IC-NRLF flDb The ruling ot the I urck aisc. explanation 01 me squares. In the first place, we have the square which incloses the entire ruled sur- face. This is made up of nine squares each i mm. square. These are the squares to use in connection with leukocyte counts with the white pipette. They may be termed the large squares. The very smallest square which can be found are those made by the intersection of the double-ruled lines in the center; they are 1/40 mm. square and are never used for any purpose. When \ve refer to the small square, those which are found in groups of sixteen bounded by double-ruled lines are intended. These squares are 1/20 mm. square and are used in the count of red cells. Another square is outlined in the more sparsely ruled large square in each of the four corners. This square includes a space equal to sixteen small squares and in addition the spaces in the frame-like space surrounding make twenty additional small squares, cr thirty-six in all. This space is not used ordinarily. DaCosta uses a space made up of the sixteen small squares and the bordering upper and left-hand double-line cells. This gives twenty - five small squares. There are 400 small squares in each large square, consequently as there are nine large squares the entire ruled surface consists of 3600 small squares. \0 ^o v V\.-va.eU><< ^«.v. Principal normal and pathological blood-cells with average size, percentage in a normal differential count and the diseases in which certain pathological cells are more or less pathognomonic. Orv^ raelct •UO^. \\.t\a,eeJroc toxxXt-t The ruling of the Turck disc. Explanation of the squares. In the first place, we have the square which incloses the entire ruled sur- face. This is made up of nin? squares each i mm. square. These are the squares to use in connection with leukocyte counts with the white pipette. They may be termed the large squares. The very smallest square which can be found are those made by the intersection of the double-ruled lines in the center; they are 1/40 mm. square and are never used for any purpose. When we refer to the small square, those which are found in groups of sixteen bounded by double-ruled lines are intended. These squares are 1/20 mm. square and are used in the count of red cells. Another square is outlined in the more sparsely ruled large square in each of the four corners. This square includes a space equal to sixteen small squares and in addition the spaces in the frame-like space surrounding make twenty additional small squares, cr thirty-six in all. This space is not used ordinarily. DaCosta uses a space made up of the sixteen small squares and the bordering upper and left-hand double-line cells. This gives twenty- five small squares. There are 400 small squares in each large square, consequently as there are nine large squares the entire ruled surface consists of 3600 small squares. Wv.ova.cux' \0 ^o vG yk Principal normal and pathological blood-cells with average size, percentage in a normal differential count and the diseases in which certain pathological cells are more or less pathognomonic. The diameter of the bottom of this Petri dish is 3 inches or 7.5 + centimeters. The area of a circle is equal to the square of the radius multiplied by * or 22/7. i 1/2 in. =radius. 11/2x11/2=2.25. 2.25 x 22/7 = 7.07 square inches. 3.75 cm. =radius. 3.75x3.75 = 14.06. 14.06 x 22/7 = 44.1 square centimeters. Number of bacterial colonies in i sq. in. averages, approximately, 75. Number in 7.07 sq. in. = 530. Number of bacterial colonies in i sq. cm. averages, approxi- mately, 12. Number in 44.1 sq. cm. = 528. In a microscopic field, if the diameter were 8 small squares (1/20 mm.), the radius would be 4 small squares and the area of such a round field would be 4 x 4 = 16 x 22/7 = 50 + . Such a field would contain 50 small squares. PRACTICAL BACTERIOLOGY, BLOOD WORK AND ANIMAL PARASITOLOGY ST I TT PRACTICAL Bacteriology, Blood Work AND Animal Parasitology INCLUDING Bacteriological Keys, Zoological Tables and Explanatory Clinical Notes v/V E. R. STITT, A. B., Ph. G., M. D. SURGEON, u. s. NAVY; GRADUATE LONDON SCHOOL OF TROPICAL MEDICINE; INSTRUCTOR IN BACTERIOLOGY AND TROPICAL MEDICINE, U. S. NAVAL MEDICAL SCHOOL; LECTURER IN TROPICAL MEDICINE, JEFFERSON MEDICAL COLLEGE WITH 86 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON'S SON & CO. 1012 WALNUT STREET 1909 COPYRIGHT, 1909, BY P. BLAKISTON'S SON & Co. Printed by The Maple Press York, Pa. PREFACE. WHILE a member of the Naval Examining Board and examiner in bacteriology and clinical microscopy, I have during the past six years had an opportunity to judge of the qualifications of several hundred graduates of the various medical schools of the country from the stand- point of practical application in the laboratory of that which they had learned as undergraduates. More particularly I have made it a point to ascertain from the suc- cessful candidates, while under instruction at the Naval Medical School, the features of their laboratory courses, which had seemed to them most practical; such methods being subsequently tested in our own class work. As a result I have endeavored to incorporate in this manual methods which have been submitted to the criticism of postgraduate students from all the leading medical schools of the country, and which have been considered by them adapted to the requirements of practical, speedy and satisfactory clinical laboratory diagnosis. For the laboratory worker the most valuable asset is common sense and he must be able to bring to mind the possibilities of the production of various artefacts and results from trivial errors in technic. It has been my object to point out where such mistakes may arise, the reasons for obtaining results differing from those ordinarily obtained and the means to employ to eliminate as far as possible such results. We are too apt to neglect the trivial details of stains, reaction of media and the like, yet it is only when every detail of technic has been rigidly carried out that we are in a position to judge of the significance of an object observed in a microscopical preparation. In bacteriology, candidates were frequently able to give the cultural and morphological characteristics of all the important pathogenic VI PREFACE. organisms, yet when it was required of them to outline the procedure by which they would differentiate members of the typhoid-colon groups when encountered in a plate made from feces, the problem appeared to them impossible. They possessed the information, but did not know how to apply it. In practical work, organisms can only be separated culturally by the use of Keys and for this reason Keys are given at the beginning of each division of bacteria. These enable one to quickly place the organism isolated in its respective group. Only methods of differentiation which are applicable in a physician's private laboratory are given. Practical methods for making the final identification by agglutination or other immunity tests are described. Technic for immunizing animals to furnish such sera is given in detail. The giving of the cultural characteristics in a systematic tabulated Key gives space in the notes for presenting the salient points in the pathological and epidemiological aspects of each organism. I have endeavored to give a scientific yet practical classification of the important pathogenic moulds, a subject about which there exists greater confusion in the minds of students than for any other. In the nomenclature, I have followed Gedoelst's " Les Champignons Para- sites." In the chapter on media making, it is believed than anyone after reading this section and following the instructions will be able to satis- factorily and without the adjuncts of a large laboratory make any kind of media. The directions as to titrations are given in detail because it is beginning to be recognized that reaction of media in bacteriology is of as great importance as staining is in blood work. The section on blood work is practical and gives a method for making a Romanowsky stain which is quick and reliable. The chapter on Normal and Pathological Blood gives in a few pages the more impor- tant points to be borne in mind in considering a possible diagnosis. While there is no difference between the laboratory requirements of medical work in the tropics and that in temperate climates, unless by reason of such measures of diagnosis being indispensable in the tropics, it has, however, been my endeavor to treat every tropical question, whether in blood work, bacteriology or animal parasitology, in a more PREFACE. Vll complete way than is usual in manuals of this character. Therefore it is believed that this little book will be of great service to the laboratory worker in the tropics. It is only from working under Doctor Charles W. Stiles in his course of laboratory instruction in Animal Parasitology in the United States Xaval Medical School, that I feel justified in presenting a concise out- line of the subjects in medical zoology which appear to me to be most important for the physician. The system of arranging tables, showing the families, genera, etc., in which each species belongs will, it is believed, greatly simplify the matter of classification for the medical student. The points given under each parasite are believed to be practical ones. When a parasite has only been reported for man two or three times, very little space is given to it. Part IV summarizes the various infections which may be found in different organs or excretions of the body and embraces both bacterial and animal parasites. Practical methods for examining material are also given. The chapter on Immunity, in which the theoretical side is im- mediately illustrated by the practical application will tend to simplify this bug-bear of the medical student. The illustrations have been selected with a view to bringing out points which are difficult to state briefly in the text, and furthermore they have been grouped together so that comparison of similar para- sites is possible without turning from page to page. I have in particular to thank Hospital Steward Ebeling of the Navy for his care in bringing out such details. By reason of the authority of Braun, it has been considered sufficient to give in the tables only the proper zoological name of the parasite as given in the 1908 German edition. The synonyms have been omitted for consideration of space. The works chiefly consulted in addition to that of Braun have been : Allbutt's System of Medicine; Osier's System of Medicine; Muir and Ritchie's Bacteriology; Mense's Tropenkrankheiten; Blanchard's Les Moustiques; Guiart and Grimbert's Diagnostic; Ehrlich's Studies in Immunity; Stephens and Christopher's Practical Study of Malaria; vlii PREFACE Daniel's Laboratory Studies in Tropical Medicine; Hanson's Tropical Diseases; Gedoelst's Les Champignons Parasites; Neveu-Lemaire Parasitologie Humaine; Chester's Determinative Bacteriology; Leh- mann and Neumann's Bacteriology. E. R. S. DECEMBER 17, 1908. CONTENTS. PART I. BACTERIOLOGY. CHAPTER I.— APPARATUS. The Microscope, i; — Apparatus for sterilization, 4; — Cleaning glassware, 8; — Concave slides, fermentation tubes. 9; — Incubators, n; — Bacterio- logical pipettes, 12. CHAPTER II.— CULTURE MEDIA. Xutrient bouillon, 15; — Standardization of reaction, 17; — Sugar-free bouillon, 20; — Glycerine bouillon, 20; — Peptone solution, 21; — Nutrient agar, 21; — Glycerine agar egg medium, 22; — Gelatine, 22; — Litmus milk, 23; — Potato, 23; — Blood serum. 24; — Blood agar, 24; — Bile and faeces media, 25. CHAPTER III.— STAINING METHODS. Loffler's methylene blue, 28; — Carbol fuchsin, 28; — Gram's method, 28; — Acid-fast staining, 30; — Neisser's stain, 31; — Capsule staining, 31; — Flagella staining, 32; — Spore staining, 32. CHAPTER IV.— STUDY AND IDENTIFICATION OF BACTERIA. GENERAL CON- SIDERATIONS. Methods of isolating bacteria, 34; — Classification, 36; — Use of keys, 38. CHAPTER V. — STUDY AND IDENTIFICATION OF BACTERIA. Cocci. Key, 41; — Streptococci, 42; — Sarcinae, 43; — Staphylococci, 44; — Pneumo- coccus, 46; — Gram negative cocci, 47. CHAPTER VI.— STUDY AND IDENTIFICATION OF BACTERLA. SPORE-BEARING BACILLI. Key, 53;— Anthrax, 54; — Cultivation of anaerobes, 57; — Malignant oedema, 59; — B. botulinus, 60; — B. tetani. 62; — B. aerogenes capsulatus, 64. CHAPTER VII. — STUDY AND IDENTIFICATION OF BACTERLA. BRANCHING, CURVING BACILLI. MYCOBACTERIA. CORNYEBACTERIA. Acid-fast bacilli, 67; — Tubercle bacillus, 68; — Leprosy bacillus, 70; — Xon acid-fast branching bacilli 71; — B. mallei, 72; — B. diphtheriae, 73; — Hofman's bacillus, 76; — B. xerosis, 77. CHAPTER VIII.— STUDY AND IDENTIFICATION OF BACTERIA. Gram negative bacilli, Hemophilic bacteria, 78; — Influenza bacillus, 79; — Friedlander's bacillus, 81; — Plague, 81; — Eberth, Gartner and Esche- rich groups, 85; — Typhoid, 86; — Dysentery 89; — Chromogenic bacilli, 92. ix X CONTENTS. CHAPTER IX. — STUDY AND IDENTIFICATION OF BACTERIA. Spirilla, 94; — Cholera, 94. CHAPTER X. — STUDY AND IDENTIFICATION OF MOULDS, 99. CHAPTER XL — BACTERIOLOGY OF WATER, AIR AND MILK. Water, 109; — Milk, 115; — Air, 117. CHAPTER XII. — PRACTICAL METHODS IN IMMUNITY. Methods of obtaining immune sera, 124;— Agglutination tests, 126; — Haemolytic experiments, 128; — Bacteriolytic experiments, 128; — Devia- tion of the Complement, 129; — Fixation of the Complement, 130; — Opsonic power and preparation of vaccines, 131. PART II. STUDY OF THE BLOOD. CHAPTER XIII. — MlCROMETRY AND BLOOD PREPARATIONS. Micrometry,i35; — Haemoglobin estimation, 138; — Counting blood, 140; — Study of fresh blood, 145; — Blood films, 146; — Staining blood films, 148; — lodophilia, 152. CHAPTER XIV.— NORMAL AND PATHOLOGICAL BLOOD. Color index, 154; — Red cells, 154; — White cells, 156; — Eosinophilia, 162; — Leukocytosis, 162; — Lymphocytosis, 163; — Diseases with a normal leukocyte count, 164; — The primary anaemias, 164; — Secondary anaemias, 166; — The leukemias, 166. PART III. ANIMAL PARASITOLOGY. CHAPTER XV. — CLASSIFICATION AND METHODS, 169. CHAPTER XVI.— THE PROTOZOA. Rhizopoda, 173; — Flagellata, 176; — Infusoria, 183; — Sporozoa, 183; — The malarial parasite, 185. CHAPTER XVII.— THE FLAT WORMS. Flukes, 194; — Liver flukes, 195; — Intestinal flukes, 197; — Lung flukes, 198; — Blood flukes, 198; — Cestodes, 200; — Somatic taeniasis, 205. CHAPTER XVIIL— THE ROUND WORMS. Filariidae, 209; — Key to filarial larvae, 213; — Trichinosis, 214; — Hook worms, 216; — Ascaridae, 218; — Leeches, 219. CHAPTER XIX.— THE ARACHNOIDEA. The mites, 222; — The ticks, 224; — The Linguatulidae, 227. CHAPTER XX.— THE INSECTS. The Pediculidae, 229; — The Diptera, 231; — Fleas, 232; — Biting flies, 234. CHAPTER XXL— THE MOSQUITOES. Dissection of the mosquitoes, 244; — Differentiation of Culicinae and Anophelinae, 246; — Classification of Culicidae, 247. APPENDIX. XI PART IV. CLINICAL BACTERIOLOGY AND ANIMAL PARASITOLOGY OF THE VARIOUS BODY FLUIDS AND ORGANS. CHAPTER XXII. — DIAGNOSIS OF INFECTIONS OF THE OCULAR REGION, 251. CHAPTER XXIII.— DIAGNOSIS OF INFECTIONS OF THE NASAL CAVITIES, 253. CHAPTER XXIV.— EXAMINATION OF BUCCAL AND PHARYNGEAL MATEIOAL, 255. CHAPTER XXV. — EXAMINATION OF SPUTUM, 258. CHAPTER XXVI.— THE URINE, 261. CHAPTER XXVII.— THE RECES, 263. CHAPTER XXVIII.— BLOOD CULTURES AND BLOOD PARASITES, 268. CHAPTER XXIX.— THE STOMACH CONTENTS, 270. CHAPTER XXX. — EXAMINATION OF Pus, 271. CHAPTER XXXI.— SKIN INFECTIONS, 273. CHAPTER XXXII.— CYTODHGNOSIS, 275. CHAPTER XXXIII.— RABIES, 277. APPENDIX. PREPARATION OF TISSUES FOR EXAMINATION IN MICROSCOPIC SECTIONS, 279. MOUNTING AND PRESERVATION OF ANIMAL PARASITES, 283. PREPARATION OF NORMAL SOLUTIONS, 284. DISEASES OF UNKNOWN ETIOLOGY, 285. BACTERIOLOGY, BLOOD-WORK AND ANIMAL PARASITOLOGY CHAPTER I. APPARATUS. THE MICROSCOPE. THE most important piece of apparatus for the laboratory worker is the microscope. Very satisfactory microscopes can be purchased in this country. Instruments of standard German make are in use in many laboratories and appear to give general satisfaction. It is impossible to do good microscopical work unless the microscope gives and continues to give good definition and the working parts remain firm. A mechanical stage is almost a necessity in connection with blood-work and its use is advantageous in bacterial preparations. For the study of tissue sections the moving of the slide with the fingers is preferable. Therefore, the mechanical stage should be capable of ready attachment or removal. A triple or quadruple nose-piece, according to the number of objectives used, is also indispensable. To meet the demands of clinical microscopy there should be three objectives, preferably a 16 mm. (2/3 in.), a 4 mm. (1/6 in.) and a 2 mm. (1/12 in.) homogeneous oil immersion. The Zeiss AA is a 17 mm. objective, and the Leitz No. 3, an 18 mm. one. The Zeiss D is about 4.2 mm. and the Leitz No. 6, a 4.4 mm. A dust-proof quad- ruple nose-piece with four objectives will be found a great convenience (in addition to the 2/3-in. and i/i2-in. objectives, a i/4-in. for urine and blood counting, with a 1/8 in. for examining hanging-drop preparations and for quick examination of blood smears). An apochromatic objective costs about three times as much as an achromatic one and, except in photographic work, has little if any advantage. As regards oculars (eye-pieces) a No. 2 and a No. 4 will best meet the requirements. For high magnification a No. 8 may be of service. 2 APPARATUS. The Zeiss oculars are numbered according to the amount they increase the magnification given by the objective; thus a No. 2 increases the magnification, given by the objective alone, twice; a No. 8, eight times. Some oculars are classified according to the equivalent focal distance, and are referred to as i/2-in., i-in. and 2-in. oculars. The oculars in common use are known as negative oculars, by which is meant an ocular in which the lower lens (collective) assists in forming the real inverted image which is focused at the level of the diaphragm writhin the ocular. When using a disk micrometer, it is supported by this diaphragm, and the outlines of the image are cut by the rulings on the glass disk, and so we are enabled to measure the size of the object being examined. The measurement of various bacteria, blood-cells and parasites is exceedingly simple and assists greatly in the study of bacteria, and is indispensable in work in animal parasitology. (For details of micrometry see section on blood-work.) When an ocular is termed positive, it refers to an ocular which acts as a simple microscope in magnifying the image, the image being formed entirely by the objective and being located below the ocular. Objectives are usually designated by their equivalent focal distance. It is important to remember that the equivalent focal distance does not represent the working distance of an objective, by which is meant the distance from the upper surface of the cover-glass to the lower surface of the objective. Thus a i/4-in. objective may have to be approached to the object so that the distance intervening may be only 1/6 in. or even less. This explains the frequent inability to focus an object when a high-power dry objective (1/6 in. or 1/8 in.) is used with a rather thick cover-glass — the objective possibly having a short working distance, so that the thickness of the cover-glass does not allow of any free working distance. Instrument makers generally specify the thickness of cover-glass to be used with a certain tube length, but as a practical matter it will be found convenient to use No. i (very thin) cover-glasses. The principal objection to these is that they are more fragile than a No. 2, but with a little practice in cleaning cover-glasses this is negligible. One of the most fruitful causes of the crushing of microscopical .objects and the overlying cover-glass or, what is far more important, Till. MICROSCOPE. 3 the breaking <>f the cover-glass of a hanging-drop preparation and consequent risk of infection is the attempt to focus with the fine ad- justment. It should be an invariable rule for the worker to bring his objective practically into contact with the upper surface of the cover- glass, then using the coarse adjustment (rack and pinion) to slowly elevate the objective, looking through the eye-piece at the same time. In other words, obtain focus with the coarse adjustment and maintain it with the fine adjustment (micrometer screw). The fine adjustment should only be used after the focus is obtained. It will be observed that objectives frequently have their numerical aperture marked on them. This is expressed by the letters N.A. From a practical stand-point this gives the relative proportion of the rays which proceeding from an object can enter the lens of the objective and form the image. Of course, the greater the number of rays, the greater the N.A., the better the definition, and consequently the better the objective. Immersion oil, having the same index of refraction (1.52) as glass, would not deflect rays coming from the object and so prevent their entering the objective, as would be the case if we used a dry objective with an intervening air .space. In this case a portion of the rays would be turned aside by the difference in the refractive index of air. As a rule, the higher the numerical aperture, the better the objective and the less the working distance. In blood counting, the cover-glass being comparatively thick, it may happen that with a 1/6 in. of high numerical aperture there may not be sufficient working dis- tance to bring the blood-cells into focus, which could be done with an objective of lower numerical aperture. Consequently, we must always consider the matter of working distance as well as that of numerical aperture. An important matter in the use of the microscope is to get all the details possible with a low power before using a higher power. This, of course, does not apply to a bacterial preparation where it is necessary to use a i/ 1 2-in. or a high-power dry lens. With tissue sections, how- ever, it is not only advisable to begin the study with the lowest power, but even an examination with the unaided eye or with a magnifying before using the microscope, will give a surprising amount of information. 4 APPARATUS. It is advisable to cultivate the use of both eyes in doing microscopi- cal work. When using one eye the other should be kept open with accommodation relaxed. It is this squinting of the unemployed eye which so often fatigues. A strip of cardboard four or five inches long, with an opening to fit over the tube of the microscope, leaving the other end to block the vision of the unused eye, will prevent the strain. This apparatus can be purchased in vulcanite. Direct sunlight or excessively bright light is to be avoided. If such conditions must exist a wrhite shade or muslin curtain drawn across the window is a necessity. Light from the north and from a white cloud is the most desirable. The technic in connection with proper illumination is probably more important than any other point; unless the light is utilized to the best advantage, the best results cannot be obtained. In examining fresh blood preparations or hanging drops the concave mirror should be used and the light almost shut off bv the iris diaphragm so as to give a contour picture. In examining a stained blood or bacterial preparation, the Abbe condenser should be properly focused so as to best illuminate the stained film. In many instruments set-screws are provided which check the elevation of the Abbe condenser when the proper focus is reached. Inasmuch as the light from the condenser should come to a focus exactly level with the object studied, it is evident that a fixed position for the condenser would not answer when slides of different thickness were used. Always use the plane mirror when examining stained bacterial or blood -films, as a color image is desired. Ordinarily in examining tissue sections, the Abbe condenser should either be put out of focus by racking down or by the use of the concave mirror and the narrowing of the aperture of the iris diaphragm. Swing-out condensers are now made which are very convenient. The proper employment of illumination only comes with experience, and one should continue to manipulate his mirrors, diaphragm and condenser until the best result is obtained. Then study the specimen. APPARATUS FOR STERILIZATION. For the purpose of sterilizing glassware, media and old cultures there are three methods ordinarily employed. The hot-air sterilizer, APPARATUS FOR STERILIZATMN. 