stab-cultures develop- ment takes place along the line of inoculation, with liquefac- tion. On agar streak-cultures there forms a moist, shining, golden-yellow raised column, and also upon potatoes. Bouillon is rendered densely turbid, and presents a yellow sediment. Milk is coagulated. In milk and bouillon, prin- cipally lactic acid, also propionic acid, valerianic acid, and ^ ^'i ^tt^ Fig, 37.— Staphylococcus pyogenes aureus, from an agar-agar culture (Giinther). isobutyric acid, are formed. Facultative anaerobic, the staphylococcus aureus generates its yellow pigment only in the presence of oxygen. Cultures retain their vitality for more than a year. They are destroyed by brief exposure to the action of live steam, and in pus dried on silk threads by exposure to the action of two or three per cent, carbolic acid for five minutes. Staphylococcus pyogenes albus (Fig. 38) is absolutely identical with the foregoing except that it does not give rise to pigment-formation. Staphylococcus pyogenes citreus generates a citron - yellow ferment, but in its other properties it is identical with the staphylococcus aureus. MORPHOLOGY OF CAUSATIVE AGENTS. 117 Both staphylococcus cere us albus and staphylococcus cereus flavus, which are uncommon, are characterized by not liquefying gelatin. The one possesses a wax-like white color, while the other generates a wax-like yellow pigment. Fig. 38. — Staphylococcus pyogenes albus (Jakob). Streptococcus Pyogenes (Erysipelatis). — This appears in the form of nonmotile micrococci arranged in chains of greater or lesser length. (Fig. 39.) They vary in size Fig- 39- — Streptococcus pyogenes (Jakob). between 0.3 At and i ai. They stain readily, and by Gram's method. The temperature-optimum is between 30° C. (86° F.) and 37° C. (98.6° F.) ; they develop also at 118 CLINICAL BACTERIOLOGY. room-temperature, which, however, must not be too low. Upon gelatin-plates the streptococcus grows in the form of white, small, granular colonies that do not cause liquefac- tion. With high powers of the microscope the chains can distinctly be seen projecting beyond the margin of the plate. In gelatin stab-culture there is no confluent growth ; the line of inoculation can be seen distinctly to be made up of individual colonies separated from one another. The stab-culture on agar presents the same appearance. Bouillon, which constitutes an admirable culture-medium for streptococci, is usually not rendered turbid, but it presents a flocculent crumbling sediment. Upon potatoes only slight growth takes place. In milk the streptococcus induces coagulation. It exhibits facultative anaerobiosis. In' cultures the streptococcus dies much more quickly than the staphylococcus — within as short a time as four months. Accordingly as the streptococci develop in bouillon into long or short chains, a distinction has been made between strepto- coccus longiis and streptococcus brevis. Besides, a streptococcus conglomeratus has been distinguished that resembles super- ficially the staphylococcus in the interlacing and adhesion of the individual chains. This differentiation, however, has had to be abandoned. In order to maintain the vir- ulence of streptococci, Petruschky recommends their re- newal in gelatin stab-cultures every five days, and their preservation in the refrigerator. Diplococcus Pneumoniae Frankel {Streptococcus Lanc- eolatus Pasteur^. — This organism is, as a rule, a nonmotile coccus, arranged in pairs, with lancet-shaped extremities, and frequently joined together in small chains. (Figs. 40, 41.) They possess a capsule, which, however, is only clearly visible in the products of disease, but which is usually ab- sent in cultures. They stain readily, and also by Gram's method. The capsule can be demonstrated by placing the cover-glass preparation for a minute in i per cent, acetic acid, drying, and then staining in aniline-water gentian- violet. Johne's method of staining capsules may also be employed (p. 108). The temperature-minimum is 22° C. (71.6° F.) ; the temperature-maximum 39.5° C. (103.1° F.) for cultures upon solid media, 42.5° C. (108.5° F.) for cul- tures in fluid media ; the temperature-optimum from 35° C. (95° F.) to 37° C. (98.6° F.). Upon gelatin the pneumo- coccus appears at temperatures above 25° C. (yj^ F.) in MORPHOLOGY OF CAUSATIVE AGENTS. 119 the form of small, delicate colonies. Upon solidified agar in slants, when only feebly alkaline, upon agar-plates and upon blood-serum it develops in the form of small, finely # i Fig. 40. — Diplococcus pneumoniae in the blood (Frankel and PfeifFer). granular colonies, resembling dewdrops. Development in bouillon presents nothing characteristic. Some varieties induce coagulation in milk. The cultures die with extra- ordinary rapidity, usually in the course of a few days. The cause of death has been attributed to the formation of lac- tic acid and formic acid, which can always be demonstrated in cultures several days old. If the cultures are neutralized with calcium carbon- ate, they may retain their vitality for several months. The diplo- coccus of Frankel thrives best upon culture-media that contain considerable blood. This is added to solid media in a thick layer, or it is mixed in considerable amounts with fluid media. The diplococcus grows well in the absence of oxygen and thus retains longer its vitality and its virulence. Diplobacillus Pneumoniae Friedlander. — Much larger than the preceding (minimum, i //), the bacterium of Fried- f4« Fig:. 41- — Diplococcus pneu- moniae: a, Cocci without cap- sules ; b, single and paired cocci with capsules ; c, chain- form ; d, colony of cocci (Zieg- ler). ®®V III 120 CLINICAL BACTERIOLOGY. lander is rod-shaped. It is arranged in pairs and in chains. (Fig. 42.) It also possesses a capsule, which is distinct, except in cultures. It does not stain by Gram's method. The bacillus grows vigorously as well at room-tempera- ture as at the temperature of the body. Upon gelatin-plates it forms small, porcelain-like points. The gela- ® ^ ^ \\\/^^ I 'I ^^^ ^^ ^^^ liquefied, but in the course ^ x^ \»\ III of time is discolored brown. A stab - culture assumes a typical nail-shape ; an agar streak-culture, a thick succu- Fig. 42. — Bacillus pneu- 1 1 t ^ ^ moniae of Friediander. lent layer, lu an agar stab-culture gas is generated. Potato exhibits a yellowish coating and gas-formation. Grape-sugar nutrient solutions undergo fermentation, with the formation of car- bon dioxid, hydrogen, ethylic alcohol, and acetic acid. Milk is not coagulated. Friedlander's bacillus retains its power of development for a long time. Bacillus Pyocyaneus. — The bacilli of green or blue pus are small, slender, exceedingly active motile rods (with a single flagellum) that in culture sometimes arrange them- selves in small chains. They do not stain by Gram's method. They grow almost as well at room-temperature as in the thermostat. They exhibit facultative anaerobiosis, but no spore-formation. On gelatin-plates flat, irregularly circumscribed colonies form, with a radiate arrangement, about which a zone of liquefaction soon forms. In gelatin stab-culture liquefaction takes place rapidly. Upon agar and potatoes there is vigorous development. Bouillon is rendered densely turbid. Milk is coagulated and pepton- ized. All cultures, especially, however, those containing grape-sugar, soon assume a green or a greenish-blue color that is imparted to the entire nutrient medium. The bacil- lus pyocyaneus generates various pigments, according to the constitution of the culture-medium, but only with free access of oxygen. The best known of these is pyocyanin, a crystallizable aromatic combination, related to anthracene (Ledderhose), and a fluorescent green pigment. Bacterium Coli Commune. — This appears as short, narrow rods, frequently with vacuoles, twice as long as wide, arranged in pairs. (Fig. 43.) Besides this simple form, there is, however, marked pleomorphism, with long rods, coccus-like bodies, and filaments. Motility is generally quite MORPHOLOGY OF CAUSATIVE AGENTS. 121 active, although it may at times be wanting completely. The motile varieties possess flagella. For staining, Loffler's methylene-blue solution or carbolfuchsin is most available. The bacteria do not stain by Gram's method. They thrive Fig- 43- — Bacillus coli communis, from an agar-agar culture ; X looo (Itzerott and Niemann). Fig. 44. — Bacillus coli communis : superficial colony two days old upon a gelatin-plate ; X 21 (Heim). best at body-temperature, although they grow well, also, at room-temperature. Upon gelatin-plates (Fig. 44) there form deeply situated, yellow, punctate colonies. The superficial colonies either spread upon the surface, possess 122 CLINICAL BACTERIOLOGY. a dense center, and are surrounded by a thin, serrated, leaf- like, bluish, iridescent coating, which, with high powers of the microscope, exhibits a delicate linear network-arrange- ment ; or they present a sharp boundary, and appear as porcelain-white points half the size of a pinhead. In gelatin stab-cultures a chain of small, white, spherical col- onies forms along the line of inoculation, while upon the surface in individual cases a roset appears, and in other in- stances a hemisphere. The gelatin is never liquefied, but soon exhibits a dense turbidity and distinct iridescence. In all gelatin-cultures ammonia and crystals of ammonio- magnesium phosphate (coffin-lid shape) are formed. In agar streak-cultures a dense yellowish deposit forms, and upon potatoes a brownish membrane. Bouillon is ren- dered turbid, and in the case of the actively motile varieties a coating is often formed. Distinct gas-formation takes place in growths upon all of the nutrient media mentioned. It is favored by the presence of reducing substances, and by anaerobic growth. Addition of potassium nitrite and sulphuric acid to peptone -bouillon cultures (i cu. cm. of a 0.02 per cent, potassium-nitrite solution and several drops of sulphuric acid to lo cu. cm. of bouillon) causes the de- velopment of a red tint (nitroso-indol reaction). Coagula- tion takes place in milk. Grape-sugar, cane-sugar, and glycerin undergo fermentation. Acid urine is not altered by the growth of the bacterium coli, while alkaline urine at times undergoes ammoniacal decomposition. The bacterium coli commune retains its vitality for a long time. Bacillus Aerogenes. — This organism is closely related to the preceding. It appears as short, nonmotile rods, forming filaments, sometimes with capsules, and with- out spores. They do not stain by Gram's method. Upon gelatin-plates they form round, porcelain-white, prom- inent colonies. In gelatin stab-cultures they form a nail- like growth, and gas-formation takes place. In agar streak-cultures a white, slimy deposit forms. Upon potato a yellowish deposit takes place. Bouillon is rendered turbid and a coating forms. Milk is coagulated. Growth takes place in urine without decomposition of urea. In all nutrient media energetic gas-formation takes place, and in those that contain sugar acetic and formic acids are gen- erated. The bacillus aerogenes is probably identical with the organism described by the Guyon school as bacillus PATHOGENIC PROPERTIES OF CAUSATIVE AGENTS. 123 pyogenes, which plays so important a role in the pathology of the genito-urinary tract. THE PATHOGENIC PROPERTIES OF THE CAUSATIVE AGENTS OF INFLAMMATION WITH RELATION TO ANIMALS. The pathogenic activity of the exciting agents of inflam- mation and suppuration with regard to animals is, on the whole, much the same for all members of the group. In- troduced subcutaneously, they cause local itijlammation, which manifests itself in simple swelling and slight febrile movement. If the virulence of the bacteria be somewhat greater, this inflammation leads to suppuration, and there results a local abscess, which may rupture outward, and undergo cure. Still greater virulence of the microor- ganisms may cause the local imflammation and suppuration to be complicated by sepsis — that is, by intoxication with bacterial poisons generated at the site of inflammation and thence absorbed ; or it may lead to pyemia — that is, to dissemination of the bacteria through the lymph-channels and the blood-vessels ; in the latter instance probably through emboli containing bacteria — and which give rise to the formation of multiple purulent foci. The introduction of the causative agents of inflammation into the serous cavities leads to the development of serous or purulent pleuritis or peritonitis. Under all of these conditions both the clinical course of the disease and the postmortem findings exhibit completely the conditions sufficiently well known from human pathology. Septicemia is exceedingly common in animal pathology, while it occurs but seldom in human beings. This may be considered as the expression of especially virulent activity on the part of the causative factors of inflammation. It occurs after intravenous introduction of the bacteria ; in some cases, however, also in connection with every other variety of inoculation — and especially the infection of rabbits with pneumococci. Characteristic of septicemia is the absence of any localization of the disease. The clinical picture has, therefore, no distinctive features. The animal is severely ill, as may be recognized from the appearance of the skin, of the eyes, and the general attitude. It does not eat, and it often suffers from diarrhea, especially after 124 CLINICAL BACTERIOLOGY. infection with bacterium coli or with another member of this group. At the same time there is always fever, which gradually increases ; and usually also dyspnea. After the disease has existed for a shorter or a longer period of time, death results, with decline of temperature, and not rarely with convulsions. Upon postmortem examination the individual organs exhibit parenchymatous cloudiness, the spleen is enlarged, there is often nephritis, but there are otherwise no special alterations. On microscopic exam- ination and cultural investigation, however, the bacteria introduced can be seen in large numbers everywhere : in eVery tissue, in the secretions, in the blood. They have multiplied in the blood, and fill all of the vessels down to the smallest capillaries, which, by reason of their numbers, they often actually plug. The bacteria are demonstrable in the blood already during life, after the disease has existed for some time, but generally not in the numbers in which they are found after death. Their greatest multi- plication appears to take place just before death, and in the cadaver. The pathogenic activity of the individual causative agents of inflammation may be stated as follows : Staphylococci : Cutaneous inoculation is unattended with ill results ; subcutaneous injection induces a local abscess in mice, guinea-pigs, rabbits ; and intravenous injec- tion, at times, pyemia in rabbits. Streptococci : If the material is rubbed upon small cutaneous wounds of the rabbit's ear, erysipelatous inflam- mation follows. Subcutaneous injections in mice and guinea-pigs are followed by septicemia, with or without local abscess ; and intravenous injection induces septicemia. Diplococcus lanceolatus Frankel is pathogenic for rab- bits, mice, and guinea-pigs. Death from septicemia occurs with certainty in rabbits after subcutaneous injection of even small amounts. Mice are somewhat less susceptible, but they die mostly of pneumococcus-septicemia ; and guinea-pigs, only after the introduction of considerable amounts. After intraperitoneal injection guinea-pigs frequently die in con- sequence of fibrinous peritonitis. In dogs the inflammation caused by the diplococcus remains local, the bacteria not entering the blood ; the dogs, however, die as a result of the poisoning (sepsis), and most readily after subcutaneous inoculation. PATHOGENIC PROPERTIES OF CAUSATIVE AGENTS. 125 Diplobacillus pneumoniae Friedlander readily destroys mice and guinea-pigs on subcutaneous and intravenous in- jection, and dogs and rabbits less readily. Postmortem examination discloses here, also, septicemia. Bacillus pyocyaneus, when employed in considerable amounts, is pathogenic for guinea-pigs and rabbits, inducing local suppuration, diarrhea, and sepsis. Bacterium coli commune : Mice, guinea-pigs, rabbits, and dogs are susceptible to subcutaneous, intraperitoneal or intravenous injection. There result local abscesses, hemorrhagic diarrhea, and septic manifestations ; post- mortem examination disclosing marked hyperemia of all the abdominal organs, and often septicemia. In conclusion, it may be again especially emphasized that the results obtained in experiments on animals with all of these common causative agents of inflammation are by no means constant. They vary quite extraordinarily, in ac- cordance with the virulence of the material employed, and the inoculations frequently enough pursue a completely negative course. The virulence of the exciting agents of inflammation varies, however, according to their source. The bacteria, for instance, cultivated from a fatal pyemia, are much more virulent than the relatively harmless bacte- ria derived from a mild suppuration of the skin, etc. It is to be emphasized, further, that all of these exciting agents of suppuration and inflammation are not exclusively pyogenic, but they may also induce purely serous pro- cesses : as, in the main, the three principal varieties of in- flammation— the serous, the fibrinous, and the purulent — differ from one another only quantitatively. THE OCCURRENCE OF THE CAUSATIVE AGENTS OF INFLAMMATION AND SUPPURATION IN HEALTHY PERSONS AND OUTSIDE THE BODY. Staphylococci have been found in dust, upon the sur- face of the earth, in the air, in household wash-water ; further, almost constantly upon the surface of the skin, in the accumulations beneath the finger-nails, in the saliva, in the mucus of the pharynx, in the nasal secretion, in the intestinal contents, in the vagina, and in the urethra. Streptococci have been cultivated from the air of hospi- 126 CLINICAL BACTERIOLOGY. tal-wards and dissecting rooms, from the mouth, the nose, the pharyilx, and the skin ; further, from the intestine, and — though selfiom — from the vagina of healthy women. The diplococcus pneumoniae Frankel is an exceed- ingly frequent inhabitant of the buccal and nasal cavities, and especially of the entire upper portion of the respira- tory apparatus ; at times it is present in the intestine. The diplobacillus Friedlander is found normally in the same situations as the diplococcus Frankel,' although its occurrence is much more seldom. The bacillus pyocyaneus has been found upon the skin, especially in the axillary cavity ; further, in the external auditory canal and in intestinal mucus ; it is not rarely encountered also in the air and in water. The bacterium coli commune and bacterium lactis aerogenes are found throughout the entire digestive tract, especially in the intestine (the aerogenes more commonly in the feces of infants) ; further, in the vulva and the vagina and on the prepuce, upon the skin, in the air, in water, and in milk. The common causative agents of inflammation and suppuration are thus found in healthy persons in the main upon the skin and the so-called open cavities of the body, the pyogenic cocci more commonly upon the surface of the skin and in the upper portion of ^the digestive and respira- tory tracts, and the pyogenic bacterium coli more com- monly in the intestine. It is, therefore, to be anticipated that the inflammatory and suppurative processes that take place upon the skin and in the neighborhood of the buccal, nasal, and pharyngeal cavities are excited especially by cocci, while those of the intestinal tract and of the genito- urinary tract and their entire neighborhood are due more commonly to the bacterium coli or the bacterium lactis aerogenes. THE OCCURRENCE OF THE CAUSATIVE AGENTS OF INFLAMMATION IN DISEASE. CUTANEOUS SUPPURATION. Furuncle and carbuncle are almost always attended with the presence of staphylococci (staphylococcus aureus and albus). That staphylococci are, in fact, the cause of OCCURRENCE OF CAUSATIVE AGENTS. 127 carbuncles has been demonstrated by Garre, in an experi- ment upon himself. He rubbed a staphylococcus-culture into the intact skin of his left forearm. Four days later a characteristic carbuncle developed, and around it several isolated furuncles. The pus from all of the lesions con- tained the same staphylococcus that had been employed for the inoculation. This experiment renders it at the same time probable that furuncles and carbuncles are probably due to infection of the excretory ducts of the glands of the skin, into which the pyogenic material is in some way forced. Practically, furuncles are often seen to develop in places subjected to pressure or friction : as, for instance, by parts of the clothing. Panaris. — In the pus from panaris staphylococci, as well as streptococci, and in rare cases also the bacterium coli com- mune have been found. Abscess and Phlegmon. — These are due to staphylo- cocci and streptococci, and in some cases also to Frankel's pneumococci, especially in childhood and in the course of genuine croupous pneumonia. In abscesses in those sick with and convalescent from typhoid fever, staphylococci and streptococci are found frequently, and not at all seldom also typhoid-bacilli and bacterium coli. Urinary pJilegmons, so frequently observed in the sequence of urinary infiltra- tion, are usually due to the bacterium lactis aerogenes or the bacterium coli commune, which play the most important role in the suppurative processes of the genito-urinary apparatus. So-called cold abscesses are generally found to be sterile. They may be considered as the product of tubercle-bacilli, and actually it has frequently been possible in experiments on animals to induce tuberculosis with the pus from such abscesses. Microscopically, however, it has been possible, only in the rarest of instances, to demonstrate in the pus the tubercle-bacilli that are present in small number. Also, in the larger, nontuberculous abscesses bacteria are some- times not found at the center of the area of suppuration, and the pus, therefore, appears sterile. It is only necessary in such cases to examine material from the periphery, the so-called abscess-membrane, in order to find the pyogenic agents without difficulty. Gas-abscesses, like ordinary abscesses, do not possess a uniform etiology. From them there have thus far been cultivated : (i) The bacterium coli commune and the bac- 128 CLINICAL BACTERIOLOGY. terium lactis aerogenes, principally from the gas-phlegmons in the neighborhood of the intestinal canal ; (2) a special bacillus — the bacillus emphysematosus. (Fig. 45.) This appears in the form of nonmotile, plump rods, forming fila- ments. These are anaerobic, and they stain by Gram's method. They grow slightly in gelatin, without causing liquefaction. In bouillon, and also in agar, they give rise to the formation of fetid gas. This bacillus is but seldom found alone in gas-abscesses, but almost always in association with the ordinary pyogenic cocci. In experiments on animals a severe nonsuppurative inflammation, with gas-formation, and which at times causes death, is induced in guinea-pigs by sub- Fig. 45. — Bacillus aerogenes capsulatus (from photograph by Prof. Simon Flexner). cutaneous injection of this bacterium. That the bacterium coli and the bacterium lactis aerogenes may at times cause gas-phlegmons is not surprising, as it is known that both are active gas-producers, especially in the absence of oxygen. In the bacteriologic investigation of a gas -abscess it is always necessary to be prepared for the presence of anae- robic microorganisms, and the choice of a culture -method must be governed with reference thereto. Impetigo. — This is a peculiar anatomic form of suppura- tion within the layers of the epidermis that leads to the formation of pustules. From the contents of the pustules the staphylococcus pyogenes albus and aureus and the OCCURRENCE OF CAUSATIVE AGENTS. 129 cereus albus are obtained. These observations with ree^ard to the common exciting agents of inflammation explain the cHnical fact that the most variable suppurative processes — furunculosis, etc. — sometimes precede and sometimes follow the actual attack of impetigo. Ecthyma. — This also is a form of cutaneous suppura- tion in which the pyogenic cocci are found. From the pus staphylococci and streptococci have been cultivated. Herpes. — Herpes zoster has been designated by Pfeiffer (Weimar) an infectious disease whose causative agent belongs to the class of protozoa. The cells believed by Pfeiffer to be protozoa have, however, not received recog- nition, and, above all things, it has as yet been impossible to cultivate them. In the vesicles that become turbid staphylococci and streptococci are always found, while the contents of the clear vesicles are often sterile. In cases of herpes labialis the vesicles contain the excit- ing agents of inflammation from the beginning, and more frequently streptococci and Frankel's diplococci than staphylococci. According to Pfeiffer, protozoa are absent in this form of herpes. As soon as the contents of the vesicles are turbid, staphylococci will be found present therein, and partly in association with streptococci, partly alone. These observations suggest that herpes labialis is not a true zoster. In general it occurs only as a complica- tion of such infectious diseases as themselves stand in etio- logic relation with the common exciting agents of inflam- mation (pneumonia, meningitis, etc.) ; it may, perhaps, be viewed as a secondary localization of the causative agent of the primary process. In herpes of the pharynx (angina herpetica) and of the larynx the same conditions seem to prevail as in herpes labialis. ERYSIPELAS. Erysipelas is excited by the streptococcus. The old discussion whether or not the streptococcus erysipelatis (Fehleisen) is distinct from the streptococcus pyogenes, the exciting agent of suppuration, can now be considered as finally decided. The two microorganisms are without doubt identical. This is demonstrated as well by their complete agreement in morphologic and cultural peculiarities, as also by the results of experiments on animals and man. 9 130 CLINICAL BACTERIOLOGY. Microscopic Arrangement of the Streptococci in the Invaded Skin. — As suggested by Fehleisen, three zones may be distinguished : (i) A central, in which the process is in retrogression ; (2) the elevated erysipelatous margin ; and (3) a peripheral zone surrounding this deeply red ery- sipelatous zone, which, microscopically, is apparently still completely normal. Streptococci are found in each of these three zones, but in much the largest number in the erysipelatous margin and in smaller number in the central and in the peripheral zone. The organisms lie in the Fig. 46 — Streptococcus erysipelatis, seen in a section through human skin ; X 500 (Frankeland Pfeiffer). lymph-spaces and the lymph-vessels of the skin and the subcutaneous fatty tissue ; the lymph-spaces are almost completely choked up by them. Sometimes they collect also about the lymph-vessels and the blood-vessels. In the vesicles in cases of erysipelas bullosum streptococci are not constantly present. On the other hand, they are never absent from the purulent products oi phlegmonous ery- sipelas. As a rule, in cases of erysipelas pursuing a favor- able course the streptococci do not gain entrance into the blood. Metastases are, therefore, rare in the symptom- OCCURRENCE OF CAUSATIVE AGENTS. 131 complex of erysipelas. Cases are, however, on record in which it was possible to cultivate streptococci from the blood of patients ; general infection, therefore, having existed. Immunity. — Recovery from one attack of erysipelas confers no immunity to subsequent attack. Erysipelas is one of the diseases attended with numerous recurrences. The blood-serum of persons just recovered from an attack of erysipelas does, however, at times possess immunizing properties ; and large amounts thereof may, according to some reports, immunize animals (guinea-pigs and mice) to streptococci. Experimental Evidence of the Etiologic Significance of the Streptococcus for the Development of Erysipelas. — The introduction of streptococcus-material into shallow, superficial wounds of rabbits' ears is followed by the devel- opment of erysipelatous inflammation. Much more im- portant are the observations that have been made in human beings. Proceeding from the clinical experience that in individuals with advanced lupus or with inoperable tumors an attack of intercurrent erysipelas may lead to the cessa- tion, or even the cure, of the original disease-process, it was ventured in such cases to undertake cutaneous inocu- lations with streptococcus-cultures derived from cases of erysipelas. These experiments yielded positive results : typical erysipelas pursuing a characteristic course occurring almost constantly. A similar result was attained by Koch and Petruschky by means of a streptococcus derived from a purely suppurative process. There is no doubt that a therapeutic influence is exerted by several streptococcus- infections upon the course of carcinoma, but the strength of the patient suffers so much in consequence of the inocu- lations with erysipelas that a practical application of this method is entirely out of consideration. The Occurrence of Other Microorganisms in Erysipe- las.— In two cases of true erysipelas (the second infected by the first) Jordan cultivated. the staphylococcus pyogenes aureus. From this it may be concluded that the other causative agents of inflammation are capable, under certain circumstances, of inducing erysipelas. The usual cause, however, is without question the streptococcus. The hacteriologic diagnosis of erysipelas is only most rarely necessary. In such an event a small scarification is made with a sterilized lancet at the border of the inflamma- 132 CLINICAL BACTERIOLOGY. tory area, and plates are prepared from the fluid that escapes ; or four or five obliquely solidified gelatin-tubes or agar-tubes are smeared therewith. In this way, in most cases, individual colonies of streptococci will be obtained probably in the first tube, and with certainty in the remaining tubes. Specific Treatment of Erysipelas and of Streptococ- cus-diseases Generally. — Marmorek increased the viru- lence of streptococci, which are readily attenuated in artificial culture, by cultivating them in bouillon to which sterile horse -serum had been added. With progressively increas- ing doses of the highly virulent cultures thus obtained he immunized horses, whose serum was then reported to exhibit immunizing and curative properties with relation to all strep- tococcus-infections also in human beings. Petruschky, who repeated the experiments of Marmorek, was, however, unable to confirm his conclusions. The serum of Marmorek obtained from the Pasteur Institute possessed neither im- munizing nor therapeutic activity, also in observations made upon human beings. The streptococcus of Marmorek was most highly virulent for rabbits, but not for human beings. p:hlebitis and lymphangitis. Lymphangitis, which arises from some peripheral in- flammation or suppuration — in some cases not demonstra- ble— is attended with the presence of the exciting agents just described, the pyogenic cocci, and at times with that of the bacterium coli. Also, the infectious form of phlebitis, which occurs as an associated manifestation of numerous in- fectious diseases, may be caused by the same microorganisms as give rise to the original disease. Thus, for instance, tuber- cle-bacilli have been found in the phlebitides of tuberculous individuals, and the streptococcus pyogenes in the phleg- masia alba dolens of puerperal women. More frequently, however, the inflammation of the vein is the expression of a secondary infection, and, therefore, its causes are, as a rule, found to be the common pyogenic cocci. The bac- terial inflammation of the vein or the lymphatics leads to the formation of a bacteria-containing thrombus. This may be conceived as originating from adhesion of the micro- organisms circulating in the vessel to a projecting point of its wall, or of a valve, or from the penetration by the bacteria of the wall of the vessel from without through the interme- OCCURRENCE OF CAUSATIVE AGENTS. 133 diation of the vasa vasorum. In the bacteriologic examina- tion of the thrombus it is important to know that the bac- teria are to be found only in the oldest, first-formed portion thereof, and on the corresponding portion of the wall of the vessel. The central and peripheral portions of the thrombus that subsequently form mechanically are often sterile. INFLAMMATIONS OF THE NOSE AND THROAT. In the secretions in cases of acute rhinitis, pharyn- gitis, and laryngitis, staphylococci and streptococci have been found, and also pneumococci and pneumobacilli, the last most frequently in the nose. In cases of fibrinous rhinitis diphtheria-bacilli have been found repeatedly. In other cases their demonstration was not successful, so that it is not certain whether all cases of croupous rhinitis are to be considered as instances of diph- theria. Ozena. — From the nasal crusts in cases of ozena a micro- organism has been cultivated by various observers (Lowen- berg, Abel, Paulsen), which presents so close a resem- blance to the bacillus pneumoniae Friedlander that a detailed description will not be necessary. The cultures are slimy and viscid, form comparatively little gas, and do not cause coagulation in milk. That this so-called ozena-bacillus is the cause of the disease is scarcely probable. The char- acteristic odor of ozena is wanting in all of the cultures. Further, the bacillus is never found in the diseased struc- tures of the nose, but only in the secretions whose peculiar putrid decomposition it may possibly cause. For the present, the ozena-bacillus may be considered a variety of Friedlander's bacillus, slightly modified in consequence of the peculiar conditions related to the ozenous nose. Rhinoscleroma. — Also the bacilli found in rhinosclero- matous tumors resemble Friedlander's bacilli. They in- duce fermentation of sugar and of milk, though in slighter degree than the pneumobacillus. On potatoes an often invisible, at times brown, gas-forming coating occurs. In the tissues the bacilli are found almost exclusively in the cells, in which they displace the nucleus and the proto- plasm to one side, themselves almost entirely filling the whole cell (Mikulicz). Noma. — Schimmelbusch described special noma-bacilli. 134 CLINICAL BACTERIOLOGY. In a case of noma studied personally the bacterium coli commune was found. Concerning these observations the same criticism may be made that has been suggested with regard to the presence of Friedlander's bacillus in cases of ozena and rhinoscleroma. ANGINA. In the inflamed mucous membrane of anginas pursuing their course without the development of membrane staphy- lococci have, as a rule, been found, as well as in the purulent plugs of follicular (or lacunar) angina. The anginas char- acterized by the presence of a membranous deposit are frequently associated with streptococci, and often besides also with staphylococci, but at times with the latter alone. Finally, a considerable number of cases have been reported in which only pneumococci were present. An attempt has been made on the basis of these bacterio- logic observations to distinguish a staphylococcus-angina, a streptococcus-angina^ and a pneumococcus-angina, each of which is held to be etiologically a distinct variety and to possess its special characteristics both clinically and from the diagnostic and prognostic point of view. Accord- ingly, staphylococcus-angina would be a relatively harmless affection, scarcely ever leading to complicating disorders. Streptococcus-angina would be severer, exhibiting more pronounced manifestations of intoxication (fever, glandular enlargement), and possibly being followed by nephritis and even general sepsis. Pneumococcus-angina, finally, would be characterized by the resemblance of its clinical course to that of pneumonia, by its stormy onset, possibly with a chill, and by high fever, with critical defervescence. It must, however, be emphasized that the clinical picture of these anginas is by no means a constant one, and that the severity of the disease depends more upon the virulence than upon the species of the bacteria present. There occur staphylococcus-anginas i^phlegmonoiis anginas), as well as anginas associated with the presence of streptococci alone, that pursue as mild a course as those with which the staphy- lococcus alone is associated. On the other hand, severe pseudo-membranous anginas, which clinically are indistin- guishable from true diphtheria, and in association with which only streptococci are demonstrable, are not rare. OCCURRENCE OF CAUSATIVE AGENTS. 135 Pneumococcus-angina, further, pursues frequently the char- acteristic course outlined, though by no means always. It is to be added that isolated cases of tonsillitis have been reported in which the bacterium coli commune exclusively has been found. Above all, however, in a not inconsiderable number of cases of angina, there is mixed infection with several of the micrococci named. Then, most commonly the two varieties of bacteria are found together from the beginning. At times, however, only streptococci can be demonstrated at first, and after some days also staphylococci, or even the latter alone. Under these conditions it must be concluded that the one coccus has associated itself with or overgrown the other. The frequency of these mixed infections, which clinically are indistinguishable from the pure infection, alone renders impossible a rigid separation of the anginas accord- ing to their causative agents. Scarlatinal angina is usually associated with the presence of streptococci. The bacteriologic diagnosis can, according to what has been said, be made only with a certain degree of probability from the clinical picture and the course of the disease. A positive diagnosis is possible only on microscopic and cul- tural investigation. The latter is made simply by rubbing a sterile swab of cotton that has been applied to the tonsil, or a bit of secretion or membrane obtained by means of a sterilized platinum loop, upon from three to five glycerin- agar tubes, or, better — in the differential diagnosis of diph- theria— upon from three to five tubes of Lofifler's serum, or upon a serum-plate. The bacterial decision will frequently be questionable on account of the impossibility of exclud- ing diphtheria with certainty by other means. Detailed reference will be made to this point in the section on Diphtheria. From what has been said, it will be clear that the dem- onstration of staphylococci, streptococci, or pneumococci, is available for purposes of prognosis only with great caution. OTITIS MEDIA. In cases of serous inflammation of the middle ear, as well as in cases of suppurative or hemorrhagic type, there have been found the diplococcus pneumoniae Frankel, the 136 CLINICAL BACTERIOLOGY. streptococcus pyogenes, the staphylococcus pyogenes, the diplobacillus pneumoniae Friedlander, the bacillus pyocya- neus, alone or in mixed infection. All of these organ- isms are more or less common inhabitants of the buccal cavity, and migrate through the Eustachian tube into the middle ear under special circumstances. Influenza-otitis is associated with the presence of influenza-bacilli ; tubercu- lous otitis, constantly with that of the tubercle-bacillus. Bacteriologic Diagnosis. — The auditory canal is cleansed carefully with mercuric-chlorid solution, paracentesis is practised with a suitable needle or knife sterilized in the flame, the escaping pus is taken up with a platinum wire bent at an angle, and from it plates are cast (agar, on account of the presence of pneumococci). After the occurrence spontaneously of perforation, the examination is of little value, as the secretion will have become contaminated by the numerous microorganisms of the external auditory canal. If tuberculosis is suspected in a case of chronic otitis media, the pus should be examined microscopically for tubercle-bacilli, and possibly animals should be inoculated with it. MENINGITIS. Inflammation of the cerebral meninges occurs, as a primary disorder in the form of epidemic cerebrospinal meningitis, and as a secondary (metastatic) condition in con- nection with pyemia and numerous infectious diseases, espe- cially in the sequence of otitis media, inflammation of the accessory cavities of the nose, and croupous pneumonia. A special position is occupied by tuberculous meningitis, which preferably involves the base of the brain. From the exudate in cases of secondary meningitis there have been cultivated the diplococcus lanceolatus Frankel, the strep- tococcus pyogenes, the staphylococcus pyogenes, the bac- terium coli, and the diplobacillus pneumoniae Friedlander. In cases of tuberculous meningitis the pus and sections of tissue contain tubercle-bacilli, partly alone and partly in association with phlogogenic cocci. These bacteria gain access to the cerebral or spinal membranes from the naso- pharyngeal space (lamina cribrosa), from the middle ear, or from the original focus of suppuration : in the last in- stance through the intermediation of the blood-stream. OCCURRENCE OF CAUSATIVE AGENTS. 137 The portal of entry for the bacteria in cases of epidemic meningitis has not yet been determined with certainty. The principal causative agent of this disease was, until recently, believed to be the diplococcus lanceolatus Fran- kel, which was found in numerous cases in pure culture. A special organism is, however, to be taken into considera- tion in connection with the etiology of epidemic cerebro- spinal meningitis — namely, the diplococcus intracellularis meningitidis (Weichselbaum, Jager). This is a biscuit- shaped organism, arranged in pairs, which is almost always contained within the cells, and which is strongly suggestive of the gonococcus. It is readily stained in the exudate, but with more difficulty in sections. The most suitable stain is Lofifler's methylene-blue. The organism does not stain by Gram's method, although Jager maintains that it does. It grows best at 37° C. (98.6° F.). On agar- plates there develop superficial gray colonies that with low powers of the microscope appear dirty-yellow at the center and become lighter toward the periphery. The deep colonies are quite small, and marked by fine granula- tion and a slightly indented border; Upon glycerin-agar there develop small gray colonies that at times coalesce to form a thin coating. Upon blood -serum a slight deposit forms. In order to continue the culture of meningococci for a protracted period they must be reinoculated from every four to six days. They are feebly pathogenic for mice and guinea-pigs on introduction into the thoracic or abdominal cavity, and for rabbits on introduction into the blood-stream. The clinical diagnosis is based upon the principle that with the simultaneous existence of some other infectious disease — as, for instance, pneumonia, otitis, tuberculosis — the causative agents of these conditions may be considered the cause of the meningitis. The absence of any such simultaneous organic disease justifies a diagnosis of epidemic cerebrospinal meningitis. A direct bacteriologic diagnosis is possible during life by means of puncture of the spinal canal, which has now been quite extensively practised. Preparations are made from the exudate, and agar-tubes and serum-tubes, or agar-plates, are inoculated, and animals are infected. If tuberculosis is suspected, the specific stain for tubercle -bacilli must be em- ployed. To obtain the causative agents from the meningeal pus after death, the same plan is pursued. As the diplo- 138 CLINICAL BACTERIOLOGY. COCCUS is frequently encountered, it is advisable to inoculate a white mouse or a rabbit immediately with the pus. BRONCHITIS. Bronchitis, whatever its nature may be, is likewise de- pendent upon the activity of the common exciting agents of inflammation : pneumococci, streptococci, staphylococci, pneumobacilli, bacterium coli. Through the action of cold or of some other injurious agency that generally leads to bronchitis, these normal inhabitants of the commence- ment of the respiratory tract become lodged in the bronchi, and there excite inflammation. Their demonstration in sputum is easy. The patient is instructed to cleanse his mouth thoroughly with a solution of boric acid or of potas- sium chlorate, and then to expectorate in a sterilized glass dish. The sputum thus obtained is rinsed carefully in sev- eral vessels of sterile water, and then a flake from the center of the mass is smeared successively upon each of several agar-tubes or upon an agar-plate (Koch). In this way the usual plate -procedure can be avoided, as only one of the species of bacteria named is, as a rule, found in the bron- chitic sputum in each case. The large number of bacteria that are found on microscopic examination of the expectora- tion are mostly derived from the mouth and the pharynx, and are adherent, therefore, to the outer layers of the sputum. In the majority of cases there develop from the expectora- tion thus treated pure cultures of staphylococci, Frankel's diplococci, or streptococci. If mixed infection is present, the colonies develop separately in the tubes last inoculated, and from these they can be readily isolated. Fetid bronchitis is associated with the presence of the same bacteria, but, in addition, also with putrefactive bacteria (proteus and others). The green color that is sometimes observed in bron- chitic sputum is in some cases due to the bacillus pyocya- neus, in others to the bacillus fluorescens, and to varieties of sarcinae. PLEURITIS. Pleuritis has no uniform bacteriology. It may be pri- mary or secondary ; in the latter event in association with diseases of the lungs, diseases of adjacent organs, trauma- OCCURRENCE OF CAUSATIVE AGENTS. 139 tism, or general infection. The pleural effusions due to general stasis (transudates attending heart-lesions, nephritis, etc.) are, naturally, sterile, providing secondary infection has not taken place, and for which the serous infiltration of the tissues offers a favorable soil. The tubercle-bacillus and all of the pyogenic micro- organisms are capable of inducing serous as well as puru- lent pleurisy. The primary pleurisies (so-called pleurisies due to cold) are most frequently dependent upon the tubercle-bacillus. Next in frequency follows the diplococcus pneumoniae Frankel, which plays a most important role, especially in the pleural inflammations of childhood ; and, further, all of the other pyogenic and phlogogenic microorganisms. Whether in all of these cases the pleuritic effusion is really primary can not always be determined with certainty, as the smallest pulmonary lesions — for instance, slight disease of the apex or a bronchopneumonia — that are scarcely sus- ceptible of diagnosis clinically may induce pleuritis. The secondary pleurisies are associated in a portion of the cases with the presence of the same bacteria that are responsible for the primary disease. In the effusions that so frequently occur in the sequence of pneumonia, in the so-called metapneumonic exudates, there is encountered the diplococcus ; in the effusions attending pulmonary tuber- culosis, the tubercle-bacillus ; in the uncommon effusions complicating typhoid fever, the bacillus of Gaff ky-Eberth ; in the pleurisies that originate in purulent processes within the abdominal cavity, the bacterium coli ; and so on. In those diseases whose causative agents are as yet unknown — as, for instance, articular rheumatism and carcinoma — the accompanying pleural effusion has been examined for bacteria, but as yet mostly with negative results. In an- other portion of the cases these concomitant pleurisies are dependent upon secondary or upon mixed infection. The pleural effusion then contains the common exciting agents of inflammation. Thus, in the empyema following scarlet fever the streptococcus pyogenes is often found ; in that following smallpox, staphylococci ; in that following influ- enza, the diplococcus lanceolatus Frankel ; and so on. Metastatic pleurisy, as part manifestation of a pyemic general infection, is caused by the staphylococcus or the streptococcus pyogenes. The same statement applies also 140 CLINICAL BACTERIOLOGY. to pleurisies that arise in consequence of wounds of the chest-wall. The putrid effusions contain, in addition to the causative agents of suppuration, also putrefactive bacteria, and generally the proteus. Method of Bacteriologic Investigation. — A hypoder- mic syringe with a capacity of from one to six cubic centi- meters is kept filled for from six to twelve hours in five per cent, carbolic acid, and is then carefully cleansed with sterilized water in order to remove all of the disinfectant ; or a Roux's syringe is sterilized by thorough boiling. The point on the chest-wall where the exploratory puncture is to be made is washed with soap, alcohol, mercuric-chlorid solution (i : looo), and ether, and the puncture, after thorough disinfection of the hands, is made in the usual way. The fluid obtained is received into a sterilized dish and each of four or five agar-tubes successively, or an agar-plate, is inoculated with a drop thereof, or a drop of the exudate is permitted to flow directly from the syringe upon the sur- face to be inoculated. The tubes are introduced into the thermostat at a temperature of 37° C. (98.6° F.). At the same time cover-slip preparations are made in the usual manner, and examined for tubercle-bacilli and other bac- teria. With the remainder of the fluid, if tuberculosis be suspected, two or three guinea-pigs may be inoculated through the peritoneum (see later). Serous effusions con- tain kw, if any, microorganisms. It is, therefore, a more reliable procedure to remove considerable amounts of the fluid, to centrifugate or sediment it, and to study the pre- cipitate only. Diagnostic and Prognostic Significance of the Bac- teriologic Findings. — Bacteriologic investigation is not of great importance in the diagnosis of serous effusions. The large majority of serofibrinous pleurisies prove to be sterile. The metapneumonic serous effusions contain at times the diplococcus lanceolatus Frankel, and before, as well as after, the crisis. The presence of pyogenic microbes in serous pleural effusions, as has been repeatedly observed, does not, in all instances, justify the conclusion that puru- lent metamorphosis into an empyema will take place. Such effusions may, under circumstances, recede completely. If a decision is to be reached in doubtful cases of serofibrinous pleurisy whether tuberculosis exists or not, it is advisable to inject into the peritoneum of guinea-pigs some of the OCCURRENCE OF CAUSATIVE AGENTS. 141 pleural fluid obtained under sterile conditions, and to wait and see whether the animals die of tuberculosis or not. This procedure, however, always occupies several weeks, and even then it is not always entirely reliable, and it fre- quently fails to resolve the doubt, as in spite of an evident tuberculous origin of the pleuritic effusion the animals often remain well. The reason for this is to be found in the small number in which the tubercle-bacilli are usually present in the effusion. It is, therefore, advisable to centrif- ugate a considerable amount of the effusion, and to inocu- late guinea-pigs with the precipitate thus obtained. Bacteriologic examination is, however, much more im- portant in the diagnosis of empyema. If the agar-tubes inoculated with the pleural pus remain sterile, this indicates that the process is, in all probability, tuberculous. Direct microscopic demonstration of tubercle-bacilli in cover-glass preparations is, however, successful only in a few cases. Not rarely the empyema of tuberculous subjects is due to secondary infection. Various observers have reached conclusions with regard to both the prognosis and the treatment of empyema from the species and the virulence of the bacteria found in the pleural pus. The presence of the diplococci is believed to indicate a much more favorable prognosis than that of the other pyogenic organisms. In this so-called pneumococcus- empyema less radical treatment is therefore necessary. Thoracotomy would, under these circumstances, be super- fluous, simple evacuation by puncture being sufficient. It can actually be admitted that the prognosis of pneumo- coccus-empyema is usually good. Nevertheless, radical operation is indicated if the effusion is not quickly absorbed spontaneously, or at least after puncture. Too much reli- ance is not to be placed upon spontaneous attenuation of the bacteria in the pus, or upon their death, although the organisms in question live usually only for a short time in artificial culture. Bacteria with fully preserved virulence have been cultivated from pleural pus after the lapse of as long as three and one-half months. Further, recovery^ from em- pyema due to staphylococci, streptococci, and even typhoid bacilli, has been reported in isolated instances as occurring spontaneously or after simple puncture. The question is yet undecided whether operation should be undertaken for the relief of purulent effusions in tuberculous subjects when 142 CLINICAL BACTERIOLOGY. they contain tubercle-bacilli or are sterile. While operation for all other varieties of empyema, even in cases of tuber- culosis, is attended with a relatively favorable prognosis, this is unfavorable in cases of empyema associated with the presence of tubercle-bacilli. Generally, healing does not take place, a fistula remains, and the chronic suppuration leads to death. Nevertheless, some surgeons recommend opera- tion even in these cases, as, without doubt, recovery may take place also under such conditions. Pneumothorax. — Pneumothorax with perforation, which occurs so commonly in tuberculosis, is, as a rule, followed by purulent effusion. The exudate contains tubercle- bacilli, demonstrable microscopically or by experiment on animals, and, in consequence of mixed infection, one or another of the pyogenic cocci, and, besides, at times, putre- factive bacteria, especially the proteus. Only one case of pneumothorax without perforation has been studied bacter- iologically. In this the anaerobic, gas-forming bacillus emphysematosus (p. 128) was found to be the exciting agent. The method of examination is the same as in the case of pleurisy. PNEUMONIA. Frankel's pneumococcus maybe looked upon as the caus- ative agent of lobar croupous pneumonia, as this organism is demonstrable in more than three-fourths of all the cases in the pulmonary tissues, which, normally, are entirely free from bacteria. In the larger proportion of cases the pneu- mococcus alone is present in the diseased tissue ; in the smaller proportion, staphylococci and streptococci besides are present. Streptococci alone, or in association with staphylococci, rarely staphylococci alone, may, however, be found in pneumonic foci. In addition, the bacillus of Friedlander (Figs. 42, 47) is found in some cases. All of these bacteria, as has been pointed out, exist in the upper air- passages. They have, further, been demonstrated, though less commonly, in the healthy larynx, but bacteria do not occur in the bronchi and in the pulmonary tissues in healthy persons. For the development of pneumonia, it is, therefore, generally necessary that an accidental, indirect cause (cold, traumatism, etc.) shall cooperate that renders it possible for the bacterium to penetrate more deeply into the air-passages, and there to give rise to inflammation. .*^^ >^ OCCURRENCE OF CAUSATIV] IZtspartjrf^ It is possible, to a certain degree, to differentiate the pneumonias according to their causative agents : I. Pneuniococcus-pneumonia corresponds with the charac- teristic cHnical picture of true croupous pneumonia, with fibrinous exudation into the alveoli, and lobar distribution of the morbid process. Clinically, this variety is character- ized by blood-streaked sputum, and especially by the sudden onset of the disease with a chill, the high, stormy course, and the critical decline of the fever. That this special type of disease is related etiologically to the activity of pneumococci is proved almost with certainty by the Fig. 47. — Bacillus pneumoniae of Friedlander, from the expectoration in a case of pneumonia ; X 1000 (Frankel and Pfeiffer). following facts : In the first place, in typical cases the diplococcus alone is always found in the diseased area, in which it is demonstrable during life by puncture. In the next place, the general symptoms characteristic of pneu- monia are also often present in marked degree, in con- junction with other localizations of the diplococcus, espe- cially pneumococcus-angina. Finally, it may be pointed out in this place that the blood of human beings that have passed through an attack of typical pneumonia with critical defervescence is capable of immunizing animals to pneumococci. There is thus furnished evidence that con- valescents from pneumonia are often immune to pneumo- 144 CLINICAL BACTERIOLOGY. cocci, and it is thus comprehensible why the symptoms dis- appear abruptly with the occurrence of the crisis, while the pneumonic infiltration remains ' unchanged. From these facts it may be inferred that a toxic action on the part of the pneumococci constituted a decisive element in the previous clinical picture ; for, when the crisis at once obliterates the disease, no change has been effected in its anatomic basis, in the infiltrate itself; only the toxic activity of the pneu- mococci has been withdrawn, and the manifestations that have just disappeared may, therefore, reasonably be referred to that. Finally, it may be mentioned that true pneumonia has been induced experimentally in favorable cases by inhalation of pneumococci. Such experiments are usually attended with difficulty, because the animals commonly employed (rabbits, mice), which are so much more susceptible to the pneumococcus than human beings, die, as a rule, in conse- quence of the septicemic general infection, and localized disease is induced in them with difficulty. 2. Streptococcus-pneumonia corresponds with the clinical picture of bronchopneumonia (cellular, catarrhal pneu- monia). The infiltrate is only lobular, usually less dense, and associated with less fibrin ; the sputum is mucopuru- lent and not rusty ; the onset is not so pronounced ; the course is more insidious and attended with remissions and intermissions in the fever (so-called streptococcus-curve) ; and, above all, the crisis is wanting. Some cases of strepto- coccus-pneumonia are characterized by especial severity iypneumonie infectieuse of the French). The differentiation of this variety is, however, not readily sustained, in the first place because — 3. Staphylococcus-pneumonia, which is exceedingly un- common as a single infection, resembles it absolutely ; and, secondly, because of the occurrence. so frequently of — 4. Mixed forms of pneumojiia, in which two or even three of the bacteria named are found together. These forms of the disease furnish clinically also a mixed picture that occupies a position between croupous pneumonia and bronchopneumonia. As a rule, the occurrence of blood- streaked sputum or the temperature-curve, even when the infiltrate is small, indicates the participation of pneumo- cocci in the morbid process ; or, in a case of apparently true croupous pneumonia, which, however, is not a pure OCCURRENCE OF CAUSATIVE AGENTS. 145 type etiologically, but is a mixed form, the crisis does not appear. On the whole, however, there are no fixed rules for these mixed types. They render impossible the clinical diagnosis with regard to the causative agent in the indi- vidual case. 5. Influenza-pnemnonia is associated with the presence of the influenza-bacillus, partly in pure culture, and partly together with streptococci or pneumococci. 6. The pneumonia complicating tuberculosis in the form of caseous pneumonia may be due to the tubercle-bacillus exclusively. On the other hand, pneumonia dependent upon streptococci, pneumococci, and influenza-bacilli is, however, not at all uncommon in tuberculous lungs. When pneumococci take part in the morbid process, the clinical picture assumes the peculiar characters that have been described, and are indicative of an acute severe infec- tion. 7. The pneumonia complicatiiig other infectious diseases is less commonly due to the cause of those diseases — as, for instance, typhoid pneumonia due to typhoid-bacilli — and more commonly to the organisms usually causative of in- flammation. The pneumonia that occurs in the sequence of diseases of the abdominal organs is often associated with the presence of the bacterium coli — as, for instance, the bronchopneumonia not rarely observed in connection with incarcerated hernia. The bacteriologic diagnosis can be made from exam- ination of the sputum, but the procedure of Koch, as de- scribed by Kitasato, for the culture of tubercle-bacilli directly from the sputum must be observed — that is, the mass of sputum must be thoroughly rinsed in dishes with sterilized water, in order to remove the large number of bacteria that have been added from the pharynx and the mouth. Then a flake is isolated from the interior of the mass, and this is smeared several times upon an agar-plate or upon several glycerin-agar tubes in slants ; that which is left is examined microscopically. Frequently direct puncture of the diseased lung has been made with a thoroughly sterilized hypodermic syringe, and a few drops of the exudate are obtained ; and these are treated bacteriologically in the manner described. This procedure — perfect asepsis being observed — need not occa- sion the slightest concern, as it is often practised involun- 146 CLINICAL BACTERIOLOGY. tarily in the search for pleuritic effusions (exploratory puncture) without ever doing any harm. If the information be sought whether the pneumococcus participates in the process, it is best to inject subcuta- neously some of the sputum or pulmonary fluid into a mouse, or, better, into the still more susceptible rabbit. If the pneumococcus is present, the animal will die in from twenty- four to forty-eight hours in consequence of diplococcus- septicemia, which is readily demonstrable microscopically in stained cover-glass preparations from the heart's blood of the animal. Prognostic significance is to be attached in only limited degree to the results of bacteriologic examination, be- cause in the case of pneumonia also much depends upon the virulence of the exciting agent. However, a crisis may be looked for with greater assurance when pneumococci alone are found than under any other conditions. It must, however, not be forgotten that even in cases of pure pneu- mococcus-pneumonia the crisis may not occur ; while, on the other hand, it may be present in cases of streptococcus- pneumonia. The general condition of the patient, evidently, is of considerable importance in this connection, and it must be given due consideration in every aspect in pre- dicting the crisis. Thus, the prognosis is unfavorable in the pneumonia of alcoholics, in that of the aged, and of those with kyphosis, even when pneumococci alone are found. The patients often die before the crisis, or the dis- ease pursues a protracted course, and terminates late with- out the occurrence of a crisis. The pneumococcus-pneu- monia of tuberculous subjects justifies a more favorable prognosis than a purely tuberculous pneumonia under the same conditions. Reference has already been made to the conception of the crisis as indicating the advent of immunity, as well as to the evidence of the immunizing property of the blood- serum after the crisis in a number of cases of pneumonia. Why this immunity is so transient, disappearing in some cases in the course of a few days, is not yet known. In any event it has been established clinically that pneumonia has a tendency to recur, and it may almost be included among those diseases of which one attack rather predisposes to subsequent attack. In a pneumococcus-culture the bacteria die in the course of a few days (from four to seven). OCCURRENCE OF CAUSATIVE AGENTS. 147 but this takes place only in cultures, and not in the body, and it is incapable, therefore, of explaining the crisis. The focus of disease in the lung, as well as the sputum, contains living bacteria both during and long after the occurrence of the crisis. Often these retain their virulence throughout the entire period. Should the virulence be diminished during the crisis, it is, however, soon again augmented, as experience has shown. It is, therefore, not the cocci, but the human organism that undergoes some change in the crisis : it be- comes insusceptible to the diplococcus-virus — that is, im- mune. Transmission of Pneumonia. — Infection with pneumonia takes place principally through the respiratory passages. Only in some cases of secondary, complicating pneumonia may the microorganisms gain entrance into the lungs through the blood-stream. Direct transmission of pneu- monia from one individual to another appears possible, and a considerable number of Jiouse-cpide7nics of pneumonia have been reported. In the majority of cases an attack of pneumonia probably results from the inhalation of pneumo- cocci with the air ; or, more frequently, pneumococci that have long been present in the mouth, the pharynx, or the nose, are permitted by some accidental occurrence to gain entrance into the lungs and there to set up inflammation. Naturally, in connection with the epidemic distribution of pneumonia, the possibility can not be excluded that the accidental influence has been operative in all cases in com- mon, that the pneumococcus need not be transmitted from one person to another, but that it was present previously in all affected. The hereditary transmission of pneumonia from the mother to the fetus is a matter of great interest. A few cases have been reported in which the children of pneu- monic mothers have been born with pneumonia. In gen- eral the pneumococcus is not disseminated outside the lungs of the infected individual. The slighter degree of susceptibility of human beings protects them, as a rule, against septicemic infection, which occurs regularly, for in- stance, in the more susceptible rabbit. In some cases, however, the pneumococcus has been found, also, in the blood of pneumonic patients during life. Such cases have always been marked by especial severity, with a fatal ter- mination, so that the discovery of pneumococci in the cir- 148 CLINICAL BACTERIOLOGY. culating blood must always be considered as of unfavorable prognostic omen. In the small number of cases of con- genital pneumonia reported the bacteria must have gained entrance into the placenta, through a lesion of which they have passed over into the fetal circulation. Without such lesion the placenta, as is quite generally admitted, is impassable to microorganisms. Like all febrile diseases, pneumonia readily induces abortion. The child that has been infected with diplococci can, however, suffer from true pneumonia only if it has already breathed. As a matter of fact, in only two of the children in the reported cases was pneumonic infiltration present ; in the others diplococci were found in the blood — there existed a sep- ticemia. ENDOCARDITIS. Endocarditis occurs usually as a secondary infection in the course of various other diseases. The most important of these is acute articular rheumatism, whose predominant etiologic significance in the generation of endocarditis is well known. The causative agents of rheumatism have not yet been isolated, and rheumatic endocarditis also is one of those infectious diseases whose intimate etiology re- mains to be cleared up. The same statement may be made with regard to chorea, whose endocarditic manifestations also are probably to be attributed to the same rheumatic etiology ; and also with regard to erythema nodosum. In the deposits upon the valvc3 of the heart in the se- quence of erysipelas the streptococcus pyogenes is demon- strable both on culture and on microscopic examination. In the endocarditis following suppurative processes (py- emia, septicemia, puerperal fever) the streptococcus or the staphylococcus pyogenes has been found in the vegetations : in that following osteomyelitis, generally the staphylo- coccus ; and after suppuration in the abdominal cavity, the bacterium coli commune. Endocarditis is comparatively frequent in the sequence of croupous pneumonia. The lesions, which preferably involve the aortic valve, contain, as a rule, the diplococcus lanceo- latus Frankel. Bacteriologic examinations with reference to the endocarditis following influe7iza are yet wanting. Diphtheric endocarditis is extremely uncommon. Only one case of true diphtheric endocarditis (mitral valve), with OCCURRENCE OF CAUSATIVE AGENTS. 149 demonstration of diphtheria-bacilli, has been recorded in the literature. Almost equally as uncommon as diphtheric endocarditis is also true typhoid endocarditis — that is, that caused by the Gaffky-Eberth bacillus. Tubercidous endocarditis has long been known. It attacks with a certain degree of exclusiveness the thin margins of the mitral leaflets, and particularly on their auricular sur- face. Tubercle-bacilli have been repeatedly demonstrated in the lesions. True gonorrheal endocarditis is dependent upon the activ- ity of the gonococcus, whose presence in the endocarditic vegetations has been demonstrated by Ley den. The causative agent of the endocarditis following measles, scarlet fever, smallpox, as well as the causes of the primary disease, are still unknown. The endocarditis complicating acute nephritis is generally caused by the same microorganisms (the exciting agents of suppuration and inflammation) that give rise to the primary morbid process in the kidneys. It is, however, to be emphasized that in all of the dis- eases named, as well as in malaria and in carcinoma, the concomitant endocarditis need not be due to the original infecting pathogenic germ, but it may be the expression of a secondary or of a mixed infection that has been engrafted upon the endocardium, whose resistance has been lowered by reason of the primary infection. Under such circum- stances staphylococci, streptococci, diplococci, or the bac- terium coli commune will be found in the vegetations upon the valves : in short, those microorganisms that have re- peatedly been shown to be the cause of secondary infection. So-called malignant, ulcercLtive endocarditis is etiologi- cally and clinically only a variety of ordinary endocarditis pursuing a malignant course, and giving rise to necrosis of the vegetations. If it is due to the common exciting agents of inflammation — staphylococci, streptococci, pneumococci, or the bacterium coli commune — it may be viewed as a special form of pyemia, characterized by the localization of the metastases on the valves of the heart. In isolated cases of this kind the microorganisms were demonstrated in the circulating blood during life. Experimental Development of Endocarditis by Means of Bacteria. — After a previous lesion of the valves (through , 150 CLINICAL BACTERIOLOGY. catheterization by way of the right carotid), it has been possible to induce endocarditic changes of maHgnant char- acter in experiments on animals by intravenous injection of the exciting agents of inflammation and suppuration. The same result may be attained without this serious pro- cedure if the bacterial material is introduced into the vein in the form of a suspension that contains coarse particles — for instance, from a potato-culture. The particles, with the contained and adherent microorganisms, lodge upon the valves, and thus form the starting-point of endocarditic changes. Bacteriologic Diagnosis. — In doubtful cases it is advis- able to examine the blood bacteriologically (p. 162). As has been mentioned, it is sometimes possible to demonstrate the microorganisms in the circulating blood, and the diag- nosis of ulcerative endocarditis is then justified. In the majority of cases, however, examination of the blood yields negative results, and even in such cases as exhibit a dis- tinctly malignant character. The clinical diagnosis may, therefore, receive but little support from bacteriologic ex- amination in connection with this disease. PERICARDITIS. Also with regard to pericarditis, the distinction between primary and secondary inflammation can not readily be made from the etiologic standpoint. The principal factor in the causation of pericarditis likewise is acute articular rhezunatism, whose exciting agent, as has been stated, is unknown. Traumatic pericarditis is either to be referred to the action of bacteria that gain entrance into the peri- cardial cavity simultaneously with the inception of the injury, or, in the case of nonpenetrating wounds of the thorax, a point of lessened resistance is established in the pericardium in consequence of rupture of blood-vessels, ecchymosis, etc., resulting from the contusion of the chest, in which microorganisms circulating in the blood lodge and give rise to inflammation. Pericarditis in the sequence of erysipelas is dependent upon the streptococcus pyogenes ; the pericarditis attending pnetimonia, upon the diplococcus of Frankel ; puerperal and pyemic pericarditis, upon the ex- citing agents of suppuration. A distinct position is occupied by tuberculous pericarditis ^ OCCURRENCE OF CAUSATIVE AGENTS. 151 which, next to the rheumatic, is probably the most com- mon variety. Its exciting agent, the tubercle-bacillus, gains entrance into the pericardium through direct exten- sion of the tuberculous process from the adjacent lung, or by way of the blood or the lymph. The bacteriologic diagnosis is possible during life only on puncture or operation for the relief of the pericardial effusion. MYOCARDITIS. Suppurative Myocarditis. — A greater or smaller number of purulent foci of varying size may be present in the heart- muscle in association with pyemic processes of diverse origin. They contain pyogenic microbes. Acute Diffuse Myocarditis. — Acute inflammation of the myocardium may complicate all infectious diseases of rapid course. Its bacteriologic relations are as yet but little known. The bacillus of Gaffky-Eberth has been repeat- edly found in the myocardium in cases of typhoid fever. Frequently, however, the infection appears to be secondary and dependent upon the common exciting agents of inflam- mation. The metabolic products of microorganisms may likewise ^w^ rise to similar myocarditic alterations — as, for instance, the toxin of the diphtheria-bacillus, which is the cause of diphtheric myocarditis. PERITONITIS. The inflammatory processes involving the peritoneum may be induced through chemic influences exerted by the metabolic products of bacteria — aseptic peritonitis, for in- stance, in consequence of absorption of decomposition- products from the intestine in cases of ileus. In the majority of cases, however, such processes result directly from the activity of bacteria {bacterial or septic peritonitis^. In accordance with the source of the exciting agents, peri- tonitis is conveniently divided into several varieties : (i) That which arises from the digestive tract — from the stomach, the duodenum, and the small intestine, as well as from the cecum, the vermiform appendix, the colon, and the rectum ; (2) that which arises from the gall-bladder and the liver ; (3) that which arises from the kidneys and the urinary bladder ; (4) that which arises from the female 152 CLINICAL BACTERIOLOGY. genito-urinary tract ; to these may be added (5) the rare cases in which the infection is of hematogenous origin ; and (6) the still rarer, in which the peritonitis is the result of operative intervention. In the first four groups named the bacteria may migrate to the peritoneum (as in the case of puerperal peritonitis, in which the exciting agents reach the peritoneum from the uterus by way of the lymphatics) in the absence of a breach in the continuity of the organ primarily infected ; or there may be such a breach in con- tinuity, and perforative peritonitis results. The latter is the more dangerous variety, because with the microorganisms other materials — as, for instance, intestinal contents — gain entrance into the peritoneal cavity through the perfora- tion, and these act as chemic and mechanical irritants, and thus make possible and easy the proliferation of the bacteria. By reason of the extraordinarily great absorp- tive power and the extent of the absorbing surface of the peritoneum, the entrance of bacteria into the peritoneal cavity in experiments on animals does not invariably lead to peritonitis, as has been demonstrated by the injection of moderate amounts of pyogenic cocci into the abdominal cavity of -dogs, rabbits, and guinea-pigs. For the develop- ment of peritonitis a predisposing factor is additionally necessary, such as especial virulence of the bacteria — -as, for instance, in general septicemia — the simultaneous en- trance of intestinal contents, and, experimentally, the con- joint introduction of considerable amounts of preformed bacterial poisons, etc. The distinction between diffuse and circumscribed peri- tonitis, which is so important clinically, is not supported by bacteriologic examination, as the causative agents are the same in both instances. The bacteriologic diagnosis can only be made in con- nection with operation or on exploratory puncture. If it is desired for special reasons in a given case to make an examination for bacteria after death, this must be done as soon as possible after dissolution has taken place, as the result may be invalidated by the migration of bacteria from the intestine after the lapse of a few hours. From the effusion or from the deposits upon the peritoneum — in ad- dition to microscopic examination — plates are simply cast or tube-slants are smeared. The many bacteriologic investigations that have been OCCURRENCE OF CAUSATIVE AGENTS. 153 carried out in the study of peritonitis have thus far not yielded practically available results. In some of the cases there existed multiple infection — that is, infection with a number of varieties of bacteria. Coli-bacteria, as well as staphylococci and streptococci, have been mostly found in the effusion. In addition, pneumococci have been ob- served, as well as a large number of other bacteria, of which the gonococcus, the proteus vulgaris, the. bacillus pyocya- neus, as well as a number of nonpathogenic varieties described by Tavel and Lanz, a bacillus resembling that of diphtheria, one resembling the tetanus-bacillus, and one the actinomyces-fungus, may be mentioned. The bacterium coli commune, which is the most common attendant upon peritonitis, is not a bacteriologic entity, but so variable in form and properties that the designation, bacterium coli, must be employed only as the collective name for a large group of related bacteria. When infection takes place through the blood-stream, only one exciting agent is found, and principally the streptococcus or the pneumo- coccus. In cases of operative peritonitis, usually strepto- cocci have been found ; and in cases of puerperal peritonitis, in addition also the other exciting agents of inflammation. Tuberculous peritonitis is caused by the tubercle-bacillus. From all that has been said it may be seen that peri- 'tonitis is not due to a single specific exciting agent, as the organ from which it arises (bladder, intestine) generally contains various bacteria. The infections that originate from the female genital organs are associated especially with cocci ; those originating from the intestine, especially with coli-bacteria. Further, the peritonitis arising from the small intestine is believed to be attended with the presence of fewer bacteria, while that arising from the large intestine is attended with the presence of more, as the contents of the large intestine exhibit a larger number of microorgan- isms than those of the small intestine. Both points of dis- tinction are, however, not conclusive, and the results of bacteriologic examination of the peritonitic exudate can not be employed without reservation as a certain guide for diagnostic purposes, nor have these results yet acquired noteworthy significance from a prognostic or a therapeutic point of view. 154 CLINICAL BACTERIOLOGY. PERITYPHLITIS. Perityphlitis, like other inflammations arising from the intestine, is due to the activity of the bacterium coli com- mune. Thus far only perityphlitic abscesses and their complications have been submitted to examination, coli- bacilli being predominantly found. Actinomycotic and tuberculous perityphlitis are associated with the presence of their special exciting agents. CHOLECYSTITIS AND CHOLANGITIS. Normally the bile is sterile, the bacterium coli and cocci being found only in the lowest portion of the choledoch duct. Every obstruction, however, that interferes with the free discharge of bile (gall-stones, etc.) renders possible in- fection of the biliary passages. The bacteria present in the bowel, particularly the bacterium coli commune, less commonly staphylococci, streptococci, and pneumococci, gain entrance into the gall-bladder and the biliary passages, where they induce inflammation and suppuration. Normal bile possesses no bactericidal activity ; when ob- tained in a sterile condition, it constitutes a fairly good nutrient medium for coli-bacilli and staphylococci. Infection of the unobstructed biliary passages takes place in human beings in individual cases of typhoid fever, cholera, and croupous pneumonia. It may be assumed that in these cases also the infection takes place through the choledoch duct. Typhoid -bacilli and cholera-bacilli are systematically present in a virulent state in the intestinal canal throughout the course of the respective diseases to which they give rise. The pneumococcus occurs but seldom in the intestinal tract, although it has been found there with certainty in individual cases of pneumonia. As a result of the constitutional disease, the function of the liver is more or less impaired, the excretion of bile is not normal, and, in consequence, infection from the intestine is possible. Infection through the blood is probably of rare occur- rence in human pathology. In experiments on animals this may be attained by introducing the bacteria in large number, or by previous injury of the biliary passages — as, for instance, by the establishment of a biliary fistula. OCCURRENCE OF CAUSATIVE AGENTS. 155 Experiments on Animals. — If, after ligation of the choledoch duct, coli-bacilli are introduced in the central portion of the duct, the animals (dogs, rabbits) die as a result of purulent cholecystitis and cholangitis. If the bacilli are injected directly into the mouth of the choledoch duct after the duodenum has been opened, the same result is obtained if the coli-cultures are sufficiently virulent. The bacteriologic diagnosis can be made only in cases of empyema of the gall-bladder, aseptic puncture being practised and plates being cast with the bile obtained. Such puncture, however, should not be undertaken unless urgently indicated, as even when made with most careful aseptic precautions, signs of peritoneal irritation, usually mild, follow. ABSCESS OF THE LIVER. So-called biliary abscesses belong in the domain of cho- langitis. Pyemic abscesses of the liver constitute part of a purulent general infection. (See Pyemia and Its Causes, p. 1 6 1.) Hepatic abscesses in the sequence of gastro-intcs- tinal lesions arise through the intermediation of the portal vein ; coli-bacilli act as exciting agents. With regard to tropical abscesses of the liver, the section on Dysentery may be consulted. In the bacteriologic diagnosis the pus obtained on ex- ploratory puncture with a sterile hypodermic syringe in the search for the abscess is subjected to examination. CYSTITIS. Numerous investigations in recent years have shown that the bacterium coli commune, or the bacterium aerogenes, plays the most important part in the etiology of cystitis. The other microbes of suppuration and inflammation also deserve consideration in this connection, but they occur- less frequently than the aerogenes and the coli. An excep- tion is formed by puerperal cystitis, following parturition, with which the streptococcus pyogenes and the staphylo- coccus pyogenes are equally associated ; also by gonorrheal cystitis, which, in part at least, is dependent upon the gono- coccus, and by tuberculous cystitis, which is caused by the tubercle-bacill u s. 156 CLINICAL BACTERIOLOGY. The bacteria gain entrance into the urinary bladder — 1. Through unclean instruments employed in catheteri- zation. 2. By ascending through the urethra — this is especially the case in women. 3. Through the intermediation of the kidneys ; some bacteria are capable of passing through the renal filter; and thus gain entrance with the urine into the bladder. Among influences favoring the development of cystitis are cold, traumatism, and retention of urine. In addition to the common exciting agents of suppuration other less common microorganisms have been found in individual cases of cystitis : the so-called micrococcus albicans am- pins, the diplococcus subflavus, the proteus, and others. These bacteria are, however, not pathogenic in themselves, and probably acquire importance only in mixed infection. The proteus causes putrid decomposition of the urine. Bacteriologic Diagnosis. — Catheterization is practised by means of a catheter sterilized by boiling, the urine being received into a sterile vessel. Men are permitted simply to pass urine spontaneously, the last portion evacuated being kept for examination, and the orifice of the urethra having previously been thoroughly cleansed. From the urine plate- cultures are rhade. Experiments on Animals. — By injecting pyogenic cocci into the bladder it is possible to induce cystitis in male ani- mals with certainty, if the escape of urine is prevented for from twelve to twenty-four hours by ligation of the penis. Ammoniacal Fermentation of Urine. — The bacterium coli possesses, in only slight degree, the faculty of causing decomposition of urine. Ammoniacal fermentation results from the action of individual varieties of the bacterium coli in the presence of a feebly acid, or an alkaline, reaction of the urine. Other varieties of the bacterium coli are incap- able of causing decomposition of urine. Ammoniacal cystitis, however, develops in the presence of the staphy- lococcus pyogenes or the proteus, whether in pure culture or in mixed infection with the coli commune. Pneumaturia. — In some cases of cystitis, especially with coincident presence of sugar in the urine, but also in the absence of glycosuria, the formation of gas may take place in the bladder. The urine under such circumstances is evacuated with an audible sound (pneumaturia). This OCCURRENCE OF CAUSATIVE AGENTS. 157 phenomenon is the result, in some cases, of fermentation of the sugar in the urine through the activity of microorgan- isms ; in other cases special varieties of coli-bacilli (aero- genes, etc.), characterized by abundant production of gas, have been cultivated from such urine. In one such case the gases formed consisted, in addition to carbon dioxid, of free nitrogen and hydrogen. NEPHRITIS. A bacterial origin has been ascribed to — 1. Primary infectious nephritis. 2. The nephritis occurring as a complication of infec- tious diseases, including the septic. Streptococci have repeatedly been found in association with acute nephritis, and both in the urine, from which they disappeared with the termination of the disease, and after death in the kidneys, in which they were visible in the vessels, in the epithelium, and in tube-casts. Acute nephri- tis has been induced also experimentally in animals by injection of streptococci into the blood. Under these con- ditions a large number of streptococci at first appeared in the urine, but these disappeared later. The disease, how- ever, progressed and terminated fatally, but no cocci were demonstrable in the kidneys after death. This observation indicates that nephritis may be of bacterial origin without bacteria being found in the kidneys after death (Mannaberg). The passage of the bacteria through the kidneys may suf- fice under certain circumstances to excite the anatomic in- flammatory process, which may then pursue its further course independently of them. In the majority of cases of acute primary nephritis bacteria are, as a rule, found in the kid- neys. A variety of inflammation of the kidney excited by bacteria appears further at times to present itself from the beginning as a chronic nephritis. In the morbid condition of slow course induced by Gharrin in rabbits by inoculation with pyocyaneus, chronic nephritis was found repeatedly. In some cases of complicating nephritis the exciting agents of the primary disease have been present. Thus, typhoid-bacilli have been found in the kidneys in associa- tion with typhoid nephritis ; diplococci, in association with pneumonic nephritis ; and spirilla of relapsing fever, in the 158 CLINICAL BACTERIOLOGY. urine of a patient suffering from nephritis complicating that disease. In other cases streptococci were found in the kidneys (smallpox, rheumatism, some cases of scarlet fever, etc.) ; under these conditions it is possible that a secondary or a mixed infection existed. Finally, complicating nephritis need not be excited directly by the bacteria, but it may be of toxic origin, resulting from the elimination through the kidneys of the toxins generated by the bacteria at the site of the primary disease. This is true peculiarly of diphtheric nephritis ; in conjunction with which, likewise, as is usual with scarlatinal nephritis, bacteria can not be found in the majority of the kidneys examined. The bacteriologic diagnosis can be made during life from examination of the urine (possibly after centrifugation), when there is no disease of the conducting passages, and especially of the bladder. Under normal conditions the urine that enters the bladder is and remains sterile, only becoming contaminated with bacteria in the urethra. For this reason the urine removed from the healthy bladder with a sterile catheter may be transferred directly to a nutrient medium, and such bacteria as develop may be con- sidered as being derived from the kidney. In men, gener- ally, irrigation of the urethra with the first half of the urine contained in the bladder is sufficient ; the last half is, as a rule, sterile in healthy individuals. Diagnostic or prognostic significance can not be at- tached to the demonstration of bacteria in the urine in cases of nephritis. According to Mannaberg, streptococci are found in the urine only in cases of true acute nephritis that pursue a rapid and favorable course, while they are wanting from the beginning in the apparently acute cases that subsequently prove to be chronic. This statement has, however, not been confirmed by other observers. PERINEPHRITIS. Perinephritis consequent upon disease of adjacent organs (kidney, intestine) is generally due to the bacterium coli. Perinephritis following traumatism, or attending general infection, may be due to any pyogenic microorganism. OCCURRENCE OF CAUSATIVE AGENTS. 159 PYELONEPHRITIS. A distinction is to be made between an ascending and a descending pyelonephritis. The first variety, by far the more common, is associated with the presence of identi- cally the same microorganisms as is cystitis, upon which it is dependent, and of which it represents the final, incurable stage. The microorganism most frequently associated is the bacterium coli or aerogenes. The descending variety, with infection from the kidney, is usually a pyemic process, and is dependent upon the related microorganism. In ascending pyelonephritis the bacteria gain entrance into the pelvis of the kidney as the result of retention of urine. No longer disturbed by the discharge of urine, the microorganisms that have caused the inflammation of the bladder wander into the ureter and, multiplying therein, they finally, by extension upward, invade the pelvis of the kidney. Chronic occlusion of the ureter may be followed by a pure pyelonephritis, without preceding cystitis, when the exciting agents of inflammation are present in the circulat- ing blood, are eliminated through the kidneys, and collect in the stagnating urine in the pelvis of the kidney. The bacteriologic diagnosis is only possible during life if operation be performed. Experiments on Animals. — After ligation of the ureter, pyelonephritis can be induced in rabbits by means of coli-bacilli or of pyogenic cocci, both by injection of these bacteria directly into the pelvis of the kidney or into the ureter above the ligature, and by intravenous iniection. INFLAMMATIONS OF THE FEMALE GENITAL ORGANS. Vulvitis. — Suppurative inflammation of the vagina is caused by the exciting agents of suppuration ; diphtheric inflammation, by the diphtheria-bacillus ; gonorrheal in- flammation, by the gonococcus. Endometritis. — Puerperal endometritis is always of bac- terial origin (streptococci, staphylococci, coli-bacilli) ; of the remaining varieties of endometritis the majority are to be attributed to gonorrhea. Salpingitis and Oophoritis. — Inflammation of the tubes and ovaries usually represents an extension of the endo- 160 CLINICAL BACTERIOLOGY. metritic process. The bacteria pass by continuity from the mucous membrane of the uterus to that of the tube, and from this to the ovary. The most frequent cause is, there- fore, gonorrhea. In addition, the presence of streptococci, staphylococci, coH-bacilli, and Frankel's pneumobaciUi has been demonstrated. A special position is occupied by tuberculosis of the tubes. Exceptionally, the oviducts are attacked by actinomycosis. The oophoritis that occurs in the course of some infectious diseases is due to the excit- ing agents of the primary disease, or they are caused secondarily by pyogenic microorganisms. Perimetritis and Parametritis. — Primary inflammation of the perimetrium and the parametral connective tissue is, almost without exception, dependent upon puerperal in- fection (streptococci, staphylococci, bacterium coli, in mixed infection also proteus), in the course of which the exciting agents of suppuration gain access to the perimetrium and the pelvic connective tissue by way of the lymph-channels. Secondary inflammation exhibits the same etiologic rela- tions as salpingitis and oophoritis, of which it constitutes a complication. Gonorrhea frequently and tuberculosis less commonly are of etiologic significance in this connection. INFLAMMATORY DISEASES OF THE EYE. Conjunctivitis. — The following microorganisms have been cultivated in cases of simple conjunctivitis : Staphylo- cocci, streptococci, pneumococci. Diphtheric conjunctivitis is excited by the diphtheria-bacillus, gonorrheal conjunc- tivitis by the gonococcus, and tuberculous conjunctivitis by the tubercle-bacillus. Keratitis. — Some cases of keratitis owe their origin to the common exciting agents of inflammation and suppura- tion, which at times gain access to the tissues through a lesion of the cornea. Hypopyon originates in a similar manner. Iritis and Choroiditis. — Inflammation of the deeper structures of the eye arises secondarily by extension of the pyogenic organisms from the cornea (contact-infection), or metastatically through dissemination of the bacteria by means of emboli (puerperal fever, pyemia, etc.). The same statement applies to panophthalmitis. Primary inflamma- tion of the choroid and retina is usually dependent upon OCCURRENCE OF CAUSATIVE AGENTS. 161 the activity of the metaboHc products of bacteria, which do not themselves advance, as a rule, beyond the cornea. Sympathetic Ophthalmia. — Deutschmann observed in cases of sympathetic ophthalmia an infiltration of the pia and the presence of phlogogenic bacteria in the optic-nerve sheath of the sympathetically affected eye. He attributed the sympathetic inflammation to direct invasion of the microbes by way of the optic-nerve path from the primarily affected eye. These observations have, however, not been confirmed, and the parasitic origin of sympathetic ophthal- mia must still be considered as undemonstrated. Chalazion is looked upon as the expression of a chronic inflammation of the tarsal connective tissue that is caused by the entrance of the exciting agents of inflammation into the excretory ducts of the Meibomian glands, and in the hair-follicles of the eyelashes. Giant-cells are invariably present in the granulation-tissue of the chalazion, and Tangl observed therein the presence of tubercle-bacilli. This observation, however, has not been repeated, and numerous inoculations of chalazion-tissue upon animals have never led to the development of tuberculosis in these. Trachoma. — Diplococci closely resembling gonococci have been repeatedly demonstrated in the contents of the trachoma-follicle. The transmission of the disease by means of these microorganisms appears to have been suc- cessful in several instances, but their specific significance is, on the whole, still doubtful. PYEMIA AND SEPTICEMIA. Pyemia and septicemia are not sharply separable from each other either clinically or etiologically. Both are caused by the exciting agents of suppuration, with the difference, however, that in relation with septicemia the element of intoxication is the more conspicuous ; whereas, in relation with pyemia dissemination of the bacteria by way of the blood-stream and development of multiple foci of suppuration (metastases) take place. So-called putrid intoxication and acute malignant edema (gangrene fou- droyante of the French) must not be considered as varie- ties of ordinary septicemia. Both of these disorders occupy a peculiar position from the etiologic point of view, as the first is due to mixed infection with putrefactive bacteria, 162 CLINICAL BACTERIOLOGY. especially the proteus of Hauser (see Proteus-infections), and the second is dependent upon the activity of a special specific cause — the bacillus of malignant edema (vibrion septique, see Malignant Edema). In connection with the pyemia and septicemia of human beings, therefore, there are to be considered the streptococcus pyogenes, the staphy- lococcus pyogenes, the diplococcus lanceolatus Frankel, the bacterium coli commune, and the diplobacillus pneu- moniae Friedlander. The starting-point of pyemia and septicemia is usually a primary focus of suppuration or of inflammation. If such a lesion can not be demonstrated, the condition is designated cryptogenetic septicemia. Under these circumstances the primary focus has, in the majority of cases, merely escaped detection because of its obscure situation. Among such concealed sources of septic dis- orders may be mentioned mediastinitis, prostatic abscesses, collections of pus in the accessory cavities of the nose (antrum of Highmore, ethmoid sinuses, etc.). Only in rare cases does pyemia or septicemia result from direct absorption of the exciting agents of inflammation into the blood from without or from an internal surface without the formation of a primary focus. Bacteriologic Diagnosis. — The bacteria can by no means be demonstrated in the blood in all of the cases. This should not occasion surprise in cases of septic intoxica- tion,-as the microorganisms are present in the blood in small number, if at all. In cases of pyemia the bacteria can be more frequently demonstrated in the blood, espe- cially at the time of the chills, when the infected thrombi are set free and give rise to fresh metastases ; but even here the results of blood-inoculation are often negative. The technic for examination of the blood is simple. The finger of the patient is cleansed by means of soap, alcohol, mercuric-chlorid solution, and ether, its tip is slightly punctured with a lancet sterilized in the flame, and the escaping blood is smeared upon culture-media by means of the platinum loop heated in the flame ; eventually, plates are made. It is better, because of the small number of bacteria circulating in the blood at any time, to employ a considerable, amount of blood. This is aspirated by means of a sterilized Roux syringe from a vein distended by compression. Metastatic abscesses are opened with aseptic precautions, OCCURRENCE OF CAUSATIVE AGENTS. 