5 in which a temperature of about 150° C. is maintained for one hour, is ordinarily used for the sterilization of Petri dishes, test-tubes, pipettes, etc. If the temperature is allowed to go too high, there is danger of charring the cotton plugs and also of causing the development of an empyreumatic oil which makes the plugs unsightly and causes them to stick to the glass. Again we must be careful not to open the door until the temperature has fallen to 60° C., otherwise here is danger of cracking the glassware. Where gas is not obtainable, the hot-air sterilizer is not a very satisfactory apparatus. The Arnold sterilizer is to be found everywhere and can be used on blue-flame kerosene-oil stoves as readily as with gas burners. The most convenient form, but more expensive, is the Boston Board of Health pattern. The ordinary pattern, with a telescoping outer portion, answers all purposes, however. In the Arnold, sterilization is effected by streaming steam at 100° C. It is usual to maintain this temperature for fifteen to twenty-five minutes each day for three suc- cessive days. The success of this procedure — fractional sterilization — is due to the fact that many spores which were not killed at the first steaming have developed into vegetative forms within twenty-four hours, and when the steam is then applied such forms are destroyed. Experience has shown that all the spores have developed by the time of the third steaming, so that with this final application of heat we secure perfect sterilization. It is customary to use the Arnold for sterilizing gelatin and milk media, even when the autoclave is at hand, the idea being that the greater heat of the autoclave may interfere with the quality of such media. The most convenient autoclave is the horizontal type, such as is to be found even-where for the sterilization of surgical dressings. The source of heat may be either gas, the Primus kerosene-oil lamp or steam from an adjacent boiler. During the past year, in the labora- tory of the U. S. Xaval Medical School, we have been using a dressing sterilizer, made by the American Sterilizer Co., with which it has been possible to most satisfactorily carry out all kinds of sterilization thus doing away with the use of the Arnold and the hot-air sterilizer. It is impossible to sterilize ordinary fermentation tubes in the autoclave on account of the boiling up of the media and wetting of the plugs. APPARATUS. This is still done with the Arnold. By use of the Durham tubes — which are to be preferred, except for gas analysis — sugar media can be thus sterilized, and glassware will come out with the wrappers as dry C . Fro. i. — Dressing sterilizer showing cylinder containing water (K) heated either by gas or Primus Kerosene lamps. and the plugs of test-tubes as stopper-like as could be effected in a hot- air sterilizer. The objection which exists in the use of some autoclaves, as regards condensation on dressings or apparatus, does not exist in this type. STERII.I/ATloN. 7 The mechanism, by which the inner and outer chambers are con- nected and disconnected, and that for vacuum production, rest in the simple turning of a lever from mark to mark. We have been able with a gas burner to obtain a pressure of fifteen pounds in less than ten minutes. In sterilizing test-tubes we place them in small rectangular wire baskets, 6x5x4 ins. These baskets are to be preferred to round ones, as they pack more satisfactorily in the refrigerator used for storing media. In sterilizing flasks, test-tubes, Petri dishes, throat swabs, pipettes, etc., it has been our custom, after exposing to 20 pound- for twenty minutes, to produce a vacuum for two or three minutes; then with the steam in the outer jacket for a few minutes to thoroughly dry the articles in the disinfecting chamber. The valve to the inner chamber is then opened to break the vacuum; the door is now opened, and the articles removed in as dry a state as if they had been in the hot-air sterilizer. PRESSURE AND TEMPERATURE TABLE. 5 pounds' pressure, 107.7° C., 226° F. 10 pounds' pressure, 115. 5° C., 240° F. 15 pounds' pressure, 121. 6° C., 250° F. 20 pounds' pressure, 126. 6° C., 260° F. 25 pounds' pressure, 130.5° C., 267° F. 30 pounds' pressure, 134.4° C., 274° F. All such articles as Petri dishes, pipettes, swabs, etc. are wrapped in cheap quality filter-paper, making a fold and turning in the ends as is done in a druggist's package. Old newspapers answer well for this purpose. The sterile swab can be used for many purposes in the laboratory. They are most easily made by taking a piece of copper wire about eight inches long, flattening one end with a stroke of a hammer, then twisting a small pledget of plain absorbent cotton around the flattened end. After wrapping, the swabs are sterilized in bunches. We not only use them for getting throat cultures, but in addition for culturing faeces, pus or other such material. The pus is obtained with a swab, which material is then distributed in a tube of sterile bouillon or water. With the same swab the surface of an agar plate is successively stroked. This method is equally as satisfactory as 8 APPARATUS. the German one of using bent glass rods for this purpose. Everyone has encountered the difficulties attendant upon the bending of platinum wires and also the possibility of destroying your organisms by an insufficiently cooled wire. CLEANING GLASSWARE. It is a routine in our laboratory for everything to go through the sterilizer at 125° C. before anything else is done. This is a safe rule FIG. 2. — i, Inoculation of tubes; 2, plugging of tubes; 3, filling tubes; 4, Smith's fermentation tube; 5, Durham's fermentation tube. when dealing with dangerous pathogenic organisms. As soon as taken out of the sterilizer the contents are emptied, and the tubes or dishes placed in a i% solution of washing soda and boiled. This thoroughly cleans them. As the washing soda slightly raises the boiling-point and also makes the spores more penetrable, it would appear that under ordinary circumstances, it would be sufficient to place all contaminated articles in a dishpan with the soda solution, CLEANING GLASSWARE. 9 and boil for at least one hour, not using a preliminary sterilization in the autoclave. The tubes are now cleaned with a test-tube brush, thoroughly rinsed with tap water and placed in a i% solution of hydrochloric acid for a few minutes; then rinsed thoroughly in water and placed in test-tube baskets, mouth downward, and allowed to drain over night. When thoroughly dry they may be plugged and sterilized. To plug a test-tube, pick out a little pledget of plain absorbent cotton about two inches in diameter from a roll. Place it over the center of the tube and with a glass rod push the cotton down the tube about an inch. The cleaning fluid commonly used in labora- tories consists of one part each of potassium bichromate and com- mercial sulphuric acid with ten parts of water. This is an excellent mixture for cleaning old slides, etc., especially when grease or balsam is to be gotten rid of. It is very corrosive, however. An efficient and less corrosive methcd for cleaning slides and covei>glasses is to leave them over night in an acetic acid alcohol mixture (two parts of glacial acetic acid to one hundred parts of alcohol). After drying and polishing out of this mixture, it is well to pass the slides and cover- glasses through the flame of a Bunsen burner or alcohol lamp to remove every vestige of grease. Ordinarily, rubbing between the thumb and forefinger with soap and water, then drying with an old piece of linen, and finally flaming will yield a perfect surface for making a bacterial preparation. CONCAVE SLIDES, FERMENTATION TUBES. The concave slide is ordinarily used for making hanging-drop prepara- tions. A substitute which is equally good may be made by spreading FIG. 3. — Hanging drop, over hollow ground slide. (Williams.} a ring or square of vaselin — smaller than the cover-glass to be used — in the middle of the slide. Then putting a loopful of salt solution in the center of the space, and inoculating with the culture to be studied, we finally cover it with a cover-glass, gently pressing the mar- gins down on the vaselin. This gives a preparation for the study 10 APPARATUS. of motility or agglutination which does not dry out for hours, and is easier to focus upon than the concave slide hanging-drop preparation. The fermentation tube with a bulb and closed arm is expensive, difficult to clean and is easily broken. As a substitute in the study of gas production and in water bacteriology, the Durham tube is to be recommended. Into a test-tube, about i x 7 in., we introduce the special sugar media, then drop down a small test-tube (1/2 x 3 in.) with its open end downward. Insert the plug of the large tube and sterilize. During sterilization the fluid enters the mouth of the smaller FIG. 4. — Blood serum coagulating apparatus. tube and fills it, and when the medium is subsequently inoculated, if gas forms, it appears in the upper part of the closed end of the smaller tube. For inspissating blood serum slants a regular inspissator is desirable. This is nothing more than a double-walled vessel, the space between the walls being filled with water. As a substitute one may take the common rice cooker (double boiler). Fill the outer part with water; and in the inner compartment pack the serum tubes properly slanted on a piece of wood or a wedge-shape layer of cotton. Place a weight on the cover of the inner compartment to sink it into the surrounding water, and allow to boil for one or two hours. This same apparatus may be used for their sterilization on two subsequent days, but it is better to sterilize in the autoclave or Arnold. As regards a working desk, it will be found convenient to have an arrangement similar to the ordinary flat-top desk, writh a tier of drawers on each side. A block INCUBATORS. H of wood with holes bored in it to contain dropping-bottles may be placed in the upper left hand drawer. In this way the stains are as accessible as if they encumbered the desk. It is advisable to paint the inside of this drawer black so that the light may not cause the staining reagents to deteriorate. Ordinary glass salt cellars will be found very useful, where the watch- glass is employed. They may also be wrapped, sterilized and used to con- . FIG. 5. — Rice cooker. tain fluids for inoculating, etc. For use in making loops and needles, platinum wire of 26 gauge will be found most suitable. The handle made of glass rocNis pref- erable to the metal ones. INCUBATORS. \\hen gas is obtainable, the maintaining of a constant temperature for the body temperature incubator (38° C.) and the paraffin oven (60° C.) is best secured by the use of some of the various types of thermo-regulators. The Reichert type is the one in general use, although there are many features about the Dunham and Roux regulators which are advantageous. If the pressure of the gas-supply varies from time to time, it is essential to regulate this by the use of a gas pressure regulator (Murrill's is a cheap and satisfactory one). Incubators, controlled electrically, can be obtained of certain foreign makers, and are quoted in catalogues of American dealers. It is probable that the Koch petroleum lamp incubator is the most satisfactory one where gas is not obtainable. They should be of all metal construction, and not with a wood casing, on account of the danger from fire. They cost from twenty-five to fifty dollars. An incubator may be extemporized by putting the bulb of an incan- descent electric lamp in a vessel of water. The proper temperature may be obtained by increasing the amount of water or by covering the opening more or less completely with a towel. The test-tubes to be 1 2 APPARATUS. incubated can be put into a fruit jar or tin can, which receptacle is placed in the vessel heated by the lamp. Emery suggests the use of a Thermos bottle as an incubator. As regards the matter of a low-temperature incubator (for gelatin work) , this is best met. by using a small refrigerator. The ice in the upper part maintains an even cold, and by connecting up an electric lamp in the lower part of the refrigerator we can easily maintain a temperature which only varies one or two degrees during the twenty-four hours. With a i6-candle-power lamp a temperature of about 25° C. is mantained (this is too high, being about the melting-point of gelatin), with an 8 candle-power, one about 21° to 23° C., and with a 4-candle- power from 18° to 20° C.; the box being about 20 x 30 x 36 inches. When much serum reaction work is done, an electrically run cen- trifuge is of the greatest convenience. A filter pump attached to the water faucet, preferably by screw threads, is almost indispensable for filtering cultures,' etc., and for cleaning small pipettes, especially the hgemacytometer pipettes. Such a filter or vacuum pump with a vacuum gauge is more easily controlled. BACTERIOLOGICAL PIPETTES. With the possible exception of the platinum loop, there is no piece of apparatus so applicable to many uses as the capillary pipette made from a piece of glass tubing. These may be made in a great variety of shapes. The one with a hooked end, the Wright tube, is the best apparatus for securing blood for serum tests. The crook hangs on the centrifuge guard and by filing and breaking the thicker part of the tube the serum is accessible to a capillary rubber bulb pipette or to the tip of a haemacytometer pipette. In this way dilutions of serum are easily made. The capil- lary pipette is made by taking a piece of 1/4 in. soft German glass tubing, about six inches long, and heating in the middle in a Bunsen flame, revolving the tubing while heating it. When it becomes soft in the center, remove from the flame and with a steady even pull separate the two ends. The capillary portion should be from eighteen to twenty inches in length. When cool, file and break off this capillary portion in the middle. We then have two capillary pipettes. By BACTERIOLOGICAL PIPETTES. using a rubber bulb, such as comes on medicine droppers, \ve have a means of sucking up and forcing out fluids by pressure with the thumb and forefinger of the right hand. The bulb should be pushed on about 1/2 to 3/4 in.; this gives a firmer surface to control the pressure on the bulb. FIG. 6. — i, 2, 3, Drawing out glass tubing; 4, 5, Wright's rubber bulb capillary pipettes showing grease pencil mark for making dilutions; 6, 7, Wright's U tubes; 8, 9, 10, Methods of drawing out test tubes for vaccines in opsonic work; n, Bac- teriological pipette. A bacteriological pipette is made by drawing out a nine-inch piece of tubing about three inches at either end, then heating in the middle we draw out and have two pipettes similar to the one shown in the drawing. A piece of cotton is loosely pushed in just above the narrow portion. These may be wrapped in paper and sterilized for future1 use. They may be made perfectly sterile at the time of drawing out. Where gas is not at hand, the Barthel alcohol lamp gives a flame similar to that of the Bunsen lamp and is equally satisfactory for heating glass tubing. CHAPTER II. CULTURE MEDIA. WHILE there are certain advantages in sterilizing the glass test- tubes prior to filling them with media, yet this may be dispensed with — the sterilization after the media has been tubed being sufficient. If a dressing sterilizer is at hand, this is preferable for sterilizing such media as bouillon, potato and agar (10 to 15 pounds' pressure for fifteen .minutes) . Milk shculd be sterilized with the Arnold, subjecting the media to three steamings for twenty minutes on three successive days. Gelatin may be sterilized in either way, but preferably in the autoclave at 7 pounds' pressure for fifteen minutes. As soon as taken out of the sterilizer it should be cooled as quickly as possible in cool water. . This procedure tends to prevent the lowering of the melting- point of the finished gelatin and also preserves its spissitude. Blood-serum is preferably solidified as slants in a blood serum inspissator. This requires one to two hours. The subsequent sterili- zation in the autoclave or Arnold should not be done immediately after making the solidified slants, but on the subsequent day. If done on the same day, many of the slants are ruined by being dis- rupted by bubbles. The preparation of blood-serum slants or slants of egg media can be conveniently carried out in a rice cooker (double boiler). Place the tubes in the inner compartment of the cooker, obtaining the slant desired by manipulating an empty test tube, or with a towel or cotton batting on the bottom. Then cover the tubes with another towel. The outer compartment should contain water alone '(no 25% salt solution). The inner compartment should be weighied down so that it is surrounded by water — the light tubes not being sufficient to sink it. Allowing the water in the outer compart- ment to boil one or two hours will inspissate or solidify the slants satisfactorily. The sterilization on subsequent days may be carried out in the same apparatus, although it is more efficient if done in an 14 M TRIKM 1501 II i 15 Arnold or an autoclave. (This sterilization in the rice cooker makes the media too dry.) In making media a rice cooker is almost essential; at any rate, it is so if ease, expedition and unfailing success in preparation are to be achieved. As it is necessary to make the contents of the inner com- partment boil, the temperature of the water in the outer compartment must be raised. This is done by using a 25% solution of common salt or a 20% solution of calcium chloride in the outer compartment instead of plain water. A 15% solution of salt raises the boiling point 2 1/2° C.; a 20%, 3 1/2° C., and a 25%, 4 1/2° C. The raising of the boiling- point by calcium chloride is about the same for similar strength solutions. Although the Bacteriological Committee of the A. P. H. Asso- ciation recommends special steps to be taken in the preparation of gelatin and agar, yet for clinical purposes it will be found satisfactory to keep on hand a stock of bouillon, and when it is desired to make agar or gelatin to simply prepare such media from the stock bouillon in the way to be subsequently given. XUTRIEXT BOUILLON. This may be made either from fresh meat or from meat extract. Media from fresh meat are usually lighter in color and possibly clearer. In the Philippines, however, certain measures employed for the preser- vation of the meat made it very difficult to prepare clear bouillon from it, so that meat extract was used entirely. There is very little differ- ence, if any, in the nutritive power of media made in either way. The chief objections to fresh meat as a base are: (i) It takes more time and trouble. (2) The reaction, due to sarcolactic acid and acid salts, is quite acid, so that it is necessary to titrate and neutralize the excess of acidity. (3) The reaction of the finished media tends to change unless the boiling at the time of making was very prolonged. (4) It is not infrequent to have a heavy precipitate of phosphates thrown down at the time of sterilization, thus making it necessarv to repeat the process of filtration and sterilization. 1 6 CULTURE MEDIA. If fresh meat is used, take about 500 grams (one pound), remove fat and cut it up with a sausage mill or purchase the meat already cut up as fora Hamburg steak. It makes little difference whether the amount be 100 grams more or less. Place the chopped-up meat in a receptacle and pour 1000 c.c. of water over it. Keep in the ice chest over night and the next morning skim off with a piece of absorbent cotton the scum of fat; then squeeze out the infusion with a strong muslin cloth, making the amount up to 1000 c.c. This meat infusion contains all the albuminous material necessary for the clarification of the bouillon. It is convenient to designate this meat base as Meat Infusion to distinguish from the base containing meat extract. Having obtained 1000 c.c. of this 50% meat infusion, we dissolve in it i% of Witte's peptone and 1/2% of sodium chloride. While there is a sufficiency of the various salts necessary for bacterial development in the meat juices, yet there is not enough to give the best results when bouillon cultures of various organisms are used for agglutination tests; and futhermore, when bouillon is used for blood cultures, disinte- gration of the red cells, with clouding of the clear medium, may occur if there be not sufficient salt present to prevent this. The salt and the peptone are best put in a mortar, and adding about one ounce of the meat infusion we make a pasty mass; then we grad- ually add the remaining infusion until solution is complete. It is sometimes recommended to use a temperature of 50° C. to facilitate the solution of the peptone. This is not necessary, and if the tempera- tare is not watched closely it might go up to 65° C. or higher and we should lose the clearing albuminous material from its coagulation. Of this rather cloudy solution take up 10 c.c. with a pipette and let it run out into a beaker. Add forty c.c. of distilled or rain water and about six drops of a 0.5% phenolphthalein solution. (Phenolphtha- lein, 0.5; dilute alcohol, 100 c.c.) Now from a burette filled with decinormal sodium hydrate solution, run in this solution until we have the development of a rich violet-pink color in the diluted bouillon in the beaker. To obtain a standard for comparison, simply add six drops of the phenolphthalein solution to 50 c.c. of water and add about i c.c. of the N/io sodium hydrate solution. As soon as we have obtained a TITRATION OF MEDIA. IJ color of the same intensity as our standard, we read off the number of c.c. or fractions of a c.c. of N/io sodium hydrate solution added to produce the color. This number gives the acidity of the bouillon in percentage of N/i acid solution.