163 and from the pus cultures are made directly, or plates are poured and cover-slip preparations are prepared. The excretory products of septic patients — urine, sweat, and saliva — at times contain the exciting agents of the ex- isting disease, and in suitable cases it is, therefore, advisable to examine these secretions also. The elimination of the bacteria through these channels constitutes a mode by which the organism spontaneously gets rid of the exciting agents of the disease. Experiments on Animals. — Experimental septicemia, such as may be induced in animals by means of the most varied microorganisms, can not without qualification be considered analogous to septicemia in human beings. In experiments on animals unlimited multiplication of the bac- teria in the blood takes place, although it is to be borne in mind that enormous multiplication sets in only a short time before the death of the animal. The conditions are much simpler in the case of pyemia, as this disorder may be induced in animals through all portals of infection and with all pyogenic microorganisms. It is a necessary con- dition, however, that the microorganisms employed in the experiment shall possess a sufficient degree of virulence. PUERPERAL FEVER. Puerperal fever is only clinically a special form of pyemia or septicemia ; but in relation to its causative agents, analo- gous to all other varieties. The streptococcus pyogenes, the staphylococcus pyogenes, and, less commonly, the bacterium coli have been found to be its exciting agents. The severity of the disorder is dependent upon the fact that the bacteria gain entrance directly into the open lumen of the vessels of the uterine mucous membrane injured in the process of parturition, and by this means into the gen- eral circulation. The process pursues a relatively favor- able course when the vessels are already occluded by thrombi, and the larger lymph-trunks are again closed. The bacteria then wander through the lymph-spaces be- tween the muscle-fibrils, reach the pelvic connective tissue, and there give rise to a localized, circumscribed, suppura- tive process, so-called puerperal parametritis. The occur- rence simultaneously, or in rapid succession, of widely separated foci of suppuration — as, for instance, the periton- 164 CLINICAL BACTERIOLOGY. itis, the empyema, the arthritis, etc., of puerperal sepsis — can only take place through the dissemination of the germs by way of the blood-stream. It may be mentioned that, in the milk of nursing women suffering from puerperal fever, pyogenic cocci can not rarely be found. OSTEOMYELITIS. Osteomyelitis is not a specific disease. Staphylococci have been most frequently found to be its exciting cause — both the aureus and the albus ; less commonly, the streptococcus pyogenes, the diplococcus Frankel, the typhoid-bacillus, and the bacterium coli commune have been cultivated from osteomyelitic foci. In accordance with these observations osteomyelitis may be considered as a form of pyemia characterized clinically by its localiza- tion in the bone-marrow. The skin and the open cavities of the body constitute portals of entry for the bacteria. It is, however, by no means necessary that primary foci (furuncle, panaris, etc.) should exist in every instance. The forms of life normally present upon the skin and mucous membranes render superfluous such an assumption in the individual case. Osteomyelitis occurs exclusively in young individuals, and it has, therefore, been designated the pyemia of the developmental period. In young in- dividuals the growing zone of bone represents a point of lessened resistance at which bacteria, when they gain entrance into the circulation from any source, may lodge and multiply. Experimental Evidence. — If young animals (rabbits or dogs) are inoculated by intravenous injection with pyogenic microorganisms, subperiosteal abscesses and purulent in- flammation of the medulla of bone occur. In older ani- mals similar results are obtained only if previously a frac- ture has been induced ; thus, to a certain degree, a point of lessened resistance has been established artificially. Under such conditions osteomyelitic changes take place in the situation of the fracture. These experimental obser- vations are in accord with the facts of human pathology — as osteomyelitis, as has been mentioned, is a disease of early life. OCCURRENCE OF CAUSATIVE AGENTS. 165 PYOCYANEOUS GENERAL INFECTION. The bacillus pyocyaneus is, in general, quite a harmless bacterium. Its presence in the pus of wounds, in cases of otitis media, etc., retards the process of healing in the re- spective diseases only inconsiderably, and gives rise to the well-known greenish or bluish discoloration of the pus and the dressings. In the bodies of children the bacillus pyocyaneus at times exhibits pernicious activity, and it appears under some circumstances capable of causing severe general infection. Neumann obtained this micro- organism from the blood and the internal organs of a new- born infant dead of hemorrhagic septicemia. H. Kossel found it in children in the meningeal exudation in the sequence of otitis, in diarrheal stools, in cases of nephritis, and in the presence of inflammatory affections of the naso- pharyngeal space. He believes that the bacillus is capable of causing serious injury to the organism of the child, either directly through the intermediation of the blood- stream or indirectly through its metabolic products. PART III. SPECIFIC DISEASES OF BACTERIAL ORIGIN. TYPHOID FEVER, Morphology of the Typhoid-bacillus. — The bacillus of typhoid fever was first observed by Koch and Eberth^ and grown in pure culture by Gaffky in 1884. Fig. 48.— Bacillus typhi, from an agar-agar culture twenty-four hours old ; X 650 (Heim). The typhoid-bacilli are small, plump rods, with rounded ex- tremities (0.5-0.9 X 1-3 /^)- In the tissues the organisms lie, as a rule, isolated, while in cultures they are arranged in pairs and not rarely in long filaments. They possess from eight to eighteen terminal and lateral flagella, and, in consequence, they are most actively motile, their movement being serpentine in character. They do not stain so well as other bacteria and they take the stain with some difficulty. It is, therefore, advisable to warm the aqueous staining solutions and also the diluted carbol- fuchsin solution. The bacilli do not stain by Gram's method. 166 TYPHOID FEVER. 167 Spore-formation. — Gaffky considered as spores certain ter- minal bright, oval bodies that are said to remain unstained. These bodies (polar granules) have, in accordance with subsequent in- vestigations, come to be looked upon as involution-forms. At any rate the bacilli that contain such bodies are not noted espe- cially for their powers of resistance, as they are destroyed with certainty by exposure for ten minutes to a temperature of 60° C. (140° F.). The typhoid-bacillus grows in the absence of oxygen, though by no means so well as in its presence (facultative anaerobiosis). The temperature-optimum for the typhoid-bacillus is that of the body. The organism thrives well, however, at room-tempera- ture. The temperature-maximum is 46° C. (114.8° F.). The Appearance of Typhoid-bacilli in Cultures. — The bacillus of typhoid fever — in contrast with most other patho- genic bacteria — exhibits vigorous growth on slightly acid nutrient media. On gelatin-plates deep colonies appear as small, punctate, sharply circumscribed dots ; with low powers of the microscope they may exhibit a brownish-yellow color and a whetstone shape. Superficial colonies are much larger, and form a bluish, iridescent, delicate coating, with an irregularly serrated border. Only the central portion of the colony appears, with low powers of the microscope, of a yellowish color, while toward the margin a delicate linear network can be observed, giving rise to a leaf- like appearance. The gelatin is not liquefied. In gelatin stab-cultures development takes place along the entire line of inoculation. Superficial growth is pronounced, and presents characteristics similar to those observed in superfi- cial colonies on plates. Gelatin Streak-cultures. — From the center the entire surface of thfe gelatin is covered by a delicate, iridescent, bluish coating. In all gelatin-cultures a peculiar milky turbidity of the nu- trient medium frequently occurs in the neighborhood of the culture. In agar streak-cultures and in blood-serum a whitish coating of inconsiderable density forms, without characteristic peculiari- ties. The appearance oi potato-cultures is of importance. In these the typhoid-bacillus grows in. an invisible layer, with an ap- pearance as if nothing at all had developed upon the surface of the potato. If, however, an attempt is made to remove material with a platinum loop, it is at once found that the potato is entirely covered by a layer of some kind. Microscopic ex- amination confirms this observation, and discloses the presence of large numbers of actively motile rods. This mode of devel- opment is quite peculiar, and occurs, so far as is as yet known. leS CLINICAL BACTERIOLOGY. only with typhoid-bacilli. It is, however, not constant. There are varieties of potato upon which the bacilli of typhoid fever develop in yellowish or brownish, raised, and sharply circum- scribed deposits, and generally upon potatoes whose surface yields a neutral or even an alkaline reaction. Such visible growth can be obtained also artificially by rendering the surface to be inoculated of alkaline reaction. Typical characteristic growth takes place only when the potatoes exhibit an acid reac- tion, which they do, as a rule. In milk the typhoid-bacillus induces slight formation of acid, but never coagulation. In Petruschky's whey (p. Z(i) the typhoid-bacillus generates not more than three per cent, of acid, whereas the bacterium coli commune generates more than seven per cent. Bouillon is rendered turbid by cultures of the typhoid-bacillus. The typhoid-bacillus does not induce fermentation in nutrient media containing grape-sugar, milk-sugar, or cane-sugar ; nor does it form indol in a solution of peptone and sodium chlorid. If potassium nitrite and sulphuric acid are added to peptone- cultures, a red coloration does not occur (p. 122, bacterium coli commune). The typhoid-bacillus, as well as the bacterium coli commune and the entire group of bacteria resembling both, is character- ized by a certain degree of resistance to carbolic acid, addition of which to the nutrient medium in the proportion of % per cent, not inhibiting the bacilli in their growth. Vital Activity of the Typhoid-bacillus. — Typhoid- bacilli retain their vitality in sterilized water iox a considerable time (up to three months) ; they may even, at least at first, multiply therein. In unsterilized water they die in the course of two weeks, in consequence of the activity of the water-bacteria, by which they are suppressed, and more quickly in running water than in stagnant water. Under favorable conditions, protected from light, evaporation, and competition, they may persist for a long time. Milk may at times contain living typhoid-bacilli for as long a period as five weeks. In the slime of streams and of wells typhoid- bacilli retain their capability of development for not less than three weeks. Buried in the superficial layers of the earth, they have been demonstrated in a living state after five and a half months. They appear also capable of per- sisting for an equal length of time in the feces — for three months and more ; naturally only when too many putrefac- tive bacteria are not present at the same time. Typhoid- TYPHOID FEVER. 169 bacilli bear cold quite well ; they are not injured by freez- ing for two or three times, and subsequent thawing. They are less resistant to heat^ as has been mentioned. (See Spore-formation.) Dried in a thin layer, typhoid-bacilli have been found to preserve their vital activity (Uffelmann) — In garden-soil for twenty-one days. In sweepings for more than thirty days. In white filter-sand for eighty-two days. Upon linen for from sixty to seventy-two days. Upon buckskin for from eighty to eighty-five days. Upon wood for thirty-two days. According to Kruse, they die in thin layers, dried, with- in from five to fifteen days. Portals of Infection and Dissemination of Typhoid- bacilli. — Dried typhoid-germs may be carried through the air with floor-dust, street-sweepings, particles of clothing, etc. They may, thus, possibly be inhaled. Infection by way of the lungs, however, is rather improbable with regard to typhoid fever, although it played an important part in the earlier theories that denied the transmission from case to case, and considered necessary the presence of the typhoid-bacilli in the earth for their complete maturation ; but this has not been demonstrated. Besides, in human beings the digestive tract appears to be the only portal of entry for the typhoid-bacillus. The typhoid-germs must be swal- lowed, and gain entrance into the intestine. To this end it is necessary that they lodge upon, and be taken up with, food. Apparent infection through the lungs may also be so interpreted that the typhoid-germs contained in dust and with this inhaled are restrained in the upper part of the respiratory tract, to be subsequently carried with food into the digestive tract. It is an important fact that the typhoid-bacillus is not destroyed with certainty by the hydrochloric acid of the gastric juice. The barrier inter- posed by the stomach thus fails to afford reliable protection against typhoid infection, even when the function of that organ is completely normal. Articles of food may be contaminated with typhoid-bacilli through the intermediation of the air ; dust containing typhoid-bacilli may be deposited upon articles of food. More frequent, however, is direct contamination by means of the feces, which become attached to the hands of the at- 170 CLINICAL BACTERIOLOGY. tendants in the emptying of bed-pans, and in the cleansing of soiled linen, and transference to food from want of clean- liness. In this way infection from case to case takes place, as may often enough be demonstrated. The disease oc- curs in epidemic distribution when a common article of food is contaminated. Thus, epidemics of typhoid fever have been caused by milk, by oysters obtained from infected water, and more frequently by drinking-water. Repeatedly, a communication between wells or sources of water-supply and neighboring cesspools into which undisinfected stools have been emptied has been found to be the cause of typhoid epidemics. In other epidemics contamination of the pubHc water-supply by means of typhoid dejections, or through the washing of infected clothes, has most proba- bly taken place. Recently, typhoijd-bacilli have been observed a number of times without a direct relation to cases of typhoid fever being demonstrable or even suspected. Losener found bacilli that corresponded in all respects with Eberth-Gafif ky bacilli in a specimen of earth obtained from an untilled field, and, further, in the water-supply of Berlin obtained from a conduit in his laboratory. Remlinger and Schneider, using perfect methods, have recently cultivated the same microorganisms from earth, dust, and water. They even found them in the intestines of five individuals who had never suffered from typhoid fever. Observations like these are, however, exceptional, and are as yet beyond explana- tion. In order that typhoid fever may develop, in addition to the taking up of the bacilli a special predisposition on the part of the individual certainly appears necessary ; or the bacilli must possess especial virulence or be taken up in excessive number. In general, human beings may be con- sidered as not particularly susceptible to typhoid fever. The requirement of an especial temporal and local predisposition (elevation of the ground- water), as demanded by the theory that the condition of the soil bears some relation to the occurrence of typhoid fever, can no longer be sustained. The Occurrence of the Bacilli in Typhoid Patients. — The bacilli are found systematically in Peyer s patches, the mesenteric glands, the spleen, the liver, and the bone-marrow of typhoid patients. They are always collected in groups that frequently exhibit a relation with blood-vessels. In TYPHOID FEVER. 171 the feces they are not found before the second week of the disease, usually after the tenth day, but, as a rule, not in large number. In most cases they disappear from the stools as early as the fourth week of the disease, and in rare cases they are demonstrable until after defervescence. The blood of typhoid patients is generally sterile, although typhoid-bacilli have been found in the blood of the rose- spots and from the veins. It is necessary to employ con- siderable amounts of blood (i cu. cm.) for the purpose of examination. The urine during life and the kidneys after death frequently contain the bacilli, especially in cases complicated by albuminuria. Rarely the bacilli have been found, beside's, in the hings in some cases of typhoid pneu- monia or bronchopneumonia, in the meninges in cases of typhoid meningitis, in the myocardium in cases of typhoid myocarditis, and in the testicles in cases of typhoid orchitis. Typhoid-bacilli have been found repeatedly in the pus of suppurative processes that develop in the course, or as a sequel, of an attack of typhoid fever : as, for instance, osteoperiostitic processes, encapsulated peritonitis, abscesses of the spleen and the liver, inflammation of joints, thyroiditis, empyema, etc. These observations show that the typhoid- bacillus is also capable of manifesting pyogenic activity. Typhoid-bacilli have been found in such posttyphoid sup- purative processes fifteen months after the termination of the disease, and in one case of old bone-disease as long as seven years afterward.* Mixed Infection and Secondary Infection. — The course of typhoid fever is frequently modified by complicating in- fections due to other microorganisms, usually those exciting inflammation. At times these complications appear at the commencement of the disease as the expression of a mixed infection ; at other times they occur later as secondary in- fections. The bacterium coli commune plays the most im- portant part in the development of these conditions. It is found to be the cause of peritonitis, cholangitis, etc. Next in importance is the streptococcus pyogenes, which has been frequently demonstrated in cases presenting secondary empyema, otitis, and bronchopneumonia. Mixed infection *Hunner, "Bulletin of the Johns Hopkins Hospital," Aug.-Sept., 1899, p. 163, has reported a case of acute suppurative cholecystitis in which typhoid- bacilli were isolated from the contents of the gall-bladder eighteen years after an attack of typhoid fever. — A. A. E. 172 CLINICAL BACTERIOLOGY. with streptococci is responsible for the development of the dangerous condition known as streptococcous typhoid septi- cemia. The investigations of Vincent have shown that the presence of the streptococcus and artificial mixed cultures materially increases the virulence of typhoid-bacilli. The staphylococcus pyogenes, the diplococcus lanceolatus Frankel, and the proteus, likewise frequently find oppor- tunity to gain lodgment in the organism enfeebled by typhoid fever, and to give rise to the development of fur- uncles, cutaneous abscesses, bronchopneumonia, catarrh of the middle ear, gangrene of the skin, and the like. Experiments on Animals, — Mice, guinea-pigs, rabbits, goats, etc., die after the introduction of virulent Eberth- Gaff ky bacilli, with decline of temperature, convulsions, and diarrhea. Considerable numbers of bacteria are necessary in subcutaneous introduction, while smaller numbers suffice in intraperitoneal and intravenous inoculation. The fatal result is brought about under these conditions through intoxication. If the virulence of the infectious agents is marked, the animals, and especially white mice, may, how- ever, be destroyed by intraperitoneal injection of quite small numbers of bacteria, although large numbers of the bacilli are found in the blood after death. It is certain that under these conditions an increase has taken place in the number of bacteria, and it can, therefore, not be denied that the disease induced in animals by typhoid-bacilli dis- plays the characters of a true infection. The development of actual typhoid fever in animals (and also mere typhoid-bacilli intoxication) in consequence of introduction of the bacteria through the mouth is attended with serious difficulties. It is, however, to be observed that a true typhoid disease appears not to exist naturally in animals. If preliminary treatment is employed similar to that adopted in the experimental development of cholera — alkalinization of the gastric contents, and injection of tinc- ture of opium — changes are at times induced in guinea-pigs by the introduction of typhoid-bacilli through the mouth that in some degree are suggestive of those of typhoid fever in human beings. Experiments on animals of a completely positive character, with the pathologic-anatomic lesions of true typhoid fever, have not been recorded in the literature. The pyogenic activity of the typhoid-bacillus can be readily demonstrated in experiments on animals. TYPHOID FEVER. 173 Etiologic Relations of the Bacilli to Typhoid Fever in Human Beings. — In view of their constant presence in all cases of typhoid fever, and of their occurrence exclu- sively in this disease, the Eberth-Gaffky bacilli may be considered as the exciting agents of typhoid fever, although experimental development of typhoid fever by means of the bacilli has not been induced. The bacteria that gain entrance into the digestive tract with the food obtain lodg- ment, in the presence of the necessary predisposition on the part of the individual, in the follicles and plaques of the intestinal wall. The period of incubation for typhoid fever is from one to three weeks. During this time the bacteria slowly give rise to the anatomic process that constitutes the basis of the disease, the swelling of the plaques, and, later, their ulceration. At the same time the bacteria multiply and gain entrance — probably by way of the lymph-paths especially — into the mesenteric glands, also into the liver and the spleen. To this point typhoid fever is a truly in- fectious disease. The toxic activity of the bacteria is, how- ever, of equally marked importance. Brieger and Frankel have demonstrated in bouillon-cultures of typhoid-bacilli a chemic poison that they place in the class of toxalbumins. R. Pfeiffer also held the view with regard to typhoid fever that the peculiar poison resides in the bodies of the bacteria. (See General Section, p. 31.) This can be readily obtained by destroying young agar-cultures carefully by means of chloroform-vapor or by exposure for an hour to a tempera- ture of 54° C. (129.2° F.). From eight to ten mg. of the bacterial mass suffice to destroy a guinea-pig. There is no doubt that also in the body of the typhoid patient a specific poison is generated by the bacteria, and is absorbed. The characteristic mental dulness, and the peculiar febrile course may be considered as expressions of this intoxication. Not rarely cases of typhoid fever are observed in which the intes- tinal lesions are subordinate to the toxic state. These are instances of typhoid fever with most profound mental con- fusion and high fever, but with mild intestinal manifestations, in which after death only a few ulcers of small extent are found in the bowel. Cases have even been reported in which during life the characteristic stools, and after death the typhoid ulcers, were entirely wanting : the diagnosis of typhoid fever being based solely upon the demonstration at the autopsy of typhoid-bacilli in the greatly enlarged spleen. 174 CLINICAL BACTERIOLOGY. Bacteriologic Diagnosis of Typhoid Fever (Differentia- tion of Typhoid-bacilli and Bacterium Coli Commune). — The bacteriologic diagnosis of typhoid fever is usually complicated by so many difficulties that it can not be con- sidered as an aid in the clinical diagnosis. Isolation of the typhoid-bacillus in the feces is a matter of great difficulty by reason of the extraordinary resemblance of the bacillus to the bacterium coli commune — a resemblance that is so marked that some observers (particularly the Lyons school) have considered the two microorganisms identical. The micro- scopic appearances, the agar-culture and the gelatin-culture of both bacteria are absolutely alike. Potato-cultures are, generally, though by no means always, different : the bacterium coli gives rise to a thick, raised, circumscribed, greasy, brownish deposit ; the typhoid-bacillus, on the other hand, to an invisible, extensive coating. In using potatoes for differential diagnostic purposes one-half of the surface must be inoculated with the suspected bacteria, and the other half with actual typhoid-bacilli. If the same growth takes place on each half of the potato, it is highly probable, providing there is agreement in other respects, that the suspected organism is really the specific Eberth- Gafifky bacillus. Among further points of differential diagnosis as between typhoid-bacilli and coli-bacilli are the following: (i) The bacterium coli coagulates milk; the typhoid-bacillus does not. (2) The bacterium coli gener- ates gas in nutrient media containing peptone, and especi- ally in those containing grape-sugar, after exposure in the thermostat for even a few hours ; the typhoid-bacillus does not. It must, however, not be overlooked that, though quite seldom, there occur varieties of coli-bacilli that do not coagulate milk and do not cause fermentation of grape-sugar, and thus can not, by any of the methods thus far known, be absolutely differentiated from the Eberth-Gaffky bacillus. A further point of differentiation consists in the fact that the typhoid-bacillus does not yield the indol-reaction, whereas the bacterium coli does. There is, however, a variety of bacterium coli that does not generate indol. The differentiation between the two bacteria thus re- mains extremely difficult, and only such bacteria can be decided with perfect certainty to be typhoid-bacilli as possess all of the peculiarities described, and as have been cultivated from the spleen of human beings who TYPHOID FEVER. 175 exhibit, or have exhibited, the dinical symptoms of typhoid fever. Recent and reliable aids in the differential diagnosis between the bacillus of Eberth-Gaff ky and the great horde of bacteria resembling the typhoid-bacillus and the bac- terium coli are furnished by the reaction of Pfeiffer and that of Gruber. (See General Section, pp. 62 and 63.) Ten times the minimal lethal dose amount of the sus- pected bacteria are mixed with a small amount — less than o. I cu. cm. — of serum obtained from an animal highly im- munized against typhoid fever, and the mixture is injected into the peritoneum of a guinea-pig. If after from ten to twenty minutes the characteristic disintegration of the bac- teria into granules becomes apparent in the fluid obtained from the abdominal cavity by means of a capillary tube, the conclusion may be reached that the organisms are actually typhoid-bacilli. If the virulence of the microor- ganisms is slight or wanting, Pfeiffer' s reaction is obviously not available. Under these conditions Gruber' s reaction is employed. The technic has been described in the general section (p. 64). If agglutination takes place, the diagnosis may be considered as positive. Typhoid-bacilli are isolable from the feces only with great difficulty on account of their close resemblance to the bacterium coli commune. The procedure most likely to prove successful is that recommended by Eisner. Plates are made in the usual manner with potato-gelatin (see Methods of Culture and of Examination, p. 81), to which shortly before use potassium iodid has been added in the pro- portion of one per cent. This culture-medium appears espe- cially adapted for the growth of typhoid-bacilli and coli-bac- teria, with the qualification that the latter grow more vigor- ously than the former. After the lapse of forty-eight hours the colonies of coli-bacilli appear as dark -brown spherical masses, while those of the typhoid-bacilli appear as small, transparent, water-like drops. The method of Eisner is, however, not absolutely trustworthy. Its appli- cation requires much practice, and it does not render superfluous further precise identification of the apparent typhoid-colonies according to all of the rules mentioned. The bacteriologic diagnosis of typhoid fever may be made quickly and easily by puncture of the spleen, and the development of cultures from the fluid obtained. If the 176 CLINICAL BACTERIOLOGY. disease present is typhoid fever, pure cultures may thus be obtained at once, which do not coagulate milk and do not generate gas in peptone-bouillon. In rare cases of mixed infection, in addition to typhoid-bacilli the streptococcus pyogenes or the staphylococcus is also found in the fluid from the spleen. The method of puncturing the spleen is the same as that employed in every other form of explora- tory puncture. On aspiration the splenic fluid, mixed with blood, is readily obtained, and this is injected into a sterilized dish, five agar-tubes being each then smeared successively with a drop of the fluid ; with the remainder plates are made, and milk-flasks and peptone-bouillon tubes are inoculated. Notwithstanding the trustworthy character of its results, puncture of the spleen is, however, not to be recom- mended. The procedure is by no means free from danger. It should not be forgotten that the typhoid-bacillus is also capable of pyogenic activity, and that the puncture-track in the spleen may give rise to purulent complications, possibly in the peritoneum. The most valuable service in the clinical diagnosis of typhoid fever appears to be rendered, however, by the pro- cedure of Widal, who, as has already been mentioned (p. 66), showed that also the blood-serum of typhoid patients yields the agghitinatioii-phenomenon of Gruber. Blood-serum from the patient suspected to be suffering from typhoid fever is obtained by aseptic puncture of the tip of a finger, or of a vein by means of a sterilized syringe. The blood thus obtained is permitted to flow into a test- tube, and to coagulate in a slanting position ; in this way the largest proportion of serum will be obtained. The test is then made according to one of the methods described on page 64 ; the one preferred is a matter of indifference. The desired information is gained most quickly through the microscope, and only by this means is it possible to determine the extreme limit of agglutinating activity — that is, the highest degree of dilution of the serum with which the reaction can still be induced. It has been emphasized that the reaction of Gruber is a quantitative one, and that, therefore, everything depends upon determining quantita- tively with precision the agglutinating activity of the serum. A proportion of i to 50 is sufficient for the posi- tive diagnosis of typhoid fever. Many thousand specimens of serum have now been examined according to the method TYPHOID FEVER. 177 of Widal, and in no instance has this dilution yielded the reaction with serum not from a case of typhoid fever. It is possible, however, that the agglutinating activity in cases of typhoid fever may be below these figures ; with dilu- tions between i : 50 and i : 10 Widal and Sicard rec- ommend that the case be considered as suspicious with regard to typhoid fever, and that the observation be re- peated in the course of a few days. The serum of typhoid patients, even when not sterile, retains its agglutinating power unchanged for several months. It may, therefore, be preserved and sent from one place to another. The time when the agglutinating property appears in the serum is of importance in diagnosis. As a rule, it is demonstrable after the seventh day, although it may appear later or earlier. The earliest that it has been observed was on the second day by C. Frankel, and the latest in the first days of convalescence by Achard. The Widal-Gruber reaction grows feebler in the first weeks or months of convalescence, finally to disappear completely in some cases. Frequently, however, it per- sists, and it may be demonstrable after the lapse of years, or even of decades, and it can thus be utilized as an evi- dence that the individual in question has previously suffered from an attack of typhoid fever. On this account it is absolutely necessary, in the application of serum-diagnosis, to learn from the patient's history whether he has not already at some time passed through an attack of typhoid fever, however mild ; otherwise, there is danger that a re- action of older date may be employed diagnostically in relation to the disease under observation. Widal and Sicard divide their cases of typhoid fever into five groups, accord- ingly as the serum exhibits a greater or lesser agglutinating activity. In the first group the agglutinating power is very slight, below i to 100; in the second group it is feeble, between i to 100 and i to 200 ; in the third group it is moderately great, up to i to 500 ; in the fourth group it is great, up to i to 2000 ; and, finally, in the fifth group it is above i to 2000. Each of these five groups includes both mild and severe cases. The agglutinating curve in individual cases of typhoid fever, observed throughout the whole course of the disease, likewise exhibits the widest variations. Each case bears in this respect, as Widal and Sicard believe, its individual impress. Its agglutinating 178 CLINICAL BACTERIOLOGY. power may appear earlier or later, and in greater or in less degree, and it may even be entirely absent, as these observ- ers have noted, although but once among 163 cases. The examination of water for typhoid-bacilli is of great practical importance, as in most epidemics the drink- ing-water is to be considered as the vehicle for the typhoid virus. For this purpose carbolic acid is employed, being added to the suspected water in such amount that this shall contain from 0.05 to 0.25 per cent, of the acid. This addi- tion is made for the purpose of inhibiting the activity of the bacteria present in water that liquefy gelatin ; the typhoid-bacilli themselves readily withstand such slight additions of carbolic acid. With the carbolized water three plates are made in the customary manner, according to the method of Eisner. As by this means only small amounts of water are subjected to examination, it is easily possible that typhoid-bacilli may escape detection, even when pres- ent. It is, therefore, well to subject considerable amounts of the suspected water to examination. To this end a sterilized, alkaline, concentrated solution of peptone and sodium chlorid is employed that contains in a specified number of cubic centimeters one gram of peptone and one gram of sodium chlorid. This amount is added to 100 cu. cm. of the carbolized water in an Erlenmeyer flask, and the mixture is placed in the thermostat for from eigh- teen to twenty-four hours. If typhoid-bacilli are present in the water, in some degree protected against the compe- tition of the other bacteria by the addition of carbolic acid, they undergo multiplication, and they can be more readily demonstrated on plates prepared from the mixed cultures. The identification with certainty of the developing suspi- cious colonies as typhoid-colonies is, however, again at- tended with considerable difficulty. The water contaminated by typhoid dejections naturally always contains also the bacterium coli commune, and besides there are frequently present in the water other nonpathogenic bacilli that bear an extraordinary resemblance morphologically and in cul- ture to the typhoid-bacillus — so-called pseudotyphoid- bacilli. These, however, sometimes yield the indol-reaction and sometimes not. A considerable number of these bac- teria also develop in the mixed cultures, and a number even much better than the specific typhoid-bacilli. A de- cision as to the presence of typhoid-bacilli in water may TYPHOID FEVER. 179 therefore be finally reached only when, after comparison with an unequivocal pure culture (from the spleen of a typhoid patient), all doubt has been removed that both the culture obtained from the water and the earlier pure culture agree in every detail, and with regard to both Pfeiffer's and Gruber's reactions. Notwithstanding these difficulties, typhoid-bacilli have been demonstrated in drinking-water in several instances by competent observers. The prophylaxis of typhoid fever, in accordance with what has already been said, consists especially in the anti- sepsis of the sick-room. The feces and the urine from typhoid patients, all materials contaminated by these dis- charges (body-clothing and bed-clothing, etc.), in fact, everything that has come in contact with the patient, must be most thoroughly disinfected, as the adherent bacilli may constitute the source of new infections. Methods of dis- infection are described in the Appendix. In the second place, the prophylaxis concerns itself prin- cipally with the hygienic relations of the drinking-water^ which must be boiled before being used whenever suspicion of contamination exists in times of epidemics. Immunity and Cure. — Typhoid fever is one of those dis- eases, as shown by clinical experience, that attack the same individual but once. Two attacks have occurred in the same individual in about two per cent, of all the cases ; the occurrence of three attacks in the same individual has, according to a recent report, been observed only five times, and four attacks in the same individual but once. It may, therefore, be concluded that recovery from an attack of typhoid fever confers a certain degree of immunity. Sup- port for this view is found in the fact that the blood-serum of some individuals who have recovered from typhoid fever exhibits immunizing properties with relation to the disease induced experimentally in animals with typhoid-bacilli. The immunization of animals to typhoid-bacilli is readily effected. Bouillon-cultures heated to a temperature of 60° C. (140° R), or agar-cultures exposed to a temperature of from 54° C. (129.2° F.) to 56° C. (132.8° F.) have been employed for this purpose, and also the filtrate of unheated virulent cultures, or of cultures in thymus-bouillon.* The simplest ■'^ According to a recent communication, Buchner and Hahn obtained im- munity with the cell -juice of typhoid-bacteria rubbed up and expressed by the method of E. Buchner (p. 32). (The plasmatic cell-juices of the bacteria 180 CLINICAL BACTERIOLOGY. method of immunization consists in the employment of the ordinary unheated and unfiltered bouillon-culture. Many experimental animals possess a considerable degree of immunity to the typhoid-bacillus, and there is no great dif- ficulty in increasing this. The animal is treated once or twice with an intraperitoneal injection of half that amount of bouillon-culture that is just necessary to cause death. After from three to five days the animal will be able to withstand this previously lethal amount, and after several days, one and a half times, then twice, thrice, etc., this dose can be injected. In this way a high degree of immu- nity can rapidly be induced. The blood-serum of the immunized animals is in turn capable of conferring immu- nity upon untreated animals. As R, Pfeififer and his pupils assume, the blood-serum of typhoid convalescents and of animals immune to typhoid fever does not contain antitoxic, but only lysogenic pro- tective substances (p. 62) — that is, the serum, injected into the peritoneal cavity of guinea-pigs simultaneously with living typhoid-bacilli, causes dissolution of the specific microorganisms. If two milligrams of a fresh, virulent agar-culture that has been sterilized by exposure for several hours in the thermostat at a temperature of 56° C. (132.8° F.) are injected into human beings, after a brief period of indisposition the blood-serum of such persons likewise possesses lysogenic properties. The strength of the lyso- genicity attains the same degree as is present in the typhoid convalescent, and the standard of the serum equals about 0.0 1 (p. 63). If the appearance of the specific bactericidal (lysogenic) substances in the blood of individuals that have suffered from typhoid fever is really, as Pfeiffer assumes, the essential cause of immunity, then, according to the experi- ments just described, it must also be possible, by means of prophylactic injections of minimal amounts of the dead bodies of typhoid-bacilli, to induce immunity of like degree and duration. The decision of this most important ques- tion must await the results of further investigation. Attempts at cure with protective serum or with milk ob- tained from an immunized animal have been made only on a small scale, and distinct success has not as yet been observed. This is in harmony with the view alrea'dy expressed that are designated plasmins, and the expressed juice of typhoid-bacilli correspond- ingly as typhoplasmin.) ASIATIC CHOLERA. 181 the serum of those immune to typhoid fever does not possess antitoxic properties. The therapeutic experiments with typhoid-cultures exposed to a temperature of 60° C. (140° F.) that have thus far been made upon human beings have also failed. This want of success can not be opposed to the possibility already suggested of prophylactic immuni- zation by means of dead bacilli, inasmuch as the already diseased and poisoned organism may react differently than the healthy body to injections of typhoid poison. ASIATIC CHOLERA. The exciting agent of Asiatic cholera is the comma- bacillus discovered by Koch in 1883. The cholera-bacilli are more or less markedly curved rods (vibrios) , from one-half to at most two-thirds as large as tubercle- bacilli (from 0.8 to 2 /a), although thicker than these. The comma-form is not well defined in all bacilli. Every preparation contains forms, especially the quite young bacilli, that appear as simple, straight, extended bacilli. The most characteristic and typical commas are present in freshly made artificial cultures. Besides, the form of the growing vibrios varies in accordance with their source in one or another epidemic. In certain epidemics the cholera-bacteria assume throughout an almost straight form. Frequently the comma-bacilli are arranged in pairs ; when two commas are so applied to each other that the corresponding curves are opposed, the so-called S-form results. If in the growth of the vibrios the individual newly formed bacteria adhere to one another after division, the so-called cholera-spirilla result. Though observed very seldom in the dejections of cholera-patients, the spirilla occur frequently in artificial cultures, especially when these have become old, the nutrient material exhausted, or if an antiseptic in dilute con- centration (as, for instance, alcohol) has been added. The spirilla occur with especial frequency in the peritonitic exudate of guinea-pigs inoculated with cholera-bacilli. The spirilla are generally looked upon as involution -forms, principally because they are thicker in cultures than the young individual commas. The cholera-vibrios exhibit extraordinarily active motility. This is especially noticeable in the hanging drop, in which their ap- pearance suggests a swarm of dancing gnats. This motility is dependent upon the presence of terminal flagella that can be readily demonstrated at one extremity by means of Loffler's method of staining. 182 CLINICAL BACTERIOLOGY. The comma-bacillus does not possess spores. The arthro- spore-formation that Hiippe assumed to exist as a result of his earlier investigations has not been confirmed by other ob- servers. The comma-bacilli are most readily stained with a saturated aqueous solution of fuchsin or with carbolfuchsin. The expo- sure to the stain should be longer than usual. The bacilli do not stain by Gram's method. Cholera-bacteria grow upon all of the usual nutrient media, and also in the absence of oxygen (facultative anaerobiosis), al- though, according to recent investigations, it appears that oxygen can never be entirely wanting. The cholera-bacilli always require a distinctly alkaline nutrient medium for their development, as they are exceedingly sensitive to the presence of even slight v.. ^ >l -^S , -i'-^d? Fig. 49.— Comma-bacilli (from the mouth) ; X 1000 (Giinther). amounts of acid. The degree of alkalinity most suitable for the cultivation of cholera-bacilli is secured by adding one gram of crystallized sodium carbonate to loo cu. cm. of carefully neu- tralized gelatin (Dahmen), or by preparing a 10.6 per cent, solution of soda (from calcined sodium carbonate) and adding 55 cu. cm. of this to one liter of gelatin (Fliigge). For ordi- nary purposes simple determination of the alkalinity by means of litmus-paper is sufficient, but the paper must be made dis- tinctly blue. The temperature-minimum for cholera-cultures is 8° C. (46,4° F.) ; the temperature-optimum from 30° C. (86° F. ) to 40° C. (104° F.). Appearances of Comma-bacilli in Cultures. — Gelatin- plates. — The plates are best permitted to develop at 22° C. ASIATIC CHOLERA. 183 (71.6° F.) — a temperature at which the gelatin still retains its solid consistence. After from twenty-four to thirty hours the colonies appear, on microscopic examination, as small, whitish- yellow dots, with an irregular, rough margin. Their contents are coarsely granular. After a time the granules become glis- tening, so that the colonies appear as if they had been strewn with small bits of glass. With the further development of the cul- ture the gelatin undergoes liquefaction, and this occurs the more rapidly the more favorable the temperature and the more nearly the degree of alkalinity approximates its optimum. At the be- ginning liquefaction progresses quite slowly; small, funnel-shaped depressions constitute the first peculiarity visible to the naked eye. Viewed with oblique light, the plate appears as if it has been punctured superficially with a fine needle. This beginning lique- faction is characterized by a bright boundary surrounding the in- dividual colonies, as viewed with low powers of the microscope. The colonies have now become somewhat darker and opaque, and their irregular margin is not rarely marked by fine, pointed processes. Later the liquefaction becomes more active, the funnel-shaped depressions become larger, and the colonies sink to the bottom of the depressions. The border surrounding the colony (area of liquefaction) is no longer bright, but filled with small, grayish masses. These consist of groups of bacteria that have become detached from the colony and admixed with the liquefied gelatin. The colony itself, a brown, irregular mass, lies at the bottom of the funnel. In order to bring it clearly into view, the tube of the microscope must be pushed downward. In employing gelatin prepared according to the method of Forster (p. 81) the plates may be exposed to a tem- perature of 25° C. (77° F.) or 26° C. (78.8° F.). Under these circumstances growth takes place much more actively, and the peculiarities of the colonies described appear much more quickly. Gelatin Stab-culture. — Growth takes place along the entire line of inoculation in the form of a white thread that grows thinner downward. After from twenty-four to forty-eight hours liquefaction slowly sets in in the upper portions, and here also leads to the formation of a funnel. This is naturally more ex- tensive than the funnel of liquefaction of the individual colonies in plates. Liquefaction takes place so slowly that at first the fluid formed has time to undergo evaporation. The upper por- tion of the liquefaction-funnel is, therefore, empty, and an ap- pearance is created as if the puncture of inoculation contained an air-bubble. The line of inoculation itself appears in slight degree liquefied, and, in consequence, somewhat enlarged. Its lower portion contains the bacterial masses that have gravi- tated thither, and which here assume the form of a spirally 184 CLINICAL BACTERIOLOGY. wound thread. In the further course of the growth — not before several weeks — the gelatin is completely liquefied. On agar-plates growth is not so characteristic as in gelatin- plates. The superficial colonies present a peculiar, light gray- ish-brown, transparent appearance. Agar streak- cultures exhibit a grayish-white, moist, glistening coating. Blood- serum is gradually liquefied. Potatoes. — In spite of the usually acid reaction of potatoes the cholera-vibrios generally thrive upon this culture-medium, but only at temperatures above 21° C. (69.8° F.). They then form a grayish or grayish-brown, thin, translucent coating. Upon some varieties of potatoes, however, the comma-bacilli do not thrive, but they can readily be cultivated if the potatoes are rendered slightly alkaline by means of a solution of soda, or if they are boiled in a three per cent, solution of sodium chlorid. Milk is coagulated by cholera-bacteria obtained in certain epidemics, but not by others. The latter appears to be the rule. Bouillon is rendered turbid, and in the majority of cases a superficial membrane forms at body-heat. Like the majority of vibrios and spirilla, the cholera-vibrios also possess the prop- erty of multiplying with especial activity in quite dilute bouillon (from six to eight times). A one per cent, aqueous solution of peptone, with addition of one-half per cent, sodium chlorid, also favors the development of the comma-bacilli. If the pep- tone is not alkaline originally, the last-mentioned nutrient medium must be rendered so by addition of soda. Cholera-red Reaction. — If a few drops of pure dilute hydro- chloric acid or sulphuric acid are added to cholera-cultures that have grown in nutritive media containing peptone, a rose-red or purple-red coloration appears within a short time. Bouillon- cultures yield this reaction after exposure for twelve hours in the thermostat. This so-called cholera-red reaction is nothing more than an ordinary nitroso-indol reaction. The comma- bacilli possess the property of forming indol and of converting into nitrites the nitrates always present, at least in small amount, in nutrient solutions. In addition to the comma-bacilli, there are other vibrios that likewise yield the nitroso-indol reaction : as, for instance, the vibrio Metschnikoff, and the vibrio bero- linensis found in the water-supply of Berlin. (See water-bac- teria. Appendix.) The Finkler-Prior bacillus and the Denecke cheese-bacillus also form indol, but no nitrite, so that addition of pure acids free from nitrous acid does not cause a red colora- tion with them. The one per cent, aqueous solution of peptone and sodium chlorid previously mentioned is especially suited for the formation of cholera-red. The reaction is at times want- ing in bouillon when too much or too little nitrate is present. ASIATIC CHOLERA. 185 Tenacity of the Cholera- vibrios. — The comma-bacilli are endowed with extremely little resistance. They can be destroyed in four minutes in ivater at a temperature of 52° C. (125.6° F.). Lower temperatures are, however, better borne, although the bacilli die in ice after the lapse of a few days. The susceptibility of comma-bacilli to small amounts of acids has already been mentioned. The ad- dition of not more than 0.07 or 0.08 per cent, of hydro- chloric or nitric acid to neutral culture-media is capable of preventing all development. These facts explain why the normal gastric juice, with its hydrochloric-acid content of about 0.2 per cent., constitutes an insurmountable obstacle to the cholera-vibrios. • If cholera-bacilli are spread in a thin layer upon a basis of any sort so that they are ren- dered completely adherent and dry, they lose their capa- bility of development in the course of three hours. Dried upon the hand, they survive only for one or two hours. The same rapid destruction (in any event within twenty -four hours) takes place in connection with the contamination of smooth swfaces — such as floors, paper, etc. — with comma- bacilli. From these considerations it may be concluded that the transmission of cholera by way of the air, through the intermediation of dried particles of dust, is scarcely possible. Surrounded by moisture the comma-bacilli may, under favorable conditions, retain their vitality for a long time, up to nine months — as, for instance, in moist linen rolled together compactly and kept in a cool place, and containing the vibrios in pure culture, so that they are not overgrown by other bacteria. They may be found alive in agar-cultures and gelatin-cultures after the lapse of six months. The usual antiseptics, even in feeble concentration, destroy the cholera-vibrios within a short time : one-half per cent, carbolic acid, for instance, in the course of a few minutes. In fresh milk the comma-bacilli retain their vitality for twenty-four hours ; in boiled milk, for two or three days ; upon articles of food protected from evapora- tion under a glass jar, for from four to eight days. The bacilli sometimes survive for weeks in the dejections of cholera-patients, although this occurs only under peculiarly favorable conditions. In sterilized water, from whatever source, living cholera-vibrios can be demonstrated after the lapse of months. In unsterilized water, on the other hand, and especially in bacterial mixtures, in which the comma- 186 CLINICAL BACTERIOLOGY. bacilli enter into contest with other microbes, the conditions vary in accordance with the external temperature and the amount of sodium chlorid present in the fluid in question. The high temperature of summer and increase in the amount of sodium chlorid favor the development of cholera-bacilli, while at low atmospheric temperatures, or in the presence of a small amount of sodium chlorid, they are quickly over- grown by the associated microorganisms. If cholera- dejections gain entrance into streams, the bacilli mostly adhere to the particles of mucus, which they utilize as a nutrient medium. They are thus often removed from the influence of the current and the competition of other bac- teria, and they not rarely maintain their vitality in streams for a long time in spite of the unfavorable temperature and the process of self-purification. The Occurrence of Cholera- vibrios. — Comma-bacilli are found constantly in all cases of Asiatic cholera, and in innumerable amount ; in part, in pure culture in the liquid intestinal contents ; further, in the intestinal walls of patients'; and only exceptionally in other organs. The vibrios can be demonstrated in the feces on an average to the tenth day after the inception of the disease ; not rarely, however, for a longer time, and sometimes after the termination of the disease (from the forty-sixth to the forty- eighth day of convalescence). Comma-bacilli have never been demonstrated in association with other diseases. Dur- ing recent epidemics true cholera-bacilli were found repeat- edly in the diarrheal stools of patients whose disease clini- cally pursued a mild course and apparently bore no relation to Asiatic cholera. It is possible, however, that these cases represented the mildest grades of cholera, caused by bac- teria of low degrees of virulence. Further, comma-bacilli were found in some cases in the solid stools of healthy per- sons subjected to bacteriologic examination only because their relations brought them into the environment of cholera-patients, or because they had drunk suspected water, but who presented no symptoms of disease whatever. It is probable that in these cases there existed a natural immunity that permitted the passage of the vibrios through the intestinal canal without injury. Outside the human body the cholera-bacillus of Koch has been found in stagnant water in India, where cholera is epidemic. In recent epidemics of cholera comma-bacilli ASIATIC CHOLERA. 187 were repeatedly demonstrated in the water-supply through conduits, in the water of improperly situated irrigation-fields (Nietleben), in the water of streams, and in the bilge-water of ships. The waters in question all bore some close relation to cholera-foci, having been contaminated by the dejections of the first cases of the disease introduced, so that in this way they were capable of constituting the source for the further spread of the disease. These facts demonstrate that the cholera-bacillus may pass a saprophytic existence. Forms of comma-bacilli have, further, been cultivated from the Spree, the Elbe, the Danube, and the Seine, that, in their microscopic and cultural appearances, closely agreed with the vibrio of Koch, but whose relation to existing cases of cholera was not so easily demonstrable. These water- vibrios are in part to be distinguished from the bacillus of Koch by the absence of pathogenicity ; others, however, are toxic for animals. Thus, virulent vibrios were found almost regularly in the contents of Paris sewers in the summer of 1892, although at the time there was not a sin- gle case of cholera in Paris. All of these bacteria, however, exhibit differences as compared with the vibrio of Koch, even though they are often slight and difficult of dem- onstration. A bacterium absolutely identical with the cholera-bacillus, but in its source without any relation to cholera, has never been found. There are two methods of making with certainty the differential diagnosis between the true exciting agent of cholera and the vibrios resembling it. These are the reac- tions of Pfeiffer and Gruber (pp. 62 and 63). In the per- formance of Pfeiffer's test the blood-serum of guinea-pigs or of other animals that have been highly immunized to cholera is diluted with ordinary bouillon in the proportion of I : TOO. To one cubic centimeter of this mixture are added about two milligrams of the vibrios to be examined, caught up with a platinum loop, and the whole is injected into the abdominal cavity of a young guinea-pig weighing about 200 grams. By means of delicate capillary glass tubes specimens of the peritoneal exudate that at once forms are removed at intervals of five minutes and examined, both stained and unstained. If the suspected organisms are true cholera-vibrios, the bacilli are shortly seen to become immobile, later collected in small spherules, and finally, within twenty minutes, completely dissolved. If, however, 188 CLINICAL BACTERIOLOGY. this phenomenon does not take place, the vibrio in question is not the cholera-organism. One source of error must, however, always be borne in mind, if Pfeiffer's reaction proves positive : It is possible that the organisms present are attenuated, to a certain degree saprophytic, vibrios, that are dissolved in consequence of the normal bactericidal activity of the organism of the guinea-pig, in the absence of any specific serum. To avoid this error, a platinum loopful of the culture in question added to one cubic centi- meter of normal serum-bouillon mixture (i : lOo) is in- jected into the peritoneal cavity of a control guinea-pig. If, after the lapse of twenty minutes, the vibrios are found living and motile in the control-animal, whereas those treated with cholera-serum are destroyed, then the diagnosis of Asiatic cholera may be made with certainty. The second test, that of Gruber, is more readily per- formed, as experiment on an animal is not required. The suspected vibrios* may be added to the serum of an animal rendered immune to cholera in the proportions of I to 50, I to 100, and above ; and the mixture is studied at once with high powers of the microscope. If the vibrios lose their motility ; if they collect in groups ; if, thus, agglutination takes place — then the suspected microorganisms are true cholera-vibrios. One of the macroscopic agglutination-tests (p. 64) may be made — e.g., bouillon is inoculated with the suspected vibrios and the serum of an animal immune to cholera is added in the pro- portion named. If, after the lapse of from sixteen to twenty-four hours, the vibrios have formed a flocculent pre- cipitate at the bottom of the test-tube, while the overlying fluid has become clear, then again the suspected organisms are true cholera-bacilli. Gruber's reaction has the advan- tage of being independent of the virulence of the micro- organisms, and, besides, it renders unnecessary the induc- tion of a high degree of immunity in the animals yielding the blood-serum. The Development of Cholera. — Infection takes place invariably by way of the mouth, the bacilli being taken up with articles of food, and principally with drinking-water. Infection through the air is possible only in the immediate neighborhood of the source of infection. The rapid death of the germs on drying renders improbable the transmission of the disease for long distances by means of dust, etc. ASIATIC CHOLERA. 189 The inhaled bacilli must also be restrained in the mouth, as infection does not take place through the lungs. From the mouth the vibrios gain entrance into the stomach. If the hydrochloric-acid content of the stomach is normal, the vibrios succumb to it. If infection takes place, it may be assumed that either large amounts of the infected water have been drunk, so that some of the bacteria escape the action of the hydrochloric acid, in consequence of the marked dilution of the gastric contents, or that the function of the stomach was impaired from some cause, and the hydrochloric-acid content was subnormal. Having gained entrance into the intestine, the bacteria multiply and give rise to the production of toxins. Mere multiplication of the bacteria in the intestine does not constitute cholera. Such an effect was observed in the well-known experiments of Pettenkofer, without the development of actual cholera. Only when sufficient toxin has been produced to cause injury of 4Jie intestinal mucous membrane and when the toxi7t is absorbed does the disease develop. While the tis- sues and the blood become impoverished in water, the pro- fuse rice-water stools take place, and with them innumer- able bacilli are evacuated. The toxic action is manifested especially in the constitu- tional symptoms (feebleness of heart, decline of temperature, etc.). Cholera-typhoid also is now generally looked upon as an intoxication, and the renal disease complicating cholera likewise depends in part upon the toxic activity of the comma-bacilli, being caused in part by the ischemia re- sulting in consequence of the withdrawal of water. Brieger and Frankel have demonstrated the presence of a toxalbumin in cholera-cultures, but greater significance in experiments on animals has been attached to the poison contained within the bodies of the bacilli themselves, and whose effects have been studied, especially by R. Pfeiffer. If a young agar streak-culture, from twenty to twenty-four hours old, is destroyed by exposure for ten minutes to the action of chloroform, and ten milligrams of the bodies of the vibrios thus destroyed are injected into the peritoneal cavity of a guinea-pig, the animal will die. After the lapse of two hours it becomes relaxed, its temperature falls below 30° C. (86° F.), and death takes place in the course of eight or ten hours, usually amid violent clonic convul- sions. The poison that thus adheres to the bodies of the 190 CLINICAL BACTERIOLOGY. bacteria is of most evanescent nature.* It does not with- stand exposure to temperatures above 60° C. (140° F.), drying and the Hke. If the cultures are boiled for several hours, according to the view of R. Pfeiffer secondary toxic substances are formed that manifest their activity only in much larger amounts. The intoxication, however, pre- sents the clinical features described. This intoxication with the intracellular cholera-poison is remarkable for the rapidity with which it appears, and for the entire absence of any period of incubation, such as is commonly observed in con- nection with the diphtheria-toxin and the tetanus-toxin. The severity of the intoxication depends upon the amount of toxin and the rapidity of absorption. The effects are most quickly manifested when the poison is introduced directly into the blood-stream. Epidemics of cholera arise, according to Koch, from the first imported case, through whose dejections, con- taining the bacilli, the disease is spread. The type of distribution may be of two kinds. The disease may spread in foci : a member in the family of the patient first attacked is seized, then others, then another family in the same house, a neighbor, a stranger accidentally present in the house, the laundress to whom the linen from the first case is sent ; in this way each disease-focus gives rise to another, and all together form a closed chain. In every instance infection has taken place through contamination with the dejections of an earlier case. Often enough the connection of the individual cases with one another is not demonstrable. Thus, for instance, insects may carry the disease-germs great distances and deposit them upon articles of food that seemingly have in no way come in contact with a focus of disease ; or apparently healthy individuals from the neighborhood of the person first attacked may disseminate the germs through their infected dejecta. On the other hand, the outbreak of the disease may take place in an explosive manner, large numbers of cases appear- ing equably and simultaneously throughout a town or a city. This occurs when the water, whether delivered through conduits or obtained from streams, is infected by cholera-dejections, and the germ is thus capable of being equably spread over the entire city, and carried into every * The poison can also be expressed from the bodies of the bacteria accord- ing to the method of E. Buchner, (See foot-note, p. 179.) ASIATIC CHOLERA. 191 household. The cholera-bacillus has never been demon- strated in the air or in the soil, which likewise might be considered as sources of such explosive outbreaks of cholera. The bacillus has been found during recent epidemics in river-water and in conduits. Epidemics of cholera do not always conform strictly to one or the other of the two types described by Koch ; one may be combined with, or pass over into, the other, etc. Individual predisposition, the quantity and quality of food, the density of population, individual and civic cleanliness, distinctly influence the distribution of the disease. The foregoing theories, representing essentially Koch's views, are, in fact, capable of explaining almost all of the manifestations of recent cholera-epidemics. One point only, however, is obscure : namely, the question why in some in- stances an epidemic does not occur. Thus, Paris escaped in 1892, although the bacilli were present in the sewers; and only a few cases occurred in Berlin in the year 1893, although the waterways were known to be infected. It is possible that the energetic intervention on the part of the authorities, the filtration of the water, as well as the careful observance of all hygienic regulations, prevented the irrup- tion of an epidemic. The thought, however, can not be en- tirely put aside that possibly the *' local predisposition " was wanting. According to Pettenkofer, for the occurrence of an epidemic a local and a temporal predisposition are necessary. *' The cholera-germ (x) forms upon the basis of the local and the temporal predisposition of the soil (j) the cholera-poison (^)." According to this view, the dis- ease can never be transmitted from one human being to an- other, but the germ must first mature in the earth, and then the poison is taken up by the lungs. In view of the facts mentioned this theory of Pettenkofer's can scarcely be main- tained any longer as against the bacillus of Koch. A number of epidemiologic facts, however, indicate that, in addition to the comma-bacillus, some other not yet suffi- ciently determined influences ' are necessary for the occur- rence of an epidemic. Experiments upon Animals and Human Beings. — Even though some uncertainty exists with regard to the last point, there can, however, be no further doubt with regard to the etiologic significance of the comma-bacillus as the exciting agent of cholera. The final evidence for this was 192 CLINICAL BACTERIOLOGY. furnished by the experiments upon human beings that were undertaken in part unintentionally (accidental laboratory- infection), in part intentionally. The introduction of the bacilli into the stomach may be unattended with any effect ; at times it gives rise to more or less intense diarrhea (auto- infection of Pettenkofer and Emmerich) ; in other cases — as, for instance, in one reported by Metschnikoff — however, it induces true dangerous cholera, with all its clinical symp- toms. The sad fate of a young Hamburg physician is well known, who died of typical cholera after a drop of peri- toneal exudate containing vibrios had entered his mouth in the performance of Pfeiffer's test. Subcutaneous inocu- lation with cholera-bacilli causes in human beings only moderate local symptoms and fever of brief duration. The blood under these circumstances acquires immunizing prop- erties (G. Klemperer). A disease resembling cholera can be induced in guinea- pigs by direct introduction of the vibrios into the duodenum, by avoidance of the stomach, after ligation of the choledoch duct ; or by introduction of the bacilli into the stomach after previous alkalinization of the gastric contents by means of soda-solution and injection of two or three cubic centi- meters of tincture of opium into the peritoneal cavity. The object of ligating the choledoch duct, as well as of the in- jection of the opium, is to inhibit the peristaltic activity of the intestine. Similar morbid manifestations — cholera-like stools, decline of temperature, etc. — can be induced in guinea-pigs and rabbits by intravenous introduction of the comma-bacilli, especially after previoiis intoxication with alcohol, or by simultaneous injection of comma-bacilli and metabolic products of varieties of proteus. Intraperitoneal injection of virulent cholera-vibrios is fol- lowed in guinea-pigs (a platinum loopful containing about 2.5 mg. of bacterial colonies scraped from the surface of agar-agar for a guinea-pig weighing from 300 to 500 grams) by death, preceded by paralytic phenomena and rapid de- cline of temperature. There takes place under these con- ditions, as has already been stated, an intoxication with the poisons contained within the bodies of the comma-bacillus (p. 189). In the digestive tract of guinea-pigs destroyed by this means special changes are usually not to be observed. At times, the large bowel is markedly injected and the small intestine is not rarely filled with grayish fluid ASIATIC CHOLERA. 193 containing large numbers of comma-bacilli. On the whole, however, it is scarcely possible to insist upon a complete analogy between the intoxication of guinea-pigs and cholera as it appears in human beings. Bacteriologic Diagnosis of Cholera. — If an attack of disease characterized by the occurrence of profuse, rice- water diarrhea, vomiting, decline of temperature, cramps in the calves, etc., arouse suspicion of cholera — a suspicion that must be considered and investigated in every instance, especially in travelers during the summer season, and par- ticularly when cholera exists — bacteriologic examination of the stools is an absolute duty. The procedure in a case suspected to be one of cholera is as follows : 1. Microscopic Examination. — Cover-slip preparations are made with a flake of mucus from the feces, and stained with dilute carbol-fuchsin solution. The diae^nosis of Asiatic cholera is rendered in the highest degree probable if the vibrios form masses " in which the individual bacilli all point in the same direction, so that an appearance is created as if a small swarm of them, somewhat like fish in a slowly flowing stream, are following one another"; or if, " in addition to isolated bacteria presenting the appear- ance of cholera-bacteria, only the bacterium coli is found. " The probable diagnosis of cholera can be made microscop- ically in about 50 per cent, of suspected cases, but in every instance it should be confirmed by cultural investigation. 2. Cidtitral Investigation. — {a) Gelatin-plate Method. — Three gelatin-inoculations are prepared, in the usual man- ner, from the feces, if possible from a flake of mucus, and the inoculated culture-media are poured into three Petri dishes ; these are kept at a temperature of from 22° C. (71.6° F.) to 26° C. (78.8° F.). If upon these plates colonies are found after from fourteen to thirty or forty- eight hours, with uneven, rough borders, strewn with bright granules resembling fragments of glass, and presenting an area of liquefaction, and if these colonies prove to be constituted of commas, a high degree of probability is given the diagnosis, and it is scarcely possible that the dis- ease under consideration is any other than Asiatic cholera. So far as is yet known, there occur in the human intestine no vibrios other than the comma-bacilli that ^w^ rise to colonies presenting the characteristics that have been de- scribed. 13 194 CLINICAL BACTERIOLOGY. (J?) Peptone -culture (Fertilizing Method, SchotteliuSy Koch). — The plate-method, which yields admirable results in the diagnosis of marked cases of Asiatic cholera, does not suffice, however, for other cases in which the dejections contain only a small number of comma-bacilli. This small number of vibrios are overrun on plates by the fecal bac- teria, and do not develop at all. It is, therefore, necessary in every suspected case to adopt, in addition to the original plate-method, a further method of investigation : namely, the preparation of peptone-cultures. It has previously been pointed out (p. 184) that the cholera-vibrios thrive especially well in a simple alkaline solution of peptone and sodium chlorid (one per cent, peptone and one-half per cent, sodium chlorid). It may be added that the vibrios, by reason of their motility and their great need of oxygen, tend toward the surface of the liquid culture- medium, and there undergo enormous multiplication. Upon both of these facts is based the method of peptone- culture, which has the further great advantage that it leads to the desired result much more rapidly than the plate- method. A platinum loopful of the suspected feces, or, if this be obtainable, a flake of mucus, is introduced into a test-tube or an Erlenmeyer flask containing the solution described, and the vessel is exposed in the thermostat to a temperature of 37° C. (98.6° F.). As soon as the fluid exhibits the slightest traces of turbidity, which usually occurs in the course of from six to ten or twelve hours, a specimen is taken from the surface and is examined in hanging drop and in dry cover-slip preparations. If the examination discloses the presence of a pure culture of cholera-vibrios, the diagnosis is almost certain. In most cases, however, the procedure is not quite so simple. In the superficial layer of the peptone-solution the vibrios are usually intermixed with other microorganisms, and most frequently with the bacterium coli commune. There then remains nothing but to make plates from the material on the surface. These plates, however, are made under much more favorable conditions. There is now no longer any danger that the small number of cholera-bacilli that were present originally in the feces will be overrun in their growth. Through the intermediation of the peptone-culture the vibrios have undergone enormous multiplication, and the Petri dishes ASIATIC CHOLERA. 195 now exhibit, in consequence, numerous characteristic colo- nies. From the gelatin-plates, finally, pure cultures are made, with which the cholera-red, and Gruber's and Pfeiffer's reactions are obtained, and experiments on animals are undertaken. When all these yield positive results, then the diagnosis of cholera-bacilli is final and certain. Instead of gelatin-plates, agar-plates frequently are made. These have the advantage that they can be kept in the thermostat at a temperature of 37° C. (98.6° F.), and can be examined after from eight to ten hours. It has already been pointed out that only the superficial colonies on agar present an approximately characteristic, light grayish- brown appearance. It is, therefore, useful, in order to ob- tain only such superficial colonies, to have in readiness agar-agar poured in Petri dishes, upon the surface of which the inoculating material (the fertilized peptone-cul- ture) is smeared by means of a platinum loop. As, how- ever, the agar-agar always expresses a certain amount of water of condensation, which renders impossible the growth of isolated colonies, it is necessary to place dishes into which this culture-medium is poured in the thermostat be- fore they are used, until the fluid has evaporated, or they are preserved in an inverted position. It is not sufficient in making a diagnosis to find light grayish-brown, trans- parent colonies, but by means of preparations it must be decided whether the bacteria constituting the colonies cor- respond also morphologically with cholera-vibrios. Examination of Water for Cholera-bacilli. — The con- ditions are somewhat different if water is to be examined for cholera-bacilli. Formerly it was the custom to dilute freely the usually much polluted water, and to make plates with a portion of a drop. Under these circumstances it was largely a matter of chance whether cholera-germs were obtained or not. It would be necessary for them to be present in the infected water in considerable number, in order to be demonstrated in this way. These difficulties may be avoided by taking large amounts of water for examination. To this end from 100 to 1000 cu. cm. of the suspected water are placed in sterilized flasks, and to each specimen one per cent, of alkaline peptone (preferably the peptone of Witte) and ^ of one per cent, of sodium chlorid are added. The peptone and 196 CLINICAL BACTERIOLOGY. sodium chlorid are kept in readiness in sterilized solutions, and, for instance, 5 cu. cm. and 2.5 cu. cm. of 20 per cent, solutions are added respectively to 100 cu. cm. of water. After testing its alkalinity, the mixture is placed in the thermostat, and it is then treated in exactly the same way as has been described for the peptone -culture. On micro- scopic examination of the surface of the peptone after eight, ten, fifteen, and twenty hours, curved bacteria that closely resemble the comma-bacilli are found in many samples of water. It can not be too strongly emphasized, however, that such a discovery is alone not demonstrative, if the vibrios are derived directly from the feces of a sick human being. In examinations of water it is still further an unavoidable postulate that plates of gelatin or of agar be made with the fertilized material from the surface. If upon these are found colonies that correspond in appearance with cholera- colonies, it is not yet demonstrated that they are true comma-bacilli and not merely similar microbes, of which a number have already been described. It is then necessary to make pure cultures, and these are to be identified by means of the indol-reaction, by experiments on animals, and especially by means of Pfeiffer's and Gruber's reactions (pp. 62,63, 187, 188). Disinfection and Prophylaxis. — Every cholera-patient must be isolated at once. Feces and vomited matters, as well as all materials soiled therewith, should be most thoroughly disinfected. The details of disinfection are given in the Ap- pendix. Those who come in contact with the patient should, further, be watched with care, and their dejections should be examined for comma-bacilli. Personal prophylaxis extends to the maintenance of the digestive tract in a state of health (avoidance of all dietetic errors), and the protection against contamination of all articles of food (only boiled water should be drunk, etc.). The general prophylaxis concerns itself with the purification of the water-supply by means of suit- able filtration, with the protection against contamination of waterways, and, above all, with the prevention of importation of the disease from abroad. The pilgrimages to Mecca are under the scrutiny of the International Sanitary Commission at Alexandria. Cholera prevails frequently among the pilgrims, and upon the slightest suspicion the pilgrim-ships must be subjected to quarantine before being permitted to pass through the Suez Canal. ASIATIC CHOLERA. 197 Immunity. — A large number of persons — according to Koch almost half — are naturally immune to cholera. Re- covery from one attack of the disease confers immunity : at least, second or more attacks have rarely been observed. Places that in one year suffer from a severe visitation of cholera generally remain exempt from the succeeding epidemic. The blood-serum of convalescents from cholera has, in some instances, exhibited a surprising degree of pro- tective influence for guinea-pigs. Thus it was possible to immunize guinea-pigs against otherwise fatal inoculation with cholera-vibrios, in one case by means of o.Oi cu. cm., and in another case by means of 0.025 cu. cm. of serum from human beings that had recovered from cholera (G. Klemp- erer, Lazarus). As Metschnikoff has shown, not the serum of all convalescents from cholera possesses such immu- nizing properties ; while, on the other hand, these may be observed occasionally also in the blood of persons that have died of cholera. Guinea-pigs, rabbits, and goats can be readily immunized to intraperitoneal and subcutaneous infection with comma- bacilli by means of cultures that have been heated to a tem- perature of from 54° C. (129.2° F.) to 60° C. (140° R), or have been attenuated by any other means. All of the methods of protective inoculation hitherto employed have, however, been efficient only with relation to intraperitoneal or subcutaneous inoculation, not affording certain protec- tion against introduction of the vibrios by the mouth. The blood-serum of immunized animals is, in turn, capable of conferring immunity. In the process of preliminary treatment the bodily fluids of the animals acquire specific bactericidal (lysogenic) properties, and the immunity to in- fection with cholera-bacilli appears to depend exclusively upon these bactericidal influences (p. 62). According to the view of R. Pfeiffer, the blood-serum of convalescents from cholera likewise contains no antitoxin, but, on the other hand, the same lysogenic substances as the serum of animals immunized artificially. R. Pfeiffer, in consequence, maintains the view also for Asiatic cholera in human beings that the immunity depends upon specific bactericidal influences (lysogenicity). It is possible in human beings to stimulate artificially the formation of the lysogenic cholera anti-bodies in the blood. It is only necessary to vaccinate the individuals in question accord- 198 CLINICAL BACTERIOLOGY. ing to the method first described by Haffkine. This ob- server injects first -^-^ of an agar-culture, twenty-four hours old, carefully devitalized by chloroform, and five days later -^^ of a living virulent culture, and, again, after the lapse of five days, yi of the last. Each inoculation is fol- lowed by an insignificant, slight, painful infiltration and mild fever, which, however, soon recede. The serum of persons thus inoculated acquires, as Kolle has shown, lysogenic properties at least equal to those of the serum of convalescents from cholera. This bactericidal activity be- gins on the fifth day, attains its maximum on the twentieth day, and then gradually diminishes. It is, however, still demonstrable after the lapse of a year. Kolle has shown, further, by experiments on human beings, that a single introduction of living or carefully devitalized vibrios is at- tended with the same success, so that repeated inoculation is not at all necessary to attain marked bactericidal activity. Little of a positive nature can yet be said with regard to the results of Hafifkine's protective inoculations ; but they are at least quite encouraging. CHOLERA NOSTRAS AND SUMMER DIARRHEA. Cholera nostras includes all of those cases of severe diarrhea presenting symptoms similar to those of Asiatic cholera, but with an absence of the comma-bacilli of Koch from the feces, and without epidemic distribution. In by far the larger number of cases the bacterium coli commune is found in the stools and in the vomited matters. Under these conditions the bacterium cpli manifests a considerable degree of virulence ; and in cultures from a case of cholera nostras it proves far more malignant in experiments on animals than in cultures from normal feces. In severe cases of cholera nostras the bacterium coli is often present in the dejections in pure culture. In some cases strepto- cocci have been found in the feces in overwhelming number, or in pure culture, and in isolated instances also vibrios presenting a superficial resemblance to Koch's bacilli, with- out, however, agreeing perfectly with these — as, for instance, the vibrio Lisbon, in Appendix. As the necessity for bacteriologic examination in cases of cholera nostras arises only in the presence of suspicious CHOLERA NOSTRAS AND SUMMER DIARRHEA. 199 conditions, when the point to be determined is whether the disease in question is true Asiatic cholera or not, the mode of procedure must in every instance be precisely that which has been described in the preceding chapter for Asiatic cholera. Closely related to cholera nostras is the so-called sum- mer diarrhea of children. The probable cause of this condition is thought to be the so-called bacteria of bitter milk, which are known under the collective name of bacillus lactis Fliigge. Fliigge has isolated twelve varieties of this organism, all of which belong to the group of the hay- bacillus, and with which they have many points of resem- blance in common. (See Hay-bacillus and Potato-bacillus in Appendix.) These microorganisms cause peptonization of the casein of milk — hence the designation peptonizing milk-bacteria — in consequence of which the milk acquires a bitter taste. Some of them give rise to toxic metabolic products, which, when fed to young dogs, cause diarrhea, muscular weakness, and decline of temperature. The whole group of these milk-bacteria is characterized by the formation of highly resistant spores that withstand boiling for several hours without injury. It is on this account that the sterilization of milk is attended with so much difficulty ; even the method of Soxhlet is incapable of destroying the peptonizing microorganisms. If such an insufficiently or, to use the common expression, partially sterilized milk is pre- served at a moderate temperature — as, for instance, 22° C. (71.6° F.) or above — the spores develop, and the poisons mentioned are generated. For this reason great care must be taken that milk prepared by the Soxhlet method is kept in a cool place until shortly before it is used, and in summer best in a refrigerator. The custom of sending with children a supply of milk for a considerable time, heated and kept in special warming bags, should be aban- doned, as in the presence of numerous spores germination and beginning multiplication may set in speedily (in the course of one or two hours). These, however, are to be considered as dangerous, as, according to the investigations of Liibbert, *' the active principle of the decomposed milk is to be looked for in the bodies of the bacilli." The anaerobic microorganisms to be found in milk — the bacillus butyricus Botkin and that of Fliigge — probably play no part in the etiology of the intestinal catarrh of infants. 200 CLINICAL BACTERIOLOGY. They occur only in small number and change the appear- ance and the odor of the milk to such a degree that it will probably not be used. PLAGUE. The exciting agent of plague was discovered by Kitasato and Yersin during an epidemic of cholera in China in the year 1894. Morphology. — The plague-bacillus is a small, nonmotile rod, with rounded extremities. It is believed to possess a capsule, which, however, is not readily demonstrated. Plague-bacilli vary extraordinarily in form, both in cultures and in the products of the disease in human beings and animals. At Fig. 50. — Bacillus of bubonic plague (Yersin). times they appear as short, compact bacteria, at other times they are suggestive of diplococci, distinct rods, short, sharply bent threads, and, finally, in older cultures, degeneration-products occur in the form of swollen spheres and clubs. The plague- bacillus does not stain by Gram's method, but, on the other hand, readily with all aniline dyes, and most strikingly with methylene-blue. Under these circumstances it exhibits distinct polar staining. The extremities are more deeply stained than the central portion, which after feeble action of the staining solution appears to be a deficiency. Spores are not present. The temperature-optimum is that of the body, although the plague-bacillus thrives well at room-temperature. PLAGUE. 201 Appearance in Culture. — On gelatin-plates the colonies appear as finely granular bodies of brownish color, with a smooth border. The superficial colonies possess a delicate mar- ginal zone. The culture-medium is not liquefied. In gelatin stab-cultures slow , uniform growth takes place along the line of inoculation, while a smooth deposit forms upon the surface. In gelatin streak-cultures a light yellowish deposit forms. On agar-plates, after twenty-four hours, delicate, dewdrop-like colonies form that appear, after the lapse of forty-eight hours, as grayish points, whose border is slightly iridescent. At times a number of large colonies are seen among the smaller. In agar stab-cultures a tough, mucoid coating develops, the water of condensation is rendered turbid, but no membrane forms. In Loffler' s blood-serum the same development takes place as in agar streak-cultures. Bouillon is rendered diffusely turbid. If, however, it be in- oculated with a coherent bacterial mass from an agar-culture, the bacilli develop at the bottom of the tube, while the over- lying fluid remains clear. In this way a growth is obtained that is suggestive of streptococci. Milk is a poor culture-medium, and is not coagulated. Upon potatoes a scanty, whitish-gray coating forms at a tem- perature of 37° C. (98.6° F.). In culture-media containing sugar the plague-bacillus does not generate gas. It does not form indol either in bouillon or in peptone-water. Wladimiroff and Kressling believe a neutral reaction of the culture-medium most favorable for growth. Addition of glycerin to the nutrient medium is rather disadvantageous. Vital Capability of Plague-bacilli. — Exposure for ten minutes to a temperature of 55° C. (131° F. ) or for five min- utes to a temperature of 80° C. (176° F.) suffices to cause the death of the bacilli. They are destroyed at once by a i : 1000 solution of mercuric chlorid, and in ten minutes by one per cent, carbolic acid or one per cent, lysol. Mineral acids are very active : Sulphuric acid, i : 2000, destroys the bacilli within five minutes ; hydrochloric acid, i : 1000, within thirty min- utes. When material containing plague-bacilli was transferred to linen, wool, earth, etc., the longest period for which life was preserved was eight days. The same observation was made with regard to preserved portions of organs. The sputum from cases of pneumonia complicating plague, kept in tubes closed with cotton, was no longer infective after sixteen days. In or- dinary tap-water the bacilli die in three days, in sterilized water in eight days, in sterilized bilge-water after five days (German Plague-commission). 202 CLINICAL BACTERIOLOGY. Portals of Infection for, and Mode of Distribution of, Plague-bacilli. — The plague-bacillus gains entrance into the human organism most frequently by way of slight injuries, scratch-wounds, etc. Infection through the skin may take place simultaneously at several different points of the body in the same case ; it has not yet been decided whether it can occur through the intermediation of insects. The related lymph-glands nearest to the portal of infection become swollen, most frequently those of the inguinal and axillary regions (primary plague-boils). Sometimes the glands of the first degree remain free or are but slightly irritated, and only those of the second and third degrees are seriously affected. Sometimes the entire chain of glands from the nearest to the most remote are intensely inflamed. At the site of infection a pustule or a carbuncle is not rarely found, and between this and the related lymphatic enlargement frequently a distinct lymphangitis can be traced. In the milder cases the buboes disappear. If, however, the gland- filter is broken through, the plague-bacilli gain entrance through the glands into the blood and the internal organs, and the condition of plague-septicemia, which almost always terminates fatally, is established. If the buboes undergo suppuration, the plague-bacilli usually die quickly. Some- times, however, secondary infection with streptococci is superadded, and this then constitutes a serious menace to the patient. In the septicemic form of the disease the bacilli gain entrance into the feces and the urine in consequence of the hemorrhages that occur so frequently. A second and less common portal of infection is consti- tuted by the lungs. Bronchopneumonic foci occur, in which the exciting agents of plague are found in pure cul- ture or in association with diplococci and streptococci. "The sputum of such patients contains the plague-bacillus. Finally, cases of primary infection through the tonsils have . been observed, rapidly giving rise to general infec- tion. The German Commission that went to Bombay in the beginning of the year 1897 for the study of plague, and from whose report the foregoing statements are taken, em- phasized that the incredible filth amid which the natives live, their crowding together in small dwellings, the frequent small injuries, especially of the bare feet, the constant scratching induced by vermin, are quite sufficient to explain PLAGUE. 203 the frightful frequency with which plague prevails among the lower classes in Bombay. Plague adheres obstinately to human habitations. It does not extend in an explosive manner over large portions of the same city, but passes from house to house. In addi- tion to human intercourse, an important part is played by the remarkable relations that exist between rats and simi- lar vermin and bubonic plague, as was already recognized in the middle ages. From numerous sources the German Plague-commission was informed that the outbreak of the epidemic was preceded by a pestilential disease among rats, with an enormous mortality. The natives believe so firmly in the connection between the plague of rats and that of human beings that some of them will at once leave their houses if they find a dead rat present. After great effort the German Plague-commission succeeded in obtaining the fresh cadaver of a rat that had been infected, and in it they found numerous plague -bacilli.* Yersin has made the note- worthy statement that he found the plague-bacillus in the dust and the filth of plague-houses. Experiments on Animals. — The animal most suscepti- ble to plague is the rat. Minimal amounts of a culture sufifice on cutaneous inoculation to cause death regularly. The same result may be attained by application of the plague-bacilli to the mucous membrane of the eye or of the nose. Rats die after eating quite small amounts of plague- infected food, or after gnawing the bodies of other rats dead of plague. The latter circumstance especially is of great im- portance. It explains the incredibly rapid spread of rat- plague, and also renders it probable that rats are responsible for the extension of the disease from house to house. Next to the rat in susceptibility to the plague is the gray ape. Plague-bacilli are pathogenic for the usual labora- tory-animals in general ; pigeons alone form an exception. At the autopsy a mucous, at times hemorrhagic, exudate is found at the point of injection. The nearest glands are most enlarged, and they undergo suppuration but seldom. The blood and the internal organs contain the bacilli, the former, however, in but small amount. Kolle calls atten- tion to a more protracted course for the disease, which takes place if rats and guinea-pigs are inoculated with cul- * It is believed that squirrels and monkeys also may be infected by plague and act as disseminators of the disease. — A. A. E. 204 CLINICAL BACTERIOLOGY. tures that have already been carried on artificially for sev- eral years. The animals then die in the course of the second week, and one or more glands, up to the size of a walnut, are found, which contain creamy pus in their cen- tral portions, in which plague-bacilli are present in large numbers. An involuntary experiment in the human being is also on record : A member of the German Plague-com- mission suffered infection in making an autopsy of a body dead of plague. Two days later a small pustule formed on the right hand, followed shortly by a lymphangitis, and a swollen gland in the axilla. The discharge from the pus- tule contained plague-bacilli. In spite of the alarming character of the disease at first, recovery took place. Etiologic Relations of the Bacilli to Plague. — The con- stant presence in all cases of plague and the positive out- come of experiments on animals indicate with certainty that the plague-bacillus is the specific exciting agent of plague. In accordance with what has been said, the disease is to be looked upon rather as an infectious disease ; but a toxic element is by no means wanting. The hyperemia and the ecchymoses of the stomach and the bowel are considered by the German Plague-commission as purely toxic effects. As an evidence of the toxic action, the case is reported of a fetus born on the third day of the disease in the mother, in which the characteristic hemorrhages were found, al- though all portions of the body were free from germs. Certain sequelae also are to be attributed to the toxic effects : paralysis of the recurrent laryngeal nerve, amauro- sis, aphasia, deafness, paraplegia, etc. The poison of the plague-bacilli is probably contained within the bodies of the bacteria. The German Commission dried cultures carefully, mixed them with water, and heated them to a temperature of 65° C. (149° F.). As much as eighty milligrams of the dry residue were introduced into the peritoneal cavity of brown apes, which exhibited only mild manifestations of disease, slight decline of temperature, want of energy, and anorexia. In brown apes, which are by no means so susceptible to plague as gray apes, this toxic effect is, therefore, but little marked. Bacteriologic Diagnosis of Plague. — The suppurating plague-buboes contain the characteristic bacteria in enor- mous number. These are readily recognized by their polar staining when treated with methylene-blue. A similar PLAGUE. 