* Percent acid means that so many c.c. of N/i acid added to 100 c.c. of the medium at the neutral point would give that percentage reaction. Thus i 1/2 c.c. of N/i HC1 solution added to 100 c.c. of medium at o, would give us i 1/2% of acidity or +1.5. Percent alkaline means so many c.c. of N/i sodium hydrate solution added to 100 c.c. of the medium at the neutral point. Thus a 1/2% alkaline medium would be one whose alkalinity would cor- respond to the addition of 1/2 c.c. of N/i NaOH to 100 c.c. of the medium at o. It is written — .5. If we took 100 c.c. of the medium and put it in a beaker and then ran in N/i NaOH solution from a burette, it will be readily under- stood that if we had to add 31/2 c.c. of N/ 1 NaOH to obtain the pink color, it would show that the acidity of the 100 c.c. of medium, being tested, corresponded to 3.5 c.c. of N/i acid solution, and that its acidity was equal to 3 1/2% of N/i acid solution, or that its reaction was +3.5. As N/ 1 NaOH solution is too corrosive for general use in a burette, and as 10 c.c. of medium is more convenient to work with than 100 c.c., we use a solution one-tenth the strength of the N/i NaOH and we take only one-tenth of the 100 c.c. of medium. In this way it is the same from a stand-point of directly reading off our percentage reaction as if we had 100 c.c. of medium and used N/i NaOH solution. The A. P. H. Association recommends 5 c.c. of the medium and the use of X/20 NaOH. As the N/io NaOH is always at hand for titrating gastric juice, the N/io is used instead. * A more satisfactory method for one with experience in titrating is to continue to add the N/io XaOH solution from the burette, drop by drop, until the addition of a drop fails to show any intensifying of the purplish violet color at the spot where it came in contact with the diluted bouillon in the beaker. This marks the end reaction. A reaction of about +.7 in the cold gives a delicate pink. In titrating at the boiling temperature (probably preferable for gelatine and agar) use a por- celain dish. and boil for i or 2 minutes. Then add the sodium hydrate solution from the burette until a faint pink develops. Then add about 4 additional drops from the burette and the reading will be fairly accurate. l8 CULTURE MEDIA. Having determined the percentage acidity of the 10 c.c. sample tested, we easily calculate the number of c.c. of N/iNaOH solution required to be added to the 1000 c.c. of bouillon to obtain a reaction corresponding to the neutral point of phenolphthalein. It is more exact to take the average of two titrations. As 100 c.c. of medium would require 31/2 c.c., 1000 c.c. would require 10 times as much, or 35 c.c. N/i NaOH solution. Having measured out and added 35 c.c. of the N/i NaOH solution to the meat infusion, containing salt and peptone, we have a solution which is exactly neutral to phenolphthalein, or o. It is usually considered that a reaction of about i percent acid is the optimum reaction for bacterial growth. Hence we should now add i % of N/i HC1 solution to the medium. This would be accomplished by adding 10 c.c. of N/i HC1 solution to the 1000 c.c. of neutralized medium, and we would have a medium with a reaction of + 1. If we desired a reaction of one percent alkalinity we would add an additional c.c. of N/i NaOH solution to every 100 c.c. of the medium at o, or 10 c.c. for the 1000 c.c. of medium. The reaction would then be — i. As a matter of convenience, we usually determine the reaction of the medium, which is always more or less acid, and then add enough N/i NaOH to reduce the acidity to the percentage we desire to set the medium, instead of neutralizing all the acidity present and then, in a second operation, restoring the acidity to the point desired. Thus finding the acidity of the medium to be 3 1/2% and desiring to give it an acidity of i%, we would add only 21/2 c.c. of N/i NaOH to every 100 c.c. of medium, or 25 c.c. for the 1000 c.c. of medium. The reaction would then be found to be -fi. The neutral point of litmus is not a sharp one, but it corresponds rather closely with a reaction of +1.5 to phenolphthalein. The recommendations of the A. P. H. Association call for making the titration with the medium boiling. This is a very difficult titration and students obtain results varying greatly, which is not the case when the titration is conducted at room temperature and a standard color is at hand. If the color of the end reaction at boiling-point be obtained, it will be found that when cool it deepens until it corresponds to the rich violet-pink of the end reaction in the cold or vice versa. i 1 1. 1. o.\. ig To summarize: Take Peptone, 10 grams Sodium chloride, 5 grams 50% meat infusion, 1000 c.c. Dissolve the peptone and sodium chloride in the meat infusion and add enough N/i NaOH to make the reaction +i. Put the solution in the inner compartment of a rice cooker and bring to the boiling-point and maintain this temperature for twenty minutes. The calcium chloride or sodium chloride in the outer compartment of the rice cooker enables us to secure a boiling temperature for the con- tents of the inner compartment. Do not stir the bouillon that is being heated, as the pultaceous membranous mass of coagulated albumin makes filtration easy. Filter. The filter-paper in the funnel should be thoroughly wet with water before pouring on the bouillon. This is to prevent clogging of the pores of the filter-paper. Make up the quantity of filtrate to 1000 c.c. by adding water. If greater exactness is demanded than answers for ordinary clinical work, it is advisable to again titrate and again adjust the reaction or to simply record the exact reaction. It is more convenient to have a counterpoise to balance the inner compartment and then to add water to the medium until a kilo weight, in addition to the weight balancing the container, is just balanced,. Then titrate, adjust the reaction (if so desired) and filter. Sterilize in the autoclave at 115° to 120° C. for fifteen minutes or in the Arnold on three successive days. The use of a balance is preferable in the preparation of bouillon, necessary in making gelatin and imperative in making agar media BOUILLON MADE FROM LIEBIG'S MEAT EXTRACT. Place in a mortar 3 grams of Liebig's extract, 10 grams of pep- tone and 5 grams of sodium chloride. Dissolve the whites of one or two eggs in 1000 c.c. of water. Then add this egg-white water, little by little, to the extract, peptone and salt in the mortar until a brownish solution is obtained. Pour this into the inner compartment of a rice cooker; apply heat to the outer compartment containing the salt or calcium chloride solution, allow to come to a boil and to continue 20 CULTURE MEDIA. boiling for fifteen to twenty minutes. Do not stir. Place inner com- partment on the scales and its counterpoise and a one-kilo weight on the other side. Add water until the two arms balance. Filter and sterilize. The reaction of media made with Liebig's meat extract rarely exceeds +.75 (from +.6 to +.9). Consequently for growing bacteria it is unnecessary to titrate and adjust reaction unless precision is demanded. SUGAR-FREE BOUILLON. Inoculate' nutrient bouillon in a flask with the colon bacillus. Allow to incubate at 37° C. over night. Pour the contents into a sauce- pan and bring to a boil to kill the colon bacilli. Put about 15 grams of purified talc (Talcum purificatum, U. S. P.) in a mortar. Add the dead colon culture, stirring constantly. Then filter through filter- paper. It may be necessary to again pass the filtrate through the same filter until the sugar-free bouillon is perfectly clear. For all ordinary purposes the very small amount of sugar in bouillon made from Liebig's meat extract may be neglected in determining gas production; so that under such conditions the various sugars could be added directly to the meat-extract bouillon. SUGAR BOUILLONS. The sugar media ordinarily used for determining fermentation or gas production are those of glucose and lactose. In special work such carbohydrates as saccharose and maltose are used. The alcohol mannite is used in differentiating strains of dysentery bacilli. To make, simply dissolve i or 2% of the sugar in sugar-free bouillon or that made from meat extract. Tube in Durham's or the ordinary fermentation tubes and sterilize in the autoclave at only about 5 pounds' pressure for 15 minutes, or in the Arnold. GLYCERIN BOUILLON. Add 6% of glycerin to ordinary bouillon. It is chiefly used in the cultivation of tubercle bacilli. MTRIENT AGAR. 21 PEPTONE SOLUTION (DUNHAM'S). Dissolve i' , of Witte's peptone and 1/2% of sodium chloride in distilled water. Filter, tube and sterilize. Peptone solution may he used as a base for sugar media instead of bouillon. It is the medium used in testing for indol production. This test is made by adding from six to eight drops of concentrated H2SO4 to a 24- to 48-hour-old peptone culture of the organism to be tested. If the organism pro- duces both indol and a nitroso body, we obtain a violet-pink coloration, "cholera red." If no pink color is produced on the addition of the sulphuric acid, add about i c.c. of an exceedingly dilute solution (i : 10,000) of sodium nitrite. XITRIEXT AGAR. In making agar medium it is preferable to use powdered agar, as this goes into solution more readily than the shredded agar. The reaction of agar is slightly alkaline, so that if i 1/2 to 2% of agar is added to nutrient bouillon having a reaction of + 1 the finished prod- uct will be found to be about -f .8. To make: Weigh 15 to 20 grams of powdered agar and place in a mortar. Make a paste by adding nutrient bouillon, little by little, and when a smooth even mixture is made, pour it into the inner com- partment of a rice cooker and add the remainder of the 1000 c.c. of bouillon. The use of the balance is preferable. The outer compartment of the rice cooker should contain the 25% salt solution. Bring to boil, and the agar will be found to have entirely gone into solution after three to five minutes of boiling. Then, using a funnel which has been heated in boiling water and which contains a small pledget of absorbent cotton, we filter the agar, tube it and sterilize it in the autoclave or Arnold. One and one-half percent agar can be readily filtered through filter-paper and gives a clearer medium. By taking of meat extract 3 grams, peptone 10 grams, salt 5 grams, powdered agar 15 grams, the white of one egg and 1000 c.c. of water, making at first a paste of all the ingredients in a mortar, then gradually adding the remainder of the 1000 c.c. of water, putting in the rice 22 CULTURE MEDIA. cooker, bringing to a boil without stirring, allowing to boil fifteen minutes and then filtering through absorbent cotton in a hot funnel, we obtain a satisfactory medium, the reaction of which will be from + .7 to +.9. GLUCOSE AGAR. Add the agar to i or 2% glucose bouillon and proceed as for ordinary agar. GLYCERIN AGAR. Add the agar to 6% glycerin bouillon instead of nutrient bouillon. GLYCERIN AGAR EGG MEDIUM. Take the white and the yolk of one egg and mix thoroughly in a vessel kept between 45° and 55° C. with an equal amount of glycerin agar. Tube the medium, inspissate in a rice cooker as for serum tubes, and sterilize as for blood-serum tubes. This makes an excellent medium for growing tubercle bacilli. As egg medium has a tendency to be dry, it is well to add i c.c. of glycerin bouillon to each slant before autoclaving. NUTRIENT GELATIN. Add about 12% (120 grams) of "gold label" gelatin to 1000 c.c. of nutrient bouillon in a rice cooker. If the bouillon had a reaction of about +i, the gelatin solution will be about +3.5, so that it is always necessary to titrate gelatin and neutralize to about +i. The pro- cedure is the same as for bouillon. As the color reaction is not quite as distinct with gelatin, it is better to make a color standard with 4 or 5 c.c. of the gelatin medium in 50 c.c. of water, instead of using the distilled water alone, as was recommended for bouillon. Having neutralized and allowed to boil for fifteen minutes, we filter through filter-paper in a hot funnel. As it is very important that gelatin should be perfectly clear, it is better to filter through filter- paper than through cotton. The filter paper should be very thoroughly wetted with very hot water before filtering gelatine or agar. Tube the medium and sterilize, either in the Arnold on three POTATO Ml.DIA. successive days or in the autoclave at 8-10 pounds' pressure for ten minutes. The tubes should be cooled as quickly as possible in cold water after taking out of the sterilizer. LITMUS MILK. Milk for media should be as fresh as possible. It should then be put in a 1000 c.c. Erlenmeyer flask, sterilized for fifteen minutes in the Arnold and set over night in the refrigerator. The next morning the milk beneath the cream should be siphoned off. The short arm of the siphon should not reach the bottom of the flask so as to avoid the sediment. Add sufficient tincture of litmus to this milk to give a decided lilac tinge; tube and sterilize in the Arnold on three suc- cessive days. POTATO SLANTS. Take Irish potatoes and scrub thoroughly with a stiff brush. Then pare off generously all the outer portion. From the white interior cut out cylinders with a cork borer. These cylinders should be of 1/2 to 3/4 of an inch in diameter. Divide a cylinder by a diagonal cut. This gives a plug with a flat base, the other extremity being a slant. These potato plugs should be left in running water over night or washed with frequent changes of water. This prevents the blackening of the plug. Into a i -in. test-tube drop a pledget of absorbent cotton well moistened with water. Then drop in the potato plug, FlG base downward. Sterilize in the autoclave at 15 pounds Potato in for fifteen to twenty minutes, to insure sterility. For glycerin potato, soak the plugs in 6% glycerin solution for about one hour. Then drop in a pledget of absorbent cotton moistened with the same glycerin solution into the test-tubes and follow it with the potato plug. Sterilize in the autoclave. BLOOD-SERUM. The blood of cattle should be collected in large pans or pails at the abattoir. This vessel of blocd should then be kept in the cold-storage 24 CULTURE MEDIA. room and the next morning the more or less clear serum will have been squeezed out from the clot. Collect this serum and keep in the ice chest for future use. If to be kept for a long time, it is advisable to add about 2% of chloroform to the serum. This will not only keep the serum, but will eventually sterilize it. To make Loffler's serum, take one part of glucose bouillon and three parts of blood-serum. Mix, tube and coagulate the albumin in the inspissator or rice cooker, giving the tubes a proper slant before heating. Sterilize the following day in the autoclave as previously directed or in the Arnold on three successive days. A SUBSTITUTE FOR ORDINARY BLOOD -SERUM. Purchase a good article of commercial blood-serum albumin and make a 15% solution of it in glucose bouillon. Tube and inspissate as for blood-serum. If made with glycerin bouillon, it makes a good medium for tubercle bacilli. As this dried blood albumin only costs about fifty cents a pound and will keep permanently, it is exceedingly convenient for those not near an abattoir. Its use was first suggested by Hospital Steward King, of this laboratory, and I cannot find that it has been previously used as a substitute for fresh blood-serum. At any rate, the results with it as a culture medium seem to be the same as with the fresh serum. This is better than the various white of egg substitutes usually recommended. HYDROCELE, AND BLOOD AGAR. To tubes of melted agar at 50° C. add from one to three c.c. of hydrocele or ascitic fluid, observing aseptic precautions. For blood agar the blood from a vein should be received into a sodium citrate salt solution to prevent coagulation, and added subsequently as for hydro- cele fluid. Allow the agar to solidify as a slant. BLOOD-STREAKED AGAR. Sterilize the lobe of the ear and puncture with a sterile needle. Collect the exuding blood on a large platinum loop and smear it over F.ECES PLATING MEDIA. 25 the surface of an agar slant. It is advisable to incubate overnight as a test for sterility. BILE MEDIA. Secure ox bile from the abattoir or human bile from cases of gall- bladder drainage in hospitals. Put about 10 c.c. in each tube and sterilize. Some prefer to add i% of peptone. This is the medium for blood cultures in typhoid, etc. PLATING MEDIA FOR RECES WORK. The media of Endo, Conradi-Drigalski and the lactose litmus agar medium are probably the most satisfactory of the numerous ones that have been proposed for plating out faeces. A convenient way of pre- paring any one or all of these, and which apparently gives media equal to that prepared according to the original formulae, is as follows: Liebig's extract, 5 grams. Salt, 5 grams. Peptone, 10 grams. Lactose (C. P.), 10 grams. Agar, 20 grams. Water to make, 1000 c.c. Prepare as for ordinary nutrient agar, with the difference that the reaction should be brought down to — .5. For Endows Medium. — Keep this lactose agar base in 100 c.c. quantities in Erlenmeyer flasks instead of test-tubes. When needed for plating, melt a flask of this agar, and while liquid add to the 100 c.c. ten drops of a saturated alcoholic solution of basic fuchsin, and then twenty drops of a freshly prepared 20% solution of sodium sulphite. The medium should be of a light flesh or pale salmon color. Colon bacilli show on this medium as vermilion colonies, which in about 48 hours have a metallic scum on them. Typhoid and dysentery colonies are grayish. For Lactose Litmus Agar. — Color the lactose agar base with tincture of litmus to a lilac color. This may be tubed, putting 10 c.c. 26 CULTURE MEDIA. in each test-tube, or put in quantities of 50 or 100 c.c. in small Erlen- meyer flasks. It is then sterilized in the autoclave (10 pounds for 15 minutes) or in the Arnold. For Conradi-Drigalski Medium. — Melt down the lactose litmus agar in the flasks holding 100 c.c. Then add to the 100 c.c. of medium, i c.c. of a solution of crystal violet (crystal violet o.i gram, distilled water 100 c.c.). The medium is then ready to put into plates. Colon colonies are pink. Typhoid and dysentery colonies, a bluish-gray. CHAPTER III. STAINING METHODS. L\ order to study a bacterial or blood specimen the first essential is a properly prepared film the matter of staining is of less importance. The slide or cover glass, after cleaning with soap and water or by special solutions, should be polished with a piece of old linen. If a glass surface is free of grease a loopful of water will smear out evenly and over the entire surface. The only quick practical way to make the slide or cover -glass grease free is to burn the surface for a moment in a Bunsen or alcohol flame. The cover -glass must not be warped. To make a preparation, apply a small loopful-of distilled water on the slide or cover glass and, touching a colony with a platinum needle, stir the transferred culture into the loopful (not drop) of water. The mistake is almost invariably made of taking up too much bacterial growth. Fluid cultures do not need dilution. Smearing the mixture over a large part of the cover-glass or over an equal area of a slide, it is allowed to dry. If very little water is used, the preparation dries readily. Otherwise it can be dried in the fingers high over a flame. As soon as dry, the cover glass should be passed three times through the flame, film side up, to fix the preparation. Slides may be fixed by passing them five times through the flame, but the method by burning aVohol recommended for fixing blood-films gives more satisfactory bacterial fixation. For routine work the stain recommended is a dilute carbol fuchsin. Drop about five to ten drops of water on the cover-glass, then add one drop of carbol fuchsin. Allow the dilute stain to act from one to two minutes, then wash in water, dry between small squares of filter-paper (4x4 in.), and mount in balsam or the oil used for the 1/12 in. immersion objective. Some prefer to mount directly in water without preliminary drying. It is good practice to make a rule to always keep the smeared side of the preparation up — never allowing it to be reversed. By this simple rule, preparations 27 28 STAINING METHODS. can be carried through the most complicated staining methods without the necessity of scratching the cover-glass, etc., to see which is the film side. In grasping a cover-glass with a Cornet or Stewart forceps, be sure that the tips are well by the margins of the glass, otherwise the stain will drain off. In staining with slides, the grease pencil and the glass tubing, as recommended under Blood Smears, will be found useful. The dilute carbol fuchsin and Loffler's methylene blue are probably the best routine stains. Loffler's Alkaline Methylene Blue. — Saturated alcoholic solution of methylene blue, 30 c.c.; one to ten thousand caustic potash solution, 100 c.c. (Two drops of a 10% solution KOH in 100 c.c. of water makes a i : 10,000 solution.) Carbol Fuchsin (Ziehl-Neelsen). — Saturated alcoholic solution basic fuchsin, 10 c.c.; 5% aqueous solution carbolic acid, 100 c.c. Gram's Method. — The most important staining method in bac- teriological techni: and the one so rarely giving satisfactory results to the inexperienced is Gram's stain. In using this method, the lollowing points must be kept in mind: 1. Laboratory cultures (subcultures) which have been carried over for years frequently lose their Gram characteristics. 2. Cultures which are several days old or dead or degenerated do not stain characteristically. 3. The aniline gentian violet deteriorates when exposed to light in two or three days — it should be kept in the dark. It should have a rich, creamy, violet appearance. 4. The iodine solution deteriorates and becomes light in color. It should be of a rich port-wine color. 5. The decolorizing with 95% alcohol -should stop as soon as no more violet stain streams out. This is best observed over a white background, washing at intervals. Do not confuse stain on forceps for that on preparation. 6. The preparation should be thin and evenly spread. Some prefer carbol gentian violet to aniline gentian violet. (Saturated alcoholic solution of gentian violet, one part; 5% aqueous solution of carbolic acid, ten parts.) This tends to overstain. The following stock solutions of Weigert are recommended: GRAMS METHOD. 29 No. i. No. 2. Gentian violet, 2 grams. Gentian violet, 2 grams. Aniline oil, 9 c.c. Distilled water, 100 c.c. Alcohol (95%), 33 c.c. These stock solutions keep indefinitely. Mix i c.c. of No. i with 9 c.c. of No. 2. Filter. This keeps about two weeks and is the solution to pour on the preparation. It may be kept on from two to five minutes. Some hasten the staining by steaming as for tubercle bacilli. Next wash the preparation with water and flood the cover- glass with Gram's iodine solution. Some bacteriologists simply pour off excess of aniline gentian violet and immediately drop on the iodine solution. It is well to repeat the application of the iodine solution a second time. The iodine solution is left on one minute or until the preparation has a coffee-grounds brown color. Gram's Iodine Solution. Iodine, i gram. Potassium iodide, 2 grams. Distilled water, 300 c.c. After washing off the excess of iodine solution at the tap, drop on 95% alcohol and decolorize until no more violet color streams out. Now wash again and counterstain either with the dilute carbol fuchsin or with a saturated aqueous solution of Bismarck brown. The Gram positive bacteria are stained a deep violet-black. Stained by Gram's method. Not stained by Gram's method. S. pyogenes aureus. Meningococcus. S. pyogenes albus. M. catarrhalis. S. pyogenes. M. melitensis. M. tetragenus. B. typhosus. Pneumococcus. B. coli communis. Anthrax bacillus. B. dysenteriae (Shiga). Tubercle bacillus. Sp. cholerae asiaticae. Lepra bacillus. B. pyocyaneus. Tetanus bacillus. B. mallei. Diphtheria bacillus. B. pneumoniae (Friedlander) . 30 STAINING METHODS. Stained by Gram's method. Not stained by Gram's method. B. aerogenes capsulatus. B. proteus. Odium albicans. B. of influenza. Mycelium of actinomyces. B. of bubonic plague. B. of chancroid. B. of Koch- Weeks. Gonococcus. Method for Staining Acid-fast Bacilli. — i. Carbol fuchsin' with gentle steaming for one to two minutes or in the cold three to five minutes. 2. Wash in water. 3. Decolorize in 95% alcohol containing 3% of hydrochloric acid (acid alcohol), until only a suggestion of pink remains — almost white. 4. Wash in water. 5. Counterstain in saturated aqueous solution of methylene blue or with LofBer's methylene blue. 6. Wash, dry and mount. A very beautiful stain for bacteria in pus, etc., is Pappenheim's solution. Sat. aqueous sol. methyl green 50 c.c. Sat. aqueous sol. pyronin. 15 c.c. The bacteria are stained red; cell nuclei blue to purple. Smith's formol fuchsin. Saturated alcoholic solution basic fuchsin, 10 c.c. Methyl alcohol, 10 c.c. Formalin, * 10 c.c. Distilled water to make 100 c.c. This gives a very sharp differentiation of bacteria and nuclear structures. It has a purplish tinge. Fixation by heat gives the best staining. Allow the stain to act for two to ten minutes. It should not be used until after standing twenty-four hours, and after standing about two weeks it appears to lose its sharp staining power. CAPSULE STAINING. 31 Neisser's stain for diphtheria bacilli. Solution No. i. Solution No. 2. Methylene blue, o.i gram. Bismarck brown, .2 Alcohol, 2 c.c. Water (boiling), 100 c.c. Glacial acetic acid, 5 c.c. Dissolve the stain in the boiling Distilled water, 95 c.c. water and filter. Dissolve the methylene blue in the alcohol and add it to the acetic acid water mixture. Filter. To stain: Fix the preparation. Pour on the dilute acetic acid methylene blue solution and allow to act from thirty to sixty seconds. Wash. Then pour on the Bismarck brown solution, and after thirty seconds wash off with water. Dry and mount. The bodies of the bacilli are brown with dark blue dots at either end. Neisser recommends only five seconds as the time of application of each solution. He also recommends that the culture be only nine to eighteen hours old and that the temperature of the incubator do not exceed 36° C. Incubation at 37° C. gives satisfactory results. Formulae for the Romanowrsky stains and for carbol thionin are given in the section on blood. Capsule Staining. — The best method for studying bacteria, as to presence of capsules, is in the hanging drop, with the greater part of the light shut off by the diaphragm. In material where capsules are wrell developed, as in pneumonic sputum, the Gram method of staining brings out the capsule perfectly. This is of diagnostic value, as the more or less nonpathogenic pneu- mococci common about the mouth do not seem to show a capsule when stained in this way. The most beautiful method of staining capsules is the latest one proposed by Muir. 1. Prepare thin film,- dry and stain in carbol fuchsin one-half minute; the preparation being gently heated (steamed). 2. Wash slightly in 95% alcohol, then wash well afterward in water. 3. Flood preparation in mordant for five to ten seconds. 32 STAINING METHODS. Mordant. — Sat. aqueous sol. mercuric chloride, 2 parts Tannic acid (20% aqueous sol.), 2 parts Sat. aqueous sol. potash alum, 5 parts 4. Wash in water thoroughly. 5. Treat with 95% alcohol for one minute. (The preparation should have a pale red color.) 6. Wash well in water. 7. Counterstain with methylene blue one-half minute. 8. Dehydrate in alcohol. Clear in xylol and mount. (May simply dry specimen with filter-paper.) Flagella Staining. — Inoculate a tube of sterile water (gently) in upper part, with just enough of an i8-to 24-hour-old agar culture, to produce faint turbidity. Incubate for two hours at 37° C. From the upper part of culture take a loopful and deposit it on a cover-glass. Dry in thermostat for one to five hours or over night. Use perfectly clean cover-glasses. To stain by. Muir's Modified Pitfield Method. — i. Flood specimen with mordant. Steam gently one minute. Mordant. — Tannic acid (10% aqueous solution), 10 c.c. Sat. aq. sol. mercuric chloride, 5 c.c. Sat. aq. sol. alum, 5 c.c. Carbol fuchsin 5 c.c. Allow precipitate to settle or centrifuge. Keeps only one week. 2. Wash well in water for two minutes. 3. Dry carefully — preferably in incubator. 4. Pour on stain. Steam gently one minute. Stain. — Sat. aq. sol. alum, 10 c.c. Sat. ale. sol. gentian violet, 2 c.c. (May use carbol fuchsin instead of gentian violet.) Stain only keeps two days. 5. Wash well in water. Dry and mount. Spore Staining.— The most satisfactory spore staining method is really the negative staining of the spore obtained "when a bacterial preparation is stained by dilute carbol fuchsin or LofHer's methylene SPORE STAINING. 33 blue. The spore appears as a highly refractile piece of glass in a colored frame. The acid-fast method, as for tubercle bacilli, gives good results. The decolorizing, however, must be lightly done, otherwise the spore will lose its red stain. CHAPTER IV. STUDY AND IDENTIFICATION OF BACTERIA— GENERAL CONSIDERATIONS. IN order to study bacteria it is necessary to isolate them in pure culture. This may be accomplished by taking one or more loopfuls of the material and mixing it in a tube of melted agar or gelatin. From this first tube one or more loopfuls are transferred to a second tube of melted agar or gelatin, and from this a third transfer is made, thereby giving us tubes in which the distribution of the bacteria is one or more hundred times less in the second than in the first tube, and equally more dilute in the third than in the second. When we pour the con- tents of the tubes into Petri dishes we would have the bacterial colonies on the first plate so thick that it would be impossible to pick up a single colony with a platinum needle without touching a different one. On the second plate the distribution might be such that we should have discrete, well separated colonies, material from which could be taken up on the point of the needle or loop without touching any other colony. If the second plate did not meet these requirements, the third would. In clinical bacteriology we work almost entirely with organisms preferring blood-heat temperature, hence it is necessary to use agar or blood-serum as standard media for the obtaining of isolated colonies. Gelatin is of little value for this purpose in medical work. In using agar it will be remembered that it solidifies at a temperature slightly below 40° C. and does not melt again until it is subjected to a tempera- ture practically that of boiling. Again, if the temperature of the media exc;eeds_ 44° C. it may affect injuriously the organisms we wish to study.- Consequently it requires careful attention and quick work to inoculate the tubes, mix, transfer and pour into plates within the limits of a temperature which injures the organisms, and one which brings about the solidification of the agar. 34 STREAKED PLATES. 35 Again, we not only have colonies developing from organisms which have been fixed at the surface as the agar solidified in the plate, but more numerous ones developing from bacteria caught in the depths FIG. 8. — Petri Agar Plate. Made by spreading scrapings from the mouth over sterilized nutrient agar; after 48 hours in the thermostat the light "colonies" develop. Streaked plate. (Delafield and Prudden.) 21363 of the media. Therefore we have superficial and deep colonies. Except to the person of great experience, all deep colonies look alike and there is at times great difficulty in deciding whether a colony is deep or superficial. It is in the matter of trying to obtain information 36 STUDY AND IDENTIFICATION OF BACTERIA. from the differences in deep colonies that the greatest difficulties in the study of bacteriology arise. By using the method of simply stroking plates along five or six parallel lines from one side of the plate to the other with a glass rod, loop or swab, we obtain colonies which are well separated and which are entirely superficial. The material as pus, feces, throat membrane, etc., should be evenly distributed in a tube of sterile water or bouillon; the swab which was originally used for obtaining the material being then pressed against the sides of the test-tube to express excess of fluid and then stroked gently over successive lines on one plate. Or, if the organisms be very abundant, over a second plate without recharging it from the inoculated tube. To obtain isolated colonies on blood-serum or blood-streaked agar, which can be touched and by transfer obtained in pure culture, we simply smear the material on a slant of either medium. Then, without sterilizing the loop, we smear it thoroughly over a second slant, and so on to a third, or possibly a fourth or fifth. At present the classification of the bacteria is very unsatisfactory from a scientific stand-point. The nomenclature abounds in instances where three or four terms are used in naming a single bacterium, in- stead of the single generic name and single specific one as is used in zoological nomenclature. This matter of nomenclature is a subor- dinate factor in the confusion when we begin to investigate and find that different names have been applied to apparently the same organ- ism. The slightest variation in morphological, locomotor or biological characteristics seems to be considered sufficient by many observers to justify the description of a new species, and, of course, the giving of a new name. Many of these names which are now retained were applied prior to the epoch-making introduction of gelatin media by -Koch (1881) and consequently at a time when the isolation of organisms in pure culture was a matter of extreme difficulty and uncertainty. One of the first facts noted by the student in taking up bacteriology is the difficulty in determining motility; this property should always be tested on young cultures in bouillon. In Brownian movement there is a sort of scintillating movement, but the bacterium does not move from that part of the field. In current movement all the bacteria CULTURAL CHARACTERISTICS. 37 swarm in the same direction, going very fast at times, and then more slowly. If in great doubt, the mounting of the organisms in a 2% solution of carbolic acid will stop movement if it be true functional motility, while Brownian and current movement are not interfered Chflncl'Vo FIG. 9. — Chart in use at the U. S. Naval Medical School. with. In true motility bacteria move in opposite and in all directions, and move away from the place where first observed unless degenerated or dead. Reaction of media is of the greatest importance in causing variation in the functions of bacteria, and is one which has until recently been 38 STUDY AND IDENTIFICATION OF BACTERIA. almost entirely neglected. In describing an organism at the present time it is always necessary to note the reaction of the media, the tem- perature at which cultivation took place and the age of the culture when examined. In the following keys the term bacterium has been used as a general designation for all schizomycetes. Migula calls motile rod-shaped organisms bacilli, and nonmotile ones bacteria. Lehmann and Neumann call spore-bearing organisms bacilli, and nonspore-bearing ones bacteria. The B. typhosus is very motile and does not possess spores. Ac- cording to Migula, it would be the Bacillus typhosus; according to Lehmann and Neumann, the Bacterium typhosum. The B. anthracis has spores and is nonmotile. Hence it would be Bacterium anthra- cis, according to Migula, and the Bacillus anthracis, according to Lehmann and Neumann. In the use of the keys at the head of each group of organisms it will be observed that the primary separation is on the basis of morphology — the cocci in one group, the bacilli in three subgroups: one for those rod-shaped organisms showing branching and curving forms, one for the spore bearers and one for the simple rods. The spirilla are grouped by themselves. An important method of differentiation is the reaction to Gram's stain. It should be remembered that organisms carried along on artificial media often lose their Gram staining characteristics; hence it is desirable to determine this staining reaction in cultures freshly isolated. Be sure that the stains, especially the aniline gentian violet and the iodine solution, have not deteriorated. There is no more important stain than this, and none which requires greater experience. The chief causes of conflicting results are (i) working with old cultures and (2) not having satisfactory staining solutions. Motility, as stated above, is at times difficult to determine. For this purpose young eighteen-hour-old bouillon cultures are preferable, and the preparation should be made by applying a vaselin ring to the slide, then putting a drop of the bouillon culture in the center of the ring (or a drop of water inoculated from an agar slant growth), then putting on a cover-glass. By this method current movement is done CULTURAL CHARACTERISTICS. 39 away with and the preparation keeps for hours. This is a convenient method for agglutination tests. Liquefaction of gelatin is a very important means of differentia- ting. When a room-temperature incubator is not at hand (20° to 22° C.), it is better to put the inoculated gelatin-tube in the body-temperature FIG. 10. — Series of stab cultures in gelatine, showing modes of growth of different species of bacteria. (Abbott.) incubator, and from day to day test the power of solidifying with ice- water. If the organism digests the gelatin (a liquefier), the medium will remain fluid when placed in ice-water — if the organism is a non- liquefier, the medium in the tube becomes solid. Of course we lose the information to be obtained from the shape of the area of liquefaction. 40 STUDY AND IDENTIFICATION OF BACTERIA. For routine work the only sugar media used are the glucose and the lactose bouillon. These are of the utmost importance in differentia- ting organisms of the typhoid and colon group. Following Ford, these intestinal bacteria have primarily been separated by their action on litmus milk — whether turning it pink or only slightly changing or not changing at all the original color. CHAPTER V. STUDY AND IDENTIFICATION OF BACTERIA— COCCI. KEY AND NOTES. Streptococcus Forms. — Cells divide to form chains. A. Gelatin not liquefied. 1. Tendency to form long chains. a. Streptococcus pyogenes. (Cocci .7 to i /*.) b. Streptococcus anginosus. 2. Tendency to form short chains. a. Streptococcus salivarius. b. Streptococcus fecalis. B. Gelatin liquefied. a Streptococcus coli gracilis. (Cocci quite small — .2 to .4/1. In feces.) Sarcina Forms. — Cells divide in three dimensions of space. (Packets.) A. No pigment production on agar. a. Sarcina alba. (Colonies finely granular.) b. Sarcina pulmonum. B. Yellowish pigment. a. Sarcina lutea. (Colonies coarsely granular.) b. Sarcina flava. (Colonies finely granular.) C. Rose-red pigment. a. Sarcina rosea. Micrococcus Forms. — Cells divide irregularly in various directions. I. Gram positive cocci. A. Cocci — round. 1. Divide in two planes at right angles. Merismopedia. a. M. tetragenus. Moist viscid colonies. No liquefaction of gelatin. Capsule. 2. Divide irregularly. a. Gelatin not liquefied. M. cereus albus. h Pelatin linuefied I M. (Staphylococcus} pyogenes albus. b. O lique ed. J M (StaphyoIocccus) pvogenes aureus. c. Gelatin very slightly liquefied. S. epidermidis albus. (Stitch coccus.) B. Cocci — biscuit-shape. Diplococcus crassus. (May be mistaken for meningococcus.) C. Cocci — lance shape. Diplococcus pneumoniae. Fraenkel's pneumococcus. Capsule. II. Gram negative cocci. A. Grow only about incubator temperature. 1. Grow only on blood or serum media. Gonococcus. 2. Grow on blood serum media, or glycerine agar. a. Diplococcus intracellularis meningitidis. (Produces acid in glucose.) 3. Grows on ordinary media. Micrococcus melitensis. B. Will grow at room temperature as well as at 37° C. a. Micrococcus catarrhalis. (Produces alkalinity in glucose media.) 41 42 STUDY AND IDENTIFICATION OF BACTERIA. STREPTOCOCCUS FORMS. Those cocci tending to arrange themselves in chains are usually described as streptococci. When we consider that certain bacilli at times assume an arrangement which we term strepto-bacilli, yet have no relationship, it would suggest that the matter of chain morphology is simply a characteristic common to many entirely different cocci. The essential point to bear in mind is that the finding of a strepto- coccus does not necessarily explain an infection, because normally streptococci are among the organisms most frequently and abundantly found in plates made from normal buccal and nasal secretions. It is FIG. ii. — Streptococcus pyogenes. (Kolle and Wassermann.) well to be very conservative when reporting streptococci as the etiologi- cal factor from lesions of the throat or nose. Probably the most practical point in the differentiation of strepto- cocci, next to that of pathogenicity, is the occurrence of long or short chains, the virulent ones tending to appear in chains of from ten to twenty cocci, while the normal inhabitants of the nose, mouth and feces generally tend to be in shorter chains. As regards virulence, this is exceedingly variable — it is soon lost, but may be restored either by inoculating streptococci along with various other organisms or by passage through successive rabbits. The rabbit is the most susceptible animal and should be inoculated in one of the STREPTOCOCCI. 43 prominent ear veins. If the needle of the syringe is not inserted in the vein it will be difficult to force in the material and a swelling will im- mediately show itself. Besides the morphological and pathogenic variations, Schottmuller has noted differences where these organisms are grown on one part of blood and three parts of agar. On this medium Strep, erysipelatis has a hemolytic action, the laking of the red cells bringing about a more or less clear ring surrounding the colony. The short- chain streptococci do not have a hemolytic halo. Some of the English authorities have introduced biochemical methods of differentiating: the Strep, pyogenes coagulating milk, reducing neutral red, and producing acid in lactose, saccharose, mannite or inulin media. When we consider the biochemical varia- tions which a single organism, as the colon bacillus, may exhibit, the value of such methods of differentiating may well be questioned. The question of the symbiotic relationship, which, when established between two or more bacteria, may cause harmless organisms to take on viru- lence, would appear to be a more important consideration. Almost without exception, streptococci are .Gram positive. Their colonies are quite small, but distinct and discrete. In appearance the colonies of streptococci and pneumococci are practically identical. In a blood- serum throat culture pneumococcus and streptococcus colonies are the smallest, diphtheria ones are quite small and discrete, but slightly flatter. (Always examine the water of condensation for streptococci.) The sarcina and staphylococcus colonies are much larger. Streptococci are commonly the cause of diffuse phlegmonous in- flammation, while the staphylococci cause circumscribed lesions. Streptococci cause necrosis and do not characteristically produce pus. The importance of the streptococcus as a secondary infection in diph- theria, tuberculosis, small-pox and even in typhoid fever must always be kept in mind. It is this infection which does not respond to diph- theria antitoxin, and not the diphtheria one. SARCINA FORMS. These are best observed in hanging-drop preparations, when they can be seen as little cubes, like a parcel tied with a string, and by noting 44 STUDY AND IDENTIFICATION OF BACTERIA. them when turning over, it will be seen that they are different from the tetrads which only divide in two directions of space. At times the packet formation is not perfect and it will be difficult to distinguish such as sarcinae. All sarcinae stain by Gram. If the staining of sarcinse be too deep it may obscure the lines of cleavage. Sarcinaae are nonmotile. Various sarcinae have been isolated from the stomach, especially when there is stagnation of stomach contents. Sarcinae have also been found in the intestines. In plates the S. lutea is frequently a contami- nating organism, being rather constantly present in the air. The demonstration of sarcina morphology should always be made from liquid media, as bouillon. Urine makes an excellent medium. MICROCOCCUS FORMS. This grouping includes all cocci which do not show chain or packet formation. It will be found convenient to divide them into two classes according to their staining by Gram. The M. tetragenus, S. pyogenes aureus and the pneumococcus stain by Gram, while the gonococcus the meningococcus, the M. catarrhalis and the M. melitensis are Gram negative. M. Tetragenus. — This organism is frequently found associated with other organisms in sputum, especially with tubercle and influenza bacilli. The colonies are white, slightly smaller than staphylococci and are quite viscid. It was formerly considered unimportant in disease, but the idea now prevails that it is responsible for many abscesses about the mouth, especially in connection with the teeth. Injected subcutaneously into mice, it produces a septicaemia and death in three or four days. The blood shows great numbers of encapsulated tetrads. It has been re- ported twice as a cause of septicaemia in man. Staphylococci. — To cocci dividing irregularly and usually forming masses which are likened to clusters of grapes the term staphylococcus is applied. While there have been experiments which show that by selecting pale portions of a yellow colony, eventually a white colony could be produced, yet, as a practical consideration, it is convenient to consider at least two types of staphylococci: the staphylococcus STAPHYLOCOCCI. 45 pyogenes aureus and the staphylococcus pyogenes albus. In culturing from the pus of an abscess or furuncle we generally obtain a golden coccus, while in material from the nose or mouth, the staphylococcus colonies are almost invariably white. As regards the common skin coccvs, this will be found to produce a white colony. A coccus which very slowly liquefies gelatin and has been sup- posed to cause stitch abscesses is the S. epi- dermidis albus. While it is customary to look for a golden colony in the case of organisms showing virulence, yet at times a cream -white colony may develop from cocci of great virulence. The S. pyogenes citreus is considered as of very feeble pathogenic power. Certain cocci whose colonies have presented a waxy appear- ance have been designated as S. cereus albus and S. cereus flavus, respectively. They are of very little practical importance. The staphylo- coccus pyogenes aureus grows readily at room temperature, but better at 37° C. It coagulates milk and renders bouillon un'formly turbid. It grows on all media, as blood-serum, agar, potato, etc. It has been proposed to distin- guish it from skin staphylococci by its power of producing acid in mannite. Ordinarily the individual cocci are about la in diameter, but they vary greatly in size according to the age of the culture and other conditions. The aureus, as it is frequently called, is not only often found in circumscribed processes, but it is a frequent cause of septicaemia, osteomyelitis, endocarditis, etc. It is the organism most frequently concerned in terminal infections. The lowered resistance of the patient permits of their passage through bar- riers ordinarily resistent. Not only should this be kept in mind when such organisms are isolated at an autopsy, but as well the fact that their entrance may have been agonal or subsequent to death. FIG. 12. — Gelatine cul- ture Staphylococcus aureus one week old. (Williams.) 46 STUDY AND IDENTIFICATION OF BACTERIA. The Pneumococcus of Fraenkel.