205 phenomenon is exhibited among the pathogenic micro- organisms only by the bacilli of chicken-cholera. These, however, are larger than the plague-bacilli, and are of absolutely no significance with relation to human beings. If suppuration has not yet taken place, the diagnosis is not so simple, although it appears most necessary just under these conditions. Puncture of the buboes for this purpose was considered by the Commission in its first communication as not unattended with danger on account of the possibility of opening a blood-vessel. It has, however, since been shown that English physicians in plague-hospitals made long incisions into the diseased glands, with subsequent antiseptic treatment, for therapeutic purposes. In connec- tion with this procedure fluid from the gland is readily obtained for the purpose of making cover-slip preparations, plates, and cultures. Microscopic examination of the blood yields successful results only in cases of general infection. Cultural investi- gation of the blood, however, yields better results. In order to cultivate plague-bacilli from sputum, and, in general, from bacterial mixtures, it is best to make gelatin streak- cultures. The suspected material is spread in several streaks upon the surface of a solidified gelatin-plate. The plague-bacilli grow well at a temperature as low as from 22° C. (71.6° F.) to 25° C. {jj'' F.) ; while the associated bacteria — the diplococcus lanceolatus and the streptococcus — grow but feebly if at all. From a diagnostic point of view it is further of the greatest significance that the blood- serum of human beings and of animals that have recovered from infection with plague possesses agglutinating activity (German Commission). It is said that the serum does not cause agglutination before the second week, and that this is most marked in the second and third weeks. In cases of mixed infection, which occur especially in conjunction with suppuration of the buboes, streptococci appear, not only in the glands, but also in the blood. The prophylaxis of plague consists in disinfection of all products of the disease that contain the specific bacillus. In the first place, general hygienic precautions should be observed, with especial regard to cleanliness and ventilation of dwellings, care of the skin, etc. In general, plague attacks preferably the lower classes of society, ex- posed to want, filth, and misery. Isolation of plague- 206 CLINICAL BACTERIOLOGY. patients and observation of those b^^ whom they are sur- rounded are naturally necessary. Immunity and Protective Inoculation. — Plague-bac- teria retain their virulence until shortly before death. It is, therefore, impossible to immunize susceptible animals with old cultures (German Commission). Slightly susceptible animals can be readily immunized by increasing doses of living bacilli. Highly susceptible animals must, however, be treated carefully with dead cultures. For this purpose it is best to employ cultures that have been exposed for an hour to a temperature of 65° C. (149° F.). In subjecting a brown ape to protective inoculation the Commission em- ployed a heated agar streak-culture. Seven days later the animal was immune to subcutaneous' infection, but only after it had recovered from this was it protected against intra- peritoneal inoculation. Filtered bouillon-cultures possess only slight immunizing properties. Haffkine has employed his method of protective inocu- lation, which he first recommended for cholera, also in the treatment of plague. A fluid preparation, made from care- fully devitalized plague-bacilli, is injected in doses of from ^ to 2j4 cu. cm., in accordance with the age of the individuals. Slight reaction follows, and the injection is repeated in a somewhat larger dose after the lapse of eight or ten days. The results obtained with Haff kine's method are not un- favorable, but a definite decision as to its value must be de- ferred for the present ; at any rate, the protection conferred is not absolute. A plague-serum is prepared in the Pasteur Institute for therapeutic purposes from the blood of highly immunized horses. In the observations of the German Commission, three cubic centimeters of this serum sufficed to protect brown apes against subsequent subcutaneous infection. As much as ten cubic centimeters, however, did not suffice to protect susceptible gray apes. The serum exhibits, also, an undoubtedly curative action (after twelve hours) in pre- viously inoculated brown apes. With regard to its efficacy in cases of plague, nothing definite can as yet be stated. DIPHTHERIA. 207 DIPHTHERIA. The diphtheria-bacillus was grown in pure culture in 1884 by Loffler. The diphthej'ia-bacillus is a rather plump rod of varying size, from I to 6 /^ long and from 0.5 to i ix thick. Its form is sub- ject to great variations in different cultures. At times it appears as a small, wedge-shaped body ; at other times as a rather long body, with a bulbous thickening at one extremity — a so-called club ; and at still other times as a double club, or a dumb-bell. Slender forms, occasionally curved slightly, are also observed, especially in membranes. Not rarely bifurcations are encountered, and upon the basis of this observation diphtheria- c^ ^/-^'^ >>1£ «^' K Fig. 51. — Bacillus diphtlieriae from a pure culture (Stengel). bacilli have been placed in relation with streptothrices, and even with hyphomycetes. The diphtheria-bacillus is incapable of independent movement. Spore -formation is wanting. Staining Properties. — The diphtheria-bacillus is best stained with methylene-blue or dilute carbolfuchsin. Gentian- violet overstains and conceals the more delicate structural rela- tions. The organism is stained by Gram's method. The small, blunt clubs stain equably, while the longer specimens exhibit unstained areas, so that the rods appear in places to consist of several segments. Fliigge has described a method of double staining, devised by M. Neisser, which is considered to possess essential differential diagnostic significance. The preparation is placed for from three to five seconds in an acetic-acid solu- tion of methylene-blue (methylene-blue i, alcohol 20, distilled water 950, glacial acetic acid 50), rinsed in water, and counter- stained for from three to five seconds in vesuvin (vesuvin 2, 208 CLINICAL BACTERIOLOGY. dissolved in boiling distilled water loco, and filtered). The bodies of the bacilli appear brown, and, as a rule, they contain two blue granules, which have at once been intensely stained by the first aniline dye, and have not yielded their color to the vesuvin subsequently employed. These are the so-called Babes- Ernst bodies (p. 19). The form, arrangement, and situation of these bodies are considered as characteristic of the diphtheria- bacillus under the following conditions : The preparations must be made only from cultures that have grown upon Loffler's blood-serum (solidified at 100° C. — 212° F. ) at a temperature of 34° C. (93.2° F. ) or 35° C. (95° F. ), and never above 36° C. (96.8° F.). The cultures must not be less than nine Fig. 52. — Bacillus diphtheriae, from a culture upon blood-serum ; X looo (Frankel and Pfeiffer). hours, and not more than from twenty to twenty-four hours, old. At one end, more frequently at both ends, of the brown rod a blue granule is then to be seen, and not rarely a third is visible in the middle. These granules are oval in shape, and possess a greater diameter than the bacillus itself, which, however, if the whole appearance is to be considered of diagnostic significance, must be distinctly visible in its entire length and form. It is further said to be characteristic of the diphtheria-bacillus that the individual bacteria are arranged side by side like pali- sades. As the most distinctive feature, M. Neisser considers the appearance of impression-preparations from a serum-plate six hours old that has developed at a temperature of from 34° C. (93.2° F.) to 36° C. (96.8° F.). In these there are DIPHTHERIA. 209 visible ** moderate-sized free masses in which the slender, rather long, slightly curved bacilli lie in characteristic irregular ar- rangement— an appearance that is to some degree represented by placing the extended fingers of one hand in varying combi- nations over or by the side of those of the other." Cultural Properties. — The diphtheria-bacillus thrives only at temperatures between 20° C. (68° F.) and 40° C. (104° F.) upon all slightly alkaline culture-media. Its temperature-opti- mum is that of the body. Upon gelatin-plates round, whitish colonies form that remain small. Microscopically these appear yellowish brown and gran- ular, with an irregular border. In gelatin stab-cultures similar small, whitish, spherical colo- nies, not exceeding a certain size, form along the line of inocu- lation. At a temperature of 24° C. (75.2° F. ) superficial growth takes place, with indications of nail-culture. The gela- tin is not liquefied. Upon agar^ and better upon glycerin-agar plates, after from twenty-four to forty-eight hours, small, grayish-white, glistening colonies form that often exhibit microscopically concentric lami- nation ; with low powers of the microscope they appear pecu- liarly granular, with an irregular border. In agar streak-cultures, after twenty-four hours, small, trans- lucent, slightly raised colonies appear. Further growth is in- considerable, and scarcely extends beyond the line of inocula- tion. In agar stab -cultures the colonies develop along the line of inoculation, and there is slight growth upon the surface. Upon Loffler s serum, after twenty-four hours, fairly large, whitish, opaque colonies of firm consistence appear. In the succeeding days these colonies increase but little in size. Loffler's blood-serum (p. 84) constitutes, all in all, the best culture-medium for diphtheria-bacilli. It is, therefore, always employed for purposes of differential diagnosis. In bouillon, after twenty-four hours some precipitate has formed, after two days slight turbidity, which increases to the fifth day, then to grow less, until finally the fluid overlying the crumbling, flocculent precipitate is completely clear. Not rarely a thin, fragile coating appears upon the surface of the bouillon. A delicate coating forms upon the surface of potato rendered alkaline. Milk constitutes a favorable nutritive medium, but is not coagulated. In boiled and unboiled egg, both white as well as yolk, the diphtheria-bacillus grows well; upon coagulated egg-albumin it not rarely exhibits branching. In slightly alkaline Loffler's meat-infusion bouillon the diph- 14 210 CLINICAL BACTERIOLOGY. theria-bacillus generates acid. The increase in acidity is dis- tinctly appreciable within twenty-four hours ; it augments from the second day, then to subside. Later, after two or three weeks, the bouillon becomes again alkaline. On addition of litmus to the culture-medium these variations can be distinctly followed by the changes in color. Tenacity of the Diphtheria-bacilli. — A solution of mercuric chlorid, i : looo, destroys cultures in a thick layer within twenty seconds ; and five per cent, potassium permanganate, five per cent, aqueous solution of carbolic acid, three per cent, solution of carbolic acid in thirty per cent, alcohol, four per cent, solution of kresol in forty per cent, alcohol, within the same time. Five per cent, potassium chlorate is still ineffective after sixty seconds (Loffler). Pure lemon-juice likewise destroys the bacilli speedily. They are destroyed by exposure for ten minutes to a temperature of 60° C. (140° F.), although in a some- what thicker layer they withstand drying for months. In the form of dust, however, they rapidly die. They with- stand cold well, although in the refrigerator the diphtheria- bacilli rapidly lose their property of generating toxins. Abel found diphtheria-bacilli on building-blocks with which a child suffering from diphtheria had played six months previously. Diphtheria-bacilli have been found, further, upon soiled bed-linen, on the rim of a drinking-glass, in the hair and on the shoes of nurses, etc. In gelatin- cultures they may survive, according to Loffler, for three hundred and thirty-one days. Diphtheric membranes dried and preserved in the dark yield cultures even after the lapse of months. Pathogenic Properties of the Diphtheria-bacillus for Animals. — Diphtheria does not naturally occur in animals. So-called spontaneous diphtheria of fowl, of pigeons, etc., are etiologically different diseases. Diphtheria-bacilli give rise to the formation of true diph- theric pseudoniembrane , with multiplication of the bacilli, in the previously injured vagina or conjunctiva of guinea- pigs, in the trachea of guinea-pigs and rabbits after trache- otomy. Most birds, especially pigeons and chickens, then young dogs, rabbits, and especially guinea-pigs, are suscep- tible to the diphtheria-bacillus. Subcutaneous introduction of the bacilli is first followed by purely local alterations : more or less extensive hemorrhagic edema of the subcuta- DIPHTHERIA. 211 neous connective tissue. Then, in the presence of fever, there develop pleuritic effusions, swelling and redness of the adrenal glands, hemorrhages into the structure of the lymphatic glands, and death, preceded by decline of the temperature, occurs in the course of from twenty-four to forty-eight hours. If the virulence of the bacillus is slight, the disease is protracted : it may last three, four, or five days, and even several weeks. In the less susceptible rabbit the slow course is the rule. Under these circumstances there occur true diphtheric palsies, at first involving the posterior extremities of the animal, then the anterior, and finally the muscles of the neck. Death takes place after marked emaciation. At the autopsy fatty degeneration of the liver and the kidneys is found, and also inflammatory changes in the spinal cord and the nerves. If the virulence is still further diminished, the constitutional symptoms (fever) and the remote manifestations (pleuritis, palsies) are less conspicuous. After recovery from the local inflamma- tory process, which terminates in necrosis of the skin, the animal may be restored to health. Physiology of the Disease in Animals. — Whether their virulence be great or slight, the bacilli remain at the site of introduction, and, as a rule, they do not penetrate more deeply into the organism. Multiplication of the bacilli at the point of injection takes place only to a small extent, and only at the beginning — in the first six or eight hours ; later, the number of bacteria rather diminishes. They may, however, survive for a long time, and they have been found alive after weeks among the necrotic portions of skin at the point of injection. The constitutional dis- ease of animals results exclusively from the poison gene- rated by the bacteria : the diphtheria-toxin. The disease developed in animals by diphtheria-bacilli is, thus, a purely toxic one. If the poison alone, without the bacilli, is intro- duced into the bodies of animals, there result the same morbid manifestations, with the exception of the false membrane, and especially the palsies, just as after inocula- tion with the bacilli themselves. In experimental intoxi- cation of animals a bouillon-culture, several weeks old, is employed for inoculation whose reaction, originally alka- line, has become acid and is again rendered alkaline, and which has been freed of bacteria by filtration through a porcelain filter (Chamberland filter in which the fluid con- 212 CLINICAL BACTERIOLOGY. taining the bacteria is under positive pressure, or Kitasato's porcelain cylinders in which suction of the filtrate takes place). Instead of filtering them, the bacilli in the culture may be destroyed by addition of carbolic acid in propor- tion of o. 5 per cent. Reference has been made to the efforts directed to obtaining the poison chemically pure from the toxin-con- taining bouillon-filtrate (p. 29). Brieger and Boer have made the greatest advances in the preparation of the diph- theria-toxin. To the toxin-containing filtrate they add twice as much of a one per cent, solution of zinc chlorid. The resulting precipitate is agitated with from a three to a six per cent, solution of ammonium carbonate, and enough ammonium phosphate is added to effect complete solution ; then a delicate white turbidity results on addition of zinc phosphate. To free the solution of metallic precipitate, it is passed through a hardened filter, is well washed, and the filtrate is saturated with ammonium sulphate. In this way a precipitate is obtained that contains the diphtheria-toxin. A portion of the peptone adherent to the toxin is separated by solution in water and precipitation with sodium sulphate, but the albumin can not be completely eliminated, even by repeated washings. Brieger and Boer have, therefore, cultivated the diph- theria-bacillus upon dialyzed urine of human beings, and have prepared from this albumin-free culture-medium by the methods just described a toxin free from peptone and albumin. This toxin is destructive to animals, and the conditions found after death are characteristic. It does not yield the usual reactions of albumin, and is unin- fluenced by reducing substances, but it is almost immedi- ately destroyed by oxidizing substances. Acids also de- stroy the purified toxin of Brieger and Boer, while feeble alkalies do not. By means of a concentrated solution of ammonium chlorid it is possible, as Brieger and Boer, further, have shown, to free the bodies of the bacteria completely of the specific poison. If the bacteria thus treated are ground to powder, and small amounts of this suspended in water are injected into guinea-pigs, the animals die with necrosis and suppuration at* the point of infection. The bodies of the bacilli are, therefore, possessed, further, of a necrotic poison. DIPHTHERIA. 213 Relations of the Bacilli to Diphtheria in Human Beings. — The diphtheria-bacillus may be found in all cases of diphtheria in the diphtheric membrane, and in cases of diphtheria of the tonsils throughout the cavity of the mouth. It lies superficially, usually in large number ; it rarely penetrates deeply. In the bodies of patients suffer- ing from diphtheria the bacillus has almost never been found at any other place than in the false membrane, and especially never in the blood. Only in the bodies of patients dead of diphtheria has it been detected on several occasions also in the blood and in the viscera. Diphtheria in human beings must thus be viewed as a foxic infectious disease. The bacillus is responsible only for the local pro- cess ; the constitutional manifestations (fever, palsy, etc.) being dependent in human beings also upon absorption of the toxic metabolic products of the bacillus. The local action of the diphtheria-bacillus consists in necrosis of the epithelium and of the uppermost layer of the mucosa, which are thus converted into diphtheric membrane. This true diphtheric inflammatory process does not occur — apart from the action of certain poisons, as, for instance, diph- theria of the large intestine in conjunction with mercurial poisoning — in the absence of diphtheria-bacilli. The diph- theria-bacillus, however, by no means always gives rise to such a necrotic inflammatory process, when present in the body. The organism has been found repeatedly also in cases of fibrinous rhinitis and of croupous conjunctivitis, the latter being clinically quite different from diphtheria of the conjunctiva. Under these conditions the diphtheria-bacilli have simply given rise to a croupous exudation without necrosis. Finally, Loffler has found virulent diphtheria- bacilli in the mouth of a healthy child, an observation that has since been repeated by others. The diphtheria-bacillus may, thus, under certain conditions, vegetate upon mucous membranes in a virulent state, without occasioning disease of those structures. Mixed Infeption in Human Beings. — The diphtheria- bacillus is rarely found alone in the membranes, being usually associated with streptococci, also with staphylococci, pneumococci, and coli commune. It is almost certain that the severe purulent and septic manifestations observed in some cases of diphtheria are to be attributed to such mixed infection with especially virulent streptococci. It is a matter 214 CLINICAL BACTERIOLOGY. of interest in this connection that Roux and Yersin have shown that attenuated diphtheria-bacilli can be rendered again virulent by simultaneous inoculation with virulent streptococci. Saprophytic Occurrence of Diphtheria-bacilli outside the human body has not yet been observed. Pseudo-diphtheria-bacilli. — Hoffmann Wellenhof, and independently of him Loffler, cultivated as early as 1887 a microorganism bearing an extraordinary resemblance to the specific diphtheria-bacillus, and which Loffler designated pseudo-diphtheria-bacillus. Since then, reports of obser- vations of this or quite similar bacteria have multiplied, so that a whole series of pseudo-diphtheria-bacilli obtained from cases of angina, rhinitis, from the healthy mucous membrane of the mouth, pharynx, and nose, and from the skin and its diseases is now known. The confusion became still greater when the xerosis-bacilli were included in this group, and when it was later shown that these bacilli are present upon the normal conjunctiva. The pseudo-diphtheria-bacillus exhibits the same varia- tions in form as the true diphtheria-bacillus. The short bacil- lus is said to occur more commonly, but this is by no means always the case. According to M. Neisser the pseudo-diph- theria-bacilli exhibit negative manifestations when double staining with methylene-blue acetate and vesuvin is em- ployed. Only rare examples of certain varieties of xerosis- bacilli take the stain. In applying this staining reaction the conditions laid down (pp. 207, 208) must be strictly observed, as this means of differentiation does not suffice for cultures several days old. Further, impression-preparations from serum-cultures six hours old (p. 208), according to Neisser, constitute a serviceable aid in differential diagnosis. The pseudo-diphtheria-bacilli are not arranged in a typical manner, and after the lapse of the short interval of time men- tioned they do not exhibit the uniformly slender, rather long form. The xerosis-bacilli develop but slightly in the course of six hours ; they adhere so firmly to the nutrient medium that typical accumulation does not appear in the impression- preparation. The bacilli exhibit thickenings, swellings — in short, appear older than diphtheria-bacilli cultivated for six hours upon serum usually do. In gelatin stab-cultures the pseudo-diphtheria-bacilli form small colonies that spread upon the surface, and after two days quickly grow larger. DIPHTHERIA. 215 In agar streak -cultures large, grayish-white colonies form, that soon give rise to extensive elevated deposits ; whereas, in the case of the diphtheria-bacillus, development extends only a short distance from the line of inoculation. On serum the colonies soon become larger, brighter, and softer than those of the diphtheria-bacillus. In bouillon they form a precipitate, clarification taking place after the lapse of three weeks. Upon alkaline potato slight growth takes place, and in milk and egg a good growth. Milk is not coagulated. The xerosis-bacilli develop but slightly upon all culture-media. The pseudo-diphtheria-bacilli usually generate no acid in their development in ordinary bouillon ; individual varieties form minimal amounts, and only one culture of xerosis- bacilli is mentioned by Neisser that exhibited as great an increase in acidity as is observed in the case of diphtheria- bacilli. On the whole, the pseudo-diphtheria-bacilli and the xerosis-bacilli are not pathogenic for guinea-pigs, although, according to Spronck and C. Frankel, there are some varieties that exhibit a certain degree of virulence for these animals (causing edema, and after inoculation of three cubic centimeters loss of weight, etc.). The guinea-pigs could not be protected against the action of these pseudo-bacilli by previous injection of diphtheria-antitoxin. The observ- ers named, therefore, reached the conclusion that diphtheria- bacilli and pseudo-diphtheria-bacilli are not identical. Roux and Yersin, however, take the position that the pseudo- diphtheria-bacillus represents an attenuated or temporarily nonvirulent diphtheria-bacillus. This view still has ad- herents, who support their contention with the fact that in every case of true severe diphtheria nonvirulent as well as virulent diphtheria-bacilli are always observed. The diph- theria-bacillus would, accordingly, be somewhat analogous to the pneumococcus, which, likewise, is a common inhabi- tant of the healthy mouth. The Susceptibility of Human Beings to Diphtheria can not be considered as great, in view of the possibility of the presence of diphtheria-bacilli upon the mucous mem- brane of healthy persons. It would appear that a special predisposition on the part of the mucous membranes or special virulence on the part of the bacilli is necessary for the development of the disease. The first years of life have 216 CLINICAL BACTERIOLOGY. distinctly a greater susceptibility than adults, but this grows less after the fifth or sixth year. The portal of infection for the diphtheria-bacillus may be constituted by any mucous membrane, as that of the nose, the pharynx, and the larynx, as well as that of the vagina, the conjunctiva, etc., and, further, every wound- surface. The larger number of cases of diphtheria are at- tributable to direct association with diphtheria-patients. The diphtheria-bacilli may, under such conditions, gain en- trance into the pharynx, where infection takes place most commonly ; as well as with the food and by way of the air- passages. The diphtheria-bacillus is probably not carried great distances through the air. It has never been found in the air, and it is rather sensitive to drying in a thin layer. In thicker layers the bacilli may survive for four months, and if the drying be incomplete, for seven months. Proba- bly the bacilli are conveyed directly through the agency of the portions of membrane coughed up by the patients. In some cases eating-utensils and drinking-utensils, handker- chiefs, toys, etc., have been the means of conveying the disease-germs from children suffering from diphtheria, as well as convalescents. Children may also be infected by adults through kissing, if these have suffered from a harm- less angina clinically free from diphtheric characteristics. In other cases direct infection can absolutely not be demon- strated, and at times such an occurrence can be actually excluded — as, for instance, when the first case of diph- theria occurs in a village cut off from all communication with the external world. Under such circumstances it must be concluded that the diphtheria-bacilli have been derived from a case of diphtheria that pursued the clinical course of a benign angina and gave rise to no suspicion of diph- theria ; or from a case already cured, in which the bacilli may be present for a long time after the disappearance of all symptoms (p. 230) ; or the conjunction of special cir- cumstances has, in accordance with the theory of Roux and Yersin, endowed with virulence pseudo-diphtheria-bacilli that have been present in the mouth of- the infected indi- vidual. What conditions may bring about this circum- stance, or whether it is at all possible, is not yet known. On the other hand, it is known, from an observation in which virulent bacilli were found in the mouth of a healthy child, that a certain predisposition, a lesion of the mucous DIPHTHERIA. 217 membrane, or the like, must be present for diphtheria to result from infection with true diphtheria-bacilli from a case of diphtheria. Method and Significance of the Bacteriologic Diag- nosis of Diphtheria. — Every case of inflammation of the throat should be examined with regard to the presence of diphtheria-bacilli, and, if these be found, the case should be treated from both the medical and the hygienic standpoint as one of diphtheria (just as every case of diarrhea with comma-bacilli in the stools should be treated as one of cholera), as the mildest case of diphtheric angina may give rise to severe infection. For diagnostic purposes, simple microscopic examination of the mucus or of the membrane in stained cover-glass preparations will suffice in some cases. Double staining, by the method of M. Neisser, which is so characteristic for preparations made from cultures, has not proved entirely reliable in the study of original preparations. If the prepara- tions, stained single or double, are not entirely convincing, cultural investigation must be additionally undertaken. A small portion of the diphtheric membrane is removed with forceps, sterilized by heat, or, directly, with a strong platinum loop ; or a sterile swab of cotton or a sterile bit of sponge is rubbed upon the suspected surface to be examined, and six or eight strokes are made upon a plate of Loffler's blood-serum. Should this not be avail- able, from three to five tubes of blood-serum or glycerin- agar, solidified in slants, are successively inoculated. Plates and tubes are placed in the thermostat at a temperature of 37° C. (98.6° F.) — according to M. Neisser best at a temperature of 34° C. (93.2° F.) or 35° C. (95° F.). In the first streak or in the first tube inoculated the colonies are too dense, and they coalesce ; in the last they are isolated, and these are then examined further. It is recommended also that the membranes, before being smeared, be rinsed for several minutes in two per cent, solu- tion of boric acid, whereby a considerable number of the saprophytic bacteria, accidentally present, are removed, so that a separation of the individual colonies is brought about in the first inoculation-smears. After six or eight hours impression-preparations are made from the serum-plate. If these display the typical collections described (p. 208), then a positive diagnosis can be made. A negative diag- 218 CLINICAL BACTERIOLOGY. nosis, however, should be made only with great reserve after the lapse of only six hours (M. Neisser). After eighteen or twenty hours smear-preparations are made, and these are treated by the method of double staining already described (p. 207). Typical granules in typical bacilli may be considered as conclusive, although Neisser himself issues a warning against dependence upon double staining alone until his observations have been amply confirmed by others. If, in accordance with older methods, dependence is placed upon simple staining of an ordinary cover-slip preparation from a plate or a test-tube of Loffler's serum, the possi- bility of confusion with pseudo-diphtheria-bacilli must con- stantly be borne in mind. It is then , necessary to scrutinize carefully the peculiarities that have been described as in- dicating either diphtheria or pseudo-diphtheria. No single feature should be accepted as conclusive in the diagnosis ; but the conjunction of all should be required. Frequently, experiments on animals must be resorted to in order to determine whether the bacilli are virulent or not. If they prove virulent, the organisms are probably without question true diphtheria-bacilli.* Immunity and Specific Therapy. — Diphtheria may attack the same child on several occasions. It is not one of those diseases that leave behind them permanent immu- nity, but rather one of those that predispose to recurrence. Nevertheless, it is certain that after recoveiy from diph- theria some degree of immunity exists temporarily. This is demonstrated by the observations of Escherich, Kle- mensiewicz, and others, who showed that the blood- serum of children convalescent from diphtheria exhibits immunizing properties. Experimental animals are readily immunized to diph- theria. Behring and, later, Roux conferred such immunity on a large scale upon horses by preliminary treatment with diphtheria-toxin whose toxicity was attenuated by addition of iodin trichlorid or solution of iodin and potassium iodid. By the introduction of gradually increasing amounts of diphtheria-toxin the immunity of these animals was in- creased to a high degree. * The method commonly practised in public laboratories consists in inocula- tion of a tube of Loffler' s serum with a previously sterilized swab applied to the suspicious membrane, cultivation in the incubator overnight, and microscopic examination of cover-slip preparations stained with Loffler' s solution. — A. A. E. DIPHTHERIA. 219 The following instance, taken from the text-book of Mace, will illustrate the immunization of a horse by the method of Roux. For the better comprehension of the description it should be mentioned, as has already been pointed out in the consideration of the physiology of the disease (p. 211 ), that the toxin is the equivalent of the fil- trate of a bouillon-culture, or of a culture in which the bacilli have been destroyed by addition of carbolic acid in a proportion of o. 5 per cent. : Toxin with Addi- Day. Injection tion of Iodin- OF potassium Iodid. I j^ cu. cm. I : 10 No reaction. 2 /^ cu. cm. I : 10 '* 4, 6, 8 .... ^ cu, cm. I : 10 *' 13, 14 I cu. cm. I : 10 " 17 J4^ cu. cm. Pure toxin Slight edema, without fever. 22 I cu. cm. *' '* " 23 2 cu. cm. " ** ** 25 3 cu. cm. *< «* *« 28 5 cu. cm. *' *< <* 30, 32, 36 . . . 5 cu. cm. '< « ** 39, 41 ... . 10 cu. cm. ** " *' 43, 46, 48, 50 . 30 cu. cm. " Marked edema, disappearing in the course of twenty- four hours. 53 60 cu. cm. " ** ** 57> 63, 65, 67 . 60 cu. cm. *< ** ** 72 90 cu. cm. ** " ** 80 250 cu. cm. '* " " Small doses of pure toxin may also be employed in the first inoculations. Some horses bear from the beginning I cu. cm. of a toxin of which ^ cu. cm. is sufficient to destroy within forty-eight hours a guinea-pig weighing 300 grams. The immunity can be looked upon as well established after the animal has received from 60 to 70 cu. cm. of this toxin. It is then possible to inject much larger amounts without injury. In order to obtain diphtheria- antitoxin it is best to employ horses, in the first place be- cause they are very readily immunized, and in the second place because they can be bled innumerable times. In immunizing the animals a normal toxin-solution must be available to begin with, and which affords a standard of comparison. Behring designates as a diphtheida normal toxin that diphtheria-solution of which i cu. cm. represents the minimal lethal dose for 100 guinea-pigs each weighing 250 grams. To indicate this normal toxin, he has intro- 220 CLINICAL BACTERIOLOGY. duced the abbreviation DTN^, and he designates a toxin of 10 times this strength as DTN^^ and one o( ^ this strength as -^^. As guinea-pigs weighing 250 grams are not always obtainable, the important minimal lethal dose is not estimated with regard to the entire experimental ani- mal, but upon the basis of each gram of living bodily weight. In order to express this relation also in simple symbols Behring designates the whole animal with an m,* and a gram of the living bodily weight with an M. The weight of the guinea-pig in each individual instance is added besides, and above the letter m (thus, for instance, m^so stands for an animal weighing 280 grams). The minimal lethal dose for i gram of body-weight — thus, for I M — is indicated by prefixing the symbol -{-. +1500M expresses that amount of toxin that just suffices to destroy 6 guinea-pigs each weighing 250 grams. One cubic centi- meter of DTN^ causes the death of 100 m^^o^ ^nd is thus equal to +25,000 M — that is, it represents 25,000 minimal lethal doses for each gram of living guinea-pig by weight. Of diphtheria-toxin 10 times the normal strength, DTN^^, I cu. cm. = + 250,000 M; i cu. cm. of diphtheria-toxin one-tenth the normal strength, -^^, ^+ 2500 M. In order to obtain an active diphtheria-toxin it is ob- viously necessary to employ highly virulent diphtheria- bacilli in preparing the bouillon-cultures. As a consequence it is frequently necessary to fortify the virulence of the diphtheria-bacilli. A diphtheria-bacillus that has com- pletely lost its property of generating toxin can not be rendered virulent again by any method thus far known. If, however, a trace of virulence remain, this may be augmented by repeated passage through animals. Six or eight hours after the injection a specimen is obtained from the area of local edema, and with it a culture is made, which then serves for the inoculation of a new animal. Roux and Yersin augmented and accelerated the production of toxin on the part of the diphtheria-bacilli by cultivating these in a shallow layer of bouillon through which a current of moist air was constantly passed. The diphtheria-bacillus thrives vigorously in nutrient media containing sugar, although it generates considerably less toxin. ■^In the original, the German capital letter 9Ji is employed, but for conve- nience sake the English small letter m is here substituted. — A. A. E. DIPHTHERIA. 221 ♦ When the horses have been rendered sufficiently immune, five or six liters of blood are withdrawn from the jugular vein by means of a trocar, eight or ten days after the last injection. The blood, after standing in the refrigerator, yields a clear serum, which exhibits its maximum activity eight or ten days after the injection of toxin. If the blood is removed earlier, the serum is considerably weaker. After the period of maximum activity, which persists for several days, a gradual reduction takes place, which may lead to complete disappearance of the antitoxin, unless meanwhile new injections of toxin are made. For purposes of preservation carbolic acid, in the proportion of 0.5 per cent, is added to the antitoxin (Behring, Hochst serum), or 0.4 per cent, trikresol (Aronsohn, Schering serum), or a bit of camphor (Pasteur Institute). It has been pointed out in the general section that toxin and antitoxin, admixed in a test-tube, mutually neutralize the activity of each other, so that when injected simultaneously into guinea-pigs, no manifestations of disease follow. In this mutual interaction a regular gradation is ob- servable. In estimating the immunizing value of the cura- tive serum Behring and.Ehrlich did not proceed from the simple lethal dose, but from ten times that amount. They designate as normal serum that of which o. i cu. cm. suffices to neutralize ten times the lethal toxic dose, so that a guinea-pig weighing 250 grams withstands the injection without injury. One cubic centimeter of this serum repre- sents a normal antitoxin-unit. The further calculation can be readily made upon this basis. A serum, for instance, of which 0.0 1 cu. cm. neutralizes 10 times the lethal dose represents 10 times the strength of normal serum — that is, I cu. cm. contains 10 normal antitoxin-units. Should o.ooi cu. cm. of a serum suffice for this purpose, it repre- sents a strength 100 times that of normal serum, and i cu. cm. contains 100 normal antitoxin-units. Behring subsequently introduced a new method of cal- culation. He designates as diphtheria normal antitoxin, DANi, that serum of which i cu. cm. neutralizes i cu. cm. of diphtheria normal toxin, DTN^, = -|- 25,000 M. These figures he converts directly into weight of guinea-pigs, in grams, M, by simply prefixing the minus sign. DAN^ thus equals —25,000 M : that is, i cu. cm. of diphtheria normal antitoxin is capable of neutralizing the lethal dose 222 CLINICAL BACTERIOLOGY. for 25,000 grams in weight of guinea-pig. As may be readily seen, this mode of calculation agrees entirely with the first described. According to that, o. i cu. cm. of serum was capable of neutralizing 10 times the minimal lethal dose ; while according to this, i cu. cm. of serum is capable of neutralizing 100 times the minimal lethal dose in the typical guinea-pig weighing 250 grams. Roux estimates the immunizing power in quite another manner. He determines how much serum must be injected from twelve to twenty-four hours previously, in order to protect a guinea-pig against the lethal dose of diphtheria- bacilli or of diphtheria-toxin that otherwise would have de- stroyed the animal in not more than thirty hours. He fixes as the power of the serum that figure which expresses the relation between the amount of serum and the body-weight of the guinea-pig. An immunizing power of 20,000 in- dicates thus that of this serum -2-fo"o"o P^^^ ^^ ^^^ body- weight of a guinea-pig must be injected in order to protect the animal against the amount of diphtheria-culture or diphtheria-toxin that would cause death in thirty hours. Spronck has proposed a special formula for converting the immunity-unit of Roux into that of Behring. B = ^ — that is, it is only necessary to divide Roux's figures by 500 in order to obtain the number of Behring units. In a communication upon the estimation of the strength of diphtheria-antitoxin and its theoretic basis Ehrlich starts with a dry diphtheria-antitoxin prepared in the Hochst works in order to obtain a sufficiently constant standard and a unit of measure for the serum that is protected from the destructive influence of water, oxygen, light, and heat. This contains 1 700 immunization-units or normal antitoxin- units to the gram. It is preserved in a small apparatus con- sisting of two communicating glass tubes, of which one con- tains the powder and the other phosphoric anhydrid as a dehydrating agent. The apparatus is most carefully ex- hausted of air, and kept in a dark, cool place. Each tube contains 2 grams of antitoxin-powder, which before being used is dissolved in 200 cu. cm. of a solution of sodium chlorid (10 per cent.) and glycerin (from 50 to 80 per cent). One cubic centimeter of this solution diluted 17 times represents the immunity-unit or normal antitoxin-unit. If to this immunity-unit are added increasing quantities of diphtheria-toxin, it is possible to fix two limits (L), which DIPHTHERIA. 223 are of great importance for the characterization of the toxin. The first (Lq) represents that amount of toxin that is neutraHzed by the serum, so that injection of the mixture is borne by the guinea-pig without injury. The second hmit (L ^) represents the amount on injection of which such a marked excess of toxin is rendered active in spite of the presence of the anti-body that death of the guinea-pig takes place within four days. According to the foregoing con- siderations, Lq represents about lOO lethal doses ; for, as has been explained, if the antitoxin-unit DAN ^ neutralizes the toxin-unit DTN i, that is i cu. cm. of that toxin of which o.oi cu. cm. destroys a guinea-pig weighing 250 grams ; and the difference (D) between the two limits (L^ and L J should be equal to the minimal lethal dose. These assumptions are, however, not justified, as Ehrlich has shown as the result of most painstaking experiment. L ^ varies be- tween 27 and 109 toxin-doses, and D between i, 7, and 28. Ehrlich, upon the basis of experiments with tetanus, had already come to the conclusion that by the action of carbon disulphid tetanus-toxin could be transformed into an in- nocuous modification, but which is still capable, both in the test-tube and in the animal body, of combining with the anti-bodies. The spontaneous attenuation of the diphtheria- toxin so frequently encountered, without the slightest dimi- nution in its neutralizing activity, is attributed by Ehrlich to a similar transformation of a portion of the toxin into such toxin- modifications, and for these he proposes the name of toxoids. Ehrlich divides the toxoids into three groups : (i) Pro- toxoids, which unite with the antitoxin more easily than the toxin ; (2) syntoxoids, which exhibit for the antitoxin the same affinity as the toxin ; and (3) epitoxoids, which possess a lesser attraction for the antitoxin. As the pro- toxoids possess a greater affinity for the anti-bodies than, and the syntoxoids the same affinity as, the toxin, they can not be displaced by the toxin from their combination with the anti-toxin. If, therefore, the neutral toxin-antitoxin -mixture (Lo) consists of toxins, protoxoids, and syntoxoids, then L ^ is produced by the addition of the simple lethal dose, or, what amounts to the same thing, D, the difference be- tween Lq and L ^. is equal to this lethal dose. The condi- tions are, however, quite different when epitoxoids are present. These must, in the first place, be displaced by the 224 CLINICAL BACTERIOLOGY. toxin added, and then there must be present, besides, a sim- ple toxin-unit in order to attain L ^ and to cause the death of the animal. D corresponds here to the epitoxoid-units plus I toxin-unit. Every normal toxin-bouillon contains the three toxoids. Their relations may be represented as follows : Diphtheria-bouillon = x-toxoids (protoxoids and syntoxoids) -j- y-toxin -f z-epitoxoid. Lq, then, = x- toxoid saturated -|- y-toxin saturated + z-epitoxoid satu- rated. To determine the equivalent of « in toxin, it is only necessary to know the number of lethal doses present in Lq. L+ then corresponds with the formula : x-toxoid saturated -|- (y + z) toxin saturated + 1 toxin free -f- z-epi- toxoid free. The number of epitoxoids /? equals, as has been seen, D — 1, but only in the event of the bouillon being constituted exclusively of toxin and epitoxoid. If additional toxoids are present, then /5 expresses a relative value, and is, therefore, designated by Ehrlich as a function of /5, thus, F (/?). The formula for the toxin-bouillon may, therefore, be expressed as follows : x-toxoids -j- « toxin -j- F (/5) epitoxoid. For the majority of diphtheria-poisons it may be shown by addition of the simple immunity-unit that they originally contained the required lOO doses of toxin. The attenua- tion is effected gradually according to the principle of thirds or halves. Of three toxin-molecules two are converted into toxoids, or 1 toxin is converted into equal parts of toxoids and toxin. It appears that in this process of de- composition in the cold no epitoxoid — which always occurs in cultures maintained at a temperature of 37° C. (98.6° F.) — is formed, but only protoxoids and syntoxoids. In the study of an especially active toxin immediately after its acquisition Ehrlich obtained the following figures : L+ = 100 doses of toxin ; L^ = 50 doses of toxin. The toxin, thus, was of half strength, and the figures obtained had, therefore, to be multiplied by 2. The value of L^., then, equaled 200, and the formula for the bouillon was as follows : 50 toxoids + 50 toxin + 100 epitoxoids. As a re- sult of this demonstration that the antitoxin-unit saturates 200 toxin-equivalents, that the poison itself is attenuated dichotomously, it was possible without difficulty to explain the previously mysterious manifestation that with freshly prepared toxins frequently just 100 toxin-equivalents are neutralized by the immunity-unit. DIPHTHERIA. 225 The total of toxoids, toxins, and epitoxoids, equals, according to Ehrlich, 200 ; a toxin possessing the a equivalent of toxin and z-epitoxoid should yield the following formula: (200 — a — z) toxoids -f « toxin + z-epitoxoids. To this the immunity-unit is added, and in this way is ob- tained the value Lq = (200-a-z) toxoid-antitoxin -f a toxin-antitoxin + z-epitoxoid-antitoxin. The value of L_|. is obtained by adding to the neutral mixture so much of the original material that the z-epitoxoid-antitoxin is decomposed by the mixture of toxoid -|- toxin. As is evident, this addition must represent a. ^^^ toxin-units. The epitoxoid toxin-units have just been found to be /9 = D — i. It results that /5 ^ «•— J— and z = -^^. If the amount of 200 — z a + P epitoxoid is estimated according to this formula, figures are obtained that stand in the simplest relation to that found for the number of immunity-units, 200 — namely, ^, y^y ;^, or i^, or ^ thereof As the outcome of these experiences, Ehrlich has sug- gested the following alterations in the directions for testing the diphtheria-antitoxin, and these have been confirmed by a decree dated March 29, 1 897 : I. As a standard for the estimation of the antitoxin an antitoxin-powder of accurately determined strength, pro- tected against the influence of oxygen and water, is em- ployed. This is contained in carefully measured quantities in specially prepared vacuum-tubes. The apparatus at the time present in the laboratory are filled each with 2 grams of a dry antitoxin 1700 times the normal strength. II. To secure the greatest possible degree of permanence the antitoxin should be dissolved in a mixture of equal parts of 10 per cent, solution of sodium chlorid and glycerin. A tube is to be opened every three months and a new solution prepared. Of the dry antitoxin at the time preserved in the laboratory the contents of a tube are dissolved in 200 cu. cm. of the mixture described, and thus a test antitoxin-solution 17 times the normal strength is prepared. III. The present test-dose of toxin is determined with the aid of an immunity-unit, such as is contained, for in- stance, in I cu. cm. of a y^ dilution of the test-antitoxin 17 times the normal strength. To this amount of antitoxin increasing amounts of toxin are added, and by means of 15 226 CLINICAL BACTERIOLOGY. most careful experimental observations the limit is deter- mined at which just that excess of toxin becomes manifest which causes death of the animal in the first four days. The amount of toxin thus obtained represents the imme- diate test-dose. By means of the same dose of serum, for the more exact characterization of the toxin, the determi- nation of a second limit is made, for the purpose of learning the dose of toxin that is just neutralized by admixture with the amount of serum named. IV. The determination of the strength of a diphtheria- antitoxin is made by means of the test-dose of toxin (see paragraph III) as follows: The test-dose of toxin in question — for instance, 0.355 ^^- cm. of tested toxin at the time present in the laboratory — is mixed with 4 cu. cm. of anti- toxin corresponding to the test-figures given. As the test- dose of toxin is estimated for i cu. cm. of antitoxin of normal strength, or for 4 cu. cm. of antitoxin }^ the normal strength, an antitoxin of x-strength will have to be diluted i^ x, and in testing an antitoxin 100 times the normal strength, -^-^. V. The mixture obtained is injected unmodified sub- cutaneously' into guinea-pigs weighing from 250 to 300 grams. If the animals die in the test-experiments made by two observers in the laboratory within the first four days, the antitoxin does not possess the required strength. Should death occur within five or six days, the antitoxin is close to the required strength, and in order to avoid the early withdrawal to be anticipated an improvement of from 5 to 10 per cent, is recommended the manufacturers. In- durations that occur in the animals experimented upon do not, however, constitute sufficient ground for objection. In the case of the dead animals an autopsy should be held, and careful attention directed to complications with previ- ously existing disease (tuberculosis, pseudo-tuberculosis, and pneumonia) that may induce undue susceptibility on the part of the test-animals. VI. Both liquid and solid toxins may be employed for test-purposes, if the limits defined in paragraph III can be accurately estimated, and the difference between them does not exceed fifteen simple lethal doses. If liquid toxins preserved in toluol are employed, this should be done only if as a result of preliminary investigation the perma- nency of the test-constants is demonstrated, if the test- DIPHTHERIA. 227 dose does not exceed i cu. cm. The examination with re- gard to the quaUties of the test-toxins should be continued. VII. The test-toxins, if hquid, are to be examined monthly with regard to their sterility by means of culture- methods. VIII. The test-poison is to be redetermined at intervals of six weeks by means of the test-dose of serum, the test- dose and the net-valuation being estimated anew. If, on reexamination, any considerable deviation in the test-dose should be detected, the toxin must be considered to be in process of decomposition, and it should be replaced by fresh toxin. IX. The manufacturers are to be informed that the test-toxin in small amounts decomposes readily, and that even brief exposure to light may induce considerable at- tenuation. It is therefore to be recommended that a new supply of toxin be obtained from the laboratory ever>- three weeks. * Diphtheria-antitoxin is prepared in Germany in four establishments — namely, the Hochst Works, the Factory of Schering, in -Berlin, the Pasteur Institute in Stuttgart, and the Factory of Sthamer, Noack & Company in Hamburg. The Hochst Works manufacture the following prepara- tions : ANTITOXIN OF 250 TIMES THE NORMAL STRENGTH. Number o, yellow, . .0.8 cu. cm. contain 200 immunity-units. Number i, green, . . 2.4 " " 600 " Number 2, white, . . 4 " " 1,000 *' Number 3, red, ... 6 " " 1,500 *' * Dr. Jos. McFarland and Dr. Chas. T. McClintock, to whom these regula- tions were submitted, kindly describe, as follows, the method of testing pur- sued by the largest manufacturers of diphtheria-antitoxin in the United States : " In order to secure uniformity in the toxin the same culture of the diph- theria-bacillus is always employed. This is grown for seven days at 37° C. (98.6° F.) in an accurately prepared alkaline two per cent, peptone-bouillon. The same degree of alkalinity is always secured (phenolphthalein being used as the indicator in titration). After a.ddition of 0.4 per cent, trikresol, this toxic bouillon is filtered through unglazed porcelain and stored in a dark, cold place. The amount of this toxin that will kill a guinea-pig weighing 250 grams on or before the sixth day is considered the minimum fatal dose. Should this toxin deteriorate ten per cent, from its original strength, it must be discarded. '* In testing antitoxin, a series of guinea-pigs, weighing from 240 to 270 grams, are injected with ten times the minimum fatal dose of toxin previously mixed with varying amounts of antitoxin. Those pigs are considered protected that do not die or lose more than 20 per cent, of their original weight in seven days."— A. A. E. 228 CLINICAL BACTERIOLOGY. STRONG ANTITOXIN OF 500 TIMES THE NORMAL STRENGTH. Number o D, yellow, . i cu. cm. contains 500 immunity-units. Number 2D, white, . 2 " contain 1,000 " Number 3 D, red, • .3 " " i,5oo " Number 4 D, violet, . 4 '* '* 2,000 ** Number 6 D, blue, . . 6 *' " 3, 000 " ANTITOXIN OF 600 TIMES THE NORMAL STRENGTH. Number 6 e, blue, . . 5 cu. cm. contain 3000 immunity- units. The Pasteur Institute, of Stuttgart, manufactures only one preparation of 50,000 Roux immunity-units. The Schering factory produces the following antitoxins : A, 100 immunity-units per cu. cm. B, 200 *' '* '* (in vials of 5 and lo cu. cm.). Strong antitoxin of 500 times the normal strength, 500 immunity- units per cu. cm. (in vials of 2 and 4 cu. cm.). The Hamburg establishment dispenses diphtheria-anti- toxin prepared by Ruete and Enoch and containing 300 immunity-units per cu. cm. In injecting the serum the rubber-ball syringe of Koch may be employed, and this is easily manipulated by one who is experienced. (Fig. 36, 2.) The antitoxin-syringe of Roux is to be recommended as especially practical, and it can be sterilized with the greatest ease. (Fig. 36, i.) It is either boiled in a one per cent, solution of soda, or it is cleansed with a five per cent, solution of carbolic acid, after which it is rinsed with a o. 5 per cent, solution in order that the antitoxin may not be injured in consequence of too great concentration of the antiseptic. The number of immunity-units to be injected in the individual case will depend entirely upon its severity, and upon the day of the disease that the patient comes under treatment. In a mild case, coming under observation on the first day, 600 immunity-units will suffice. Severe cases, seen as late as the third day, will require 1000 im- munity-units. In still more severe cases, seen at a late stage, 1500 immunity-units and more are employed. The results that have thus far been obtained in the treatment of diphtheria with the antitoxin have been ex- ceedingly favorable. The mortality from diphtheria has been reduced about half under the influence of the antitoxin. In support of this statement we may quote the results of DIPHTHERIA. 229 the collective investigation with regard to the diphtheria- antitoxin for the period from April, 1895, to March, 1896, conducted by Dieudonne in the Imperial Health- office. There were treated with antitoxin in hospitals 9581 cases, among which death occurred in 1589 — 15.5 per cent. If the cases still under treatment at the conclusion of the report be excluded, the proportion of deaths to recoveries was 3 to 16. In the eleven years preceding the introduction of the antitoxin (from 1883 to 1893) the pro- portion of deaths to recoveries averaged 6 to 16. Nearly one-half of the cases included in this study were designated in the reports as severe. The mortality among children under two years old (11 89) equaled 39.1 per cent, and that of those subjected to tracheotomy (2744) 32.3 per cent.* In the cases submitted to treatment with antitoxin the disease pursued, in general, a milder and more favorable course. Existing manifestations of stenosis improved in a large number of cases, so that tracheotomy was avoided. Serious sequelae with certainty attributable to the antitoxin have thus far not been observed. In rare cases unpleas- ant complications of a transient character occur — viz., in- filtration at the point of injection, pains in the joints and ex- tremities, urticaria, exanthemata, possibly also albuminuria. These symptoms are not at all dependent upon the antitoxin itself, but are to be attributed exclusively to the serum of the horse. Like amounts of antitoxic serum induce the same results in this respect, whether they contain a larger or a smaller number of immunity-units. For this reason it is desirable to obtain as highly concentrated an antitoxin as possible in order that as small amounts of serum as pos- sible need be injected. Behring succeeded in preparing an antitoxin of 1200 times the normal strength, but, unfortu- nately, it was soon found that such highly concentrated solutions could not be preserved for as long a time as less concentrated solutions, and that in the course of time the immunizing activity diminished considerably. Behring was, therefore, led to the preservation of serums of greater than 500-strength by converting them into a dry form by a special procedure. The dry powder, containing * These results have been amply confirmed not only on the Continent of Europe, but also by observations in England and in America, with reference • to both nonoperative and operative cases. — A. A. E. 230 CLINICAL BACTERIOLOGY. sodium chlorid and albumin, can be preserved for an in- definite time without any addition, and it is readily soluble in water. One gram contains at least 5000 immunity-units, and in some preparations as much as 10,000 immunity- units. From Yt ^^ y^ gram thus represents a simple cura- tive dose ; from ^ to ^^ gram, twice as much, for severe cases ; and from % to yi gram, four times as much, for advanced cases. Behring considers it desirable that the preparation of these solutions should be undertaken by pharmacists, and he is hopeful that the dried serum may in due time be incorporated into the Pharmacopeia. Prophylaxis. — It is important to know that after com- plete disappearance of the membrane virulent diphtheria- bacilli may persist in the mouth and pharynx of children until the fifth week, and sometimes even much longer. The children must, therefore, be isolated for from five to six weeks after recovery from the disease, and, above all, be kept from school during that time. Those who surround the patient should also be kept under medical observation. Careful disinfection of the sick-room is a matter of impor- tance, as it is known that diphtheria-bacilli will persist for a long time and most tenaciously in a house in which there has been a case of diphtheria. With regard to personal prophylaxis by means o^ injections of antitoxin, this has not yet been employed upon a large scale. An injection of 250 normal antitoxin-units confers protection lasting three or four weeks. This will suffice in most cases, but if the danger of infection continues for a longer time, a second prophylactic injection will become necessary. The main objection that has hitherto stood in the way of this form of diphtheria-prophylaxis was the fear of disagreeable complications due to the serum. Such fear would, however, be removed by the use of a highly con- centrated antitoxin in powder-form, as 0.025 gram of the powder with a strength of 10,000 contain the requisite 250 immunity-units. TETANUS. The exciting agent of tetanus was recognized by Nicolaier, of Gottingen, in 1885, as a bristle-like rod with a terminal bulbous spore, but it was first isolated by Kitasato in 1889 from the foreign bacteria always associated with it TETANUS. 231 in infectious earth and in pus from a case of tetanus and grown in pure culture. The tetanus-bacillus is a long, slender rod, from 0.3 to 0.5 /a thick and from 3 to 5 /a long, with rounded extremities and with feeble, but distinct, motility, which, however, ceases immediately in the presence of oxygen. It frequently grows in filaments, the individual elements of which are not always distinctly dif- ferentiable from one another. The temperature-optimum is from 36° C. (96.8° F.) to 38° C. (100.4° F.), although the bacillus thrives also at room-temperature. It does not develop FJg* 53.— Bacillus tetani; X looo (Friinkel and Pfeiffer). below a temperature of 14° C. (57.2° F.), while at 42° C. Qio7.6° F.) or 43° C. (109.4° F.) it presents distinct invo- lution-forms, and at 60° C. (140° F. ) it is destroyed quite rapidly. The tetanus-bacillus is . an, anaerobic organism, although it still grows in the presence of small amounts of oxygen. Protected from air and light the tetanus-spores retain their vitality and their virulence in cultures after the lapse of a year. Spore-formation. — At a temperature of 37° C. (98.6° F.) after the lapse of thirty hours, and at room-temperature within a week, the tetanus-bacillus forms spherical spores always situated at one extremity. The bacillus swells at one extremity like a drumstick, and there results the characteristic head-bearing bacillus (pin-shaped or note-shaped). The organism is always 232 CLINICAL BACTERIOLOGY. nonmotile. The diameter of the spores measures from i to i . 5 /a. They are extremely resistant to the action of heat. They are not injured by exposure for an hour to a temperature of 80° C. (176° F.), and they are not destroyed by live steam with a temperature of 100° C. (212° F. ) before the lapse of five or eight minutes. The spores are also rather resistant to chemic disinfectants. They are not destroyed in five per cent, carbolic acid before the lapse of fifteen hours ; and they die in one per cent, solution of mercuric chlorid within three hours. Staining. — The tetanus-bacilli stain readily with the usual dyes, and they can be stained also by Gram's method. The spores can be demonstrated by the usual methods for staining spores. Pure Cultures. — The tetanus-bacillus is encountered in nature (garden-soil, dust, animal excrement) and also in the pus from wounds infected with tetanus always in association with numerous other bacteria, some of which are anaerobic and others aerobic. On account of the difficulty of isolating the tetanus-bacillus from its associates, the attempt to obtain it in pure culture long failed. Kitasato overcame this difficulty by availing himself of the great resistance of the tetanus-spores. He inoculated tetanus-pus upon agar-tubes ; after exposure for two days in the thermostat, in addition to the other bacteria there were found also numerous characteristic bristle-like rods with bulbous heads. The mixed culture was now heated in the water-bath at temperature of 80° C. (176° F. ) for an hour, when all of the bacteria, including the tetanus-bacilli, were de- stroyed, and only the tetanus-spores survived. These could now be grown in pure culture without difficulty by the various culture-methods employed for other anaerobic bacteria. It ought, however, not occasion surprise if a pure culture should not succeed even by this method, if, as happens rarely, other resistant spores are present in the material submitted to exam- ination. Under these circumstances anaerobic plates must be made, by means of which, further, it is possible to isolate the tetanus-bacilli in the discharge (pus). Cultural Properties of the Tetanus-bacillus. — The tetanus-bacillus grows upon all the customary culture-media with exclusion of oxygen, and to which grape-sugar (two per cent.) may be advantageously added. All tetanus-cultures pos- sess in common a peculiar, rather disagreeable odor of burned material. On gelatin-plates at room-temperature there appear about on the fifth day, slowly growing, small colonies, with radiate pro- cesses that give to the whole a feathery or thistle-like appear- ance. Microscopic examination discloses in the center a dense, yellowish mass, with numerous delicate, ciliary fibers and pro- TETANUS. 233 cesses arranged radially at the brighter margin. The gelatin is slowly liquefied. In high gelatin stab-cultures growth confined to the lower por- tion of the line of inoculation appears after about a week. From the grayish-white bacterial mass innumerable small, pointed processes extend in every direction into the gelatin, giving the culture a characteristic appearance suggestive of a large-branched fir-tree. In the second week liquefaction sets in and obliterates this appearance. The process advances slowly, until gradu- ally the entire culture is converted into a turbid, grayish-white, viscid mass, of which the upper portion subsequently becomes clear, while the bacilli sink to the bottom as a cloudy gray mass. On agar-plates delicate colonies develop, which with low powers of the microscope appear to consist of a network of fine threads such as is not usually seen in anaerobic cultures. On agar stab-cultures the growth is similar to that upon gela- tin, although not so pronounced. It takes place much more rapidly at the temperature of the body. Stab-cultures in a high layer of grape-sugar agar develop within from twenty-four to forty-eight hours close to the surface. They exhibit the character- istic odor, and are usually attended with abundant gas-formation. In glucose-bouillon growth at 37° C. (98.6° F.) is very ener- getic. On account of the abundant formation of gas it is well not to close the bouillon-flasks too tightly. The bouillon is at first rendered quite turbid ; after standing for weeks the bac- terial masses settle to the bottom as a grayish-white layer, so that on careful suction of the overlying clear fluid a solution of toxin free from bacteria is obtained. This may be secured with greater certainty by filtering the bouillon-cultures through porce- lain cylinders. The tetanus-bacillus develops in milk without causing any change. Upon potatoes a moist invisible deposit forms similar to that caused by typhoid-bacilli (Vaillard and Vincent). Tetanus in Animals. — Tetanus occurs under natural conditions in horses, sheep, and neat cattle ; it has not been observed in dogs, and especially not in fowl. The mouse is the animal best adapted for the experimental development of tetanus. Of an old bouillon-culture (freed of bacilli by filtration, or by careful decanting) o.ooi cu. cm., and frequently still much smaller amounts, suffice on subcu- taneous injection to cause death in white mice (from twelve to fifteen grams in weight) within twenty-four hours. The guinea-pig is almost equally susceptible. The rabbit is much less susceptible : not less than from o. 5 to i cu. cm. 234 CLINICAL BACTERIOLOGY. of the culture mentioned being required to cause tetanus in an animal weighing about looo grams, with death after the lapse of several days. The dog may be looked upon as being naturally immune to tetanus, being affected only on in- oculation with large doses of toxin : from 5 to lo cu. cm. and more. Birds are highly immune, withstanding inoculations of from 10 to 20 cu. cm. of highly toxic tetanus-bouillon ; still larger amounts of toxin, naturally, give rise to fatal tetanus both in the hen and in the pigeon. Frogs also may suffer from tetanus if after inoculation they are kept permanently in a heated room ; but relatively large doses of toxin are necessary, and the disease develops only after two or three weeks. Introduction of the tetanus-virus into a wound or injec- tion into the large cavities of the body or directly into the veins acts in the same way as subcutaneous inoculation. Experimental infection can not be induced by way of the digestive or the respiratory tract. The period of incubation varies in accordance with the susceptibility of the animal and the virulence and amount of toxin inoculated : between one and two days, in the mouse ; and between eight and fourteen days, in the rabbit. Tetanus in animals manifests itself in the form of extensor spasm, which gives rise to a clinical picture entirely analo- gous to that exhibited by tetanus in human -beings. The spasm appears first in the neighborhood of the site of inocu- lation ; thus, when the root of the tail is inoculated, in the hind extremities and also in the tail ; when the nape of the neck is inoculated, in the fore extremities and the muscles of the neck. In the further development of the disease the entire muscular system is involved in the spasm, which quickly leads to death. After intraperitoneal and intra- venous inoculation the spasm is general from the outset. The longer the period of incubation, the slower generally is the course of the disease, and the shorter the period of incubation, the more unfavorable is the prognosis. After the disease has existed for some time recovery not rarely takes place. This ensues always but slowly, the rigid extremities relaxing in the course of weeks or months. On postmortem examination, only slight changes are found at the site of in- oculation when a pure culture has been employed in the inoculation — viz., slight infiltration or hemorrhage, and nothing else. If the inoculation has induced no extensive TETANUS. 235 injury, the site of inoculation may escape observation at autopsy. If a mixed culture was inoculated — e. g., tetanus- infected earth, or pus or fragments of tissue from a wound in a case of tetanus — a focus of suppuration will be found at the site of inoculation. No changes are demonstrable in the remaining organs of animals dead of tetanus ; above all, tetanus-bacilli have never been found present, and, likewise, not in the blood. The bacilli have been demonstrated only at the site of inoculation, and in rare instances in the nearest adjacent lymph-glands ; but even in these places they are present only in small number, and they usually can be found only with great difficulty. The Nature of Tetanus. — The postmortem findings just described are explained by the fact that tetanus is an ex- quisitely toxic-infectious disease. The convulsions and death are due to a poison generated by the tetanus-bacilli and rapidly absorbed from the site of inoculation. The evidence in favor of this view has been derived from vari- ous sources. In the first place, exactly the same clinical picture can be developed with the germ -free filtrate of a tetanus bouillon-culture, which thus contains only the dis- solved chemic tetanus -toxin, as by means of the bacilli themselves. In the next place, Kitasato has inoculated mice with tetanus-bacilli at the root of the tail, and has excised and cauterized the site of inoculation throughout a large extent after one-half, one, and one and a half hours, and longer, so that there was no chance for the bacilli, which do not penetrate beyond the site of inoculation, to be retained within the body, and only the toxin absorbed could in any way be responsible for the further develop- ment of the disease. Only those animals operated on half an hour after inoculation remained well, while all of the others were attacked with tetanus. This means that as early as an hour after the inoculation so much toxin was absorbed that the bacilli were no longer necessary to the development of the attack of tietanus. The subordinate position taken by the bacilli in their sig- nificance for the clinical picture, as compared with the toxins to which they give rise, led Vaillard and Vincent to the conclusion that the bacilli, through themselves and their spores, are not alone capable of causing the disease, and that in a pure state they are entirely inactive ; and only the layer of toxin which adheres to them externally gives rise to the 236 CLINICAL BACTERIOLOGY. outbreak of tetanus. If this is removed, for instance by washing with large amounts of water or by exposure to a temperature of 65° C. (149° F.) or by growth of the cul- tures at a temperature of 20° C. (68° F.) or 22° C. (71.6° F.) — under which conditions the bacilli generate no poison for the first six days — such toxin-free bacilli will be in- capable of inducing tetanus." These views have in general been confirmed. The introduction of toxin-free bacilli does not, as a rule, give rise to infection ; but the spores freed of toxin reacquire their virulence, and tetanus will develop with certainty if with them there are introduced other bacte- ria not giving rise to tetanus, or if simultaneously trauma- tism is inflicted, or a splinter of wood is forced beneath the skin, or, finally, a chemic agent — for instance, lactic acid, trimethylamin, etc. — is injected at the same time. These experimental data are of the highest importance for a com- prehension of tetanus developing spontaneously. From them the probable conclusion may be drawn that under natural conditions tetanus is not induced by the tetanus- bacilli alone, but that the presence of favoring bacteria — thus a mixed infection — is necessary, or that a severe trau- matism, or the like, must attend the infection. The question has been studied experimentally as to how the extensor spasm arises in cases of tetanus, and whether the tetanus-toxin has a central or a peripheral action. Tizzoni and Vaillard divided all the nerves of an extremity in an animal before inoculation with tetanus. This member remained relaxed, whereas the remainder of the muscular system became rigid. Buschke curarized a tetanized frog, and the tetanus ceased at once. The toxin, thus, can not act upon the muscles themselves or upon the peripheral nerves. After removal of the brain the tetanized frog remained rigid. A direct influence of the poison upon the motor centers through application to the cerebral cortex was without effect upon rabbits. It thus appears that the brain also is not the part attacked by the toxin. Gradual destruction of the spinal cord in tetanized animals causes disappearance of the rigidity in the parts corresponding to the respective sections of the cord. Accordingly, the activity of the toxins appears to be localized in the spinal cord, as is the case also with strychnin. The Tetanus- toxin. — The germ-free filtrate of a teta- nus-culture from two to four weeks old that has been TETANUS. 237 grown upon feebly alkaline glucose-bouillon contains the specific tetanus-toxin in an exceedingly active state. Often as little as 0.000005 ^u. cm. of such a filtrate will suffice to cause death in a white mouse. The toxin is destroyed in solution by exposure to a temperature of 65° C. (149° F.) for five minutes, and it is gradually enfeebled in the thermostat. In a cold compartment (refrigerator) and protected from light it retains its toxicity unchanged for months. For the preservation of such a toxic filtrate it is advisable to add glycerin in equal amount, or carbolic acid in proportion of 0.5 per cent., and it may then be employed for months in the same way as the solution of a chemic poison of known strength. Efforts have not been wanting to isolate the toxin in a pure state. Brieger obtained from cultures two basic sub- stances— tetanin and tetanotoxin — both of which caused death in animals, preceded by manifestations like those of tetanus. Neither, however, can have any important bear- ing upon the occurrence of the disease, as relatively large amounts were required in order to induce disease in animals. The actual tetanus-toxin, however, must, in accordance with what is known regarding the toxicity of the bouillon- filtrate, be active in minimal amounts. Brieger and Frankel have, according to their method, by precipitation with alcohol, obtained a toxalbumin that is much more active. Even this albuminous powder does not represent the tetanus-toxin chemically pure ; the toxic substance is merely adherent to it. The attempts to obtain the virus in its chemic purity have been pursued unremittingly, and it has been obtained in a quite concentrated form. Reference was made in the general section (p. 30) to the investigations of Brieger and Boer, from which it appears that the toxin of the tetanus- bacillus is not an albuminous substance at all. These investigators precipitated the tetanus-toxin by the original toxalbumin-method of Brieger and Frankel with the aid of ammonium sulphate, and then by means of a weak solution of mercuric chlorid (from 10 to 20 cu. cm. of a 0.05 per cent, solution to 10 cu. cm. of tetanus-toxin fluid) precipitated the redissolved toxin. The carefully washed precipitate is well rinsed with water, and decom- posed by successive treatment with ammonium carbonate, ammonium phosphate, and ammonium sulphate. Brieger 238 CLINICAL BACTERIOLOGY. and Boer conclude the description of this rather complicated procedure with the following statement : If the readily- soluble specific toxin has been cleansed as thoroughly as possible and deposited upon the filter, it will be completely absorbed by hardened filters, and there will be nothing to suggest its presence except a little salt, or the yellowish discoloration of the filter, which is often quite considerable, especially in the case of the tetanus-toxin. The activity of these substances will, therefore, not be expressed by the sum of decimals, but to a certain degree only as integers. For quantitative studies of the tetanus-toxin the bouillon- filtrate appears for the present, therefore, to be the best suited. Tetanus-infection in Human Beings. — The portal of entry for tetanus-bacilli in human beings may be con- stituted by any lesion of the external integument. The tetanus-bacillus has been repeatedly demonstrated in the earth, especially in that which has been manured, and more particularly in the ' uppermost layers; in the dust of the crevices of floors ; in manure from horses and cows. If a certain amount of any of these substances be introduced subcutaneously in guinea-pigs, there result, usually, ma- lignant edema and, rarely, tetanus. This result depends upon the fact that the bacilli of malignant edema, frequently present in earth and in manure, overrun and suppress the tetanus-bacilli. In order to determine whether tetanus- bacilli are present in suspected material (earth, etc.), bouillon mixed cultures may be made, according to the suggestion of Sanfelice, and kept for a considerable length of time in the thermostat, and then filtered through porcelain cylin- ders. The results of injection of the filtrate will decide whether the material examined contains tetanus-bacilli or not. Tetanus in human beings is a toxic disease, just as is tetanus in animals. In human beings also the bacilli never enter the blood or the viscera, but remain confined to the site of original infection. On the other hand, the presence of the tetanus-poison in the blood in cases of tetanus has been demonstrated by successful intoxication of mice with the blood-serum from such cases. Likewise, a substance has been obtained from the liver, the spleen, and the spinal cord of a patient dead of tetanus by precipitation with al- cohol, and which, dissolved in water, is capable of destroying TETANUS. 239 small animals immediately, after the development of tetanic manifestations. Tetanus-toxin has been found on several occasions in the urine of tetanus-patients, but never in the sweat or in the saliva. The portal of infection is ascertainable in the majority of cases. Often it is a wound of considerable extent, which can not escape observation — as in puerperal tetanus the wounded surface of the uterus, in tetanus of the new-born usually the umbilical wound, in cephalic tetanus (hydro- phobic tetanus) an injury of the head. The source of infec- tion, also, may often be discovered without difficulty. Not rarely a relation between the patient and horses can be elicited, as these animals, according to Verneuil, occupy a central position in the etiology of tetanus and they may convey the tetanus-bacillus to the ground with their manure ; or the patient may have introduced beneath the skin a splinter to which the bacilli were adherent ; or in working about a garden he may have contaminated an already existing wound with earth ; and the like. It should here be mentioned that tetanus-virus retains its virulence for an exceedingly long time. An observation has been recorded in which a splinter of wood that had already given rise to infection again caused tetanus after the lapse of eleven years. Bandages and dressings from tetanus-wounds likewise preserve their infectivity for a long period, and tetanus-cadavers also retain for a considerable time their capability of causing tetanus. Direct contagion has also been observed, tetanus having been transmitted in a hospital-ward from one patient to his neighbor, or several patients occupying successively the same bed each developing tetanus. In some cases, however, the portal and the mode of infection are ascertainable with difficulty. Cases have been reported in which operation-wounds, after antiseptic treatment, healed by primary union and without complication, and later' tetanus developed {cicatrix-tetanus). Further, there occur cases of so-called idiopathic or rheumatic tetanus in which apparently no lesion exists anywhere, and also at autopsy no suppuration can be found. The information gained from experiments on animals furnishes a complete explanation for these cases. The portal of entrance may be the smallest abrasion or fissure of the skin, which will escape observation, and which perhaps has already healed some time before the 240 CLINICAL BACTERIOLOGY. convulsions occur. If the infection is single — that is, if pyogenic cocci do not gain entrance into the wound at the same time as the tetanus-bacilli — there will be no focus of suppuration at autopsy to indicate the portal of infection. In an animal experiment recorded by Vaillard spores healed without reaction in the wound induced artificially, and only some time later, when the affected member was irritated, did tetanus occur. The cases of tetanus that occur in connection with wounds treated antiseptically are suscep- tible of a similar interpretation. The tetanus-bacillus may long remain latent ; the antiseptic employed not being power- ful enough to destroy the tetanus-spores with which the wound is infected, and these are included in the healing wound, to proliferate and to induce the disease later in con- sequence of some predisposing influence. The period of incubation of tetanus in human beings varies from one to twenty-two days ; in the case of an injury induced in the laboratory with tetanus-toxin it was four days. The course of the disease is the more violent, and the prognosis the more unfavorable, the shorter the period of time that has intervened between the infliction of the injury (infection) and the outbreak of the disease. Recovery took place in only slightly more than three per cent, of cases with an in- cubation-period of from one to ten days ; in twenty-five per cent, of those with an incubation-period of from ten to twenty -two days ; and in as high as fifty per cent, of those with longer periods of incubation. The susceptibility of human beings to the toxin of tetanus must, therefore, be looked upon as pronounced. Bacteriologic Diagnosis. — If it is desired to demonstrate the bacilli in the pus, or in the granulation-tissue of the wound in a case of tetanus, or if a specimen of earth or a splintef of wood is to be examined for the presence of tetanus-bacilli in order to discover the source of infection, the suspected material may be directly introduced into a pocket of skin at the root of the tail of a mouse. If the animal dies of tetanus in the course of a few days, the pus at the site of inoculation is treated in the manner described for the purpose of obtaining a pure culture (p. 232). The material to be examined may also be intro- duced into bouillon, through which a current of hydrogen is passed, and which is then placed in the thermostat for from three to five days. The mixed culture that has TETANUS. 241 developed in the bouillon is exposed over a water-bath for an hour at a temperature of 80° C. (176° F.), and from it a new anaerobic bouillon-culture is made at once. If after several days' development this culture still contains foreign bacteria, anaerobic plates are prepared. Immunity and Cure of Tetanus in Animals. — The laws of immunity and immunization have been more thoroughly studied in tetanus than in any other disease. The reason for this resides, in the first place, in the exceedingly sharp and certain reaction of animals to tetanus-infection, and, in the second place, in the possibility of testing quantitatively the degree of immunity by means of treatment with the toxic bouillon-filtrate. Immunity to a toxic-infectious disease is indicative of proof to intoxication : an animal is protected from infec- tion with tetanus if the tetanus-toxin is incapable of mani- festing its toxic activity within the body of the animal. Animals slightly susceptible (dog, hen) can be further im- munized simply by injection of gradually increasing amounts of tetanus-toxin. Their serum, in the hen, likewise the egg- yolk, also acquires thereby immunizing properties. From the serum of dogs subjected to such preliminary treatment Tizzoni and Cattani have precipitated their so-called anti- toxin by means of alcohol. Behring's method of immunization by means of tetanus bouillon-cultures attenuated with iodin trichlorid is applica- ble to the immunization of susceptible animals (mice, rab- bits, horses, sheep). The animals are treated first with a bouillon-culture containing 0.25 per cent, iodin trichlorid, and, finally, with undiluted bouillon, of which the animals — smaller at intervals of from three to five days, and larger at intervals of eight days — then receive steadily increasing doses, until, finally, they bear quite large amounts of pure toxin. A fluid suitable for immunization may be secured also by warming the cultures (Vaillard heats the filtrate at first to 60° C— 140° R, later to 55° €.—131° F., and, finally, to 50° C. — 122° F.) ; further, by addition of iodin-water or of lactic acid to the cultures, by cultivation of the bacilli in thymus-bouillon, etc. The serum of immunized animals transmits immunity to animals not previously treated ; the higher the original degree of immunity, the more active is the blood-serum. 16 242 CLINICAL BACTERIOLOGY. This acts by rendering the animal toxin-proof; it contains an antitoxin that enters into innocuous combination with the tetanus-toxin. Also after infection, and even after the development of symptoms of tetanus, the serum of the animal may afford protection ; it thus exerts a curative action. To effect cure in animals, however, a much larger amount of serum, or a much more highly active serum, is required than for pro- phylactic immunization ; and the amount of serum required becomes the greater the longer the period of time that has intervened between the intoxication and the employment of the serum. According to observations of Donitz, little more serum is required for the protection of the animal four minutes after the intoxication than in the test-tube for the neutralization of the amount of toxin employed (in the test-tube a serum-dilution of i : 2000, in the body after four minutes a dilution of i : 1 200) ; after the lapse of eight minutes six times the amount of serum will be required ( I : 200) ; after the lapse of fifteen minutes twelve times the amount (i : 100) ; and after an hour twenty -four times the amount of serum. Serum-therapy in Human Beings. — It is obvious that the outlook for serum-therapy in the case of tetanus in human beings is from the outset less promising than it is in that of diphtheria. As it can not be determined from inspec- tion that a wound is infected with tetanus-bacilli, the disease comes under medical observation only after the toxin has already invaded the central nervous system, and has induced more or less extensive and in part perhaps irreparable injury. The cases of tetanus, therefore, thus far treated with serum have not, on the whole, yielded good results. The expla- nation for this may be found in the weakness of the serum ; in view of the lateness of application of the treatment the serum should be of especial strength, Behring has, there- fore, and also in conjunction with Knorr, sought mainly to increase the activity of the serum. To obtain tetanus-toxin he employs horses, which are immunized in the same way as to diphtheria-toxin (p. 218). The tetanus normal toxin, Tet N T^, with which Behring starts out, is so strong that one gram contains 1,000,000,000 lethal minimal doses for one gram by weight of guinea-pig (-f 1,000,000,000 M), 1 50,000,000 lethal doses for one gram by weight of mice (-{- 150,000,000 Ms), 1,000,000 lethal minimal doses for TETANUS. , 243 one gram by weight of rabbit (-{- 1,000,000 K). That serum serves provisionally as tetanus normal antitoxin, Tet AN^, whose curative activity Behring and Knorr demonstrated at a meeting of the Physiologic Society in Berlin, on January 13, 1893. This serum, injected on several days successively in doses of 0.04 cu. cm., cured mice that had been infected from twenty -four to twenty- eight hours previously with the lethal minimum dose, and that already presented distinct symptoms of tetanus. One cubic centimeter of this serum contains one normal anti- toxin-unit. For the future it is, however, probable that that serum will be designated as normal antitoxin of which I cu. cm. will render innocuous i cu. cm. of tetanus nor- mal toxin. The Hochst Works have placed two preparations on the market. The first occurs in the form of a dry powder packed in 5 -gram vials. This antitoxin has 100 times the strength "of the normal (Tet AN ^^^) ; each vial thus con- tains 500 tetanus normal antitoxin-units. The dried serum keeps well, without addition of an antiseptic, in well-closed bottles, and remains of uniform strength. The 500 anti- toxin-units constitute a curative dose for human beings and horses. They are dissolved in 45 cu. cm. of sterile, luke- warm water, not above 40° C. (104° F.), and are injected, in the case of horses, directly into a vein. By introduction into the blood-stream a much more energetic action is obtained, and this, in addition, appears twenty-four hours earlier than after subcutaneous inoculation. For this reason, Behring and Knorr recommend also in human beings, in urgent cases, that the injection be made into a vein. Recovery may be expected after subcutaneous injec- tion in acute cases only when the serum is employed before the lapse of the first thirty -six hours after the symptoms of tetanus have set in. The time lost is less readily com- pensated for by increase of the dose in the case of tetanus than in that of diphtheria. The second preparation dispensed by the Hochst Works is in solution in vials of 5 cu. cm. ; i cu. cm. contains 5 normal antitoxin-units — the equivalent of five times the normal antitoxin (Tet AN^). As in the case of the diph- theria-antitoxin, o. 5 per cent, of carbolic acid is added to prevent decomposition. This dissolved antitoxin may be employed prophylactically in human beings and animals 244 CLINICAL BACTERIOLOGY. after such injuries as experience has shown may be followed by tetanus. The size of the dose (from 0.5 to 5 cu. cm.) is governed by the period of time that has elapsed since the infliction of the injury. If it be desired to make an injection of antitoxin in advance of an operation that is frequently followed by tetanus in animals — as, for instance, preceding castration — 0.2 cu. cm. will suffice. ' Recent observations have afforded noteworthy support for the justification and the utility of serum-therapy with relation to tetanus. Donitz established the fact in the Institute for Serum-therapy that the tetanus-toxin, as soon as it enters the blood, is taken up by the tissues of the body, and that, in cases of severe intoxication, the simple lethal dose is bound within from four to eight minutes. The serum, however, separates the toxin from its combination with the tissues, and neutralizes it. The dissolution of the toxin-combination is effected with the greater difficulty the severer the intoxication and the longer the period of time that has elapsed before the serum is employed. Of especial significance is the discovery by Goldscheider and Flatau that certain degenerative processes affecting nerve-cells that are caused by tetanus-intoxication recede under the influence of the serum. We have thus anatomic evidence for the curative activity of the serum.* As to the results of tetanus-therapy, a definite opinion can not yet be given, as the new preparations have been generally available for too short a time. As bearing upon the value of prophylactic inoculations in veterinary medicine, we have the statistics of Nocard, covering the period from August I, 1895, to June i, 1897. Altogether, 2707 animals were vaccinated. Of this number, 2300 received an injec- tion of serum immediately after the operation (castration, amputation of the tail, etc.). Not one of these animals developed tetanus. The remainder were inoculated from one to four days after the operation or after the infliction of a traumatism. Only one horse exhibited symptoms of tetanus, but these rapidly subsided. Nocard considers these results as quite remarkable, as the animals were derived from stables in which tetanus had prevailed shortly before. On the other hand, the sixty -three veterinary surgeons who *The results appear, further, to be more promising when the serum is in- troduced beneath the dura mater than after any other method of introduction. —A. A. E. BOTULISM. 245 had furnished Nocard the material in question, observed 259 cases of tetanus in the same time in animals that had not been inoculated. BOTULISM. In spite of numerous investigations with regard to the exciting agent of meat-poisonirig this whole question was involved in considerable doubt until recently, when, through the discovery by Van Ermengem, in an epidemic at EUe- zelles (Belgium), of a specific anaerobic microbe, the bacillus botiillnus, renewed attention was directed to this important subject. Under the designation of meat-poisoning are included two entirely distinct symptom-complexes, which should properly be rigidly differentiated from each other. The one variety, which is most appropriately designated the gastro-intestinal, simulates the clinical picture of cholera nostras — a simple or hemorrhagic gastro-enteritis. Among associated manifestations may be mentioned fever, albumin- uria, and cutaneous exanthemata of most varied kind and in- tensity. These gastro-intestinal lesions arise after the inges- tion of decomposed meat or of meat derived from diseased animals, the diseases especially concerned being pyemia, septicemia, and puerperal fever. In most cases of this kind the exciting agents are bacteria of the colon-group, and in other cases, occurring less commonly, bacteria of the proteus-group. The second variety is identical with so-called sausage- poisoning. It is characterized especially by nervous symp- toms of central origin, secretory and motor disturbances, cessation of salivary secretion, dryness and redness of the mucous membrane of the mouth and pharynx, dysphagia, hoarseness, barking cough, paralysis of accommodation, mydriasis, ptosis, diplopia, etc. For this form of the dis- ease the designation meat-poisoning should no longer be employed, and it has been given the name of botulism. Botulism may arise after the ingestion of special kinds of sausage — ^as, for instance, blood-sausage or liver-sausage — which are prepared particularly in certain parts of Wiir- temberg and Baden. It is further caused by decomposed salt fish, by smoked meat, ham, preserved meat, venison, old roasts, and the like. These are articles of food that 246 CLINICAL BACTERIOLOGY. are intended for consumption after a considerable length of time, and that, according to Van Ermengem, are, by reason of their mode of preparation, exposed to the danger of undergoing anaerobic fermentative processes. Van Ermen- gem believes the actual cause to be the anaerobic bacillus botulinus, which he isolated in the epidemic at EUezelles from a piece of ham that gave rise to typical cases of in- toxication. As opportunity has not been afforded since Van Ermengem made his communication for investigating cases of botulism, the following description will closely follow his statements. Fig. 54. — Bacillus botulinus ; eight-day culture in glucose-gelatin ; X 1000 (Van Ermengem). Morphology of the Bacillus Botulinus (Fig. 54). — The bacillus is a large, slightly motile rod, from 4 to 6, a long and from 0.9 to 1.2 ;tjt thick, with slightly rounded extremities, and possess- ing from 4 to 8 flagella. The formation of filaments is but rarely observed ; more frequently involution-forms are present, the bacilli becoming smaller, presenting deficiencies, and being at times arranged in filaments. Both in cultures and in the body the bacillus botulinus generates spores of oval form, gen- erally polar, rarely central, and exceeding the thickness of the bacillus. The bacillus is readily stained, and also by Gram's method, but the exposure to alcohol must not be too protracted. Cultural Peculiarities. — The temperature-optimum is be- tween 20° C. (68° F.) and 30° C. (86° F.). Below a temper- ature of 16° C. (60.8° F.) the bacillus botulinus grows but slowly, while above 35° C. (95° F.) it no longer generates spores, does not itself thrive so well, and exhibits involution- BOTULISM. 247 forms. The organism is strictly anaerobic, so that, in order to cultivate it, anaerobic methods of investigation must be pursued. The culture-media must always be distinctly alkaline, and the growth is favored by the addition of two per cent, of grape- sugar. Gelatin-plates exhibit between the fourth and the sixth day round, transparent, brownish-yellow colonies, composed of thick, glistening granules, in constant movement. The colonies are surrounded by a slight zone of liquefaction. Later, the margin becomes irregularly radiate, and, finally, processes of the most varied form extend out from it. High gelatin stab-cultures exhibit no peculiarity. Round, whitish masses develop along the line of puncture, at times sending processes out into the adjacent gelatin. The gelatin is not liquefied, but active gas-formation takes place. In agar stab-culture the growth, apart from liquefaction, is quite similar. Glucose-bouillon is rendered deeply turbid. In milk slight development takes place, without alteration in the nutrient medium. On potato no growth takes place. All of the cultures emit an odor of butyric acid. In addition the bacillus generates in nutrient media containing glucose still other fatty acids, butyl alcohol, hydrogen, carbon dioxid, and methane. Tenacity of the Bacillus Botulinus. — The cultures retain their capability of development for more than a year if they are kept at a temperature below 30° C. (86° F.). At temperatures above 35° C. (95° F.) they die in the course of a few weeks. The spores possess relatively little resistance. They are destroyed at a temperature approxi- mating 85° C. (185° F.) within a quarter of an hour, and at a temperature of 80° C. (176° F.) with certainty within an hour. Five per cent, carbolic acid causes their destruc- tion in less than twenty-four hours. Dry spores exposed to diffuse daylight exhibit capability of germination after three months. The growing forms in emulsion with distilled water die under these conditions within three or four weeks. Pathogenic Properties. — The bacillus botulinus proves pathogenic when fed to guinea-pigs, mice, and monkeys. One or two drops of a liquefied gelatin-culture placed upon a bit of bread or in milk suffice to cause the death of the animal within one or two days, after paretic manifestations. 