— This is by far the most com- mon cause of pneumonia, whether it be of the croupous, catarrhal or septic type. It is also frequently found in meningitis, empyema, endocarditis and otitis media. It should not be confused with the pneumobacillus of Friedlander, which, although possessing a capsule like the pneumococcus, differs from it by being Gram negative, being a bacillus and having large viscid colonies. The pneumococcus is the cause of more than 80% of the cases of pneumonia. It does not grow below 20° C. and is best cultivated on blood-serum, or blood-streaked . «*%» , .; * - FIG. 13. — Pneumococcus, showing capsule, from pleuritic fluid of infected rabbit, stained by second method of Hiss. (Williams.) agar. On plain agar it grows as a very small dew-drop-like colony, which is slightly grayish by reflected light. It is smaller and more transparent than a streptococcus colony. In sputum or other patholog- ical material it is best recognized by the presence of a capsule inclosed in which are two lance-shaped cocci with their bases apposed. In arti- ficial culture we rarely get the capsule. It also sometimes grows in short chains like a streptococcus. The best medium for differentiating is the serum of a young rabbit, in this it grows as a diplococcus, while streptococci show chains. The best method of isolating it in pure GRAM NEGATIVE COCCI. 47 culture is to inject the sputum into the marginal ear vein of a rabbit or subcutaneously into a mouse. Death results from septicaemia in about two days and the blood teems with pneumococci. Usually the pneumococcus quickly loses its virulence, and also dies out in a few days unless transferred to fresh media. The best medium for it preservation is rabbit's blood agar; this also maintains the virulence. On this medium the colonies are larger than on agar and they present a greenish appearance with a hemolytic zone. It is a well-known fact that the pneumococcus is a frequent inhabitant of the nasal and buccal cavities. The explanation of infection is either on the ground of lowered resistance of the patient or enhanced virulence of the organ- ism. Oscar Richardson has reported an organism in cases of lobar pneumonia, cerebrospinal meningitis, mastoid disease, etc., bearing resemblance to both pneumococci and streptococci — the Streptococcus capsulatus. It differs from the pneumococcus in that the colonies on blood-serum are viscid and like irregular flecks of mucus. The characteristic culture is a glucose agar stab. (Reaction must not exceed + .5.) From the line of puncture there are flail like projections extending outward from one-fifth to one-fourth of an inch. The capsule persists on culture media. This organism resembles the Streptococcus of Bonome of the French. Diplococcus Crassus. — This is a Gram positive, biscuit-shaped diplococcus, which might be confused with the M. catarrhalis or the meningococcus by ordinary staining methods. In throat cultures I have isolated on several occasions a Gram positive diplococcus which is at times biscuit-shaped, at times irregularly spherical. It possesses two or three metachromatic granules, so that in a Neisser stain for diphtheria the appearance of these granules may be confusing. Gram Negative Cocci. — It is important to bear in mind that there are many cocci of varying shapes, which in cultures or in smears from the throat, nose or feces are Gram negative. These are not well classified or described. To distinguish the three important biscuit- shaped diplococci, it can be most easily accomplished by cultural methods, using hydrocele agar (ascites or blood agar will answer), ordinary blood-serum and plain agar. The gonococcus will only grow on the hydrocele agar; the meningococcus will grow on this, but 48 STUDY AND IDENTIFICATION OF BACTERIA. likewise grows on ordinary blood-serum. The M. catarrhalis will grow on plain agar as well as on the other media. Gonococcus (Neisser, 1879). — This organism is characteristically a diplococcus, the separate cocci being plano-convex with their plane surfaces apposed. (Biscuit shape, coffee-bean shape.) They are generally found grouped in masses of several pairs, most strikingly in pus cells or epithelial cells, but also found extracellularly. Except in the height of the disease, there is a great tendency for the organisms to show involution forms, so that instead of biscuit-shaped diplococci we have FIG. 14. — Gonococcus. Film from urethral pus. (Coplin.) round, irregular and uneven cocci. It is therefore advisable in search- ing smears from chronic gonorrhoea to continue the search of Gram stained specimens until some fairly typical diplococci are found. There is nothing requiring greater discrimination than a diagnosis from such a smear. At the commencement of a gonorrhcea the epithelial cells are abundant and gonococci are found adhering to them or lying free. Later on, at the acme of the discharge (the creamy, abundant discharge), it is in the pus cells we find them and they may be so abundant that 10 to 20% of the pus cells may contain them. In GONOCOCCUS. 49 the subacute stage the epithelial cells, which practically disappear when the discharge is so abundant, begin to reappear, and in the chronic stage the epithelial cells are the chief ones, and are the ones, in which we find an occasional gonococcus, often distorted in shape. The best method of diagnosis in cases of chronic gonorrhoea is to have the patient drink beer and eat the stimulating food previously interdicted, inject a weak solution of silver nitrate and massage the prostate or seminal vesicles. The smears made from the resulting discharge or centrifuged urine will probably contain gonococci if they are present in the urethra. In the female the favorite sites are the urethra and the cervix uteri. In municipal examinations it is custom- ary to make two smears: one from the urethral meatus and a second from the cervix. The vagina is not a suitable soil for their develop- ment. In female children it is most often found in the discharge of the vulvovaginitis. In addition to the genital organs, the gonococcus may at times invade and be isolated from the eye (gonorrhceal ophthalmia), the joints, rarely as a cause of endocarditis and possibly as the factor in septicaemia. Grown upon hydrocele or ascites agar, or blood streaked agar, or upon blood agar from man or the rabbit, the colonies appear as irregular, minute, dew-drop spots. By the second or third day the involution forms are abundant, and within four to seven days the culture will probably be found to be dead. Unless frequent transfers are made, it will be best kept alive on blood agar. The organism grows best at 37° C, and will not grow below 25° C. It will not grow on plain or glycerin agar or ordinary blood-serum unless the transfer of considerable pus in inoculating the slants give it a suitable culture medium. In material from joints, it is in the fibrin flakes that the gonococci are most apt to be found, if found at all. Diplococcus IntracellularisMeningitidis (Weichselbaum, 1887). —This is the organism of epidemic cerebrospinal meningitis, and is frequently termed the meningococcus. The diplococcus is Gram negative and biscuit shaped and is, like the gonococcus, chiefly con- tained in pus cells. It is also found free in the cerebrospinal fluid withdrawn from spotted fever cases. There is a greater tendency to variation in size and shape than is the case with the gonococcus, which 4 50 STUDY AND IDENTIFICATION OF BACTERIA. latter, in fresh material, show a striking uniformity morphologically. The meningococcus is at times not abundant — early in the case, how- ever, the picture may be similar to that of gonorrhoea. On blood- serum the colonies appear after 24 to 48 hours as discrete, very slightly hazy colonies, about one-eighth of an inch in diameter. On serum agar, as ascites or hydrocele agar, they grow best. Unless considerable cerebrospinal fluid is transferred with the inoculating loop, they do not grow on plain agar. They will grow at times on glycerin agar. It ferments dextrose and only grows at blood temper- FIG. 15. — Diplococcus intracellularis meningitidis and pus-cells (X 1000.) (Williams.} ature, thus distinguishing it from the M. catarrhalis. It is scarcely pathogenic for laboratory animals when injected subcutaneously. Intradural injections give results. The cultures die out very rapidly, so that it is necessary to make transfers every one or two days. The meningococcus has been isolated from the nasal secretions of patients. The possibility of these organisms being the M. catarrhalis must be considered. The meningococcus has very slight resistance to sun or drying so that its aerial transmission seems doubtful. It is supposed to effect an entrance by the nares, thence reaching the cerebral meninges. Infection is probably by direct contagion. Several cases have been MENINGOCOCCUS. 51 reported where with a high leukocytosis the cocci have been found in the polymorphonuclears of blood smears and in cultures from the blood. By the use of alternate injections into horses of living diplococci, then seven days later of an autolysate made from different strains; seven days later again injecting living diplococci; thus alternating material every week, an antiserum of value has been obtained by Flexner. The immunization requires about one year. In using, withdraw about 20 c.c. of cerebrospinal fluid with a syringe, and then inject, through the same needle, an equal quantity of the serum. The injection is repeated every day for three or four days. For diagnosis, make smears and cultures from cerebrospinal fluid. The sediment from the centrifuged material gives better results. In tuberculosis the lymphocytes preponderate ; in cerebrospinal meningitis the polymorphonuclears. It has been stated that a point of difference between the phagocyto- sis with the gonococci and the meningococci is that the meningococci invade and at times destroy the nucleus of the polymorphonuclear, which is not true of gonococci. The appearance of large phagocytic endothelial cells, often containing polymorphonuclears, in the centri- fuged cerebrospinal fluid is a favorable prognostic sign. At times there does not appear to be any relation between the number of pha- gocytic polymorphonuclears and the severity of the case. Micrococcus Catarrhalis (Seifert, 1890).— This organism has been specially studied by Lord. It resembles the meningococcus strikingly and can only be differentiated by cultural procedure. It grows on plain agar and at room temperature, and does not produce acid in glucose media. It not only occurs in the nasal secretions of healthy people, but appears to be responsible for certain coryzas resembling influenza. It also is responsible for certain epidemics of conjunctivitis. The colonies are larger, more opaque and have a more irregular wavy border than the round colonies of the meningo- coccus. Micrococcus Melitensis (Bruce, 1887).— This is the organism of Malta or Mediterranean fever, sometimes called undulating fever, on account of successive waves of pyrexia running over several months. 52 STUDY AND IDENTIFICATION OF BACTERIA The djsease has a very slight mortality (2%), and the lesions are chiefly of the spleen, which is large and diffluent. The organisms can best be isolated from the spleen. M. melitensis is only about .3^ in diameter. The characteristics are its very small size and the dew-drop minute colonies on agar, which at incubator temperature only show themselves about the third to the sixth day. It is nonmotile and Gram negative. In bouillon there is a slight turbidity. The organism occurs in the peripheral circulation, it having been cultivated from blood very successfully by Eyre. He takes blood at the height of the fever, and in the afternoon. Formerly it was custom- ary to isolate by splenic puncture. Infection is chiefly by means of the milk of goats. The organisms are excreted in the urine of patients, and a diagnostic point is to make plates from the urine. Such urine applied to abraded surfaces causes infection. The serum of patients shows agglutinating power as early as the fifth day of the disease, and this may persist for years after recovery. A dilution of 1 130 or i : 50 is recommended. CHAPTER VI. STUDY AND IDENTIFICATION OF BACTERIA. SPORE- BEARING BACILLI. KEY AND NOTES. A. Grow aerobically. 1. Stab culture in gelatin has branches growing out at right angles to line of stab. a. Has no membrane on bouillon or liquefied gelatin. Projecting branches from line of stab only at upper part of line of growth. Absolutely nonmotile. Ends sharply cut across or concave. ANTHRAX GROUP. b. Has thick whitish membrane on bouillon and surface of liquefied gelatin! Projecting branches all along the line of stab. Sluggishly motile. MYCOIDES GROUP. (B. mycoides. B. ramosus.) 2. Stab cultures in gelatin do not show projecting branches. a. Potato cultures do not become wrinkled. At first slightly moist, later dry and mealy. SUBTILIS GROUP. (Hay bacillus group.) Actively motile. The yellow subtilis is at times found in water. The colonies on potato are of a cheese-yellow color. The bacilli are very large and show a slugglish, worm-like motion. b. Potato cultures become wrinkled. VULGATUS GROUP. (Potato bacillus.) Two water bacilli belonging to this group are the B. mesentericus fuscus (brown growth) and B. mesentericus ruber (red growth). NOTE. — The following cultural characteristics are common to all the above spore bearers. 1. Liquefaction of gelatin. 2. Milk slowly coagulated with very little change in reaction. Later the coagulum is digested. 3. No gas in either glucose or lactose. 4. No indol. 5. All are Gram positive. 6. All digest blood-serum. B. Grow only anaerobically. 1. Rods very little swollen by centrally situated spores. a. Motile. B. cedematis maligni. (Gram negative.) b. Nonmotile. B. aerogenes capsulatus. (Capsule.) 2. Spores tend to be situated between center and end. a. No liquefaction of gelatin. B. butyricus. b. Gelatin liquefied slowly. B. botulinus. Milk not coagulated. B. anthracis symptomatici. B. enteritidis sporogenes. Milk coagulated with abundant gas. c. Gelatin liquefied rapidly. B. cadaveris sporogenes. Very motile. 3. Spores situated at end of rod. Drum-stick sporulation. TETANUS GROUP. 53 54 STUDY AND IDENTIFICATION OF BACTERIA. The following table taken from Lehmann and Neumann, based on pathogenic effects, is of great practical value. After inoculation of some animal subcutaneously with the suspected material we have: A. No particular symptoms at site of inoculation. Absorption of the soluble toxin causing: (1) General symptoms of tetanus. B. tetani. (2) Botulism poisoning symptoms. Pupillary symptoms. Cardiac and respiratory failure. B. Local symptoms marked at site of inoculation. Hemorrhagic emphysema- tous oedema. (1) Motile. (a) Gram negative. B. cedematis maligni. (b) Gram positive. B. anthracis symptomatic!. (2) Nonmotile. B. aerogenes capsulatus or B. phlegmonis emphysematosae. SPORE-BEARING AEROBES. Bacillus Anthracis (Pollender discovered 1849. Davain re- cognized nature 1863. Koch proved 1876). — Of the aerobic spore- bearing bacilli this is the only one of particular medical importance. FIG. 16. — Anthrax bacilli. Cover-glass has been pressed on a colony and then fixed and stained. (Kolle and Wassermann.} Anthrax is an important disease in domestic animals, especially sheep and cattle. The characteristic postmortem change in animals is the greatly enlarged, friable, mushy spleen. Man is much less susceptible than these animals, but is more so than the goat, horse or ANTHRAX. 55 pig. The Algerian sheep has a high degree of immunity, as has the white rat. The brown rat is^quite susceptible. The disease in man chiefly occurs among those working with hides, wool or meat of infected cattle. The two chief types in man are: (i) Malignant pustule and (2) Woolsorter's disease. An intestinal type is also recognized. Malignant pustule results from the inoculation of an abrasion or cut; thus it frequently shows on the arms and the backs of those unloading hides. It first appears as a pimple, the center of which becomes vesicular, then necrotic. A ring of vesicles surrounds FIG. 17. — Anthrax bacilli growing in a chain and exhibiting spores. (Kolle and Wassermann.} this central eschar and a zone of congestion the vesicles. The lymphatics soon become inflamed as well as neighboring glands. The bacilli are found especially in the vesicles or the lymphatics. If the pustule is not excised and death occurs, there is not much enlargement of the spleen and the bacteria are not abundant in the kidneys, etc., as with animals. Man seems to die from a toxaemia rather than a septicaemia. In woolsorter's disease there is great swelling and oedema of the bronchial and mediastinal glands. The lungs show oedema, which about the bronchi is hemorrhagic. The bacillus is 5 to 8// by i to i i/2fi. It has square cut or concave ends and is often found in chains. It is Gram positive. Colonies, by interlacing waves of strings of bacteria, show Medusa head appearance. 56 STUDY AND IDENTIFICATION OF BACTERIA For cultural characteristics see key. Spores develop best at incubator temperature. Stiles thinks that animals are infected by eating the bones of animals which have died of anthrax, cutting buccal mucous membrane, and so becoming infected. Spores do not form in an intact animal body, but they do form after a postmortem or the disintegration of the body by maggots. For this reason it is better not to open up the body of the animal, but to make the diagnosis by cutting off an ear. FIG. 18. — Bacillus anthracis in blood of rabbit. (Coplin.} Dried spores will live for years and will withstand boiling temperature for hours. In vaccinating animals against anthrax, Pasteur used two vaccines. The first is attenuated fifteen days at 42.5° C., The second, attenuated for only ten days, is given twelve days later. In taking material from a malignant pustule before excision, be careful not to manipulate it roughly, as bacteria may enter the circula- tion. Make cover-glass preparations, staining by Gram. Make culture on agar. Blood cultures are usually only positive later in the disease. Inoculate a guinea-pig or a mouse subcutaneously. SPORE-BEARING ANAEROBES. There are three very important pathogens in this group — that of malignant oedema; that of botulism, and the organism of tetanus. The B. enteritidis sporogenes is of importance in connection with ANAEROBES. 57 indications of fecal contamination of water. In connection with B. aerogenes capsulatus, there is some question as to whether the extensive oedema produced by it may not usually be from a terminal or cadaveric infection. To Cultivate Anaerobes. — Probably the apparatus giving the most perfect anaerobic conditions is the Novy jar, in which the air has been replaced by hydrogen. The difficulties attending the method are : 1. Unless a special ap- paratus (Kipp's) is at hand, there may be difficulty in pre- venting the sul- phuric acid from frothing over when poured on the zinc. It should, at first, be added in small quantities at a time — well diluted (i to 6). 2. Various wash-bottles are required : one containing silver nitrate solution for traces of AsH3 and one with lead acetate for H2S and another with pyrogallic acid and caustic soda for any oxygen that may come over. 3. Mixtures of hydrogen and air explode. Consequently, in determining whether all air has been expelled and in its place an atmosphere of hydrogen exists, it is necessary to see if the escaping gas burns with a blue flame. Unless this is collected in a test-tube and examined, we may have an explosion. 4. Except in a large laboratory, where the apparatus is set up and ready for use, too much time would be required. 5. Simpler methods appear to give as good results. FIG. 19. — Novy jar. STUDY AND IDENTIFICATION OF BACTERIA. The Method of Liborius. — In this it is necessary to have a test- tube containing about four inches of a one percent glucose agar. Glucose acts as a reducing agent and furnishes energy. It is conven- ient to add about one-tenth of one percent of sulphindigotate of soda ; the loss of the blue color at the site of a colony enabling us to pick them out. The tube of agar should be boiled just before using to expel remaining oxygen from the tube. Now rapidly bring down the temperature to about 42° C., by placing the tube in cold water, and inoculate the material to be ex- amined. A second or third tube may be inoculated from the first, just as in ordi- nary diluting methods for plate cultures. Having inoculated the tubes, solidify them as quickly as possible, using tap- water or ice-water. The anaerobic growth develvops in the depths of the medium. Some pour a little sterile vaselin or paraffin, or additional agar on the top of the medium in the tube as a seal from the air. Others have recommended the inoculation of some aerobe, as B. prodi- giosus, on the surface. This latter method is not advisable. A deep stab culture is often sufficient. The Method of Buchner.— In this method one gram each of pyrogallic acid and caustic potash or soda for every 100 c.c. of space in the vessel containing the culture is used to absorb the oxygen. It is convenient to drop in the pyrogallic acid; then put in place the inoculated tubes or plates; then quickly pouring in the amount of caustic soda, in a 10% aqueous solution, to immediately close the con- taining vessel. A large test-tube in which a smaller one containing the inoculated medium is placed, and which may be closed by a FIG. 20. — Arrangement of tubes for cultivation of anae- robes by Buchner's method. (Williams.) CULTIVATION OF ANAEROBES. 59 rubber stopper, is very convenient. A good rubber-band fruit jar is satisfactory. A desiccator may be used for plates. An excellent method for anaerobic plates, either in a desiccator with the pyrogallic acid and caustic soda, or less satisfactorily in the open air, is to sterilize the parts of the Petri dish inverted; that is, the smaller part is put bottom downward in the inverted cover (as one would set one tumbler in another). Then, in using, unwrap the Petri dish, lift up the inner part, pour in the inoculated medium into the upturned cover. Then immediately press down the inner dish, spreading out a thin film of medium between the two bottoms. J. H. Wright's Method. — Make a deep stab culture in glucose agar or gelatin, preferably boiling the media before inoculating. Then flame the cotton plug and press it down into the tube so that the top lies about three-fourths of an inch below the mouth of the test-tube. Next fill in about one-fourth of an inch with pyrogallic acid; then add 2 or 3 c.c. of a 10% solution of caustic soda, and quickly insert a rubber stopper. This method is one of the most convenient and practical, and is to be strongly recommended. Method of Vignal. — In this a section of glass tubing (1/4 in.) is drawn out at either end, as in making a bacteriological pipette, with a mouth-piece containing a cotton plug. The liquid agar or gelatin is then inoculated and the medium drawn up into the tube. In a very small flame the capillary narrowings are sealed off, and we have inside the tube very satisfactory anaerobic conditions. To get at the colonies, file a place on the tube and break at this point.