248 CLINICAL BACTERIOLOGY. mydriasis, aphonia, dysphagia, and blepharoptosis. Cats withstand the action of considerable amounts of the cul- ture without injury when introduced by the stomach ; but subcutaneous injection, on the other hand, leads to death amid such typical manifestations that the cat may be con- sidered as the physiologic reagent for the bacillus botulinus. After considerable doses (of from five to ten cubic centi- meters) cats die within one or two days, but after smaller doses (of from one to two cubic centimeters), only after from eight to twelve days. After an incubation-period gen- erally of thirty-six hours the animal becomes depressed, no longer moves about, and refuses nourishment. On the third day it exhibits a peculiar physiognomy. The facial expression is dull, and the blinking and licking move- ments that healthy cats never fail to exhibit are abol- ished ; the eyes are almost completely immobile, and the pupils widely dilated. The dilatation of the pupils be- comes, in the course of a few days, quite enormous. The tongue hangs out of the mouth and can scarcely be drawn in again. Besides, aphonia sets in and, further, dysphagia, which finally may increase to total aphagia. Urine and feces are retained. Death occurs usually in consequence of paralysis of respiration and circulation. Small doses of the bacilli give rise to a form of marasmus, as a result of which the cats die after the lapse of several weeks amid paralytic manifestations and with degeneration of the parenchymatous organs. Rabbits, guinea-pigs, and mice die in consequence of subcutaneous injection of minimal amounts, amid pa- retic manifestations, salivation, dysphagia, etc. After injec- tions of one or two cubic centimeters of the culture pigeons exhibit at first paresis of the wings, and then general par- alysis. Intravenous injection leads to the same results as subcutaneous inoculation, while intraperitoneal injection is followed by less marked consequences. The pathologic-anatomic alterations found at autopsy consist in more or less marked hyperemia of most of the viscera ; in an acute, sometimes interstitial, sometimes par- enchymatous, hepatitis, with fatty degeneration ; in des- quamative parenchymatous nephritis ; in fatty degenera- tion of the muscular fibers of the heart and of the ocular muscles. Of especial interest are the degenerative changes in the central nervous system, which are especially marked in the spinal cord, and less so in the medulla oblongata. BOTULISM. 249 These are entirely wanting in the central nerves, and almost entirely in the brain. In the spinal cord the gray substance is involved almost exclusively, and particularly the anterior horns ; in the medulla oblongata the nuclei of the hypo- glossus, the dorsal nucleus of the vagus, the middle small- cell nucleus of the oculomotor nerve — in short, the nuclei of the cerebral nerves affected. Physiology of Botulism. — Microscopic examination of the organs and of the blood of animals dead of botulism fails to disclose the presence of the bacilli anywhere ; even at the site of injection they are to be found in only small numbers after the lapse of three or four hours, and then distinctly in process of degeneration. After intravenous injection, cultures prepared from the fresh organs exhibit, as early as twelve hours, only a limited number of colonies. If, however, these organs are placed in the thermostat at a temperature of 30° C. (86° F.), numerous microorganisms can be cultivated from them after the lapse of from twelve to twenty-four hours. Botulism, therefore, represents essen- tially an intoxication and not a true infection. If the cul- tures are freed by filtration of the bodies of the bacilli, and if animals are inoculated with the toxic solution thus ob- tained, precisely the same morbid manifestations are induced as follow inoculation of the bacilli themselves. Van Er- mengem attempted, in the same way as Vaillard and Rouget in the case of tetanus, to free the spores of the layer of toxin surrounding them by washing them in water, and he found that the spores thus treated were much less active. He was unable, however, to free the spores entirely of the toxin, and he, therefore, came to the conclusion that the protoplasm of the microbes must retain a certain amount of toxin. Alkalies, and even sodium bicarbonate, destroy the toxin of botulism. If spores are permitted to macerate for a day in an alkaline solution that is so feeble as not to destroy their germinating capability — as, for instance, a saturated solution of sodium bicarbonate — and are then exposed for two hours to a temperature of 50° C. (122° F.), and animals are inoculated therewith, the latter continue to live. The spores, whether introduced under the skin or into the stomach, are incapable of multiplying and generating toxin ; outside the living body, however, the spores retain the capability of generating their toxin in ordinary strength.. 250 CLINICAL BACTERIOLOGY. Cultures made with them also prove equally toxic with those from spores not exposed to the action of an alkaline solution and of heat. The influence to which is due the fact that the bacillus botulinus is incapable both of multi- plying and of generating toxic metabolic products within the animal body is not yet thoroughly understood. Per- haps it is due to a high degree of saprophytism, which does not permit the microbe to adapt itself to the animal body ; and, perhaps, further, to the fact that the bodily tem- perature, which is above 35° C. (95° F.), is not favorable to its development. It has been shown experimentally that, at a temperature of 37° C. (98.6° F.) and above, involution- forms occur, and no toxin is generated. The bacillus botu- linus thus occupies a position of its own. It is pathogenic for human beings only by reason of its toxin, which it forms outside the body upon dead substances (articles of food). Van Ermengem proposes that it be designated as toxico- genic. The toxin of botulism is to be placed upon the same plane as that of diphtheria and that of tetanus. It can be obtained by precipitation with alcohol, tannic acid, and neutral salts. It was further separated by Brieger and Kempner by treating the toxin-containing filtrate of the culture according to the method proposed by Brieger and Boer for diphtheria and tetanus. Occurrence of the Bacillus Botulinus. — The organism has hitherto never been observed apart from its discovery by Van Ermengem in ham and in the body of a patient dead of botulism. Mixed Infection. — The associated bacteria that were found together with the bacillus botulinus appear neither to favor nor to inhibit the generation of toxin. Bacteriologic Diagnosis. — Articles of food that have given rise to botulism are examined by means of culture (plate -procedure) for the presence of anaerobic bacteria. In the case of Van Ermengem the spores of the bacillus botulinus could be demonstrated in the ham microscopically. These were present principally in the red portion of the ham, and in smaller number in the fat. They were irregularly distributed, and were entirely absent in some places. In addition, it is well to inoculate animals with an aqueous maceration (four parts of chopped ham and five parts of water). In the bacteriologic examination of the TUBERCULOSIS. 251 bodies of individuals dead of botulism or of animals that have succumbed to injection of the maceration, it is to be borne in mind that the viscera and the blood contain the bacilli only in small numbers. It is, therefore, necessary to examine large quantities, or to place entire organs in the thermostat at a temperature of from 20° C. (68° F.) to 30° C. (86° F.), in order to bring about fertilization. Van Ermengem has succeeded in isolating the bacillus from the spleen and from the gastric and intestinal contents from the body of a patient dead of botulism. Immunity and Specific Therapy. — Immunization of animals to botulism was effected by Kempner with the filtrate of a bouillon-culture in the same way as in the case of diphtheria and of tetanus. The serum of immunized animals is highly antitoxic — a further evidence of the fact that the toxin of botulism is closely related to that of diphtheria and that of tetanus. When Kempner injected his serum in doses of from one to five cubic centimeters within from three to twenty-four hours after inoculation of guinea-pigs, the animals survived. As investigations of Kempner and Pollack show, changes in the central nervous system have already taken place after the intoxication has existed for twenty-four hours. These changes in the nerve-cells, as the anatomic evidence seems to show, are neutralized by the serum. TUBERCULOSIS. The exciting agent of all tuberculous processes is the tubercle-bacillus discovered and cultivated by Robert Koch in the year 1882. The first evidence of the infectious nature of tuberculosis was furnished in 1865, by Villemin, who rendered healthy animals tuberculous by inoculation of tuberculous material. These ob- servations were confirmed by Cohnheim, and they were supple- mented by inoculation into the anterior chamber of the eye. Cohnheim, upon the basis of his experiments, formulated the doctrine of the specific etiology of tuberculosis, and this opinion was crystallized into absolute certainty by the classic bacteri- ologic investigations of Koch (**Arbeiten aus dem Reichsge- sundheitsamt," 11). Morphology and Staining of Tubercle-bacilli. — The tubercle-bacilli are delicate, slender rods, from 0.2 to 0.4 /a 252 CLINICAL BACTERIOLOGY. thick, and, on the average, from 3 to 4 ix long. They are slightly curved, nonmotile, and as a rule lie singly, but in cultures at times in small chains of from 4 to 6. In rare cases they exhibit bulbous terminal enlargement and bifurcation, in consequence of which a certain relationship with the actinomyces-group (p. 25) is suggested. Tubercle-bacilli are distinguished from all other bacteria by the fact that they stain with extreme difficulty, but, having once taken up the stain, they retain it with great tenacity. The simple solutions of aniline dyes ordinarily employed for staining other bacteria do not suffice for tubercle- bacilli unless the exposure be very prolonged. For this reason tubercle-bacilli are stained with aniline-water staining solutions (Koch-Ehrlich stain) or with the more commonly employed carbolfuchsin (Ziehl stain). Upon cover-slip preparations, made in the usual way from pure cultures and fixed, freshly prepared aniline-water fuchsin / ^ 1 \ Fig- 55- — Tubercle-bacilli: i, Forms suggesting sporulation ; 2, forms described as beaded (the open spaces in the fragmented rods are sometimes mistaken for spores). (or aniline-water gentian-violet or aniline-water methyl-violet), or carbolfuchsin is dropped ; then heat is applied by means of a small gas-flame or spirit-flame until vapor of steam arises, and after a minute the excess of stain is washed off with water. The tubercle-bacilli are now stained, and if the specimen is treated for several seconds with dilute acid — for instance, fifteen or twenty per cent, nitric acid — and alcohol — from 60 to 70 per cent. — they will not yield up their color. Stained in this way the tubercle-bacilli frequently exhibit bright deficiencies that have not taken the stain. These were at first looked upon as spores, and, later, as degenerative manifestations, but both views are incorrect. Originally, the tubercle-bacilli were stained in cold solutions, in which they were permitted to remain for several hours ; but by application of heat the duration of the exposure can be re- duced to a few minutes. The cause of this characteristic behavior in staining was con- TUBERCULOSIS. 253 sidered by Ehrlich to depend upon the fact that the tubercle- bacilli possess an especially resistant cell-memdrane, which permits the stain to enter the body of the cell only with the aid of mordants (aniline water, carbolic acid, etc.). This membrane, later in the process of decolorization, prevents the entrance of acids into the interior of the bacillus, so that the microorganisms do not give up their stain. The tubercle-bacil- lus can be stained by Gram's method. According to recent investigations by Robert Koch, the tubercle-bacilli contain two unsaturated fatty acids, one of which is soluble in dilute alcohol, and is saponified by sodium hy- droxid ; whereas the other is not saponifiable, and is soluble only in boiling absolute alcohol and ether. Both fatty acids take the specific stain of the tubercle-bacilli ; but as one of them is soluble in alcohol, only the other remains after decolorization, and this fixes the stain and must be looked upon as the medium of the color-reaction. By means of hot sodium hydroxid the fatty acids may be slowly driven out of the bodies of the bacilli and their escape in the form of tingible drops uniting into larger drops can be observed under the microscope. According to Koch, these fatty acids form a coherent layer within the bodies of the bacilli, to which they afford protection against external influences. Culture of Tubercle-bacilli. — Pure culture of tubercle- bacilli is difficult, and mainly because the microorganisms develop very slowly and require a temperature of 37° C. (98.6° F.) for their growth. Their temperature-minimum is 29° C. (84.2° F.), their temperature-maximum 41° C. ( 105.8° F.), and their temperature-optimum 37° 0.(98.6° F.) or 38° C. (100.4° F. ). Tubercle-bacilli thrive well upon blood- serum, upon from four to six per cent, glycerin -agar, and upon glycerin-bouillon. The preparation of glycerin-agar plates for the purpose of isolating tubercle-bacilli from the mixture of bacteria in tuberculous sputum is scarcely possible, as the tubercle- bacilli develop so slowly that they are overgrown and suppressed by the colonies of the other bacteria. Their cultivation will, therefore, be successful only if the material examined is uncon- taminated. To this end the following plan of procedure is pursued : Several guinea-pigs-^animals that are extremely sus- ceptible to tuberculosis — are inoculated with material containing tubercle-bacilli. After the lapse of about four weeks the first of the inoculated animals will die, the autopsy revealing marked tuberculosis of the abdominal viscera. One of the remaining guinea-pigs is now killed, its skin cleansed most carefully by means of hot water and i : 1000 solution of mercuric chlorid, the skin turned back with a knife sterilized in the flame, the peritoneum opened with another instrument similarly treated. 254 CLINICAL BACTERIOLOGY. and the spleen is drawn forward with forceps sterilized in the flame, as this organ appears to be involved in greatest degree in this mode of infection. A bit of the spleen containing a tuber- culous nodule is excised with sterilized scissors, the nodule is compressed between two aseptic scalpels or glass slides, in order to set the tubercle-bacilli free, and the material thus obtained is transferred by means of a strong platinum wire to the surface of blood-serum tubes. The whole procedure must be carried out with the utmost celerity and with the most scrupulous cleanliness, for should a foreign microorganism gain entrance into the serum- tube, it will soon overgrow the tubercle-bacilli. For the vSake of greater security, several tubes are always treated in the manner described. As the tubes must be kept for a long time in the thermostat at a temperature of 37.5° C. (99.5° F.), they are closed with rubber caps that have been sterilized in mercuric- chlorid solution. After the lapse of fourteen days, if the cul- ture be successful, the first signs of growth are observable in the serum-tubes. In the neighborhood of the expressed material there form gray, dry, small scales, which, with low powers of the microscope, appear to be made up of delicate curved lines. Development then progresses slowly, and after the lapse of from four to six weeks it is possible to continue inoculations from this culture. For further culture blood-serum tubes likewise are used. Also in this second generation growth is first observed distinctly only after the lapse of two weeks. Subsequent generations, usually after the fifth or sixth, grow more vigorously and more rapidly; and, particularly when a special thermostat is employed and is saturated with steam, so that the rubber caps can be dispensed with, the entire surface of the serum, after from seven to fourteen days, is found strewn with the characteristic dry scales. From the fifth serum-generation trans- ference to glycerin nutrient media may be readily effected. Upon gfycerin- agar develoipment is much more abundant than upon blood-serum. The bacilli form upon this a grayish, dry deposit of coarse particles, with the same wavy, slightly raised outline. This coating extends downward, and envelops the water of condensation present, without, however, rendering it turbid ; and if the culture is kept long enough in the thermostat, the deposit grows even upon the free surface of the test-tube, where no nutrient medium is present, and for a considerable distance upward. As a fluid culture-medium veal-bouillon with six per cent, gly- cerin is preferably selected, and this is poured into Erlenmeyer flasks. In inoculating bouillon the dry scales must be intro- duced into the nutrient fluid in such a way that they float upon its surface. The tubercle-bacilli have a strong avidity for oxygen, and they develop luxuriantly only where air has suffi- TUBERCULOSIS. 255 cient access. Upon veal glycerin-bouillon the tubercle-bacilli grow in the form of a superficial membrane, which exhibits the same characters as the deposit upon glycerin-agar. In this also, after the lapse of weeks — under favorable conditions, after ten days — they reach the walls of the vessel, and likewise grow for a certain distance upward upon the glass. The underlying bouillon remains perfectly clear — a feature that is characteristic of the growth of tubercle-bacilli. Upon potatoes whose lower extremity projects into a solution of glycerin (5 per cent.) and sodium chlorid (0.5 per cent.), the tubercle-bacilli grow exceedingly well. Upon the surface of the potato they form a dense deposit. The glycerin-solution remains clear, and it is covered by the well-known coating. In the preparation of such cultures it is best to employ the cyl- inders of potato recommended by Roux. In the year 1892 Kitasato described a procedure suggested by Koch for obtaining tubercle-bacilli directly from the sputum of tuberculous patients. The patient should rinse his mouth care- fully with an antiseptic gargle, and then expectorate into a ster- ilized double dish. One of the masses of sputum ejected is washed in several sterilized dishes containing sterile water, and is thus freed from the bacteria adherent to its surface. From the central portion of such a mass a flake is removed, by means of a platinum wire, and smeared upon blood-serum in tubes. The cultures that develop under these conditions exhibit quite a different appear- ance than do those cultivated from the animal body. There develop round, and rather whitish and translucent colonies, which in subsequent generations grow in precisely the same manner as the bacilli obtained by the method first described. Resistance of the Tubercle-bacilli. — Tubercle-bacilli are destroyed only after exposure for ten minutes to a tem- perature of 70° C. (158° F.) ; for one minute, at a temper- ature of 95° C. (203° F.) ; for an hour, at a temperature of 60° C. (140° F.) ; and for four hours, at a temperature of 55° C. (131° F.). They are, thus, more resistant to heat than most other varieties of vegetative bacteria, which are generally destroyed by brief exposure to temperatures between 54° C. (129.2° F.) and 60° C. (140° F.). Tubercle- bacilli resist for only a relatively short time the influence of direct sunlight — in accordance with the density of the bacterial mass, from several minutes to several hours. Diffuse daylight causes death after the lapse of a week. Tubercle-bacilli can be cultivated for many years through a series of generations, without loss of vitality, although they gradually become somewhat less virulent. 256 CLINICAL BACTERIOLOGY. Spontaneous and Experimental Tuberculosis in Ani- mals.— Spontaneous tuberculosis occurs in animals, with frequency only among cattle and in monkeys living in captivity, although there is scarcely a domestic animal that is immune to tuberculosis. Among laboratory-animals the guinea-pig is the most susceptible, a few tubercle-bacilli sufficing to induce infection in them. Next in suscepti- bility is the rabbit, and then the field-mouse. Less sus- ceptible, though by no means immune, are white mice and dogs. Young animals exhibit a much greater predisposi- tion to tuberculosis than old animals. Typical tubercu- lous disease is induced experimentally in animals (guinea- pigs, rabbits, field-mice) by means of subcutaneous injec- tion, by inoculation of the anterior chamber of the eye, by intrapleural, intraperitoneal, and intravenous injection, or by inhalation of moist tubercle-bacilli in the form of dust. The last remaining portal of entry for tubercle-bacilli into the organism, the gastro-intestinal tract, also was utilized suc- cessfully by Koch in experiments on animals. Susceptible animals that are given tubercle-bacilli in considerable num- ber with their food die of intestinal tuberculosis. The re- sulting tuberculous process is, apart from the results of intravenous injection, at first always local, restricted to the site of infection ; it extends, however, steadily, but slowly, by way of the lymph-paths. The bacilli quickly gain entrance into the lymph-glands nearest the portal of infection. As early as three days after inoculation of tuberculous material into the anterior chamber of the eye Baumgarten found that the bacilli had advanced as far as the auricular lymph-glands. When the bacilli are injected into a vein, general miliary tuberculosis takes place at once. Of great assistance in the comprehension of some tuber- culous local processes in human beings are the observations of Schiiller, who inoculated tuberculous material at some indifferent portion of the body of the animal and then in- flicted traumatism in the neighborhood of the knee-joint, and found that the infection localized itself at this point. The chronic cold abscesses, which upon bacteriologic ex- amination are found free from pyogenic microbes, are cer- tainly to be attributed to tubercle-bacilli. Dead tubercle- bacilli, destroyed by live steam or by other means, exhibit pyogenic activity experimentally. They are positively chemotactic : they attract leukocytes. The pyogenic sub- TUBERCULOSIS. 257 Stance contained within the bodies of the bacilli can be extracted only with great difficulty. If dead tubercle-bacilli are inoculated into rabbits by in- travenous injection, and the animals are subsequently killed (some of them die spontaneously), the lungs and the liver are found strewn with small nodules, consisting of round cells and epithelioid cells, giant-cells, which contain dead tubercle- bacilli, and which are thus indistinguishable from true tubercles. Prudden and Hodenpyl, who were the first to make these observations, believed the formation of the tubercle to be a result of the activity of a specific bacterial proteid contained within the protoplasmic body of the tuber- cle-bacilli. Baumgarten does not agree with this opinion, considering the formation of nodules by the dead bacteria as tuberculosis due to a foreign body (p. 276). The agency through which tubercle-bacilli in general give rise to the formation of tubercles, and whether a toxic influence is operative, are questions that also have not yet been deter- mined with certainty with regard to the living bacilli. It is undoubted, however, that the tubercle is a direct result of the presence of the tubercle-bacillus, for this -is found in every tubercle, and wherever it is introduced into animals, there tubercles develop with certainty. It is noteworthy that in the clinical picture of tuberculosis manifestations of general intoxication are not conspicuous so long as the process is in its incipiency, is localized, and mixed infec- tion has not yet taken place. Pathologic Anatomy of Tuberculosis in Animals. — In different varieties of animals the bacilli cause anatomic changes of a widely different character. Thus, in the liver and the spleen of guinea-pigs coagulation-necrosis occurs, without true caseation ; in monkeys rapid softening takes place, with the formation of a thin, diffluent purulent secre- tion ; in the tuberculosis of cows there is simultaneous cal- cification and caseation. The most common product of tubercle-bacilli in animals is, however, the tubercle, which is also always encountered in connection with the experi- mental development of tuberculosis, and which resembles in every respect the tuberculous nodule of human tuber- culosis to be described shortly. Portals of Entry for the Tubercle-bacillus in Human Beings. — The tubercle-bacillus most frequently gains en- trance into the human body through the respiration by way 17 258 CLINICAL BACTERIOLOGY. of the lungs. The frequency of occurrence of other forms of tuberculous disease falls far behind that of involvement of the lungs. The next most important portal of entry is the digestive tracts which plays an important role especially in the etiology of tuberculosis in early life. Under these conditions the infection gives rise, on the one hand, to swelling of the cervical lymph-glands, often with conse- quent suppuration (scrofulosis), and on the other hand — and more frequently without primary involvement of the intestine — to caseation of the mesenteric glands, or to chronic peritonitis. In adults the digestive tract is rela- tively seldom the portal of entrance for tubercle-bacilli, which in that event give rise to ulcerative processes in the intestine. Breaches in the continuity of the ski?t constitute a third and not at all uncommon medium of infection with tubercle-bacilli. Cases of cutaneous tuberculosis and of tuberculosis of wounds have in recent years been reported in considerable number. The so-called postmortem tubercle must be included in this category, as careful investigation has shown. Further, the relationship of lupus to tubercu- losis is scarcely any longer a matter of serious doubt. Koch has succeeded in isolating tubercle-bacilli in pure culture from lupus-nodules. The Pathogenic Activity of the Bacilli in the Hu- man Body. — The most characteristic product of tuber- cle-bacilli is the tubercle. In this the bacilli lie mainly within the interior of the giant-cells^ partly at the center, partly at the periphery ; but the bacilli are to be found also in and among the round cells constituting the tubercle. At times the rods within the giant-cells appear not to be distinctly stained, but disintegrated. Metschnikofif considers such appearances as the remains of tubercle-bacilli, and, in consequence, believes the giant-cells to be phagocytes. The specific nodules persist only for a short time, and soon disintegrate into cheesy-necrotic material. Besides giving rise to the formation of nodules, the tubercle-bacilli exhibit varied other pathogenic activity. At times they give rise to serous, purulent, or hemorrhagic inflammation, and in rare cases even to fibrinous exudation. They may further cause cheesy-necrotic inflammation without the previous formation of nodules. In some cases the inflammation caused by the bacilli has from the begin- ning a tendency to the formation of connective tissue, so TUBERCULOSIS. 259 that it soon leads to indurative cicatricial processes. This great variability in the anatomic alterations induced by the tubercle-bacilli explains the difference in the point of view- taken by the pathologic anatomist and by the clinician in part with regard to tuberculosis. The anatomist, in de- scribing the various processes, undertakes to separate them, in spite of their common etiology, while to the clinician the etiologic standpoint is alone productive, and therefore decisive ; and he considers all diseases tuberculous in whose products tubercle-bacilli can be demonstrated. Susceptibility of Human Beings to Tuberculosis (Predisposition). — Tubercle-bacilli are widely distributed throughout the inhabited world. About one-seventh of mankind is attacked by tuberculosis. The susceptibility of human beings to the disease is, therefore, not especially great. This view receives strong support from the obser- vation that has now been repeatedly made that the bron- chial glands of apparently healthy persons dying suddenly by accident contain living and virulent tubercle-bacilli (Loomis, Pizzini). In these cases the bacilli have passed the lungs without giving rise to disease. From the ana- 'tomic-bacteriologic point of view such individuals in perfect health as harbor tubercle-bacilli in the bronchial glands or in some other part of their body may be designated tuberculous ; while from the clinical standpoint only those may be considered tuberculous that exhibit the clinical manifestations of tuberculosis. From these observations the significance of the general predisposition to tuberculosis must be clear. It is evident that only a healthy and resistant body may harbor the bacilli without danger, whereas every debilitating influence facilitates the proliferation of the bacilli within the body. The time of invasion by the bacilli and the appearance of the disease do not always coincide. Only a poorly nour- ished organism enfeebled by grief and worry or by pro- tracted disease will suffer immediately after the entrance of the bacilli. A special predisposition is conferred by the abundance of sugar in the tissues in cases of diabetes. Not rarely contusions of the lung determine the mobilization of previously latent tubercle-bacilli (traumatic tuberculosis). Resistance to invasion by the bacilli appears to be strengthened in human beings by elevated climates. Ele- vations of above 2000 meters (6500 feet) are measurably 260 CLINICAL BACTERIOLOGY. free from tuberculosis. In the large cities of Mexico and Pueblo, situated between 2000 (6500 feet) and 2500 meters (8200 feet) above the level of the sea, tuberculosis is said to occur but rarely, in spite of the density of the popula- tion. Localization of Tuberculosis in Human Beings. — In human beings tuberculosis usually remains localized to the lungs. Primary tuberculosis of other organs is rare in adults. From the lungs, in the course of the disease, propagation of the bacilli frequently takes place, partly, through contact, to the pleura or other organs, partly, through the intermediation of expectorated or swallowed sputum, to the larynx and the intestine. In these organs also the tuberculosis pursues the course of a localized disease. The bacilli are carried by way of the lymph-paths and the blood-stream to the membranes of the brain, the genito- urinary apparatus, the bones and the joints, where they also give rise to local disease. Highly febrile and rapidly fatal general tuberculosis occurs when tubercle-bacilli gain entrance into the pul- monary veins in large number, and rapid dissemination through the whole body follows (iniliary tuberculosis^. Under these conditions tubercle -bacilli can be found in the blood. Mixed Infection. — The symptom-complex of tubercu- losis in human beings is at times quite materially changed by the presence and proliferation of other microorganisms in addition to the specific exciting agents. Especially in pulmonary cavities is this almost regularly the case. In the walls of such cavities sarcinae, streptococci, staphy- lococci, tetragenus, the bacilli of blue pus, varieties of proteus, and others, may lodge. There thus result septic and pyemic disturbances that are foreign to the tuber- culous process as such. Thus, the marked intermittent fever observed in so many cases of tuberculosis is proba- bly dependent principally upon the activity of streptococci (streptococcus-curve). Occurrence and Distribution of Tubercle-bacilli. — The tubercle-bacilli are found especially in the lungs and in the sputum in cases of pulmonary tuberculosis ; further, in all tuberculous lesions, including lupus. The blood contains the bacilli only in cases of general miliary tuberculosis and then only in small number. Infection may take place TUBERCULOSIS. 261 through all of the products of the disease. Most danger- ous in this respect naturally is tuberculous sputum. Of less importance are the feces in cases of intestinal tuber- culosis, the urine in cases of genito-urinary tuberculosis, and the pus in cases of bone-tuberculosis. By means of the sputum of tuberculous patients who, instead of using a spit-cup, expectorate upon the floor or into handker- chiefs, the tubercle-bacillus is disseminated in the environ- ment of the patient. The microorganism exhibits consid- erable resistance to drying, and in dried sputum, for in- stance, retains its vitality and infectivity for six months. The dried and powdered sputum is readily carried as dust in currents of air, and the tubercle-bacilli may in this way enter the air-passages of other individuals and there give rise to infection.* The credit for pointing out this mode of distribution for tubercle-bacilli belongs to Cornet, who was able to in- fect guinea-pigs with tuberculosis by means of dust from the walls, the floors, the furniture, etc., of hospital -wards and dwelling-apartments occupied by tuberculous patients careless with regard to the disposition of their sputum. Dust from localities not invaded by tuberculous patients invariably proved free from tubercle-bacilli. Reference must, however, be made at this place to the observations of Kitasato, who showed by culture-methods that not rarely the tubercle-bacilli in the sputum of tuber- culous patients have died. To what extent current views as to the danger from tuberculous sputum must be quali- fied by this fact can not yet be determined with certainty. A further source of infection, whose importance must not be underestimated, is constituted by the milk of tuber- culous cows. Tuberculosis in cattle is a manifestation of the activity of the tubercle-bacillus — a tuberculous process that differs from human tuberculosis only in the occurrence of calcification coincidently with caseation. The milk of cows suffering from tuberculosis contains tubercle-bacilli * According to observations made by Fliigge, the larger particles of sputum, both in the fresh and in the dry state, are less dangerous. The main danger is believed to reside in the fine particles of fluid that are ejected simultaneously with the sputum, and which may float in the air for a long time and be inhaled by those with whom the patient comes in contact. These small drops, as Fliigge has demonstrated experimentally, contain bacilli. To what extent this fact should influence the attitude of the physician with regard to the sputum from the prophylactic standpoint (p. 266) can not yet be defined. 262 CLINICAL BACTERIOLOGY. with extraordinary frequency (in fifty per cent, of diseased animals), even when no tuberculous changes are appreciable in the udder. If the frequency of tuberculosis in cows is considered — in some districts it occurs in from twenty to fifty per cent, of all the cows — the danger from the use of unboiled or insufficiently boiled milk, especially the mixed milk of large cities, and particularly for children, must be obvious. The hydrochloric-acid content of the stomach does not afford sufficient protection. The tubercle-bacilli pass the barrier interposed by the stomach, and gain entrance, at lea^t in part, uninjured, into the intestine: A large pro- portion of the cases of tuberculosis of the intestine and the peritoneum, which is so common in childhood, may be ^ Fig. 56. — Tubercle-bacilli in sputum; Zeiss' homogeneous immersion ^5, Oc. 4; X about 1000 diameters. attributed to the use of such infected milk. The use of meat from tuberculous cows probably gives rise to the development of intestinal tuberculosis only exceptionally. The parts that contain nodules are not permitted to be offered for sale, and the parts free from nodules contain no bacilli. Only in cases of acute general miliary tuberculosis could meat free from tubercles contain bacilli, carried to it through the blood. The diagnostic demonstration of tubercle-bacilli is of the highest importance for the early recognition of tuber- culosis. Owing to the specific behavior of tubercle-bacilli with relation to stains, the demonstration of the micro- organisms can be made with the greatest certainty by means of the microscope. TUBERCULOSIS. 263 (a) Demonstration of the Bacilliin Sputum {Pus). — For con- venience of observation the sputum is spread upon a black plate or upon a glass dish with a black background (of paper). The well-known yellowish masses ("lentils") are sought for and one of these or some other purulent portion of the sputum is placed, by means of forceps, upon a cover-slip, and spread as uniformly as possible upon its surface. After the cover-slip prep- aration has dried in the air, it is passed, in the usual manner, with the aid of the forceps, three times through the flame ; then a few drops of a freshly prepared aniline-water fuchsin-solution or of a carbol-fuchsin solution are added, and the preparation is heated over the flame until the vapor of steam distinctly escapes. After the lapse of a minute the cover-slip is moved to and fro F'&' 57- — Tubercle-bacillus in sputum (Fr^nkel and Pfeiffer). for several seconds in dilute nitric acid (from fifteen to twenty per cent.), for purposes of decolorization, and it is then intro- duced into alcohol (seventy per cent.) for the removal of the coloring-matter dissolved by the nitric acid. These two man- ipulations are repeated until the preparation appears scarcely stained. In the preparation everything has now been decolor- ized, and only the tubercle-bacilli, if these be present, have retained the stain. The alcohol is removed with distilled water. In order that the stained tubercle-bacilli may be more sharply differentiated from their surroundings, the preparation is coun- terstained by exposure for a short time to the action of a dilute aqueous solution of methylene-blue or vesuvin. It is then again rinsed with distilled water, and is finally mounted in the 264 CLINICAL BACTERIOLOGY. usual way for examination. The microscopic examination is best made by means of an oil-immersion lens ; slightly curved bacilli stained red are looked for, and if these are found, they can be tubercle-bacilli only. At times the spores of mold-fungi and of bacilli, portions of hair, fragments of horny epithelial cells, cholesterin-plates, crystals of fatty acids, and similar ele- ments, stained red, are observed, which have resisted decolor- ization ; but a little experience will prevent confusion of these with tubercle-bacilli. Decolorizaiion and counterstaining may be practised together (B. Frankel-Gabbet) by adding the decolorizing acid to the solution employed in counterstaining. After staining with hot carbol-fuchsin solution the cover-slip preparation, rinsed in water, is placed in the following solution : Nitric acid 20, alcohol 30, water 50, methylene-blue to saturation. After washing with water the preparation is dried and mounted in the usual manner. In order to find the tubercle-bacilli when these are present in small number, it is advisable, according to the suggestion of Biedert, to dilute the sputum in a test-tube with water, to add potassium or sodium hydroxid, and to continue the application of heat until the fluid assumes a homogeneous appearance. Sed- imentation is then permitted to take place, and the bacilli, by reason of their weight, sink to the bottom of the tube. The sediment is then examined by one of the methods just de- scribed. {b') Examination of Feces. — A flake of mucus or of pus is selected from the feces, and treated precisely in the manner de- scribed for sputum. {/) Demonstration of Tubercle-bacilli in Urine. — The urine, which is usually turbid from the presence of pus, is permitted to settle in a conical glass, or it is centrifugated. The sediment is placed upon a cover-slip in a somewhat thicker layer than the sputum, but in other respects it is treated in exactly the same manner. In urine the tubercle-bacilli are prone to lie together in small masses (nests). {d ) Pus fro7n cold abscesses and fluid from pletiral effusions suspected to be tuberculous are often examined in vain for tuber- cle-bacilli. After intraperitoneal injection of such material (possibly after centrifugation) guinea-pigs not rarely die of experimental tuberculosis. In this way it is possible, although after the lapse of weeks, to make a diagnosis of the presence of tubercle-bacilli. (^) Staining of Tubercle-bacilli in Sections. — The staining of sections in hot solutions is not practicable. The preparations are, therefore, kept in the aniline-water staining solution or the carbolfuchsin for from twelve to twenty-five hours at room-tem- TUBERCULOSIS. 265 perature, or from one or two hours at 37° C.(98.6° F.) in the thermostat. They are decolorized in ten per cent, nitric acid for about two minutes until they appear greenish blue, then in seventy per cent, alcohol until they appear pale rose. Next, they are introduced into water, counterstained with a dilute aqueous solution of methylene-blue or vesuvin for two or three minutes, dehydrated in absolute alcohol, cleared in cedar-oil, and mounted in Canada balsam. (/) Examination of Milk for Tubercle-bacilli. — The milk is advantageously first centrifugated. Before being stained the cover-slip preparations are placed for from four to six minutes in chloroform for extraction of the fat. On their removal the chloroform is permitted to evaporate. Fig. 58. — Tubercle-bacilli in the urine; from a case of tuberculous cystitis (Jakob). The demonstration of tubercle-bacilli in the sputum ren- ders unequivocally certain the diagnosis of pulmonary tuberculosis. A negative result permits of a definite con- clusion only when frequently repeated. The demonstration of tubercle-bacilli in the stools permits of a diagnosis of intestinal tuberculosis only in the absence of primary pul- monary tuberculosis. If this is present, it may be con- cluded that sputa have been swallowed, the bacilli of which appear in the feces unchanged. For this reason examination of the stools but rarely yields practical results. If urinary sediment is stained from a suspicion of genito- urinary tuberculosis, the possibility of confusion with smegma-bacilli (see Syphilis) must be considered, as these bacteria also, when exposed to acids, retain tenaciously the 266 CLINICAL BACTERIOLOGY. stain taken up. Smegma-bacilli are, however, readily to be distinguished from tubercle-baciUi in that they are decolor- ized within a minute in absolute alcohol, whereas the tubercle-bacilli retain their stain under like conditions. Prophylaxis. — The principal source of tuberculous in- fection is the sputum of tuberculous patients. If it were possible to render all sputum containing tubercle-bacilli in- nocuous, complete suppression of tuberculosis might be conceivable. The too zealous pursuit of this ideal aim would, however, lead to hardship on the part of tuberculous patients, so that in application the principle must be modi- fied in accordance with humane considerations. Besides, a certain degree of moderation is permissible in this connec- tion, as the liability to the disease may be diminished in not less degree by increasing the individual resistance — that is, by improving the general conditions of life — than by Fig- 59.— Pasteboard spit-cup for receiving infectious sputum. After being used, the pasteboard can be removed from the steel frame and burned. destruction of the bacilli. In any event, however, the fol- lowing principles should be enforced, or at least impressed upon the general public : The tuberculous patient should expectorate only into a spit-cup of glass or porcelain, filled with water, in order that there may be no opportunity for the sputum to dry and to be converted into dust. In this conversion into dust resides the greatest danger, and for this reason the addition to the cup of all substances capa- ble of generating dust, such as sand, ashes, etc., should be avoided. Disinfection of the sputum can not be accom- plished by means of antiseptic agents, as most of these sub- stances cause coagulation of the albuminous constituents of the sputum and thus give rise to a dense membrane that surrounds the mass and shields the tubercle-bacilli in the interior from contact with the disinfectant. In general, TUBERCULOSIS. 267 it will suffice to empty the sputum-cup into the water- closet, as the tubercle-bacilli are sure to die in putrid mix- tures. In hospitals it will be best to sterilize the sputa, together with other excretions, by boiling. (See Disinfec- tion, Appendix.) Especial attention should be' paid to the tuberculous patient out of doors. Sputum expectorated in- discriminately upon the street is especially dangerous, be- cause when reduced to powder, innumerable persons are exposed to the liability of infection. The use of the hand- kerchief to receive the expectoration is likewise not without risk, because here, also, there is danger of drying and con- version into dustj. For this reason it has been wisely re- commended that tuberculous patients carry with them small pocket-flasks for the reception of the expectorated matters — for instance, Dettweiler's spit-cup. It must, however, not be overlooked that certain insurmountable difficulties stand in the way of a general employment of such flasks, and that a careful use of the handkerchief may yet be permitted. The rooms occupied by tuberculous patients should be cleansed quite frequently with moist materials, in order to prevent the conversion into dust of the sputum that has possibly become deposited on the floor or on the furniture. After the death of a tuberculous patient the rooms previ- ously occupied and, besides, all articles with which he came in contact should be disinfected with the same care that is" observed in other infectious diseases. In hospitals, prisons, etc., those suffering from tuberculosis should be separated from the others. In cases of intestinal and genito-urinary tuberculosis and of tuberculous suppuration care should be taken that the feces or the urine or the pus is rendered innocuous. Sexual i7itercourse should be forbidden those suffering from genito-urinary tuberculosis, as the disease may be trans- mitted through the spermatic fluid. Finally, it may be stated that milk should never be drunk unboiled, but should always be boiled before being used. It is best to forbid the use of meat from a tuberculous ani- mal, or at least to permit its sale only when thoroughly cooked, if the disease is not strictly localized to a single organ. Heredity of Tuberculosis. — A distinction is made between direct inheritance of tuberculosis — the disease 'being transmitted as such to the offspring — and indirect 268 ' CLINICAL BACTERIOLOGY. inheritance — only the tendency or the predisposition to the disease being transmitted. With indirect inheritance the children of a tuberculous father or mother have inherited a feeble body, a small chest — in brief, the tuberculous habitus. They are not tuberculous, but are greatly predis- posed to tuberculosis (sujets tuberculisables, Peter). Cases of direct inheritance — that is, of congenital tuber- culosis— are known both in human pathology and in vet- erinary medicine, but their number is extremely small in comparison with the wide distribution of the disease. The same statement is applicable to tuberculosis in the first few days of life. On the other hand, the mortality from tuber- culosis in early infancy, especially in the first year of life, is quite enormous. Most observers assume that this high mortality is readily explained by the numerous sources of infection to which the child of tuberculous ancestry is ex- posed after birth. The kiss of a tuberculous father, the milk of a tuberculous mother, are equally dangerous, inde- pendently of the fact that with neglect in the treatment of the sputum on the part of the parents there is also afforded frequent opportunity for inhalation of the tubercle-bacilli. That the disease is so prevalent and so fatal in the first year of life is to be explained by the fact that at this period the children are more predisposed, and less resistant, to the tubercle-bacillus than later. The relation in these cases would thus be one of indirect inheritance and not one of true congenital tuberculosis. Baumgarten, probably the most earnest advocate of the doctrine of direct inheritance of tuberculosis, takes the ground that the tubercle-bacilli are transmitted to the em- bryo during fetal life. The inherited germ, however, does not proliferate because the tissues of the new-born child, through their vital activity, offer considerable resistance. The bacilli thus to a certain degree remain latent in the lymph- glands, the bone-marrow, etc., later, under the favoring influence of traumatism, intercurrent disease, or diminution in the vital energy of the cells, to unfold their deleterious activity. This view is supported by the observations of Landouzy, Martin, Birch-Hirschfeld, Schmorl, and others, who found tubercle-bacilli in apparently healthy organs from fetuses born of tuberculous mothers. On the other hand, Gartner lays emphasis upon the fact that the infantile cells do not exert an especially injurious influence upon TUBERCULOSIS. 269 the bacilli. Tuberculosis pursues a more rapid course in youth than at a later age, and experimentally no difference can be observed between young and old animals. On the other hand, Gartner is of the opinion that in the act of par- turition, perhaps in consequence of tears in the placenta, the bacilli pass over from the mother to the child. The possibility of such a transfer of the tubercle-bacilli to the embryo Gartner has himself demonstrated with certainty in his experiments upon mice, canary-birds, and rabbits. Fetal infection, according to this view, takes place late, and in all probability it is effected through only one or a few bacilli. Consequently, the disease can not be manifest at birth, but it becomes evident only after the child has lived for some time outside the uterus. Also, the cases of pri- mary tuberculosis of the skin, the joints, the bones, the glands, the spleen, the liver, which are not uncommon in early childhood, indicate a hematogenous fetal infection. The objection that the frequency of pulmonary tuberculosis is opposed to the preponderant occurrence of hematog- enous fetal transmission, is, according to Gartner, justified ; but it is to be borne in mind that, in spite of exhibiting the most conspicuous changes, the lungs are by no means always the seat of the primary tuberculous lesion. The lungs of human beings are, *'by reason of their constitu- tion, their situation, and their chemistry, especially adapted for the lodgment and development of tubercle-bacilli," which may gain entrance from any primary focus (perhaps of fetal origin). The question whether tuberculosis may be transmitted to the embryo from the father Gartner an- swers in the negative. He rendered male rabbits and guinea-pigs tuberculous by injection of bacilli into the tes- ticles, and he then brought them in contact with females, but the offspring were not tuberculous ; whereas when the bacilli were present in the seminal fluid in considerable numbers, the females were infected. Therapeutic Experiments. — Many efforts have been made to confer immunity to tuberculosis, and to effect the cure of tuberculosis by the same means. This end was sought by the use of the blood-scrum of animals that are relatively refractory to the disease, such as dogs (Richet and Hericourt) and goats. Further, attempts have been made to establish immunity by means of attenuated or sterilized cultures of tubercle-bacilli. All of these endeavors, how- 270 CLINICAL BACTERIOLOGY. ever, have thus far proved unsuccessful. Richet maintained for a time that he had succeeded in vaccinating dogs by inoculating them with the bacilli of fowl-tuberculosis, or with small amounts of mammalian tubercle-bacilli, but his observations have not been confirmed. R. Koch endeavored to secure a direct and specific in- fluence upon the tuberculous lesions by means of tuberculiji. This product was obtained by concentrating mature bouillon-cultures of tubercle-bacilli (from six to eight weeks old) to one-tenth of their volume over the water- bath, and freeing the culture-fluid of the bodies of the dead bacilli by means of filtration through porcelain or gravel. Experimental observation showed that healthy guinea-pigs withstand without harm subcutaneous injec- tions of as much as two cubic centimeters of tuberculin, whereas tuberculous animals, infected four weeks previ- ously, succumb to a dose of 0.6 cu. cm. On autopsy in cases of animals dead after injections of tuberculin the following conditions were found : The site of tuberculous inoculation was markedly reddened, as well as the adjacent lymph-glands. In the viscera enormous dilatation of the capillaries surrounding the tuberculous foci was discernible microscopically. The hemorrhage-like spots on the sur- face of the liver were quite pathognomonic. In tuber- culous guinea-pigs treated with small doses of tuberculin (at first I mg., increasing to o. i and 0.2 gram) a striking effect upon the morbid condition was observed. Improve- ment in the primary area of inoculation in the abdominal wall was observed, with diminution in size of the adjacent tumid lymph-glands, but complete recovery, as later reports from the Koch Institute have shown, occurred only excep- tionally. The animals survived for a longer time than tuberculous control guinea-pigs not treated with tuberculin. Postmortem examination in the case of animals treated disclosed marked retrogression, in part cicatrization of the tuberculous lesions in the abdominal viscera, but eventually death resulted from pulmonary tuberculosis. The curative influence of tuberculin is explained by Koch on the assumption that as a result of its action the necrotic substance, which is always present in the neighborhood of a tuberculous lesion, is increased in amount, so that coag- ulation-necrosis occurs throughout a wide extent, and this hinders the bacillus in its further growth, and at TUBERCULOSIS. 271 times causes its death. Opposed to the original opinion of Koch is the fact that the action of tuberculin upon tuber- culous tissues is, however, not specific, and not confined to it alone. As Romer was the first to show, the products derived from other microorganisms by boiling (proteins) induce precisely the same reaction. The influence of tuberculin upon healthy and diseased human beings has been tested from both a diagnostic and a therapeutic standpoint in so large a number of cases that a definite opinion can now be expressed. Diagnos- tically, tuberculin has proved an exceedingly delicate reagent for the detection of tuberculous lesions. Subcu- taneous injection of five milligrams is followed by no appreciable effect in healthy persons. In tuberculous in- dividuals, however, this dose gives rise to fever of moderate degree, lasting for several hours, while at the same time a local reaction, manifested by redness and swelling, takes place in the tuberculous lesions in so far as these are acces- sible to examination. Initial doses of ten milligrams cause in tuberculous subjects greater and more protracted eleva- tion of temperature, together with headache, nausea, and' vomiting. Similar doses cause also in healthy persons febrile disturbance and general manifestations. The undoubted diagnostic titility of minimal doses of tuberculin is rendered practically unavailable from the fact that the injection is followed by febrile movement in all persons who harbor tubercle-bacilli, even when these are latent and encapsulated — as, for instance, in lymphatic or bronchial glands. It has been found, in numerous cases, that apparently healthy persons develop fever after injec- tions of tuberculin in the same way as tuberculous patients. From what has been said with regard to the frequent occurrence of tubercle-bacilli in healthy persons, this mani- festation should not occasion surprise ; but a diagnosis of tuberculosis is not, in a clinical sense, justifiable when merely the presence of tubercle-bacilli within the organism has been demonstrated. The conditions are quite different in the diagnosis of tuberculosis than in that of diphtheria or of cholera. It is of the greatest sanitary importance to recognize the existence of diphtheria or of cholera in per- sons in whom, though apparently in perfect health, the exciting agents of those diseases are present in the saliva or in the dejections, because further cases of infection may 272 CLINICAL BACTERIOLOGY. arise through them. In the case of tuberculosis, however, only those individuals are dangerous who present actual symptoms of disease— that is, those who throw off tubercle- bacilli with the sputum or other excretions. In the case of tuberculosis thus the diagnostic requirements on the part of both physician and sanitarian are identical ; whereas in the case of cholera and of diphtheria these diverge. From the foregoing considerations the employment of injections of tuberculin for diagnostic purposes should be restricted to cases of actual disease of obscure etiology. Frequently, how- ever, under such conditions also this test has been abstained from because experience in some cases has demonstrated the possibility of the bacilli being disseminated from a pre- viously circumscribed focus throughout the entire body as the result of an injection of tuberculin. The tJierapeutic employment of tuberculin in tuberculous individuals has yielded the following results : Lupus is at times favorably influenced by tuberculin. Extensive areas of lupus undergo necrosis and are exfoliated, so that com- plete recovery may take place ; but no case is yet known in which recurrence has not taken place. Ulcers of the larynx clear in a most remarkable manner after injections of tuberculin, and undergo healing ; but in these cases also recurrence generally takes place. Intestinal and peritoneal tuberculosis appears to pursue a relatively favorable course when treated with injections of tuberculin. Under all of these conditions, however, the fact stands out that the results are not final, because tuberculin lacks immunizing properties. Tuberculous disease of bones and joints is not at all influenced by the treatment. With relation to pul- monary tuberculosis, it may be stated with certainty that in the presence of advanced infiltration, of cavity-formation, and of mixed infection success can not be attained with tuberculin-treatment ; and it is questionable, further, whether even incipient tuberculosis can be cured by the original method of Koch. A large number of unfavorable results are opposed to a small number of undoubted cases of in- cipient tuberculosis treated with favorable results. The objection may be raised that the results obtained in these cases are not attributable to the specific agent, but to the nutritive and general therapeutic measures employed simul- taneously. Tuberculin has been almost entirely abandoned by physicians. In veterinary medicine, especially in France, TUBERCULOSIS. 273 it is still employed on a large scale for diagnostic purposes, and, it appears, with great success. The New Tuberculin-preparations, TO and TR. — After the failure of the original tuberculin, Koch applied himself unremittingly to the improvement of tuberculin-prepara- tions. He found that immunity could not be conferred upon animals by subcutaneous injection of unchanged tubercle-bacilli. He, therefore, undertook to disintegrate the tubercle-bacilli mechanically, in order to render them the more easily absorbable. The cultures were dried in a vacuum and rubbed up in an agate mortar without addition until only a small number of bacilli were visible micro- scopically. The powder was mixed with distilled water,. and centrifugated for from a half to three-quarters of an hour. In this way Koch effected a separation into two layers — an upper transparent, opalescent layer, which con- tained no bacilli, and a densely adherent lower layer. The latter was again dried, rubbed up, and centrifugated in the manner described, and the entire process was repeated until all the bacilli had practically been reduced to solution. Koch then determined that the solutions thus obtained were readily absorbable, and that they did not give rise to the formation of abscesses. It was found, however, that the fluid obtained from the first centrifugation exhibited reactions different from that obtained from the second and from subsequent centrifugations. Koch designated the first tuberculin O {obere, upper — TO) ; and all of the others, which were alike in their reaction, tuberculin R (residue — TR). TO contains the constituents of the tubercle-bacilli soluble, and TR those insoluble, in glycerin. It can be readily understood that the properties of TO are compar- able, on the whole, with those of the original tuberculin. TR, however, according to Koch, exhibits distinct immuniz- ing properties, and gives rise to reaction in tuberculous subjects only when used in large doses. Its action is entirely independent of the reactions that played so impor- tant a part when the original tuberculin was used. Koch states that in conferring immunity with TR the reactions may be entirely avoided, and, by carefully increasing the dose, tuberculous subjects may be habituated quite rapidly, without any reaction, to considerable amounts of the new remedy. When this has been accomplished, the organism may be considered as immune to the original i8 274 CLINICAL BACTERIOLOGY. tuberculin and to TO — that is, to all of the bodily con- stituents of the tubercle-bacilli. The new preparations are put upon the market by the Hochst Works. For purposes of preservation 20 per cent, of glycerin is added. One cubic centimeter of the new tuberculin TR contains 10 mg. of solid substance. After appropriate dilution with sterile, physiologic solution of sodium chlorid (if the daughter-solution is to be preserved for some, time, 20 per cent, of glycerin is added), the treat- ment is begun by injecting -^-^ mg. The next higher dose is to be administered on the second day, and it should be of such an amount that temperature -elevations of more than ^° do not take place. Should these occur, the next injection must be deferred until the temperature has again become normal. As a rule, the treatment is suspended when the dose reaches 20 mg., and, if no reaction follows, the same amount is repeated at considerable intervals. If it is desired to immunize healthy animals, as large a quantity is injected at first as is well borne by them — for instance, in the case of guinea-pigs, 2 or 3 mg. In this way Koch was able to immunize a considerable number of guinea-pigs to highly virulent tubercle- bacilli. The height of the immunity is attained two or three weeks after ad- ministration of the large dose. From this it appears that in therapeutic experiments on artificially infected guinea- pigs, which rapidly succumb to the disease, the treatment must be instituted quite early — not later than two weeks after the introduction of the virus. Koch recommends that this new remedy be employed only in recent, pure cases of tuberculosis, uncomplicated by mixed infection. Patients who exhibit a temperature above 38° C. (100.4° F.) are not adapted to the new method of treatment. In cases of lupus Koch obtained considerable improvement without noteworthy local re- action. At the conclusion of his communication Koch emphasizes the fact that perhaps combinations of TO and TR with serum-preparations made from TO and TR may more quickly lead to the desired results. The clinical reports that have thus far been made with regard to the treatment of tuberculosis with TR show the freedom from danger that attends the use of the remedy, but little as to its therapeutic utility. Personally, no noteworthy results have been obtained in fifteen cases of pulmonaiy tubercu- FOWL-TUBERCULOSIS. 275 losis thus treated. Behring is of the opinion that tuber- cuHn R is less well adapted for therapeutic employment in the case of human beings than for the fundamental inocu- lation of animals for the purpose of further immunization. His own experiments, in connection with von Lingelsheim, in the preparation of a serum for tuberculosis, according to his statement read before the Fifteenth Congress for In- ternal Medicine, appear promising. Behring and von Lin- gelsheim in their experiments employed dry, highly viru- lent, pure cultures of tubercle-bacilli. According to Beh- ring, the tubercle-bacilli contain various substances, but only one of the tuberculosis-toxins appears to possess im- munizing properties. With the aid of this toxin it may be hoped that a curative serum or an antitoxin will be pro- duced, as in the case of diphtheria and of tetanus ; but ac- cording to Behring, years may elapse before this serum will prove sufficiently powerful to be introduced into general practice. FOWL-TUBERCULOSIS. The bacillus of fowl-tuberculosis is closely related to the bacillus of human and mammalian tuberculosis. It is somewhat longer and thinner, and exhibits more frequently bulbous and branched variations. It is more easily stained, but it retains the stain with similar tenacity. It is not so fastidious with regard to culture-media, and it develops upon ordinary agar and upon or- dinary bouillon. The addition of glycerin, however, materially favors its growth, which, on the whole, is more rapid than that of the bacillus of mammalian tuberculosis. The cultures are not so dry, but more moist, and on solid media they form a coherent coating that bridges over the water of condensation. All cultures constantly exhibit a yellowish discoloration. At temperatures of 42° C. (107.6° F.), 43° C (109.4° F.) or 45° C. (113° F. ) the bacilli of fowl-tuberculosis thrive as luxur- iantly as at a temperature of 37° C. (98.6° F.). This is their most radical distinguishing feature as compared with the bacilli of human tuberculosis, which will not develop at this temperature. If the two varieties are considered identical, this difference must be explained by the fact that by reason of their residence in the body of birds, which naturally have a higher temperature (41° 0.-105.8° F., or 42° €. — 107.6° F.), the bacilh have adapted themselves to a higher temperature. The bacilli of fowl-tuberculosis are even more resistant to heat than those of human tuberculosis, being destroyed by exposure for fifteen minutes to a temperature of 70° C. (158° F.). 276 CLINICAL BACTERIOLOGY. Occurrence of the Bacilli. — The bacilli are present in the tuberculous lesions of fowl, which consist of dense, tumor-like masses containing calcareous deposits. Giant- cells are present in but small number. Fowl-tuberculosis has been observed in rare cases also in human beings and in mammals. Experimental Development of Fowl- tuberculosis. — Most birds are highly susceptible, and infection may take place through all portals of entry. According to Baum- garten fowl-tuberculosis occurring spontaneously is con- genital in almost all instances. Rabbits, also, succumb to infection with fowl-tuberculosis. Guinea-pigs and dogs prove rather refractory, without, however, being entirely immune. On the whole, the bacilli of fowl-tuberculosis thrive poorly in mammals, and mammalian tuberculosis, conversely, develops only with difficulty in birds. Diagnosis. — The examination of tuberculous masses for bacilli is conducted in precisely the same manner as in the case of human tuberculosis. PSEUDaTUBERCULOSIS. '^y pseudo-tuberculosis is understood certain pathologic processes that exhibit the external appearance of tubercles, but are dependent upon other causes than the tubercle- bacillus. The etiology of pseudo-tuberculosis is quite varied. Among the causative factors may be mentioned : 1. Inanimate foreign bodies. 2. Animal parasites. 3. Bacteria. 4. More highly organized vegetable parasites. The tuberculosis due to foreign bodies may be developed experimentally with ease through the agency of all possible substances. It is, however, not transmissible from animal to animal. Pseudo-tiiberculosis due to auimal parasites is observed almost exclusively in animals. Among the varieties that are well known is the tuberculosis of the cat, caused by ollulanus tricuspis ; that of the sheep, due to pseudalius ovis pulmonalis ; that of the calf, due to strongylus ruf- escens ; that of the dog, due to strongylus vasorum. Miura alone has observed a single case in a human being. PSEUDO-TUBERCULOSIS. 277 This occurred in a man dying of beri-beri, in whose omen- tum were found fibrous tubercles caused by the ova of dis- toma. Bacterial pseudo-tuberculosis has been often described in animals. First designated zooglear tuberculosis by Malas- sez and Vignal, this disorder has since been studied by numerous observers, and most carefully by Preisz. The cause of this affection consists in thick, short rods, fre- quently resembling cocci, and forming filaments. Spores do not develop, and Gram's method fails to stain. Upon gelatin-plates colonies form resembling those of the typhoid-bacillus, but not causing liquefaction. Upon gela- .J . •..> Fig. 60. — Bacillus pseudo-tuberculosis from agar-agar; X 1000 (Itzerott and Niemann). tin stab-cultures a flat, nail-like growth takes place ; upon agar a grayish, fetid coating forms, and upon potatoes a yellowish deposit. In bouillon there is at first flocculent turbidity, then the formation of a precipitate and a clear- ing up of the supernatant fluid. The bacillus pseudo- tuberculosis is pathogenic for all rodents ; in slighter degree, also, for dogs and horses. The postmortem findings are strongly suggestive of true tuberculosis, particularly in the abdominal organs, which are especially attacked by pseudo- tuberculosis. The differential diagnosis is made with great ease The readiness with which the bacilli stain and their rapidity of growth permit of a decision without difficulty. 278 CLINICAL BACTERIOLOGY. Pseudo-tuberculosis due to more highly organized vegetable parasites likewise occurs preferably in animals. Various kinds of streptothrices and aspergilli act as the etiologic factors under these conditions, particularly aspergillus glaucus and fumigatus. Pigeons frequently succumb to a form of miliary pseudo-tuberculosis, in which the aspergil- lus fumigatus is found inclosed within giant-cells in the in- terior of the granulations. In individuals engaged in the fattening of pigeons pulmonary affections have been ob- served apparently dependent upon the same microbes ; at least the aspergillus fumigatus has been found in the spu- tum of such patients. It is assumed that the parasite adheres to the grain used in feeding the pigeons. Eppinger has observed in, and cultivated from, a case of pseudo-tuberculosis a variety of streptothrix that he designates cladothrix asteroides. With pure cultures of this organism he succeeded in inducing the same disease in. animals. (See Actinomycosis.) LEPROSY* The bacillus of leprosy was discovered by Armauer Hansen. It has thus far not been successfully cultivated. In morphologic appearance the bacilli closely resemble tubercle-bacilli. Often, they appear somew^hat shorter, and, like these, they are nonmotile. With regard to tingi- bility, the bacilli of leprosy occupy a position midway be- tween tubercle-bacilli and other bacteria. As a rule, they prove susceptible only to the methods of staining that are applicable to tubercle-bacilli. They stain and decolorize somewhat more readily, however, than tubercle-bacilli, and they can be stained also by simple aqueous solutions of aniline dyes, especially violet and fuchsin, at room- temperature (Baumgarten). They can be stained, further, by Gram's method. In the leprous new-formations (leprous nodules) the leprosy-bacilli lie mainly within the tissue-cells, in the so- called leprosy-cells. They have been found in the blood only during the febrile periods. E. Levy has cultivated from a case of leprosy a bacte- rium that in glycerin-agar cultures bears some resemblance to the bacillus of mammalian tuberculosis, but that after LEPROSY. 279 Staining proves resistant to neither acid nor alcohol. Micro- scopically, it bears a distinct resemblance to the anaerobic actinomyces. It presents bulbous enlargements and rami- fications. Distribution of Leprosy. — Leprosy, which formerly was indigenous to all of Europe, is now restricted to Nor- way, Livonia, Turkey, the Crimea, and Southern Italy. Of late, isolated cases of the disease have again been encountered in Eastern Prussia. In countries outside of ^P^.IS? Fig. 6i. — Bacilli shown in a section of the tongue in a case of tubercular leprosy ; X 600. The bacilli ar« extracellular: B, Bacilli in groups; Z, Z, zooglear masses, large rounded masses of bacilli ; C, bacilli in chains (Leloir). Europe leprosy is quite common. The small number of cases that are observed in other places may be explained by importation from leprous localities. The disappearance of leprosy is undoubtedly to be attributed to the isolation — formerly practised with barbaric severity — of those affected in special leprosy-houses (leproseries). From this disappearance of the disease it may be concluded that it is contagious, but this has not been demonstrated with cer- tainty, and is doubted by some physicians. Experiments on Animals. — The experimental develop- 280 CLINICAL BACTERIOLOGY. ment of leprosy in animals has thus far not succeeded. The only observations that can be considered as positive are those of Melcher and Ortmann, in which, after inocu- lation of the anterior chamber of the eye, general infection developed in rabbits ; but these are interpreted by most observers as indicating that in the case or cases from which the infecting material was obtained there existed mixed infection with leprosy and tuberculosis, and that the inoculated animals died of tuberculosis. All of the attempts at inoculation of human beings have proved equally unsuc- cessful, or at least not above criticism. Danielsen, who inoc- ulated himself and others with nodular masses, blood, etc., from leprous patients, obtained completely negative results. On the other hand, the experiments of Arning terminated positively ; this observer succeeded in transmitting leprosy to a prisoner condemned to death. This case has, how- ever, been seriously doubted, as the period of incubation was strikingly short, being only sixteen months, whereas the whole course of the disease covered only five years, and as, besides, the inoculated person was a member of a race highly susceptible to leprosy, and cases of leprosy had already occurred in his family. Bacteriologic Diagnosis. — Cover-slip preparations are made from the contents and the tissue-juice of leprous nodules, and are stained in precisely the same way as tuber- cle-bacilli. Great care must be taken in the process of decolorization. The sections of leprous tissue are not per- mitted to remain for so long a time in the staining solution as sections of tissue containing tubercle-bacilli ; half an hour is quite sufficient. In most cases of leprosy, as R. Koch was the first to ob- serve, the nasal mucus contains leprosy-bacilli. These are derived from areas of ulcerated or swollen mucous mem- brane on the cartilaginous portion of the nasal septum. The bacilli may, however, be present when such visible changes are absent. Heredity of Leprosy. — According to Baumgarten, lep- rosy, like tuberculosis, is transmissible by inheritance. If this opinion is correct, a long period of latency must be as- sumed for the inherited leprosy -bacilli, as the disease never appears before the second or the third year of life. Prophylaxis. — The best prophylaxis unquestionably consists in isolation of leprous patients in special hospitals. INFLUENZA. INFLUENZA, On the first occurrence of influenza in the winter of 1889—90 the numerous bacteriologic investigations under- taken failed to yield conclusive results. In the secretions of influenza-patients the ordinary exciting agents only of inflammation, especially streptococci and lanceolate diplo- cocci, were encountered. Of the latter it was stated that in appearance and growth they presented certain points of distinction as compared with ordinary pneumococci. A specific bacterium was, however, not found. In subsequent epidemics (189 1 and 1892) Pfeiffer, in the Hygienic Institute at Berlin, recognized a special bacillus as the exciting agent of influenza, and developed it in pure culture. The state- ments of Pfeiffer have since been completely confirmed. Morphology of the Influenza-bacillus. — The influenza- bacillus is an extremely small organism (0.2 // thick and 0.5 ;a long), in thickness not quite equaling the slender bacilli of mouse-septicemia, and being only twice or thrice as long as wide. Its extremities are rounded. Rarely in the sputum and more commonly in recent pure culture, the bacilli form short pseudo-filaments. Long bands in cultures three or four days old are to be considered as beginning involution-manifestations. The influenza-bacilli possess no capsule, and are without move- ment of their own. Frequently, two especially short bacilli lie close to each other (division-forms). This may readily give rise to confusion with diplococci. Influenza-bacilli appear not to possess spores. Spore-like formations have never been found in the secretions or in cul- tures, and, besides, the bacillus is but little resistant to the influ- ence of temperature, drying, etc. Staining of Influenza-bacilli. — The bacilli take the stain with considerable difficulty. Loffler's solution of methylene- blue may be employed, and, still better, a dilute, pale-red solu- tion of carbolfuchsin in water. The preparation must be ex- posed to the action of the stain for from five to ten minutes. If the exposure is of shorter duration, or if other stains are employed, the central portion of the bacillus is often more feebly stained than the extremities. The bacilli are not stained by Gram's method. Cultivation of the Influenza-bacillus. — The influenza- bacillus is strictly aerobic, and it develops only in the presence of hemoglobin or of leukocytes. The latter fact explains why cultivation of the influenza-bacilli remained for so long a time 282 CLINICAL BACTERIOLOGY. unsuccessful. Pfeiffer also was often able to cultivate the bacilli from the sputum or pus from the lungs directly upon agar, but this did not take place invariably, and at times it was quite impossible to continue the growth of such cultures in any way. The explanation for this fact is that the bacilli developed in the first culture when with the infecting material a trace of blood was simultaneously transferred to the culture-medium. Growth failed to take place, however, when the blood was wanting, and thus also in all daughter-cultures. The development of influenza-bacilli takes place regularly, and the culture obtained may be continued indefinitely, if the infecting material is inoculated upon a culture-medium contain- ing blood, and best upon blood-agar tubes (p. 82). For the \ Fig. 62. — Bacillus influenzae, from a gelatin-culture; X looo (Itzerott and Niemann). development of pure cultures Pfeiffer recommends the following method : The infecting material — bronchial sputum or fluid from a bronchopneumonic infiltrated portion of the lung in a case of influenza-pneumonia — is rubbed up with one or two cubic centimeters of bouillon to a homogeneous emulsion. By means of a platinum loop some of this is inoculated upon blood-agar, and also for control-purposes upon ordinary glycerin-agar, tubes, care being taken to secure uniform distribution of the infecting material upon the entire surface. The dilution of the sputum in the bouillon is intended, in the first place, to separate the influenza-bacilli to such a degree that isolated colonies will form upon the blood-agar tubes. In the second place the hemoglo- bin that may be present in the infecting material will be so greatly diluted that the influenza-bacilli will be incapable of INFLUENZA. 283 developing in the control-tubes not previously treated with blood. The inoculated test-tubes are placed in the thermostat. After the lapse of twenty-four hours the influenza-colonies will become visible in the blood-agar tubes as densely packed, watery drops, whereas the control-tubes will be either sterile or present isolated colonies of streptococci, diplococci, or other bacteria, which were present in the infecting material in addi- tion to the influenza- bacilli. The water-like drops of influenza-colonies are usually so small that they are only visible with the aid of a lens. They exhibit little tendency to become confluent. Should they be especially numerous and close together, they coalesce to form larger drops with curved margins, but these permit a recognition of the aggre- gation of individual colonies. If the colonies are isolated and widely separated from one another, they may grow to the size of a pinhead, but in this case also they retain a vitreous trans- parency. The water of condensation of influenza-cultures gen- erally remains clear. When mixed with blood derived from the oblique surface of the culture, delicate white flocculi form in it. In bouillon mixed with blood and spread in a thin layer the influenza-bacillus thrives quite abundantly. The plate-procedure is greatly to be recommended for the iso- lation of influenza-bacilli and for diagnostic purposes, if some blood is added to the liquefied agar before inoculation, or if agar is permitted to solidify in Petri dishes and blood is added, and the diluted sputum, etc., is smeared upon the sur- face in several streaks. The colonies present the same appear- ance as those in agar-tubes. The temp&rature-opti7num for the cultivation of influenza- bacilli is that of the body. The upper limit for growth is 42° C. (107.6° F.), the lower between 26° C. (78.8° F.) and 27° C. (80.6° F.). The bacilli do not grow at room-temperature. Oxygen is always necessary for the development of the influ- enza-bacilli ; they do not grow in an atmosphere of hydrogen or of carbon monoxid, even in the presence of blood. Pfeiffer undertook to determine what constituent of the blood the influenza-bacilli require for their development. On making transfers from blood-serum or blood-fibrin to agar-tubes no growth took place. Red blood-corpuscles were invariably necessary, and particularly the hemoglobin contained in them, as was later shown, was the active substance. Hemoglobin- agar (p. 82) is equally adapted with blood-agar for the cultiva- tion of influenza-bacilli. Pfeiffer endeavored to associate this Indispensability of hemoglobin for the growth of influenza- bacilli at first with its relations to oxygen, with its faculty of acting as an oxygen -carrier. He succeeded, however, in ob- taining growth upon an agar-layer in the presence of carbon- 284 CLINICAL BACTERIOLOGY. monoxid hemoglobin. Exposure of blood-agar tubes to a tem- perature of 70° C. (158° F. ), and even boiling of the hemo- globin, failed to prevent entirely the development of the in- fluenza-bacilli. Pfeiffer was then led to believe that the iron- content of the hemoglobin was the important factor, but he was unable to cultivate the bacilli in culture-media containing iron other than that of the blood. It may be mentioned further that all kinds of blood exhibit the same specific activity with relation to influenza-bacilli. Pfeifl'er obtained growth upon the blood of rabbits, guinea- pigs, pigeons, and fish, and in more luxuriant degree and more speedily on pigeon's blood — which is rich in hemoglobin — than on human blood. Resistance of Influenza-bacilli. — Influenza-bacilli are destroyed in a kw minutes when exposed to a temperature of 60° C. (140° F.). They cease to develop at a temper- ature of 43° C. (109.4° F.), but they are only coagulated, not destroyed, for if tubes that have been exposed for forty-eight hours to a temperature of 43° C. (109.4° F.), and have remained sterile, are then exposed to a tempera- ture of 37° C. (98.6° F.), colonies will yet develop abun- dantly. In unsterilized drinking-water the bacilli die quickly — in from twenty-four to thirty-six hours. Upon blood-agar and in bouillon they retain their vitality for from fourteen to eighteen days, and in moist sputum they appear to preserve their infectivity for at least fourteen days. The influenza-bacilli are quite sensitive to drying. When dried in blood or sputum at a temperature of 37° C. (98.6° F.), they succumb in an hour or two, and when dried at room- temperature, within not more than from thirty-six to forty hours. Occurrence of Influenza-bacilli. — The influenza-bacilli occur regularly in the secretions of influenza-patients. In the secretion of the nasal cavities the specific bacilli have been found in enormous numbers ; although generally associated with other microorganisms, yet, however, in preponderating number. The secretion in a case of ordi- nary coryza, on the other hand, is remarkably free of bac- teria, being almost sterile. The sputum in cases of bron- chitis and pneumonia complicating influenza is viscid, mucopurulent, globular, and not seldom also purulent and confluent, in color often yellowish green, not rarely pure white and only seldom rusty brown, and it contains the INFLUENZA. . 285 influenza-bacilli in almost absolutely pure culture, and always in surprising number. The bacilli usually lie in the mucous ground-substance arranged in nests and groups ; they are found also within the pus-corpuscles, at the begin- ning of the disease in small number, and during convales- cence in preponderating number. In the latter condition they surround the nucleus and are not included within it. Sputa containing influenza-bacilli are often ejected for days and months. Especially in cases of tuberculosis have such chronic complications of influenza with broncho- pneumonic localization not been rare. The bacilli have been found in the bronchopneumonic foci in cases of influenza-pneumonia, rarely in the pus in cases of influenza- empyema. Canon obsei-ved in the blood of influenza- patients delicate bacilli resembling those of influenza. According to Pfeiffer's investigations, these organisms, if influenza-bacilli at all, are exceptional, for, as a rule, the organisms are not present in the blood. With regard to the localization of the bacilli in the gastric and nervous forms of influenza, unequivocal investigations are wanting. Likewise, the numerous complications and sequelae of influenza have thus far been little studied from the bacteriologic standpoint, so that it has not yet been de- termined whether they represent results of the activity of the influenza-bacillus or its toxin, or are secondary infec- tions. In a case of influenza-aortitis Pfeiffer found, in addi- tion to the diplococci of Frankel, numerous influenza- bacilli, and in the exudate from a case of influenza-meningitis diplococci exclusively. The bacillus of influenza has never been found outside the human body, in the earth, or in water. It could scarcely persist for a long time under these conditions on account of its feeble powers of resistance. Portals of Infection for, and Distribution of, the In- fluenza-bacillus.— The influenza-bacillus is probably taken up by the air-passages exclusively. Its distribution by means of dried and powdered sputum can play an etiologic role only in restricted degree, as the bacillus withstands drying so badly. The ordinary mode of conveyance is certainly by means of the moist nasal and bronchial secre- tions of influenza-patients. The widespread and often pan- demic distribution of influenza may be explained by the fact that in the first place the susceptibility of human beings to 286 . CLINICAL BACTERIOLOGY. the disease is quite considerable ; further, that the disease appears in many cases in the form of a mild coryza and of a harmless bronchial catarrh, and these at the beginning of the epidemic are not immediately recognized as influenza ; that even after the epidemic character of the disease is established they do not confine patients to the house, so that opportunity is thus afforded, in sneezing and in coughing, to disseminate innumerable influenza-bacilli among those not yet infected. The sudden recrudescence of apparently extinct epidemics is rendered comprehensible by the fact just mentioned, that there are cases of chronic influenza that act for a long time as carriers of bacilli capable of inducing influenza. Experiments on Animals. — Even in the most exten- sive epidemics of influenza domestic animals escape the dis- ease. Success in the transmission of influenza-bacilli to animals was therefore improbable from the outset. Pfeififer undertook experiments on mice, rats, guinea-pigs, rabbits, swine, cats, dogs, and monkeys. Only in the last-named animal was it possible to induce an infection resembling in- fluenza, and then only by introducing the bacteria through the chest-wall directly into the lung, and also — which was more important^in a monkey, by introducing an influenza- culture into the uninjured nose. The disease manifested itself by some cough and fever of several days' duration. Multiplication of the inoculated bacteria was, however, not observed. By introducing large amounts death can be caused in rabbits quite rapidly, with antemortem depres- sion of temperature (to 32.2° C. — 90° F.) ; under these circumstances the symptoms are probably the result of in- toxication. Symptoms of intoxication (fever, muscular paresis) also appeared in rabbits after intravenous injection of considerable amounts of bacteria. Further, cultures de- vitalized by chloroform proved quite toxic. The influenza- bacillus thus appears to generate an active toxin, a fact that sheds considerable light upon the nervous manifestations frequently observed in cases of influenza in human beings. Immunity. — The monkeys in Pfeiffer's experiments re- acted much less vigorously to a second injection of influenza- bacilli than to the first ; and Pfeiffer believes this to be an indication of immunity. Human beings certainly can be attacked several times by influenza, even in the course of the same epidemic. This fact does not, of course, exclude the possibility that a certain degree of immunity follows an ANTHRAX. 287 attack of influenza, but this must, however, be considered as only temporary. Pseudo-influenza-bacilli. — In a number of broncho- pneumonic foci in patients not suffering from influenza (but from diphtheria) Pfeififer found bacilH that in form and stain- ing properties resembled influenza-bacilli, and that, like these, developed exclusively upon blood-agar. Similar bacilli have since been isolated by various observers from cases of otitis media and of influenza. Pfeiffer believes these organisms to be related to influenza-bacilli, and he desig- nates them pseudo-influenza-bacilli. They are to be dis- tinguished from true influenza-bacilli by culture, in which, after the lapse of twenty-four hours, they appear consider- ably larger in all dimensions, and they exhibit a marked tendency to the formation of long pseudo-filaments. In similar cultures of the true bacillus the latter are wanting entirely or appear but exceptionally. Bacteriologic Diagnosis of Influenza. — The bacterio^ logic diagnosis is made from examination of the sputum derived from the deepest possible portions of the air- passages, and thus preferably from examination of the bronchial sputum. Microscopic examination alone is usu- ally insufficient, but, as a rule, cultivation of the bacillus is necessary, the method for which has already been described in detail (p. 283, Plate-procedure). Bacteriologic examina- tion may acquire differential diagnostic significance. Thus, Borchardt relates a case in which the diagnosis oscillated for a long time between typhoid fever and influenza, until bacteriologic examination decided in favor of the latter. ANTHRAX. Pollender, in Germany, in 1849, and Rayer and Davaine, in France, in 1850, were the first, independently of each other, to detect bacilli in the blood of animals suffering from anthrax. The growth of the anthrax-bacillus in pure culture and the experimental development of the dis- ease by means of the bacillus were successfully accom- plished first by Koch in 1876. Morphology of the Anthrax-bacilli. — Anthrax-bacilli appear as transparent, homogeneous, nonmotile rods. They are from i to i-S fJ- thick, and from 5 or 6 to 10 /x long, but they 288 CLINICAL BACTERIOLOGY. are subject to great variations in size. In cultures the bacillus is prone to be considerably longer than in the animal organism. In the blood of human beings it is shorter than in that of ro- dents; in cattle it is shorter than in white mice and in guinea- pigs. The broad side of the bacillus is slightly rounded. The surfaces of two adjacent bacilli in direct contact are, however, plane. In the blood of animals suffering from anthrax the bacilli are at times collected into small filaments of two or four, or at most five, members. Only attenuated bacilli, just capable of causing death in experimental animals, form long filaments in the organs of the animals. Such filaments are usual in the body of the frog. In cultures, on the other hand, Fig. 63. — Bacillus antliracis, stained to show the spores ; X 1000 (Frankel and PfeiflFer). the anthrax-bacillus exhibits an especial tendency to the forma- tion of long, intertwining chains. Frequently, bacilli obtained from the lesions of the disease present a bright border that some observers have looked upon as a capsule. This supposed capsule may be demonstrated by the staining method of Johne in the following manner: The preparation made from the blood or visceral fluid and dried in the air is passed three times through the flame ; stained for from fifteen to thirty seconds in a slightly warmed, two per cent, aqueous solution of gentian-violet ; washed in water ; exposed for ten minutes to the action of a one per cent, solution of acetic acid ; and ex- amined in water. It is important to make the examination in water, as the mucoid capsule is not visible when the specimen is ANTHRAX. 289 mounted in Canada balsam. The bacillus can be stained with all aniline dyes and also by the method of Gram. Cultivation of Anthrax-bacilli. — The anthrax-bacillus is extremely indifferent with regard to its nutrient material. It will develop in the absence of oxygen, but it does not then gen- erate a peptonizing ferment. The temperature-minimum is 12° C. (53.6° F.); the temperature-optimum, 35° C. (95° F.) ; the temperature-maximum, 45° C. (113° F. ). On gelatin-plates, with a magnification of from 80 to 100, the superficial colonies appear as round discs of yellowish color, constituted of a tangle of threads which form a dense, impene- trable convolution at the center. Especially the border presents Fig. 64. — Bacillus anthracis: colony three days old upon a gelatin-plate; impress- preparation; X 1000 (Frankeland Pfeiflfer). quite distinctly this filamentous structure, and from it fre- quently passes off a delicate network of convoluted and curled processes, which gives the colonies their characteristic appear- ance. The gelatin in the neighborhood is softened, and begins slowly to undergo liquefaction. In gelatin stab-cultures the line of inoculation forms a whitish band, from the circumference of which numerous ramifying processes extend into the gelatin. Liquefaction of the culture- medium progresses slowly, the nonmotile bacilli, by reason of their weight, sinking to the bottom of the liquefied area. In bouillon small fragments appear and fall to the bottom ; they represent a union of the anthrax-bacilli for the formation of convolutions of filaments. 19 290 CLINICAL BACTERIOLOGY. On agar-agar a dense, creamy, coherent deposit forms ; and Vi^on potatoes a dry, whitish-gray layer. Blood-serum is lique- fied. Mi Ik is coagulated and peptonized. Sporulation. — If anthrax-cultures are exposed to tempera- tures above i8° C. (64.4° F.), the bacilli soon exhibit spore- formation. Slight development of spores takes place, besides, down to the temperature-minimum. The anthrax-spore in- variably lies exactly in the middle of the mother-cell, being much shorter, but as wide, and possessing an oval shape. After a time the germinating bacillus disintegrates, and the spore is set free. If the spore is introduced into a sterile hanging drop of bouillon, gelatin, or agar, its germination can be accurately followed under the microscope (possibly with the employment of a warm stage). The spore first loses its glis- tening appearance, and increases in volume. Its membrane then ruptures at one extremity and permits the escape of the newly formed bacillus. The young bacillus enlarges in the direction of the long axis of the spore, and soon throws off the still adherent spore-membrane. Spore-formation takes place only in the presence of free oxygen — thus never in the*animal body and never in the uninjured cadaver. The higher the temperature the more rapidly do the spores de- velop— at 37° C. (98.6° F.), as early as twenty hours; at 21° C. (69.8° F.), not before seventy-two hours; while above 42° C. (107.6° F.) the anthrax-bacilli do not generate spores at all. It is possible artificially to deprive the anthrax-bacilli of their capability of generating spores. For this purpose it is only necessary to add certain antiseptic substances to the nutri- ent medium (Chamberland and Roux) : for instance, carbolic acid in the proportion of from i : 600 to i : 1000, potassium bichromate in the proportion of i : 2000. The bacilli culti- vated upon such nutrient media remain sporeless permanently in all subsequent generations, and in this way special asporogenous anthrax-bacilli are developed. Resistance of Anthrax-bacilli and of Anthrax- spores. — Anthrax-spores, like all other spores, are exceedingly re- sistant structures ; while the fully developed bacilli suc- cumb after exposure for a quarter of an hour to tempera- tures in the neighborhood of 60° C. (140° F.), the spores die in compressed steam at a temperature of 107° C. (224.6° F.) only after the lapse of five minutes ; and in live steam at a temperature of 100° C. (212° F.) only after from twelve to fifteen minutes. Five per cent, carbolic acid destroys the mature forms of anthrax-bacilli in ten seconds, but the spores not before from thirty-seven to forty days. In ANTHRAX. 