* B. (Edematis Maligni (Pasteur, 1877). — This is the vibrion septique of Pasteur. It is found in garden soil and in street sweepings. It is the cause of an acute cellular necrosis attended with serous san- guinolent exudation and with more or less emphysema. The organ- ism only becomes generalized in the blood about the time of death and postmortem. Therefore, it is not a septicaemia, as is anthrax. The bacillus is an organism about the size of anthrax (7// by .8), but is narrower and does not have the same square cut or dimpled ends. * To obtain material for examination and isolation in pure culture from the deep agar stab-tube, it is best to loosen the medium at the sides of the tube with a heated platinum spud or a flattened copper wire. Then shake the mass out into a sterile Petri dish. It is dangerous to break the tubes with a hammer as some do. 60 STUDY AND IDENTIFICATION OF BACTERIA. Furthermore, it is motile, Gram negative and an anaerobe. The guinea pig is very susceptible, and about the time of death and post- mortem there may be seen long flexile motile filaments, 15 to 40^ long, which move among the blood-cells as a serpent in the grass (Pasteur). In cultures it grows out very slightly from the line of stab, giving a jagged granular line, differing from tetanus. Spores form best at 37° C. — requiring about 48 hours. It liquefies gelatin. In examining an exudate from a suspected case, note the presence of spores centrally situated. Inoculate a guinea-pig. Death occurs in about two days. There is intense hemorrhagic emphysematous oedema at the site of FIG. 21. — Bacillus of botulism. (Kolle and Wassermann.} inoculation. The subcutaneous tissue contains fluid and gas. There is present the foul odor of an anaerobe. Examine for the long filaments showing flowing motility. Be sure to stain by Gram. (Negative.) For cultures, heat the material (either from a wound or from a guinea- pig) which shows spores to a temperature of 80° C. for from 15 minutes to one hour. Then inoculate glucose agar stab culture and grow anaerobically. Courmont differentiates anthrax from malignant oedema by injecting into ear vein of rabbit. The injection of malig- nant cedema in this way, instead of subcutaneously, tends to immunize. B. Botulinus (Van Ermengem, 1896). — This is the organism which produces botulism, a form of meat poisoning. It is a spore- BOTULISM. 6 1 bearing anaerobe and must not be confused with another organism associated with meat poisoning — the B. enteritidis of Gartner. There are dysphagia, paralysis of eye-muscles and cardiac and respiratory symptoms (medulla). The symptoms are due to the elaboration of a soluble toxin of the same nature as that of diphtheria and tetanus. There is no fever and consciousness is preverved. It has been isolated from sausage and ham. It is a large bacillus — 5 to iQfji x ifjL. It is motile and stains by Gram. It produces gas in glucose media. When the toxin is introduced, it requires a period of incubation of twelve hours. An important point is that ham may not FIG. 22. — Symptomatic anthrax (Rauschbrand) bacilli showing spores. (Kolle and Wassermann.) appear decomposed and yet contain many bacilli and much toxin. It is a very potent toxin — as little as one-thousandth of a c.c. may kill a guinea-pig. In man the toxin is apparently absorbed from the alimentary canal. For diagnosis inject an infusion of the ham or sausage which was eaten of into a guinea-pig, and characteristic pupillary symptoms with death by cardiac and respiratory failure will result. Cultures may be made in glucose agar. The characteristic point is the production of a powerful soluble toxin which produces symptoms when no bacilli are present. 62 STUDY AND IDENTIFICATION OF BACTERIA. B. Tetani (Nicolaier, 1885; Kitasato, 1889).— This is the most important organism of the anaerobic spore bearers. Its character- istics are the tetanic symptoms produced by the toxin and the strictly terminal drum-stick spores. Spores are difficult to find in material from wounds infected with tetanus, but readily develop in cultures. Prior to the formation of spores the organism is a long thin bacillus (4 x .4 ft). It is motile and Gram positive. It liquefies gelatin slowly and does not coagulate milk. Theobald Smith recommends growing it in fermentation tubes containing ordinary bouillon, but to which a piece of the liver or spleen of a rabbit or guinea-pig has been FIG. 23. — Tetanus bacilli showing end spores. (Kolle and Wassermann.) introduced at the junction of the closed arm and the open bulb. By this method spores develop rapidly in from twenty-four to thirty-six hours. Sporulation is most rapid at 37° C. As there is always lia- bility to postmortem invasion of viscera by ordinary saprophytes, Smith recommends that great care be taken not to handle the animal roughly in chloroforming and in pinching off pieces of the organ at autopsy. The animal must be healthy, and the tubes to which the piece of tissue is added must be proven sterile by incubation. Smith calls attention to the uncertainty of the temperature at which tetanus spores are killed. He shows that some require temperatures only possible with an autoclave. In view of the danger of tetanus, it is TETANUS. 63 advisable to carefully autoclave all material going into bacterial vaccines, such as salt solution, bottles for holding, etc. Tetanus seems to grow better in symbiosis with aerobes; hence a lacerated dirty wound with its probable contamination with various cocci, etc., and its difficulty of sterilization, offers a favorable soil. The tetanus bacillus gives rise to one of the most powerful poisons known; it is a soluble toxin like diphtheria toxin, and it is estimated that 1/300 of a grain is fatal for man. Rosenau has established an antitoxin unit for tetanus which has the power of neutralizing one-thousand minimal lethal doses. Con- sequently it is a unit ten times as neutralizing as the diphtheria anti- toxin one. The antitoxin of tetanus is less efficient than that of diphtheria for the following reasons: 1. There is about three times as great affinity in vitro between diphtheria toxin and antitoxin than is the case with tetanus. 2. The tetanus toxin has greater affinity for nerve-cells than for antitoxin. 3. Treatment with antitoxin is successful after symptoms of diphtheria appear. With tetanus it is almost hopeless after the disease shows itself. Hence the importance of the early bacteriological examination of material from a suspicious wound (rusty nail). 4. The tetanus toxin ascends by way of the axis cylinder, and the antitoxin being in the circulating fluids cannot reach it, whereas with diphtheria both toxin and antitoxin are in the circulation. Diphtheria also selects the cells of parenchymatous and lymphatic organs which are more tolerant of injury than the nerve cells. That the disease is due to toxin is shown not only experimentally, but also if spores are carefully freed of all toxin by washing, and then introduced they do not cause tetanus — the polymorphonuclears engulfing them. The importance of the presence of ordinary pus cocci in a tetanus wound may be that the activity in phagocytizing them allows the tetanus bacillus to escape phagocytosis. This would also explain the importance of necrotic tissue in a lacerated wound — the phagocytes taking this up instead of tetanus bacilli. The toxin is digested by the alimentary canal juices and infection by that atrium is improbable. The infection occurs especially through skin wounds, 64 STUDY AND IDENTIFICATION OF BACTERIA. but also from those of mucous mem- brane. The usual period before symptoms occur is fifteen days. The shorter the period of incubation, the more probably fatal the disease. The horse is the most susceptible animal, next the guinea pig, then the mouse. Fowls are practically immune. In examining for tetanus, scrape out the material from the suspected wound with a sterile Volkmann spoon and put it in a tube containing blood- serum. Place this in an incubator. We have here the principle of the septic tank — the cocci and other aerobes grow luxuriantly and enable the tetanus bacillus to develop. From day to day smell the culture, and if an odor similar to the penetrating, sour, foul smell of the stools of a man who has been on a debauch be detected, it is suspicious. The nondevelopment of a foul odor is against tetanus. Also make smears from the material and examine for drum- stick spores. If these are found, heat the material to 80° C. for one-half hour, to kill non- sporing aerobes and facultative anae- robes, and then inoculate a deep glucose agar tube and cultivate by Wright's method. The fusiform lateral outgrowth about the middle of the stab is characteristic, ric. 24. — B. aerogenes capsulatus ^ i agar culture showing gas formation. B- AerOgCnesCapSUlatuS (Welch, 1891).— This bacillus is ap- parently widely distributed. It is possibly the same organism as GAS BACILLUS. 65 Klein's B. enteritidis sporogenes, which is constantly present in feces. It is a large capsulated organism, which does not form chains. Spores are produced on blood-serum. These are frequently absent on other media. It is questioned whether its pathogenicity is other than exceedingly feeble, the presence of the bacillus in emphysematous findings at postmortem being attributed to terminal or cadaveric invasion. Cases, however, in the Philippines, have been reported following caribou horn wounds, in which most serious and fatal results attended emphysematous lesions showing this bacillus. CHAPTER VII.^ STUDY AND IDENTIFICATION OF BACTERIA. MYCO- BACTERIA AND CORYNEBACTERIA. KEY AND NOTES. Key for Bacilli. — Having branching characteristics, as shown by parallelism, branching, curving forms, V-shapes, clubbing at ends, segmental staining, etc. Acid-fast. Mycobacterium. { I. Grow rapidly on ordinary media at room temperature. Examples: Timothy grass bacillus of Moeller (B. phlei). Mist bacillus. Butter bacilli as reported by (i) Rabino- witsch and (2) Petri. II. Only grow at about incubator temperature. Scanty growth or none at all on ordinary media. Media of preference are: (a) solidified blood- serum, (b) glycerin agar and (c) glycerin potato. 1. Cultures fairly moist, luxuriant and flat. Op. temp. 43° C. a. Bacillus of avian tuberculosis. 2. Cultures scanty, wrinkled and dry. Appear in 10 to 14 days. Op temp. 38° C. Bacilli longer, narrower and more beaded. a. Bacillus of human tuberculosis. Cultures as above, but even more scanty. Bacilli shorter, thicker, more even in staining and more regular in size. b. Bovine tubercle bacilli. 3. Very difficult to cultivate (Czaplewski). Smegma bacilli of various animals. III. Noncultivable (at present). i. B. leprae. Found chiefly in nasal mucus and in juice from lepra tuber- cles. Less often in nerve leprosy. Nonacid-fast. Corvnebac.eriun, { %?%?£*£* ^ I. Do not stain by Gram's method. i. B. mallei (Glanders). Characteristic culture is that on potato. Growth-like layer of honey by third day. Becomes darker in color, until on eighth day is reddish-brown or opaque with greenish-yellow margin. II. Gram positive. 1. Very luxuriant growth on ordinary media. Colonies often yellow to brownish. B. pseudodiphtheriae. Shorter, thicker and stain uniformly. 2. Moderate growth on ordinary media. B. diphtherias. Best media are blood-serum (LofSer's) or glycerin agar. Has metachromatic granules at poles. 3. Scanty and slow growth on nutrient media. B. xerosis. 66 TUBERCULOSIS IN GUINEA PIG. 67 THE GROUP OF ACID-FAST BRANCHING BACILLI. There is a large and ever-increasing number of organisms which have the same staining reactions as the tubercle bacilli, but which differ in four important essentials of — 1. Growing readily on any media. 2. Showing more or less abundant growth or colonies in twenty-four hours. 3. Having no pathogenic power for guinea-pigs when inocu- lated subcutaneously. 4. Not requiring body temperature for development, but growing at room temperature. Many of these organisms, if injected intraperitoneally into guinea- pigs, will produce a peritonitis with false membrane. Some also produce granulation tissue nodules which may be confused with true tubercles. For this reason it is well to study the lesions in experi- mental tuberculosis in the guinea-pig. Injected subcutaneously, on either or both sides of the posterior abdomen with the needle pointing toward the inguinal glands, we may have caseation and ulceration at the site of inoculation. The glands in relation enlarge and caseate. Smears from these show T. B. The marked and characteristic change is the enormous enlargement of the spleen, which is studded with grayish and yellow tubercles. Make smears and cultures from the spleen. The death of the guinea-pig usually occurs in about two months. The lesions may be looked for at three to five weeks. These nonpathogenic acid-fast bacilli are of greatest importance by reason of their possible confusion with the true tubercle bacilli. Their colonies correspond more or less with different types of tubercle bacilli colonies, being either dry and wrinkled like human or moist and irregularly flat as avian. Eventually the moist colonies become dry and wrinkled. They have been isolated from— 1. Butter and milk. 2. From grasses, especially in timothy grass infusion. 3. In various excretions of animals, as in dung, urine, etc. 4. Normally in man — from skin, nasal mucus, cerumen and tonsillar exudate. 68 STUDY AND IDENTIFICATION OF BACTERIA. FIG. 25. — Bacillus tubercu- losis; glycerine agar-agar cul- ture, [several months old. (Curtis.} It is important to remember that such organisms have very rarely been reported from pulmonary lesions, and when pres- ent they have been considered as proba- bly causative. Consequently the finding of tubercle bacilli in sputum has practi- cally as great value as it had before we knew of these various acid fast bacteria. Tubercle Bacillus (Koch, 1882).— This is a rather long, narrow rod, 3 x. 3//.. In the human type it tends to show a beaded appearance, this not being due to spores, however. In the bovine type the staining is .more solid, the organism shorter and thicker, and shows even a more scanty growth than human T. B. It has been established that many of the tuberculous affections of man, especially those of the skin, bone and mesenteric glands, are of the bovine type, while, as a rule, pulmonary and laryngeal lesions are of the human type. Experiments by various commissions in different countries have shown that human and bovine types are very closely related and that not only may a bovine strain affect man, but that human T. B. may infect young calves. It is a question whether the avian type is absolutely distinct; many experiments having indicated the impossibility of in- fecting fowls with human T. B. Nocard, by inserting collodion sacs containing bouillon suspensions of human T. B., claims to have changed these to the avian type. The avian type grows at 43° C. fairly luxuriantly, as a moist, more or TUBERCULINS. 69 less spreading culture. There is also a fish tuberculosis. This organism grows much more rapidly than the other types (3 to 4 days), and grows best at 24° C., growth ceasing at 36° C. The colonies are round and moist. The best culture media for primary cultures are blood-serum or, better, a mixture of yolk of egg and glycerin agar. Dorset's egg medium is also used. In subcultures, either glycerin agar, glycerin potato or glycerin bouillon make good media. In inoculating media from tuberculous material, as, say, from a tuberculous gland or, more practically, from the spleen of a guinea-pig, the material must be thoroughly disintegrated or rubbed on the surface of the media so that individual bacilli may rest on the surface of the culture media. In growing in flasks in glycerin bouillon a surface growth is desired. The cylindrical flask of Koch gives a better support to the pellicle than an Erlenmeyer one. In inoculating, a scale of such a surface growth or a grain from the growth on a slant should be deposited on the surface of the glycerin bouillon in the flask. Inasmuch as the filtrate from cultures has little toxic effect, the poison is assumed to be intracellular. Koch's old tuberculin, which wras simply a concentrated glycerin bouillon culture killed by heat, is now principally used in veterinary diagnosis. Koch's tuberculin "R" or new tuberculin was introduced in 1897. In this, virulent bacilli are dried In vacuo, ground up in water and centrifuged. The first supernatant fluid is put aside as tuberculin O. Subsequent trituration and centrifugalization, preserving each time the supernatent suspension, gives the new tuberculin. It has been found at times to contain virulent T. B. Koch's bazillen emulsion has been recently introduced by Koch. This is simply a suspension of ground-up bacilli in 50% glycerin solu- tion. It contains both T. R. and T. O. In the use of T. R. and of bazillen emulsion, Sir A. Wright recom- mends doses of 1/4000 of a milligram, and he rarely goes beyond i/ 1000 of a milligram in treatment. These products come in i c.c. bottles containing 10 mgs. of bacillary material. It is convenient to remove i/io of a c.c., containing i mg. Add this to 10 c.c. of glycerin 70 STUDY AND IDENTIFICATION OF BACTERIA. salt solution with 1/4% of lysol. Each c.c. contains i/io mg. One c.c. of this stock solution added to 99 c.c. of salt solution, with 1/4% of lysol, would give a working solution, each c.c. of which would contain i/iooo mg. of tuberculin. For diagnostic reactions we have, besides the method of injecting tuberculin and noting presence or absence of fever, four more recent diagnostic tuberculin tests : (i) Variations in opsonic index. (2) Instillation into one eye of a drop of 1/2% or i% -solution of purified tuberculin. Reaction is shown by redness, especially of inner canthus, in 12 to 24 hours (Calmette). (3) The cutaneous inoculation method similar to ordinary vaccination methods. The appearance of bright red papules in 24 hours indicates reaction (von Pirquet). (4) Ointment tuberculin test. Rub in 50% ointment of tuberculin in lanolin. Reaction is shown by dermatitis with reddened papules in 24 to 48 hours. (5) Inoculation of bovine and human tuberculin to diagnose type of infection (Detre). In staining it is better to use the Ziehl-Neelsen method, decolor- izing with 3% hydrochloric acid in 95% alcohol. The alcohol, for all practical purposes, enables us to eliminate the smegma and similar bacilli, these being decolorized by such treatment. There are two objections to the Gabbett method, where decolorizer and counter- stain are combined: (i) We cannot judge of the degree of decolor- ization — we are working in the dark; and (2) the matter of elimination of smegma bacilli is impossible. Pappenheim's method, in which corallin and methylene blue are dissolved in alcohol, does not appear to have any advantage over acid alcohol. As a practical point when the question of tuberculosis of the genito-urinary tract is involved, inoculate a guinea-pig with urinary sediment. It must be remembered that in young cultures of tubercle bacilli many of the rods are nonacid-fast, taking the blue of the counter- stain, while older rods are acid-fast. This frequently causes suspicion of a contaminated culture. Bacillus Leprae (Hansen, 1874).— This is the cause of leprosy. In nodular leprosy the organism is readily and in the greatest abun- dance found in the juice of the tubercles of the skin, nasal and pharyn- LEPROSY. 71 geal mucosa. In the skin they are chiefly found in the derma, espe- cially in connective-tissue cells and endothelial cells. They are also found in the glands in relation to the superficial lesions. The bacilli are found in smaller numbers in the liver and spleen. In anaesthetic or nerve leprosy they are found in small numbers in the granuloma tissue which affects the interstitial connective tissue of the peripheral nerves. Also, rarely, in the anaesthetic spots of nerve leprosy. In morphology and staining reactions they are almost identical with the tubercle bacillus. The main points of distinction are: (i) The fact of the leprosy bacilli being found in enormous numbers, especially in large vacuolated cells (lepra cells), and lying in the lymph spaces. They are frequently beaded and lie in masses which have been likened to a bundle of cigars tied together. It is necessary to examine for long periods of time, smears made from tuberculous lesions of skin before finding a single organism. (2) Leprosy bacilli have not surely been cultivated. (3) Injected into guinea-pigs, they do not produce lesions. Recently a leprosy -like disease of rats has been reported in which there are two types: (i) A skin affection and (2) a glandular one. In this disease acid-fast bacilli, alike in all respects to leprosy bacilli, have been found. Deane has obtained a diphtheroid-like organism in culture, which is nonacid-fast. This same finding has been obtained in cultures considered positive in human leprosy. Quite recently it has been claimed that the bacillus has been cultivated by excising aseptically the subcutaneous portion of lepromata and dropping the leprous tissue into salt solution. For diagnosis we should use both smears from the nasal mucus and from ulcerated lepromata or from the scrapings from intact tubercles. Some advise centrifuging with salt solution, but this is rarely necessary. The best method is to excise a small portion of skin or mucous membrane, fix it in absolute alcohol or Zenker's fluid. Cut thin sections in paraffin. Stain with carbol-fuchsin, decolorize with acid alcohol, and then stain with haematoxylin. This gives the location of the bacilli. This is also a good method for tuberculous tissues. It is claimed that the B. leprae stains more easily and loses its color more rapidly than the tubercle bacillus. Some prefer to stain the 72 STUDY AND IDENTIFICATION OF BACTERIA. leprosy bacillus by Gram's method, it as well as the tubercle bacillus being Gram positive. NONACID-FAST BRANCHING BACILLI. Bacillus Mallei (Lofifler and Shutz, 1882). — This is the cause of a rather common disease of horses. When affecting the superficial lymphatic glands, it is termed "farcy;" when producing ulceration of nasal mucous membrane, the term "glanders" is used. In man there are two types of glanders — chronic and acute. In the chronic form an abrasion becomes infected from contact with glanders material and an intractable foul discharging ulceration results. This may persist for months with lymphatic involvement or may become acute. The acute form may also develop from the start and the cases are usually diagnosed as pyaemia. Death invariably results in acute glanders. The bacillus is a narrow, slightly curved rod, about 3 x .^IJL. It is nonmotile and Gram negative. It at times presents a beaded appearance. In subculture on agar or blood-serum the growth is somewhat like typhoid, but more translucent. In original cultures from pus or tissues the colonies may not show themselves for 48 hours. The characteristic culture is that on potato. Grown at 37° C., we have a light brown mucilaginous growth, which by the end of a week spreads out and takes a cafe au lait color. The potato assumes a dirty-brown color. This and the inoculation of a guinea-pig are the chief diag- nostic measures. If the material is injected intraperitoneally into a male guinea-pig, marked swelling of the testicles is noted within 48 hours. The best stains are carbol thionin and formol fuchsin. In sections stained with carbol thionin the bacilli are apt to be decolorized by the subsequent passage of the section through alcohol and xylol. This may be avoided by blotting carefully after the thionin, then clearing with xylol or some oil, and mounting. Nicolle's tannin method is a good one. Mallein is prepared by sterilizing cultures that have grown in glycerin bouillon for about a month by means of heat (100° C.). The dead culture is then filtered through a Berkefeld filter and the filtrate constitutes mallein. It is chiefly used as a means of diagnosing DIPHTHERIA. 73 the disease in horses. The reaction consists in rise of temperature and local oedema. The dose is about i c.c. Bacillus Diphtheriae (Klebs discovered, 1883; Loffler cultivated, 1884). — The diphtheria bacillus is found not only in the false mem- brane which is so characteristic of the disease, but may be found in abundance in the more or less abundant secretions of nose and pharynx. In studying the epidemiology of diphtheria, especial attention must be gh'en to the examination of nasal discharges. The B. diphtheria? may be in pure culture lying entangled in the fibrin meshes or contained FIG. 26.— Bacillus of diphtheria. (X 1000.) (Williams.') within leukocytes in the membrane or be associated with staphylococci, pneumococci or especially streptococci. These latter complicate unfavorably and cause the suppurative conditions about the neck. In fatal cases the diphtheria bacillus may be found in the lungs. Ordi- narily, however, it remains entirely local and does not get into the circu- lation or viscera. It produces soluble absorbable poisons which are designated toxin in the case of the one responsible for the acute intoxication, parenchy- matous degeneration and death; and toxone for the poison which pro- 74 STUDY AND IDENTIFICATION OF BACTERIA. duces oedema at the site of inoculation and postdiphtheritic palsy. The injection of the soluble poisons alone without the bacilli produce the symptoms of the disease. The bacilli tend to appear as slighlty curved rods, showing varying irregularities in staining, as banding or beading, and in particular the presence at either end of small, deeply staining dots (metachromatic granules). These are well seen with Loffler's blue, but better with Neisser's method. In culture- they also show swelling at one or both ends or clubbing. In secretions or in culture they show V-shapes or false branching and, what is most characteristic, the parallelism — four FIG. 27. — Diphtheria bacilli involution forms. (Kotte and Wassermann.} or five bacilli lying side by side like palisades. Being a Gram positive organism while the majority of the other pathogenic bacteria are Gram negative, it is of greatest importance to stain smears by this method. It is not so strongly tenacious of the gentian violet as the cocci, so decolorization should not be carried too far. The best medium for growing it is Loffler's blood-serum. Coagu- lated white of egg answers equally well, as will a hard-boiled egg — the shell at one end being cracked and the white cut with a sterile knife. This smooth side is then inoculated and the egg placed cut side down- ward in a sherry glass. If an incubator is not at hand a tube may be carried next the body in a pocket. The bacillus grows better on glycerin DIPHTHERIA. 75 agar than on plain agar. On such plates they appear as small, coarsely granular colonies with a central dark area. In size the colonies resemble the streptococcus. On blood-serum the colonies are larger — 1 1 12 to 1/8 inch in diameter. In bouillon it tends to form a surface growth. It is at the surface that the toxin function is most marked, hence in growing diphtheria for toxin formation we use Fernbach flasks which expose a large surface to the air. It is a marked acid producer — bouillon with a + i reaction becoming +2.5 to +3 in 36 hours. The nitrate from a twro- or three-weeks-old broth culture is highly toxic, and is usually referred to as diphtheria toxin. It is used in injecting horses to produce antitoxin. Ehrlich uses as a standard to measure the toxicity of toxin the minimal lethal dose (M. L. D.) This is the amount of toxin which will kill a 350 gram guinea-pig in just four days. Some toxins have been produced whose M. L. D. was 1/500 c.c., so that one c.c. of such toxin would kill 500 guinea-pigs.)^ Theoreti- cally, the measure of an antitoxin unit is the capacity of neutralizing 200 units of a pure toxin.^( (On exposure to light, etc., toxin loses its toxic power and is termed toxoid.) Inasmuch, however, as toxone and toxoid are also present, we may practically consider an antitoxin, or immunizing unit (i.e., Immunitatseinheit), as about capable of making innocuous 100 M. L. D. In obtaining material from a throat, be sure that an antiseptic gargle has not been used just prior to taking the throat swab. The part of the swab which touched the membrane or suspicious spot should come in contact with the serum slant. This is best accom- plished by revolving the swab. An immediate diagnosis is possible in probably 35% of cases by making a smear from a piece of membrane. In doing this Neisser's stain is the most satisfactory. If there is any doubt about the nature of an organism in a throat culture, always stain: (i) with Loffler's alkaline methylene blue for two minutes; (2) with Gram's method, being careful not to carry the decolorization too far, and (3) by Neisser's method. With Loffler's you obtain a picture which, after a little experience, is characteristic ; at times the polar bodies show as intense blue spots in the lighter blue bacillus. One is liable to confuse cocci lying side by side for diphtheria bacilli with segmental or banded staining. This difficulty is not ap- 76 STUDY AND IDENTIFICATION OF BACTERIA. parent when Gram's staining is used. This gives us great information, as the diphtheria and the pseudodiphtheria are the only small Gram positive bacilli usually found in the mouth. The cocci are also well brought out. Neisser's stain gives a picture which, when satisfactory, is almost absolutely characteristic. You have the bright blue dots lying at either end of the light brownish-yellow rods. When first isolated from a throat, the diphtheria bacillus is apt to stain characteristically by Neisser. Later on, in subculture, there may be no staining of the polar bodies. Neisser originally recommended five seconds' applica- tion, with an intermediate washing, for each of his two solutions. FIG. 28. — B. diphtheria stained by Neisser's method. (Williams.) Thirty seconds for each is probably preferable. Some authorities recommend five to thirty minutes. It is well to bear in mind that about 2% of the people in apparent health carry diphtheria bacilli in their throats. Diphtheroid Bacilli. Pseudodiphtheria Bacillus. Hofman's Bacillus. — Under these terms various Gram positive bacilli have been described as occuring in nose and in skin diseases: i. They very rarely give the blue dot staining at the two ends. Exceptionally they may give a dot at one end. Neisser DII'HTHEROID ORGANISMS. 77 attaches importance to the dots at both ends as showing diphtheria. 2. They tend to stain solidly or at most with only a single unstained segment. They are shorter, thicker and do not curve so gracefully as the true diphtheria bacillus. They are stockier. 3. They produce very little acid in sugar media, not one-half that produced by true diphtheria. 4. They are nonpathogenic for guinea-pigs. 5. Many of them grow quite luxuriantly and often show chromogenic power. Xerosis Bacillus. — This organism is frequently found in normal conjunctival discharges. There is question as to its pathogenesis, and the finding of this organism should not exclude the previous presence of strictly pathogenic organisms, such as the gonococcus or the Koch- Weeks. It resembles the diphtheria bacillus in being Gram positive and showing parallelism, but differs (i) in being nonvirulent for guinea-pigs; (2) in requiring about two days for the appearance of colonies; (3) in not showing Neisser's granule staining, and (4) in producing very little acid in sugar media. CHAPTER VIII. STUDY AND IDENTIFICATION OF BACTERIA. GRAM NEGATIVE BACILLI. KEY AND NOTES. KEY to the recognition of nonspore-bearing, nonchromogenic, non-Gram staining, nonbranching bacilli. (NOTE. — Some books say that the proteus group is Gram positive. It is, however, usually negative.) Do not grow on ordinary media. Require blood or serum agar (hemophilic bacteria). Minute dew-drop colonies. 1. Influenza bacillus. 2. Koch- Weeks bacillus (conjunctivitis). Serum agar best medium. 3. Muller's bacillus of trachoma. Like Koch- Weeks bacillus, but easier to cultivate. 4. Morax diplobacillus of conjunctivitis. Produces little pits of liquefac- tion in serum. 5. Pseudoinfluenza bacillus (whooping-cough?). 6. Ducrey's bacillus (soft chancre). Requires almost pure blood. Grow well on ordinary media. I. Cultures in litmus milk. PINK. A. Nonmotile. Lactis aerogenes group. B. lactis aerogenes. Produce gas in glucose lactose, or saccharose. No liquefaction of gelatin. Short, stubby bacteria. B. Motile. 1. Nonliquef action of gelatin. a. B. coli group. Coagulation of milk. No subsequent pep- tonization. Gas in glucose and lactose, none in saccharose. Indol produced. Neutral red reduced. 2. Liquefaction of gelatin. a. B. cloacae group. Gas in glucose, slight in lactose. Slow coagulation of milk. Subsequent peptonization. II. Cultures in litmus milk. LILAC. A. Nonmotile bacilli. i. No gas generated in glucose or lactose bouillon. a. Hemorrhagic septicaemia group. These are oval bacilli with tendency to bipolar staining. Examples: B. pestis, B. suisepticus, B. cholera? gallinarum (chicken cholera). b. Dysentery gioup. Divided into two classes according as mannite is acted on: Those not giving acid — nonacid group — (Shiga-Kruse) . Those giving acid— acid group — (Flexner-Strong). 78 INFLUENZA. 79 2. Gas generated in glucose bouillon not in lactose. a. Friedlander group. Give very viscid, porcelain-like colonies. Tendency to capsule formation in favorable media. Examples: B. pneumoniae, B. capsulatus mucosus, B. rhinoscleromatis. P.. Motile burilli. 1. Do not liquefy gelatin. a. DQ not produce gas in either glucose or lactose bouillon. Typhoid, or Eberth group. No imlol. No coagulation of milk. No reduction of neutral red. b. Gartner group. This includes: Pathogenic types for man; as, B. enteritidis, B. icteroides, B. paratyphoid B. B. psittacosis. Nonpathogenic for man; as B. cholerae suum (hog cholera). 2. Liquefy gelatin. a. Proteus group. Colonies amoeboid, spreading. Produce gas in glucose, not in lactose. GRAM NEGATIVE BACILLI REQUIRING SPECIAL MEDIA. Bacillus Influenzas (Pfeiffer, 1892). — This organism is the type of the so-called haemophilic bacteria — organisms whose growth is re- stricted to media containing haemoglobin. The influenza bacillus seems to grow better on slants freshly streaked with blood than on those which have been made for some time, and they appear to grow better on this surface smear of blood than on a mixture of agar and blood. The influenza bacilli are most likely to be isolated from the sputum of bronchopneumonia due to this organism. It has also frequently been found in the nasal secretions of influenza patients. Exception- ally, it is present in the blood, and has been isolated in cases of menin- gitis. It is a very small bacillus which tends to show itself in aggre- gations, especially centering about M. tetragenus. It stains rather faintly when compared with cocci, so that a smear of sputum stained with formol fuchsin shows a deep violet staining for the M. tetragenus or other cocci, and scattered around in a clump like aggregation we see these minute, rather faintly stained rods. They also tend to stain more deeply at either end so that they sometimes appear as diplococci. Gram's method, counterstaining with formol fuchsin, is excellent for their demonstration. The red baclli and the violet black cocci are easily distinguished. To cultivate them, rub the sputum or the material from a lung on a 8o STUDY AND IDENTIFICATION OF BACTERIA. slant smeared with human blood (pigeon's blood is also satisfactory), and then without sterilizing the loop, innoculate a second blood slant; then a third, and possibly a fourth. The colonies appear as very minute dew drop-like points which seem to run into each other in a wave-like way. Lord distinguishes them from pneumococcus colonies by their disappearing from view as you shift from reflected light to direct transmitted light. To test such colonies we should transfer a single colony to plain agar and blood-serum, trying not to carry over any blood. If the least trace of blood is carried over, they may grow on agar or blood serum. Organisms resembling the influenza bacillus FIG. 29.— The Koch- Weeks Bacillus. (Hansett and Sweet.) have been isolated from whooping-cough. Such organisms have also been found in the fauces of well persons. In many epidemics of influenza the bacillus has not been isolated, or success has obtained in only a small proportion of the cases. The influenza bacillus seems to grow best in symbiosis with some other organism, especially with S. pyogenes aureus. Koch-Weeks Bacillus (Koch, 1883). — This produces a severe conjunctivitis. It is very common in Egypt and is also a frequent cause of conjunctivitis in the Philippines and in temperate climates. Smears made from conjunctival secretion show large numbers of small Gram negative bacilli, especially contained within pus cells, FRIEDLANDER'S BACILLUS. 81 but also lying free. They are more difficult to cultivate than the influenza bacillus, but the same general methods hold. Diplobacillus of Morax. — This organism causes mild conjunc- tivitis chiefly at the internal angle of the eye. They are about i or 2 // long and tend to occur in pairs or short chains. Some claim that they are Gram positive. They are haemophilic. Bacillus of Chancroid (Ducrey, 1889).— These are short cocco- bacilli, occurring chiefly in chains. They show bipolar staining. They grow best in a mixture of blood and bouillon. GRAM NEGATIVE BACILLI GROWING ON ORDINARY MEDIA. Bacillus Pneumoniae (Friedlander, 1882). — This organism is responsible for about 5% of the cases of pneumonia. It is usually termed the pneumobacillus to distinguish it from the pneumococcus; at other times Friedlander's bacillus. The name of Fraenkel attaches to the pneumococcus. Morphologically, it is a short, thick bacillus, and in pathological material, as sputum, shows a wide capsule. -It is nonmotile and Gram negative. The colonies on agar are of a pearly whiteness and are markedly visci'd.' ' On potato it shows a thick viscid growth containing gas bubble^. The characteristic culture is the nail culture of a gelatin stab. The growth at the surface is heaped up like a round-headed nail, the line of puncture resembling the shaft of the nail. It does not liquefy gelatin. It does not produce indol, and does not produce gas in lactose bouillon — differences from the colon bacillus — with which it may be confused in cultures, as it does not then possess a capsule. If in doubt, inject a mouse at the root of the tail. Death from septicaemia occurs in two days. The peritoneum is sticky and numerous capsulated bacilli are present in the blood and organs. The organisms which have been isolated from rhinoscleroma and ozcena are practically identical with the B. pneumonias. This group of organisms are generally referred to as the Friedlander group. Similar organisms have been isolated from the discharges of middle- ear diseases and in anginas. Cases have been reported where the B. pneumoniae was the cause of septicaemia in man. Bacillus Pestis (Kitasato, Yersin, 1894). — This is the organism of plague. It is the member of the group of hemorrhagic septicaemias 82 STUDY AND IDENTIFICATION OF BACTERIA. (Pasteurelloses), from which man suffers. It is primarily a disease of rats. Other Pasteurelloses are chicken cholera and swine plague. Where the plague bacilli are found chiefly in the glands, we have bubonic plague; when in the lungs, pneumonic plague, and when as a septicaemia, septicaemic plague. An intestinal type is recognized by c-ome authors. It must be remembered that in all forms of plague the lymphatic glands show hemorrhagic oedema; it is in bubonic plague, however, that the areas of necrosis with periglandular oedema are prominent. Where the symptoms are slight, mainly buboes, the term pestis minor is sometimes used; the typical disease being termed pestis major. In pneumonic plague we have a bronchopneumonia. FIG. 30. — Colonies of plague bacilli forty-eight hours old. (Kolle and Wassermann.} In smears from material from buboes, from sputum or in blood smears, as well as from blood or spleen smears from experimental animals, we obtain the typical morphology of a coccobacillus 1.5 x .$fj. with very characteristic bipolar staining; there being an interme- diate, unstained area. Inoculating tubes of plain agar and 3% salt agar with this same material, wre obtain in plain agar cultures organisms which are typically small, fairly slender rods, which do not stain characteris- tically at each end and are not oval. The smear obtained from the salt agar presents most remarkable involution forms — coccoid; root- shaped, sausage shaped forms, ranging from three to twelve microns PLAGUE 83 in length, more resembling cultures of moulds than bacteria. Another point is that on the inoculated plain agar we are in doubt at the end of twenty-four hours whether the dew drop-like colonies are really bac- terial colonies or only condensation particles. By the second day, however, these colonies have an opaque grayish appearance, so that now, instead of questioning the presence of a culture, we consider the possibility of contamination. The plague bacillus grows well at room temperature — its optimum temperature being 30° instead of 37° C., as is usual with pathogens. Next to the salt agar culture, the most characteristic one is the stalactite FIG. 31. — Pest bacilli from spleen of rat. (Kolle and Wassermann.) growth in bouillon containing oil drops on its surface. The culture grows downward from the under surface of the oil drops as a powdery thread. These are very fragile, and as the slightest jar breaks them, it is difficult to obtain this cultural characteristic. In diagnosing always use animal experimentation. Albrecht and Ghon have shown that by smearing material into the intact, shaven skin of a guinea-pig, infection occurs. This is the most crucial test. Mice inoculated at the root of the .tail quickly succumb. Rats, this being a primary disease of rats, are of course susceptible. In natural plague of rats, the lesions, which establish a diagnosis even without the aid of a microscope, are subcutaneous injection of the flaps of the 84 STUDY AND IDENTIFICATION OF BACTERIA. abdominal walls as they are turned back, fluid in the pleural cavities, ccdematous haemorrhagic swelling of the neck glands, and in particu- lar a creamy, mottled appearance of the liver. Smears from the spleen will show the oval bacilli. Recent investigations in India have definitely determined the fact that the flea (Pulex cheopis) is the intermediary in the transmission of plague from rat to rat and from rat to man. In pneumonic plague the infective nature is very great and appears to be by the respiratory atrium. This was the terrifying type of plague in the black death of the fourteenth centurv. FIG. 32. — Pest bacillus involution forms produced by growing on 3% salt agar. (Kolle and Wassermann.} For diagnosis make smears and cultures from material drawn from a bubo by a syringe. (At a later stage, when softening begins, there may not be any bacilli present.) Also, if pneumonic plague, from the sputum. Blood cultures and even blood smears may be employed in septicaemia plague. Formol fuchsin makes a satisfactory stain. Always inoculate a guinea-pig with the material either by rubbing it in with a glass spatula on the shaven skin or by subcutaneous injection. For prophylaxis the most important method is that of Haffkine. Stalactite bouillon cultures of plague are grown for five to six weeks. These are killed by a temperature of 65° C. for one hour. Lysol (J%) is added to the preparation and from 0.5 to 4 c.c. injected, according to TYPHOID COLON GROUPS. 85 tin- age and size of the individual treated. Susceptibility is reduced about one-fourth, and of those attacked after previous vaccination, the mortality is only about one-fourth of what it is among the noninocu- lated. 'Strong prepares a prophylactic vaccine from living plague cultures rendered avirulent. Yersin's serum, made by injecting horses with dead plague cultures and afterward with living ones, is of value prophylactically and has possibly considerable curative power. The Eberth, Gartner and Escherich Groups. — From a stand- point of cultures in litmus milk and sugar bouillon we can divide the organisms related to typhoid at one extreme and the colon at the other into three groups. 1. The Eberth or typhoid group. There are three important pathogens in this group: the B. typhosus, the B. dysenteriae, and the B. fecalis alkaligenes. The color of litmus milk is practically unal- tered, and there is no gas production in either glucose or lactose bouillon. No coagulation of milk. No reduction of neutral red. The B. typhosus and the B. fecalis alkaligenes are actively motile, while the B. dysenteriae is nonmotile or practically so. During the first 24 to 48 hours there is a moderate acid production by typhoid, so that the milk culture is less blue, while with the B. fecalis alkaligenes the alkalinity is intensified from the start so that the blue color is deepened. 2. The Gartner or hog cholera group. Besides organisms im- portant for animals and probably at times for man, such as B. cholerae suum and B. psittacosis and B. icteroides (interesting historically as having been reported as the cause of yellow fever by Sanarelli), we have two pathogens: (i) B. enteritidis (Gartner's bacillus) and (2) B. paratyphoid B. These organisms cannot be separated culturally, but only by immunity reactions. They do not tarn litmus milk pink. They produce gas in glucose bouillon, but not in lactose. They very powerfully reduce neutral red with the production of a yellowish fluorescence. They do not coagulate milk. There is a transient acidity in the litmus milk, but becoming shortly afterward alkaline, the lilac-blue color is intensified. Both organisms are motile. 3. The Escherich or colon group. These turn litmus milk pink, coagulate milk, reduce neutral red and show varying degrees of 86 STUDY AND IDENTIFICATION OF BACTERIA. motility. The three groups of organisms just described are non- liquefiers of gelatin. Two intestinal organisms, the B. cloacae and the Proteus vulgaris, differ in liquefying gelatin. Bacillus Typhosus (Eberth, 1880; Gaffky, 1884).— This organism may be isolated from the stools, urine and the blood of typhoid patients. At postmortem it can be best isolated from the spleen, but is also present in Peyer's patches which have not ulcerated. When ulceration has occurred contamination with B. coli is almost sure. Cultures may also be obtained from the liver. In sections made from spleen the Gram negative bacilli are apt to be decolorized. Thionin, FIG. 33. — Seventy-two hour old culture of typhoid bacillus on gelatine. (Kolle and Wassermann.} then blotting and clearing in oil or xylol, shows the clumps of bacilli lying between the cells. Formerly it was supposed that by the differences in the thickness of the film of a colony or by its varying shades of grayish-blue, we pos- sessed data of importance in differentiating typhoid from related organisms. Growth on potato was also considered as affording in- formation. At present, the biochemical reactions give us information assisting in differentiation, and the agglutination or bacteriolytic phenomena, the final diagnosis. The various plating media are con- sidered under the studv of feces. TYPHOID. 