291 I : looo solutions of mercuric chlorid the spores are not destroyed earlier than after the lapse of twenty hours (Gep- pert). In sterilized distilled water or tap-water anthrax- bacilli survive only for three days, but the spores for from one hundred and fifty-four days to a year. The lower, fur- ther, the temperature, the better do the anthrax-organisms withstand the injurious influence of the water. Anthrax- bacilli in distilled and other water have been demonstrated to undergo sporulation at a temperature of 20° C. (6S° F.). The bacilli are rapidly destroyed by the influence of putre- faction, while the spores have been found alive in putrefac- tive mixtures after a month. The statements made apply only to robust spores, the descendants of highly virulent bacilli. Anthrax-spores of indifferent source do not invariably react alike ; thus, von Esmarch was able to render some anthrax-bacilli in- nocuous by means of 5 per cent, carbolic acid within two days, and by means of live steam at a temperature of 100° C. (212° F.) within three minutes. The Occurrence of Anthrax in Animals. — The animal most susceptible to anthrax is the sheep, and it is often attacked by the disease spontaneously. There is, however, one variety of sheep that is immune — namely, the Algerian sheep. This immunity is not dependent upon climatic con- ditions, for European sheep transported to Algiers were as readily attacked by anthrax as at home. White mice and guinea-pigs succumb likewise with regularity to experi- mental anthrax-infection ; rabbits are somewhat more resist- ant ; but none of these three species of animals is scarcely ever exposed to natural infection. Cows and calves not rarely succumb to spontaneous anthrax, although they prove rather refractory to experimental subcutaneous infec- tion. Horses, antelope, deer, goats, at times acquire an- thrax ; swine more rarely. With regard to the much- discussed immunity of white rats to anthrax, it appears that young animals die of anthrax after experimental infection, whereas old animals usually exhibit only a local lesion, from which they recover. Carnivorous ani- mals (dogs, cats, etc.) rarely are attacked spontaneously by anthrax. Experimentally, the disease can be induced in mature animals only by intravenous injection of the bacilli. New-born or quite young dogs, on the other hand, prove extremely susceptible. Birds, reptiles, batrachians, 292 CLINICAL BACTERIOLOGY. possess a considerable degree of immunity to anthrax. If, however, on the one hand, the high temperature of birds is reduced artificially ; or if, upon the other hand, the low temperature of reptiles is elevated artificially, these animals react precisely like other animals — that is, they succumb to inoculation with anthrax. Pigeons may also be rendered susceptible to anthrax-infection by starvation. Occurrence of Anthrax in Human Beings. — Human beings are infected, as a rule, through contact with animals suffering from anthrax, and with the cadavers of those dead of the disease. Not only the fresh cadaver is infectious, but also, owing to the presence of spores, which develop rapidly in summer with unobstructed access of oxygen, every individual portion of the body — wool, hair, horns, etc. — -even after the lapse of a long time. For this reason tanners are exposed to danger in manipulating the skins of animals dead of anthrax. Even after the hide has been tanned, the spores are not always destroyed with certainty, and shoemakers, furriers, and harness-makers have been known to be infected by means of such leather. Workers in horsehair, turners of horn, brush-makers, dealers in hides, etc., are likewise exposed to the danger of infection with anthrax in so far as the raw material with which they work is derived from animals suffering from anthrax. The following instructive example, in this connection, has been reported by Einike : An ox dies of anthrax. Two indi- viduals who partake of its meat die of the same disease. The hide of the animal, after it has been macerated for a time in a small lake, is used by a harness-maker for making halters. This man is attacked by anthrax. The two horses that wear the halters likewise succumb to this disease. Of a herd of sheep that bathe in the small lake mentioned twenty are attacked by anthrax. The transmission of anthrax from animals to human beings may further be effected by certain varieties of flies that possess a stiff and pointed sting (stomoxys, glos- sina, Ixodes). In the bodies of such insects as have fed upon anthrax-cadavers anthrax-bacilli have been repeatedly demonstrated. Human beings are not especially susceptible to anthrax. For this reason only a local lesion generally forms at first — the so-called mali£^?iant pus tti/c (a.nthra.x-ca.rhunc\e) — which frequently terminates in recovery. In other cases the car- ANTHRAX. 293 buncle is succeeded by septicemic general infection, leading to death. Anthrax may be further transmitted from one human being to another. In the nature of things such an occurrence is rare, although it has been reliably observed. Jacobi has reported four cases in which the disease was conveyed by means of a hypodermic syringe that had pre- viously been employed in a case of anthrax. The infectivity of malignant pustule was formerly denied, and upon the basis of experiments in which the serous con- tents of the pustules were inoculated in healthy individuals, without the development of any reaction whatever. Such experiments are, however, not conclusive, as the serous contents of the vesicle, which surround the central eschar, contain only a small number of bacilli. Natural Portals of Entry for the Anthrax-bacillus. — (a) Skin. — Infection through the skin is possible only in the presence of a breach in continuity, however slight. In human beings this mode of infection is the most common, and particularly in individuals that come in contact with anthrax-cadavers or constituent parts thereof The course of infection has already been outlined. At the point of infection a carbuncle develops, and this leads either to recovery or to general infection. (U) Digestive Tract. — Spontaneous anthrax of herbivor- ous animals almost always involves the intestine (pasture- anthrax). If the herds graze on some meadow-lands in certain districts (champs maudits), anthrax is sure to occur among them. In Germany such regions exist in some parts of Saxony and in the Bavarian Alps, in France especially in the district Beauce, in Austria in the Hun- garian lowlands, and in Russia in the neighborhood of Novgorod. It is believed that the earth in these meadow- lands contains spores, which are swallowed by the animals in grazing. The question naturally arises. How are the anthrax-spores brought to the surface of these fields ? Pasteur, who was the first to occupy himself with this question, found anthrax-spores in the earth of graves in which years before anthrax-cadavers had been. buried. It was his opinion that these spores are carried to the surface from the depth by earth-worms. The worms swallow the contaminated earth, later crawl upward to the surface, and there deposit the spores with their excrement. Such a possibility has been demonstrated experimentally, and that 294 CLINICAL BACTERIOLOGY. it may actually be realized would appear from the observa- tions of Bollinger, who succeeded in finding anthrax-spores in the bodies of worms collected from the anthrax-fields of the Bavarian Alps. The view of Pasteur was vigorously attacked by Koch. At the depth at which anthrax-cadavers are usually buried (from i/^ to i meter) the temperature, even in the hottest summer-months, is only between 14° C. (57.2° F.) and 18° C. (64.4° R), a temperature that is extremely unfavorable for sporulation. According to Soyka, the addition of porous particles of earth to artificial cultures furthers spore -production quite materially. Then, it is further to be borne in mind that the temperature of the buried cadavers is certainly raised somewhat in consequence of the putrefactive processes taking place. Kitasato buried gelatin-cultures and agar-cultures of anthrax in the earth and showed that at a depth of one meter sporulation pro- ceeded in the months of June, July, and August. The conditions provided experimentally and artificially are, how- ever, not entirely comparable with those observed prac- tically. Animals dead of anthrax are, or were formerly, first dissected and skinned, the secretions, the blood, etc., being spilled indiscriminately ; burial also is not effected immediately ; in brief, abundant time and opportunity are afforded the bacilli, at least in summer, to form spores. The cadaver, which is now buried, contains not only mature forms, but also permanent forms ; quite apart from the fact that numerous anthrax-bacilli are already present upon the surface of the earth in consequence of the various manipulations at the place where the autopsy was held. It is true that if the animal is buried at a depth of two or three meters immediately after death, without further manipulation, the formation of spores would certainly be entirely prevented ; for, then, one of the most important conditions for spore-formation — namely, unobstructed ac- cess of oxygen — would be completely wanting. In addition to earth-worms anthrax-spores may be dis- seminated by means of snails, flies, etc. Further, the diseased animal furnishes during life sufficient material for subsequent infection through the feces and the urine, which contain the bacilli in large number. In these, and also in vegetable culture -media, the bacilli, which are so indifferent with relation to the nature of the nutrient material, multiply abundantly, and in summer also form spores. The excre- ANTHRAX. 295 ment of animals fed with anthrax-matter contains spores regularly. Also, the feces of healthy animals (sheep) that have grazed upon anthrax-fields contain spores under cer- tain conditions. The introduction of anthrax -bacilli into the gastro-intestinal tract does not invariably induce anthrax. The spores in some cases pass through the digestive tract without causing injury, although they remain as infective for other animals as they were formerly. Also, this mode of infection presupposes the existence of an injury or a lesion of the mucous membrane. With hay obtained from anthrax-fields the spores gain entrance into stables and give rise to so-called stable -epidemics. As a result of floods in the infected districts, the germs are carried far and wide, and thus give rise to cases of anthrax at places where previously the disease was unknown. In human beings gastro-intestinal anthrax is by no means so common as malignant pustule ; it was described by earlier observers as intestinal mycosis. Probably it occurs more commonly than current reports would indicate. The cases pursue the clinical course of dysentery or of typhoid fever, and if bacteriologic examination is omitted at the autopsy, they may readily escape diagnosis. ic) Lungs.- — Pulmonary anthrax has been repeatedly observed in England (Bradford) in individuals who pull sheep's wool or work with goat's or camel's hair and the like {wool-sorter s disease^, and in Germany in those who as- sort rags (ragpicker' s disease^. Experimental Development of Anthrax. — All of the modes of infection that have been mentioned as possible in accordance with clinical experience may be imitated in ex- periments on animals. Susceptible animals die of anthrax- septicemia after cutaneous and subcutaneous inoculation. The spleen in such animals is quite considerably enlarged at autopsy, its consistency is soft and friable, its color dark. If spore-containing material is fed to sheep, four among ten animals, on an average, die after the introduction of small amounts ; if large amounts are used, almost all die. The spores, thus, escape uninjured the influence of the acid gas- tric juice. Anthrax occurs but seldom in rabbits, guinea- pigs, and white mice as a result of feeding-experiments. Buchner has made careful observations with regard to the occurrence of pulmonary anthrax. When the spores are inhaled experimentally, the animals die of general infection. 296 CLINICAL BACTERIOLOGY. The permanent forms penetrate the intact mucous mem- brane of the alveoli, and gain entrance into the lymphatics and blood-vessels, in which they germinate. After inhala- tion of anthrax-bacilli, however, such migration does not take place. The bacilli remain inactive, and cause only local irritation — circumscribed inflammation of the lungs. In this connection it may be noted that in quite rare cases of anthrax the disease has also in human beings pursued the clinical course of septicemia-^that is, without local in- fection and without malignant pustule. The portal of infection remained undiscovered in these cases, although it is possible that this part was taken by the lungs. Distribution of Anthrax-bacilli in the Infected Body. — In cases of general infection with anthrax the bacilli are found in the blood, and in only a small number of instances in the larger vessels, the greater amount being present in the capillary area. The capillaries of all the organs, especially the spleen and the liver, are filled by the bacilli, sometimes actually packed with them, so that their lumen appears to be occluded. Most of the bacilli lie with their long axes parallel to the walls of the vessel — that is, they are directed in the course of the blood-stream. In the glomeruli of the kidneys and in the intestinal villi the mass of bacteria fre- quently induces capillary hemorrhages. The microorgan- isms in this way gain entrance into the uriniferous tubules, although generally they do not progress beyond the con- ♦voluted tubes (Koch). Anthrax in animals may, therefore, be considered as the type or paradigm of a true infection, the factor of intoxication remaining entirely in the background and the most prominent feature being the enormous multi- plication of the exciting agents. On examining the viscera of animals dead of anthrax the impression is gained that death has resulted from a flooding of the capillary area with the bacilli. The anthrax-bacilli are, however, by no means found in the blood immediately after infection, but several hours always elapse before they appear. The animal, how- ever, will previously have been sick. This indicates that the formation of toxin on the part of the bacteria is not en- tirely wanting — a circumstance whose certainty is estab- lished by the course of the disease in less susceptible ani- mals (and in human beings), in which the lesion remains localized. In cases of malignant pustule the bacilli are present in ANTHRAX. 297 considerable number only before the wall of leukocytes that separates the eschar from the subjacent tissues. They surround the hair-follicles and the sweat-glands, but their distribution bears no relation to the blood-vessels. The infiltrates in cases of intestinal anthrax are, both histologically and bacteriologically, to be placed upon the same plane as malignant pustule. The mesenteric glands are swollen and filled with the parasites. In cases of pulmonary anthrax if a local lesion has developed, the bacilli are found in the perivascular lymph- spaces. If a lesion is wanting at the portal of entry, the swollen bronchial glands nevertheless constantly contain the bacilli. Death occurs in every variety of anthrax, usually as a result of general infection. In addition to the blood the parasites pass over into the milk, the bile, the saliva, and the feces. They are found less commonly in the urine. Toxins of Anthrax-bacilli. — Practically nothing is known with regard to the metabolic products of the anthrax- bacillus. Hankin obtained a toxic albumose from pure cultures. With regard to the relation between this albumin- ous substance and the actual anthrax-toxin, the same state- ment may be made as that with regard to the toxalbumins of Brieger and Frankel (pp. 29, 30). Mixed Infection. — In cases of anthrax mixed infection is not without significance. The limiting suppuration, which brings about the exfoliation of the malignant pus- tule or of the intestinal filtrate, may be excessive and give rise to phlegmons and to septicemia or pyemia ; many a pa- tient who has survived the anthrax-infection proper has died later of these secondary suppurative processes. From the blood and from the internal viscera staphylococci and streptococci may then be cultivated. Heredity. — In the case of anthrax it has been demon- strated experimentally that the bacillus passes from the mother to the fetus only when changes have taken place in the placenta — minimal hemorrhages will suffice. Intrauter- ine infection with anthrax has also been observed in human beings (Marchand). As bearing upon the question whether the fetus in utero may infect its mother, the experiments of Lingard are interesting. This observer infected rabbit- fetuses in utero with anthrax. As a rule, the mothers were not infected, but they later proved immune to the disease. 298 CLINICAL BACTERIOLOGY. the immunity lasting for more than eight months. In this way would be demonstrated the law for anthrax that CoUes propounded for syphilis (p. 318). Bacteriologic Diagnosis. — From the deeper parts of a suspected pustule some tissue-fluid is removed and plates are made therefrom. If suspicious colonies develop, pure cultures are made and animals infected therewith. If intes- tinal anthrax is suspected, the vomited matters and the feces should be examined bacteriologically. In cases of pulmon- ary anthrax the abundant frothy sputum at times contains the bacilli. Examination of the blood will yield informa- tion as to whether general infection with anthrax is present or not. Such examination is, therefore, always of the greatest importance in the formulation of the prognosis. It must, however, always be borne in mind that the bacilli are present especially in the capillary area. For sanitary purposes it is frequently necessary to decide whether an animal has died of anthrax or not. If death has taken place but a short time previously, microscopic examination of fluid from the spleen and of the blood will suffice. The presence of the bacilli with capsules, demon- strable by the method of Johne (p. 288), renders the diag- nosis positive. If, however, one or more days have elapsed since the death of the animal, then there develop in the body of animals dead of anthrax cadaver-bacilli that can be dif- ferentiated from the specific exciting agents of anthrax only with exceeding difficulty. Under such conditions gelatin- plates must be made, and mice and guinea-pigs inoculated with fluid from the spleen. In inoculating the animals the possibility of the presence of the bacillus of malignant edema must be borne in mind, as this microorganism likewise is not rarely found in the bodies of dead animals. On subcutaneous in- oculation the latter predominates, and the animals die of malignant edema notwithstanding the presence of anthrax- bacilli. This source of error can be avoided by inoculat- ing the animals cutaneously with the suspected material. The anthrax-bacillus alone will then develop, and the diagnosis can not go astray. If putrefaction has pro- gressed too far in the body to be examined, then the anthrax-bacilli may be completely suppressed by the com- petition of the other varieties of bacteria, so that the diag- nosis is no longer possible. ANTHRAX. 299 Immunity and Vaccination, — Recovery from malignant pustule does not confer immunity, at least none of any duration. It has been repeatedly observed that the same individual has been attacked two or three times with malignant pustule, and even at intervals of a few months. The second attack often pursues even a more virulent course than the first. Nevertheless, it is possible experi- mentally to immunize animals to anthrax. The anthrax- bacillus is the microbe with which Pasteur demonstrated for the first time that the action of heat diminishes the virulence materially. Pasteur cultivated anthrax-bacilli at a temperature of 42° C. (107.6° F.). They still developed at this temperature, but gradually suffered in virulence from day to day. If after the lapse of several days the bacilli thus attenuated are transferred to a new nutrient medium and are exposed to a more favorable temperature (35° C. — 95° F.),they then grow luxuriantly again, but their virulence remains attenuated ; the bacilli do not recover. Upon the basis of these fundamental facts Pasteur prepared two varieties of attenuated anthrax-bacilli, one of which had been exposed for from fifteen to twenty days to a tempera- ture of 42° C. (107.6° F.), and which was capable of destroying only guinea-pigs not more than twenty-six hours old (Vaccine I). The other had been exposed from ten to twelve days to a temperature of 42° C. (107.6° F.), and it was capable of destroying guinea-pigs, white mice, and rarely rabbits (Vaccine II). According to the method of Pasteur, animals (rabbits, sheep, cows) are inoculated first with Vaccine I. They be- come sick and exhibit fever of greater or less intensity. After the disease-symptoms have disappeared, the animals are inoculated with Vaccine II, and the same symptoms are repeated. After the animals have recovered from this second disturbance they are thoroughly protected against inoculation with highly toxic anthrax-material. The attenuated anthrax-cultures generate alkaline sub- stances upon the nutritive media ; whereas the virulent cultures generate a considerable amount of acid. The vaccines are distinguished from the highly virulent cultures, further, by the fact that they give rise to febrile movement. This fact is usually interpreted as indicating that the fever represents a reaction of the organism to the invading parasites. As a matter of fact, it has for a long time been 300 CLINICAL BACTERIOLOGY. a prognostic rule clinically, peculiarly in the case of anthrax, that complete apyrexia is a sign of ill omen. Pasteur's method of vaccination confers complete and se- cure protection against subcutaneous anthrax. As to its protective power with regard to field-anthrax, opinions have differed. Koch, Lofifler, and Gaffky express themselves with some reserve in this connection. They do not believe that the protective inoculation confers absolute immunity to intestinal anthrax. Statistics, however, appear unquestion- ably to favor the view of Pasteur. Since the practice of vaccinating the herds has been instituted in France, the mortality from anthrax among grazing cattle has been materially reduced. The blood-serum of animals immunized to anthrax does not, so far as is known, transmit immunizing properties to other animals. Prophylaxis. — The prophylaxis against anthrax consists in rendering innocuous the bodies of animals and human beings dead from the disease. This end is attained in the simplest manner by incinerating the bodies as a whole. The danger of infection, however, is maintained by the introduction and manipulation of suspicious material (hair, wool, rags, leather) from foreign countries in which the sanitary precautions observed by most intelligent nations are not enforced. In England, the home of wool-sorter's disease, regulations have been put in force requiring that wool, which is derived principally from Asia, should be boiled before it is assorted. Since, the disease has mate- rially diminished. In the same spirit the disinfection of skins, hair, wool, etc., of suspicious origin should be everywhere insisted upon before being manipulated. GLANDERS. The exciting agent of glanders was discovered in 1882 by Lofifler and Schiitz. Morphology of the Glanders-bacilli. — The glanders- bacilli are small, slender, nonmotile rods, at times curved, with rounded extremities, from 2 to 3 ^t long, and from 0.2 to 0.4/ji thick. As a rule, they lie singly. The question whether glanders-bacilli form spores is still an open one, though answered in the affirmative by Baumgarten and Rosenthal, who succeeded in staining spores, and in the negative by most observers, because GLANDERS. 301 the germination of the so-called spores has not yet been ob- served. The glanders-bacillus takes all stains without difficulty, but it is decolorized with equal facility. The best specimens are ob- tained in cover-slip preparations by treatment with hot Loffler's solution or hot carbolfuchsin, and decolorization with distilled water. The organisms do not stain by Gram's method. In stained preparations the bacilli exhibit almost regularly un- stained deficiencies. The bacilli frequently appear in the form of short structures resembling cocci. They are facultative anaerobic. The temperature-minimum is 25° C. (77° F. ) ; the temperature-optimum, 37° C. (98.6° F.) or 38° C. (100.4° F.) j the temperature-maximum, 42° C. (107.6° F.). i ■'.'S- r-^^' 'V;-/nSpii^ Fig. 65. — Bacillus mallei, from a culture upon glycerin agar-agar; X 1000 (Frankel and Pfeiffer). Cultural Properties. — On glycerin-agar plates glistening, granular colonies, with a yellowish tinge and a smooth border, appear. Streak- cultures upon Glycerin-agar. — Along the line of inocu- lation a moist, whitish coating forms. Upon blood-serum iso- lated, translucent, yellowish drops appear, which do not liquefy the culture-medium. Upon gelatin slight growth takes place at 25° C. (77° F.), with slight liquefaction. Bouillon is rendered densely turbid. The growth of glanders-bacilli upon potato is characteristic. The inoculated surface exhibits after two days a thin, honey-yellow deposit, which after a week becomes quite dark, brownish-red, and surrounded by a slightly blue, irides- cent zone. The best culture-medium is glycerin-agar. 302 CLINICAL BACTERIOLOGY. Resistance of Glanders-bacilli. — For the continued cultivation of glanders-bacilli it is important to know that the organisms rapidly undergo natural attenuation as early as the fourth or fifth generation. If, therefore, it is desired to obtain virulent bacilli, it is necessary to interpolate inocu- lations of animals every two or three culture-generations. In the dry state the glanders-bacilli, according to Lofifler, retain their vitality for three months. Other observers, however, have found them dead within ten days when dried in a thin layer. Toward heat the glanders-bacilli prove quite resistant. They are destroyed in two minutes at a temperature of ioo° C. (212° F.) ; in five minutes at a temperature of 80° C. (176° F.) ; in fifteen minutes by I : 1000 mercuric chlorid ; and in an hour by five per cent, carbolic acid. Susceptibility of Animals to Glanders. — Among do- mestic animals there are susceptible in diminishing degree asses, mules, horses, goats, cats, sheep, dogs, swine. Cattle are immune. Among experimental animals field- mice, wood-mice, and guinea-pigs exhibit the most pro- nounced, and rabbits a much slighter, predisposition. White mice and domestic mice prove entirely insusceptible to glanders. If, however, white mice are previously pois- oned with phloridzin, they succumb to infection with glanders (Leo). Birds, with the exception of pigeons, are refractory to glanders. In all animals glanders is at first a local disease, but later it becomes generalized and attacks all of the viscera. Occurrence and Distribution of the Bacilli in the Products of the Disease. — From a pathologic-anatomic standpoint glanders belongs to the group of diseases that, like tuberculosis, give rise to the formation of nodules that exhibit a marked tendency to disintegration and softening. The small, often only submiliary, grayish new- formations consist of epithelioid cells and mainly of leuko- cytes. The glanders-bacilli are present especially at the center of the granulations. They are demonstrable with difficulty by staining. The best mode of procedure con- sists in placing the sections for from six to eight hours in carbol-methylene-blue (methylene-blue i, absolute alcohol 10 cu. cm., carbolic acid, 5 per cent., 100 cu. cm.) or car- bolfuchsin, next decolorizing in dilute acetic acid, and then in distilled water, drying upon a slide, and, after clearing in GLANDERS. 303 xylol, mounting the preparation in xylol Canada balsam. The sections may also be treated according to Weigert's method (p. io6), Loffler's alkaline methylene-blue solution being selected for staining. In this way positive results are obtained only with the relatively recent nodules. If necrosis has taken place, the bacilli are only rarely found in the products of disintegration. Experiments on Animals. — Small numbers of bacilli inoculated subcutaneously into susceptible animals (gener- ally guinea-pigs or field-mice), or larger numbers rubbed into the uninjured skin, cause death with considerable cer- tainty. The mice die quickly — within three or four days — and the spleen, the liver, and the lungs are filled with an enormous number of nodules scarcely visible to the naked eye. Guinea-pigs are better adapted for the study of the course of infection. In them a local infection first develops, an infiltration, that soon is converted into an ulcer with indurated margins. Then follow swelling and suppuration of the adjacent lymphatics and lymph-glands, and finally general infection with the characteristic new-formations. The process advances by way of the lymph-paths. Apart from cases pursuing a very acute course, the blood almost never contains the bacteria. The infection of the lymphatic apparatus extends with exceeding rapidity. As early as an hour after inoculation of a superficial skin- wound cauter- ization of the latter will no longer suffice to prevent the development of the disease. The urine, the seminal fluid, the sweat, the saliva, and the aqueous humor of infected animals may contain the parasites. The spleen and the bile are said to be free from them. Portals of Entry For and Course of Glanders. — In human beings, who, in the vast majority of cases, are infected by contact with horses suffering from glanders, the skin, with- out doubt, constitutes the principal portal of infection. The individuals in question, usually hostlers, coachmen, farmers, cavalrymen, and the like, may be infected through the in- termediation of the most superficial and most insignificant wounds of the skin. Laboratory-infection with glanders, in manipulating infective material and glanders-bacilli, has been observed repeatedly. Infection sometimes takes place also through the mucous membranes. Cases are on record in which hostlers have acquired the disease by drinking from the same pail as their sick horses, and the 304 CLINICAL BACTERIOLOGY. like. Whether or not infection may take place by way of the respiratory apparatus has not yet been decided with certainty. Nevertheless, it is remarkable that in horses glanders, -in its initial stage, is seated in the nasal cavity (glanders-ulcer). Bollinger believes that, especially in those attacked by glanders who present general symptoms in advance of the local manifestations, the virus has gained entrance into the body through the respiratory passages. The use of meat from animals suffering from glanders may, likewise, give rise to the disease. At least a number of feeding-experiments in animals are in favor of this view. Such experiments have yielded positive results in cats, dogs, lions, and bears. On the other hand, other experiments with the feeding of glanderS-material have yielded negative results. The muscles themselves do not harbor the para- sites, but rather the lymphatic vessels and glands lying within them or near them. TJie clinical picture of glanders in human beings is a rather variable one, in accordance with the site of infection. Usu- ally there occurs locally at the portal of infection a swelling, and this is soon followed by tumefaction and suppuration of the neighboring lymphatics. Then multiple abscesses form in the skin, the muscles, and the internal viscera, and often suppuration takes place in joints. The clinical picture re- sembles that of pyemia. Upon the mucous membranes, and particularly in the nose, characteristic glanders-nodules appear, which soon disintegrate and give rise to ulcers. Death results from the general infection, which takes place in human beings by way of the lymphatics. An acute and a chronic variety of glanders have been observed. In the former suppuration is the more likely to occur ; in the latter — so-called farcy — tissue-proliferation is the more conspicuous. Heredity. — The transmission of glanders-bacilli from the mother to the fetus has been observed repeatedly. The following observation by Loffler is interesting. A female guinea-pig that had recovered from inoculation with glan- ders gave birth to offspring five months later. Apparently healthy at birth, the young animal died of visceral glanders at the age of a week. Bacteriologic Diagnosis of Glanders. — The bacterio- logic diagnosis has to contend with the difficulty that in the course of suppurative destruction in the foci of disease the MALIGNANT EDEMA. 305 bacilli also are destroyed. In suspicious cases, however, agar-plates should always be made with the pus from the ulcers in question. In addition male guinea-pigs should receive intraperitoneal injections of the suspected secretion. If the disease be glanders, swelling of the testicles takes place in the course of two or three days. This is absolutely characteristic, and is subsequently followed by suppuration of the organs (Straus). The injection must always be made in the median line, as otherwise there is danger of injuring the seminal vesicles, and as by direct injection into these structures other microorganisms also may give rise to swelling and suppuration of the testicles. . Mallein. — In exactly the same manner as tuberculin is prepared, von Preusse and Kalning obtained from cultures of glanders-bacilli a lymph — mallein — representing a proteid substance of the glanders-bacilli. Mallein is employed in veterinary medicine for diagnostic purposes. Horses suf- fering from glanders react to injection of the lymph with fever. Prophylaxis. — The most efficient prophylaxis against glanders consists in the destruction of animals suffering from the disease, and the incineration of their carcases. The remaining horses in a stable in which the disease has appeared must be subjected to rigid quarantine, and the attendants must have their attention called to the danger of infection and be enjoined to careful disinfection. MALIGNANT EDEMA. The exciting agent of malignant edema, which was de- scribed in 1 88 1 by Koch, is identical with the vibrion sep- tique of Pasteur. The bacillus of malignant edema (vibrio septicus) is a slender bacillus about as long as the anthrax-bacillus, but some- what narrower (from o.S.to i //), with rounded extremities. It possesses spontaneous motility, which is due to from three to twelve lateral flagella. The vibrio septicus has a tendency to form long filaments, both in culture and also — in contradis- tinction from the anthrax-bacillus — in still greater degree in the living body. The chains not rarely present pretty, arch- like curves. The temperature-optimum is 37° C. (98.6° F.), although the bacillus will grow at room -temperature. Above 306 CLINICAL BACTERIOLOGY. 20° C. (68° F. ) it is capable of sporulation. The spore, situ- ated centrally, is at times wider than the bacillus, which then assumes a spindle-shaped, distended appearance. The vibrio septicus is a rigidly anaerobic organism. It stains without difficulty with all of the aniline dyes, but not by Gram's method. Cultural Properties. — O^ gelatin-plates the colonies present the appearance of glistening, hollow globules, filled with fluid. At their center, with a magnification of from 80 to 100, a dense network of intimately intertwined threads may be seen, with radiating processes at the periphery. On more careful exami- nation it will be observed that the colony is in motion. Fig. 66. — Bacillus of malignant edema, from the body-juice of a guinea-pig inoculated with garden-earth; X looo (Frankel and Pfeiffer). On agar-plates small, irregular, whitish, translucent colonies appear, with a dense center, from which innumerable delicate ramifications proceed. In high gelatin stab- culture liquefaction and turbidity of the underlying gelatin take place, with abundant formation of gas, especially on addition of reducing substances. In high agar stab-cultures the line of inoculation exhibits ser- rated branched margins. Abundant gas-production takes place. Bouillon is rendered turbid, and later a precipitate forms. The reaction is not changed, but carbon dioxid and hydrogen develop. All cultures generate a disagreeable, fetid odor. MALIGNANT EDEMA. 307 Experiments on Animals. — Horses, swine, sheep, goats, dogs, pigeons, ducks, hens, rabbits, mice, and guinea-pigs are susceptible to mahgnant edema. In guinea-pigs sub- acute infection pursues quite a characteristic course. The animals cower, their hair stands erect, and signs of great fear set in. On the slightest touch the animals cry out. Death takes place within twelve hours. On autopsy there is found at the site of inoculation quite con- siderable bloody edema, with a small number of gas- bubbles, involving the subcutaneous connective tissue and the superficial muscles. In this area the bacilli with spores are found in large numbers. On the other hand, the blood and the internal viscera are always free from bacteria if the autopsy is performed immediately after death. As long as the animal continues to live, the anae- robic edema-bacilli are not capable of multiplying in the oxygen-containing blood. Only after death has taken place do they advance, and soon the viscera and the blood are filled with them. The spleen of the animal is large and diffluent. The liver and the lungs are pale, and the latter are of a peculiar grayish-red color. In mice the bacilli gain entrance into the blood and the internal viscera during life. In the main, malignant edema represents an intoxication. As a matter of fact, precisely the same clinical picture can be developed experimentally in animals if the mature bouillon-culture or the serum from the edema freed of bac- teria by passage through a porcelain filter is injected intra- peritoneally into guinea-pigs in rather considerable amount. Occurrence of the Vibrio Septicus. — The bacilli of malignant edema, or rather their resistant permanent forms, are widely distributed in nature. They are the attendants of putrefactive processes, especially those that take place in the absence of oxygen. They are found present in manure, in dust, and in the earth of gardens and of fields. If a small amount of earth be introduced into a pocket beneath the skin in a guinea-pig or a rabbit, the animal will die of malignant edema. The picture developed under these con- ditions is, however, not a pure one. In addition to the septic vibrios a large number of other microbes will be found present in the edema, and as a result of this mixed infection the exudate is not merely bloody and serous, but putrid and fetid. 308 CLINICAL BACTERIOLOGY. Malignant Edema in Human Beings. — Malignant edema occurs at present but seldom in human pathology. In preantiseptic times it was more common and more alarming. The so-called septicemie gangreneuse and the gangrene gazeuse of the French were in part manifestations of the ac- tivity of the vibrio septicus, and identical with our malig- nant edema. In order that the bacillus of malignant edema may exhibit its activity, the wounds through which infection takes place must be deep, because the strictly anaerobic parasite has no opportunity for development upon the sur- face. The bacillus may, further, be associated in mixed infec- tion with other microorganisms, which consume the oxygen available, and thus artificially produce an atmosphere free from this gas. It may be mentioned that in two patients suffering from typhoid fever malignant edema has been ob- served after subcutaneous injection of tincture of musk. The portal of infection is always constituted by a breach in continuity of the external integument. Bacteriologic Diagnosis. — Plates are made from the putrid exudate in an atmosphere of hydrogen, and at the same time guinea-pigs are inoculated subcutaneously. Immunity. — Malignant edema is one of those bacterial diseases in which artificial immunization through metabolic products was first effected. Chamberland and Roux im- munized guinea-pigs by means of intraperitoneal injections of bouillon-cultures sterilized by exposure for ten minutes in the autoclave at a temperature of between 105° C. (221° F.) and 110° C. (230° F.). Immunity to malignant edema is also established without difficulty in animals by all other methods. PROTEUS-INFECTIONS. Morphology of the Proteus. — The proteus-bacteria, dis- covered by Hauser in 1885, are small, motile rods of ex- ceeding activity and variable size. The proteus-bacteria are usually arranged in pairs, and not rarely also in longer filaments. In addition to these funda- mental forms coccus-like bodies and long, winding threads (spir- ulins) also are encountered. The proteus is characterized by the possession of an unusually large number of flagella surround- ing the body of the cell. It is stained readily with carbolfuch- sin, and less well with watery solutions of aniline dyes. It does PROTEUS-INFECTIONS. 309 not stain by Gram's method. The proteus thrives equally well at room-temperature and at the temperature of the body. The temperature-optimum is between 20° C. (68° F.) and 25° C. (77° F. ). The organism does not give rise to spores, and is destroyed by exposure for five minutes to a temperature of 55° C. (i3i°F.). Cultural Properties. — On gelatin-plates small, round, yellowish colonies form at first, with a dense center and an irregular margin, from which bristle-like processes pass off. Other colonies are bounded by a zone of filaments surrounding the central opaque mass, in part circularly, in part in the most / Fig. 67. — Swarming islands of proteus-bacilli on the surface of gelatin ; X 650 (Hauser). varied loops and convolutions. Extensive and rapid liquefaction of the gelatin takes place. Into the adjacent nutrient medium extend processes, both straight and tortuous, which frequently are cut off from the mother-area and move about as free islands in the partially liquefied gelatin. These conditions may be espe- cially well observed upon five or six per cent, gelatin. There thus result peculiar figures and designs, to which the proteus owes its name of ^* bacillus figurans. ' ' Gelatine stab-culture is liquefied with exceeding rapidity. In agar streak-culture a grayish, moist coating forms. On potato a dirty grayish deposit appears. Bouillon is rendered homogeneously turbid. 310 CLINICAL BACTERIOLOGY. All nutrient media emit a disagreeable odor. Formerly, Hauser distinguished three distinct varieties of proteus, the proteus vulgaris, the proteus mirabilis, and the pro- teus Zenkeri ; but later he abandoned this differentiation. Experiments on Animals. — If rabbits or guinea-pigs receive intraperitoneal or intravenous injections of consider- able amounts (three cubic centimeters) of proteus-culture, the animals die of acute enteritis and peritonitis. Intravenous injection of from five to ten cubic centimeters of a bouillon- culture is followed by much more typical phenomena in the dog. The animal suffers from bloody vomiting and bloody diarrhea attended with severe tenesmus. The temperature is elevated, and the sclerae are distinctly icteric. On autopsy the entire digestive tract, from the cardia to the anus, is the seat of an intense hemorrhagic inflammation. The blood and the internal viscera of the dog contain none or but a few bacteria. Exactly the same result is obtained with fil- tered cultures and with the bodies of the bacilli carefully destroyed. In mice, which, likewise, succumb to proteus- inoculation, the bacilli may be cultivated from the viscera. The organisms become the more virulent the oftener they are passed through the bodies of mice. Occurrence of the Proteus. — Proteus-bacteria are present in all putrefactive processes, and also in the gastro- intestinal canal. In human beings the proteus, in mixed infection with the ordinary exciting agents of inflammation, constitutes the cause o{\h& putrid, fetid phlegmons at times observed in the sequence of cadaveric infection. Besides, the proteus gives rise to so-called putiid intoxication, by subsequently penetrating into a primary focus of suppura- tion or a traumatic lesion, multiplying there, and generating metabolic products that are absorbed. According to H. Jager certain forms o{ febrile icterus, known as Weil's dis- ease, are caused by the proteus. Jager succeeded in culti- vating a fluorescent proteus from the urine, and after death also from the viscera of individuals suffering from Weil's disease. Infection had taken place in these cases from bathing in streams the water of which was contaminated by proteus. On the banks of a tributary brook an epidemic had occurred among fowl, the exciting agent of which was like- wise found to be the proteus fluorescens. Further, the pro- teus was found by E. Levy to be the cause of hemorrhagic GONORRHEA. 311 gastro-enteritis from which seventeen persons suffered after the use of spoiled meat. Bacteriologic Diagnosis. — Plates should be made with pus from the putrid phlegmons and possibly also with the urine obtained with sterile precautions from cases of Weil's disease. GONORRHEA. Morphology of Gonococci. — The exciting agents of gonorrhea, the gonococci, discovered by Neisser, in 1879, are cocci, which are separated into two distinct hemispheres by means of a dividing fissure. The individual members Fig. 68.— Gonococci in urethral pus; X 1000 (Frankel and PfeifFer). suggest the appearance of a kidney or a biscuit. The cocci can be stained with all aniline dyes, but not by Gram's method. They are from o:"^ to 1.6 /^ long, and from 0.6 to 0.8 /z thick. Occurrence of Gonococci. — Gonococci are constantly present in the secretion from all gonorrheal inflammations (gonorrheal urethritis, cervical catarrh, blennorrhea, etc.). Their position within the pus-cells, in the immediate neigh- borhood of the nucleus, is characteristic. In cases that are no longer rare gonococci have been found also in the visceral 312 CLINICAL BACTERIOLOGY. manifestations of gonorrhea (peritonitis, salpingitis, oophor- itis, endocarditis, rheumatism, myelitis). The reported demonstration of gonococci in the normal urethra — thus their saprophytism — can not be considered as established. Culture of gonococci (Wertheim) is possible only at a temperature of 37° C. (98.6° F.). Plate-procedure. — Gonorrheal pus is introduced into a tube containing liquid human blood-serum at a temperature of 40° C. (104° F.), and from this two dilutions are prepared in two new blood-serum tubes, likewise at a temperature of 40° C. (104° F.). Into each of the three tubes a like amount of liquefied two per cent, peptone-agar, cooled at 40° C. (104° F.), is then intro- duced, thorough admixture practised, and three plates are made, which are at once placed in the thermostat. As early as twenty- four hours later isolated gonorrheal cultures will have developed. The superficial colonies present a dark, punctate center, from which a delicate, finely granular deposit extends toward the periphery ; the deeper colonies, of whitish-gray color, possess a nodular appearance, and, in the course of two or three days, assume the shape of blackberries. If inoculations are made from the colonies, it will be found that they consist of a mucoid, viscid mass. Streak- culture upon Solidified Blood-serum Agar Slants (one part of liquid human blood-serum at a temperature of 40° C. (104° F. ), mixed with three parts of liquefied agar-agar also at a temperature of 40° C. (104° F. ), and solidified in an oblique position). — Luxuriant growth takes place at first in the form of individual gray colonies that later coalesce into a moist, glisten- ing, viscid-mucous deposit, from the periphery of which a thin, veil-like coating extends. The water of condensation is covered by a membrane. A good liquid culture-medium is constituted by human blood- serum, with the addition of twice as much peptone-bouillon. Upon this a superficial membrane forms, while the culture- medium itself remains almost entirely clear. In the preparation of culture-media animal blood-serum may be employed in place of human blood-serum, although the gono- cocci do not thrive so well upon the former as upon the latter. If, also, gonorrheal pus is smeared upon the surface of several agar-tubes covered with a thin layer of human blood (blood-agar, p. 82), a pure culture of gonococci may be obtained. A mix- ture of blood-serum with urine has also been employed with suc- cess in the cultivation of gonococci ; further, a mixture of 2 parts of peptone-agar with i part of human acid urine, or of i part of ascites-fluid with i part of agar nutrient medium of the following constitution : Five per cent, peptone, 2 per cent. GONORRHEA. 313 glycerin, 3^- per cent, agar, 0.5 per cent, sodium chlorid. Wassermann has recommended the following culture-medium for gonococci : To 15 cu. cm. of swine blood-serum, from 30 to 40 cu. cm. of water, from 2 to 3 cu. cm. of glycerin, and 0.8 gram of nutrose are added, and all are thoroughly mixed and boiled for fifteen minutes. It is best to repeat the boiling on the follow- ing day. The fluid thus obtained is heated to a temperature of between 50° C. (122° F.) and 60° C. (140° F.), and mixed at the same temperature with 2 per cent, peptone-agar in equal amount. Microscopically, the gonococci thus cultivated present exactly the same appearances as those present in gonorrheal pus. The organisms survive in the cultures for from four to six weeks. They are exceedingly sensitive to drying, disinfectants, and temperatures above 42° C. (107.6° F.). The specific pathogenic significance of gonococci is demonstrated by the fact that pure cultures introduced into the normal human urethra (of paralytics) give rise to true gonorrhea. Animals do not acquire gonorrhea. Bacteriologic Diagnosis of Gonorrhea. — Microscopic examination of the urethral secretion is of the greatest importance, and in doubtful cases almost indispensable. With the suspected discharge (gonorrheal threads) dry cover-slip preparations are made, passed three times through the flame, and simply stained with an aqueous solution of methylene-blue. The cocci and the nuclei of the pus-cells are stained blue, the former more deeply than the latter. The characteristic shape of the cocci (kidney-shaped or biscuit-shaped), their position in the leukocytes, and their failure to stain by Gram's method justify a positive diag- nosis of gonorrhea. Dotible staining may be successfully effected if the prepa- rations are first stained with an alcoholic solution of eosin (the eosin being absorbed with bibulous paper), and then subsequently treated with methylene-blue. Cocci and cell- nuclei will then appear blue, the cell-bodies red. Cultivation of gonococci for diagnostic purposes is not necessary. Although positive evidence of the presence of gono- cocci in microscopic preparations renders the diagnosis of gonorrhea certain, negative evidence from examination of the urethral secretion must be accepted with caution. It is known that when gonorrhea has existed for a consider- 314 CLINICAL BACTERIOLOGY. able time gonococci are not always demonstrable in the rather mucoid secretion then present, but that, neverthe- less, the gonorrhea may persist and still be infective. The gonococci are situated in the depth of the mucous mem- brane, and the superficial secretion may be entirely free from them. It is, therefore, advisable in doubtful cases to induce irritation of the urethral mucous membrane (through the use of beer, etc.), and thereby to stimulate the secretion. Only when, after repeated examination and after previous irritation, the secretion is found free from gonococci — it then generally still contains other cocci in abundance — may the gonorrhea be considered as terminated, and only a catarrhal urethritis remains. Prophylaxis. — Gonorrheal infection occurs almost ex- clusively through sexual intercourse with those suffering from the disease. It is a noteworthy fact that intercourse with an individual suffering from gonorrhea does not neces- sarily give rise to infection. It is, however, not necessary to invoke a special predisposition to the disease for those who are infected, and, on the other hand, a special immunity for those who escape. The gonococcus must encounter a lesion of the mucous membrane in order to gain lodgment and to give rise to gonorrhea. When such a lesion is wanting, infection may not take place, especially if the gon- ococci taken up are soon removed mechanically by subse- quent irrigation (perhaps through the urine in micturition). Recovery from gonorrhea predisposes to repeated attack. Rigid sanitary supervision of prostitutes alone is capable of securing certain prophylaxis against gonorrhea. As a prophylactic measure against blennorrhea of the new-born, instillations of astringents into the conjunctivae are in gen- eral employ. SYPHILIS. The exciting agent of syphilis is not yet known. Of the numerous bacteria that have been found and to which etiologic significance has been attached, the bacilli described by Lustgarten seem worthy of mention. Lustgarten in 1884 found in syphilitic lesions and discharges special bacilli that he demonstrated by means of the following method of staining : Sections of tissue hardened in alcohol, or cover-slip preparations made from discharges and passed but once SYPHILIS. 315 through the flame, are kept for from twelve to twenty-four hours at room-temperature and then in a solution of aniline- water gen- tian-violet for two hours more at a temperature of 40° C. (104° F. ) . They are next decolorized in absolute alcohol, and are then exposed for ten seconds to the action oi ij4 per cent, aqueous solution of potassium permanganate, from which they are trans- ferred to an aqueous solution of chemically pure sulphurous acid. They are then washed in water and again immersed in the po- tassium-permanganate solution, but now only for three or four seconds, and from this they are transferred to the solution of sulphurous acid ; and this is repeated until the preparation ap- pears entirely decolorized, which usually is brought about after the manipulations have been repeated three or four times. The Fig. 69. — Bacillus of syphilis (Lustgarten), from a condyloma; X looo (Itzerott and Niemann). preparations are then dehydrated in alcohol, cleared in oil of cloves, and mounted in xylol Canada balsam. Lustgarten believed that by this method of decolorization all bacteria but the syphilis-bacilli, the tubercle-bacilli, and the leprosy-bacilli yield up their stain. The last two, however, are distinguished by their resistance to hydrochloric acid and nitric acid, both of which rapidly decolorize the syphilis-bacilli. Lustgarten found the bacilli stained in this way in all syphilitic infiltrates, and in smaller number at the center and in larger number at the periphery, as well as in the adjacent apparently healthy tissues. They rarely lie free, but singly or in groups of from two to nine within large lymphoid cells. On one occasion Lustgarten encountered them in the lumen of a large lymphatic. 316 CLINICAL BACTERIOLOGY. On examination of syphilitic papules he found the bacilli between the prickle-cells of the rete Malpighii. The bacilli are from 3.5 to 4. 5 IX long and about i /jt thick, straight or bent, in part irregu- larly curved, and their surface presents a wavy contour. They are found constantly in the syphilitic lesions in varying number, but, on the whole, not abundantly. A number of observers have confirmed Lustgarten's state- ments, while athers have failed to find his bacilli. The signifi- cance of Lustgarten's observations was severely shaken when Matterstock and Alvarez and Tavel found bacilli in smegma that could be stained by the method of Lustgarten, and that also closely resembled the bacilli of syphilis morphologically. Doutrelepont maintained, subsequently, that after staining for forty-eight hours in aqueous methyl-violet solution, and decolori- zation with solution of ferric chlorid and alcohol, the smegma- bacilli yielded up their color, whereas the Lustgarten bacilli retained theirs. Nevertheless, the opinion is quite generally held that the so-called bacilli of syphilis are identical with the smegma-bacilli, of which not a few take the stain characteristic for tubercle-bacilli. This circumstance must, further, be borne in mind in examinations for genito-urinary tuberculosis. Con- fusion between tubercle-bacilli and smegma-bacilli may be avoided by a knowledge of the fact that the latter exhibit less resistance to the action of hydrochloric acid and nitric acid than the former. The smegma-bacilli exhibit still less resistance to the action of alcohol. The bacillus of Lustgarten can not, therefore, be consid- ered as the exciting agent of syphilis, although it may possibly bear some relation to this disease. Whether bac- teria are the cause of syphilis at all is wholly doubtful. A bacterial etiology for syphilis is generally assumed be- cause of the varied resemblance of its clinical manifesta- tions to those of other bacterial infectious diseases — as, for instance, tuberculosis and leprosy. Perhaps, however, the exciting agent of syphilis is of an entirely different nature. It is only certain that it is an organized body, a contagium vivum, but nothing further is at present known with regard to it. Investigation in this field is attended with great diflficulties because syphilis is not transmissible to animals, and experimentation fails. According to some observers, monkeys are said to be capable of acquiring syphilis, while others deny this assertion. At least, it appears that not all varieties of monkeys are susceptible to the disease. SYPHILIS. 317 Syphilitic infection takes place through the conveyance of the syphilitic virus, which is contained in the degener- ated products of the syphilitic sclerosis, as well as in all secondary lesions, and in the course of florid syphilis also in the blood of syphilitic patients. This transmissibility has been demonstrated repeatedly by experiments on human beings, and first through the famous experiments of the palatinate Anonymus. Inoculations with sweat, saliva, urine, milk, and seminal fluid of syphilitics have been un- successful. The conveyance of the infecting material takes place either directly, usually through sexual intercourse, less commonly through kissing, through the nursing of a syphilitic child, through contact with syphilitic lesions by the fingers (physicians, micjwives), and the like ; or indirectly through instruments, articles, etc., contaminated by the syphilitic virus (house-epidemics through eating-utensils and drinking- utensils, infection through cigar-tubes, gloves, etc.). A most important role is played by hereditary transmission^ which will be fully discussed later. The portal of infection may be constituted by any por- tion of the skin or mucous membrane where a slight breach of continuity, any lesion of the epithelial covering, exists, so that the syphilitic virus can penetrate deeply. The virus remains localized at the site of infection, where it manifests itself as the so-called prijuary lesion, or hard chancre. In accordance with the varying frequency of the different modes of infection already named, this lesion is most com- mon on the genitalia, but it not rarely occurs also in other situations : on the lips, on the tonsils, on the nipples, on the fingers, etc. After a period of incubation of not less than from three to six weeks, and frequently much longer, the virus is disseminated throughout the entire body, and the disease becomes generalized. The exciting agents them- selves are contained in the blood and in the secondary lesions. A poison alone, absorbed perhaps from the site of the initial lesion, could not give rise to these secondary lesions, as the disease is transmissible indefinitely through them. The exciting agents further must possess an extra- ordinary length of life. They must survive throughout the whole duration of the secondary stage, extending over many years, for the disease is transmissible throughout this entire period. Only in the tertiary stage is this transmis- 318 CLINICAL BACTERIOLOGY. sibility wanting, and the manifestations of this stage can not be dependent upon the virus in a living form, or at least capable of multiplication, and infectious. In the case of hereditary syphilis the virus is taken up by the blood, and the primary lesion, which ordinarily indicates the portal of infection, is wanting, and the disease sets in at once with the secondary manifestations. Immunity. — All ages and all races are equally suscep- tible to syphilitic infection. Natural immunity to the disease in human beings does not exist. Syphilis is characterized by a marked tendency to relapses, which often appear after long periods of latency. On the other hand, one attack of the disease confers protection against subsequent attack — that is, it gives rise to immunity. Renewed infection (rein- fection) occurs — even after disappearance of all previous morbid manifestations — only exceptionally. According to a law laid down by Colles, the mother who gives birth to a child syphilitic through the father, without herself being attacked, is rendered immune by the fetus. By analogy with other immunizing procedures, it is easy to understand that the disease-germs do not pass over from the child to the mother, so that the mother is not infected, because the placental barrier is impassable to the microor- ganisms, but that, on the contrary, the toxins dissolved in the blood pass through this barrier from the child into the maternal circulation, and thus confer immunity upon the mother. The mothers of syphilitic children are actually immune. They may nurse their offspring at the breast without being infected, whereas healthy nurses nursing the same children would be infected with syphilis. Such mothers are thus certainly immune. The only point for discussion is whether they have been immunized by the children. According to the view of a number of syphilog- raphers, they are immune because they have been, or are, themselves syphilitic. Their condition of health is only apparent. We shall return to this point later, in the dis- cussion of heredity. According to the view just expounded, immunity to syphilis would always be acquired through previous infection. The specific therapy of syphilis has never been essen- tially cleared up. Whether potassium iodid and mercurials operate by destruction of the infecting microorganisms — as seems the more probable — or by immunizing infected indi- SYPHILIS. 319 viduals, can not be determined with certainty, so long as the exciting agent of syphiHs is not known. The efforts to check florid syphilis by means of the serum of individuals who have recovered from syphilis, or of animals into whom syphilitic products have been injected, have each and all failed ; and for the present, at least, they are without any experimental justification. Heredity of Syphilis. — Children of syphilitic parents are frequently syphilitic at birth, or present sooner or later evidences of hereditary syphilis. The mode of transmitting the disease may, under such conditions, be as follows : /. Through congenital transmission from the father — that is, transmission of the disease by means of the spermato- zoids {paternal infectiori). According to the opinion of most observers, this mode of transmission is actually operative, the spermatozoid of the syphilitic father carrying the disease-germ into the earliest indication of the fetus. This view, however, has not been proved. Experimental attempts to induce syphilis in healthy individuals by inoculation of the seminal fluid of syphilitic men have been made, but without inducing syphilis in the persons experimented on. The assumption of a direct paternal in- fection is thus based solely upon the clinical experience that syphilitic men may procreate syphilitic children with- out infection of the mother. The majority of clinicians accept this as a fact. According to some, the disease of the fetus is always to be ascribed to the father, and should the mother suffer at all, she is believed to have first acquired the infecting material from the child infected by the father (retroinfection, choc en retour). It has already been men- tioned that the validity of this proposition has been attacked from various sides. Distinguished syphilographers are of the opinion that no child is syphilitic in the absence of syphilis in the mother (A. Wolff) ; that healthy women who give birth to syphilitic children, and are thus immune, are only apparently healthy, but in reality are infected ; that often, even though primary or secondary manifestations are not observed, they later suffer from tertiary manifestations. If this be true — that is, if in a given case the mother of a syphilitic child is herself syphilitic — then the illustration can not be employed in support of paternal infection ; the disease of the child may then arise also through the mother. 320 CLINICAL BACTERIOLOGY. From all that is known the question as to the direct inheri- tance of syphilis by the child from the father, although this is theoretically quite conceivable, and is accepted by the large majority of clinicians as actually occurring, must yet be considered an open one. 2. Congenital transmission from the mother — -that is, transmission of the disease with the ovum — is accepted upon all sides as a possible and frequent mode of con- veyance. As a matter of fact, the offspring of syphilitic women, if the disease has not advanced to the tertiary stage, are almost without exception syphilitic, independently of whether the father is syphilitic or healthy. J. Intrauterine Infection. — If the mother, healthy at the time of conception, is infected with syphilis during the period of gravidity, the child also becomes syphilitic. Only when the infection of the mother takes place in the last two months of pregnancy are healthy children at times born. The fetus, under these conditions, must acquire the disease- germ from the mother through the placental circulation. Whether a lesion of the placenta is necessary for this to take place, as is assumed for all other forms of intrauterine infection, has not yet been decided. According to some observers, the offspring of a healthy woman may be infected in the uterus through sexual intercourse with a syphilitic man, and the mother, in turn, be infected by the fetus ; but this is as yet undemonstrated. The reverse procedure — infection of the woman, who then infects the fetus — is the probable one. ^. Extrauterine infection during parturition or in the first days of life, if the mother be suffering from recent syphilis and the child has remained uninfected until the moment of birth, is altogether conceivable, and probably also occurs. Whether, however, it plays an important part, it is difficult to decide, as probably the majority of such cases, in which a child develops syphilitic general manifestations about six weeks after birth, are designated as hereditary-syphilitic. Extrauterine infection of the new-born can be demonstrated by the presence of a primary lesion, which is wanting in placental (intrauterine) infection. HYDROPHOBIA. 321 HYDROPHOBIA (LYSSA; RABIES). The exciting agent of hydrophobia is not yet known. Nevertheless, the specific treatment of this disease has been successful, owing to the genius of Pasteur. The susceptibility for hydrophobia exists among all warm-blooded animals. Human beings are infected through the bites, in the first place, of rabid dogs, then of cats, wolves, foxes, jackals, and other animals, and, in rare cases, of human beings suffering from hydrophobia. The saliva must thus contain the virus of the disease, and as a matter of fact this had already been demonstrated experi- mentally at the beginning of the last century by inocula- tion of dogs from human beings. The parotid is the gland most concerned, while the remaining salivary glands, though virulent, are not so constantly so as the parotid. The saliva of dogs contains the virus of rabies as early as two days before the appearance of the first symptoms of the disease. The lacrimal glands, the adrenal glands, the pancreas, and the mammaiy glands of rabid animals are further infectious ; the milk, at times is so ; the blood, never. Besides, the central nervous system is virulent — the brain and the spinal cord and, in a conspicuous and constant manner, the medulla oblongata. Experiments on Animals. — The saliva is not used for inoculation-experiments because, in addition to the virus of rabies, it always contains a number of pyogenic micro- organisms that act as a disturbing factor. The medulla oblongata of animals or individuals that have died of rabies is used exclusively for inoculation-purposes. A wateiy emulsion is made from a small portion of the medulla ob- longata, and a few drops thereof are injected beneath the dura mater or into the anterior chamber of the eye in dogs, rabbits, etc. After a period of incubation of from twelve to fifteen days the animals develop, with almost absolute certainty, the symptoms of rabies. Subcuta- neous injection is not quite so trustworthy. In order to obtain positive results the injection must be made deeply, and preferably into the exposed and divided muscle-bundle. Direct injection of the virus into a periph- eral nerve is likewise attended with success. Healthy mucous membranes (as of the nose and the conjunctiva) also absorb the virus. The possibility of intrauterine * 322 CLINICAL BACTERIOLOGY. transmission of rabies has been established experimentally in a small number of instances. Infection takes place by way of the nervous system. If the spinal cord of a dog is divided transversely, and the virus of rabies is injected into a nerve of the hind-paw, only the cord below the point of division proves virulent after the death of the animal. The reverse conditions prevail after inoculation of a fore-paw. Having reached the central ner- vous system from the periphery (site of inoculation or of bite) through the intermediation of the nerves, the virus de- scends into the peripheral nerves of the opposite side. For this reason, if the disease has developed slowly, the nerves of the uninjured side are also found poisonous in experi- ments on animals. The as yet unknown excitant of rabies appears to exert its influence through its metabolic . products. At least, according to Italian observers, the filtrate through porce- lain of an emulsion of the spinal cord from animals suffer- ing from rabies induces paralytic manifestations in dogs. Rabies may, therefore, as suggested by Romberg, be desig- nated a toxoneurosis. The dissemination of the virus of rabies in the course of the nerve -paths explains why in human pathology the prog- nosis of the disease varies so widely in accordance with the number, the seat, and the depth of the bite -wounds. Every- thing depends upon whether the virus gains entrance into a nei-ve or not. Deep wounds are, therefore, much more dangerous than superficial ones ; injuries in regions with an abundant nerve-supply (as, for instance, the finger-pulp) more so than in other parts of the body. The greatest dan- ger is involved in wounds of the head and the face. From these the virus of rabies quickly reaches the medulla oblongata, the main seat of the disease. The morbidity and the mortality of rabies (the developed disease is in- curable) are, according to the most reliable statistics, about 1 6 per cent, of those bitten. Incubation. — The duration of the period of incubation depends upon the same factors that have been mentioned as significant in prognosis. The period is the shorter the nearer to the head the portal of infection is situated. The usual duration of the incubation-period is from twenty to sixty days. The trustworthy minimum observed has been fourteen days ; the maximum, eighteen months. HYDROPHOBIA. 323 Resistance of the Virus of Rabies (Medulla Oblongata of Dogs Dead of Rabies). — The virus is destroyed by ex- posure for an hour to a temperature of 50° C. (122° F.) ; further, to 5 per cent. carboHc acid for fifty minutes ; to mercuric chlorid, i : 1000 ; to acetic acid ; to potas- sium permanganate. The spinal cord of (Paris) rabbits dead of rabies, kept in dry air and protected from putre- faction, loses its toxicity only after fourteen or fifteen days. The smaller the animal, the thinner the spinal cord, the more rapidly does the loss in virulence take place. Immunization and Vaccination. — Pasteur showed that the virus of canine rabies slowly diminishes in intensity when inoculated from dogs upon monkeys in a progressive series. This gradual loss of virulence is distinctly appreci- able in the increase in the period of incubation. If the infecting material is reconveyed from monkeys to rabbits, an increase in virulence takes place, which constantly aug- ments on further inoculation into rabbits. The period of incubation becomes shorter ; and, finally, after the hun- dredth passage through the body of the rabbit, the period is not longer than seven days. It was impossible to pro- duce a more active virus. The virus retained its virulence now unchanged, and Pasteur, therefore, designated it virus fixe, Pasteur in this way prepared a series of rabies-viruses that, beginning with the spinal cord of monkeys and progressing to the spinal cord of rabbits that succumbed to the virus fixe, possessed a steadily increasing virulence. On inoculating successively dogs subcutaneously with these spinal cords, of from the lowest to the highest degree of viru- lence, the animals treated failed to develop rabies, but be- came immune even to subdural infection with the virus fixe and to the bites of other dogs suffering from ordinary rabies. A year later, in 1885, Pasteur and his collaborators, Chamberland and Roux, developed a still more practical method of immunization. Proceeding from the fact already mentioned that the medulla of animals suffering from rabies is completely deprived of its virulence in from fourteen to fifteen days by desiccation, they dried the spinal cords of rabbits that had succumbed to the virus fixe for from one to fourteen days with all aseptic precautions in high ster- ilized glass cylinders. The spinal cord fourteen days old had lost all its virulence ; that thirteen days old and that 324 CLINICAL BACTERIOLOGY. twelve days old, in part ; and the others, successively less and less. Through successive inoculations with these gradations of spinal cords Pasteur established complete im- munity in dogs. Upon the basis of these facts Pasteur proceeded to the vaccination of human beings therapeuti- cally against rabies. In view of the long period of incuba- tion of rabies in human beings the attempt was justified and hopeful ; for, if it were possible to establish immunity through vaccination immediately after the bite of the rabid animal and before the period of incubation had elapsed, then it could be hoped that the disease would not break out. The results have completely confirmed Pasteur's anticipa- tions. At first, the injections were so made that on the first day the spinal cord of rabbits kept for fourteen days, on the second day that of rabbits kept for thirteen days, and so on for ten days until the cord of rabbits kept for five days was reached, were successively injected subcutaneously into the subject to be treated (methode simple). Pasteur, however, soon recognized that this procedure was not sufficient for the severe cases with deep and numerous wounds. Vaccination is at present practised in the Pasteur Insti- tute in the following manner (methode intensive) : A piece of spinal cord about three millimeters long is rubbed up in sterile bouillon and injected beneath the skin in the hypo- chondrium, and on the first day in the morning medulla fourteen and ten days old — an injection being made on either side — in the evening medulla twelve and eleven days old ; on the second day in the morning medulla ten and nine days old, in the evening medulla eight and seven days old ; on the third day two injections of medulla six days old, and from now on an injection every twenty-four hours of the more toxic spinal cords up to that three days old. With the spinal cord three days old a new series is begun, commencing with the medulla five days old. Upon this succeeds a third, and possibly a fourth, series of like character. Vaccine may be prepared also by dilution with sterile water (Bardach) instead of by desiccation. This fact indicates that also in the dried spinal cord the virus is not actually attenuated, but only diminished in amount. As a matter of fact, Pasteur, after injecting desiccated medulla into a guinea-pig and the animal dying after thirty days, observed the medulla of this animal destroy a second guinea-pig SMALLPOX. 325 in exactly seven days ; the virus thus remained a virus fixe. The immunity to rabies appears to persist for a long time — in dogs for two years. Results of Pasteur's Procedure. — The great utility of the method of vaccination against rabies is no longer seri- ously doubted by any one. From 1886 to January i, 1894, 14,430 persons in all were treated in the Pasteur In- stitute, of whom J 2 died. In the year 189 1, 394 persons were treated, the diagnosis of rabies in the biting animals being established with all possible certainty ; and not a single patient developed the disease. In 1892, 128 persons were treated, with i death. In 1893, 132 bitten persons were treated, of whom none died. In the year 1894, 1387 persons were vaccinated, of whom 7 died, and in the year 1 895, 1 520 were vaccinated, with 2 deaths.* These statistics require no comment : they speak for themselves. SMALLPOX (VARIOLA). Smallpox, like syphilis and rabies, is one of the diseases whose exciting agents are yet unknown. The virus of smallpox resides in the contents of the variolous pustules, in the desiccating scales of the skin, in the sputum, and in the nasal secretion of those suffering from the disease. It is transported with the linen and the clothing of the sick. Also the air in the neighborhood of the sick must, from clinical experience, be considered a source of infection. Under suitable conditions the con- tagium may retain its vitality for an exceedingly long time, apparently for years. The portal of infection for the contagium of smallpox has not yet been definitely determined. According to the common opinion, the disease is generally acquired through direct or indirect contact with the sick, and the skin would thus seem to constitute the portal of infection. In other cases the disease may be attributed to simple inhalation in the neighborhood of smallpox-hospitals, etc. Finally, articles of food (such as milk) and insects are thought to convey the infection, which, under these conditions, would *The figures for 1896 were 1388, with 4 deaths; for 1897, 1521 cases, with 6 deaths.— A. A. E. 326 CLINICAL BACTERIOLOGY. take place through the mouth or the lungs. Naturally, however, under any circumstances other modes of infection can not be excluded. Predisposition and Immunity. — The susceptibility to smallpox exists at all ages. Even new-born children have been observed to present the disease. In the great epidemics of smallpox that traversed Europe before the institution of compulsory vaccination the morbidity varied in different localities. The local predisposition was the greater the more impoverished the community. It thus ap- pears that not merely the telluric conditions themselves, but rather the vital conditions and the mode of life of the people living upon a given soil, constituted the cause for the varying distribution of the disease. The marked increase in small- pox that regularly takes place in winter has been considered evidence of a temporal predisposition. This may, however, be readily explained by the changed mode of life in winter (with greater confinement in closed rooms, the wearing of heavier and a greater amount of clothing, the greater difficulty of cleanliness, etc.), without the necessity for in- voking a direct influence of the weather upon the germ of the disease. Recovery from an attack of smallpox confers immunity that lasts on the average about ten years. A second attack of the disease before the expiration of ten years is most exceptional, but has been frequently observed after a longer period. Third attacks of smallpox have been re- ported nine times in the literature, and Cantani reports one case in which seven attacks occurred. Upon the ancient experience that immunity is acquired by recovery from smallpox is based the procedure of vario- lation— that is, the inoculation of healthy persons with true smallpox for purposes of immunization, which was formerly practised and entailed not a few sacrifices. Vaccinia and Vaccination. — Edward Jenner, a physician of Berkeley, in England, between 1749 and 1823, con- vinced himself, after study and investigation pursued for years, that the pock-disease of cows (vaccinia), conveyed to human beings, protected them from infection with variola. On May 14, 1796, Jenner vaccinated a youth with cowpox- lymph obtained from the hand of a maid who had infected herself in milking a cow suffering from cowpox of the udder. The vaccinated subject was protected against sub- SMALLPOX. 327 sequent variolation. After a large series of further suc- cessful vaccinations Jenner, in 1798, published his famous communication, in which he announced the established fact of the protective influence of cowpox-lymph against small- pox. Since then, vaccination against smallpox has been gradually adopted in all civilized countries ; in Germany it has been made obligatory through a law passed in 1874. In those countries in which the general practice of vaccina- tion against smallpox is regulated by law, variola, which formerly was responsible for a large proportion of the mor- tality, has almost completely disappeared. Devastating epidemics now occur only in uncivilized countries. Vacci- nation against smallpox is practised in Germany in the first and twelfth years ; it consists in the cutaneous introduction of the contents of the pocks of young calves in a fresh state or rubbed up with glycerin (animal vaccination). The lymph obtained from the vesicles of inoculated persons has the same effect (humanized lymph). Of late, however, animal lymph is almost universally preferred, as the danger of simultaneous transmission of other disease-products (syphilis, etc.) can not with certainty be excluded in the use of humanized lymph. Animal lymph is obtained in Germany by systematic vaccination of calves in institutes under State supervision. In accordance with existing knowledge, it can be defi- nitely accepted that cowpox is identical with variola in human beings, and that vaccinia is only a form of variola mitigated by passage through the body of the cow. Fischer, of Karlsruhe, succeeded in grafting the virus of variola on the body of the calf by collecting and mixing together the liquid and the contents obtained by cureting from vario- lous pustules in human beings in their various phases, from development to suppuration. The mixture was then rubbed into the largest possible surfaces (crucial incisions and scarification). Fischer was able in this way to develop in the calf directly with the virus of variola typical pustules whose contents, subsequently upon reconveyance to human beings, from the third generation, proved to be vaccine. Vaccination against smallpox thus readily adapts itself to current views regarding immunity, inasmuch as it represents a protection against disease induced through preventive inoculation of an attenuated though similar virus. As to the nature of the virus of variola, numerous 328 CLINICAL BACTERIOLOGV. investigations have been made with variola and with calf- lymph. In spite of all the efforts that have been made the exciting agent of smallpox has not yet been discovered. It is possible that it does not at all belong to the class of bacteria, and in any event its requirements of the culture- media are different than are those of the bacteria whose gowth has thus far been successful. On ordinary bacteri- ologic examination staphylococci, pseudo-diphtheria-bacilli, and rarely streptococci are found in animal lymph. It has been maintained by Landmann that the evidences of irrita- tion that are sometimes apparent in marked degree around the pustules are due to the activity of these pyogenic cocci. On the other hand, the Commission for the Investigation of the Vaccine Question ( 1 896) emphasized that the staphy- lococci found were of moderate, and only exceptionally of considerable, pathogenicity for animals, and that virulent streptococci could no longer be found in the lymph after the lapse of eighteen days. The streptococci found in older specimens are to be looked upon as only harmless skin-epiphytes, such as can not rarely be isolated from bac- terial mixtures. Frosch, the reporter of the Commission mentioned, reached the conclusion that no etiologic relation exists between the bacteria of the lymph and the irritative and inflammatory manifestations of the inoculation-pustule, and that in the preparation of animal lymph any noteworthy reduction in the number of germs or their complete exclu- sion even with the observance of strict antisepsis is not attainable ; that, therefore, it is impossible to obtain an unirritating lymph-supply. Varicella (chickenpox) has nothing to do with the ex- citing agent of variola and of vaccinia, and recovery from the disease does not protect against smallpox. ACUTE EXANTHEMATA. The exciting agents of the acute exanthemata are, like- wise, thus far unknown. Measles is contagious in an extraordinary degree. The susceptibility of human beings to the disease is exceedingly great between the second and tenth years of life ; it is less in the first year and in adults. Natural immunity to measles practically appears not to occur. The contagium is con- ACUTE EXANTHEMATA. 329 tained in the nasal mucus, the conjunctival secretion, and the sputum of those suffering from measles, and, as experimental inoculation appears to have shown, also in the blood. The contagium is taken up through contact with the sick, or — as is generally accepted clinically — through inhalation of the exciting agent. The persistence of the virus of measles is not so marked as that of small- pox or of scarlet fever. In a dry state the virus of measles is said to persist for about six weeks. Measles is rarely conveyed by means of articles of clothing, linen, etc., and not for long distances. The infectivity of measles exists as early as the last days of the period of incubation — which is between eight and ten days — and in the stage of eruption. After the exanthem has entirely appeared, the danger of in- fection rapidly subsides. In measles also a large number of bacteria have been found, but, above all, again cocci, which have also been demonstrated in the blood. None of these observations is worthy of consideration, because they depend upon accidental or secondary infection or upon contamination. The hnmunity established by recovery from an attack of measles is rather active and enduring. Only thirty-six cases are recorded in the literature in which two attacks occurred, and only one with three attacks. Scarlet Fever. — The virus of scarlet fever is much more resistant than that of measles. It adheres for months to the clothing worn and the rooms occupied by the sick. It appears, further, to possess considerable resistance to tem- perature-influences. Infection with scarlet fever, in accord- ance with clinical experience, occurs principally through direct contact with the sick or with articles belonging to them, but also through breathing of the air in rooms satur- ated with the virus of the disease. The period of incubation is of varying duration (from two to twenty -four days). Even toward the end of this period, according to Gerhardt, scarlet fever is infective, and it remains so until the cessation of desquamation and for weeks longer. Wherein the infective material resides, whether in the secretions, in the blood, or in the cutaneous scales, is not known. • Some observers claim to have transmitted scarlet fever by inoculation, while others have failed to induce the disease experimentally by means of the blood, the scales, etc. There is much in the clinical picture of scarlet fever, especially the nephritis, in 330 CLINICAL BACTERIOLOGY. favor of the view that the disease is a toxic infection, and that the exciting agent is not itself disseminated into the viscera. The relations between scarlet fever and diphtheria are well known. Scarlet fever is sometimes complicated by true diphtheria with diphtheria-bacilli in the membranes. More frequently, however, scarlatinal angina is due to streptococci. Altogether scarlet fever exhibits a tendency to be complicated by streptococci, and the secondary sup- purative processes that not rarely occur are attributable to these microorganisms. The susceptibility of human beings to scarlet fever is greatest between the third and eighth years, though not so great as that of measles. After the tenth year the disease is less common, and still less so in the first year of life. Like measles, scarlet fever is more common in winter than in summer. Certain local influences in the distribu- tion of scarlet fever can not be ignored. Although the dis- ease is, on the whole, everywhere endemic in uniform dis- tribution, noteworthy variations occur. Thus, some cities have remained free from scarlet fever for thirty, and even for fifty, years, although in constant communication with other infected cities. The immunity after recovery from scarlet fever is active and enduring. Only twenty-nine cases in all of second attacks and four of third attacks are recorded in the litera- ture. The large number of bacteria found in cases of scarlet fever, both micrococci and bacilli, require no particular mention. Streptococci have been found most frequently, and these, as in the case of diphtheria, appear to constitute a frequent, perhaps a regular, complication of the virus of scarlet fever. WHOOPING-COUGH (PERTUSSIS). In its infectivity whooping-cough stands close to mea- sles. Our knowledge of the virus of the former disease is yet quite scanty. The contagium is contained in the breath and, especially, in the expectoration of children suf- fering from whooping-cough. With the sputum it finds its way upon the linen, and it may also be conveyed through healthy persons. Probably infection takes place always through the respiratory tract. Children between ARTICULAR RHEUMATISM. 331 one and five years of age are most susceptible to whooping- cough ; the disease is less common after the tenth year of life, although it occurs also in adults, and not altogether rarely in the first year of life. Whooping-cough has been observed repeatedly in new-born children whose mothers have suffered from the disease toward the end of gravidity. The disease is especially common in spring and autumn. As a rule, it attacks human beings but once. A large num- ber of bacteria have been cultivated from the sputum of patients suffering from whooping-cough, and animal para- sites also have been found therein ; the demonstration of any etiologic significance for any of these is yet lacking. ARTICULAR RHEUMATISM. Although the exciting agent of articular rheumatism is entirely unknown, there is, however, no doubt that this disease is infectious. The febrile course, with the general manifestations, the frequent complicating inflammations of serous membranes and the endocardium, and the secondary nephritis, point forcibly to the infectious nature of poly- arthritis. The disease is well known to be dependent in a high degree upon meteorologic influences, but the necessity for certain influences increasing the predisposition also in the etiology of other infectious diseases has already been demonstrated. Acute articular rheumatism is in no wise contagious. Rheumatic polyarthritis is one of those infec- tions after whose termination immunity lasts for only a short time, if it occur at all, to be replaced soon by heightened predisposition. Frequently the same individual suffers from repeated attacks of this disease. It is doubtful if all cases that are grouped clinically under the designation of acute articular rheumatism are associated etiologically. The fact that one-quarter of the cases are refractory to the influence of the known specifics (salicylic acid, antipyrin) appears indicative of etiologic multiplicity. In any event it is to be borne in mind that the clinical picture of poly- arthritis may also be produced by the exciting agents of other diseases (gonorrhea, scarlet fever). 332 CLINICAL BACTERIOLOGY. RELAPSING FEVER. The cause of relapsing fever was, in 1873, found by Obermeier, a former assistant of Rudolph Virchow, and who died prematurely, to be a special spiral bacterium, which he designated the spirocheta of relapsing fever. Obermeier' s discovery was the more far-reaching because with it an organism of the class of bacteria was for the first time recognized as the cause of a human infectious disease. The spirilla of relapsing fever (^spirilla Obermeieri) are delicate, wavy threads, with numerous turns (from ten to twenty), varying in length from 16 to 40 }x, with distinctly pointed extremities. In form and size they closely resemble Fig. 70. — Bacillus of relapsing fever, from human blood; X looo (Giinther). cholera-spirilla, though they are only from one-half to one- quarter as thick as these, and they never appear in the form of a comma, or in S-shaped segments of a screw, but always only in the form of a complete screw. The spiral thread can not be differentiated into individual members. The spirilla possess flagella and are actively motile. They glide in rapid, twisting movements from one side to the other across the field of vision. With regard to their propagation, nothing certain is known ; probably this takes place by division. The formation of spores has not been observed. Artificial cultivation of the spirilla of relapsing fever upon nutrient media has thus far not been successful. The spirilla must be looked upon as strict parasites that flourish only within RELAPSING FEVER. 333 the animal body. In leeches that have sucked themselves full of the blood of patients suffering from relapsing fever, and have been preserved upon ice, the spirilla remained alive for ten days. Little is known with regard to their temperature-relations. In blood from patients suffering from relapsing fever kept in glass tubes at a temperature of from i6° C. (60.8° F.) to 22° C. (71.6° F.) the spirilla survive for as long as fourteen days ; at a temperature of 37° C. (98.6° F.), for about twenty hours; at a temperature of from 39.5° C. (103.1° F.) to 41.7° C. (107.1° F. ), only from four to twelve hours ; and at a tempera- ture of 42.5° C. (108.5° F.), scarcely three hours (Heyden- reich). Conclusions with regard to the growing bacteria in the living blood can not be drawn from these observations, as the blood removed from the body is no longer a favorable nutrient medium for the spirilla. Nothing definite is known also with regard O©^/^^ ^ to the requirements of the spirilla Q P-w^ C^ for oxygen. /f\^ The relapsing spirilla can be q ^^^^ \ j/ ^ readily stained by means of watery ^^^ f > ® solutions of aniline dyes ; they do ^ ' not take acid stains. For the demonstration of relapsing spirilla in blood-preparations the method riptff^ ffi^ ^ of Giinther is useful ; this consists ^"^^U^ "q © in introducing the dried cover-slip ^.^ ,,_spi,och^t^ obermeieri preparation, fixed by heatmg, be- in the blood (v.jaksch). fore staining with gentian-violet or fuchsin, for ten seconds in five per cent, acetic acid, in order to extract the hemoglobin from the blood-corpuscles. The relaps- ing spirilla are not so resistant as all other bacteria to the action of dilute potassium hydroxid or concentrated acetic acid. In their reactions they resemble the protoplasmic rather than the nuclear substances. The spirilla do not stain by Gram's method. Occurrence of Spirilla of Relapsing Fever. — The spirilla are present, and in varying number, only in the blood of those suffering from relapsing fever, in which they appear a short time before the onset of the fever, and they undergo considerable multiplication during the continuance of the fever, to disappear, a short time before the critical deferves- cence, until the onset of the next febrile paroxysm. During the afebrile period the spirilla have been found in the blood in but one case by Naunyn. They are not ordinarily found in the secretions and excretions of patients suffering from relapsing fever, and with equal rarity outside the 334 CLINICAL BACTERIOLOGY. human body. They have been found once in the urine in a case of relapsing nephritis. The relapsing spirilla are, therefore, most rigid blood-parasites. Transmission of Relapsing Fever. — Relapsing fever has been repeatedly transmitted to healthy human beings, and with complete success to apes, by means of blood con- taining spirilla (Koch, Carter, and others). The spirilla can thus with certainty be considered as the exciting agents of relapsing fever. How they induce the disease, and whether the fever is the result of a poison generated by them, can not yet be decided positively. Equally little is known with regard to the manner in which the disease is transmitted naturally. . The disease is contagious ; as, how- ever, the spirilla soon lose their vitality outside of the body — in water, on articles of food, in the air — the ordinary modes of infection, by way of the respiratory or the digestive tract, are scarcely to be considered. The disease can be conveyed only with the blood, as the spirilla apparently do not leave this fluid. Klebs refers to the possibility of conveyance through blood-sucking cutaneous parasites, but this has not been established with certainty. Intrauterine transmission of the spirilla to the fetus has been observed. The mode of recovery from relapsing fever, which, as is known, thus terminates in the majority of cases, is essen- tially not yet understood. According to Heydenreich, the bacilli die as a result of the high temperature of the patient in the febrile attack. His observations, however, as has been pointed out, do not furnish adequate evidence in support of this view. According to Metschnikoff, the spirilla collect in the spleen during the precritical elevation of temperature, and are there destroyed by phagocytosis. Baumgarten does not consider this a healing process, but believes that the spirilla are deposited in the spleen, and also in the liver and the bone-marrow, where they undergo destruction, in part through inclosure in leukocytes, and in part indepen- dently of this process ; but this takes place only because they have ceased proliferating, because their vital activity has been impaired, and they are nearly or already dead. The cause of this attenuation Baumgarten believes to re- side in spontaneous exhaustion of the proliferating power of the infecting microbes, which are endowed with only a short duration of life in consequence of immanent vital laws. The successive febrile cycles are attributed by RELAPSING FEVER. 335 Baumgarten to new series of generations, which arise from individual spirilla that have not been destroyed. Final recovery may depend upon the development of an insus- ceptibility of the blood. PART IV, L MYCOSES (INFECTIONS WITH HLAMENTOUS AND BUDDING FUNGI) • An incomparably lesser role in the etiology of dis- ease is played by the filamentous fungi (molds) and the budding fungi than by bacteria (fission-fungi). The dis- eases generated by the former are designated mycoses. MORPHOLOGY AND BIOLOGY OF THE FILAMENTOUS AND THE BUDDING FUNGI. The filamentous fungi are chlorophyl-free, thread-like cells that exhibit progressive apical growth, and that, singly or branched, usually divided into segments by internal septa, unite to form a deposit and at times a dense felt- work of closely interlaced threads {Jiyphce). This so-called vegetative portion of the fungus is known as thallus (fungous deposit) or myceliwn. Distinct from this is the fructifying portion, the fruit-beareVy which arises from the mycelium and bears the fruit {spores or conidid). The spores again grow into threads ; the enlargement takes place steadily through progressive apical growth of the threads, which in turn give rise to new spores. The structure of the fruit- bearing apparatus is so peculiar in a number of filamentous fungi that this external feature has been made the basis of their classification. Of the numerous species of filamentous fungi, running up into thousands, only the following are of pathologic interest : I. The Aspergilli {Bulbous Molds'). — The fruit- hyphae ex- hibit no division ; they swell into the form of a club at the extremity. This bulbous enlargement is densely occupied by short, flask -shaped structures, arranged radially, the in- 336 FILAMENTOUS AND BUDDING FUNGI. 337 termediate fruit -bearers {sterig7natd), upon which the con- secutive spores are seated. 2. The Penicillii {Bntsh-7nolds). — The fruit-bearers, which generally arise vertically from the mycelium, are trans- formed, through manifold forked divisions in their upper third, into dense tufts of brush-shaped, ramifying, delicate processes (basidia), upon whose extremities the conidia are seated in long rows in the form of globules, J. The Miicorini {Glohdar Molds). — The fruit-bearers, which are mostly unsegmented and undivided, arise verti- cally from the mycelium and present at their extremity a large spore-mother-cell (sporangiuin), which is separated from the fruit-hypha by a septum markedly convex upward ©o Fig. 72. — A, Aspergillus glaucus; B, aspergillus niger ; C, ripe fructiferous head of aspergillus niger throwing off spores. Fig- 73' — Fungi (penicillium glaucum). {columella). The sporangium contains within its interior, separated by septa, the large cylindric-oval spores. 4. The streptothrices form, to a certain degree, a transi- tion between the filamentous fungi and the bacteria. They consist of long, cylindric filaments, dividing by budding, and from which finally a true mycelium is formed. In many of these air-hyphae develop, which simply give off special fruit- heads, spores {segmentatioit). These streptothrix-spores must not be placed upon the same plane as the permanent forms of the bacteria. In older cultures the streptothrix- threads disintegrate into degenerative products resembling bacteria, like rods, cocci, spirilla {fragineniation). If these structures are transplanted upon fresh nutrient material, a 22 338 CLINICAL BACTERIOLOGY. true filamentous network will at once redevelop. The peculiarities of the strep tothrices will be referred to in detail in the discussion of actinomycosis. 5. Oidia {segmented molds) are likewise simple in arrange- ment. They also possess no special fruit-heads, the spores being detached directly from the fruit-bearers that arise from out of the mycelium. Their most frequent represen- tative is the oidium lactis, the white milk-mold, which vegetates upon sour milk. The oidia form a transition to the so-called budding fungi. The budding fungi or yeast-fungi are chlorophyl-free cells of roundish or oval shape that multiply by budding — that is, a small, roundish, bud-like projection grows from the periphery of the mother-cell and, gradually increasing in size, assumes the shape of the mother- cell, from which it ultimately becomes detached. The newly formed cell under- goes the same process. If the various generations of cells remain attached to one another, long rows of yeast-cells result — the so-called strings of buds. Fig. 74.— Yeast-fungi. Under special nutritive conditions — for instance, upon solid culture-media of alkaline reaction or deficient in sugar — the budding fungi also form true mycelial threads. (See Thrush, p. 351.) The best known of the budding fungi are the yeasts (saccharomyces cerevisiae) and the mold of wine-must (mycoderma vini). Filamentous and budding fungi flourish at room-temper- ature. For their nutrition they require constantly pre- formed organic substances, water, and, further, oxygen as a rule ; a number of the filamentous fungi may develop also in the absence of oxygen. Acid culture-media are pre- ferred, although the filamentous and the budding fungi grow also upon alkaline media. These fungi are present everywhere in nature, and are always numerous in the air and upon articles of food. They are capable of inducing fermentation (especially, though not exclusively, the bud- ding fungi) and decomposition. Putrefaction, which is usually of bacterial origin, inhibits the development of fila- mentous and budding fungi in general. Microscopic examination for filamentous and budding fungi is made on the whole in the same way as that for FILAMENTOUS AND BUDDING FUNGI. 339 bacteria. The filamentous fungi, with the exception of the streptothrices, which behave exactly as do bacteria with regard to stains, do not stain well in general, although they can be demonstrated with the aid of Loffler's methylene- blue. Generally, it is preferable to examine these fungi unstained. The filamentous fungi do not take up water, so that they are generally mounted in glycerin. It is advisa- ble to undertake the demonstration of teased preparations from fungous vegetations in 50 per cent, alcohol containing a few drops of ammonia, in order to avoid the disturbing influence of air-bubbles ; or the teased preparations are made in Unna's solution : gelatin i, alcohol 25, solution of ammonia 25, glycerin 25, water 35. The following recom- mendation of Unna is useful : The cover-slip preparations are placed for a minute in 5 per cent, potassium hydroxid ; then, after rinsing in water for five minutes, in 5 per cent, acetic acid ; and finally they are exposed to the action of a strong aniline stain (for instance, gentian-violet), possibly under the influence of heat. Yeast is best stained with a dilute aqueous solution of vesuvin, as the other aniline dyes readily give rise to overstaining. The cultivation of molds and budding fungi is carried out in the same way as that of bacteria. Isolation is effected by means of the plate -procedure, preferably with acid gela- tin or agar (the gelatin or the agar is dissolved in acid fruit-decoctions, beer-wort, or potato- water (p. 81) ), instead of alkaline bouillon. For further cultivation bread-pap is well adapted (p. 86). PATHOGENIC ACTIVITY OF FILAMENTOUS AND BUDDING FUNGI FOR ANIMALS. Although the majority of filamentous and budding fungi vegetate only upon dead organic material, a small number of varieties may flourish in the animal body and thus give rise to disease. A special position is occupied by the streptothrices, the most important representative of which, the streptothrix actinomyces (p. 354), will be fully con- sidered. The best known among the remaining pathogenic filamentous fungi are the aspergillus fumigatus and fla- vesce7is, and the mucor corymbifer and rhizopodiformis. If an emulsion of these fungi in bouillon is injected into the 340 CLINICAL BACTERIOLOGY. ear-vein of a rabbit, the animal will die after the lapse of two or three days in consequence of a general mold-myco- sis. In all of the organs, most abundantly in the kidneys and in the liver, small, whitish-gray nodules are present, which, when viewed microscopically, are found to consist of a dense network of mycelial threads. These fungous vegetations never, however, contain fruit-bearers or co- nidia. Careful investigation has shown that germination of the injected spores has taken place, but that fruit-formation never takes place in the animal organism. By reason of its deficiency in free oxygen and its alkaline reaction the organism does not constitute a suitable nutrient medium for most molds. The overwhelming majority of these die when introduced into the body, whether through the breath or in any other way. The few varieties that survive at all prove pathogenic only by germinating, and acting as foreign bodies inducing disturbances mechanically through irritation and vascular occlusion, etc. They do not undergo fructification or actual multiplication. Noth- ing likewise is known with regard to any chemic activity on the part of molds or budding fungi in the body. In a certain sense the mold-mycoses are, accordingly, not true infectious diseases, as they are not attended with multipli- cation of the exciting agent, and with intoxication. Suc- cess in inoculations with pathogenic molds depends, there- fore, also upon the number of spores injected. The number of disease-foci corresponds exactly with the number of spores introduced, and the animals die as a result of the extent of the foci of disease alone. The pathogenic molds are, besides, inherently pathogenic, and likewise the non- pathogenic molds are always nonpathogenic. Augmenta- tion or attenuation of pathogenicity can not be effected. The pathogenic varieties are not equally pathogenic for all animals. Thus, the mucor corymbifer, which destroys rab- bits, is harmless in dogs. Pathogenic molds are quite widely distributed in nature. It is only necessary, for instance, to expose unsteriHzed bread-pap in a closed beaker for one or two days to a tem- perature of from 30° C. (86° F.) to 40° C. (104° R), in order to see a dark-green fungous coating form, consisting of aspergillus fumigatus. The culture is generally pure, as at a temperature of between 30° C. (86° F.) and 40° C. (104° F.) the aspergillus overgrows all other molds. If, FILAMENTOUS AND BUDDING FUNGI. 341 in addition, other molds be present, a pure culture can be readily obtained by injecting the fungous mass into an animal. The body of the animal at once differentiates pathogenic and nonpathogenic molds, inasmuch as the latter are destroyed with certainty. In spite of this distri- bution of the pathogenic molds, mycoses are not common in the animal kingdom. Spontaneously they occur rela- tively seldom. Only in the lungs of birds are fatal asper- gillus-mycoses and mucor-mycoses found at all frequently, so that a certain susceptibility must be assumed for this particular tissue, and which, otherwise, is generally wanting in the animal body. Of pathogenic yeasts but a small number are thus far known. Experimentally in animals they induce local sup- puration, tumor-like swellings, and at times septic manifesta- tions. DISEASES IN HUMAN BEINGS INDUCED BY FILA- MENTOUS AND BUDDING FUNGI. DERMATOMYCOSES (PARASITIC DISEASES OF THE SKIN). Favus. — The cause of favus was recognized by Schonlein in 1839 as a filamentous fungus, which, in his honor, has been named achorion ScJionleinii, and which was the first of all organized causative agents of disease discovered. The disease is characterized by a peculiar crust or scutulum. On microscopic examination this is found to consist of a layer of cornified epithelial cells, beneath which there is a mass of fungous elements, in the form of concentrically arranged mycelial threads, which give off conidia toward the center of the favus-body. The center consists of conidia alone ; in addition bacilli and cocci are always found. If a frag- ment of this scutulum be examined in water or in glycerin, the mycelia will be recognized as numerously partitioned or segmented ramifying filaments of varying thickness. The Conidia are of variable form and size, roundish, oval, angular, in part provided with a yellowish nucleus, in part without a nucleus, but with granular turbid contents. This fungus, which from its form belongs to the oidium-group, is the cause of favus. It is found in association with both favus of the hairy scalp and in portions of the body free from hair, and with favus of the nails. It has been grown in 342 CLINICAL BACTERIOLOGY. pure culture, and with this favus has been induced in animals and in human beings. It may be mentioned that Quincke succeeded in cultivating three distinct favus-fungi. More recent investigations have, however, rendered it quite cer- tain that in all varieties of favus but one fungus is found, which, however, is not identical with the exciting agents of the other parasitic skin-diseases. The favus-fungus grows upon all nutrient media, both at the temperature of the body and at room-temperature ; it grows best somewhat beneath the surface, while only a small number of air-hyphse are formed. The cultures are at first whitish, but later they be- come yellow, and from the periphery radiating processes Fig. 75. — Achorion Schonleinii : X 45° ; showing simple mycelium in various stages of development, and free spores (after Duhring). extend into the depth of the nutrient medium. Microscop- ically, a mycelium of branched hyphae is visible. Individual hyphae swell at their free extremity into the form of a roll, while others form lateral buds — the so-called yellow bodies of Krai — which burst, so that their contents escape as a free body. At these points the deep processes form. The filaments themselves finally disintegrate into oval, cell- shaped structures. Cultural Properties of the AcJiorion. — On gelatin-plates snow-white, star-shaped colonies, with an irregularly thick- ened center, form. The culture-medium is quickly liquefied. In gelatin stab-cultures a thick, wavy, superficial deposit appears, which is stained yellow beneath the surface. FILAMENTOUS AND BUDDING FUNGI. 343 On agar a moderate, whitish, wavy coating forms, the lower surface of which exhibits a sulphur-yellow color. Upon agar and gelatin the mycelium remains sterile. Fig. 76. — Achorion invading root-sheaths and bulb of the hair (Kaposi). Upon blood-serum, at a temperature of 30° C. (Z6^ F.), formation of conidia takes place. Favus occurs in dogs, cats, and mice, as well as in human beings. It is generally transmitted to human be- 344 CLINICAL BACTERIOLOGY. ings from others, less commonly through animals. On the whole, the disease is but slightly contagious. Whether an especial predisposition on the part of the skin is neces- sary for the vegetation of the favus-fungus is doubtful. Youth appears especially predisposed to favus. Infection with favus probably occurs only when the fungus is deposited upon a macerated area of the epidermis or gains entrance into a hair-follicle. The morbid process takes place beneath the epidermis. It does not extend deeply, and the favus-fungus only exceptionally • reaches the subcutaneous connective tissue. In rare instances favus has been observed upon the mucous membrane of Fig- 77- — Trichophyton, X 450, as found in epidermic scrapings of ringworm, showing mycelium and spores (after Duhring). the stomach. Under these circumstances it is probable that the fungus has been swallowed, as opportunity is scarcely ever afforded for embolic dissemination. The diagnosis of favus is made upon both clinical and microscopic data. Culture is not necessary therefor. If it is desired to make a culture, the scutulum is rubbed up in a sterile mortar with sterile silicic acid, and plates are made. Herpes Tonsurans. — The various forms of herpes ton- surans upon the hairy scalp and upon parts of the body free from hair are, like parasitic sycosis, eczema margina- tum, onychomycosis trichophytina, and a number of other affections of the skin, due to the presence of the trichophy- FILAMENTOUS AND BUDDING FUNGI. 345 ton tonsurans (discovered in 1845 by Gruby and Malm- sten), which, partly alone and partly in mixed infection with bacterial excitants of inflammation, is the cause of the disease. The fungus appears especially in the form of extended, slightly branched and straight mycelia, whose width is less and whose segments are comparatively much shorter than those of the favus-fungus. The conidia of the trichophyton resemble those of the achorion, al- Fig. 78. -Root of hair in tinea barbse, showing early invasion of trichophyton in root- sheaths and upper part of bulb. though they are somewhat smaller and, above all, less numerous. These fungous elements lie between the epi- dermic scales, between and in the sheaths of the roots of the hair. In the less changed hairs the mycelium pre- ponderates ; when the trichophytic infiltration is consider- able, the spores are more abundant. The trichophyton may be conveyed to human beings from animals. It occurs in dogs, cats, cattle, and horses. 346 CLINICAL BACTERIOLOGY. More frequently, however, transmission takes place from one human being to another. Of all the dermatomycoses, herpes tonsurans is the most contagious. The predisposi- tion of the skin for diseases due to the trichophyton is in general greater than that for favus. Infection is facilitated by all of the conditions favoring the vegetation of molds, such as a moist season of the year, living in damp cellars, and the like. The variability in the clinical picture of the dis- eases due to the trichophyton depends in the first place Fig. 79. — Microsporon furfur, fungus of pityriasis versicolor; X 700 (Kaposi). upon the varying reactivity of the skin and the varied local- izations of the fungus, and in the second place also upon the simultaneous activity of various bacterial excitants of inflammation. The common cause of all varieties of tricho- phytosis has, however, been determined experimentally with certainty. In every instance the trichophyton was cultivated, and with a pure culture thereof all the varied forms of the disease were reproduced. Cultural Pi'operties of the Trichophyton. — Also in culture FILAMENTOUS AND BUDDING FUNGI. 347 the trichophyton tonsurans closely resembles the favus- fungus. The two are, however, not entirely alike. Lique- faction of gelatin takes place much more quickly with the trichophyton, and the deposits are much greater, than with the achorion. At a depth the trichophyton also develops a yellow color in growth. For microscopic demonstration of the trichophyton the epidermic scales, removed with a sharp curet, or the epilated hairs, are treated with potassium hydroxid, preferably after previous treatment with chloroform and ether for the re- moval of fat. Pityriasis Versicolor. — The cause of pityriasis is the microsporon furfur (first described by Eichstedt, in 1846), Fig. 80.— Microsporon minutissimutn ; X 1000 (after Riehl). a fungus resembling the favus-fungus and the trichophyton, and, likewise, an oidium. The fungous masses, which are at once recognizable in the horny lamella, removed by means of the finger-nail or .a sharp curet and mixed with six per cent, potassium hydroxid upon a glass slide, consist of unusually large and uniform conidia, which form regularly distributed masses, each of thirty or more, and of slightly ramified, short mycelia, which connect the conidial masses with one another. The contagiousness of pityriasis is exceedingly slight. The microsporon obviously requires a quite special predis- position on the part of the skin. It is often found in tuber- 348 CLINICAL BACTERIOLOGY. culous subjects. Experimental transmission of the dis- ease has succeeded in several instances, but the culture of the fungus has not. Erjrthrasma, a circumscribed erythema of the skin, is caused by the microsporon mijiutissirnum, the small, round spores of which lie in the superficial horny cells, whereas the mycelium, consisting of small, delicate, convoluted and branched filaments, in part interlacing and with short segments, extends more deeply. Psoriasis is no longer included by dermatologists among the parasitic diseases, its supposed excitant, the epidermo- phyton, having been recognized as a factitious product (Ries). PHARYNGOMYCOSES. In the pharynx mycoses are not at all rarely observed in certain situations. The fungous vegetations appear as small, porcelain-white plugs, mostly in the lacunae of the tonsils. They consist, however, as a rule, not of true molds, but more commonly of filaments of the leptothrix buc- calis (inycosis pJiaryngis leptotHricid)^ whose botanic position has not yet been clearly made out, but which is ordinarily included among pleomorphic bacteria. Miller differentiates a leptothrix buccalis innominata, maxima, and gigantea. The leptothrix is a constant inhabitant of the oral cavity ; and its fine threads, from 0.5 to 0.8 /i thick, are frequently found on examination of the sputum. The straight, wavy, or spiral threads of the leptothrix are made up of rod-like or spiral segments. In the filaments a base and an apex may often be distinguished ; at their free extremities spher- ical formations appear, which in part have been considered arthrospores. The appearance of this fungus is thus quite similar to that of the simple varieties of molds. Accord- ing to some observers, these detached *' spores" may, like true cocci, divide in pairs. In that event the fungus would belong to the bacteria, and would be included among the pleomorphic varieties. The leptothrix stains yellow with solution of iodin and potassium iodid. Abundant opportunity is afforded for the entrance of the fungus into the lacunae of the tonsils, as the inhaled air and articles of food always contain fungi. In the secretion of the oral cavity leptothrix and filamentous fungi are always present normally. The vegetation of the fungus in the FILAMENTOUS AND BUDDING FUNGI. 349 pharynx does not penetrate deeply and does not cause de struction. Frequently, it gives rise to no subjective symp- toms whatever, and only accidentally comes under observa- tion. In other instances it acts as a mechanical irritant, giving rise to the sensation of a foreign body, and also to signs of slight inflammation. The fungi adhere quite firmly ; they can scarcely be removed by brushing and the like, but the plugs must be pulled out individually or removed with the galvanocautery. KERATOMYCOSES, OTOMYCOSES. Keratomycoses have been observed after injury of the intact cornea with soiled articles (pitchforks, etc.), their Fig. 8i. — Aspergillus fumigatus; X 500 (Frankel and PfeifFer). cause always appearing to be the aspergillus fumigatus. The spores introduced through the agency of the trauma- tism develop into filaments whose unlimited growth is capable of causing destruction of the cornea. The fungous mass is surrounded by a wall of leukocytes. Fructifica- tion does not take place under these circumstances, nor have dissemination of the germs and the formation of met- astases been observed. Otomycoses, or myringomycoses, likewise occur, and are also mostly due to the aspergillus. The presence of 350 CLINICAL BACTERIOLOGY. inflammatory processes in the auditory canal favors the lodgment of the fungi, which not only vegetate in the secretions accumulated in the external auditory canal, but also penetrate the living tympanic membrane. PNEUMONOMYCOSES. Pneumonomycoses have been observed repeatedly in human beings, and are due to both the aspei^gillus and the mucor. Mold-vegetations have not rarely been found in bronchopneumonic foci at autopsy, and also during life ; filamentous fungi have been found in the sputum, which is then sometimes characterized by a putrid odor. In general the occurrence is, however, rare. In view of the frequency of occurrence of pathogenic molds in the air, and of the abundance of opportunity offered for the entrance of fungi into the lungs, it must be assumed that the human pulmonary tissue possesses an especially slight predisposition for molds. It has been men- tioned, on the other hand, that the lungs of birds form a more suitable nutrient medium for the vegetation of fila- mentous fungi, and that birds not rarely succumb to spon- taneous mycoses. The pulmonary mycosis in human beings presupposes in general a primary disease, a hemorrhagic infiltration, a necrosis, etc., of the pulmonary tissue. The fungi pro- liferate, as a rule, only in such foci of disease. This mycosis also is unattended with a tendency to extension, and it is unassociated with evidences of general infection. When the primary disease undergoes recovery, the tissues are quite capable of disposing of the mycotic vegetations. Pneumonomycosis often terminates in recovery. VISCERAL MYCOSES. Mold-vegetations in internal organs (kidney, liver, etc.) have only exceptionally come under observation. The reason for this is readily made clear in experiments on ani- mals. The opportunity for infection is wanting. The fungi can reach the organs in question only through the blood, and they are scarcely ever taken up into the blood- stream from existing mycoses, from the intestine, and wherever else in the body fungi are present. THRUSH. 351 THRUSH. Thrush is a local" disease, preferably attacking mucous membranes lined with pavement epithelium, and resulting from the lodgment and proliferation of the thnish-ftingus. The lodgment of the fungus leads to the formation of milky-white deposits varying from the size of a millet-seed to that of a lentil. These gradually undergo enlargement toward the periphery, and in the absence of therapeutic intervention coalesce finally into large membranes. Microscopic Examination of Thrush-deposits. — Mi- croscopic examination of the white points that represent the first stage of the thrush-eruption discloses, in addition to squamous epithelium, molds and bacteria of various kinds, always in large number, the two phases of the thrush-fungus {oidiiun albicans) : the mycelial threads and the conidia. The former are threads of double contour and of varying thickness and length, with transverse septa and indentations, from which often lateral branches of equal or less thickness pass off. The conidia, which grow from the mycelia, at their extremities or in the neighbor- hood of the septa, are more or less spherical, separate readily from the mycelia, and lie among them, at times iso- lated, at times in groups. Occurrence of Thrush. — Thrush occurs most commonly in new-born children, principally about the second week of life. In older children and in adults thrush occurs only when protracted disease (typhoid fever, tuberculosis, etc.) has induced general enfeeblement of the organism. In the new-born, however, an eruption of thrush by no means presupposes a particular impairment of the general health. The mucous membrane of the infant is obviously quite espe- cially predisposed to the vegetation of the thrush-fungus. In experimental transmission of thrush the disease will develop upon the healthy mucous membrane of well- nourished children. The preferable seat of thrush is the mucous membrane of the mouth, and here the tongue, the inner surface of the lips and of the cheeks, are most commonly invaded. Next in frequency deposits of thrush appear upon the gums, in the pharynx, and also in the upper portion of the esopha- gus, on the anterior and upper surface of the epiglottis, and exceptionally upon the true vocal bands. In all of these 352 CLINICAL BACTERIOLOGY. situations pavement epithelium is present, and it has been assumed that the thrush-fungus is capable of proliferating only upon mucous membrane covered with such epithelium. The thrush-fungus is not rarely encountered in the vagina of pregnant women, which likewise is lined with pavement epithelium. It occurs, however, also upon mucous mem- branes lined with other kinds of epithelium — as, for instance, that of the stomach, the posterior surface of the epiglottis, the deeper portions of the respiratory tract, although but seldom in all of .these situations. The thrush-fungus has been demonstrated repeatedly in bronchopneumonic foci, and in numerous instances upon the mammary glands and mammillary areolae of nursing women whose children suf- fered from thrush, and in one instance in encephalitic sup- purative foci in a child suffering from thrush. Course of Thrush. — The thrush-fungus penetrates the uninjured epithelial surface, and develops beneath the up- permost layer of epithelium, within the deeper portions of the mucosa. Only rarely does it invade the submucosa. The abundant fungous vegetation raises the upper epithelial layer, and gradually separates it from the connection with the lower layers necessary for its nutrition, so that it under- goes necrosis and exfoliation. The thrush-membrane is then exposed to view. The thrush-proliferation usually gives rise to mild local -irritative manifestations. The mucous membrane generally appears somewhat altered at the site of the thrush-deposits. It is dark red, slightly swollen, dry, and distinctly sensitive to touch. The signs of an actual inflammation are, however, not discoverable microscopi- cally. It has been assumed that the thrush-fungus can find lodgment only upon mucous membranes the seat of catarrhal inflammation. This, however, does not appear to be correct, but the slight evidences of irritation may really be the result of the purely mechanical action of the fungous vegetation. The saliva of patients suffering from thrush invariably has an acid reaction. There is no sug- gestion of chemic activity on the part of the thrush-fungus, of any production of toxin. There is an absence of fever and, in fact, of all constitutional manifestations. The symptoms are of a purely local nature. The fungous proliferation may become dangerous only when, by reason of especially luxurious development, it obstructs the esophagus or the larynx ; and, further, in small children, THRUSH. 353 through impairment of the nutrition, the tenderness of the mouth interfering with nursing. In general, however, the disease is a thoroughly benign one. The thrush- vegeta- tions have no especial tendency to extend, and only excep- tionally have extension of the thrush-fungus into the interior of blood-vessels and transmission through emboli been observed. The vegetations can be readily removed by rubbing the thrush-plaques with soft cloths dipped in a solution of borax (sodium biborate 2.0, glycerin 4.0, dis- tilled water 34.0). Sources of Infection for Thrush. — The thrush-fungus is present in the air of dwelling-rooms, in the feces of in- fants, upon rubber nipples, upon saccharine and amylaceous articles of food, etc., and it may gain entrance into the mouth with all of these. Frequently, as has already been mentioned, the thrush-fungus is found in vaginal mucus, and thrush in the infant has been attributed to conveyance of the fungus from the maternal genital canal during the act of parturition. This is, however, certainly not the ordinary mode of infection, as the period of incubation of thrush, as determined by numerous inoculation-experiments, is only four or five days, and the disease usually does not appear in infants before the second week. Culture and Botanic Position of the Thrush-fungus. — In accordance with the appearance of the thrush-fungus in the thrush-membrane described, the fungus bears a close resemblance to the varieties of oidia that cause the derma- tomycoses, and it has, therefore, been given the name oiditim albicans. Grawitz, then, demonstrated subsequently by culture that the thrush-fungus is not a mold, but rather a budding fungus. As numerous later investigations have shown, the thrush-fungus grows upon culture-media of acid reaction and containing an abundance of sugar (prune- decoction agar) in filaments of actively budding yeast-cells. If these be transferred to ordinary meat-peptone agar — thus to an alkaline nutrient medium, deficient in sugar — distinct threads develop in addition to the budding cells. Retrans- ferred to an acid medium, the threads again develop almost exclusively into yeast-conidia. The oidium albicans is thus a budding fungus that, under certain nutritive conditions, forms hyphae. Other observers, however, maintain that the thrush-fungus is a mold. The organism does not liquefy gelatin. Upon gelatin-plates whitish colonies form. 354 CLINICAL BACTERIOLOGY. and in stab-cultures whitish-yellow granules that send radiating processes into the nutritive medium. Upon agar a whitish-yellow, wrinkled deposit forms ; upon potatoes a dense, white coating, and upon bread-pap a thin, white de- posit. The temperature-optimum is 37° C. (98.6° F.). It has been demonstrated experimentally that the thrush- fungus is the actual cause of thrush. It has been possible to develop thrush upon healthy mucous membranes both by means of the bands of buds, and with the filamentous cells. Rabbits die in from twenty-four to forty -eight hours after intravenous injection of emulsions of the thrush- fungus, a general mycosis resulting, and networks of thrush-vegetations being found in the kidneys, the liver, etc. (G. Klemperer). This pathogenic action of the thrush- fungus is, however, not constant. The diagnosis of thrush is made by microscopic exami- nation of the thrush-deposit ; cultivation of the fungus is not necessary. ACTINOMYCOSIS. The actinomycosis of animals was recognized as a dis- tinct disease in 1877 by Bollinger, and in its lesions pecu- liar vegetable structures (actinomyces, ray-fungus) were invariably found. Actinomycosis is a disease peculiar to cattle, having been but seldom observed in other animals (swine, dogs). The usual seat of the disease in cattle is the lower jaw, less commonly the upper jaw, or also the adjacent soft parts, especially the tongue. The disease of the jaws leads to the formation of tumors of greater or less extent, which subsequently rupture outward through the skin, less commonly into the mouth, and thus come into view as ulcerated nodules. On section the tumor appears pale yellow ; it is in general soft, but presents, in places, espe- cially softened, yellowish areas of varying size, from which on scraping with the knife a substance resembling pus and numerous yellowish granules as large as grains of sand are obtained. Examined microscopically the tumor-mass represents granulation- tissue; the yellow coloration is dependent upon abundant fatty degeneration of the cellular elements. The essential feature and the peculiar characteristic of the actinomycotic nature of the new- formation consist in the yellow granules present, the so-called actinomyces-granules, to whose structure reference will be made later. These are fungous formations, and represent the cause ACTINOMYCOSIS. 355 of the disease, as has been established with certainty by the successful transmission to calves of actinomycosis by means of such granules (Ponfick and others). The disease in animals presents, in the first place, the charac- ters of an inflammatory new-formation, which develops around the fungous proliferation as a foreign body; besides, there is considerable destructive tendency — the proliferating fungi in their growth displacing and destroying all opposing tissues. Infection in animals probably takes place principally through the mouth in feeding. The ray-fungus is said to be present in feed, in wheat-grains, which play an important part in the pro- cess of infection. Infection through the superficial integument, through the lungs, etc. , is possible also, but occurs less commonly. A case of actinomycosis of the udder has been observed in a pig, and another of miliary pulmonary actinomycosis in a cow. Fig. 82. — Actinomyces (von Jaksch). Actinomycosis in Human Beings. — Isolated cases of actinomycosis in human beings had already been observed by B. V. Langenbeck (1845) and by Lebert (1857), but the disease was first clearly recognized and accurately described as an independent affection by J. Israel in 1878. Actino- mycosis in hunian beings differs from the same disease in cattle in its slighter tendency to tumor-formation, and its marked tendency to insidious, widespread extension, which may finally lead to involvement of all of the viscera. The chronic inflammation that takes place around the fungous deposit is identical with that which occurs in animals. The newly formed granulation-tissue undergoes fatty degenera- tion more quickly, however, with disintegration or suppura- tion, while the fungous proliferation progresses and gives rise to fistulous formation, undermining the skin, penetrat- 356 CLINICAL BACTERIOLOGY. ing the muscles, and advancing without restraint. Thus, the fungus extends from the lower jaw, along the neck to the pleurae and the lungs, and through the diaphragm into the abdominal cavity. Even invasion of the heart and the brain has been observed. In addition to the continuous advance of the disease, which usually is its dominating feature, the dissemination of the fungus may take place through emboli, in consequence of penetration of actino- myces into the lumen of the vessels. The peculiar and the single characteristic feature of all actinomycotic lesions is also in human beings the presence of the actinomyces-gramiles. They vary from the size of a grain of sand to that of a mustard-seed, and are coarse, dense, at times calcareous, bodies of yellow color. Viewed with low powers of the microscope they appear as dark, finely granular globules, of roundish or irregularly nodular shape. If slight pressure is made upon the cover-glass beneath which the granules lie, they are broken up into a number of smaller portions. Among the latter certain radiate structures are especially characteristic, the so-called actinomyces-druses (spheres), from which the fungus has ac- quired its name of ray-fungus. These, however, can be observed only with higher magnifications, and best after staining the preparation. From a dense center there radiate in every direction uniform, glistening, variously ramified filaments, which enlarge toward the periphery and terminate in bulbous, club-shaped swellings. There thus result star-shaped figures, which have been compared with closed crystalline druses or filled asters. In addition to these, however, there are always present simpler structures radially branched, consisting of only a small number of filaments. These appear microscopically as gray, viscid, sometimes more consistent, granules. They are juvenile forms, which are found with especial frequency in softened foci. The filaments at times exhibit divisions, and are suggestive of strings of bacilli. Finally, there are present also collections of spherical bodies that have been con- sidered as masses of cocci. Gram's method, as modified by Giinther (p. 107), is well adapted for staining the fungus, as is also exposure for half an hour to the action of heated carbol-fuchsin solution. The portal of infection for the ray-fungus is also in human beings generally the commencement of the diges- ACTINOMYCOSIS. 357 tive tract, and most frequently, probably, carious teeth, or the wound left after extraction of a tooth. The frequent occurrence of the disease in the neighborhood of the jaw, and upon the neck, is suggestive of this site of invasion, although any other part of the body may constitute the portal of entry. Cases of actinomycotic perityphlitis have been reported, and Chiari has recorded one of primary actinomycotic disease of the intestinal mucous membrane in an insane person, and the like. It is certain that an injury of the skin or of the affected mucous membrane is necessary for infection to take place. The source of the infecting actinomyces-material is, however, only rarely demonstrable. The chewing of wheat-grains is often assigned as a cause. Some observers suggest the possi- bility of direct transmission from cattle, although this has not yet been demonstrated. In a considerable number of cases of actinomycosis a history of any industrial or acci- dental association with cattle is completely wanting. The course of actinomycosis is exceedingly insidious. The extension of the chronic inflammatory process that attends the vegetation of the fungus can be restrained only by complete removal of the latter. The disease frequently ieads to death, from the invasion of vital internal organs (kidneys, lungs, etc.) by the fungous vegetation. Fre- quently, secondary acute inflammation, amyloid degen- eration, etc., are superadded. Secondary infection with the excitants of suppuration appears especially common. The extensive phlegmons, and especially the pyemia, which often complicate the disease, can be attributed to this source, although thorough investigations in this connection are wanting. The question has not yet been studied whether the actinomyces is capable of generating toxic substances and whether it gives rise in the body to intoxication, or whether its action is purely mechanical — that is, whether it impairs the functions of organs by invading their structure. It is noteworthy in this connection that some severe cases of actinomycosis are entirely unattended with fever. Pure culture of the actinomyces has been attained by M. Wolff and J. Israel (1890). These investigators cultivated the ray-fungus in the absence of air, both upon agar and in hens' eggs. Upon the surface of agar there form at a tern- 358 CLINICAL BACTERIOLOGY. perature of 37° C. (98.6° F. ) numerous small, isolated nodules resembling dewdrops, the first indications of which are visible with a lens as early as after two days, but which do not appear distinctly before from the third to the fifth day. These project above the surface as spheres, though at times they are not entirely round. They grow quite slowly, often only reaching the size of a pinhead after eight days, and they usually do not grow larger. At the same time they become opaque. As a rule, they do not become confluent, remaining isolated for weeks and months. If, after inoculation, a veil-like turbidity appears on the surface of the agar, the nodules described often become differentiated subsequently. At times, however, the nodules coalesce into a whitish coating. The actinomyces-granules used for inoculation undergo enlargement as a whole, if not carefully rubbed upon the inoculated surface, and form a whitish area in which, however, at times, also a differentiation into nodules is discernible. In addition to these fine nodules there are present in small number white nodules, rather larger than lentils, with a blunt, club-shaped, projecting center, and which become covered over toward the periphery, and frequently ex- hibit roundish depressions at quite regular intervals. These nodules in roset-form, whose periphery, further, is not always regular, but at times is only slightly excavated or cleft, are also characterized by their growth into the body of the culture- medium ; they send root-like processes into the agar. These^ large nodules are not common, and are usually encountered only in small number in culture. As, in addition to them, the smallest nodules first described are often present also, and as, further, nodules of intermediate size — transition-forms — with root-like processes appear, Wolff and Israel consider the char- acteristic large nodules only as the highest macroscopic form of development of the inoculated actinomyces-fungus. In stab-cultures, in addition to enlargement of the intro- duced granules, there develops throughout the entire length of the puncture a delicate, gray, veil-like turbidity, and this likewise contains numerous small nodules. At times large, flat, white plaques form at the site of puncture. The cultures described develop typically only in the absence of oxygen ; otherwise the growth is but scanty. A tempera- ture of 37° C. (98.6° F.) is always necessary for the cultivation of the fungus, and no development takes place at temperatures between 16° C. (60.8° F.) and 20° C. (68° F.). For this reason growth does not take place upon gelatin. In bouillon growth is quite scanty. After from three to five days white scales and dots appear ; the bouillon itself, like the water of condensation in agar-cultures, remains clear. Culture in Eggs. — The actinomyces thrives well in pigeons* ACTINOMYCOSIS. 359 and hens' eggs, and when raw, as well as when boiled for three or four minutes. The shell of the egg is carefully cleansed by- means of mercuric chlorid and sterile water, an opening is bored into one end by means of a flamed needle, and a platinum wire mounted with actinomyces-material is introduced deeply into the egg, in which it is moved to and fro several times. The opening made into the inoculated egg is closed with sealing- wax, and the egg, with the perforated end upward, is placed in the thermostat. After the lapse of from nine to twenty-eight days the egg will be found odorless, without formation of gas, and not discolored. There will be seen small, opaque, whitish clumps and dots ranging in size up to that of a pinhead, in the raw egg, in both the white and the yolk, and in the boiled egg at the junction between the two ; or there may be present in the liquid white of the unboiled egg lines and streaks of a turbid mass resembling nasal mucus; or, finally, the line of inoculation and the central surface of the coagulated albumin may become occupied by a smeary, granular mass. On microscopic examination Wolff and Israel found the agar- cultures made up of short rods, mostly straight, but often, also, comma-shaped, or even more markedly curved. These vary in length and thickness, plump and thick rods lying side by side with short and thin and long and thick rods. Often the rods present a globular or an olive-shaped enlargement at one ex- tremity. In addition to the rods, there are found a small num- ber of filaments, rarely rectilinear, mostly wavy and curved, or also spiral. While these are uncommon upon agar, the occur- rence of beautiful, long, filamentous networks is usual in egg-cultures. The filaments, also, whose convolution often exhibits a radial arrangement at the periphery, at times present a knob-like enlargement at the extremity. They, as well as the short rods, together with the terminal enlargements, can be stained both by Gram's method and with fuchsin. Filaments stained for an hour with heated carbolfuchsin exhibit at "times segmentation into longer or shorter rods, ranging in size down to that of the shortest coccus-like bodies, arranged irregularly, and separated by unstained intervals of varying length. In addition to the filamentous network, the rods are also found in the eggs. Finally, there are found in agar-cultures, as well as in egg-cultures, also micrococcus-like bodies, at times free, at times in dense masses. These are of varying size, in part spherical, in part oval, in part rather irregular and angular ; they stain deeply with gentian-violet and also by the method of Gram. They correspond completely with the granules, in which the differentiation of the stained filaments can often be made out, and the rods also when stained exhibit similar granu- lation. Wolff and Israel do not consider these coccus-like 360 CLINICAL BACTERIOLOGY. bodies as spores, on account of their irregular shape and the readiness with which they take stains, but as disintegrated fila- ments and rods. They do not, however, represent dead detritus, for when inoculated upon fresh nutrient media, they again de- velop into rods and filaments. Experimental Development of Actinomycosis with Pure Cultures. — The various cultures thus present the same structures as the actinomyces-masses in the foci of disease : groups of cocci, rods, and filaments ; the fully de- veloped clubs alone are wanting. Culture shows, further, that the cocci, rods, and filaments represent various phases of development of one and the same organism. Each form is capable of further development in as many generations as may be desired, and in this process the characteristic features always appear. The actinomyces retains its vitality in culture for a long time — as long as nine months. That the fungus described is the true ray-fungus, in spite of the absence of typical clubs, is demonstrated by the successful inoculation of animals with cultures. Wolff and Israel infected i8 rabbits, 3 guinea-pigs, and i sheep, in part with agar-cultures, in part with egg-cultures, by intraperitoneal injection, or by injection of a suspension of the fungus into the liver. All of the animals, after the lapse of from four to seven weeks, exhibited multiple tumors in the abdomen, which contained typical actinomyces- druses. With these tumors actinomycosis could be again inoculated. Further, from their contents new cultures could readily be obtained, presenting all of the peculiarities of the original culture, including absence of typical club- forms. The chain of evidence in favor of the specific etiologic significance of the actinomyces-fungus cultivated by Wolff and Israel is thus conclusively established. The anaerobic actinomyces-fungus described in the fore- going was obtained by Wolff and Israel in two cases of actinomycosis in human beings. According to Kruse, the actinomyces of Wolff and Israel has thus far not been culti- vated by others. On the other hand, it must be pointed out that one of the authors of this work has succeeded in obtaining the same cultures with precisely the same char- acteristics in five cases of actinomycosis in human beings. A second species of actinomyces (aerobic actinomy- ces), which is quite distinct from the actinomyces of Wolfif ACTINOMYCOSIS. 361 and Israel, has been cultivated by Bostrom, Affanassieff, and others. This second variety of actinomyces grows more energetically in the presence than in the absence of air — an important differential diagnostic point in relation to that already described. The temperature-optimum is 37° C. (98.6° F.), although the fungus will grow well also at ordinary room-temperature. Upon gelatin-plates, and still better upon agar-plates, there form at first fine, radiat- ing colonies, which develop with comparative activity to opaque nodules, whose periphery presents a delicate fila- mentous network. In agar streak-culture a continuous deposit does not form, as a rule, but a series of more or less closely approximated nodules. The latter not rarely exhibit a brick-red color upon blood-serum, and become covered by a whitish, down-like coating consisting of air- hyphae. From these there develop, through a process of segmentation, a series of roundish bodies arranged like chains, the so-called actinomyces-spores. These are de- stroyed only after exposure- for five minutes to a tempera- ture of 75° C. (167° F.), whereas the mature forms die at as low a temperature as 60° C. (140° F.). When kept in the thermostat for some time, a wrinkled, coherent mem- brane eventually develops upon blood-serum, while the nutrient medium is softened. Upon potato the aerobic actinomyces grows as a brick-red deposit, and likewise with air-hyphae. In bouillon floating and in part granular mem- branes form that sink to the bottom, while the overlying fluid remains clear. Milk is gradually peptonized. With regard to the microscopic appearances, the granules and the druses, which constitute the characteristic feature of the morbid process from which the aerobic actinomyces is obtained, resemble in every detail those already described, so that it is only necessary to refer to the account of these. Preparations made from pure cultures, however, display great differences. Long filaments with rectangular branches may be seen that have arisen through the process of budding. Terminal enlargements are encountered but rarely in old colonies that have developed in the depth of the culture- media. Transmission of the aerobic ray-fungus to animals has not yet been effected with certainty — a third point in con- tradistinction from the anaerobic species of Wolff and Israel. With regard to the botanic position of the actinomy- ces, it was formerly included among the pleomorphic bac- 362 CLINICAL BACTERIOLOGY. teria. Kruse (Fliigge's Microorganisms) includes it among the streptothrices. These represent, to a certain degree, a connecting Hnk between molds and bacteria. They consist of long, cylindric filaments, dividing by budding, and eventually developing a true mycelium. Individual species produce fruit-bearers, which, after the fashion of oidia (see p. 338), constrict off spores directly, without especial fruit- heads. These spores must, however, not be placed upon the same plane as the similarly designated permanent forms of the bacteria, as they succumb to exposure for five min- utes to a temperature of 70° C. (158° F.), as has been mentioned in connection with the aerobic actinomyces. In old cultures the branching filaments break up into bodies resembling bacteria, bacilli, cocci, and even spirilla. If, however, these disintegrated products are transferred to new culture -media, true filamentous networks will again develop from them. According to our own investigations, we have likewise reached the conclusion that the entire group of actinomyces is to be included among the hyphomycetes. The bacteriologic diagnosis of actinomycosis requires only microscopic demonstration of the actinomyces-druses in the pus. Culture is not necessary to establish the diagnosis. Treatment. — According to recent statements, actinomy- cosis is specifically influenced by potassium iodid. It is said that recovery from the disease will take place without any surgical intervention upon administration of moderate doses of this drug — ^from two to three grams daily. PATHOGENIC STREPTOTHRICES. In connection with the actinomyces, brief mention will be made of the streptothrix Eppinger and the streptothrix farcinica. The former was found by Eppinger in an abscess of the brain. The fungus presented a branch mycelium. Air-hyphse and spores are, however, found only upon potatoes. The fungus can be stained by Gram's method. It grows best at a tem- perature of 37° C. (98.6° F. ) in the absence of air. Upon gelatin elevated, wart-like yellow colonies form, which do not liquefy the culture-medium. Upon glucose-agar a wrinkled, orange-colored deposit forms, and upon potatoes a thin, yellowish- red deposit. Bouillon remains clear, although flocculent islands develop. A variety of pseudo-tuberculosis develops in guinea- pigs and rabbits inoculated with the streptothrix Eppinger. PATHOGENIC YEASTS. 363 The streptothrix farcinica (also designated *'bacille du farcin des boeufs Nocard") likewise gives rise to the formation of a branched mycelium, with air-hyphae and spores. Growth takes place between 30° C. (86° F.) and 40° C. (104° F.) in the presence of air. The fungus can be stained by Gram's method. Upon agar whitish, dry scales form that subsequently become yellow and finally confluent. Bouillon is not rendered turbid, but presents flocculi. A similar change takes place in milk, which is not otherwise altered. The farcin des boeufs is a pseudo-tuberculous affection of the skin and the internal viscera in cattle. Experimentally the streptothrix farcinica induces pseudo-tuberculosis in cows, sheep, and guinea-pigs. PATHOGENIC YEASTS. The recognition of the pathogenic yeasts has been made only within recent years, and is due especially to the labors of Busse, Sanfelice, Curtis, and Rabbinowitsch. The following varieties of pathogenic yeasts have been found in human beings: The saccharomyces hominis has been observed in an infectious disease that began with subperiosteal inflammation of the tibia, and finally terminated in the clinical picture of pyemia. The organism appears in the form of round or oval cells, with double contour and a capsule. Upon gelatin -plates it forms round, projecting colonies that do not cause liquefaction ; upon agar, a whitish deposit ; upon potatoes, a grayish-brown deposit ; in bouillon, marked turbidity, with the formation of a membrane; and upon blood-serum, a dewdrop deposit. The saccharomyces hominis possesses the property of inducing fermentation of grape- sugar, with the generation of alcohol and carbon dioxid. It is pathogenic for guinea-pigs, inducing local suppuration, and for mice, which die exhibiting septic manifestations. The saccharomyces subcutaneus tumefaciens has been cultivated from a myxomatous tumor of the thigh. It consists of oval or round cells that frequently possess a large, transparent cap- sule. In gelatin stab-cultures development takes place in the form of small colonies ; the culture-medium is not liquefied. Upon agar a thick, creamy deposit forms, and upon potatoes a whitish deposit, later becoming brown ; upon beerwort-agar a brownish coating, and upon beerwort, a dense sediment without development of membrane-formation. The fungus has slight fer- mentative activity for saccharose, generating ethylic alcohol and acetic acid. This yeast is pathogenic for white mice and rats, in which extensive local vegetation occurs. Microscopically, the tumor-like formations present no true structure, but are found to consist of enormous parasitic infiltrations. 364 CLINICAL BACTERIOLOGY. n* INFECTIONS WITH THE LOWEST FORMS OF ANIMAL LIFE* The protozoa, the lowest forms of animal life, have hitherto taken part in the etiology of but a small number of diseases in human beings. Perhaps the future will show that they occupy a larger field of activity. In a number of infectious diseases whose exciting agents are as yet un- known, animal parasites are believed to have been observed — as, for instance, in whooping-cough, in carcinoma, and others. The connection of disease with the lowest forms of animal life has, however, been established with certainty only for two affections — namely, dysentery and malaria. DYSENTERY (AMEBIC ENTERITIS) AND TROPICAL AB- SCESS OF THE LIVER. The cause of dysentery was recognized by Losch in 1 87 1 as peculiar animal parasites belonging to the class of protozoa — ainebcE — which were found in dysenteric stools. Koch, on the occasion of his cholera-expedition to Egypt, noted the same organisms in the base of the ulcers of the intestine in four fatal cases of dysentery. Kartulis, Ogata, and others, and in Germany recently Kruse and Pasquale, and, further, Quincke and Roos, have since studied the amebae carefully, and confirmed their etiologic relation to dysentery. Th amebaB of dysentery are unicellular organisms of varying size, with ameboid movement. The smallest cells are about 10// in diameter ; the largest, 50/^ [giant amebce) ; the majority, at rest, from 20 to 25//. The protoplasmic body of the amebae can be differentiated on movement into an outer zone, the ectoplasm, Avhich is homogeneous and less refractive, and the entoplasm, which in part is appar- ently almost structureless, containing a small number of disseminated granules and differentiated from the ectoplasm only by its greater refractive power, and in part highly granular, and completely filled with irregular, mostly quite fine granules (^granular plasma), or, finally, exhibiting a greater or lesser number of large and small vacuoles. The two layers are distinctly differentiable from each other only when the ameba is in motion, while the differentiation is DYSENTERY. 365 lost in a state of rest. Frequently, foreign bodies, especi- ally red blood-corpuscles, also bacteria, much less com- monly leukocytes, may be seen within the entoplasm ; at times the protoplasmic body is literally stuffed with blood- discs. The ameba invariably contains a nucleus, which, however, is not always clearly visible in the moving cells. The nucleus is usually eccentric, and with change in shape, often near the periphery. It has a diameter of between 6 and 8 fi, is round in shape, generally with a sharp contour, and a punctate nucleolus. At times the contents of the nucleus are slightly granular. The characteristic of the ameba is its mode of movement, which is designated ameboid. The ectoplasm is extended at Fig, 83.— Amoebae coli in intestinal mucus (after Losch). any given point in the form of a blunt, roundish, homo- geneous process, and the protoplasm flows after it. In this way an actual change in position can be brought about by a slow, at times backward, crawling movement. The process may further be retracted, to appear immediately at the same or at another point! The ameba is thus engaged in constant change in shape. At. times the ectoplasmic processes move around the central mass in waves without locomotion taking place. Little is known regarding the nutrition of the amebae. In general the foreign bodies so frequently seen in the ento- plasm, especially the red blood-corpuscles, are considered as nutrient material. These are taken up by a process of 366 CLINICAL BACTERIOLOGY. intussusception. The ameba sends out processes — pseudo- pods — around the foreign body, and in a certain sense thus surrounds it. Nothing is yet known with certainty as to the need of oxygen on the part of the amebae. Propagation of the amebae probably takes place by dichotomous division, although direct division of cell and nucleus has not yet been observed. Spore -formation, such as has been assumed by some, has thus far not been dem- onstrated. Of importance are certain permanent forms, which have acquired especial resistance as a result .of en- capsulation ; these are designated permanent cysts or en- cysted amebae. They are generally small — from lo to \2 fx long — have a much sharper, at times distinctly double, contour, and possess a translucent luster. The nucleus is only indistinctly recognizable. In form the bodies are round, and they are nonmotile. DeatJi of the amebae is manifested principally by cessa- tion of movement, the greater or lesser activity of w^hich also is an expression of a corresponding degree of vitality. With the advent of loss of motility the ameba invariably assumes a round shape, the differentiation between ecto- plasm and entoplasm disappears, and the nucleus becomes distinctly visible. The dead amebae after a time undergo degeneration : either they become homogeneous, with a fat-like luster, or they disintegrate into granules, often after having become constricted into several round masses. In cover-slip preparations the amebae, after cessation of movement — that is, after death — do not remain visible for more than two days ; in the stools they have already dis- appeared within twenty -four hours ; only the encysted forms persist for a longer time, being still distinctly recog- nizable after twenty days in cover-slip preparations, as well as in preserved stools, but no longer after four weeks (Quincke). The resistance of the amebae to thermal and chemic in- fluences has been but little investigated. The most suitable temperature is that of the body. Outside of the organism the parasites rapidly lose their motility, which at room-tem- perature is lost after the lapse of a few hours — exceptionally eight hours, and in one instance twenty-four hours. Ele- vation of the temperature to that of the body (examination upon a warm stage) increases the motility and conduces to the survival of the amebae for a longer time. It has, how- DYSENTERY. 367 ever, thus far been impossible, even under the most favor- able conditions of preservation, to keep them alive for more than twenty-four hours. Efforts at cultivation of the amebae upon any nutrient medium have, likewise, thus far been unsuccessful. Even when they were inoculated in pure culture no growth developed. (See Liver-abscess, p. 370.) Kartulis has reported successful cultivation of dysentery-amebae in infusions of hay. His observations have, however, turned out to be incorrect, as the supposed dysentery-amebae were hay-amebae, which always develop abundantly in infusions of hay not sufficiently sterilized. It may yet be mentioned that the amebae are rather in- different to the action of tannic acid (0.3 per cent.) and of boric acid (i per cent), while quinin, in a solution of i : 5000, speedily causes their death. With regard to staining the amebae, reference may be made to the sections dealing with the intestinal changes below and with examination of the stools for amebae (p. 371). Occurrence of Amebae in the Stools of Dysenteric Patients. — The amebae described are regularly present in the stools in cases of tropical dysentery, and especially in the mucous elements thereof. They vary in number. In the albuminoid masses of mucus, often streaked with blood, found in recent cases, they are present actually in hordes ; in older cases, sometimes only in small number. Medicinal treatment may reduce the number materially, and eventually cause their temporary disappearance. The amebae can be demonstrated only when the fresh stool is examined. Ac- cordingly, a negative result in a single examination of the stools can not be accepted as evidence of the absence of the amebae. Only continuous and repeated examination of all the intestinal evacuations, especially at the beginning of the disease, will yield definite information as to the presence or the absence of the amebae. The Intestinal Alterations of Dysentery. — The clinical symptoms of dysentery are those of hemorrhagic catarrh of the large bowel. The pathologic-anatomic conditions in the intestine include the presence of the dysenteric ulcer, with elevated, wall-like, undermined borders. The ulcera- tive process responsible for these alterations originates pri- marily in the submucosa, which undergoes a peculiar necrotic transformation. If a section of the intestinal wall, after hardening in absolute alcohol, is stained with Loffler's 368 CLINICAL BACTERIOLOGY. solution or by Gram's method, the amebse will be found regularly in the ulcers. They lie almost exclusively in the submucosa, at times more deeply, upon the serosa. The smaller forms, about lyi times or twice as large as white blood-corpuscles, are constantly found in the intestinal wall. They appear roundish, without a distinct nucleus ; their bodies stain a deep blue, and vacuoles are often indicated. In addition to the amebae, bacteria are always found in the floor of the ulcer. In the intestinal wall also the amebae, as in the stools, are only demonstrable when the tissues are subjected to examination in a fresh state. At autopsies made four and a half hours after death the amebae were present in the in- testinal ulcers in enormous number, while at autopsies made eighteen hours after death but few were present. Pathogenic Activity of the Amebae of Dysentery for Animals. — Dysenteric affections, with the evacuation of hemorrhagic stools containing amebae, and with character- istic ulcerative changes in the large intestine, in which also amebae are present, have been induced experimentally in large numbers of cats by injection of dysenteric stools through the anus. Losch had induced dysentery in a dog in the same way, by the introduction of large numbers of amebae, both by the mouth and by the anus. In other animals, however, infection has thus far not been in- duced. It is noteworthy that Quincke and Roos ob- served that of four cats that received by the mouth exclu- sively stools containing amebae, with an abundance of the encysted varieties, two were seized with typical amebic enteritis. The encysted varieties thus appear to be able to escape the action of the hydrochloric acid of the stomach. Occurrence of Bacteria in Dysentery. — In amebic stools, in addition to amebae, bacteria are always present ; and especially and in preponderant number, streptococci ; not rarely, also the bacterium coli ; and, further, the bacillus pyocyaneus, staphylococci, etc. In the intestinal wall also the amebae are found constantly in association with bacteria, and here bacilli rather than streptococci are present. The bacteria lie within and around the amebae. At times they penetrate independently into the tissues, and more deeply than the amebse. The association of bacteria and amebae occurs with such regularity that some connection must exist between the two. It is possible that the condi- DYSENTERY. 369 tion is one of mixed infection. This is, however, not prob- able, as all of the bacteria found were not virulent for the animal on infection by enema, although the pus from a liver-abscess that contains only amebae and no bacteria will induce dysentery in cats. It is more probable that secondary infection with these bacteria, all of which are regular inhabitants of the intestine, has taken place. What modification of the clinical picture of the disease and what portions in the anatomic alterations are to be attributed to the secondary bacterial infection, and how much is due to the amebic infection alone can not for the present be de- cided. Portals of Infection for and Transmission of the Amebae. — Dysentery is, according to clinical experience, only transmissible in a limited degree from one person to another, and from one place to another. It has not yet been determined through what medium the amebae gain entrance into the body. The results of the experiments of Quincke, already alluded to, render introduction by the mouth possible. Accordingly, infection from case to case could take place directly through the intermediation of the stools. In general, however, direct contagion is not con- sidered probable by clinicians. It is rather assumed that, as a rule, the amebae are taken up from surrounding nature. Drinking-water is believed often to play the role of the intermediary. As has been pointed out, the amebae do not persist in their original form in the stools, but they quickly undergo degeneration. Whether they persist in some as yet unknown vegetative intermediate stage in nature or in another host, or whether after leaving the body they develop permanent forms, has not yet been cleared up. Kruse and Pasquale froze stools containing amebae for a quarter of an hour in a cold mixture. After thawing, no amebae could be detected, not even encysted forms. The stools, however, proved pathogenic for cats, hemorrhagic enteritis, with abundant proliferation of amebae, taking place after introduction into the bowel. With the disappearance of the ordinary forms of amebae observed the parasites evi- dently do not wholly perish. It is an open question how the amebae, when introduced into the intestine, penetrate the mucous membrane and gain access to the submucosa, and whether a lesion of the mucosa is necessary for this to take place, or whether, per- 24 370 CLINICAL BACTERIOLOGY. haps, with the cooperation of the bacteria, they are capable of passing through the intact mucous membrane. Accord- ing to chnical observation, exposure to cold and disorders of digestion favor the development of dysentery. It thus appears that an especial local predisposition for amebic in- fection is necessary in precisely the same way as for bac- terial infection. Abscess of the Liver. — Abscess of the liver, which occurs so frequently in tropical regions, has long been associated clinically with dysentery. It has mostly been considered as a sequel of the latter disease. As a matter of fact, the pus and the abscess-membrane in cases of tropical abscess of the liver almost invariably contain the same amebae that cause dysentery. When the amebae are absent from the pus, there is generally no relation to dysen- tery ; so that the presence of amebae may be utilized as a point in the differential diagnosis between idiopathic and dysenteric abscess of the liver. Naturally, careful and repeated examination of the contents of the abscess is necessary, as not every specimen of the pus will contain the amebae, which are present in the abscess in varying number. Bacteriologic examination of the pus from hepatic ab- scesses containing amebae has yielded a positive result in the majority of cases,, streptococci, staphylococci, colon- bacilli, and other bacteria being found present also. Only a small proportion of the abscesses are sterile. If the pus free from bacteria contains living amebae, practically a pure culture is present, which may then be employed experi- mentally. It is an open question, further, what role in the devel- opment of abscesses of the liver is played by the bacteria, as all of them possess pyogenic activity. It is possible that the amebae injure the liver-tissue, and that the bacteria then secondarily induce suppuration. It is a question, also, as to how the amebae gain entrance into the liver. In this connection the portal circulation, the peritoneum, the lym- phatic vessels, and the biliary passages, must be considered as possible media of infection. Dissemination of the amebae through the blood-stream appears the more prob- able, as they have been found repeatedly in the intestinal wall, within the lumen of vessels. Penetration of the in- testinal wall and infection from the peritoneum also appear AMEB^ IN NORMAL INTESTINAL CONTENTS. 371 probable. Amebae have been encountered in peritonitic exudates, and hepatic abscesses are frequently quite super- ficial and preferably in the right lobe. Dissemination through the lymph-channels appears improbable by reason of the numerous lymph-glands interposed. Finally, some observers consider hepatic abscesses as not always the sequel of dysentery, but both diseases as due to a common cause, and of which one may at times pre- cede the other, and vice versa. If primary amebic ab- scesses of the liver actually occur, and are, perhaps, fol- lowed subsequently by dysentery, migration of the amebae through the biliary passages would naturally have to be assumed under such conditions. Amebae in Normal Intestinal Contents. — In the stools of healthy persons there occur, usually in small number, and sometimes in rather considerable number, amebae that are indistinguishable microscopically from those that give rise to dysentery. Thus, amoeba coli (Kruse and Pasquale) or amoeba intestini vulgaris (Quincke and Roos) is not pathogenic for animals (cats), whereas the amoeba dysenteriae causes dysentery in animals. Whether this difference in virulence is permanent, or whether the two amebae are identical, the one merely having lost its viru- lence temporarily, or the other having acquired virulence temporarily, can not be decided at present in the absence of a method of culture. Quincke observed as the causa- tive agent in a case of rather mild dysentery of autoch- thonous origin in Kiel an ameba closely resembling that of dysentery, but equally nonpathogenic for cats as the intestinal ameba. He places this amoeba coli mitis between the amoeba intestini vulgaris and the ameba of dysentery (amoeba coli (Losch)). (See Fig. 83.) Such transitions render it probable that the virulence of the amebae is no more constant than is that of the bacteria. Examination of the Stools for Amebae. — In examining the stools for amebae the most important requirement is that the stools should be quite fresh and be examined as soon as possible after evacuation. It is best to receive the stool in a vessel previously heated (in water at a tempera- ture of 40° C. — 104° F.). If the stool is thin, a drop is simply placed beneath the cover-glass ; when blood-tinged flocculi of mucus are present, these are examined. If the stool is mushy, a dilution with a warm solution of sodium 372 CLINICAL BACTERIOLOGY. chlorid (from 35° €.—95° R— to 40° €.—104° R) is made. If the stool is formed, only the superficial layer of mucus is examined. To fix and to preserve the amebae, a portion of the material containing them is spread in as thin a layer as possible upon a cover-slip, which is at once in- troduced into absolute alcohol before it dries. The amebae take stains less well than the bacteria ; so that in cover- slip preparations they are distinguished by their pallor. Methylene-blue is best suited for staining. The nucleus stains more deeply than the protoplasm. More, however, is to be learned from examination of fresh material than from that of stained preparations, and if the observation cover a protracted period, a warm stage is a useful adjunct. MALARIA. The exciting agent of malaria y^^,^ discovered in 1880 by Laveran in the blood of patients suffering from that dis- ease. The parasites of 7nalaria are unicellular forms of life that at an early stage are endowed with ameboid move- ment. They belong to the class of protozoa, the lowest forms of animal life ; but there is no unanimity of opinion with regard to their position in the zoologic scale. Metsch- nikoff included them in the class of sporozoa, and con- sidered them as coccidia. Others placed them among gregarines, Kruse among the earliest, and he designated them as hemogregarines. Still others believe them to be amebae, and include them among the rhizopods. Mingazinni suggests that the entire group of parasites that invade the red blood-corpuscles be designated hemosporidia. In con- sequence of this diversity of opinion a suitable name for the parasites of malaria is wanting. The designation malarial Plasmodia, proposed by Marchiafava and Celli, is inappro- priate, as Plasmodium indicates a body resulting from the confluence of numerous amebae, all of which preserve their nuclei. Morphology and Biology of the Parasites of Mala- ria (Plate I). — The malarial parasites vary in size from one to ten microns in accordance with the age of the individual cell. The juvenile forms are in general flattened and disc-like, and their shape varies with their ameboid movement. The mature 3 U^^ -y ^^^ "^ ^'i-^ "V
  • ff 27 ■it'- Various forms of malarial parasites (Thayer and Hewetson) : Figs. 1 to 10, in- clusive, tertian organisms ; Figs. 11 to 17, inclusive, quartan organisms; Figs. 18 to 27, inclusive, estivo-autumnal organisms. Fig. 1. — Young hyaline form ; 2, hyaline form with beginning pigmentation ; 3, pigmented form ; 4, full-gx-own pigmented form ; 5, 6, 7, 8, segmenting forms ; 9, extracellular pigmented form ; 10, flagellate form. Fig. 11. — Young hyaline form; 12, 13, pigmented forms; 14, fully-developed pigmented form ; 15, 16, segmenting forms ; 17, flagellate form. Figs. 18, 19, 20.— Ring-like and cross-like hyaline forms; 21, 22, pigmented forms ; 23, 24, segmenting forms ; 25, 26, 27, crescents. MALARIA. 373 cells are spherical. A distinct cell-membrane, visible as a double contour, is not peculiar to most varieties. The proto- plasmic body is relatively small in the juvenile forms, the nucleus preponderating. In the older cells these conditions are reversed. In the living parasites, especially at an early period, nucleus and plasma are usually not differentiable. In stained preparations (p. 393) the unstained nucleus is clearly distin- guishable from the deeply stained cell-body. Differentiation into an outer layer of cytoplasm (ectoplasm) and a central portion (entoplasm) is, on the whole, not practicable. The plasma appears mostly homogeneous and hyaline, although ma- ture parasites at times present dense, slightly refractive granula- tions. In the older cells the substance of the plasma generally contains from a brownish-red to a black pigment — the malarial pigment, or melafiin, which is considered a digestive product of hemoglobin. This assumes the form in part of fine, dust-like particles, in part of coarser granules; or it appears as delicate needles up to i /t in length.' The pigment often exhibits a peculiar dancing and wriggling movement, which only resembles, but is not identical with, the Brownian molecular movement, and is believed to be an active motor procedure. In addition to the ameboid movement and that of the pigment the malarial para- sites are characterized by a third mode of motion — namely, that dependent upon flagella. From the fully developed round parasite arise flagella that move actively, in part become free, and then retain their motility. According to Kruse, these structures must be considered as evidences of degeneration. In addition to pigment the cell-body of the malarial parasites fre- quently contains vacuoles, mostly small and few. Often difficult of differentiation from these is the relatively large, vesicular, generally eccentric nucleus, which in the living cell is only recognizable by its distinct contour, and in stained preparations exhibits no nuclear . membrane, but a distinct deep-black nucleolus at the periphery. The nucleolus is often surrounded by a feebly stained zone, while the nucleus itself does not take the stain. Kruse, upon the basis of his investigations, does not admit the significance of this large nucleus, but he considers the nucleolus as the nucleus. Laveran' s Crescents. — Morphologically distinct from these simple forms of malarial parasites (corps spheriques of Laveran) are the crescents of Laveran (corps en croissant) and the spher- ical and spindle-shaped bodies related to them (spheres of the crescentic series). The crescents have the shape of a half- moon, are from eight to ten microns long, at the middle from two to three microns thick, and are characterized by a considerable degree of refractive power. They always contain pigment, usually in abundance, but sometimes only a few granules. This 374 CLINICAL BACTERIOLOGY. is usually collected at the center, frequently in the form of a figure of eight, although it may be distributed throughout the entire crescent ; in the latter event granules are frequently in tremulous movement. The concentrated pigment is invariably still. The crescentic bodies possess a membrane (Mannaberg), which, however, is not visible in all specimens. At times their poles are united on the concave aspect by a delicate curved line (remainder of the host-cell). The crescents are not endowed with ameboid movement ; they have, however, the faculty of slowly changing their form and becoming spindle-shaped, oval, and finally quite round bodies. This transformation, which can be observed under the microscope, takes place quite gradually, in the course often of several hours. In fresh blood-prepara- tions containing numerous malarial parasites, there are always present a few spindles, ovals, and spheres, and Mannaberg is of the opinion that these changes in the shape of the crescents are dependent exclusively, or almost exclusively, upon the removal of the parasite from the human body, and that they do not occur at all, or but exceptionally, within the blood-vessels. By no means all crescents, further, undergo the changes in shape de- scribed, but the larger number retain their shape in blood-prepara- tions preserved in a moist chamber. In the sphere resulting from the crescent the pigment is generally arranged in the shape of a wreath. After some time it begins to move, and, with tremu- lous movement, to intermingle, and soon the moving granules of melanin occupy the entire cell-body. Flagella also then form, and the entire body suddenly engages in jerking movements, while at the periphery projections and retractions take place. After some time processes resembling in form the finger of a glove appear, which are formed by the membrane of the body. This membrane ruptures, the processes retract, and long, slender fila- ments are thrust out from them, which actively lash themselves about. These flagella usually present a bulbous enlargement at their free extremity, and they often exhibit in their course knob- like swellings that appear to change place. From one to five filaments develop from a single sphere. Their active movement soon diminishes, and has entirely disappeared after from fifteen to thirty minutes. The resting filaments remain attached to the body; although often they separate and then move actively about free in the blood. The membrane ruptured in the exit of the flagella becomes rolled together and occupies the per- iphery of the sphere in the form of a globule or a ringlet. Such globules distinguish the spheres resulting from the crescents from the other spherical elements of the malarial parasites already described. Further, the spherical bodies resulting from the crescents have, also after the loss of their membrane, when, therefore the double contour is no longer present, still a much MALARIA. 375 more sharply defined boundary-line than the other spherical forms. Some crescents undergo segmentation, usually dividing transversely through the middle of the body. Relation of the Parasites to the Red Blood-corpus- cles.— The parasites described appear in part free in the blood- plasma, and in part in association with the red blood-corpuscles. This association is considered by Laveran as one of simple adhe- sion of the parasites to the blood-cell, whereas Marchiafava and Celli, in opposition to this extraglobular mode of life, assume an actual penetration of the parasite into the blood-cell, an endoglobular mode of life. Both views are probably correct. The smaller forms appear impressed into the surface of the red blood-corpuscles, to which they are merely closely attached, whereas the older forms certainly occur within the bodies of the blood-corpuscles. This latter occurrence can be observed especially when the parasites leave the infected blood-corpuscle. In this process the residuum of the blood-corpuscle can be seen to rupture, while its contents, stained with hemoglobin, are dis- tributed in fine drops in all directions. The endoglobular arrangement is the rule, especially for the crescents of Laveran, as Marchiafava and Celli have demonstrated. The infected blood-corpuscles are usually soon decolorized, while the para- sites at the same time take up pigment. In the presence of some varieties of the parasite hypertrophy of the infected blood- corpuscle is observed to take place, while others cause diminu- tion in size and shrinking of the blood-discs in which they occur. Sporulation. — The propagation of malarial parasites takes place through spores that form in the mature cells. In the fully developed parasite a greater or smaller number of bodies develop, each of which exhibits complete cell-structure. This process of sporulation terminates the existence of the spore-form- ing bodies ; the spores disperse, and of the mother-cell only a dead residual body remains, consisting mainly of the pigment of the mother-parasite. The systematic arrangement of the spores within the sphere around a mass of pigment often seated cen- trally gives rise to peculiar appearances that have been compared with those of daisies or sunflowers. Every spore contains a visible nucleolus, while the formation of the nucleus often takes place later. Development of the Parasites. — According to Golgi, in the development of the parasites the unpigmented spore moves about free for a time in the blood-plasma, and meanwhile pos- sibly grows somewhat ; it then becomes attached to a red blood- corpuscle, in which it finds the conditions for its further devel- opment. The young parasite, into which the spore within the blood- 376 CLINICAL BACTERIOLOGY. corpuscle has, without especial external alteration, been trans- formed, grows and deprives the red blood-corpuscle of nutritive material. It converts the hemoglobin of the host into melanin, which it collects in the outer layer of its protoplasmic body. It grows until it reaches the height of its development, when it sometimes entirely fills the red blood-corpuscle, and it then gives rise to new spores, in the setting free of which the residuum of the blood-cell is ruptured. With regard to the relation of the crescentic bodies to this process of development of the parasites, it is commonly assumed that the crescents result from the small ameboid parasites, of which Laveran considers them to be the encysted form, while Councilman believes them to be their spores. Mannaberg con- siders the crescents as copulation-bodies (syzygia), which result from the union of two ameboid malarial parasites. The two parasites do not completely coalesce (pseudo-conjugation), and they may subsequently separate (segmentation). Kruse believes the crescentic bodies to be no longer capable of infection, but he considers them the harmless residue of the infectious process. Polymorphism or Multiplicity of the Malarial Para- sites.— The appearances presented by the blood in the dif- ferent types of malarial fever are most variable, and for a long time there has been a difference of opinion as to whether there are various forms of malarial parasites, with a special species of parasite for every type of fever, or whether the various forms of the parasite are but different phases of the same organism. Laveran and his adherents consider the parasites as polymorphous. They believe that the various types of fever are induced not by different species of parasites, but as a result of variations in the predisposition of the organism attacked. The Italian school, on the other hand, considers the exciting agents of the various types of fever as belonging to different species. Golgi makes three varieties : the parasites of quartan, those of tertian, and those of irregular, febrile type. According to his view, the quotidian type is dependent upon either two generations of parasites of the tertian or three generations of the quartan type, of which each generation is separated in its development from the other by a period of twenty- four hours. Golgi has described the cycle of development for these three varieties. Marchiafava and Celli recognize Golgi's tertian and quartan parasites. As the exciting agent of the irregular fever, however, they consider the small ameboid forms from which the crescents are derived. MALARIA. 377 At times these also give rise to typical quotidian fever. The different parasites appear to Marchiafava and Celli, however, as varieties of a single parasite. Grassi and Feletti have gone so far as to describe five species. As cultivation of the malarial parasites is at present not possible, the question whether the forms of parasites are constant and variable, or whether each form gives rise to a definite type of fever, can only be determined by experi- mental conveyance of the parasites with the blood of malarial patients. Such inoculation- experiments have been repeatedly made in human beings. The results, as sum- marized by Mannaberg, are as follows : Of sixteen care- fully performed experiments, there was in fourteen a com- plete concurrence between the forms of parasites from the source of inoculation and those from the inoculated person ; whereas in two cases the inoculated person exhibited other forms than those from the source of inoculation. The two cases, however, that appear to support the view of poly- morphism are, on more careful scrutiny, as Mannaberg shows, not entirely free from criticism, so that the results of the sixteen experiments render it highly probable that the individual varieties of parasites represent distinct spe- cies, which do not undergo a transformation into other varieties. From the experiments, the conclusion is to be drawn further that an unmistakable relation exists between the type of fever and the species of parasites. Classification of the Malarial Parasites. — The funda- mental types of intermittent fever are the quotidian, the tertian, and the quartan. In addition there occur fre- quently irregular X-y^^s, which pursue their course in part continuously, in part with distinct remissions, or with attacks following one immediately upon another. Of the quotidian variety two forms have long been distinguished — namely, the double tertian and the triple quartan. The different types are dependent upon different forms of para- sites. The following are among those best known : I. The Quartan Parasite. — The juvenile form of the quartan parasite is an unpigmented body, with slow, ame- boid movement, visible as a small, bright spot within the infected blood-corpuscle. It grows but little during the first twelve or twenty-four hours. Then pigment appears in the outer layer in the form of coarse granules and rods ; this is nonmotile. With increasing pigmentation the para- 378 CLINICAL BACTERIOLOGY. site loses its previous torpid motility, becoming spherical and filling the blood-corpuscle one-third or one-half. It then continues to grow slowly until it has attained the full size of the blood-corpuscle, so that no portion of this remains visible, and the parasite appears to be free. Sporu- lation now takes place, the pigment-granules concentrating at the center, while a radiate striation makes its appear- ance in the plasma, first at the periphery, subsequently also at the center, gradually becoming more distinct and dividing the parasite into from eight to twelve segments. These segments separate more distinctly from one another as oval bodies (daisy-form), and each contains a bright spot — the nucleolus. Spore -formation is now completed, and the spores are set free by rupture of the mother-cell. The entire process of development has occupied seventy- two hours. Segmentation takes place before and during the febrile attack. About three hours before the occur- rence of the chill the first complete sporulation-bodies are visible in the blood. The red blood-corpuscles that have been infected with quartan parasites do not undergo change in size. The parasites at times progress to sporulation before they have attained the size of the blood-corpuscle. They then generally give rise to from four to six spores. The extrusion of flagella is observed seldom and only in the younger forms. In the regular progress of development of the quartan parasites it is relatively easy to distinguish several genera- tions from one another when these are present. The quartan parasite induces typical quartan fever (looiooiooi) ; two or three generations separated by an interval of twenty-four hours give rise to double quartan (120 1 20 1 20) or triple quartan (123 123 123). The last may be considered a false quartan. If several generations of the quartan parasite are present whose development is separated not by intervals of twenty-four hours, but by shorter or longer intervals, irregular types of fever re- sult. 2. The tertian parasite completes its cycle of develop- ment within forty-eight hours. The juvenile form re- sembles that of the quartan parasite : presenting a small plasmic body, with a nucleus free from pigment, in the living MALARIA. 379 state visible only as a bright spot within a red blood-cor- puscle. It is actively motile, and extrudes numerous pseu- dopods. In the first twenty-four hours (first phase of Golgi) it develops gradually, and collects finely granular pigment within itself, which continues in active vibratile movement, with a preference for the outer layer of the plasmic body. With increasing deposition of pigment the activity of ameboid movement diminishes, without, however, ceasing entirely. After twenty -four hours the parasite has attained about half the size of the red blood-corpuscle, which itself has lost in color and has undergone increase in size — often to a considerable degree. " The blood-corpuscles in- fected with tertian parasites are frequently distended and chlorotic." After forty -eight hours, when the parasite has attained almost the size of the blood-corpuscle and is " no longer in motion and the pigment also is at rest, sporula- tion takes place. Usually, the pigment moves again toward the center, the plasma divides into from fifteen to twenty round, highly refractive globules, which sometimes ar- range themselves in two concentric rows (Golgi's sun- flowers), but frequently lie together in the shape of berries, rosets, or grapes. The round globules are smaller than the spores of the quartan parasites. The nucleolus is only with difficulty to be distinguished in them. The spores then become free, and after a time infect new blood-cor- puscles, in turn themselves to repeat the same process of development. By no means all parasites undergo sporula- tion, just as is the case with the quartan parasite. A large number of tertian parasites remain sterile. These barren elements generally become as large as the parasites that undergo propagation, or larger. In them, however, the pigment remains actively motile. Laveran considers them as hydropic and involved in degeneration. They may still be visible in the blood for hours after the attack, and even on the afebrile days. The act of sporulation corresponds also in tertian fever with the febrile paroxysm. Golgi has shown that as early as three hours before the chill the temperature begins to rise, and that then the first spores make their appearance in the blood. They are, however, most numerous at the time, of the chill. In the mature tertian parasites the formation of flagella can often be ob- served. Kruse considers this process, likewise, as evidence of degeneration. 380 CLINICAL BACTERIOLOGY. The tertian parasite causes typical tertian (qyqy (^loioioi). Two generations of the parasite may give rise to a false quotidian, a so-called double tertian (12 12 121). Several generations not separated from one another by intervals of twenty-four hours give rise to irregular fever. J. The Quotidian Parasite. — The commencement of the cycle of development, which in its entirety occupies twenty- four hours, is like that of the other parasites. The juvenile form, unpigmented, consisting of plasmic body and nucleus, infects the red blood-corpuscle. The parasite is actively motile, and is often recognized by this property, while by reason of its extremely delicate contour and its color, which is only slightly less pale than that of the red blood-corpus- cle, it is scarcely distinguishable from this. On removal of the blood the parasite soon loses its motility, at the latest after the lapse of an hour. A whitish ring is then seen to form, with a reddish center. In consequence of a pro- jection at one point of the periphery the ring frequently resembles a seal-ring. Mannaberg believes that these forms are only closely attached to the red blood-corpuscles, and are not contained within them. The red spot in the center is thought to depend upon dilution of the plasma and the appearance of the underlying red blood-corpuscle as seen through this. The annular form may return to the ameboid. The ameboid parasite does not grow much — alto- gether to only about one-third the size of the red blood- cell. At the same time a fine pigment, which is often only reddish and is but slightly motile, collects at the periphery. After the lapse of twenty-four hours the pigment becomes concentrated at the center or at the periphery in the form of dark, resting clumps, and the parasite disintegrates within the. red blood-corpuscle into the smallest spores (from five to ten). This process of sporulation takes place, according to Marchiafava and Celli, only in the internal viscera of the body, and scarcely at all in the peripheral blood. For this reason the spores are encountered in abundance in the blood obtained from the spleen, whereas they are found not at all, or only isolated, in blood, from the finger-tip. The red blood-cells infected with the quotidian parasites con- tract and become brass-colored (brassy bodies.) After the quotidian parasite has been present in the blood for several days the crescents already described invariably appear, with MALARIA. 381 their secondary form, the spindle-shaped (cigar-shaped) bodies and the spheres. With regard to the nature of the connection between these forms and the ameboid parasite there is as yet no unanimity of opinion. The various views with regard to the source and the destiny of the crescents have already been briefly outlined (p. 376). The quotidian parasite gives rise to typical quotidian fever, and, when present in several generations, to continued, or irregular, fever. The fever induced by the quotidian parasite differs clinically from that due to the tertian and quartan parasites in its malignancy. It recurs obstinately, and often gives rise to profound anemia, and to other per- nicious manifestations (diarrhea, cachexia, coma, etc.). The recurrences take place usually from seven to fourteen days after the first febrile paroxysm. The crescents are generally held responsible for the recurrences. These parasites are present in the blood in the afebrile interval, and are believed to be capable, through segmentation or true spore -formation, of giving rise to new ameboid forms. The new paroxysms would, thus, not be true recurrences, but, as Golgi believes, only the expression of a type with long intervals. This is denied by others, and the crescents are considered to be only degenerative forms that are incapable of contributing further to the formation of new individuals. It is true that with the presence exclusively of crescents and their spheres in the blood, fever is generally not present. On the other hand, the demonstration of these bodies in the blood indi- cates with certainty that fever existed a short while previ- ously, and with great probability that new paroxysms will occur within a short time. It is to be mentioned, finally, that there is an unpig- mented quotidian parasite (Marchiafava and Celli), which is distinguished from the ordinary quotidian parasite only by the complete absence of pigment. This parasite, like- wise, forms crescents, but these are unsupplied with pig- ment. The clinical course of the infection with these unpigmented parasites is in no wise different from that de- scribed for infection with the pigmented quotidian para- site. ^. The malignant tertian parasite has been separated by Marchiafava and Bignami as a special species. It stands close to the quotidian parasite, but is believed to differ from this in the fact that its cycle of development occupies 382 CLINICAL BACTERIOLOGY. forty-eight hours, that it becomes somewhat larger (at the time of sporulation it is one-half or two-thirds the size of the blood-corpuscle), and that it remains unpigmented for twenty-four hours and then begins to acquire pigment, without, however, losing its motihty. This parasite also forms annular bodies, and especially crescents. It gener- ally develops from eight to fifteen spores. The blood- corpuscles infected by it become brass-colored, and usually shrink, but never increase in size. The parasite is distin- guished by these last-named peculiarities from the ordinary tertian parasite (Golgi), which, besides, in all correspond- ing stages is larger, is provided with more pigment, and forms larger spores (from fourteen to twenty). The malig- nant tertian parasite gives rise to severe tertian types of fever, and also to peculiar temperature-curves^ with quite short afebrile intervals often lasting only a few hours, and with regular pseudo-crises, and, finally, continued, or ir- regular, fever. All of the varieties of fever induced by it are characterized by the malignancy peculiar also to the quotidian parasite. Mixed Infection. — In a number of cases of intermittent fever several of the parasites described are found together in the blood, and with especial frequency pigmented quo- tidian parasites, with unpigmented, and not rarely also quartan with tertian parasites. Golgi found in one patient three generations of the quartan parasite and two of the tertian parasite. All of the various components may find definite expression in the temperature-curve, or the fever may pursue an irregular course. It may be, further, that the temperature-course corresponds with only one variety of parasite, while the other appears to exert no influence. Diagnosis of the Malarial Parasites (Method of Blood-examination. — A drop of blood is taken, as in ordinary examination, from the lobule of the ear or the tip of the finger, after previous cleansing by means of brush, soap, mercuric chlorid, alcohol, and ether. Slides and cover-glasses must be cleansed (with alcohol and ether) and dried with especial care. It is advisable not to manipulate them with the fingers at all, but to grasp them with the aid of suitable forceps (Ehrlich). A puncture is made with a lancet, the extruded drop of blood is received upon the cover-slip, and this is inverted simply upon the slide. It is especially important that the drop obtained be not too MALARIA. 383 large. The blood-cells must lie side by side somewhat scattered, as arrangement in rouleaux would conceal the parasites. The preparation is then examined with a good oil-immersion lens. If the examination is to be continued for some time, evaporation is prevented by surrounding the margin of the cover-slip with wax, or the uniformly dis- tributed drop of blood is examined on an excavated sHde, at the bottom of which is a drop of water. To stimulate ameboid movement, a warm stage should be employed. Examination in a warm room is possible, however, for an hour or more, without the aid of this device. In the preparation of dry specimens also it is important to obtain quite a small drop of blood. The cover-slip is then quickly drawn over the surface of a second cover-slip, and both, protected from dust, are permitted to dry in the air. The dried preparations are placed for fixation for from five to thirty minutes in a mixture of equal parts of abso- lute alcohol and ether. They are now dried between lay- ers of bibulous paper, and are stained — either in dilute aqueous solution of methylene-blue, and then, after rinsing with water, in two per cent, alcoholic (sixty per cent.) solu- tion of eosin ; or the specimen is exposed to the action of both stains at the same time. For this purpose Plehn recommends the following mixture : Coi;icentrated aqueous solution of methylene- blue, 60 One-half per cent, solution of eosin (in sev- enty-five per cent, alcohol), 20 Distilled water, 40. The preparation is kept in this solution for from five to ten minutes, and is then rinsed in water, dried, and examined in xylol Canada balsam. The hemoglobin-containing blood- cells are thereby stained red ; the plasmic body of the para- sites, more or less deeply blue. For .rapid examination, simple treatment with aqueous methylene-blue suffices. For special examinations and for the determination of the finer structural relations, a large number of methods of fixation and staining have been proposed. For diagnostic purposes, examination of fresh specimens and of stained preparations by the method described is quite sufficient. With regard to the diagnostic utility of the results of the examination, the following statements may be made : 384 CLINICAL BACTERIOLOGY. The presence of even a single undoubted malarial parasite establishes the diagnosis with certainty. Should the result be negative, it must be borne in mind that after the action of quinin the parasites at times can not be found in the blood ; further, that they are sometimes wanting after quite recent infection during the first days of the disease. Negative results from a single observation are, therefore, not conclusive, even when a large number of specimens have been examined. Repeated examinations must be made, preferably from three to ten hours in advance of the par- oxysm, when the parasites have reached the height of their development. In the determination of the variety of parasites, and with regard to the prognosis, it is especially important to decide whether crescents and their spindles or spheres are present. The characteristics of these have been described. The recognition of the species, as well as the decision whether several generations of the parasite are present, naturally demands continued observations, to be repeated in the course of several hours. The differentiation of free bodies moving about in the blood is attended with consider- able difficulty, and opportunity is here afforded for confusion from several sources. The parasitic nature of the bodies contained within the red blood-corpuscles is, however, more readily appreciated. The presence of pigment is here especially of significance. Only the unpigmented juvenile forms may occasion difficulty, as they may be readily mis- taken in living preparations for vacuoles of the red blood- corpuscles. The structure, which can not be overlooked, especially in stained preparations (nucleus), is decisive in this connection ; the vacuoles of the blood-discs are struc- tureless. Explanation of the Symptoms of Malaria by the Presence of the Parasites. — The parasites of malaria are present exclusively in the blood. Even in the viscera they are found only within the capillaries. The melanemia of malarial patients, which has long been known, is explained by the conversion of the hemoglobin into melanin within the parasites. The melanin is set free in the process of sporulation, when it floats about in the plasma and is taken up by the leukocytes. The infection of the blood-corpus- cles by the parasites furnishes a direct explanation for the anemia that arises in the course of malaria. The infected MALARIA. 385 blood-discs are destroyed in the process of development of the parasites. At the same time an injurious influence is exerted upon the uninfected blood-corpuscles by a parasitic poison dissolved in the blood-plasma. A chemic toxic action on the part of the parasites has, it is true, not yet been established. It is, however, rendered probable by the fact that the urine and the sweat of malarial patients, both of which secretions do not contain the parasite itself, are poisonous to rabbits, and are capable of causing death in these animals. The presence of a poison is also necessary for the explanation of the febrile attack. The paroxysm always sets in with sporulation — that is, with disintegration of the completely developed parasite. It is generally as- sumed that in this process, simultaneously with the spores, a poison is set free that is thrown into the blood and gives rise to the fever. Malaria would thus be a form of proto- zoan septicemia presenting analogies with ordinary bacte- rial septicemia. The toxic action would then without diffi- culty explain also the other symptoms — the diarrhea, the dyspnea, the ecchymoses, and especially the nervous symp- toms. The bone-pains are usually attributed to the increased demands upon the blood-forming activity of the bone- marrow, and comparable with those observed in leukemia. Coma may be induced by the occlusion of the cerebral vessels with the parasites themselves, and this has been demonstrated microscopically in a number of cases. Mode of Infection with Malaria. — Cultivation of the malarial parasites has thus far not been successful. Trans- mission of the disease to animals has likewise not been successful, although it has been attempted with numerous varieties. The only positive result that has been obtained 'in this connection consists in the survival for forty-eight hours of the parasites within the bodies of leeches that had been applied to malarial patients (Rosenbach). The malarial parasites are known only as blood-parasites of human beings. No conception has hitherto been possible as to where and in what form they exist outside of the human body. For this reason existing knowledge of the manner in which malarial infection takes place, and which is mainly based upon empiric observation, is not very ex- tensive. Malaria may be transmitted from one human being to another by means of the blood. Gerhardt was the first to demonstrate this by subcutaneous injections of 25 386 CLINICAL BACTERIOLOGY. malarial blood. Subsequently, infection was repeatedly- transmitted with the blood of malarial patients by subcuta- neous and intravenous injection. Malaria is, however, not a contagious disease. It does not pass from one human being to another under natural conditions. This could only be possible if the blood of a malarial patient gained en- trance into the body of another individualj and probably few opportunities for this are afforded. The parasites are not present in the secretions and excretions of malarial patients ; they appear to be present in the contents of the herpetic vesicles that malarial patients often exhibit. At least, malaria has been inoculated by means of the contents of such vesicles. The malarial parasites must, however, be present some- where in nature. They must live in some form in air, earth, or water, in certain swampy regions in which the disease is endemic, and at certain times becomes epidemic. The view is generally held that the parasites are inhaled ; some believe that they are taken up with the drinking-water. Transmission through the bites of insects is theoretically possible, and is admitted by some observers, but it is not yet demonstrated.* The period of incubation is in most cases from eight to fourteen days ; cases are, however, known in which the disease appeared months after infection, and others in which the period of incubation was only one or two days, or even hours. These differences may be explained by the num- ber of the infecting germs, and by the varying individual predisposition of those infected. That the germ is taken up in a form that must pass through certain variations within the body in order actually to become the malarial parasite is negatived by the cases with a short period of incubation. The Action of Quinin and Spontaneous Recovery from Malaria. — Laveran has determined that addition of even a very dilute solution of quinin to a blood-preparation at once causes cessation of the ameboid movements of the parasites. Within the human body, also, the parasites un- dergo visible alterations after administration of quinin : they suffer in motility and undergo degeneration, and, in many, sporulation does not take place in the normal manner. * Recent observations have demonstrated most conclusively that malaria is transmitted by some varieties of mosquitoes. — A. A. E. LEYDENIA GEMMIPARA SCHAUDINN. 387 According to Golgi, the spores are most susceptible to quinin, and the mature forms, before the beginning of the process of segmentation, are somewhat less so, and the en- doglobular, juvenile forms still less so. The crescentic bodies are considered as entirely insensitive to the action of quinin. The parasites altered by the action of quinin are designated as ** quinin-varieties." It is generally ac- cepted that quinin is a specific fatal poison for the malarial parasites (Binz). Quinin certainly exerts a curative effect, and, according to some observers, given in small doses, it is also of prophylactic utility. In explanation of spontaneous recovery from malaria Metschnikoff considers phagocytosis a most important factor. Probably this plays a not insignificant role, but other influences, besides, must certainly be taken into con- sideration. As has been mentioned, in every case of malaria a number of normally developed parasites regularly do not undergo sporulation. The remaining sterile elements are still visible in the blood for from twenty-four to forty- eight hours, when they undergo degeneration. Upon what this sterility depends is not known. It may, however, readily contribute to the process of recovery, should a considerable number of the parasites not undergo propa- gation. Further, the fever itself, as in the bacterial diseases, is believed to exert an injurious influence upon the parasites. LEYDENIA GEMMIPARA SCHAUDINN. In connection with the protozoa of dysentery and of malaria mention must be made of an observation by V. Leyden, who found an organism in the sterile ascites fluid obtained from two patients suffering from carcinoma. This organism is an ameba^ which the discoverer investi- gated in conjunction with the zoologist Schaudinn. It is spherical or irregular in shape, and from 3 to 36 // long. In hot weather it retains its motility for four or five hours with- out the employment of a warm stage. The ectoplasm sends out pseudopods, in whose formation the entoplasm also soon takes part. The latter possesses a honeycomb struc- ture, and contains yellowish, refractive granules, perhaps hemoglobin derived from the inclosure of red blood-cor- 388 . CLINICAL BACTERIOLOGY. puscles. Within the entoplasm of the processes vacuoles, food-residua, and excrementitious granules resembling crys- tals are also found. Among the vacuoles one exhibits pul- sation, undergoing contraction about every fifteen minutes. The nucleus is about one-fifth as large as the resting ameba. The pseudopods of adjacent bodies frequently unite, and there thus result plasmodia consisting of as many as forty segments. Multiplication takes place through division or budding. Leydenia belongs to the rhizopods. Von Leyden expresses himself with reserve in regard to its etiologic significance. APPENDIX. L BACTERIOLOGIC EXAMINATION OF SOIL, AIR, AND WATER. Although air and soil do not play the important role in the transmission of disease assigned to them by the path- ology of the past (pp. 169, 191), they nevertheless fre- quently act as media for the transmission of disease-germs, as has been shown in the preceding chapters. Bacterio- scopic examination of air and soil is frequently demanded of the hygienist when it is desired to determfne the avail- ability of ground for any public purpose. In individual instances such examination may become the duty of the phy- sician, when suspicion is aroused as to a relation between existing disease and soil or air. Of much greater signifi- cance in the development of the infectious diseases is water, concerning whose essential participation in the causation of epidemics reference has been made (pp. 170, 190). Bacteriologic examination of water often devolves upon- the physician. SOIL. Method of Investigation. — By means of a sterilized platinum spoon of known capacity (about -^^ cu. cm.) a specimen of the earth to be examined is taken up, and with this Esmarch gelatin roll-tubes are made, in which subse- quently the number of colonies that have developed are counted and their nature is determined with the aid of a microscope. Ordinary plates may, likewise, be prepared. In order that a portion of the fragments of earth shall not remain behind in the test-tube when poured out, and the result of the estimation be, thereby, rendered quite uncer- tain, especial care must be taken to secure uniform distribu- 389 390 CLINICAL BACTERIOLOGY. tion of the sample. To this end mortar and pestle are sterilized, and one gram of earth diluted with ten times the amount of sterile 0.6 per cent, solution of sodium chlorid is rubbed up. By means of a graduated platinum spiral a definite amount is removed, and with this gelatin-tubes (with three or four dilutions) are made m the usual man- ner. In order to obtain deeper layers of earth a special boring instrument (Fig. 84), devised by C. Frankel, is employed ; this can be opened at the desired depth by a rotatory movement, and, when it is filled, is again closed and removed. By these methods, however, informa- tion is gained only with regard to the aerobic bacteria present in the earth. As, however, anaerobic microorganisms occur in the soil, especially in manured garden-earth, in not inconsiderable num- ber, it is advisable in making bacterio- logic examinations of earth always to prepare also anaerobic plates. If it be desired to isolate a certain bacterium from the earth, animal experimentation is resorted to if the organism is patho- genic. If the bacterium sought pos- sesses resistant permanent forms, the sample of earth may be distributed in water and heated for a considerable length of time to between 80° C. (176° F.) and 90° C. (194° F.), and then plates are made. The Bacteria of the Soil. — The superficial layers of earth, even when uncultivated, contain large numbers of bacteria — about 100,000 to the cubic centimeter, and more. The further progress is made in depth the smaller the number of bacteria present in earth, and unless coarse gravel be present, germs will no longer be found at a depth of between ^ and i j^ meters, thus at the ground-water area. Porous soil filters the air, as well as liquids, in a perfectly reliable manner. This is naturally not the case in regions where fissures and breaks of any origin are present. Bacilli especially are found in earth. Of innocuous mi- Fig:. 84. — Frankel's instrument for obtain- ing earth from various depths for bacteriologic study. SOIL. 391 crobes, the hay -bacillus, the potato-bacillus, and the root- bacillus (p. 404) are cultivated from earth with great fre- quency. Upon the bacteria of earth devolves the function of disintegrating dead organic substances, and converting them into materials from which new organic substances (plants) are formed. The carbon of organic substances is trans- formed into carbon dioxid ; the nitrogen, into ammonia ; and the hydrogen, into water. Certain bacteria of the earth oxi- dize the ammonia into nitrites (ferments nitreux, nitroso-bac- teria) ; others, the nitrites into nitrates (ferments nitriques, nitro-bacteria) ; the entire procedure of the transformation of organic nitrogen into nitric acid is designated nitrification. This transformation-process, which is brought about by the bacterial flora of the earth, is absolutely indispensable to the vegetable world. If plants are placed in soil that con- tains all necessary nutritive elements, but has been sterilized artificially — that is, rendered bacteria-free — they will de- velop but incompletely, and soon begin to die (Duclaux). It must be pointed out that these nitrifying bacteria develop also in cultures that contain no trace of organic carbon- compounds, and that they, therefore, obtain their carbon- requirement directly from the carbon dioxid, without the aid of chlorophyl and light. In the superficial layers of the earth, in addition to the bacilli, vcvaxvy permanent spores are present, which in part possess extraordinary resistance, and withstand for four or five hours the action of live steam. Favorable conditions for spore -formation must, therefore, be present in the super- ficial layers of the soil. Perhaps the circumstance is of significance that the individual particles of earth are sur- rounded by a capillary zone of fluid, and are thus protected from drying. It has been shown that anthrax-bacilli in cultures mixed with porous particles of earth undergo- sporulation much more quickly and actively than otherwise.. Anthrax-spores retain their infectivity for years in earth in: which dissected animal carcases have been buried. Of pathogenic bacteria, cultivated and manured earth fre- quently contains, besides, the bacillus of tetanus and that of malignant edema. The malarial parasites, likewise, must reside in the earth in certain localities ; at least, numerous clinical facts support such a view. Their demonstration therein has, however, as yet not been successful. Other infectious agents have 392 CLINICAL BACTERIOLOGY. up to the present, been found in the earth only when but a short time has elapsed after the bacteria in question have been introduced at the given point with the disease-pro- ducts (cholera or typhoid dejections, etc.). Typhoid-bacilli deposited at a depth of J^ meter retain their capability of development, under favorable conditions, for five and a half months, and perhaps still longer. On the whole, it may be stated that pathogenic germs may multiply upon and within the surface of the earth if the atmospheric temperature be high ; but that, as a rule, they are suppressed by the competition of saprophytes. In the deeper layer the conditions for their propagation are much less favorable. The burial of the bodies of individuals that have died from infectious diseases is scarcely capable of giving rise to infection. The accompanying bacteria are overrun by the saprophytes, and even if a small number — ^^as, for instance, of tubercle-bacilli — may persist for months, perhaps for years, the opportunity is, on the whole, seldom afforded for their being carried from a depth to the surface of the earth or into the subsoil water. AIR. Method of Examination. — /. Procedure of Hesse. — A glass tube 70 cm. long and 3.5 cm. in diameter is sterilized and coated with gelatin, which, in the same way as in Esmarch's tubes, is distributed uniformly upon the inner surface by rotation in cool water. By means of an aspirator air is then drawn 'through this tube at the rate of a liter in from two to four minutes. At this slow rate of speed the germs of the air are deposited upon the gelatin, in which they subsequently develop into colonies. This method permits of examination of only relatively small quantities of air (from 10 to 20 liters). 2. Procedure of Petri. — A sand-filter, 3 cm. thick, sup- ported upon two wire nets, is fastened in a short, glass tube, with a diameter of from 1.5 to 2 cm. ; the whole is steril- ized, and air is permitted to stream through in a rapid cur- rent. The sand, which should have a grain of from ^ to ^ mm. in size, filters with certainty all of the germs con- tained in the air. After from 50 to 100 liters of air have been drawn through the apparatus, the entire filter is intro- AIR. 393 duced into liquefied gelatin or agar, and plates are made. The amount of air is measured by means of a gas-meter. 3. If a precise quantitative result is not desired, gelatin- plates may simply be exposed to the air for a certain period of time, and the germs deposited may be permitted to de- velop into colonies. Bacteria of the Air. — One cubic meter of air contains, on the average, from 500 to 1000 germs, including from 100 to 200 bacteria. Molds constitute by far the majority of the germs ; next in frequency are yeast-fungi, and last, Fig. 85.— Hesse's apparatus for collecting bacteria from the air. Fig. 86.— Petri's sand-filter for air-examination. bacteria, which are usually represented only by micrococci and sarcinae. The number of germs present in the air is in marked degree dependent upon local and temporal variations. In inhabited localities, where dust is being constantly created and blown about, the air contains a larger number of micro- organisms than in deserts. On uninhabited mountains the air is almost entirely free from germs, as it is also at sea. At a short distance from land sea-air contains germs carried thither from the land by currents of air. After a heavy 394 CLINICAL BACTERIOLOGY. rainfall and during the winter the number of germs in the air is considerably diminished. In the still air of rooms comparatively few germs are found, even in densely occu- pied quarters — as, for instance, in hospital-wards. As soon, however, as dust is set in motion, the number of microorganisms in the air increases enormously — to as many as i6,ooo in the cubic meter. The majority of the bacteria, however, by reason of their weight, are soon again, after from half an hour to an hour, deposited upon the floor and the walls with the dust, and the air will then contain almost only the light mold-spores. Sources of Aerial Germs (Air-infection). — The pres- ence of spores of molds in the air in such large number is explained by the fact that generally the fruit-bearers of the fungous deposits project upward from the mycelium, and the spores can, therefore, be readily conveyed by currents of air. Bacteria frequently find their way into the air with the small fibers separated from clothing, handkerchiefs, linen, etc. With regard to the possibility of air-infection, recent in- vestigations by Fliigge and his pupils have yielded a wealth of facts that are well adapted to modify the views hitherto held. The earlier supposition that no germs enter the air from liquids or from moist surfaces is, according to Fliigge, valid only in so far as the surface of the fluid remains un- moved and intact. A moderate wind (a velocity of four meters in a second) suffices, however, to set free germ- containing small drops from water-surfaces, moist articles, and the like. The conditions favoring the conversion of fluids into small drops in the air are often present, as in the open air, in a high degree in the neighborhood of the surging sea, of water-mills, etc., in lesser degree with every current of air that sets the leaves of trees in motion. In closed apartments, according to * Fliigge, small drops that enter the air are set free more frequently than is generally believed in sprinkling, in washing, in manipulating wet linen, and — upon which especial weight must be placed — in speaking, sneezing, and coughing. These droplets, whether they contain germs or not, are, as Fliigge further demonstrated, carried away by the slightest currents of air for considerable distances, and not only in a horizontal, but also in a vertical direction. In order to demonstrate the AIR. 395 dissemination of such droplets in speaking, coughing, and sneezing, Fliigge had the person under observation intro- duce a suspension of prodigiosus into the mouth. All the observations yielded positive results : The plates exposed at a distance of several meters became covered by char- acteristic colonies, and they remained sterile only after quiet speaking in a low tone. With regard to the separation of desiccated germs, it may be said that this may likewise occur frequently. Fliigge found that the movement of the finest dust already begins with a current of air having a velocity of one meter. In the open air the most varied mechanical influences (wagon- wheels, pedestrians) cause the separation of the smallest particles, which are then readily borne by the wind. In rooms particles of dust and fibers are set free by the vibra- tion of the floor, by manipulation of utensils, furniture, clothing, etc. The smallest of these floating particles con- taining germs are, however, as Fliigge has shown, moved in a horizontal direction by minimal currents of air of 0.2 mm., and in a vertical direction by currents of from 0.3 to 0.4 mm. per second. In the course of his experiments Fliigge succeeded with the prodigiosus in demonstrating the distribution of dust-germs in all possible parts of a closed room. Germ-containing dust is not completely removed from rough surfaces even by the strongest cur- rents of air. Airing of infected clothing, as frequently practised, does not, therefore, entirely attain the object desired. Upon the basis of the results of these observations Fliigge expresses the view that in the case of all infectious diseases air-infection may take place through the smallest droplets separated from fluid sources of infection. In cholera and typhoid fever this will occur but rarely — for in- stance, in the sprinkling of polluted water, in the washing of linen, and the like. The principal role in these affections is played by contact-infection, the dissemination through drinking-water, articles of food, etc. In the infectious dis- eases of the nose, the pharynx, and the respiratory organs, however, the possibility of transmission through small drops must be given greater consideration than it has re- ceived in the past. In diphtheria, influenza, whooping- cough, pneumonia, and pulmonary tuberculosis, droplets containing germs are sent into the air in coughing, 396 CLINICAL BACTERIOLOGY. sneezing, and loud talking ; they are set in motion by min- imal currents of air, and in a quiet room can be demon- strated floating in the air for as long as five hours. In cases of air-infection through dry particles of dust, only the finest particles, capable of being carried by any movement of air, demand consideration. Only such diseases are transmitted naturally in this way whose exciting agents are still capable of surviving in the dry state. For the acute exanthemata, which have always been considered as diseases due to volatile contagia, such a possibility must be accepted. Pulmonary tuberculosis has hitherto been generally accepted as disseminated through inhalation of ejected dried and pulverized sputum containing tubercle-bacilli ; Fliigge opposes this view. Attempts to infect animals through inhalation of dried tuberculous sputum have never quite succeeded ; and, further, the tubercle-bacilli do not appear at all capable of being carried by the finest particles of dust. Fliigge expresses a warning against going to the other extreme of attaching too great importance to the danger of infection through drops of spray or even of con- sidering it the only source of infection. Infection with tuberculosis is dependent upon a large number of factors : upon the environment of the patient, upon the presence of tubercle-bacilli in the saliva, which forms fine droplets much more readily than the viscid sputum proper, and upon other conditions. WATER. Method of Examination. — The water to be examined is collected in sterilized Erlenmeyer flasks, and is investi- gated as speedily as possible. If the examination is deferred for only a few hours, the indifferent saprophytic bacteria that reside in water will have undergone multiplication, and the estimation of the number of water-germs will not yield reliable results. If the specimens of water to be examined are sent from a distance, the water must be forwarded in sterilized flasks, provided with glass stoppers, and packed in ice. In obtaining water for examination care should be taken that the material to be examined has not stagnated in the conduit from the well or spring or other source of supply. For this reason a certain amount of water is first WATER. 397 permitted to escape. Of the specimen obtained, i, ^, and i^ cu. cm. are removed by means of sterilized pipets, and introduced into liquefied gelatin, and plates are made. Greatly polluted water should be diluted with sterilized water from another source, and no more than -^, yi^, and Y^-Q-Q CU. cm. should be employed. Before introducing the pipets the specimen is vigorously agitated, as the bacteria, by reason of their weight, quickly settle to the bottom. In the process of water-examination Koch's old method of making plates is .still frequently followed, because these permit better than the Petri dishes the distribution of the gelatin in as uniform a layer as possible, \yhen the plates have developed, the colonies are examined and counted, if present in large number by means of a special enumerator (a glass plate with etched squares). An estimate is made of the number of germs to the cubic centimeter of water, and the number as well as the identity of the varieties present is determined. The Number of Bacteria Present in Water. — Spring- water, as well as subsoil-water, is free from germs at the point where it escapes from the earth. It has been men- tioned that the earth at the level of the subsoil-water no longer contains bacteria. According to the most trust- worthy investigations, pure tap-water and spring-water con- tain, on the average, from 2 to 50 bacteria per cubic centi- meter ; pure pump-water, from lOO or 200 to 500 germs ; unfiltered water from streams kept unpolluted, from 6000 to 20,000 ; filtered river-water, from 50 to 200 ; polluted wells, as many as 100,000 germs ; and river tap-water, when the filtering apparatus is out of order, the same num- ber. Drain-water and greatly contaminated streams contain from 2,000,000 to 40,000,000 germs to the cubic centimeter (Fliigge). In the summer and after a heavy rainfall the number of bacteria in water is increased. The Bacteria of Water.— The microorganisms living in water are mainly bacilli. A large number of them liquefy gelatin ; others generate offensive gases, and still others beautiful pigments. Of great interest are the so-called typhoid-like water-bacteria (p. 402) and the water-vibrios, which in a number of points resemble the comma-bacilli of Asiatic cholera (p. 404). Among pathogenic bacteria typhoid-bacilli and cholera- vibrios have been repeatedly found in water. The method 398 CLINICAL BACTERIOLOGY. for cultivating these bacteria from water has been consid- ered in the discussion of typhoid fever (p. 178) and of cholera (p. 195). At this point it may be briefly repeated that by the addition of one per cent, peptone and ^ per cent, sodium chlorid, the water to be examined itself constitutes a nutrient medium, so that in this way large quan- tities of the fluid can be employed, whereas in earlier investigations a fraction of a drop had to suffice, and, as a result, the pathogenic germs were readily overlooked. The saprophytic water-bacteria multiply in water to an unlimited degree. For the pathogenic bacteria, however, while the possibility of preservation often exists, but rarely is opportunity for proliferation in water afforded. To this end there are necessary a favorable external temperature (summer) and, further, solid particles of vegetable or ani- mal origin, to which the bacteria adhere, which serve as nutrient medium, and which, at the same time, afford the bacteria protection from the competition of saprophytes. The exciting agents of typhoid fever and of cholera appear to survive in ordinary water for days, and even for weeks. Some observers even believe that cholera-vibrios may at first undergo multiplication in water. As a rule, however, the pathogenic germs are suppressed sooner or later in water in consequence of overgrowth by the saprophytes. Self-purification of Water. — The microorganisms pres- ent in water are derived from the surface of the earth, the air, from waste water and the sewage of cities, emptying into the water-courses, from cesspools communicating with imperfectly constructed subsoil-wells, etc. The pollution of streams by cities is a most prominent factor ; the Seine at Ivry, for instance, contains 32,500 germs to the cubic cen- timeter ; below Paris, in Asnieres, the number is 12,800,000. Fortunately, if renewed contamination does not take place, streams purify themselves (Pettenkofer). The microor- ganisms settle and are carried to the bottom, partly with the constituents suspended in the water and with the in- soluble earthy combinations that form from calcium and magnesium bicarbonates after escape of the carbon dioxid. Light also in a high degree exerts an injurious influence upon the microorganisms present in water down to a depth of about two meters. The organic substances con- tained in water are gradually consumed by bacteria and algae. WATER. 399 Sources and Mode of Water-supply. — In the practical application of bacteriologic examination of water for the construction of conduits, wells, etc., it is, in the first place, important to determine whether the water used contains pathogenic bacteria or not. If a given water contains many putrefactive bacteria (proteus) or the bacterium coli commune, it should be entirely excluded from use ; for such conditions indicate with certainty that the source of supply is polluted. Formerly, great weight was attached to the number of germs present. Some difficulty was en- countered in this connection in the establishment of a limit, beyond which water should no longer be considered pure. Some observers made this limit one hundred, others fifty, and still others five hundred. The absolute number of germs contained in a given water depends, however, upon such varied factors that an opinion based solely upon the number of colonies formed is not to be depended upon. It was then proposed to attach the greatest importance to the number of different varieties present. A large number of different species, it was thought, would, to a certain degree, be an indication of the greater probability of the occur- rence of contamination of the water. This belief is not without justification, but too much significance must not be attached to the number of different varieties of bacteria in water. The fact can not be evaded that bacteriology has thus far not attained the importance in the hygienic deter- mination of the usefulness of water that was originally attributed to it. As a central source of water-supply for large communi- ties bacteria-free spring-water or subsoil-water is most to be recommended, when this is obtainable in at all sufficient quan- tities. In the selection of a source of supply care should be taken to avoid undue proximity to communities or manured land. If spring-water or subsoil-water is not available, there is no alternative but to employ river-water or lake-water, which naturally must be obtained from a point above the city to be supplied. Such water is, as has been pointed out, exposed to numerous sources of contami- nation, and must, therefore, unconditionally be subjected to some process o^ filtration before being used. This maybe effected by means of sandfilti'ation, which is carried out in large, cemented, covered reservoirs. The utility of sand-fil- ters has been studied in recent years with especial care by 400 CLINICAL BACTERIOLOGY. C. Frankel and Pief ke. At the bottom of a filtering basin large cobblestones should be placed to a height of 305 mm., upon these a layer of small cobblestones, 102 mm. high, then 76 mm. of coarse gravel, 127 mm. gravel of medium size, upon this 51 mm. of coarse sand, and, finally, 559 mm. of fine sand. Filtering properties reside solely in the layer of sand. Before the process of filtration is begun the reservoir must be permitted to remain filled with water for twenty-four hours. As a result, there will form a coating of sediment, and a slimy covering for the pores of the filter, which constitute the essential factors in the purifica- tion of the water. The rate of filtration should not ex- ceed 100 mm. an hour. The form of filter described does not yield perfectly sterile water, but this will be found to contain between 50 and 200 germs in the cubic centimeter, which it must be noted are derived in largest part from the lower layers of the filter. The mechanism can be disturbed by tears in the cover of the filter, when the filtration- pressure, which will increase with increasing sliminess of the filter, becomes too great. The filter must, therefore, be cleansed from time to time. The working of the filter altogether demands unremitting attention. The water of every filter must be examined bacteriologically every day, and as soon as more than 100 bacteria are contained in the cubic centimeter, use of the water should not be permitted, and the filter should be renewed. Of house -jilt ei's for the purification of water for domestic use numerous varieties have been recommended. Of all not one properly serves its purpose, as they, are soon sat- urated with bacteria. For domestic purposes suspected water is rendered harmless in the simplest manner by boil- ing for five minutes (after the vapor of steam has arisen). Of wells the best as a source of water-supply are of the tubular variety, in which an iron tube leads to the subsoil- water area, and thus furnishes germ-free water. Ice.- — Natural ice, which is obtained in winter from streams and ponds, contains numerous bacteria — on an average 2000 to the cubic centimeter in ice-water, with a minimum of 50 and a maximum of 25,000 germs. Quite a number of bacteria, among them the cholera-vibrios, offer considerable resistance to freezing. Some even are capable of multiplication at this temperature (p. 22). Artificial ice prepared from distilled water contains from none to 10 BACILLI IN WATER. 401 germs in the cubic centimeter of ice- water. Distilled water itself, after standing for some time, contains many water- bacteria, but these are among those that do not bear freezing. Artificial carbonated waters are often rich in bacteria, even after standing for months, and it has been shown ex- perimentally that typhoid-bacilli, for instance, may survive for from days to weeks in such water. Care, therefore, should be taken to prepare artificial waters only from pure drinking-water or from distilled water. THE BACTERIA PRINCIPALLY FOUND IN SOIL, AIR, AND WATER* L BACILLL I. NOT LIQUEFYING GELATIN. (a) Chromogenic. 1. Bacillus Aurantiacus . — This is a short, plump rod, with slight spontaneous movement. Upon plates it appears in the form of an orange-colored, knob-like deposit. In gelatin stab- cultures it exhibits a glistening, orange-colored growth. Its appearance in bouillon is characteristic. The fluid itself remains clear, while upon the surface a membrane forms, presenting a small number of orange-colored spots, and at the bottom a somewhat lighter sediment collects. 2. Bacillus Constrictus. — This organism derives its name from the peculiar appearance it presents when stained by the method of Zimmermann. The rods exhibit a slight constriction between the individual segments, which are united into short chains, and they resemble biscuits in shape. In plates the colo- nies present the appearance of granular discs, with eroded mar- gins. The color is between yellowish-gray and light sulphur- yellow. J. Bacillus Fluorescens Nonliquefaciens. — This is a delicate, short, actively motile rod. Upon gelatin the colonies present a peculiar mother-of-pearl luster, which also exhibits fluores- cence. Upon agar-agar the growth acquires a greenish tint. A subvariety has been described as bacillus fiuorescens nonliquefa- ciens immobilis, which is distinguished by the absence of motility and of flagella. 4. Bacillus Fuscus. — This is a medium-sized rod, sometimes curved, which takes its name from the dark-brown pigment to 26 402 CLINICAL BACTERIOLOGY. which it gives rise in all nutrient media. In gelatin stab- cultures a nail-shaped growth develops at first, the head subse- quently extending. 5. Bacillus Rubefaciens. — This is a fine rod, consisting of two or more segments. Gelatin-cultures exhibit a pale rose-red color. Upon potatoes the substratum appears of a rose-red color, while the colony itself is between yellowish -gray and brownish-red. 6. Bacillus Subflavus. — Cultures of this organism give rise to a pale-red pigment; upon plates they exhibit a mother-of- pearl luster. The pigmentation is most distinct in agar-agar cultures. Several bacilli often lie attached to one another. The individual organism is from two to four times as long as it is thick. 7. Bacillus Brunneus. — The colonies of this small, nonmotile bacterium are characterized by diffusing a brownish pigment into the surrounding culture-medium. (b) Nonchromogenic. 8. Ty phoid- like , Bacilli (^Weichselbaum) . — Under this desig- nation is included a group of motile bacilli that resemble the bacillus of Eberth-Gaffky and the bacterium coli commune in both morphologic and cultural properties. On plates the colonies present easily the appearances of those of the typhoid- bacillus and the colon-bacillus (pp. 167, 120). Upon potatoes a deposit forms, at times brownish, at other times yellowish, at still other times scarcely visible. Coagulation is induced in milk. Some varieties cause fermentation of grape-sugar, while others do not. The nitroso-indol reaction is positive with some, and negative with others. These bacteria are free from all pathogenic activity in experiments on animals. That this group is constituted of a series of bacteria differing among themselves is demonstrated by the fact that any one variety is incapable of conferring immunity to any other. The attempt, also, to cause agglutination in the cultures of one variety with the blood- serum of animals that have been immunized to another variety has invariably failed. 2. LIQUEFYING GELATIN. (a) Chromogenic. 9. Bacillus Arborescens. — This is a slender bacillus that fre- quently forms wavy filaments. It is incapable of spontaneous movement, and it is characterized by branch-like ramifications in gelatin plate-cultures, and by iridescence of its colonies. It gives rise to a yellowish or yellowish-red pigment, especially upon potatoes. 10. Bacillus Fluorescens Liquefaciens . — This is a motile BACILLI IN WATER. 403 organism that closely resembles the bacillus pyocyaneus. It liquefies gelatin rapidly, with the formation of a greenish-yellow pigment, which is vividly fluorescent. Typical cultures form upon glycerin-agar, which acquirer from an olive-green to a dark olive-brown color. 11. Bacillus Rubidus. — This is a medium-sized rod, actively motile, and arranged in threads of considerable length. It generates a brownish-red pigment, both in gelatin and in agar, and also in potato-cultures. Other than pigment-formation it presents scarcely anything else characteristic. 12. Bacillus Violaceus. — This is a small, slender, actively motile rod, that upon agar forms central spores. In its growth upon gelatin-plates a bluish-violet bacterial mass appears in the liquefied culture-medium. Upon agar-agar and potatoes the formation of pigment is intense, and the color is dark violet, almost black. I J. Bacillus Viscosus. — This organism closely resembles the bacillus fluorescens liquefaciens, from which, however, it is differentiated by the formation of a chocolate-colored coating. 14. Bacillus lanthinus. — This is a motile bacillus of medium size. Its appearance in growth upon gelatin-plates is usually compared with the appearance of a drop of ink that has fallen upon them. It forms a violet pigment in all nutrient media. i^. Bacillus Helvolus. — This appears in the form of motile rods of varying length, which are oftea united into short threads. These generate from a yellowish to a sulphur-yellow pigment. Upon plates the colonies appear as circular, bright-yellow discs that lie in a funnel of liquefaction. Upon agar an abundant deposit of intensely yellow hue forms. 16. Bacillus Prodigiosus. — This is a small rod (formerly designated micrococcus prodigiosus or monas prodigiosa), often collected in small chains, of slight motility, occurring not rarely in the air, less commonly in water, rather frequently upon amy- laceous nutrient media (bread, potatoes), upon meat, and in milk. It grows upon all nutrient media, with the development of a bright-red pigment ; this is most intense on potatoes, which present a blood-red coating. Upon gelatin-plates rather deep, small, white dots appear, and . also superficial, roundish, red colonies with an irregular border. Gelatin is energetically liquefied. Upon agar-agar a coating of moderately dark-red color appears, while the nutrient medium itself is not discolored. On cultivation at a temperature of 37° C. (98.6° F. ) the pro- digiosus loses its red color in the course of several generations. In cultures, especially upon potato, in addition to the red pigment, trimethylamin forms (odor of pickled herring). Coagulation is induced in milk. Saccharine nutrient media undergo fermen- tation. The prodigiosus will thrive also in the absence of oxy- 404 CLINICAL BACTERIOLOGY. gen, but then without the development of red pigment. It is somewhat pathogenic, inoculated animals dying with toxic symptoms after introduction of large amounts. The name pro- digiosus is derived from the fact that the bloody appearance of the so-called miraculous holy wafers is attributed to infection with this microorganism. 17. Among other pigment-producing bacteria characterized only by the color of their cultures may be mentioned the bacillus ruber balticus, bacillus ruber aquatilis, bacillus coeruleuSy bacillus pavoninus, bacillus amethystinus. (b) Nonchromogenic. 18. Bacillus Liquefaciens . — This is one of the most wide- spread of the water-bacilli. It is an actively motile rod, often joined in short chains of four or more segments. It rapidly liquefies gelatin. In plates it assumes the form of a dish, upon the base of which lies a gray, bacterial mass. In stab-cultures the growth assumes the form of a stocking, with a dilated upper portion. The odor of the culture is highly disagreeable. The bacillus exhibits facultative anaerobiosis. In nutrient media containing nitrates it generates nitrous acid. The following likewise are included among water-bacteria : zp. Bacillus Liquidis. — This is a short, plump, slightly motile bacillus, which also rapidly liquefies gelatin. In tubes the gray, liquefied gelatin becomes covered with a thin membrane, which sinks to the bottom on agitation. 20. Bacillus A quatilis. — This is a slender rod, with spontane- ous motility. It liquefies gelatin slowly, and according to some observers not at all. It grows in gelatin upon the surface in the form of small, yellowish colonies, and upon potatoes with a scanty yellow deposit. In the earth and in certain articles of food the following bacteria are always to be found, which are characterized by especial resistance of their spores : 2j. Root-bacillus. — This is a large, thick bacillus, with rounded extremities, and possessing slight motility. It develops central spores, and growth takes place only in the presence of oxygen. The whitish-gray colonies that form consist of a network of fine, interlacing threads. They liquefy gelatin. In stab-cultures also filaments and processes form, and an appearance results re- sembling an inverted fir-tree. Upon agar-agar a network forms suggesting the ramifications of the roots of a tree. 22. Bacillus Subtilis {^Hay -bacillus'). — This is a large, deli- cate bacillus that often develops into long, straight threads. The bacillus subtilis is a strictly aerobic organism, and it quickly liquefies gelatin. The temperature-optimum is 30° C. (86° F.); the temperature-minimum, 10° C. (50° F.); the temperature- maximum, 45° C. (113° F.). Upon plates the bright, grayish- MICROCOCCI IN WATER. 405 white colony appears surrounded by a sparkling crown. Upon agar-agar the growth is peculiar, a stiff, wrinkled, readily detached deposit forming. The hay-bacillus forms central spores, which are somewhat thicker, but considerably shorter than the mother-cells. It is found in the air, water, dust, feces, hay, etc. In order to obtain the organism in pure culture hay is. cut up into small pieces, which are covered with water in an Erlenmeyer flask, and exposed to a boiling temperature for fifteen minutes. In this way all of the germs are destroyed, with the exception of the resistant spores of the hay-bacillus. These then grow, and after two or three days they form a super- ficial membrane upon the hay-infusion. 2j. Potato-bacillus (^Bacillus Mesentericus). — Three varieties of this microorganism are distinguished : bacillus mesentericus vulgatus, fuscus, and ruber. The last, especially, which im- parts a rose tint to potatoes upon which it grows, possesses per- manent forms of extraordinary resistance, which withstand boiling for from five to six hours. In proportion to the cell the spore is quite large. The cultural peculiarities of the organism resemble those of the hay-bacillus. Upon potatoes the bacillus gives rise to a wrinkled coating. Milk is coagulated and pep- tonized. 24. Bacillus Spinosus. — This is a strictly anaerobic, motile rod. Its colonies in gelatin form iridescent globules, with thorn- like processes. Gelatin is liquefied, with the formation of gas. Stab-cultures, before liquefaction takes place, present the appear- ance of a prickly caterpillar (Liideritz). The bacillus spinosus grows both at room-temperature and at the temperature of the body. It forms central spores, the rod becoming at the same time spindle-shaped (Clostridium). The bacillus is usually found in garden -earth. n, MICROCOCCL I. NOT LIQUEFYING GELATIN. (a) Chromogenic. 25. Micrococcus Aurantiacus. — This is a round or oval coccus, arranged in groups. The cultures are yellow, slimy, knob- shaped, and they do not extend greatly in width. 26. Micrococcus Versicolor. — This is a small coccus, arranged in groups or in pairs. It occurs with extraordinary frequency in the air. The colonies are irregular in shape, with a yellowish- green color. They exhibit, especially upon gelatin, a mother- of-pearl iridescence, and they cause fermentation in nutrient media containing glucose. 406 CLINICAL BACTERIOLOGY. (b) Nonchromogenic. 27. Micrococcus Candicans. — This is a round coccus of mod- erate size. Its most distinctive feature is its growth in gelatin stab-cultures, in which a nail-shaped growth appears, with a porcelain- white, glistening head. 28. Micrococcus Concentricus. — This is characterized by the concentric extension of its colonies upon gelatin-plates and in stab-culture. The colonies present from a whitish-gray to a bluish-gray color, and are superficially serrated. The cocci themselves are small and are arranged like grapes. 2p. Micrococcus Rosettaceus. — This is a coccus of moderate size. Its growth is mainly superficial. Roset-like deposits form, with irregular margins. The colonies are grayish-white in color, but darker in the center, up to brown. 30. Micrococcus Aquatilis. — The colonies are round, and possess a mother-of-pearl luster. The margins appear serrated ; the color is light gray. Viewed with low powers of the micro- scope, the colony appears in the form of a berry. 2. LIQUEFYING GELATIN. (a) Chromogenic. 31. Micrococcus Cremoides. — This is a small coccus, arranged in groups, and giving rise to a cream- colored pigment. At first, the colonies upon gelatin are from yellowish- white to brownish- gray, granular, circular ; while later the discs appear eroded, and they lie in a liquefied excavation. 32. Sarcina Lutea {Yellow Sarcind). — This coccus, strictly aerobic, is arranged in so-called balls of twine. Upon gelatin- plates it forms rounded, slightly granular, S^^ ^% yellow colonies. In stab-cultures marked aD0#» fiSft superficial growth takes place. The cul- ••''^A ^^ ffl» tures generate a citron -yellow pigment. «flvft^A «^ ffl^ Liquefaction occurs quite late ; then the So^^ ^^ £S clear, liquefied gelatin overlies the citron- yellow precipitate. In addition to the yel- Fig. 87.-Sar_dn^^; X 600 j^^^ garciua there are also white, orange, and red sarcinse, which are distinguished from that described only by the difference in color. The varieties of sarcinae are present in the air. jj. Micrococcus Agilis {AH Cohen). — This actively motile organism (flagella), cultivated from drinking-water, grows upon all nutrient media, with the formation of a rose-red pigment. It slowly liquefies gelatin, (b) Nonchromogenic. J4. Micrococcus Radiatus. — This is a small coccus, without typical arrangement. Upon plates it forms colonies surrounded VIBRIOS IN WATER. 407 by a sparkling crown. In stab-cultures the colonies, likewise, display horizontal radiation. Gelatin is slowly liquefied. m. VIBRIOS. Since the great epidemic of cholera at Hamburg in the year 1892, a large number of vibrios more or less closely resembling that of cholera have been described, which have been cultivated in part from river-water, in part from other sources, and of which the most important will be mentioned. jj'. Vibrio Aquatilis Gilnther. — This organism is scarcely to' be confounded with the comma-bacillus, on account of its cir- cular, finely granular colonies, with smooth borders, even inde- Fig. 88. — Spirillum aquatilis, from an agar-agar culture ; X 1000 (Itzerott and Niemann). pendently of the absence of the cholera-red reaction. It at first grew feebly in liquid nutrient media, but as a result of re- peated inoculation it acquired, in consequence of adaptation to such media, the property of growing upon bouillon and peptone- water. 36. Vibrio Berolinensis. — This organism, which was cultivated by Neisser (1893) from the water-supply of Berlin, bears a close resemblance to the cholera-bacillus in regard to form and flagella. Upon gelatin-plates the border of the colonies is, however, mostly smooth. The colonies themselves exhibit a much more finely granular appearance than those of the comma- bacillus. Gelatin is slowly liquefied, and the cholera-red reac- 408 CLINICAL BACTERIOLOGY. tion appears. Guinea-pigs died after intraperitoneal injection, with precisely the same symptom-complex as appeared after introduction of true comma-bacilli. Similar vibrios have been cultivated from Munich well-water by Weibel; from Peene water, by Loffler ; from the harbor of Groningen, by Fokker ; and from the harbor of Altona, by Kiesling ; and, further, from the Seine. In forming an opinion as to the identity of these and the following vibrios with the true exciting agents of Asiatic cholera, Pfeiffer's reaction (p. 187) and the agglutination-test must be given preeminent importance. Both tests yield nega- tive results. J/. Vibrio Metschnikoff. — This was first cultivated in an epi- demic among fowl, and subsequently from the water of the Spree. :^ Fig. 89.— Spirillum berolinensis, from an agar-agar culture ; X looo (Itzerott and Niemann). It is somewhat thicker and shorter than the cholera-vibrio, and sometimes almost coccus-shaped. In hanging drop it exhibits active motility. The cultures resemble those of the cholera- bacillus, except that liquefaction is more marked. The nitroso- indol reaction appears within twenty-four hours. This vibrio, in contradistinction from that of cholera, is pathogenic for both pigeons and guinea-pigs. j<5*. Vibrio Gindha. — This organism was cultivated by von Pasquale from well-water in Gindha near Massowah. It is a rather long, slightly curved rod, actively motile (with a single terminal flagellum). It is but slightly pathogenic, and it does not yield the nitroso-indol reaction. jp. Vibrio Lissabon. — This has been cultivated in the course VIBRIOS IN WATER. 409 of an extensive epidemic of cholerine in Lisbon, in which, however, but a single death occurred. On gelatin-plates cir- Fig. 90. — Spirillum Metschnikoff, from an agar-agar culture ; X 1000 (Itzerott and Niemann). Fig. 91. — Spirillum Metschnikoff; stab-culture in gelatin forty-eight hours old (Frankel and Pfeiffer). cular, sharply circumscribed, whitish-yellow colonies form that cause little liquefaction. The culture does not yield the nitroso- indol reaction. 410 CLINICAL BACTERIOLOGY. 40. Vibrio Phosphorescens Dunbar. — This may be con- founded with the cholera-bacillus on account of its morphologic and cultural appearances, but is distinguishable by its phos- phorescence. 41. Vibrio Massowah. — This possesses from two to four flagella, whereas the comma-bacillus possesses but a single ter- minal flagellum. It yields the nitroso-indol reaction, and is quite pathogenic for pigeons, guinea-pigs, and rabbits. /' V, , .•*.-'•> T- ^ .^' \.-r%''^ ^ \.\ :i.>.'rv.^ V ; -. ""■^-.;*_ * Fig. 92.- —Spirillum Dunbar, from agar-agar; X 1000 (Itzerott and Niemann), n. DISINFECTION. To afford protection against the importation of epidemics, which mostly threaten Europe from the Orient, resort is had to supervision of maritime intercourse by means of guarantiite-stations, the International Sanitary Bureau, and other official arrangements. General hygienic efforts also, through drainage of the soil, through the provision of a pure water-supply, through care for healthful dwellings, etc., to render the sanitary conditions of the country during periods free from pestilence such that epidemics, when imported, do not find the soil upon which they thrive, must be made by the public authorities. The practis- ing physician can cooperate in these to only a slight degree. The conditions, however, are different when once the epi- demic has been introduced ; then the relations of the phy- sician with regard to it are identical with those that exist DISINFECTION. 411 with regard to endemic diseases, such as typhoid fever, whooping-cough, tuberculosis, etc. These also may attain epidemic distribution. The duty of notification, which de- volves upon the physician, renders it possible for the authorities to have cognizance of every extraordinary in- crease in the prevalence of any infectious disease, and to recognize the epidemic at the outset, to trace its causes and eventually to remove them. An important part of the prophylactic measures against disease that already prevails lies within the control of the physician. He should en- deavor to prevent the increase of an endemic disease to the proportions of an epidemic, and, when the disease is intro- duced from without, to prevent its further extension by suppressing the source of infection, by rendering the indi- vidual case harmless. Disinfection, the destruction of dis- ease-germs, with which every case of disease threatens its immediate and remote environment, is an integral element of all disease-prophylaxis. In the preceding chapters it has been pointed out in de- tail how the germs of disease pass over into the secretions and excretions, how they adhere to beds, to linen, and to sick-rooms, etc. It has also been pointed out how they become deposited upon articles of food and with these gain entrance into other organisms. All of these carriers of disease-germs should be subjected to disinfection in every case of disease. The agents for disinfection act mechanically or chemically. Those of the first group are aimed at the removal of the disease-germs mechanically (by brushing, rubbing, washing, rinsing, scouring, etc.), or at their destruction directly (by drying, exposure to the sun, or to the action of heat). Of these mechanical means the first, washing with brushes and soap, etc., are in common employ. Exposure to the sun (airing of beds, etc.) is probably efficient, but can influence only the bacteria lying superficially. Heat may be em- ployed in the form of hot or boiling water, of hot air, and of steam. Boiling water is an active disinfectant, destroy- ing bacteria generally within a few seconds, and particu- larly resistant forms — apart from the spores of harmless potato-bacilli (mesentericus) — in from two to five minutes. Hot air is much less efficient. According to Koch and Wolff hiigel, the most resistant of the spore-free bacteria are destroyed by exposure for one and a half hours to air 412 CLINICAL BACTERIOLOGY. at a temperature of 80° C. (176° F.), and spores only after exposure for three hours at a temperature of from 140° C. (284° F. ) to 160° C (320° F.). Steam may be employed still or streaming, and further under tension and super- heated. Live steam is most available for large disinfecting apparatus. These represent in principle nothing more than the Koch steam-chest. Into a large vessel or an air- tight chamber, steam -vapor at a temperature of 100° C. (212° F.) is so introduced that it displaces all of the air and comes in contact with every portion of the articles to be disinfected. As in the steam-chest, in these larger apparatus also complete sterilization is effected in from fifteen to thirty or sixty minutes, in accordance with the size of the articles. Steam under pressure (120° C. — 248° F.) is employed for purposes of disinfection, especially in France. With its aid sterilization is effected quickly and with absolute certainty when provision is made for the escape through a valve of all the air contained in the appa- ratus. Cold inhibits the development of, but scarcely destroys, resistant microorganisms. Anthrax-spores retain their vitality and virulence for more than twenty-four hours at a temperature of 130° C. (266° F.). Of greater importance are chemic dismfectants. These consist of all sorts of chemic agents (acids, alkalies, salts, aromatic substances, etc.), and the large majority cause enfeeblement and inhibit the development of the bacteria, and in sufficient concentration effect their destruction. As the chemic disinfectants are, in the nature of things, almost invariably employed in aqueous solution, recent in- vestigations in the domain of physical chemistry with regard to the nature of the solutions have not only acquired sig- nificance for the comprehension of observed phenomena, but they have also yielded valuable aid in the selection and application of disinfectants. In the course of these observations, which were begun by Dreser, then continued especially by Paul and Kronig, and at the same time also by Scheurlen and Spiro, it has developed that the changes which the dissolved substances undergo in the solution through the action of the solvent are of decisive import- ance with regard to the mode of action. According to the views of Arrhenius, now almost generally accepted as cor- rect, a dissociation (division) of the dissolved salts, acids, DISINFECTION. 413 and bases, into electropositive and electronegative con- stituents— into their ions — takes place in watery solutions. While in general the salts in watery solution are much more actively dissociated than the acids and the bases, the degree of dissociation of the various salts of a metal is dependent upon the nature of the acid-ion, and that of the same salt in turn upon the dilution. As the chemic and physical properties of solutions are dependent upon the degree and the character of the ioniza- tion, it has developed that also the disinfectant activity of equimolecular solutions is influenced by the dissociation, so that, for instance, solutions of mercurial salts are the more active the more mercury they contain, not by weight- percentage, but in the form of ions. For this reason solu- tions of mercuric chlorid exceed in activity solutions of all other mercurial salts. In the course of their investigations Paul and Kronig discovered another series of laws. Thus, the acids act in general accordance with their electrolytic dissociation (ex- cept hydrofluoric acid, nitric acid, trichloracetic acid), and likewise the bases in accordance with the concentration of the hydroxyl-ions contained in the solution. It has also been shown that the oxidizing agents — nitric acid, chromic acid, chloric acid, persulphuric acid, and permanganic acid — are active in accordance with the position that they occupy in the scale of oxidizing agents by reason of their electric activity. The halogens — chlorin, bromin, iodin — whose disinfecting activity on the whole, in correspondence with their chemic activity, diminishes with increase in atomic weight, play a specific role. Whereas ionization has thus proved itself an index for both the chemic and the disinfectant activity of salts, acids, and bases — in general, the inorganic disinfectants — there exists, according to Scheurlen and Spiro, another large group of disinfectants, whose activity is not dependent upon the action of ions, but upon ** molecular action," and in connection with which the entire undissociated molecule must be taken into consideration. The mode of action of this class of substances, the principal representative of which is carbolic acid, can be explained, as Spiro has recently shown, by analogy with the laws for the division of a body between two solvent agencies : that is, the condi- tion present is not, as in the first class of disinfectants, one 414 CLINICAL BACTERIOLOGY. of true chemic reaction, but one of solution manifestations, similar to those better-known processes, of which, for in- stance, we have long had knowledge in a whole series of stains. The distinction between the two classes of disinfectants is of significance also for practical purposes. It thus be- comes clear why, on addition of other substances — for instance, acids — the action of the one class (chemic disin- fectants) is usually lessened, while that of the other — for instance phenol — is increased, whereas addition of alcohol has the reverse effect. An explanation is afforded also for the practically significant phenomenon that the disinfection of organic or albuminous nutrient media (such as feces, sputum, etc.) is- effected more readily with the second class of agents than with the first. In order to determine the activity of a disinfecting agent, according to Koch's procedure highly resistant bacteria — anthrax-spores that have been dried upon short pieces of sterile silk thread are most commonly employed as test- objects — are introduced into a solution of the agent and from time to time it is noted in specimens that are removed and transferred to nutrient medium whether the bacteria have already been destroyed or not. Geppert has called attention to a possible source of error attending this mode of procedure. With the silk thread not alone the spores are transferred to the new nutrient medium, but always, even when the thread is rinsed with water after removal, also a certain amount of the disinfecting agent. This diffuses from the silk into the nutrient medium, and renders it unsuitable for the culture of the bacteria. Growth, there- fore, may readily fail to take place, although all of the bac- teria may not be destroyed. Koch has himself studied the inhibition of growth in culture-media to which antiseptics were added in varying amount, and in which then silk threads with anthrax-spores were introduced. He found Distinct Retardation Complete Cessation OF Growth of Growth On Addition of Mercuric chlorid in the strength of I : 1,600,000 I : 300,000 Thymol *' I : 80,000 Oil of turpentine ** 1:75,000 Potassium-soap ** i : 5000 i : looo DISINFECTION. 415 Distinct Retardation Complete Cessation OF Growth On Addition of OF Growth lodin in the strength of i : 5000 Salicylic acid ** I 13300 I : 1500 Hydrochloric acid ** I : 2500 I : 1700 Camphor *' I : 2500 More than i : 1250 Borax " I : 2000 I : 700 Potassium permanganate '* I : 1400 Boric acid " I : 1250 1:800 Carbolic acid " I : 1250 I :850 Quinin *' I : 830 I : 625 Potassium chlorate " I : 250 Alcohol " I : 100 1:12.5 Sodium chlorid *' I : 64 From the foregoing it will appear that even quite slight additions of the disinfectant may nullify the results of the experiment when the method of investigation first named — of introducing the silk threads into the nutrient medium — is pursued. When Geppert, by means of dilute solutions of ammonium sulphate, removed completely the disinfectant from the anthrax-spores that had lain in mercuric-chlorid solution, I : 1000, before their transference to the nutrient medium, he still observed growth after exposure to the action of the mercuric chlorid for fifteen minutes, and also after exposure for five hours, and in one instance even after exposure for twenty-four hours. The spores were still infective. Animals that Geppert inoculated therewith fre- quently died of anthrax. Solutions of mercuric chlorid of I : 100 did not with certainty destroy the spores in from six to twelve minutes. From this it can be seen that the disinfecting activity of the agents tested by the method of Koch has probably been often placed too high. The figures obtained by Koch and numerous subsequent investi- gators still retain their value for practical purposes, as Koch himself endeavored to reduce to a minimum the sources of error indicated by making the silk threads as short, and using as large an amount of culture-medium, as possible, and rinsing in distilled water, alcohol, etc., the threads removed from the antiseptic solution before trans- ference to the nutrient solution. The following table from Fliigge's " Text-book " (1897) 416 CLINICAL BACTERIOLOGY. will give an idea of the utility of the better known anti- septics : Destroy Strepto- Anthrax-bacilli, Bactericidal cocci AND Staphylo- cocci. Typhoid-bacilli, Cholera-bacilli. Agents. Anthrax-spores. Within Five Min- utes. Within Five Minutes. In from Two to Twenty- four Hours. Hydrogen dioxid . Concen- trated I :200 1:500 1: 100 after an hour. Chlorin O.I per O.I per ... Fresh chlorin- water cent. cent. 0.2 per cent, in an hour. lodin trichlorid . . I : 200 I : 1000 Potassium iodid . . ... I : 10 Sulphuric or hydro- chloric acid . . . I : 10 I : 100 1:1500; ty- phoid, 1:700 I : 50 in ten days. Sulphurous acid . . I : 300, gas 10 vol. per cent, (only superficially) Arsenic acid .... ... ... ... I : 1000 after ten days. Boric acid ... ... 1:30 Concentrated after six days incom- pletely. Potassium hydroxid . 1:5 ... I :300 Ammonia ... I :300 Soda ... ... I :40 Ammonium carbon- ate ... ... I : 100 Calcium hydroxid . ... I : 1000 Silver nitrate . . . I : 1000 ... I :4000 Mercuric chlorid . . I : 10,000 -I : 1000 I :2000 ... I :2000 Copper sulphate . . ... ... ... I : 20 (five days). Potassium perman- ganate I :200 ... ... I : 20 (one day). Potassium bichromate ... ... ... I : 1700 Chlorinated lime . . I :500 I : 20 (one hour). Ferric chlorid . . . ... ... I : 20 (six days). Alcohol 80 per ct. ... ... ... Acetic acid, oxalic acid, etc 1 : 200 or 300 Chloroform .... I : 14 Carbolic acid . . . iViSo Choi era, I : 200; glanders, anthrax, I : 100; typhoid, 1:50 I : 300 1 : 20 in from four to forty-five days. DISINFECTION. 417 Destroy Bactericidal Agents. Strepto- cocci AND Staphylo- cocci. Anthrax-bacilli, Typhoid-bacilli, Cholera-bacilli. Anthrax-spores. Within Five Min- utes. Within Five Minutes. In from Two to Twenty- four Hours. Salicylic acid . . . Creosote . . . . . Creosote sulphate . Creolin Aseptol Quinin Oil of turpentine . . I : lOOO 1:300 1 : 500 I : 100 From 3 to 5 per cent. 1:3000; ty- phoid, 1 : 250 I : 20 in six hours. 10 per cent, in thirty minutes. 1: 100 after ten days. Concentrated in five days. Upon the basis of a comparison of the results of Koch's observations with those of numerous subsequent investiga- tors Schimmelbusch arranges the disinfectants in accord- ance with the time in which they destroy anthrax-spores, in the following order : II. Mercuric chlorid, \ lodid, chlorin, and bromin, / j ^ ^i, •*!,• lodin trichlorid (Behring), 7enT-four^hours Kresol rendered soluble by addition j ^* of sulphuric acid (C. Frankel), / Five per cent, carbolic acid, creolin, 1 j . .1 • u 4.4 j Crude wood-vinegar, | ^^^^^^^ ^^^ ^P^''^^ ^" ^^^"^ *^° ^^y'' Chlorinated lime, five per cent, ^ Oil of turpentine, f Ammonium sulphate. Formic acid, Ferric chlorid, five per cent., Chlorpicrin, five per cent. , Quinin, one per cent., with hydro- chloric acid, Arsenic acid, one in the thousand, i Hydrochloric acid, two per cent., ) Ether destroys the spores in about thirty days. III. Anthrax-spores are still not destroyed after the lapse of a month in absolute alcohol, distilled water, chloroform, glycerin, benzoic acid, ammonia, concentrated solution of sodium chlorid, five per cent, potassium chlorate, alum, borax. destroy the spores in about five days, destroy the spores in about six days, y destroy the spores in about ten days. The figures obtained experimentally, however, constitute no trustworthy index of the practical utility of an agent. For practical purposes the greater or lesser solubility of a 27 418 CLINICAL BACTERIOLOGY. disinfectant, its capacity for penetrating the objects to be disinfected — for this reason oily solutions of even active an- tiseptics are almost ineffective, because the oil does not penetrate into the organisms — its chemic constitution (upon which is dependent the extent to which the object to be disinfected suffers in the process of disinfection), and much besides must be taken into consideration. The cost of a dis- infecting agent is also not without bearing upon its practical applicability upon a large scale. It would carry us too far to discuss individually the in- numerable antiseptics that have been tested in recent years, and that have been recommended for one purpose or another. We shall limit ourselves to the following obser- vations : DISINFECTION OF THE HANDS. Disinfection of the hands is practised at surgical clinics in the following manner, in accordance with the regulations of Fiirbringer : The hands are washed as clean as possible in tepid water with soap and vigorous brushing for five minutes, then rinsed in fresh water ; the nails are cleaned, especially the spaces beneath the nails, by means of a metal- lic nail-cleaner ; the hands are then rubbed in alcohol for three minutes ; next they are rinsed and rubbed for a minute in from ^ to i : looo solution of mercuric chlorid. In the event of marked contamination of the skin, it is first rubbed with ether before the disinfectants are used, or the entire procedure is performed twice. Before every operative procedure this process of rigid disinfection must be carried out completely at the site of operation, as well as on the hands of the operator. A simpler mode of disinfection of the hands — ^washing with soap and brushing, rinsing with mercuric chlorid or alcohol — should be practised by physicians and attendants always after con- tact with a patient, and especially before each meal. In houses in which an infectious disease exists, especially in times of epidemic, disinfection of the hands before eating is a general duty. Instead of mercuric chlorid, the following may also be employed : Carbolic acid, from 3 to 5 per cent. ; creolin, 3 per cent. ; lysol, i ^ per cent. The sub- stances last named, however, have, as has been pointed out, less disinfecting power than mercuric chlorid. DISINFECTION. 419 DISINF5CTION OF MUCOUS MEMBRANES. The disinfection of mucous membranes is far more diffi- cult than that of the external integument. Simple irriga- tion with a disinfectant will not yield the desired result — quite apart from the danger of intoxication. Vigorous brushing, however, and alcohol and ether are naturally not applicable here in the same degree as upon the exter- nal integument. The greatest stress is, therefore, to be placed upon mechanical removal of the germs, and this may be effected by brushing and rubbing with the fingers or with swabs of cotton or of gauze. Irrigation of the loos- ened mucus and debris may be practised with simple warm water, or with nonirritant solutions (weak solutions of boric acid, potassium permanganate, aluminum sulphate, physiologic salt-solution, infusion of chamomile, etc.). DISINFECTION OF INSTRUMENTS AND DRESSINGS. Metallic instruments are boiled for five minutes in water, preferably after addition of one per cent, of soda, whereby rusting is avoided. Cotton, gauze bandages, and the like are sterilized in the steam-chest or the dry chamber. DISINFECTION OF FECES AND CESSPOOLS, Lime is best adapted for disinfection of the stools. In the bed-pan in which the infectious stool (especially in cases of typhoid fever and of cholera) is received, so much milk of lime is previously introduced as will just cover the bottom of the vessel. After defecation an amount of milk of lime is added equal to the bulk of the feces, and the mixture is vigorously agitated and permitted to stand for an hour. The mixture must be highly alkaline (Pfuhl). The milk of lime should always be freshly prepared : To unslaked lime in stone jars or wooden pails so much water is added as will be taken up. The slaked lime is diluted with four times the amount of water. If chlorinated lime be employed, it must be added in an amount equal to one per cent, of the mixture of urine and feces. It may be added in the form of powder or of a paste 420 CLINICAL BACTERIOLOGY. consisting of about twenty grams of chlorinated lime and lOO of water. After thorough admixture the stool need stand for only fifteen minutes. For the disinfection of cesspools Pfuhl likewise recom- mends milk of lime. When the tun-system is used, 3 grams = 6 cu. cm. of pulverulent slaked lime, or, better, 30 cu. cm. of milk of lime, and in the case of pits 2 grams = 4 cu. cm. of pulverulent lime, or, better, 20 cu. cm. of milk of lime should be allowed daily for each person. The seat as well as the funnel-shaped basin and the discharge -pipe must be thoroughly irrigated with the disinfecting solution. If, in addition to feces, which may be estimated at 400 cu. cm. for each person daily, also the total amount of urine is thrown into the receptacle, the mass of material to be dis- infected may be estimated at between 1 500 and 2000 cu. cm., and four or five times as much of the disinfectant should be employed for each person. Under these conditions also disinfection is attained only when the privy-contents exhibit a distinctly alkaline reaction. Of other disinfectants for water-closets, carbolic acid, sulphocarbolic acid, lysol, and saprol, may be mentioned. These must be introduced in solution, and in an amount equal to two per cent, of the material to be disinfected ; about eight grams daily may, therefore, be estimated for each person. All of these substances are more expensive than milk of lime, without possessing especial advantages. For hospitals it may be recommended that the entire fecal accumulation be boiled in a suitable vessel with addition of deodorizing substances (potassium permanganate). DISINFECTION OF BATH-WATER, WASH-WATER, ETC Bath-water, when contaminated by patients, is disinfected by means of milk of lime (six liters to a bath of 300 liters) or of carbolic acid. Mercuric chlorid is to be avoided when metallic tubs are used. Wash-water is freed from typhoid-bacilli and cholera- bacilli, according to Pfuhl, within an hour if i. 5 to one thous- and of calcium hydroxid be added, and if constant agitation is maintained. Koch permitted milk of lime to be introduced into the drainage-fluid at Nietleben until the fluid appearing in the DISINFECTION. 421 main discharge -conduit at the lower end of the irrigation- field presented a marked alkaline reaction. For the disinfection of water-conduits dilute milk of lime, carbolic acid, or a mineral acid, is employed. At Nietleben Koch disinfected the service by means of carbolic acid, per- mitting three per cent, carbolic acid to be driven from the pumping well into all divisions of the service, and to remain in the conduits for twenty-four hours. Then the pipes were irrigated with wholesome water. This procedure is attended with the objection that the water retains for a con- siderable time the taste of carbolic acid. On the other hand, however, it does not carry with it the danger of ob- struction of the conduits that attends the employment of milk of lime. DISINFECTION OF URINEL The urine is usually disinfected in common with the feces. It is generally not so infective as the stools. If it is to be disinfected alone, this may be accomplished by addition of milk of lime, carbolic acid, or mercuric chlorid. DISINFECTION OF SPUTUM. The sputum must be received and preserved in a moist condition, preferably in sputum-cups containing a layer of water. So long as the sputum is contained within such cups it is relatively harmless. A greater danger resides in the conversion into dust and into spray of sputum evacu- ated into handkerchiefs, upon floors, etc. On emptying the vessel the sputum must be disinfected. Crude carbolic acid (from 5 to 10 per cent.) or mercuric chlorid (i or 2 : 1000) would be suited for this purpose if the disinfectant penetrated the sputum. Generally, however, the albumin on the outer surface of the mass of sputum is coagulated, and the bacilli contained within do not come in contact with the antiseptic at all. For this reason the sputum must at least be thoroughly rubbed up in the disinfecting solution, and remain therein for a long time. Lysol (10 per cent.) and crude solutol (from 5 to 10 per cent.) do not coagulate the sputum, and are therefore better adapted for disinfec- tion. It is further useful to disinfect the sputa by heat. If 422 CLINICAL BACTERIOLOGY. they are not too abundant and are at the same time viscid, they can be simply burned in a stove. Otherwise they are introduced, together with the sputum-cup, into a spe- cially constructed disinfector, resembling the steam-chest (Kirchner), in which they are exposed for half an hour to the action of steam at a temperature of ioo° C. (212° F.). The apparatus has the disadvantage that a number of the cups will be broken, and in families it can not often be pro- vided. Under such circumstances the sputa can be poured into a pot and boiled for half an hour in water. Usually, however, one will have to be satisfied with simply emptying them into the water-closets, where the pathogenic germs are simultaneously destroyed, in part with the disinfection of the feces and in part by the process of putrefaction. The sputum-cups should be sterilized by means of hot water and carbolic acid, and preferably likewise boiled. DISINFECTION OF BODY-CLOTHING AND BED-LINEN. Uncontaminated linen is boiled for half an hour in petro- leum soap-water (two bucketfuls of water — about thirty liters — to 250 grams of soft soap and two spoonfuls of petro- leum ; then, after removal of the soap-water, rinsed in cold water ; next washed with soap in clean hot water ; then again rinsed in cold water, permitted to remain over night in clean water, and finally dried in the open air. Instead of this procedure the linen may also be disinfected in the steam disinfecting apparatus. Contaminated linen requires removal of feces, mucus, or pus before application of heat, as otherwise burned spots will appear. Such linen must immediately after removal be placed in a sheet moistened with mercuric chlorid, i : 2000, then in strong, moist sacks, and sent away for disinfection. The sacks, unopened, are introduced into the disinfecting fluid, three per cent, soft-soap solution, in which the linen is treated for three hours at a temperature of 50° C. (122° F.), and then remains for an additional forty-eight hours during the process of cooling ; or mercuric-chlorid salt solution (of 0.5 to one thousand mercuric chlorid and 6 to one thousand sodium chlorid). After disinfection in this way the linen is further treated in the same manner as uncon- taminated linen. DISINFECTION. 423 DISINFECTION OF BEDS AND CLOTHING. Articles of clothing and beds are best disinfected in suit- able apparatus in live steam. If such an apparatus is not available, exposure to air and sun must be resorted to or rubbing with three per cent, carbolic acid. To purify arti- cles by exposure to air, they must be hung for days in a dry room and exposed as uniformly as possible upon all sides to the rays of the sun. Even then the disinfection is not trustworthy. The bedstead, articles of leather, and the like, are rubbed off with five per cent, carbolic acid. For the disinfection of clothing formalin (a forty per cent, solu- tion of formaldehyd) has been warmly recommended. The clothing should be loosely packed in a chest, and be- tween its layers are placed strips of goods saturated with for- malin. From thirty to fifty grams of formalin are required for a suit of clothing. Disinfection is said to be completed within two hours, and the disagreeable odor may be removed by means of ammonia. DISINFECTION OF ARTICLES OF FOOD. The keeping clean of articles of food is especially a pro- phylactic measure. These should not be permitted to stand about in the sick-room, but they should be kept covered, etc. Portions of food left unused by the patient are burned, as well as articles of food that are known to have been in- fected in other ways. Milk and other liquids are drunk only boiled, as should also be drinking-water in times of cholera-epidemic. DISINFECTION OF THE SICK-ROOM. A sick-room should not contain pictures, curtains, etc., in fact any superfluous articles that only serve as dust-col- lectors. After the termination of the illness the room of the patient, and possibly the entire dwelling, should remain undisturbed for about ten hours, to permit the dust to settle, and then it should be thoroughly disinfected. In some large cities this is undertaken by special establishments. Such disinfecting stations should be established everywhere, for 424 CLINICAL BACTERIOLOGY. only with their aid and through their trained attendants can thorough disinfection of dweUings be carried out. All portable articles that will withstand the action of moist steam are wrapped in cloths moistened with three per cent, carbolic acid or mercuric chlorid, i : looo, and sent to the disinfecting station, where they are placed in a steam-appa- ratus. The walls and ceilings are thoroughly rubbed down with bread-crumbs, and these are eventually burned in the stove. Walls painted with oil-colors are washed with five per cent, carbolic acid or painted with milk of lime. White- washed walls are given a new coat. The furniture is rubbed off with three per cent, carbolic acid and then rubbed dry. Polished articles can be rubbed off with bread. Up- holstered furniture, when possible, is sterilized in the steam- apparatus ; otherwise rubbed down with carbolic acid and brushed. Articles of leather, metal, glass, and the like can be vigorously rubbed with carbolic acid. Mantels, the upper surfaces of stoves, etc., are first freed of dust by means of moist cloths, then soaped and rubbed with three per cent, carbolic acid. Finally, the floor is disinfected by scouring with warm water and soap and then with carbolic acid. Of late formaldehyd, which has already been mentioned as a disinfectant for clothing, has also been employed for the dis- infection of dwellings. The following mode of procedure is pursued : A forty per cent, aqueous solution of formal- dehyd (also known as formalin or formol) is mixed with chlorinated lime and water — to each liter of formalin 200 grams of chlorinated lime in 400 cu. cm. of water ; the mixture is designated formochloral — and the mixture is evaporated in a Trillat autoclave, with four atmospheres of pressure. One liter of formochloral is sufficient for 200 cubic meters of air-space. The disinfection is, however, essentially only superficial, the bacteria in thick articles, such as beds, mattresses, clothing, etc., not being destroyed thereby. After disinfection has been completed the vapor of formaldehyd is removed by means of a spray of ammonia and exposure to air. Greater advantages than the Trillat apparatus, whose manipulation is always difficult, are pos- sessed by the formalin-lamp placed upon the market by Schering,* with the aid of which the disinfecting vapor is generated in a most simple manner by the burning of * Numerous forms of serviceable apparatus can now be obtained. DISINFECTION. 425 formalin-pastils. Two pastils, each of one gram, are em- ployed for every cubic meter of space. According to our experience, trustworthy superficial disinfection can be effected by this means for all bacteria without spores. DISINFECTION OF SHIPS, VEHICLES, RAILWAY-CARS, ETC. Entire ships, cars, etc., are disinfected in a manner similar to that employed in disinfection of the sick-room. The burning of sulphur, which was formerly much practised (20 grams of sulphur to each cubic meter of space, moistened with alcohol before ignition), has been shown by Koch's investigations to be relatively useless, and injures many articles rather seriously. INDEX Abscess, 127 complicating typhoid fever, 127 local, 123 of liver, 155, 370 pyemic, 155 tropical, 155 Abscesses, cold, 127 Absolute immunity, 56 Achorion, cultural properties of, 342 Schonleinii, 341 Acquired immunity, 50 Actinomyces, aerobic, 360 botanic position of, 361 pure culture of, 357 Actinomyces-druses, 356 Actinomyces-granules, 356 Actinomycosis, 354 bacteriologic diagnosis of, 362 course of, 357 experimental development of, 360 in human beings, 355 of animals, 354 treatment of, 362 Active immunization, 55, 68 Aerial germs, sources of, 394 Aerobic bacteria, 26 Agar streak-plates, 94 Agar-agar, plates of, 94 preparation of, 81 Agar-plates, 95 Agglutination, 62 Agglutination-phenomenon in typhoid fever, 176 Air, bacteria in, 393, 401 bacteriologic examination of, 389, 392 exclusion of, anaerobic pure culture with, loi germs in, sources of, 394 Hesse's procedure for examination of, 392 Air, hot, disinfection by, 41 1 method of examination of, 392 Petri's procedure for examination of, 392 Air-infection, 394 Albuminoids, culture-media free from, 87 Alexins, 60 Amebse, examination of stools for, 371 in normal intestinal contents, 371 Amebic enteritis, 364 Amount of infectious material, 39 ^ Amphitrocha, 19 Anaerobic bacteria, 26 cultivation of, 98 facultative, 26 plate-culture of, 98 ' pure culture by the method of Buchner, loi in a high layer, loi in an atmosphere of hydrogen, loi in test-tubes, loi raw eggs for, 102 with complete exclusion of air, loi Angina, 134 bacteriologic diagnosis of, 135 due to mixed infection, 135 herpetica, 129 prognosis of, 135 scarlatinal, 135 Anginas, phlegmonous, 134 pseudo- membranous, 1 34 Aniline water as a mordant, 104 Animal experimentation, determina- tion of pathogenicity (or speci- ficity) of bacteria by, no life, infections with lowest forms of, 364 Anthrax, 287 bacteriologic diagnosis of, 298 427 428 INDEX. Anthrax, experimental development of, 295 heredity of, 297 immunity to, 299 mixed infection in, 297 occurrence of, in animals, 291 in human beings, 292 prophylaxis of, 300 vaccination against, 299 Anthrax-bacilli, cultivation of, 289 distribution of, in infected body, 296 infection with, through digestive tract, 293 through kmgs, 295 through skin, 293 morphology of, 287 natural portals of entry for, 293 resistance of, 290 sporulation of, 290 toxins of, 297 Anthrax-carbuncle, 292 Anthrax-spores, destruction of, by dis- infectants, 417 resistance of, 290 Anti-bodies, 59 Antiseptics, utility of, 416 Antitoxic power of blood-serum, 70 Antitoxin -immunity, 71 Antitoxins, 67 Arthrosporous bacteria, 23 Artificial immunity, 50 Ascending pyelonephritis, 159 Aseptic peritonitis, 151 Asiatic cholera, 181 Aspergilli, 336 Aspergillus flavescens, 339 fumigatus, 339 Attenuation-method of immunization, 54 Babes-Ernst bodies, 19 Bacillary filaments, 20 Bacilli, 17 Bacillus aerogenes, 122 amethystinus, 404 aquatilis, 404 arborescens, 402 aurantiacus, 401 botulinus, 245 cultural peculiarities of, 246 morphology of, 246 occurrence of, 250 pathogenic properties of, 247 tenacity of, 247 brunneus, 402 coeruleus, 404 Bacillus constrictus, 401 fluorescens liquefaciens, 402 nonliquefaciens, 401 immobilis, 401 fuscus, 401 helvolus, 403 ianthinus, 403 liquefaciens, 404 liquidis, 404 mesentericus, 405 pavoninus, 404 prodigiosus, 403 pyocyaneus, 120, 125, 126 rubefaciens, 402 ruber aquatilis, 404 balticus, 404 rubidus, 403 spinosus, 405 subflavus, 402 subtilis, 404 violaceus, 403 viscosus, 403 Bacteria, aerobic, 26 anaerobic, 26 cultivation of, 98 arthrosporous, 23 biology of, 17 chemic activities of, 28 combinations due to, 26 classification of, 17 determination of pathogenicity (or specificity) of, by animal experi- mentation, no diagnostic significance of metabolic products of, 28 endosporous, 23 facultative anaerobic, 26 ground-substance of, 19 identification of, 28 in soil, air, and water, 401 influence of oxygen on, 26 light in relation to, 26 metabolic products due to, 29 microscopic examination of, 102 morphology of, 17 multiplication of, 20 nonpathogenic, 33 pathogenic, 23 permanent forms of, 22, 23 pigment formed by, 26 pleomorphic, 24 rod- shaped, 17 spheric, 17 spiral, 17 staining of, 102 sustenance of, 25 thermophilic, 22 ubiquity of, 25 Bacteriacese, 17 INDEX. 429 Bacterial diseases, susceptibility to, 45 peritonitis, 151 poison, specific, 30 proteids, 31 Bactericidal substances, 59 Bacterium coli commune, 120, 125, 126 differentiated from typhoid- bacilli, 174 encapsulated, 19 lactis aerogenes, 126 Basidia, 337 Bath-water, disinfection of, 420 Bed-linen, disinfection of, 422 Beds, disinfection of, 423 Beggiatoa, 27 Biliary abscesses, 155 Birth, infection during, 49 Blood, typhoid-bacilli in, 171 Blood-agar, preparation of, 82 Blood- serum agar, 84 antitoxic power of, 70 glycerin-agar, 84 human, preparation of, 84 liquid, preparation of, 83 of immunized animals, immuniza- tion with, 54 Bodies, permanent, 22 Body-clothing, disinfection of, 422 Bone-marrow, typhoid-bacilli in, 170 Botulism, 245 bacteriologic diagnosis of, 250 immunity to, 251 mixed infection in, 250 ' physiology of, 249 specific therapy of, 251 Bouillon, preparation of, 78 Bread-pap, 86 Bronchitis, 138 fetid, 138 Brush-molds, 337 Buchner, method of, for anaerobic pure culture, loi Budding fungi, 338 infections with, 336 Bulbous molds, 336 C. Capsules, staining of, 108 Carbolic acid as a mordant, 104 Carbuncle, 126 Causative agents of inflammation in disease, 126 in healthy persons, 126 morphology of, 115 outside the body, 126 Causative agents of inflammation, pathogenic properties of, with relation to animals, 123 of suppuration in healthy per- sons, 125 outside the body, 125 Causes of immunity, 57 Cavities of the body, inoculation of, III Cell-capsule, 19 Cell-membrane, 19 Cesspools, disinfection of, 419 Chain, closed, extension of cholera in, 190 continuous, disease-extension by, 47 Chain -cocci, 20 Chalazion, 1 61 Chemic activities of bacteria, 28 combinations due to bacteria, 26 disinfection, 412 Chemotaxis, negative, 58 positive, 58 Chickenpox, 328 Cholangitis, 154 bacteriologic diagnosis of, 155 experimental development of, 155 Cholecystitis, 154 bacteriologic diagnosis of, 155 experimental development of, 155 Cholera, Asiatic, 181 cause of, 181 bacteriologic diagnosis of, 193 development of, 188 disinfection of, 196 epidemics of, 190 experimental development of, 191 explosive extension of, 190 extension of, in closed chain, 190 immunity to, 197 local predisposition to, 191 nostras, 198 prophylaxis of, 196 temporal predisposition to, 19I toxins of, 189 Cholera-bacilli, 181 cultural investigation of, 193 examination of water for, 195 gelatin plate -culture of, 193 microscopic examination of, 193 peptone-culture of, 194 Cholera-red reaction, 184 Cholera-vibrios, occurrence of, 1 86 tenacity of, 185 Chorea, endocarditis secondary to, 148 Choroiditis, 160 Cicatrix-tetanus, 239 Circumscribed peritonitis, 152 Clostridium, 23 430 INDEX. Clothing, disinfection of, 423 Coccacese, 17 Cocci, 17 Cold abscesses, 128 action of, on bacteria, 412 Columella, 337 Comma-bacilli, appearance of, in cul- tures, 182 Condensation, water of, 82 Congenital pneumonia, 148 syphilis, 319, 320 tuberculosis, 268 Conidia, 336 Conjunctivitis, 160 Contagion, ^^ ' Contagious disease, ^^ miasmatic disease, 34 Continuous chain, disease-extension by, 47 Cover-glass specimen, preparation of, 104 staining of, 104 Crescents, Laveran's, 373 Cryptogenetic septicemia, 162 Cultivation of anaerobic bacteria, 98 Culture, methods of, 74, 87 pure, 74, 87 Culture-media free from albuminoids, 87 Cure, 49 immunity and, relations between, 71 Cutaneous inoculation, no Cystitis, 155 bacteriologic diagnosis of, 156 experimental development of, 156 gonorrheal, 155 puerperal, 155 tuberculous, 155 D. Death-point, thermal, 22 Dermatomycoses, 341 Descending pyelonephritis, 159 Diagnostic significance of metabolic products of bacteria, 28 Diarrhea, summer, 199 Diffuse peritonitis, 152 Digestive tract, infection with an- thrax-bacilli through, 293 typhoid fever through, 169 peritonitis arising from, 15 1 Dilution-method of immunization, 54 Diphtheria, 207 bacteriologic diagnosis of, 217 immunity to, 218 mixed infection in, 213 Diphtheria, physiology of, in animals, 211 prophylaxis of, 230 relations of diphtheria-bacillus to, 213 specific therapy of, 218 susceptibility of human beings to, 215 Diphtheria-antitoxin, complications from use of, 229 methods of testing, 225 Diphtheria-bacillus, 207 cultural properties of, 209 pathogenic properties of, 2IO portal of infection for, 216 relations of, to diphtheria, 213 saprophytic occurrence of, 214 staining properties of, 207 tenacity of, 210 Diphtheria-toxin, active, 220 Diphtheric endocarditis, 148 Diplobacilli, 21 Diplobacillus Friedlander, 126 pneumoniae Friedlander, 119, 125 Diplococci, 21 Diplococcus lanceolatus Frankel, 124 pneumoniae Frankel, 118, 126 Direct immunization, 55, 68 infection, 48 Discontinuous sterilization, 77 Disease, causative agents of inflam- mation in, 126 contagious, 33 miasmatic, 34 explosive distribution of, 47 infectious, 33 miasmatic, 33 Disease-extension by continuous chain, 47 Disinfectant, activity of, 414 Disinfection, 410 by boiling water, 41 1 by heat, 411 by hot air, 41 1 by hot water, 411 by steam, 411 chemic, 412 in cholera, 196 mechanical, 411 of bath-water, 420 of bed-linen, 422 of beds, 423 of body-clothing, 422 of cess- pools, 419 of clothing, 423 of dressings, 419 of feces, 419 of food, 423 of hands, 418 INDEX. 431 Disinfection of instruments, 419 of mucous membranes, 419 of railway-cars, 425 of ships, 425 of sick-room, 423 of sputum, 421 of urine, 421 of vehicles, 425 of wash- water, 420 of water-conduits, 421 Double staining, 107 Dressings, disinfection of, 419 Drinking-water, infection with ty- phoid fever through, 170 Drumstick-bacteria, 22 Dysentery, 364 amebse of, 364 death of, 366 in the stools of dysenteric pa- tients, 367 movement of, 365 nutrition of, 365 pathogenic activity of, 368 permanent forms of, 366 portals of infection for, 369 propagation of, 366 resistance of, 366 staining of, 367 transmission of, 369 intestinal alterations of, 367 occurrence of bacteria in, 368 E. Ecthyma, 129 Ectoplasm, 364 Edema, malignant, 305 bacillus of, 305 cultural properties of, 306 occurrence of, 307 bacteriologic diagnosis of, 308 experimental development of, 307 immunity to, 308 in human being, 308 portal of infection for, 308 Eggs, raw, for anaerobic culture, 102 Eisner's modification of potato-gela- tin, 81 Endemic occurrence of infectious dis- eases, 47 Endocarditis, 148 bacteriologic diagnosis of, 150 complicating acute nephritis, 149 diphtheric, 148 experimental development of, 149 gonorrheal, 149 malignant, 149 Endocarditis, secondary, 148 to acute articular rheumatism, 147 chorea, 148 croupous pneumonia, 148 erysipelas, 148 erythema nodosum, 148 influenza, 148 osteomyelitis, 148 puerperal fever, 148 pyemia, 148 scarlet fever, 149 septicemia, 148 smallpox, 149 suppurative processes, 148 tuberculous, 149 typhoid, 149 ulcerative, 149 Endometritis, 159 Endosporous bacteria, 23 Enteritis, amebic, 364 Entoplasm, 364 Epidemic distribution of pneumonia, 147 of typhoid fever, 1 70 occurrence of infectious diseases, 47 Epidemics of cholera, 190 of disease, 47 Erysipelas, 129 bacteriologic diagnosis of, 13I bullosum, 130 endocarditis secondary to, 148 etiologic significance of streptococ- cus for, 131 immunity to, 131 microorganisms in, 131 pericarditis secondary to, 150 phlegmonous, 130 specific treatment of, 132 Erythema nodosum, endocarditis sec- ondary to, 148 Erythrasma, 348 Esmarch's modification of Koch's procedure, 94 Examination, methods of, 74 Exanthemata, acute, 328 Experimentation, animal, determina- tion of pathogenicity (or specificity) of bacteria by, no Explosive distribution of disease, 47 extension of cholera, 190 Eye, anterior chamber of, inoculation of. Ill inflammatory diseases of, 160 Farcy, 304 Favus, 341 432 INDEX. Favus, diagnosis of, 344 Feces, disinfection of, 419 tubercle-bacilli in, 264 typhoid-bacilli in, 171 Fetid bronchitis, 138 Fibrinous rhinitis, 133 Filamentous fungi, infections with. Filaments, bacillary, 20 Filtration of water, 399 Fission-fungi, 17, 336 Fixed phagocytes, 58 Flagella, 19 staining of, 109 Food, disinfection of, 423 infection with typhoid fever through, 169 Fowl -tuberculosis, 275 bacillus of, 275 diagnosis of, 276 experimental development of, 276 occurrence of bacilli of, 276 Fractional sterilization, 77 streak, 95 Fragmentation, 337 Fruit-bearer, 336 Fungi, budding, 338 biology of, 336 cultivation of, 339 diseases due to, 341 infections with, 336 microscopic examination of, 338 morphology of, 336 pathogenic activity of, 339 filamentous, biology of, 336 diseases due to, 341 infections with, 336 microscopic examination of, 338 morphology of, 336 pathogenic activity of, 339 Furuncle, 126 G. Gall-bladder, peritonitis arising from, 151 Gas-abscesses, 127 bacteriologic investigation of, 128 Gastro-enteritis, hemorrhagic, 311 Gastro-intestinal lesions, abscess of liver secondary to, 155 tract, inoculation through. III Gelatin, preparation of, 80 General predisposition, 45 Genital organs, female, inflammations of, 159 Genitourinary tract, female, peritoni- tis arising from, 151 Giant-ameba, 364 Glanders, 300 bacteriologic diagnosis of, 304 clinical picture of, 304 course of, 303 experimental development of, 302 heredity of, 304 portals of entry for, 303 prophylaxis of, 305 susceptibility of animals to, 302 Glanders-bacilli, cultural properties of, 301 distribution of, in products of dis- ease, 302 morphology of, 300 occurrence of, in products of dis- ease, 302 resistance of, 302 Globular molds, 337 Gonococci, cultivation of, for diagnos- tic purposes, 313 culture of, 312 double staining of, 313 morphology o^ 311 occurrence of, 311 specific pathogenic significance of, Gonorrhea, 311 bacteriologic diagnosis of, 313 prophylaxis of, 314 Gonorrheal cystitis, 155 endocarditis, 149 Gram's method of staining, 106 Giinther's modification of, 107 Weigert's modification of, 107 Growth, variability in form of, 25 Gruber's reaction, 63 Giinther's modification of Gram's method of staining, 107 H. Hands, disinfection of, 418 Hay, infusion of, d)"] Hay-bacillus, 404 Healthy persons, causative agents of inflammation in, 125 of suppuration in, 1 25 Heat, disinfection by, 41 1 dry, 74 . Hemoglobin-agar, 82 Hereditary syphilis, 319 transmission of pneumonia, 147 Heredity of anthrax, 297 of glanders, 304 of immunity, 56 INDEX. 433 Heredity of infectious diseases, 48 of leprosy, 280 of syphilis, 319 of tuberculosis, 267 Herpes, 129 labialis, 129 of larynx, 129 of pharynx, 129 tonsurans, 344 zoster, 129 Hesse's procedure for examination of air, 392 High layer, anaerobic pure culture in, loi House-epidemics of pneumonia, 147 House-filters, 400 Hydrogen, atmosphere of, anaerobic pure culture in, loi Hydrophobia, 321 experimental development of, 321 immunization to, ^^, 323 incubation of, 322 results of Pasteur's treatment of, 3'f5 . .. susceptibility to, 321 vaccination against, 323 Hyph?e, 336 I. Ice, 4CX) artificial, 400 natural, 400 Icterus, febrile, 310 Identification of bacteria, 28 Idiopathic tetanus, 239 Immediate immunization, 55, 68 Immunity, 49 absolute, 56 acquired, 50 and cure, relations between, 71 artificial, 50 causes of, 57 heredity of, 57 natural, 49 to measles, 328 quantitative limitations of, 56 reaction of, 66 relative, 56 specificity of, 56 to anthrax, 299 to botulism, 251 to cholera, 197 to diphtheria, 218 to erysipelas, 131 to influenza, 286 to malignant edema, 308 to plague, 206 28 Immunity to smallpox, 326 to syphilis, 318 to tetanus, 241 to typhoid fever, 179 Immunization, 49 active, 55, 68 attenuation-method of, 54 dilution-method of, 54 direct, 55, 68 immediate, 55, 68 indirect, 55, 68 mediate, 55, 68 methods of, 50 passive, 55, 68 to hydrophobia, 323 with blood- serum of immunized animals, 54 Immunization-therapy, 72 Impetigo, 128 Indirect immunization, 55, 68 Infected organism, susceptibility of, 44 Infection, 23^ 3^ direct, 48 during birth, 49 infectious, 34 intrauterine, 49 mixed, 41 period of, reaction of, 66 portals of, 42 secondary, 46 toxic, 34 Infections with budding fungi, 336 with filamentous fungi, 336 with lowest forms of animal life, 364 Infectious agent, virulence of, 37 disease, S3 diseases, endemic occurrence of, 47 epidemic occurrence of, 47 heredity of, 48 pneumonia complicating, 145 infection, 34 material, amount of, 39 purity of, 39, 41 Inflammation, 115 causative agents of, in disease, 126 in healthy persons, 125 morphology of, 115 outside the body, 125 pathogenic properties of, with relation to animals, 123 local, 123 Inflammations of female genital or- gans, 159 Inflammatory diseases of the eye, 160 Influences, predisposing, 46 434 INDEX. Influenza, 281 bacteriologic diagnosis of, 287 endocarditis secondary to, 148 experimental development of, 286 immunity to, 286 Influenza-bacillus, cultivation of, 281 distribution of, 285 localization of, 285 morphology of, 280 occurrence of, 284 resistance of, 284 staining of, 281 Influenza-pneumonia, 145 Inhalation, inoculation by, ill Injection, intravenous, no Inoculation by inhalation, in cutaneous, no of the anterior chamber of the eye, III of the cavities of the body, in protective, against plague, 206 subcutaneous, no subdural, in through gastro-intestinal tract, in Instruments, disinfection of, 429 Intestinal mycosis, 295 Intoxication, putrid, due to proteus, 310 typhoid, 172 Intrauterine infection, 49 Intravenous injection, no Involution-changes, 25 Irids, 160 K. Keratitis, 160 Keratomycoses, 349 Kidneys, typhoid-bacilli in, 171 Koch procedure, Esmarch's modifica- tion of, 94 Koch's method of plate-making, 88 Laryngitis, acute, 133 Larynx, herpes of, 129 Laveran's crescents, 373 Leprosy, 278 bacillus of, 278 bacteriologic diagnosis of, 280 distribudon of, 279 experimental development of, 279 heredity of, 280 prophylaxis of, 280 Leptothrix buccalis, 348 Leydenia gemmipara Schaudinn, 387 Light in relation to bacteria, 26 Limitations, quantitative, of immu- nity, 56 Liver, 370 abscess of, 155 bacteriologic diagnosis of, 155 examination of pus from, 370 abscesses of, secondary to gastro- intestinal lesions, 155 peritonitis arising from, 151 pyemic abscesses of, 155 tropical abscess of, 155, 364 typhoid-bacilli in, 170 Local abscess, 123 inflammation, 123 predisposition, 45 Localization, secondary, 46. Lophotrocha, 19 Lungs, infection with anthrax-bacilli through, 295 with typhoid fever through, 169 typhoid-bacilli in, 171 Lustgarten, bacillus of, 314 Lymphangitis, 132 Lysogenic activity of immune serum, 62 Lyssa, 321 M. Macrophages, 58 Malaria, 372 exciting agent of, 372 Malarial fever, continued, 381, 382 double quartan, 378 tertian, 380 explanation of symptoms of, by presence of parasites, 384 false quotidian, 380 irregular, 378, 380, 381, 382 malignant tertian parasite of, 381 mode of infection with, 385 parasites of, 372 biology of, 372 classification of, 377 development of, 375 morphology of, 372 multiplicity of, 376 polymorphism of, 376 relation of, to red blood-cor- puscles, 375 sporulation of, 375 period of incubation of, 386 prognosis of, 384 quartan parasite of, 377 quotidian parasite of, 380 severe tertian, 382 * spontaneous recovery from, 386, 387 tertian parasite of, 378 INDEX. 435 Malarial fever, triple quartan, 378 typical quartan, 378 quotidian, 381 tertian, 380 infection, mixed, 382 parasites, diagnosis of, 382 in soil, 391 pigment, 373 Malignant edema, 305 bacillus of, in soil, 39I endocarditis, 149 pustule, 292 Mallein, 305 Malt-gelatin, 81 Measles, 328 contagium of, 328 endocarditis following, I49 immunity to, 329 infectivity of, 329 natural immunity to, 328 Meat-poisoning, 245 with gastrointestinal symptoms, 245 Mediate immunization, 55, 68 Melanin, 373 Meninges, typhoid-bacilli in, 171 Meningitis, 136 bacteriologic diagnosis of, 137 clinical diagnosis of, 137 epidemic cerebrospinal, 136 metastatic, 136 primary, 136 secondary, 136 Mesenteric glands, typhoid-bacilli in, 170 Metabolic products due to bacteria. Metastatic pleurisy, 139 Miasm, 34 Miasmatic disease, ^^ Micrococci, 17 Micrococcus agilis, 406 aquatilis, 406 aurantiacus, 405 candicans, 406 concentricus, 406 cremoides, 406 radiatus, 406 rosettaceus, 406 versicolor, 405 Microphages, 58 Microscopic examination of bacteria, 102 Microsporon furfur, 347 minutissimum, 348 Milk, 86 tubercle-bacilli in, 265 Mixed infection, 41 Mobile phagocytes, 58 Molds, 336 bulbous, 336 cultivation of, 339 globular, 337 segmented, 338 Monotrocha, 19 Morphology of the causative agents of inflammation, 1 15 Mucor corymbifer, 339 rhizopodifoi-mis, 339 Mucorini, 337 Mucous membranes, disinfection of, 419 Multiple purulent foci, 123 Mycelium, 336 Mycoderma vini, 338 Mycoses, 336 visceral, 350 Mycosis, intestinal, 295 pharyngis leptothricia, 348 Myocarditis, 151 acute diffuse, 15 1 suppurative, 15 1 Myocardium, typhoid-bacilli in, 1 71 » Myringomycoses, 349 N. Natural immunity, 49 predisposition, 44 Negative chemotaxis, 58 Nephritis, 157 acute, 157 endocarditis complicating, 149 bacteriologic diagnosis of, 158 complicating, 157 Nitrification, 391 Nitrobacteria, 27 Nitrosobacteria, 27 Nitrosococcus, 27 Nitroso-indol reaction, 122 Nitrosomonas, 27 Noma, 133 Nonpathogenic bacteria, ;^^ Nose, inflammations of, 133 Notification, 41 1 •Nutrient media, preparation of, 78 Oidia, 338 Oidium albicans, 351, 353 Oophoritis, 159 Ophthalmia, sympathetic, 161 Organism, infected, susceptibility of, 44 Osteomyelitis, 164 436 INDEX. Osteomyelitis, endocarditis secondary to, 148 experimental development of, 164 Otitis media, 135 bacteriologic diagnosis of, 136 Otomycoses, 349 Oxygen, influence of, on bacteria, 26 Ozena, 133 P. Panaris, 127 Pandemics of disease, 47 Parametritis, 160 Parasites, ^^ facultative, ^3 Passive immunization, 55, 68 Pasteur's treatment of hydrophobia, 323 results of, 325 Pathogenic bacteria, 33 properties of causative agents of inflammation with relation to animals, 123 Pathogenicity of bacteria, deteimina- tion of, by animal experimentation, 1 10 Penicillii, 337 Peptone-water, 86 Perforative peritonitis, 152 Pericarditis, 150 bacteriologic diagnosis of, 15 1 primary, 150 puerperal, 150 pyemic, 150 secondary, 150 to acute articular rheumatism, 150 to erysipelas, 150 to pneumonia, 150 traumatic, 150 tuberculous, 150 Perinephritis, 158, 160 Peritonitis, 151 arising from the digestive tract, 151 the female genito-urinary tract, 151 the gall-bladder and liver, 15 1 aseptic, 151 bacterial, 151 bacteriologic diagnosis of, 152 circumscribed, 152 diffuse, 152 due to operative intervention, 152 of hematogenous origin, 152 perforative, 152 septic, 151 Peritrocha, 19 Perityphlitis, 154 Permanent bodies, 22 forms of bacteria, 22, 23 Pertussis, 330 Petri's procedure for examination of air, 392 Peyer's patches, typhoid-bacilli in, 170 Pfeiffer's reaction, 62 Phagocytes, fixed, 58 mobile, 58 Phagocytic theory, 58 Phagocytosis, 36 Pharyngitis, acute, 133 Pharyngomycoses, 348 Phlebitis, 132 Phlegmonous anginas, 134 erysipelas, 1 30 Phlegmons, putrid, fetid, due to pro- teus, 310 urinary, 127 Pigment formed by bacteria, 26 malarial, 373 Pigment-bacteria, 26 Pityriasis versicolor, 347 Plague, 200 bacteriologic diagnosis of, 204 etiologic relations of plague-bacilli to, 204 experimental development of, 203 immunity to, 206 prophylaxis of, 205 protective inoculation against, 206 Plague-bacilli, appearance of, in cul- ture, 201 etiologic relations of, to plague, 204 mode of distribution of, 202 morphology of, 200 portals of infection for, 202 vital capability of, 20I Plasma, granular, 364 Plate-cocci, 21 Plate-culture of anaerobic bacteria, 98 Plate-making, Koch's method of, 88 Plates of agar-agar, 94 Pleomorphic bacteria, 24 Pleural effusions, tubercle-bacilli in fluid from, 264 Pleurisy, metastatic, 139 primary, 139 secondary, 139 Pleuritis, 138 bacteriologic investigation of, 140 diagnostic and prognostic signifi- cance of bacteriologic findings in, 140 Pneumaturia, 156 INDEX. 4.;7 Pneumococcus-angina, 134 Pneumococcus-pneumonia, 143 Pneumonia, 142 bacteriologic diagnosis of, 145 complicating infectious diseases, 145 tuberculosis, 145 congenital, 148 croupous, endocarditis secondary to, 148 direct transmission of, 147 epidemic distribution of, 147 hereditary transmission of, 147 . house-epidemics of, 147 mixed forms of, 144 pericarditis secondary to, 150 prognostic significance of bacterio- logic findings in, 146 transmission of, 147 Pneumonomycoses, 150 Pneumothorax, 142 Poison, specific bacterial, 30 Portals of infection, 42 Positive chemotaxis, 58 Potassium hydroxid as a mordant, 104 Potato-bacillus, 405 Potatoes, preparation of, 84 Potato-gelatin, 81 Eisner's modification of, 81 Predisposing influences, 46 Predisposition, 44 general, 45 local, 45 temporary, 44 to smallpox, 326 Preparation of cover-glass specimens, 104 Primary pericarditis, 150 pleurisy, 139 Prophylactic inoculations against te- tanus, 244 Prophylaxis of anthrax, 300 of cholera, 196 of diphtheria, 230 of glanders, 305 of gonorrhea, 314 of leprosy, 280 of plague, 205 of tuberculosis. 266 Proteids, bacterial, 31 Proteus, cultural properties of, 309 febrile icterus due to, 310 hemorrhagic gastro-enteritis due to, 3" morphology of, 308 occurrence of, 310 putrid, fetid phlegmons due to, 310 Proteus, putrid intoxication due to, 310 Proteus-infection, bacteriologic diag- nosis of, 311 experimental development of, 310 Proteus-infections, 308 Protozoa, 364 Prunes, decoction of, 87 Pseudo-diphtheria-bacilli, 214 Pseudo-filaments, 20 Pseudo-influenza-bacilli, 287 Pseudo-membranous anginas, 134 Pseudo-tuberculosis, 276 bacterial, 277 due to animal parasites, 276 to foreign bodies, 276 to highly organized vegetable parasites, 278 etiology of, 276 Psoriasis, 348 Ptomains, 28 Puerperal cystitis, 155 fever, 163 endocarditis secondary to, 148 pericarditis, 150 Pure culture, 87 Purity of infectious material, 39 Purulent foci, multiple, 123 Pus from cold abscesses, tubercle- bacilli in, 264 tubercle-bacilli in, 263 Pustule, malignant, 292 Pyelonephritis, 159 ascending, 159 bacteriologic diagnosis of, 159 descending, 159 experimental development of, 159 Pyemia, 123, 161 bacteriologic diagnosis of> 162 endocarditis secondary to, 148 experimental development of, 163 Pyemic abscesses of the liver, 155 pericarditis, 150 Pyocyaneous general infection, 165 Q. Quartan parasites of malaria, 377 Quinin, action of, 386 R. Rabies, 321 resistance of virus of, 323 Rag-pickers' disease, 295 Railway-cars, disinfection of, 425 Ray-fungus, 356 portal of infection for, 350 438 INDEX. Reaction, Gruber's, 63 of immunity, 66 of infection, 66 Pfeiffei's, 62 Relapsing fever, 332 occurrence of spirilla of, 333 recovery from, 334 spirilla of, 332 transmission of, 334 Relative immunity, 56 Rheumatic tetanus, 239 Rheumatism, acute articular, endo- carditis secondary to, 148 pericarditis secondary to, 150 articular, 331 Rhinitis, acute, 133 fibrinous, 133 Rhinoscleroma, 133 Rice-pap, 87 Rod-shaped bacteria, 17 Roll-plate, 94 Root-bacillus, 404 S. Saccharomyces cerevisise, 338 hominis, 363 subcutaneus tumefaciens, 363 Salpingitis, 159 Sand-filtration of water, 399 Saprophytes, S3 facultative, 33 Sarcina lutea, 406 yelloM^, 406 Sarcinse, 21 Sausage-poisoning, 245 Scarlatinal angina, 135 Scarlet, fever, 329 endocarditis following, 149 incubation of, 329 virus of, 329 Scrofulosis, 258 Secondary endocarditis, 148 infection, 46 localization, 46 pericarditis, 150 pleurisy, 139 Sections of tissue, staining of, 106 tubercle-bacilli in, 264 Segmentation, 337 Segmented molds, 338 Self-purification of water, 398 Sepsis, 123 Septic peritonitis, 151 Septicemia, 34, 123, 161 bacteriologic diagnosis of, 1 62 cryptogenetic, 162 endocarditis secondary to, 148 Septicemia, experimental develop- ment of, 163 Serum, immune, lysogenic activity of, 62 Serum-immunity, 71 Serum-plates, preparation of, 83 Serum-therapy, 72 of tetanus, 242 Ships, disinfection of, 425 Sick-room, disinfection of, 423 Skin, infection with anthrax-bacilli through, 293 parasitic diseases of, 34I Smallpox, 325 endocarditis following, 149 immunity to, 326 portal of infection for, 325 predisposition to, 326 virus of, 325 Soil, bacilli in, 390 bacillus of malignant edema in, 391 of tetanus in, 390 bacteria in, 390, 401 bacteriologic examination of, 389 malarial parasites in, 391 method of investigation of, 389 permanent spores in, 391 Specific therapy, 72 of botulism, 251 of diphtheria, 218 Specificity of bacteria, determination of, by animal experimentation, no of immunity, 56 Spheric bacteria, 17 Spiral bacteria, 17 Spirilla, 17 Spirillaceae, 17 Spleen, puncture of, in typhoid fever, 175 typhoid-bacilli in, 170 vSporangium, 337 Spore-formation, 23 arthrogenous, 23 endogenous, 23 Spore-germination, 23 Spore-membrane, 22 Spores, 22, 23, 336 staining of, 108 Sputum, disinfection of, 42I tubercle-bacilli in, 263 Stab-culture, 96 Stained preparations, examination of, 103 Staining, double, 107 Gram's method of, 106 Giinther's modification of Gram's method of, 107 INDEX. 439 Staining of bacteria, I02 of capsules, io8 of cover-glass specimens, 104 of flagella, 109 of sections of tissue, 106 of spores, 108 Weigert's modification of Gram's method of, 107 Staphylococci, 21, 124, 125 Staphylococcus cereus albus, 117 flavus, 117 pyogenes albus, 1 16 aureus, 115 citreus, 1 16 Staphylococcus-angina, 134 Staphylococcus-pneumonia, 144 Steam, disinfection by, 41 1 live, sterilization by, 75 Sterigmata, 337 Sterilization, 74 by dry heat, 74 by live steam, 75 discontinuous, 77 fractional, 77 Streak, fractional, 95 Streak-culture, 96 Streak-plates, agar, 94 Streptococci, 20, 124, 125 microscopic arrangement of, in skin, 130 Streptococcus brevis, 118 conglomeratus, 118 etiologic significance of, for erysip- elas, 131 lanceolatus Pasteur, 118 longus, 118 pyogenes (erysipelatis), 117 Streptococcus-angina, 134 Streptococcus-diseases, specific treat- ment of, 132 Streptococcus-pneumonia, 144 Streptothricege,'24 Streptothrices, 337 pathogenic, 362 Streptothnx Eppinger, 362 farcinica, 363 Subcutaneous inoculation, no Subdural inoculation, in Sulphur-bacteria, 27 Summer diarrhea, 190 Suppuration, 115 causative agents of, in healthy per- sons, 125 outside the body, 125 Suppurative myocarditis, 151 processes, endocarditis secondary to, 148 typhoid-bacilli in, 171 Susceptibility of infected organism, 44 Susceptibility to bacterial diseases, 45 Symbiosis, 41 Sympathetic ophthalmia, 161 Syphilis, 314 bacillus of, 314 congenital, transmission of, from father, 319 of, from mother, 320 conveyance of, 317 extrauterine infection with, 320 hereditary, 319 transmission of, 317 heredity of, 319 immunity to, 318 intrauterine infection with, 320 paternal infection with, 319 portal of infection for, 317 specific therapy of, 318 Syphilitic infection, 317 Temperature-maximum, 21 Temperature- minimum, 21 Temperature-optimum, 21 Temporary predisposition, 44 Testicles, typhoid-bacillus in, 171 Tetanus, 230 bacillus of, in soil, 391 bacteriologic diagnosis of, 240 cure of, 241 direct contagion in, 239 idiopathic, 239 immunity to, 241 in animals, 233 nature of, 235 period of incubation of, 234 portal of infection for, 239 prophylactic inoculations against, 243 rheumatic, 239 serum- therapy of, 242 Tetanus-bacillus, 231 cultural properties of, 232 pure cultures of, 232 Tetanus-infection in human beings, Tetanus-toxin, 236 Tetrad arrangement, 21 Tetragenus, 21 Thallus, 336 Therapeutic experiments in tubercu- losis, 269 Therapy, specific, 72 Thermal death-point, 22 Thermophilic bacteria, 22 440 INDEX. Thiothrix, 27 Throat, inflammations of, 133 Thrush, 351 course of, 352 diagnosis of, 354 occurrence of, 351 sources of infection for, 353 Thrush-deposits, microscopic exami- nation of, 351 Thrush-fungus, botanic position of, 353 culture of, 353 Tissue, sections of, staining of, 106 Tissue-immunity, 71 Toxalbumins, 30 Toxic infection, 34 Toxins of anthrax-bacilli, 297 of cholera, 189 specific, 29 Toxoids, 223 Trachoma, 161 Traumatic pericarditis, 150 Trichophyton, cultural properties of, .346 microscopic demonstration of, 347 tonsurans, 345 Tropical abscess of liver, 155 Tubercle-bacilli, diagnostic demon- stration of, 262 distribution of, 260 in feces, 264 * in fluid from pleural effusions, 264 in milk, 265 in pus, 263 from cold abscesses, 264 in sections, 264 in. sputum, 263 in urine, 264 occurrence of, 260 Tubercle-bacillus, 251 culture of, 253 morphology of, 25 1 pathogenic activity of, 258 portals of entry for, 257 resistance of, 255 staining of, 251 Tuberculin, curative influence of, 270 diagnostic utility of, 171 new preparations of, 273 O, 273 R, 273 therapeutic employment of, 272 treatment of tuberculosis with, 270 Tuberculosis, 251 congenital, 268 due to foreign bodies, 276 experimental, in animals, 256 general, 260 Tuberculosis, general miliary, 256 heredity of, 267 localization of, 260 miliary, 260 mixed infection in, 260 pathologic anatomy of, 257 pericarditis, 150 pneumonia complicating, 145 predisposition to, 259 prophylaxis of, 266 spontaneous, in animals, 256 susceptibility to, 259 therapeutic experiments in, 269 treatment of, with tuberculin, 270 Tuberculous cystitis, 155 endocarditis, 149 Typhoid endocarditis, 149 fever, 166 abscess complicating, 127 agglutination-phenomenon in, 76 bacteriologic diagnosis of, 174 cure of, 1 79 epidemic distribution of, 170 etiologic relations of typhoid- bacilli to, 173 experimental development of, 172 immunity to, 179 infection with, by way of diges- tive tract, 169 by way of lungs, 169 through articles of food, 169 through drinking-water, 1 70 mixed infection in, 171 prophylaxis of, 179 puncture of spleen in, 175 secondary infection in, 171 special predisposition to, 170 temporal and local predisposition to, 170 intoxication, 173 patients, occurrence of typhoid- bacilli in, 170 Typhoid-bacilli differentiated from bacterium coli commune, 174 dissemination of, 169 etiologic relations of, to typhoid fever, 173 examination of water for, 1 78 in blood, 1 71 in bone-marrow, 1 70 in feces, 171 in kidneys, 171 in liver, 170 in lungs, 171 in meninges, 171 in mesenteric glands, 1 70 INDEX. 441 Typhoid-bacilli in myocardium, 1 71 in Peyer's patches, 170 in spleen, 170 in testicles, 171 in typhoid patients, 170 in urine, 171 Typhoid-bacillus, appearance of, in cultures, 167 morphology of, 166 portals of infection for, 169 pyogenic activity of, 172 vital activity of, 168 Typhoid-like bacilli, 402 U. Ulcerative endocarditis, 149 Urinary phlegmon, 127 Urine, ammoniacal fermentation of, 156 bacteria in, diagnostic and prog- nostic significance of, 158 disinfection of, 421 tubercle-bacilli in, 264 typhoid-bacilli in, 17 1 V. Vaccination, 326 against anthrax, 299 hydrophobia, 323 Vaccinia, 326 Varicella, 328 Variola, 325 nature of virus of, 327 Variolation, 326 Vehicles, disinfection of, 425 Vibrio aquatilis Giinther, 407 Berolinensis, 407 Gindha, 408 Lissabon, 408 Vibrio, Massowah, 410 Metschnikoff, 408 phosphorescens Dunbar, 410 septicus, occurrence of, 307 Vibrios, 17 Virulence of infectious agents, 37 Visceral mycoses, 350 Vulvitis, 159 W, Wash-water, disinfection of, 420 Water, bacteria in, 401 bacteriologic examination of, 389, 396 boiling, disinfection by, 41 1 examination of, for cholera-bacilli, 195 for typhoid-bacilli, 178 filtration of, 399 hot, disinfection by, 411 method of examination of, 396 number of bacteria in, 397 of condensation, 82 sand-filtration of, 399 self- purification of, 398 Water-conduits, disinfection of, 421 Waters, artificial carbonated, 401 Water-supply, sources and mode of, 399 Weigert's modification of Gram's method of staining, 107 Weil's disease, 310 Whey, 86 Whooping-cough, 330 Wool-sorters disease, 295 Y. Yeast-fungi, 338 Yeasts, pathogenic, 341, 363 Yellow sarcina, 406 CATALOGUE OF THE MEDICAL PUBLICATIONS OF W. B. SAUNDERS, No* 925 WALNUT STREET, PHILADELPHIAc Arranged Alphabetically and Classified under Subjects* THE books advertised in this Catalogue as being sold by subscription are usually to be obtained from travelling solicitors, but they will be sent direct from the office of pub- lication (charges of shipment prepaid) upon receipt of the prices given. All the other books advertised are commonly for sale by booksellers in all parts of the United States; but books will be sent to any address, carriage prepaid, on receipt of the published price. Money may be sent at the risk of the publisher in either of the following ways : A post- office money order, an express money order, a bank check, and in a registered letter. Money sent in any other way is at the risk of the sender. See pages 30^ Z\y for a L^t of Contents classified according to subjects* LATEST PUBLICATIONS. International Text-Book of Surgfery. See page 32. American Text-Book of Surgery — Third (Revised) Edition* Seepages. American Text-Book of Dis* of Eye, Ear, Nose, and Throat* Page 3* American Text-Book of Genito-Urinary and Skin Diseases* Page 4* Heisler's Embryology* See page 32. Nancf ede's Principles of Surgery* See page 32. Jackson's Diseases of the Eye* See page 32. Kyle on the Nose and Throat* See page 15. Pryor's Pelvic Inflammations* See pages J9 and 32. Abbott's Hygiene of Transmissible Diseases* See page 32. Anders' Practice of Medicine — Third (Revised) Edition* See page 6* Vierordt's Medical Diagnosis — ^Fourth (Revised) Edition* See page 29. Church and Peterson's Nervous arid Mental Diseases* See page 8. Da Gjsta's Surgery — Revised and Enlarged Edition* See page JO. Saunders' Medical Hand-Atlases* See page 2. Griffith on the Baby — ^Revised Edition. See page J2. Butler's Materia Medica and Therapeutics — Third (Revised) Ed* Page 8* De Schweinitz's Diseases of the Eye — Third (Revised) Ed. See page JO. Vecki's Sexual Impotence* See page 28. Stoney's Materia Medica for Nurses. See page 28. McFarland's Pathogenic Bacteria — ^Revised Edition* See page J 7. American Pocket Medical Dictionary — ^Second (Revised) Ed* Page JO. Stengel's Text-Book of Pathology* Second Edition* See page 26. Hirst's Text-Book of Obstetrics* See page 13. SAUNDERS' MEDICAL HAND-ATLASES. The series of books included under this title consists of authorized translations into English of the world-famous Lehmann Medicinische Handatlanten, which for sci- entific accuracy, pictorial beauty, compactness, and cheapness surpass any similar volumes ever published. Each volume contams from 50 to 100 colored plates, executed by the most skilful German lithographers, besides numerous illustrations in the text. There is a full and appropriate description of each plate, and each book contains a condensed but adequate outline of the subject to which it is devoted. One of the most valuable features of these atlases is that they offer a ready and satis- factory substitute for clinical observation. To those unable to attend important clinics these books will be absolutely indispensable. In planning this series of books arrangements were made with representative publishers in the chief medical centers of the world for the publication of translations of the atlases into nine different languages, the lithographic plates for all these editions being made in Ger- many, where work of this kind has been brought to the greatest perfection. The expense of making the plates being shared by the various publishers, the cost to each one was materially reduced. Thus by reason of their universal translation and reproduction, the publish- ers have been enabled to secure for these atlases the best artistic and professional talent, to produce them in the most elegant style, and yet to offer them at a pl-ice heretofore unapproached in cheapness. The success of the undertaking is demon- strated by the fact that the volumes have already appeared in nine different languages — German, English, French, Italian, Russian, Spanish, Danish, Swedish-, and Hungarian. In view of the striking success of these works, Mr. Saunders has contracted with the publisher of the original German edition for one hundred thousand copies of the atlases. In consideration of this enormous undertaking, the publisher has been enabled to prepare and furnish special additional colored plates, making the series even handsomer and more complete than was originally intended. 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Illus- trated. Prices per volume : Cloth, ^5.00 net ; Sheep or Half Morocco, $6.00 net. Sold by Subscription. " I am quite sure it will commend itself both to practitioners and students of medicine, and become one of our most popular text-books." — Alfred Loomis, M.D., LL.D., Pro- fessor of Pathology and Practice of Medicine, University of the City of New York. •* We reviewed the first volume of this work, and said : * It is undoubtedly one of the best text-books on the practice of medicine which we possess.' A consideration of the second and last volume leads us to modify that verdict and to say that the completed work is in our opinion the best of its kind it has ever been our fortune to see. " — New York Medical JourncU. lUtsstrated Catalos^e of the ** American Text-Books'' sent free tfpon application* 6 Medical Publications of W, B, Saunders. AN AMERICAN YEAR-BOOK OF MEDICINE AND SURGERY. A Yearly Digest of Scientific Progress and Authoritative Opinion in all branches of Medicine and Surgery, drawn from journals, monographs, and text-books of the leading American and Foreign authors and investigators. Collected and arranged, with critical editorial com- ments, by eminent American specialists and teachers, under the general editorial charge of George M. Gould, M.D. One handsome imperial octavo volume of about 1200 pages. Uniform in style, size, and general make-up with the ** American Text-Book" Series. Cloth, $6.50 net; Half Morocco, $7.50 net. Sold by Subscription. ** It is difficult to know which to admire most — the research and industry of the distin- guished band of experts whom Dr. Gould has enlisted in the service of the Year-Book, or the wealth and abundance of the contributions to every department of science that have been deemed worthy of analysis. . . . It is much more than a mere compilation of abstracts, for, as each section is entrusted to experienced and able contributors, the reader has the advantage of certain critical commentaries and expositions . . . proceeding from writers fully qualified to perform these tasks. . . . It is emphatically a book which should find a place in every medical library, and is in several respects more useful than the famous *Jahrbucher' of Germany." — London Lancet. THE AMERICAN POCKET MEDICAL DICTIONARY. [See D or I and' s Pocket Dictionary ^ page 10.] ANDERS' PRACTICE OF MEDICINE. Third Revised Edition. A Text-Book of the Practice of Medicine. By James M. Anders, M.D., Ph.D., LL.D., Professor of the Practice of Medicine and of . Clinical Medicine, Medico- Chirurgical College, Philadelphia. In one handsome octavo volume of 1292 pages, fully illustrated. Cloth, $5.50 net; Sheep or Half Morocco, ^^6.50 net. "It is an excellent book, — concise, comprehensive, thorough, and up to date. It is a credit to you ; but, more than that, it is a credit to the profession of Philadelphia — to us." James C. Wilson, Professor of the Practice of Medicine and Clinical Medicine^ Jefferson Medical College ^ Philadelphia. ASHTON'S OBSTETRICS. Fourth Edition, Revised. Essentials of Obstetrics. By W. Easterly Ashton, M.D., Pro* fessor of Gynecology in the Medico-Chirurgical College, Philadelphia. Crown octavo, 252 pages; 75 illustrations. Cloth, ^1.09; interleaved for notes, ^1.25. [See Saunders* Question- Compends, page 21.] " Embodies the vphole subject in a nut-shell. We cordially recommend it to our read- ers."— Chicago Medical Times. BALL*S BACTERIOLOGY. Third Edition, Revised. Essentials of Bacteriology ; a Concise and Systematic Introduction to the Study of Micro-organisms. By M. V. Ball, M.D., Bacteriol- ogist to St. Agnes' Hospital, Philadelphia, etc. Crown octavo, 218 pages; 82 illustrations, some in colors, and 5 plates. Cloth, $1.00; interleaved for notes, $1.25. [See Saunders* Question- Compends, page 21.] " The student or practitioner can readily obtain a knowledge of the subject from a perusal of this book. The illustrations are clear and satisfactory." — Medical Record, New York. Medical Publications of W. B. Saunders, 1 BASTINGS BOTANY. Laboratory Exercises in Botany. By Edson S. Bastin, M.A., late Professor of Materia Medica and Botany, Philadelphia College of Pharmacy. Octavo volume of 536 pages, with 87 plates. Cloth, ^^2.50. **It is unquestionably the best text-book on the subject that has yet appeared. The work is eminently a practical one. We regard the issuance of this book as an important event in the history of pharmaceutical teaching in this country, and predict for it an unquali- fied success." — Alumni Report to the Philadelphia College of Pharmacy. "There is no work like it in the pharmaceutical or botanical literature of this country, and we predict for it a wide circulation." — American Journal of Pharmacy, BECK'S SURGICAL ASEPSIS. A Manual of Surgical Asepsis. By Carl Beck, M.Do, Surgeon to St. Mark's Hospital and the New York German Poliklinik, etc. 306 pages; 65 text-illustrations, and 12 full-page plates. Cloth, ^1.25 net. "An excellent exposition of the *very latest' in the treatment of wounds as practised by leading German and American surgeons." — Birmingham (Eng.) Medical Review, "This little volume can be recommended to any who are desirous of learning the details of asepsis in surgery, for it will serve as a trustworthy guide." — London Lancet. BOISLINIERE'S OBSTETRIC ACCIDENTS, EMERGENCIES, AND OPERATIONS. Obstetric Accidents, Emergencies, and Operations. By L. Ch. BoiSLiNiERE, M.D., late Emeritus Professor of Obstetrics, St. Louis Medical College. 381 pages, handsomely illustrated. Cloth, ;^2.oo net. ** It is clearly and concisely written, and is evidently the work of a teacher and practi- tioner of large experience." — British Medical Journal. " A manual so useful to the student or the general practitioner has not been brought to our notice in a long time. The field embraced in the title is covered in a terse, interesting way." — Yale Medical Journal. BROCKWAY'S MEDICAL PHYSICS. Second Edition, Revised. Essentials of Medical Physics. By Fred J. Brockway, M.D., Assistant Demonstrator of Anatomy in the College of Physicians and Surgeons, New York. Crown octavo, 330 pages; 155 fine illustrations. Cloth, ^i.oo net ; interleaved for notes, ^1.25 net. [See Saunders* Que stion-Comp ends, page 21.] " The student who is well versed in these pages will certainly prove qualified to com. prehend with ease and pleasure the great majority of questions involving physical principles likely to be met with in his medical studies." — American Practitioner and News. **We know of no manual that affords the medical student a better or more concise exposition of physics, and the book may be commended as a most satisfactory presentation of those essentials that are requisite in a course in medicine." — New York Medical JoumaU ** It contains all that one need know on the subject, is well written, and is copiously illustrated." — Medical Record^ New York. BURR ON NERVOUS DISEASES. A Manual of Nervous Diseases. By Charles W. Burr, M.D., Clinical Professor of Nervous Diseases, Medico-Chirurgical College, Philadelphia; Pathologist to the Orthopedic Hospital and Infirmary for Nervous Diseases; Visiting Physician to St. Joseph's Hospital, etc. In Preparation. 8 Medical Publications of W. B. Saunders. BUTLER'S MATERIA MEDICA, THERAPEUTICS, AND PHAR- MACOLOGY. Third Edition, Revised. A Text-Book of Materia Medica, Therapeutics, and Pharma- coJog^y. By George F. Butler, Ph.G., M.D., Professor of Materia Medica and of Clinical Medicine in the College of Physicians and Surgeons, Chicago ; Professor of Materia Medica and Therapeutics, Northwestern University, Woman's Medical School, etc. Octavo, 874 pages, illustrated. Cloth, ^^4.00 net; Sheep, ^5.00 net. ** Taken as a whole, the book may fairly be considered as one of the most satisfactory of any single-volume works on materia medica in the market," — Journal of the American Medical Association. CERNA ON THE NEWER REMEDIES. Second Edition, Revised. Notes on the Newer Remedies, their Therapeutic Applications and Modes of Administration. By David Cerna, M.D., Ph.D., formerly Demonstrator of and Lecturer on Experimental Therapeutics in the University of Pennsylvania ; Demonstrator of Physiology in the Medical Department of the University of Texas. Rewritten and greatly enlarged. Post-octavo, 253 pages. Cloth, ^1.25. " The appearance of this new edition of Dr. Cerna's very valuable work shows that it is properly appreciated. The book ought to be in the possession of every practising physi- cian."—iV^ York Medical Journal. CHAPIN ON INSANITY. A Compendium of Insanity. By John B. Chapin, M.D., LL.D., Physician-in-Chief, Pennsylvania Hospital for the Insane ; late Physi- cian-Superintendent of the Willard State Hospital, New York ; Hon- orary Member of the Medico-Psychological Society of Great Britain, of the Society of Mental Medicine of Belgium. i2mo, 234 pages, illustrated. Cloth, $1.25 net. " The prafctical parts of Dr. Chapin's book are what constitute its distinctive merit. We desire especially to call attention to the fact that on the subject of therapeutics of insanity the work is exceedingly valuable. It is not a made book, but a genuine condensed thesis, which has all the value of ripe opinion and all the charm of a vigorous and natural style." — Philadelphia Medical Journal. CHAPMAN'S MEDICAL JURISPRUDENCE AND TOXICOLOGY. Second Edition, Revised. Medical Jurisprudence and Toxicology. By Henry C. Chapman, M.D., Professor of Institutes of Medicine and Medical Jurisprudence in the Jefferson Medical College of Philadelphia. 254 pages, with 55 illustrations and 3 full-page plates in colors. Cloth, ^1.50 net. "The best book of its class for the undergraduate that we know of." — New York Medical Times. CHURCH AND PETERSON'S NERVOUS AND MENTAL DISEASES. Nervous and Mental Diseases. By Archibald Church, M. D., Professor of Mental Diseases and Medical Jurisprudence in the North- western University Medical School, Chicago ; and Frederick Peter- son, M. D., Clinical Professor of Mental Diseases, Woman's Medical College, N. Y.; Chief of Clinic, Nervous Dept., College of Physi- cians and Surgeons, N. Y. Handsome octavo volume of 843 pages, profusely illustrated. Cloth, ;^5.oo net; Half Morocco, ;^6.oo net. Medical Publications of W. B. Saunders. 9 CLARKSON'S HISTOLOGY. A Text-Book of Histology, Descriptive and Practical. By Arthur Clarkson, M.B., CM. Edin., formerly Demonstrator of Physiology in the Owen's College, Manchester; late Demonstrator of Physiology in Yorkshire College, Leeds. Large octavo, 554 pages; 22 engravings in the text, and 174 beautifully colored original illustra- tions. Cloth, strongly bound, ^4.00 net. " The work must be considered a valuable addition to the list of available text- books, and is to be highly recommended." — JVeza York Medical Journal. "This is one of the best works for students we have ever noticed. We predict that the book will attain a well-deserved popularity among our students." — Chicago Medical Recorder. CLIMATOLOGY. Transactions of the Eighth Annual Meeting of the American Climatological Association, held in Washington, September 22-25, 1891. Forming a handsome octavo volume of 276 pages, uniform with remainder of series. (A limited quantity only.) Cloth, ^1.50. COHEN AND ESHNER'S DIAGNOSIS. Essentials of Diagnosis. By Solomon Solis-Cohen, M.D., Pro- fessor of Clinical Medicine and Applied Therapeutics in the Philadel- phia Polyclinic ; and Augustus A. Eshner, M.D., Professor of Clinical Medicine in the Philadelphia Polyclinic. Post-octavo, 382 pages; 55 illustrations. Cloth, $1.50 net. [See Saunders' Question- Compends, page 21.] **We can heartily commend the book to all those who contemplate purchasing a 'com- pend.' It is modern and complete, and will give more satisfaction than many other works which are perhaps too prolix as well as behind the times." — Medical Review, St. Louis. CORWIN'S PHYSICAL DIAGNOSIS. Third Edition, Revised. Essentials of Physical Diagnosis of the Thorax. By Arthur M, CoRWiN, A.M., M.D., Demonstrator of Physical Diagnosis in Rush Medical College, Chicago ; Attending Physician to Central Free Dis- pensary, Department of Rhinology, Laryngology, and Diseases of the Chest, Chicago. 219 pages, illustrated. Cloth, flexible covers, $1.25 net. ** It is excellent. The student who shall use it as his guide to the careful study of physical exploration upon normal and abnormal subjects can scarcely fail to acquire a good working knowledge of the subject." — Philadelphia Polyclinic. '*A most excellent little work. It brightens the memory of the differential diagnostic signs, and it arranges orderly and in sequence the various objective phenomena to logical solution of a careful diagnosis. ' ' — Journal of Nervous and Mental Diseases. CRAGIN'S GYNAECOLOGY. Fourth Edition, Revised. Essentials of Gynaecology. By Edwin B. Cragin, M. D., Lecturer in Obstetrics, College of Physicians and Surgeons, New York. Crown octavo, 200 pages; 62 illustrations. Cloth, ^i.oo ; interleaved for notes, $1.25. \^QQ Saunders' Question- Compends, page 21. J "A handy volume, and a distinct improvement on students' compends in general. No author who was not himself a practical gynecologist could have consulted the student's needs so thoroughly as Dr. Cragin has done," — Medical Record, New York. 12 Medical Publications of W, B, Saunders. GRAFSTROM'S MECHANO=THERAPY. A Text=Book of Mechano-Therapy (Massage and Medical Gym- nastics). By Axel V. Grafstrom, B. Sc, M. D., late Lieutenant in the Royal Swedish Army ; late House Physician City Hospital, Black- well's Island, New York. i2mo, 139 pages, illustrated. Cloth, ^i.oo net. GRIFFITH ON THE BABY. Second Edition, Revised. The Care of the Baby. By J. P. Crozer Griffith, M.D., Clini- cal Professor of Diseases of Children, University of Pennsylvania; Physician to the Children's Hospital, Philadelphia, etc. i2mo, 404 pages, with 67 illustrations in the text, and 5 plates. Cloth, ;^i.5o. " The best book for the use of the young mother with which we are acquainted. . . . There are vpry few general practitioners who could not read the book through with advan- tage. ' ' — Archives of Pediatrics. "The whole book is characterized by rare good sense, and is evidently written by a master hand. It can be read with benefit not only by mothers but by medical students and by any practitioners who have not had large opportunities for observing children." — Ameri- can Jourtial of Obstetrics. GRIFFITH'S WEIGHT CHART. Infant's Weight Chart. Designed by J. P. Crozer Griffith, M. D. , Clinical Professor of Diseases of Children in the University of Penn- sylvania, etc. 25 charts in each pad. Per pad, 50 cents net. A convenient blank for keeping a record of the child's weight during the first two years of life. Printed on each chart is a curve representing the average weight of a healthy infant, so that any deviation from the normal can readily be detected. GROSS, SAMUEL D., AUTOBIOGRAPHY OF. Autobiography of Samuel D. Gross, M.D., Emeritus Professor of Surgery in the Jefferson Medical College, Philadelphia, with Remi- niscences of His Times and Contemporaries. Edited by his Sons, Samuel W. (jROSS, M.D., LL.D., late Professor of Principles of Sur- gery and of Clinical Surgery in the Jefferson Medical College, and A. Haller Gross, A.M., of the Philadelphia Bar. Preceded by a Memoir of Dr. Gross, by the late Austin Flint, M.D., LL.D. In two handsome volumes, each containing over 400 pages, demy octavo, extra cloth, gilt tops, with fine Frontispiece engraved on steel. Price per volume, ^2.50 net. ** Dr. Gross was perhaps the most eminent exponent of medical science that America has yet produced. His Autobiography, related as it is with a fulness and completeness seldom to be found in such works, is an interesting and valuable book. He comments on many things, especially, of course, on medical men and medical practice, in a very interest- ing way," — The Spectator, London, England. HAMPTON'S NURSING. Second Edition, Revised and Enlarged. Nursing : Its Principles and Practice. By Isabel Adams Hamp- ton, Graduate of the New York Training School for Nurses attached to Bellevue Hospital ; late Superintendent of Nurses and Principal of the Training School for Nurses, Johns Hopkins Hospital, Baltimore, Md. 12 mo, 512 pages, illustrated. Cloth, ^2.00 net. " Seldom have we perused a book upon the subject that has given us so much pleasure as the one before us. We would strongly urge upon the members of our own profession the need of a book like this, for it will enable each of us to become a training school in him- self."— Ontario Medical Journal. Medical Publications of W. B, Saunders. 13 HARE'S PHYSIOLOGY. Fourth Edition, Revised. Essentials of Physiology. By H. A. Hare, M.D., Professor of Therapeutics and Materia Medica in the Jefferson Medical College of Philadelphia. Crown octavo, 239 pages. Cloth, ^i.oo net; inter- leaved for notes, ^^1.25 net. [See Saunders* Question- Compends, page 21.] " The best condensation of physiological knowledge we have yet seen." — Medical Record, New York. HARrS DIET IN SICKNESS AND IN HEALTH. Diet in Sickness and in Health. By Mrs. Ernest Hart, formerly Student of the Faculty of Medicine of Paris and of the London School of Medicine for Women ; with an Introduction by Sir Henry Thompson, F.R.C.S., M.D., London. 220 pages.. Cloth, ^1.50. " We recommend it cordially to the attention of all practitioners ; both to them and lo their patients it may be of the greatest service." — New York Medical Journal. HAYNES' ANATOMY. A Manual of Anatomy. By Irving S. Haynes, M.D., Adjunct Professor of Anatomy and Demonstrator of Anatomy, Medical Depart- ment of the New York University, etc. 680 pages, illustrated with 42 diagrams in the text, and 134 full-page half-tone illustrations from original photographs of the author's dissections. Cloth, ^^2.50 net. " This book is the work of a practical instructor — one who knows by experience the requirements of the average student, and is able to meet these requirements in a very satis- factory way. The book is one that can be commended." — Medical Record, New York. HEISLER'S EMBRYOLOGY. A Text=Book of Embryology. By John C. Heisler, M.D., Pro- fessor of Anatomy in the Medico- Chirurgical College, Philadelphia. Oc- tavo volume of 405 pages, handsomely illustrated. Cloth, $2.50 net. HIRST'S OBSTETRICS. A Text=Book of Obstetrics. By Barton Cooke Hirst, M. D., Professor of Obstetrics in the University of Pennsylvania. Handsome octavo volume of 848 pages, with 618 illustrations, and 7 colored plates. Cloth, ^5.00 net; Sheep or Half Morocco, ;^6.oo net. "The illustrations are numerous and are works of art, many of them appearing for the first time. The arrangement of the subject-matter, the foot-notes, and index are beyond criticism. As a true model of what a modern text-book on obstetrics should be, we feel justified in affirming that Dr. Hirst's book is without a rival." — New York Medical Record. HYDE AND MONTGOMERY ON SYPHILIS AND THE VENEREAL DISEASES. Syphilis and the Venereal Diseases. By James Nevins Hyde, M.D., Professor of Skin and Venereal Diseases, and Frank H. Mont- gomery, M.D., Lecturer on Dermatology and Genito-Urinary Diseases in Rush Medical College, Chicago, 111. 618 pages, profusely illustrated. Cloth, $2.50 net. •* We can commend this manual to the student as a help to him in his study of venereal diseases. ' ' — Liverpool Medico- Chirurgical Journal. "The best student's manual which has appeared on the subject." — SL Louis Medical and Surgical Journal. 14 Medical Publications of W, B, Saunders, JACKSON AND GLEASON'S DISEASES OF THE EYE, NOSE, AND THROAT. Second Edition, Revised. Essentials of Refraction and Diseases of the Eye. By Edward Jackson, A.M., M.D., Professor of Diseases of the Eye in the Phila- delphia Polyclinic and College for Graduates in Medicine ; and — Essentials of Diseases of the Nose and Throat. By E. Bald- win Gleason, M.D. , Surgeon-in-Charge of the Nose, Throat, and Ear Department of the Northern Dispensary of Philadelphia. Two volumes in one. Crown octavo, 290 pages; 124 illustrations. Cloth, ^i.oo; interleaved for notes, ^1.25. [See Saunders' Question- Compends, page 21.] " Of great value to the beginner in these branches. The authors are both capable men, and know what a student most needs." — Medical Record, New York. KEATING'S DICTIONARY. Second Edition, Revised. A New Pronouncing Dictionary of Medicine, with Phonetic Pronunciation, Accentuation, Etymology, etc. By John M. Keating, M.D., LL.D., Fellow of the College of Physicians of Phila- delphia; Vice-President of the American Paediatric Society; Editor "Cyclopaedia of the Diseases of Children," etc.; and Henry Hamilton, Author of *•' A New Translation of Virgil's ^neid into English Rhyme," etc.; with the collaboration of J. Chalmers Da- Costa, M.D., and Frederick A. Packard, M.D. With an Appendix containing Tables of Bacilli, Micrococci, Leucoma'ines, Ptomaines; Drugs and Materials used in Antiseptic Surgery; Poisons and their Antidotes; Weights and Measures; Thermometric Scales; New Official and Unofficial Drugs, etc. One volume of over 800 pages. Prices, with Denison's Patent Ready-Reference Index; Cloth, ^5.00 net; Sheep or Half Morocco, ^6.00 net; Half Russia, $6.50 net. Without Patent Index: Cloth, $4.00 net; Sheep or Half Morocco, ;^5.oo net. '* I am much pleased with Keating's Dictionary, and shall take pleasure in recommend- ing it to my classes." — Henry M. Lyman, M.D., Professor of the Principles and Practict of Medicine, Rush Medical College, Chicago, III. " I am convinced that it will be a very valuable adjunct to my study-table, cpnvenient in size and sufficiently full for ordinary use." — C. A, Lindsley, M.D., Professor of the Theory and Practice of Medicine, Medical Dept. Yale University. KEATINQ»S LIFE INSURANCE. How to Examine for Life Insurance. By John M. Keating, M. D., Fellow of the College of Physicians of Philadelphia; Vice- President of the American Paediatric Society; Ex- President of thje Association of Life Insurance Medical Directors. Royal octavo, 211 pages ; with two large half-tone illustrations, and a plate prepared by Dr. McClellan from special dissections ; also, numerous other illustra- tions. Cloth, $2.00 net. " This is by far the most useful book vv^hich has yet appeared on insurance examination, a subject of growing interest and importance. Not the least valuable portion of the volume is Part II., which consists of instructions issued to their examining physicians by twenty-four representative companies of this country. If for these alone, the book should be at the right hand of every physician interested in this special branch of medical science." — The Medical News. Medical Publications of W, B. Saunders, 15 KEEN ON THE SURGERY OF TYPHOID FEVER. The Surgical Complications and Sequels of Typhoid Fever. By Wm. W. Keen, M.D., LL.D., Professor of the Principles of Sur- gery and of Clinical Surgery, Jefferson Medical College, Philadelphia; Corresponding Member of the Societe de Chirurgie, Paris ; Honorary Member of the Societe Beige de Chirurgie, etc. Octavo volume of 386 pages, illustrated. Cloth, $3.00 net. " This is probably the first and only work in the English language that gives the reader a clear view of what typhoid fever really is, and what it does and can do to the human organism. This book should be in the possession of every medical man in America." — American Medico-Surgical Bulletin. KEEN'S OPERATION BLANK. Second Edition, Revised Form. An Operation Blank, with Lists of Instruments, etc. Required in Various Operations. Prepared by W. W. Keen, M.D., LL.D., Professor of the Principles of Surgery in Jefferson Medical College, Philadelphia. Price per pad, containing blanks for fifty operations, 50 cents net. KYLE ON THE NOSE AND THROAT. Diseases of the Nose and Throat. By D. Braden Kyle, M.D., Clinical Professor of Laryngology and Rhinology, Jefferson Medical College, Philadelphia; Consulting Laryngologist, Rhinologist, and Otologist, St. Agnes' Hospital. Handsome octavo volume of about 630 pages, with over 150 illustrations and 6 lithographic plates. Price, Cloth, 114.00 net; Half Morocco, $5.00 net. LAINE'S TEMPERATURE CHART. Temperature Chart. Prepared by D. T. Laine, M.D. Size 8 x 13^ inches. A conveniently arranged Chart for recording Temperature, with columns for daily amounts of Urinary and Fecal Excretions, Food, Remarks, etc. On the back of each chart is given in full the method of Brand in the treatment of Typhoid Fever. Price, per pad of 25 charts, 50 cents net. " To the busy practitioner this chart will be found of great value in fever cases, and especially for cases of typhoid." — Indian Lancet ^ Calcutta. lockwood's practice of medicine. A Manual of the Practice of Medicine. By George Roe Lock- wood, M.D., Professor of Practice in the Woman's Medical College of the New York Infirmary, etc.. 935 pages, with 75 illustrations in the text, and 22 full-page plates. Cloth, $2.50 net. "Gives in a most concise manner the points essential to treatment usually enumerated in the most elaborate works." — Massachusetts Medical Journal. LONG'S SYLLABUS OF GYNECOLOGY. A Syllabus of Gynecology, arranged in Conformity with ** An American Text=Book of Gynecology." By J. W. Long, M.D., Professor of Diseases of Women and Children, Medical College of Virginia, etc. Cloth, interleaved, $1.00 net. " The book is certainly an admirable resumi of what every gynecological student and practitioner should know, and will prove of value not only to those who have the ' American Text- Book of Gynecology,' but to others as well." — Brooklyn Medical Journal. 16 Medical Publications of W. B. Saunders. MACDONALD'S SURGICAL DIAGNOSIS AND TREATMENT. Surgical Diagnosis and Treatment. By J. W. Macdonald, M.D. Edin., F.R.C.S., Edin., Professor of the Practice of Surgery and of Clinical Surgery in Hamline University; Visiting Surgeon to St. Barnabas' Hospital, Minneapolis, etc. Handsome octavo volume of 800 pages, profusely illustrated. Cloth, $5.00 net; Half Morocco, ;^6.oo net. ** A thorough and complete work on surgical diagnosis and treatment, free from pad- ding, full of valuable material, and in accord with the surgical teaching of the day." — T^e Medical Nezvs, New York. •* The work is brimful of just the kind of 'practical information that is useful alike to students and practitioners. It is a pleasure to commend the bock because of its intrinsic value to the medical practitioner." — Cincinnati Lancet- Clinic . MALLORY AND WRIGHT'S PATHOLOGICAL TECHNIQUE. Pathological Technique. A Practical Manual for Laboratory Work in Pathology, Bacteriology, and Morbid Anatomy, with chapters on Post-Mortem Technique and the Performance of Autopsies. By Frank B. Mallory, A.m., M.D., Assistant Professor of Pathology, Harvarn University Medical School, Boston; and James H. Wright, A.M., M.D., Instructor in Pathology, Harvard University Medical School, Boston. Octavo volume of 396 pages, handsomely illustrated. Cloth, ;g2.5o net. *' I have been looking forward to the publication of this book, and I am glad to say that I find it to be a most useful laboratory and post-mortem guide, full of practical, information, and well up to date." — William H. Welch, Professor of Pathology, Johns Hopkins Uni- versity, Baltimore, Md. MARTIN'S MINOR SURGERY, BANDAGING, AND VENEREAL DISEASES. Second Edition, Revised. Essentials of Minor Surgery, Bandaging, and Venereal Diseases. By Edward Martin, A.M., M.D., Clinical Professor of Genito-Urinary Diseases, University of Pennsylvania, etc. Crown octavo, 166 pages, with 78 illustrations. Cloth, ^i.oo j interleaved for notes, ^1.25. [See Saunders' Question- Compends, page 21.] "A very practical and systematic study of the subjects, and shows the author's famil- iarity with the needs of students." — Therapeutic Gazette. MARTIN'S SURGERY. Sixth Edition, Revised. Essentials of Surgery. Containing also Venereal Diseases, Surgi- cal Landmarks, Minor and Operative Surgery, and a complete de- scription, with illustrations, of the Handkerchief and Roller Bandages. By Edward Martin, A.M., M.D., Clinical Professor of Genito- Urinary Diseases, University of Pennsylvania, etc. Crown octavo, 338 pages, illustrated. With an Appendix containing full directions for the preparation of the materials used in Antiseptic Surgery, etc. Cloth, ^i.oo; interleaved for notes, $1.25. [See Saunders' Question- Compends, page 21.] *' Contains all necessary essentials of modern surgery in a comparatively small space. Its style is interesting, and its illustrations are admirable." — Medical and Surgical Reporter. Medical Publications of W. B. Saunders. 17 McFARLAND'S PATHOGENIC BACTERIA. Second Edition, Re- vised and Greatly Enlarged. Text-Book upon the Pathogenic Bacteria. By Joseph McFar- LAND, M. D. , Professor of Pathology and Bacteriology in the Medico- Chirurgical College of Philadelphia, etc. Octavo volume of 497 pages, finely illustrated. Cloth, $2.50 net. ** Dr. McFarland has treated the subject in a systematic manner, and has succeeded in presenting in a concise and readable form the essentials of bacteriology up to date. Alto- gether, the book is a satisfactory one, and I shall take pleasure in recommending it to the students of Trinity College."— H. B. Anderson, M.D., Professor of Pathology and Bac- teriology^ Trinity Medical College, Toronto. MEIGS ON FEEDING IN INFANCY. Feeding in Early Infancy. By Arthur V. Meigs, M.D. Bound in limp cloth, flush'edges, 25 cents net. " This pamphlet is worth many times over its price to the physician. The author's experiments and conclusions are original, and have been the means of doing much good." — Medical Bulletin. MOORE'S ORTHOPEDIC SURGERY. A Manual of Orthopedic Surgery. By James E. Moore, M.D., Professor of Orthopedics and Adjunct Professor of Clinical Surgery, University of Minnesota, College of Medicine and Surgery. Octavo volume of 356 pages, handsomely illustrated. Cloth, ^2.50 net. "A most attractive work. The illustrations and the care with which the book is adapted to the wants of the general practitioner and the student are worthy of great praise." — Chicago Medical Recorder. "A very demonstrative work, every illustration of which conveys a lesson. The work is a most excellent and commendable one, which we can certainly endorse with pleasure." — St. Louis Medical and Surgical Journal. MORRIS'S MATERIA MEDICA AND THERAPEUTICS. Fifth Edition, Revised. Essentials of Materia Medica, Therapeutics, and Prescription- Writing. By Henry Morris, M.D., late Demonstrator of Thera- peutics, Jefferson Medical College, Philadelphia; Fellow of the College of Physicians, Philadelphia, etc. Crown octavo, 288 pages. Cloth, ;^i.oo; interleaved for notes, $1.25. [See Saunders' Question- Compends, page 21.] " This work, already excellent in the old edition, has been largely improved by revi- sion." — American Practitioner and News. MORRIS, WOLFF, AND POWELL'S PRACTICE OF MEDICINE. Third Edition, Revised. Essentials of the Practice of Medicine. By Henry Morris, M.D., late Demonstrator of Therapeutics, Jefferson Medical College, Phila- delphia ; with an Appendix on the Clinical and Microscopic Examina- tion of Urine, by Lawrence Wolff, M.D., Demonstrator of Chemistry, Jefferson Medical College, Philadelphia. Enlarged by some 300 essen- tial formulae collected and arranged by William M. Powell, M.D. Post-octavo, 488 pages. Cloth, ^2.00. [See Saunders' Question- Compends, page 21.] " The teaching is sound, the presentation graphic ; matter full as can be desired, and style attractive." — American Practitioner and News. 18 Medical Publications of W. B, Saunders. MORTEN'S NURSE'S DICTIONARY. Nurse's Dictionary of Medical Terms and Nursing Treat- ment. Containing Definitions of the Principal Medical and Nursing Terms and Abbreviations ; of the Instruments, Drugs, Diseases, Acci- dents, Treatments, Operations, Foods, Appliances, etc. encountered in the ward or in the sick-room. By Honnor Morten, author of ^' How to Become a Nurse," etc. i6mo, 140 pages. Cloth, ^i.oo. ** A handy, compact little volume, containing a large amount of general information, all of which is arranged in dictionary or encyclopedic form, thus facilitating quick reference. It is certainly of value to those for whose use it is published." — Chicago Clinical Review. NANCREDE'S ANATOMY. Sixth Edition, Thoroughly Revised. Essentials of Anatomy, including the Anatomy of the Viscera. By Charles B. Nancrede, M.D., LL.D., Professor of Surgery and of Clinical Surgery in the University of Michigan, Ann Arbor. Crown octavo, 420 pages; 151 illustrations. Based upon Gray's Anatomy. Cloth, $1.00 net; interleaved for notes, $1.25 net. [See Saunders' Question- Compends, page 21.] " For self-quizzing and keeping fresh in mind the knowledge of anatomy gained at school, it would not be easy to speak of it in terms too favorable." — American Practitioner. NANCREDE'S ANATOMY AND DISSECTION. Fourth Edition. Essentials of Anatomy and Manual of Practical Dissection. By Charles B. Nancrede, M.D., LL.D., Professor of Surgery and of Clinical Surgery, University of Michigan, Ann Arbor. Post-octavo ; 500 pages, with full-page lithographic plates in colors, and nearly 200 illustrations. Extra Cloth (or Oilcloth for dissection-room), ^2.00 net. " It may in many respects be considered an epitome of Gray's popular work on general anatomy, at the same time having some distinguishing characteristics of its own to commend it. The plates are of more than ordinary excellence, and are of especial value to students in their work in the dissecting room." — Journal of the American Medical Association. NORRIS'S SYLLABUS OF OBSTETRICS. Third Edition, Revised. Syllabus of Obstetrical Lectures in the Medical Department of the University of Pennsylvania. By Richard C. Norris, A.M., M.D., Demonstrator of Obstetrics, University of Pennsylvania. Crown octavo, 222 pages. Cloth, interleaved for notes, $2.00 net. "This work is so far superior to others on the same subject that we take pleasure in calling attention briefly to its excellent features. It covers the subject thoroughly, and will prove invaluable both to the student and the practitioner." — Medical Record, New York. PENROSE'S DISEASES OF WOMEN. Second Edition, Revised. A Text=Book of Diseases of Women. By Charles B. Penrose, M.D., Ph.D., Professor of Gynecology in the University of Pennsyl- vania; Surgeon to the Gynecean Hospital, Philadelphia. Octavo volume of 529 pages, handsomely illustrated. Cloth, ^3.50 net. ** I shall value very highly the copy of Penrose's * Diseases of Women* received. I have already recommended it to my class as THE BEST book." — Howard A. KELLY, Professor of Gynecology and Obstetrics, Johns Hopkins University, Baltimore, Md. *< The book is to be commended without reserve, not only to the student but to the general practitioner who wishes to have the latest and best modes of treatment explained with absolute clearness." — Therapeutic Gazette. Medical Publications of W. B, Saunders. 19 POWELL*S DISEASES OF CHILDREN. Second Edition. Essentials of Diseases of Children. By William M. Powell, M.D., Attending Physician to the Mercer House for Invalid Women at Atlantic City, N. J. ; late Physician to the Clinic for the Diseases of Children in the Hospital of the University of Pennsylvania. Crown octavo, 222 pages. Cloth, ;^i.oo; interleaved for notes, ^1.25. [See Saunders' Question- Compends, page 21.] "Contains the gist of all the best works in the department to which it relates."— American Practitioner and News. PRINGLE'S SKIN DISEASES AND SYPHILITIC AFFECTIONS. Pictorial Atlas of Skin Diseases and Syphilitic Affections (American Edition). Translation from the French. Edited by J. J. Pringle, M.B., F.R.C.P., Assistant Physician to the Middlesex Hospital, London. Photo-lithochromes from the famous models in the Museum of the Saint-Louis Hospital, Paris, with explanatory wood- cuts and text. In 12 Parts. Price per Part, $3.00. Complete in one volume. Half Morocco binding, ^40.00 net. ** I strongly recommend this Atlas. The plates are exceedingly well executed, and will be of great value to all studying dermatology." — Stephen Mackenzie, M.D. "The introduction of explanatory wood-cuts in the text is a novel and most important feature which greatly furthers the easier understanding of the excellent plates, than which nothing, we venture to say, has been seen better in point of correctness, beauty, and general merit." — New York Medical Journal. PRYOR— PELVIC INFLAMMATIONS. The Treatment of Pelvic Inflammations through the Vagina. By W. R. Pryor, M.D., Professor of Gynecology in New York Poly- clinic. i2mo, 248 pages, handsomely illustrated. Cloth, ^2.00 net. " This subject, which has recently been so thoroughly canvassed in high gynecological circles, is made available in this volume to the general practitioner and student. Nothing is too minute for mention and nothing is taken for granted ; consequently the book is of the utmost value. The illustrations and the technique are beyond criticism." — Chicago Medical Recorder^ PYE'S BANDAGING. Elementary Bandaging and Surgical Dressing. With Direc- tions concerning the Immediate Treatment of Cases of Emergency. For the use of Dressers and Nurses. By Walter Pye, F.R.C.S., late Surgeon to St. Mary's Hospital, London. Small i2mo, with over 80 illustrations. Cloth, flexible covers, 75 cents net. "The directions are clear and the illustrations are good." — London Lancet. ** The author writes well, the diagrams are clear, and the book itself is small and port- able, although the paper and type are good." — British Medical Journal. RAYMOND'S PHYSIOLOGY. A Manual of Physiology. By Joseph H. Raymond, A.M., M.D., Professor of Physiology and Hygiene and Lecturer on Gynecology in the Long Island College Hospital; Director of Physiology in the Hoagland Laboratory, etc. 382 pages, with 102 illustrations in the text, and 4 full-page colored plates. Cloth, $1.25 net. " Extremely well gotten up, and the illustrations have been sel-ected with care. The text is fully abreast with modern physiology. "—^r//w>^ Medical Journal, Arranged in Question and Answer Form> XJlJiO 1 VUi^ ntHE MOST COMPLETE AND BEST r^rMi;nD"civmc illustrated series of ^^'^•IVlJrlliNlJo COMPENDS EVER ISSUED. Now the Standard Authorities in Medical Literature . . ♦ • with Students and Practitioners in every City of tlie United States and Canada* Saunders' Q ^ OVER 175,000 COPIES SOLD* THE REASON WHY* They are the advance guard of "Student's Helps" — that DO help. They are the leaders in their special line, well and authoritatively written by able men, who, as teachers in the large colleges, know exactly what is wanted by a student preparing for his examinations. The judgment exercised in the selection of authors is fully demonstrated by their professional standing. Chosen from the ranks of Demonstrators, Quiz-masters, and Assistants, most of them have become Professors and Lecturers in their respective colleges. Each book is of convenient size (5x7 inches) , containing on an average 250 pages, profusely illustrated, and elegantly printed in clear, readable type, on fine paper. The entire series, numbering twenty-three volumes, has been kept thoroughly revised and enlarged when necessary, many of the books being in their fifth and sixth editions. TO SUM UP* Although there are numerous other Quizzes, Manuals, Aids, etc. in the market, none of them approach the " Blue Series of Question Compends;" and the claim is made for the following points of excellence : 1. Professional distinction and reputation of authors. 2. Conciseness, clearness, and soundness of treatment. 3. Quality of illustrations, paper, printing, and binding. Any cf these Compends will be mailed on receipt of price (see next page for List)* Saunders^ Question-Compend Series* Price, Qoth, $J.OO per copy, except when otherwise noted. •'Where the work of preparing students' manuals is to end we cannot say, but the Saunders Series, in our opinion, bears off the palm at present."— A^(fzc/ York Medical Record. 1. ESSENTIALS OF PHYSIOLOGY. By H. A. Hare, M.D. Fourth edition, revised and enlarged. (^I.oo net.) 2. ESSENTIALS OF SURGERY. By Edward Martin, M.D. Sixth edition, revised, with an Appendix on Antiseptic Surgery. 3. ESSENTIALS OF ANATOMY. By Chari.es B. Nancrede, M.D. Sixth edition, thoroughly revised and enlarged. ($i.oo net.) 4. ESSENTIALS OF MEDICAL CHEMISTRY, ORGANIC AND INORGANIC. By Lawrence Wolff, M.D. Fifth edition, revised. ($i.oo net.) 5. ESSENTIALS OF OBSTETRICS. By W. Easterly Ashton, M.D. Fourth edition, revised and enlarged. 6. ESSENTIALS OF PATHOLOGY AND MORBID ANATOMY. By C. E. Armand Semple, M.D. 7. ESSENTIALS OF MATERIA MEDICA, THERAPEUTICS, AND PRE- SCRIPTION-WRITING. By Henry Morris, M.D. Fifth edition, revised. 8,9. ESSENTIALS OF PRACTICE OF MEDICINE. By Henry Morris, M.D. An Appendix on Urine Examination. By Lawrence Wolff, M.D. Third edition, enlarged by some 300 Essential Formulae, selected from eminent authorities, by Wm. M. Powell, M.D. (Double number, ^2.00.) 10. ESSENTIALS OF GYNAECOLOGY. By Edwin B. Cragin, M.D. Fourth edition, revised. 11. ESSENTIALS OF DISEASES OF THE SKIN. By Henry W. Stelwagon, M.D. Fourth edition, revised and enlarged, (^i.oo net.) 12. ESSENTIALS OF MINOR SURGERY, BANDAGING, AND VENEREAL DISEASES. By Edward Martin, M.D. Second ed., revised and enlarged. 13. ESSENTIALS OF LEGAL MEDICINE, TOXICOLOGY, AND HYGIENE. By C. E. Armand Semple, M.D. 14. ESSENTIALS OF DISEASES OF THE EYE, NOSE, AND THROAT. By Edward Jackson, M.D., and E. B. Gleason, M.D. Second ed., revised. 15. ESSENTIALS OF DISEASES OF CHILDREN. By William M. Powell, M. D. Second edition. 16. ESSENTIALS OF EXAMINATION OF URINE. By Lawrence Wolff, M.D. Colored **Vogel Scale." (75 cents.) 17. ESSENTIALS OF DIAGNOSIS. By s'. Solis Cohen, M.D., and A. A. Eshner, M.D. (^1.50 net.) 18. ESSENTIALS OF PRACTICE OF PHARMACY. By Lucius E. Sayre. Second edition, revised and enlarged. 20. ESSENTIALS OF BACTERIOLOGY. By M. V. Ball, M.D. Third edition, revised. 21. ESSENTIALS OF NERVOUS DISEASES AND INSANITY. By John C. Shaw, M.D. Third edition, revised. 22. ESSENTIALS OF MEDICAL PHYSICS. By Fred J. Brockway, M.D. Second edition, revised. {^I.oo net.) 23. ESSENTIALS OF MEDICAL ELECTRICITY. By David D. Stewart, M.D., and Edward S. Lawrance, M.D. 24. ESSENTIALS OF DISEASES OF THE EAR. By E. B. Gleason, M.D. Second edition, revised and greatly enlarged. Pamphlet containing specimen pagest etc sent free upon application* Saunders' New Series for Students and of Manuals P'-actmoners. 'TTtlAT there exists a need for thoroughly reliable hand-books on the leading branches of Medicine and Surgery is a fact amply demonstrated by the favor with which the SAUNDERS NE^ SERIES OF MANUALS have been received by medical students and practitioners and by the Medical Press* These manuals are not merely condensations from present literature, but are ably written by w^ell-known authors and practitioners, most of them being teachers in representative American colleges* Each volume is concisely and authoritatively written and exhaustive in detail, without being encumbered with the introduction of ** cases,*' w^hich so largely expand the ordinary text-book. These manuals will therefore form an admirable collection of advanced lectures, useful alike to the medical student and the practitioner: to the latter, too busy to search through page after page of elaborate treatises for what he grants to know, they w^ill prove of inestimable value ; to the former they will afford safe guides to the essential points of study* The SAUNDERS NEW SERIES OF MANUALS are conceded to be superior to any similar books now on the market. No other manuals afford so much infor- mation in such a concise and available form. A liberal expenditure has enabled the publisher to render the mechanical portion of the work worthy of the high literary standard attained by these books* Any of these Manuals will be mailed on receipt of price (see next page for List)* Saunders^ New Series of Manuals^ VOLUMES PUBLISHED. PHYSIOLOGY. By Joseph Howard Raymond, A.M., M.D., Professor of Physiology and Hygiene and Lecturer on Gynecology in the Long Island College Hospital; Director of Physiology in the Hoagland Laboratory, etc. Illustrated. Cloth, ^1.25 net. SURGERY, General and Operative. By John Chalmers DaCosta, M.D., Clini- cal Professor of Surgery, Jefferson Medical College, Philadelphia; Surgeon to the Philadelphia Hospital, etc. Second edition, thoroughly revised and greatly enlarged. Octavo, 911 pages, profusely illustrated. Cloth, $4.00 net ; Half Morocco, $5.00 net. DOSE-BOOK AND MANUAL OF PRESCRIPTION=WRITING. By E. Q. Thornton, M.D., Demonstrator of Therapeutics, Jefferson* Medical College, Phila- delphia. Illustrated. Cloth, ^1.25 net. SURGICAL ASEPSIS. By Carl Beck, M.D., Surgeon to St. Mark's Hospital and to the New York German PohlUinik, etc. Illustrated. Cloth, ^1.25 net. MEDICAL JURISPRUDENCE. By Henry C. Chapman, M.D. Professor of Insti- tutes of Medicine and Medical Jurisprudence in the Jefferson Medical College of Phila- delphia. Illustrated. Cloth, ^1.50 net. SYPHILIS AND THE VENEREAL DISEASES. By James Nevins Hyde, M.D., Professor of Skin and Venereal Diseases, and Frank H. Montgomery, M.D., Lecturer on Dermatology and Genito-Urinary Diseases in Rush Medical College, Chicago. Profusely illustrated. Cloth, ^2.50 net. PRACTICE OF MEDICINE. By George Roe Lockwood, M.D., Professor of Practice in the Woman's Medical College of the New York Infirmary; Instructor in Physical Diagnosis in the Medical Department of Columbia College, etc. Illustrated. Cloth, ^2.50 net. MANUAL OF ANATOMY. By Irving S. Haynes, M.D., Adjunct Professor of Anatomy and Demonstrator of Anatomy, Medical Department of the New York University, etc. Beautifully illustrated. Cloth, ^2.50 net. MANUAL OF OBSTETRICS. By W. A. Newman Dorland, M.D., Assistant Demonstrator of Obstetrics, University of Pennsylvania ; Chief of Gynecological Dis- pensary, Pennsylvania Hospital, etc. Profusely illustrated. Cloth, ^2.50 net. DISEASES OF WOMEN. By J. Bland Sutton, F. R. C. S., Assistant Surgeon to Middlesex Hospital and Surgeon to Chelsea Hospital, London; and Arthur E. Giles, M. D., B. Sc. Lond., P\R.C.S. Edin., Assistant Surgeon to Chelsea Hospital, London. Handsomely illustrated. Cloth, 1^2.50 net. VOLUMES IN PREPARATION. NERVOUS DISEASES. By Charles W. Burr, M.D., Clinical Professor of Nervous Diseases, Medico-Chirurgical College, Philadelphia ; Pathologist to the Orthopaedic Hospital and Infirmary for Nervous Diseases ; Visiting Physician to the St. Joseph Hospital, etc. *#* There will be published in the same series, at short intervals, carefully-prepared works on various subjects by prominent specialists. Pamphlet containiiig specimen pages, etc* sent &ee upon application* 24 Medical Publications of W, B, Saunders. SAUNDBY'S RENAL AND URINARY DISEASES. Lectures on Renal and Urinary Diseases. By Robert Saundby, M.D. Edin., Fellow of the Royal College of Physicians, London, and of the Royal Medico-Chirurgical Society ; Physician to the General Hospital ; Consulting Physician to the Eye Hospital and to the Hos- pital for Diseases of Women; Professor of Medicine in Mason College, Birmingham, etc. Octavo volume of 434 pages, with numerous illus- trations and 4 colored plates. Cloth, $2.50 net. " The volume makes a favorable impression at once. The style is clear and succinct. We cannot find any part of the subject in which the views expressed are not carefully thought out and fortified by evidence drawn from the most recent sources. The book may be cordially recommended.' ' — British Medical Journal. SAUNDERS' MEDICAL HAND=ATLASES. This series of books consists of authorized translations into English of the world-famous Lehmann Medicinische Handatlanten. Each volume contains from 50 to 100 colored lithographic plates, besides numerous illustrations in the text. There is a full description of each plate, and each book contains a condensed but adequate outline of the subject to which it is devoted. For full description of this series, with list of volumes and prices, see page 2. " Lehmann Medicinische Handatlanten belong to that class of books that are too good to be appropriated by any one nation." — Journal of Eye, Ear, and Throat Diseases. " The appearance of these works marks a new era in illustrated English medical works." — The Canadian Practitioner. SAUNDERS' POCKET MEDICAL FORMULARY. Fifth Edition, Revised. By William M. Powell, M.D., Attending Physician to the Mercer House for Invalid Women at Atlantic City, N. J. Containing 1800 formulae selected from the best-known authorities. With an Appen- dix containing Posological Table, Formulae &nd Doses for Hypo- dermic Medication, Poisons and their Antidotes, Diameters of the Female Pelvis and Foetal Head, Obstetrical Table, Diet List for Various Diseases, Materials and Drugs used in Antiseptic Surgery, Treatment of Asphyxia from Drowning, Surgical Remembrancer, Tables of Incompatibles, Eruptive Fevers, Weights and Measures, etc. Hand- somely bound in flexible morocco, with side index, wallet, and flap. ;^i.75net. " This little book, that can be conveniently carried in the pocket, contains an immense amount of material. It is very useful, and, as the name of the author of each prescription is given, is unusually reliable." — Medical Record, New York. SAYRE'S PHARMACY. Second Edition, Revised. Essentials of the Practice of Pharmacy. By Lucius E. Sayre, M.D., Professor of Pharmacy and Materia Medica in the University of Kansas. Crown octavo, 200 pages. Cloth, $1.00; interleaved for notes, $1.25. [See Saunders' Question- Compends, page 21.] '*The topics are treated in a simple, practical manner, and the work forms a very useful student's manual." — Boston Medical and Surgical Journal. Medical Publications of W. B, Saunders. 25 SEMPLE'S LEGAL MEDICINE, TOXICOLOGY, AND HYGIENE. Essentials of Legal Medicine, Toxicology, and Hygiene. By C. E. Armand Semple, B. A., M. B. Cantab., M. R. C. P. Lond., Physician to the Northeastern Hospital for Children, Hackney, etc. Crown octavo, 212 pages; 130 illustrations. Cloth, ;^i. 00; interleaved for notes, ^1.25. [See Saunders' Question- Compends, page 21.] •' No general practitioner or student can afford to be without this valuable work. The subjects are dealt with by a masterly hand." — London Hospital Gazette. SEMPLE'S PATHOLOGY AND MORBID ANATOMY. Essentials of Pathology and Morbid Anatomy. By C. E. Armand Semple, B.A., M.B. Cantab., M.R.C.P. Lond., Physician to the Northeastern Hospital for Children, Hackney, etc. Crown octavo, 174 pages; illustrated. Cloth, $1.00; interleaved for notes, $1.25. [See Saunders' Question- Compends, page 21.] " Should take its place among the standard volumes on the bookshelf of both student and practitioner." — London Hospital Gazette. SENN'S GENITO-URINARY TUBERCULOSIS. Tuberculosis of the Genito-Urinary Organs, Male and Female. By Nicholas Senn, M.D., Ph.D., LL.D., Professor of the Practice of Surgery and of Clinical Surgery, Rush Medical College, Chicago. Handsome octavo volume of 320 pages, illustrated. Cloth, ^3.00 net. *' An important book upon an important subject, and written by a man of mature judg- ment and wide experience. The author has given us an instructive book upon one of the most important subjects of the day." — Clinical Reporter. " A work which adds another to the many obHgations the profession owes the talented author." — Chicago Medical Recorder. SENN'S SYLLABUS OF SURGERY. A Syllabus of Lectures on the Practice of Surgery, arranged in conformity with ** An American Text-Book of Surgery." By Nicholas Senn, M.D., Ph.D., Professor of the Practice of Surgery and of Clinical Surgery in Rush Medical College, Chicago. Cloth, ^2.00. " This syllabus will be found of service by the teacher as well as the student, the work being superbly done. There is no praise too high for it. No surgeon should be without it." — New York Medical Times. SExNN'S TUMORS. Pathology and Surgical Treatment of Tumors. By N. Senn, M.D., Ph.D., LL.D., Professor of Surgery and of Clinical Surgery, Rush Medical College ; Professor of Surgery, Chicago Polyclinic ; Attending Surgeon to Presbyterian Hospital ; Surgeon-in-Chief, St. Joseph's Hospital, Chicago. Octavo volume of 710 pages, with 515 engravings, including full-page colored plates. New and Revised Edi- tion in Preparation. ** The most exhaustive of any recent book in Engnsh on this subject. It is well illus- trated, and will doubtless remain as the principal monograph on the subject in our language for some years. The book is handsomely illustrated and printed, and the author has giyen a notable and lasting contribution to surgery." — Journal of the American Medical Association. Medical Publications of W. B, Saunders, SHAW'S NERVOUS DISEASES AND INSANITY. Third Edition, Revised. Essentials of Nervous Diseases and Insanity. By John C. Shaw, M.D., Clinical Professor of Diseases of the Mind and Nervous System, Long Island College Hospital Medical School ; Consulting Neurologist to St. Catherine's Hospital and to the Long Island College Hospital. Crown octavo, i86 pages; 48 original illustrations. Cloth, ^i.oo ; interleaved for notes, $1.25. [See Saunders' Question- Compendsy page 21.] "Clearly and intelligently written." — Boston Medical and Surgical Journal. " There is a mass of valuable material crowded into this small compass." — American Medico- Surgical Bulletin. STARR'S DIETS FOR INFANTS AND CHILDREN. Diets for Infants and Children in Health and in Disease. By Louis Starr, M.D., Editor of ''An American Text-Book of the Diseases of Children." 230 blanks (pocket-book size), perforated and neatly bound in flexible morocco. $1.25 net. The first series of blanks are prepared for the first seven months of infant life ; each blank indicates the ingredients, but not the quantities, of the food, the latter directions being left for the physician. After the seventh month, modifications being less necessary, the diet lists are printed in full. Formulae for the preparation of diluents and foods are appended. STELW AGON'S DISEASES OF THE SKIN. Fourth Ed., Revised. Essentials of Diseases of the Skin. By Henry W. Stelwagon, M.D., Clinical Professor of Dermatology in the Jefferson Medical College, Philadelphia; Dermatologist to the Philadelphia Hospital; Physician to the Skin Department of the Howard Hospital, etc. Crown octavo, 276 pages; 88 illustrations. Cloth, $1.00 net; inter- leaved for notes, $1.25 net. [See Saunders'' Question- Co7npends, page 21.] ** The best student's manual on skin diseases we have yet seen." — Times and Register^ STENGEL'S PATHOLOGY. Second Edition. A Text-Book of Pathology. By Alfred Stengel, M.D., Professor of Clinical Medicine in the University of Pennsylvania ; Physician to the Philadelphia Hospital ; Physician to the Children's Hospital, etc. Handsome octavo volume of 848 pages, with nearly 400 illustrations, many of them in colors. Cloth, ^4.00 net; Half Morocco, ^5.00 net. STEVENS' MATERIA MEDIC A AND THERAPEUTICS. Second Edition, Revised. A Manual of Materia Medica and Therapeutics. By A. A. Stevens, A.M., M.D., Lecturer on Terminology and Instructor in Physical Diagnosis in the University of Pennsylvania; Professor of Pathology in the Woman's Medical College of Pennsylvania. Post- octavo, 445 pages. Flexible leather, ^2.25. "The author has faithfully presented modern therapeutics in a comprehensive work, and, while intended particularly for the use of students, it will be found a reliable guide and sufficiently comprehensive for the physician in practice." — University Medical Magazine, Medical Publications of W. B. Saunders. 27 5TEVENS' PRACTICE OF MEDICINE. Fifth Edition, Revised. A Manual of the Practice of Medicine. By A. A. Stevens, A. M., M. D., Lecturer on Terminology and Instructor in Physical Diagnosis in the University of Pennsylvania; Professor of Pathology in the Woman's Medical College of Pennsylvania. Specially intended for students preparing- for graduation and hospital examinations. Post- octavo, 519 pages; illustrated. Flexible leather, ^2.00 net. " The frequency with which new editions of this manual are demanded bespeaks its popularity. It is an excellent condensation of the essentials of medical practice for the student, and maybe found also an excellent reminder for the busy physician." — Buffalo Medical Journal. STEWARTS PHYSIOLOGY. Third Edition, Revised. A Manual of Physiology, with Practical Exercises. For Students and Practitioners. By G. N. Stewart, M.A., M.D., D.Sc, lately Examiner in Physiology, University of Aberdeen, and of the New Museums, Cambridge University ; Professor of Physiology in the Western Reserve University, Cleveland, Ohio. Octavo volume of 848 pages; 300 illustrations in the text, and 5 colored plates. Cloth, $3.75 net. ** It will ma,ke its way by sheer force of merit, and amply deserves to do so. It is one of the very best English tekt-books on the subject." — London Lancet. ' * Of the many text-books of physiology published, we do not know of one that so nearly comes up to the ideal as does Prof. Stewart's volume." — British Medical Journal. STEWART AND LAWRANCE'S MEDICAL ELECTRICITY. Essentials of Medical Electricity. By D. D. Stewart, M.D., Demonstrator of Diseases of the Nervous System and Chief of the Neurological Clinic in the Jefferson Medical College; and E. S. Lawrance, M.D., Chief of the Electrical Clinic and Assistant Demon- strator of Diseases of the Nervous System in the Jefferson Medical College, etc. Crown octavo, 158 pages; 65 illustrations. Cloth, ^i.oo; interleaved for notes, ^1.25. [See Saunders' Question- Compends^ page 21.] ** Throughout the whole brief space at their command the authors show a discriminating knowledge of their subject." — Medical News. STONEY'S NURSING. Second Edition, Revised. Practical Points in Nursing. For Nurses in Private Practice. By Emily A. M. Stoney, Graduate of the Training-School for Nurses, Lawrence, Mass.; late Superintendent of the Training-School for Nurses, Carney Hospital, South Boston, Mass. 456 pages, illustrated with 73 engravings in the text, and 8 colored and half-tone plates. Cloth, 1 1. 75 net. " There are few books intended for non-professional readers which can be so cordially endorsed by a medical journal as can this one." — Therapeutic Gazette. " This is a well-written, eminently practical volume, which covers the entire range of private nursing as distinguished from hospital nursing, and instructs the nurse how best to meet the various emergencies which may arise, and how to prepare everything ordinarily needed in the illness of her patient." — American Journal of Obstetrics and Diseases of Women and Childi'en. " It is a work that the physician can place in the hands of his private nurses with the assurance of benefit," — Ohio Medical Journal. 28 Medical Publications of W. B. Saunders. STONEY'S MATERIA MEDICA FOR NURSES. Materia Medica for Nurses. By Emily A. M. Stoney, Graduate of the Training-School for Nurses, Lawrence, Mass. ; late Superintendent of the Training-School for Nurses, Carney Hospital, South Boston, Mass. Handsome octavo volume of 306 pages. Cloth, ;^i.5o net. The present book differs from other similar works in several features, all of which are intended to render it more practical and generally useful. The general plan of the contents follows the lines laid down in training-schools for nurses, but the book contains much use- ful matter not usually included in works of this character, such as Poison-emergencies, Ready Dose-list, Weights and Measures, etc., as well as a Glossary, defining all the terms used in Materia Medica, and describing all the latest drugs and remedies, which have been generally neglected by other books of the kind. SUTTON AND GILES' DISEASES OF WOMEN. Diseases of Women. By J. Bland Sutton, F.R.C.S., Assistant Surgeon to Middlesex Hospital, and Surgeon to Chelsea Hospital, London; and Arthur E. Giles, M.D., B.Sc. Lond., F.R.C.S. Edin., Assistant Surgeon to Chelsea Hospital, London. 436 pages, hand- somely illustrated. Cloth, ^2.50 net. "The text has been carefully prepared. Nothing essential has been omitted, and its teachings are those recommended by the leading authorities of the day." — Journal of the American Medical Association. THOMAS'S DIET LISTS AND SICK=ROOM DIETARY. Diet Lists and Sick=Room Dietary. By Jerome B. Thomas, M.D., Visiting Physician to the Home for Friendless Women and Children and to the Newsboys' Home ; Assistant Visiting Physician to the Kings County Hospital. Cloth, $1.50. Send for sample sheet. THORNTON'S DOSE=BOOK AND PRESCRIPTION-WRITING. Dose=Book and Manual of Prescription=Writing. By E. Q. Thornton, M.D., Demonstrator of Therapeutics, Jefferson Medical College, Philadelphia. 334 pages, illustrated. Cloth, $1.25 net. "Full of practical suggestions; will take its place in the front rank of works of this sort. " — Medical Record^ New York. VAN VALZAH AND NISBET'S DISEASES OF THE STOMACH. Diseases of the Stomach. By William W. Van Valzah, M.D., Professor of General Medicine and Diseases of the Digestive System and the Blood, New York Polyclinic; and J. Douglas Nisbet, M.D., Adjunct Professor of General Medicine and Diseases of the Digestive System and the Blood, New York Polyclinic. Octavo volume of 674 pages, illustrated. Cloth, $3.50 net. " Its chief claim lies in its clearness and general adaptability to the practical needs of the general practitioner or student. In these relations it is probably the best of the recent special works on diseases of the stomach." — Chicago Clinical Review. VECKrS SEXUAL IMPOTENCE. The Pathology and Treatment of Sexual Impotence. By Victor G. Vecki, M.D. From the second German edition, revised and en- larged. Demi-octavo, about 300 pages. Cloth, ^2.00 net. The subject of impotence has seldom been treated in this country in the truly scientific spirit that it deserves. Dr. Vecki's work has long been favorably known, and the German txx)k has received the highest consideration. This edition is more than a mere translation, for, although based on the German edition, it has been entirely rewritten in English. Medical Publications of W, B. Saunders, 29 VIERORDT'S MEDICAL DIAGNOSIS. Fourth Edition, Revised. Medical Diagnosis. By Dr. Oswald Vierordt, Professor of Medi- cine at the University of Heidelberg. Translated, with additions, from the fifth enlarged German edition, with the author's permission, by Francis H. Stuart, A. M., M. D. Handsome royal octavo volume of 603 pages; 194 fine wood-cuts in text, many of them in colors. Cloth, $4.00 net; Sheep or Half Morocco, $5.00 net. " A treasury of practical information which will be found of daily use to every busy practitioner who will consult it." — C. A. Lindsley, M.D., Professor of the Theory and Practice of Medicine^ Yale University. " Rarely is a book published with which a reviewer can find so little fault as with the volume before us. Each particular item in the consideration of an organ or apparatus, which is necessary to determine a diagnosis of any disease of that organ, is mentioned ; nothing seems forgotten. The chapters on diseases of the circulatory and digestive apparatus and nervous system are especially full and valuable. The reviewer would repeat that the book is one of the best — probably the best — which has fallen into his hands." — University Medical Magazine. WARREN'S SURGICAL PATHOLOGY AND THERAPEUTICS. Surgical Pathology and Therapeutics. By John Collins Warren, M.D., LL.D., Professor of Surgery, Medical Department Harvard University; Surgeon to the Massachusetts General Hospital, etc. Handsome octavo volume of 832 pages; 136 relief and lithographic illustrations, 33 of which are printed in colors, and all of which were drawn by William J. Kaula from original specimens. Revised and Enlarged Edition in Preparation. "There is the work- of Dr. Warren, which I think is the most creditable book on Surgical Pathology, and the most beautiful medical illustration of the bookmaker's art, that has ever been issued from the American press." — Dr. Roswell Park, in the Hai-vard Graduate Magazine. " The handsomest specimen of bookmaking that has ever been issued from the American medical press." — Atnerican Journal of the Medical Sciences. *' A most striking and very excellent feature of this book is its illustrations. Without exception, from the point of accuracy and artistic merit, they are the best ever seen in a work of this kind. Many of those representing microscopic pictures are so perfect in their coloring and detail as almost to give the beholder the impression that he is looking down the barrel of a microscope at a welbmounted section." — Annals of Surgery. WOLFF ON EXAMINATION OF URINE. Essentials of Examination of Urine. By Lawrence Wolff, M.D., Demonstrator of Chemistry, Jefferson Medical College, Philadelphia, etc. Colored (Vogel) urine scale and numerous illustrations. Crown octavo. Cloth, 75 cents. [See Saunders'' Question- Compends, page 21.] ** A very good work of its kind — very well suited to its purpose." — Titties and Register. WOLFF'S MEDICAL CHEMISTRY. Fifth Edition, Revised. Essentials of Medical Chemistry, Organic and Inorganic. Containing also Questions on Medical Physics, Chemical Physiology, Analytical Processes, Urinalysis, and Toxicology. By Lawrence Wolff, M.D., Demonstrator of Chemistry, Jefferson Medical College, Philadelphia, etc. Crown octavo, 222 pages. Cloth, $1.00 net; inter- leaved for notes, $1.25 net. [See Saunders' Question- Compcnds, page 21.] "The scope of this work is certainly equal to that of the best course of lectures on Medical Chemistry." — Fharvtaceutical Era. CLASSIFIED LIST Medical Publications W. B. SAUNDERS, 925 Walnut Street, PhiladelpKia* ANATOMY, EMBRYOLOGY, HISTOLOGY. Clarkson — A Text-Book of Histology, 9 Haynes — A Manual of Anatomy, ... 13 Heisler — A Text- Book of Embryology, 1 3 Nancrede — Essentials of Anatomy, . . i8 Nancrede — Essentials of Anatomy and Manual of Practical Dissection, ... 18 Semple — Essentials of Pathology and Morbid Anatomy 25 BACTERIOLOGY. Ball — Essentials of Bacteriology, ... 6 Crookshank — A Text-Book of Bacteri- ology, 10 Frothingham— Laboratory Guide, . . II Mallory and Wright — Pathological Technique, 16 McFarland — Pathogenic Bacteria, . . 17 CHARTS, DIET-LISTS, ETC. Griffith — Infant's Weight Chart, ... 12 Hart — Diet in Sickness and in Health, . 13 Keen — Operation Blank, 15 Laine — Temperature Chart, 15 Meigs^ — Feeding in Eariy Infancy, . . 17 Starr — Diets for Infants and Children, . 26 Thomas — Diet-Lists and Sick-Room Dietary, 28 CHEMISTRY AND PHYSICS. Brockway — Essentials of Medical Phys- ics, 7 Wolff — Essentials of Medical Chemistry, 29 CHILDREN. An American Text-Book of Diseases of Children, . 3 Griffith — Care of the Baby, 12 Griffith — Infant's Weight Chart, ... 12 Meigs — Feeding in Early Infancy, . . 17 Powell — Essentials of Dis. of Children, 19 Starr — Diets for Infants and Children, . 26 DIAGNOSIS. Cohen and Eshner— Essentials of Di- agnosis, . 9 Corwin — Physical Diagnosis, .... 9 Macdonald — Surgical Diagnosis and Treatment, l6 Vierordt — Medical Diagnosis, .... 29 DICTIONARIES. Dorland — Pocket Dictionary, .... 10 Keating — Pronouncing Dictionary, . . 14 Morten — Nurse's Dictionary, .... 18 EYE, EAR, NOSE, AND THROAT. An American Text- Book of Diseases of the Eye, Ear, Nose, and Throat, . 3 De Schweinitz — Diseases of the Eye, . lo Gleason — Essentials of Dis. of the Ear, 1 1 Jackson — Manual of Diseases of Eye, . 32 Jackson and Gleason — Essentials of Diseases of the Eye, Nose, and Throat, 14 Kyle — Diseases of tlie Nose and Throat, 15 QENITO=URINARY. An American Text-Book of Genito- urinary and Skin Diseases, 4 Hyde and Montgomery — Syphilis and the Venereal Diseases, ...... 13 Martin — Essentials of Minor Surgery, Bandaging, and Venereal Diseases, . 16 Saundby — Renal aiid Urinary Diseases, 24 Senn — Genito- Urinary Tuberculosis, . 25 Vecki — Sexual Impotence, 28 GYNECOLOGY. American Text- Book of Gynecology, Cragin — Essentials of Gynecology, Garrigues — Diseases of Women, . Long — Syllabus of Gynecology, . Penrose — Diseases of Women, . . Pryor — Pelvic Inflammations, . . Sutton and Giles — Diseases of Women, 4 9 II 15 18 32 28 MATERIA MEDICA, PHARMACOL- OGY, AND THERAPEUTICS. An American Text-Book of Applied Therapeutics 3 Butler — Text-Book of Materia Medica, Therapeutics and Pharmacology, ... 8 Cerna — Notes on the Newer Remedies, 8 Griffin — Materia Med. and Therapeutics, 12 Morris — Essentials of Materia Medica and Therapeutics, 17 Saunders' Pocket Medical Formulary, 24 Sayre — Essentials of Pharmacy, ... 24 Stevens — Essentials of Materia Medica and Therapeutics, 26 Stoney — Materia Medica for Nurses, . . 28 Thornton — Dose-Book and Manual of Prescription-Writing, 28 MEDICAL JURISPRUDENCE AND TOXICOLOGY. Chapman— Medical Jurisprudence and Toxicology, ... .... 8 Semple — Essentials of Legal Medicine, Toxicology, and Hygiene, 25 UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL LIBRARY