87 Not only do we find hyperplasia of the endothelial cells in the lymphoid tissue of Peyer's patches and the mesenteric glands and the spleen, with subsequent necroses, but focal necroses of the same character are found in the liver. A striking feature of the pathology of typhoid fever is the long- continued persistence of the organisms in the gall-bladder and else- where. It is beginning to be believed that a previous typhoid infection, possibly so mild as to have passed unnoticed, is at the basis of gall- bladder infections and resulting gall-stones. Various bone infections, especially osteomyelitis, have shown the typhoid bacilli in pure culture. Formerly it was supposed that the typhoid bacillus brought about its lesions by a local infection centered in the ileum. The present view is that typhoid bacilli effect an entrance into the blood stream through some lymphoid channel, as by tonsil or other alimentary lymphoid structure. It multiplies in the blood during the period of incubation and it is only when bactericidal properties are developed in the blood that we have destruction of the bacilli and liberation of the intracellular toxins which lead to the development of symptoms. That this is probable is shown by the fact that typhoid bacilli can be isolated from the blood during the period of incubation. It is a practical point that the time to isolate the bacteria from the blood is in the first days of the attack. The diagnosis by agglutination is only expected after the seventh to tenth day. Agglutination may not appear until during con- valescence, and in about 5% of the cases it is absent. It, as a rule, disappears within a year. Very little success has been obtained with curative sera. Chantamesse, by treating horses with a filtrate from cultures of typhoid bacilli on splenic pulp and human defibrinated blood, claimed to have obtained a curative serum possessing antitoxic power. Wright's method of prophylactic inoculation is now being employed in the British army with apparent success. In this, 24- to 48- hour-old cultures are killed at 53° C. ; 1/4% of lysol is then added. An injection of 500 million bacteria is made at the first inoculation, and ten days later an injection of one billion. The British prefer to inject subcutaneously in the infraclavicular region and at the insertion of the deltoid. The Germans consider three injections as conferring greater immunity. 88 STUDY AND IDENTIFICATION OF BACTERIA. A very important discovery is that certain persons, who may have had only a slight febrile attack, may eliminate typhoid bacilli for years in their feces (typhoid carriers). The bacilli are also eliminated for considerable periods in the urine. Distinction is now being made between acute carriers (convalescents) and chronic carriers. Or- dinarily, it is very difficult to isolate typhoid bacilli from the stools. For laboratory diagnosis, blood cultures during the first week and „• -^ v1 - " \ HE •'• S ? V^"- - O S ^C/:;;..? FIG. 34. — Bacillus of typhoid fever, stained by Loffler's method to show flagella. (X 1000.) (Williams.) agglutination tests during the second week and onward are the practical methods. B. typhosus appears in the blood in relapses. Paratyphoid Bacilli (Achard and Bensaude, 1896; Schottmiiller, 1901). — Cases resembling mild attacks of typhoid occasionally show agglutination for paratyphoid bacilli. These organisms have also been isolated from the blood, as with typhoid. Two types have been recognized: the paratyphoid A and the paratyphoid B. The latter occurs in 80% of such cases. Culturally, paratyphoid B cannot be PARATYPHOID AND DYSENTERY. 89 r]>a rated from Gartner's bacillus. In paratyphoid A there is less gas produced in glucose bouillon than with paratyphoid B, and the primary acidity of litmus milk is not succeeded by a subsequent alkalinity. It does not seem practical to draw a fine distinction between these two strains. Bacillus Enteritidis (Gartner, 1888). This organism has been frequently isolated from cases of gastroenteritis from ingestion of infected meat. This organism is very pathogenic for laboratory animals, producing a hemorrhagic enteritis and at times a septicaemia. Where meat has been contaminated with Gartner's bacillus toxins may have been produced, and symptoms of poisoning with acute gastro- enteritis would occur shortly after ingestion. It is interesting to note that this toxin is not destroyed by the boiling temperature, thus differ- ing from the toxin of the other important meat poisoning (botulism) bacillus — B. botulinus — which is rendered innocuous by a temperature of 65° or 70° C. If there is only a little toxin introduced with the con- taminated meat, the symptoms will be delayed one or two days. Such organisms have been isolated in pure culture from cases with high fever, marked intestinal derangement, with considerable blood in the rather fluid stools. In two cases studied the disease was at first diagnosed as a severe typhoid infection. Klein thinks the organism of Danysz's virus (to kill rats during plague epidemics) may be identical with B. enteritidis. Proteus Vulgaris. — This organism is often encountered in plates made from feces. It is common in decaying meat or cheese, and cases of even fatal poisoning with marked gastrointestinal symptoms and cardiac failure have been reported. At times it is the cause of cystitis. The colonies on agar are moist and unevenly spreading (amoeboid). The bacilli are very motile and, as a rule, Gram negative. It digests blood serum and is a rapid liquefier of gelatin. The cultures generally have a putrefactive odor. In infective jaundice (Weil's disease) this organism has been reported as the cause. Bacillus Dysenteriae (Shiga, 1898). — Dysentery bacilli produce a coagulation necrosis of the mucous membrane of the large intestine and occasionally of the lower part of the ileum. Polymorphonuclears are contained in the fibrin exudate. It was formerly thought that these lesions were of local origin, but the present view is that toxins are QO STUDY AND IDENTIFICATION OF BACTERIA. produced which, being absorbed, are eliminated by the large intestine with resulting necrosis. Flexner, by injecting rabbits intravenously with a toxic autolysate, produced characteristic intestinal lesions. There are two main types of dysentery bacilli : 1. Those producing acid in mannite media — the acid strains (Flexner-Strong types). 2. Those not developing acid in mannite (Shiga-Kruse types). Ohno finds that fermentative reactions do not correspond to immunity ones. Thus an acid strain used to immunize a horse may produce a serum more specific for a nonacid strain. The Shiga type is very toxic in cultures, while the Flexner type does not seem to possess a soluble toxin. At the Lister Institute injections of a soluble toxin produced a serum of marked antitoxic power. Such a dysentery serum, which is probably both antitoxic and antimicrobic, is of curative value. Shiga immunized horses with polyvalent cultures and obtained a polyvalent serum which has reduced the death-rate about one-third. The dysentery bacillus is present in the mucous stools during the first five or six days of the disease. By the tenth day it has probably disappeared. In all cultural respects the dysentery bacillus resembles the typhoid, and the only practical method of distinguishing these two organisms, other than by agglutination reactions, is by the nonmotility or exceedingly slight motility of the dysentery bacillus. The dysentery bacilli do not form those threads or whip-like filaments so characteristic of typhoid cultures. The dysentery bacillus is not found in the blood and hence is not eliminated in the urine. It is found in mesenteric glands. In dysentery patients agglutination phenomena do not show themselves until about the twelfth day from the onset. Hence, this procedure is of no particular value in diagnosis. It is of value, how- ever, to identify an organism isolated from the stools at the commence- ment of the attack, using serum from an immunized animal or a human convalescent for the agglutination test. Park and Collins, in 1904, separated various strains of dysentery bacilli into three groups: i. Original Shiga strain. No indol. No fermentation of mannite, maltose or saccharose. THE ROLE OF THE COLON BACILLUS. 91 2. A type fermenting mannite, but not maltose or saccharose. No indol. 3. An indol producer which actively ferments mannite and maltose, B. COLI, B. LACTIS AEROGENES, B. CLOACAE. While the colon bacillus chiefly inhabits the large intestine, the B. lactis aerogenes is to be found in the upper part of the small intestine. While they may be separated on the ground of motility, yet it is by the greater fermentative activity of the B. lactis aerogenes that they are best separated. Some consider them as only representing different strains of the same organism. Some consider that the B. coli produces a bactericidal substance which inhibits the growth of, or destroys pathogenic bacteria which may have passed the destructive influences of the gastric fluid; others that this effect is due to their free growth and the development of phenol and various putrefactive substances. The probable importance of the colon bacillus in protecting the organ- ism is shown by the fact that where numerous colonies of pathogenic organisms may be cultivated from feces we may find a diminution in number or absence of the colon bacillus. This condition may be observed in infections with the organisms of dysentery, cholera, typhoid and paratyphoid. While its normal function is probably protective, yet the B. coli is an important pathogenic agent, it being frequently the organism isolated from purulent conditions within the abdominal cavity, especially in appendicitis and lesions about the bile ducts. It is particularly prone to cause lesions of the bladder and pelvis of the kidn.ey. In the treatment of colon cystitis by vaccines of dead colon bacilli, the most brilliant results in opsonic therapy have been obtained. Sir A. Wright thinks that certain cases of mucous colitis may be due to colon infection and that vaccination may cure them. The colon bacillus is fully considered under the bacteriology of water. B. cloacae was isolated first from sewage by Jordan. It is a rapid liquefier of gelatin, and in its reaction^ with sugars and litmus milk resembles the colon bacillus. 92 STUDY AND IDENTIFICATION OF BACTERIA. CHROMOGENIC BACILLI. These are identified by the color of their colonies on agar. The B. pyocyaneus is the most important one of them in medicine, but the B. prodigiosus is also of interest medically. A violet chromogen, the B. violaceus, which is motile and liquefies gelatin, has been de- scribed under many names. It has been found in water. An orange-yellow chromogen, the B. fulvus, is nonmotile and varies as to its liquefaction of gelatin. B. pyocyaneus (Gessard, 1882). — This organism is frequently termed the bacillus of green or blue pus. It is a small (2.5 x .$/*) FIG. 35. — Bacillus pyocyaneus. (Kolle and Wassermann.) motile Gram negative bacillus. It grows readily at room or incubator temperature. It liquefies gelatin rapidly. The green color diffuses through the agar or gelatin on which it grows, so that we not only have the green-colored colony, but the medium as well is colored. Upon potato the colonies are more of a dirty brown. It is widely distributed in water and air, and is frequently isolated from faeces. The B. fluorescens liquefaciens of water seems to be simply a strain of B. pyocyaneus. The B. pyocyaneus is frequently associated with other pus organisms in abdominal abscesses. In addition to having an endo toxin, it produces a soluble toxin similar to diphtheria toxin. CHROMOGfiNIC BACILLI. 93 This toxin differs from those of diphtheria and tetanus in that it can stand a temperature of 100° C., while those of diphtheria and tetanus are destroyed at about 65° C. The fact that the union between toxin and antitoxin is only of a binding, neutralizing nature is best shown by taking a mixture of pyocyaneus toxin and antitoxin, which is innocu- ous and heating it. This destroys the antitoxin, but does not injure the toxin. We now find that the original toxicity has returned. The antitoxins of diphtheria and tetanus are more stable than the corre- sponding toxins; hence, this experiment would be impossible with them, as upon heating we should fir >t destroy the toxin. B. prodigiosus. This is a very small coccobacillus which shows motility in young bouillon cultures. It is Gram negative. The colonies on agar or other solid media show a rich red color. The pigment only develops at room temperature; it is absent in cultures taken out of the incubator. The B. prodigiosus is frequently found on food stuffs, especially bread, where it may simulate blood. It liquefies gelatin rapidly and gives a diffuse turbidity to bouillon. It is probable that B. indicus and B. kilensis are strains of B. prodigiosus. Coley's fluid, which has been used in cases of inoperable sarcoma and other malignant growths, is a culture prepared by growing very virulent streptococci in bouillon for ten days. This streptococcus culture is now inoculated with B. prodigiosus, and after another ten days the mixed culture is killed by heat at 60° C. and the sterile product injected. Coley injected about one-twentieth of a c.c. of this vaccine. CHAPTER IX. STUDY AND IDENTIFICATION OF BACTERIA. SPIRILLA. KEY AND NOTES. Key to recognition of gelatin-liquefying, motile and Gram negative spiral or comma-shaped organisms. A. Do not give the nitroso-indol reaction with sulphuric acid alone in 24 hours. 1. Produce an abundant moist cream-colored growth on potato at room temperature. a. Finkler and Prior's spirillum. Liquefaction of gelatin very rapid. No air-bubble appearance at top of liquefied area. Cultures have foul odor. Milk coagulated. Thicker spirillum than cholera. Isolated from cholera nostras. 2. Scanty growth or none at all on potato at room temperature. Only a moderate yellowish growth when incubated about incubator temperature. a. Spirillum tyrogenum. Does not liquefy gelatin so rapidly as Finkler Prior. Thinner and smaller spirillum than cholera. B. Give the nitroso-indol reaction with sulphuric acid within 24 hours. 1. Very pathogenic for pigeons. a. Spirillum metschnikovi. Liquefies gelatin about twice as rapidly as cholera. Gives bubble appearance at top of stab. 2. Scarcely pathogenic for pigeons. a. Spirillum cholerse asiaticas. Milk not coagulated. Nonmotile, nonliquefying gelatin and Gram positive spirilla have also been described. There is also a large group of phosphorescent spirilla. Spirillum Choleras Asiaticae (Koch, 1884). — Typically, the morphology of this organism is that of the comma (Comma bacillus of Koch). It also frequently shows S shapes, and often appears in long threads showing turns. When freshly isolated from cholera material they, as a rule, show a fairly typical morphology, but after subcultures in the laboratory variations are common, so that red forms and round involution shapes give a picture altogether at variance with the comma shape. Even in recent cultures of undoubted cholera we may have different types, as coccoid forms and slender rods. The cholera spirillum is very motile and liquefies gelatin fairly 94 CHOLERA. 95 rapidly, although more slowly than any of the spirilla mentioned in the key. The colony on gelatin was formerly considered charac- teristic, but like most cultural characteristics, it is now considered as FIG. 36. — Cholera spirilla. (Kolle and Wassermann.} being only of confirmatory value; it is not specific. These colonies show in 24 hours as small granular white spots which have a spinose periphery. An encircling ring of liquefaction now makes its appear- FIG. 37. — Involution forms of the spirillum of cholera. (Van Ermengem.) ance and the highly refractile (as if fragments of sparkling glass) colony can be separated into a granular center, a striated periphery and a clear external ring of liquefaction. 96 STUDY AND IDENTIFICATION OF BACTERIA. On gelatin stabs the liquefaction produces a turnip -like hollow at the top of the puncture — the air bubble appearance. It gives the nitroso-indol reaction with sulphuric acid alone (cholera red). Kraus attaches importance to the fact that cholera does not produce a haemolytic ring on blood agar as do the pseudo- cholera spirilla ; a difficulty is that many pseudo- spirilla do not haemolize. It grows very rapidly on peptone solution and this is the medium for the enrichment test to be later described. On this it may form a pellicle. On agar the colony is more opalescent than the typhoid. It does not grow on potato except at incubator temperature. It does not coagulate or turn acid litmus milk. The spirilla are found in myriads in the rice-water discharges, these white flakes being desquamated epithelial cells. They penetrate the crypts of Lieberkuhn, but rarely extend to the submucosa. The symptoms are due to an endotoxin. Cholera may be transmitted from water supplies, when the outbreak is apt to be widespread and in great numbers from the start. Also by indirect contagion, as by flies or on lettuce, etc. A very important point is that we have well persons whose faeces contain virulent cholera spirilla (cholera carriers). To identify such spirilla immunity reactions are necessary: 1. Injected intraperitoneally into guinea-pigs, it produces a peritonitis and subnormal temperature. This reaction exists for spirilla other than the true cholera spirillum. 2. Intramuscular injections into pigeons are only slightly pathogenic, if at all. 3. The agglutination test is the most practical. In this we use serum from an immunized animal, in dilution of from 50 to 1000. Serum of cholera convalescents may show agglutination as early as the tenth day; it is usually best shown about the third week. Dunbar's quick method is very practical. Make two hanging- drop preparations, FIG. 38.— Spiril- lum of cholera, stub - culture in gelatin, two days old. (Frankel and Pfeiffer.} CHOLERA. 97 using mucus from the stool as the bacillary emulsion. To one add an equal amount of a 1:50 normal serum; to the other a i : 500 dilution of immune serum. Cholera spirilla remain motile in the control, but lose motility and become agglutinated in the preparation with the immune serum. 4. Pfeiffer's phenomenon. If cholera spirilla are introduced into the peritoneal cavity of immunized guinea-pigs (or if together with a i : 1000 dilution of immune serum the mixture is injected intraperitoneally into normal guinea- pigs) and at periods of ten to sixty minutes after injection, material is removed by a pipette from the peritoneal cavity, the spirilla have lost motility, have become granular and degenerated. Pseudospirilla are unchanged. This reaction may be carried on in a pipette, using fresh serum. Antisera for the treatment of cholera have not proved successful. Prophylactically, there are two prominent methods: (T) That of Haffkine, where live cholera spirilla are injected subcutaneously; and (2) Strong's cholera autolysate. In this cholera cultures are killed at 60° C. The killed culture is then allowed to digest itself in the incubator at 37° C. for three or four days (peptonization). The preparation is then filtered and from 2 to 5 c.c. of the filtrate is injected. For diagnosis take a fleck of mucus, make a straight smear and fix; stain with a i : 10 carbol fuchsin. The comma shaped organisms appear as fish swimming in a stream. 2. Inoculate a tube of peptone solution. The cholera spirilla grow so rapidly, and being strong aerobes, they grow on the surface of the fluid so that by taking a loopful from the surface, we may in eight hours obtain a pure culture. Inoculate a second tube from this first and, if necessary, a third (enrichment method). 3. Test for cholera red reaction. (Simply adding from three to five drops of concentrated sulphuric acid in the first or second peptone culture after 18 to 24 hours' growth.) STUDY AND IDENTIFICATION OF BACTERIA. At times we only get the cholera red when we have a pure culture of cholera. 4. Smear a fleck of mucus or, better, the surface growth on peptone, on a dry agar surface in a Petri dish. From colonies developing, make agglutination and, if desired, cultural tests. It is by immune reactions that we identify cholera spirilla. The surface moisture of plates is best dried by the filter-paper top. 5. For methods with water, see Water Analysis. CHAPTER X. STUDY AND IDENTIFICATION OF MOULDS. CLASSIFICATION OF THE FUNGI. Order Suborder Phycomycetes Zygomycetes Gymnoascus Ascomvcetes Carpoascus Hyphomycetes Family Genus Species Mucor ' M. corymbifer M. mucedo f S. cerevisiae Saccharomyces J S. anginae [ S. blanchardi Saccharo- mycetes Endomyces E. albicans Cryptococcus f C. gilchristi C. hominis T. sabouraudi T. tonsurans ' Trichophyton T. violaceum T. mentagro- Gymno- phytes asci T. cruris Microsporum M. audouini Achorion A. schoenleini Penicillium P. crustaceum rA. fumigatus Perisporia- Aspergillus A. concentricus ceae '> A. pictor A. niger Discomyces < D. bovis D. madurae Madurella M. raycetomi Malassezia M. furfur Microsporoides M. minutissimus Trichosporum T. giganteuin THE FUNGI. The Thallophyta are plants in which there is no differentiation between root and stem. The classes of Thallophyta which are of interest medically are (i) the Algae and (2) the Fungi. 99 100 STUDY AND IDENTIFICATION OF MOULDS. Some include Lichenes as a separate class. These are really symbiotic organisms — Fungi parasitic on Algae. The Algae contain chlorophyll, with the exception of Cyanophy- ceae. To the order Cyanophyceae it is considered that the family of Bacteria belong. The fungi do not possess chlorophyll. They are in their simplest forms ramifying filaments called hyphae. The vegetative hyphae which intertwine in tangled threads, as a support, are termed the mycelium, while those which project upward are called the aerial hyphae and are the ones wrhich bear the conidia or spores. The orders of the class fungi which are of interest medically are: (i) the Phycomycetes; (2) the Ascomycetes; (3) the Hyphomycetes. Phycomycetes. — These produce a copious mycelium, bear conidia, and reproduce in the case of the suborder Oomycetes by heterogamy. (Dissimilar sexual cells — a smaller male, antheridium, and a larger female, oogonium. By fertilization by antherozoids from the anther- idium penetrating the oosphere we have oospores.) The suborder Zygomycetes reproduces either asexually or by isogamy (two similar sexual cells conjugate and form on fusion a Zygospore.) Of these wre have two species of the genus Mucor: (i) Mucor mucedo and (2) Mucor corymbifer. These moulds develop especially in external cavities. Pulmonary and generalized infections have also been reported. The pathogenic species have smaller spores and grow best at 37° C. The thick, coarse, cotton-like mould seen on horse manure is a Mucor. The sporangium, the organ of fructification, contains the spores within its interior. The M. mucedo has thick silver-gray mycelium, with large sporangia, 150/4 in diameter, contain- ing oval spores, 5 x 9/4. The M. corymbifer, which has been re- ported from a generalized infection, considered as typhoid, shows a snow-white mycelium. The sporangia are 20 to 40/4 and the spore about 3/z in diameter. Ascomycetes. — In this order are included many of the parasitic moulds. The most distinctive characteristic is the formation of ascospores in an ascus (little sac). It is an elongated sporangium in which a definite number of spores, usually eight, is formed. The ascus usually ruptures at its tip. Other members of the order are TEASTS. 10 I formed from hyprue by the separation of cells in succession from the free ends. The order is divided into those with naked asci (Gymnoascus) and those having a perithecium or investing layer (Carpoascus). Belonging to the suborder Gymnoascus we have (i) the family of Saccharomycetes, which reproduce by budding and in which the asci are without any semblance of a sheath, and (2) a family in which there is an indication of the formation of a perithecium — this may be termed the Gvmnoasci familv. FIG. 39 — Yeast cells. Saccharomyces cerevisiae. (Coplin.) Saccharomycetes. — There are three genera: Saccharomyces, En- do myces and Cryptococcus. Saceharomyces. — These have ascospores. • — Sr- cerevisiae. — This is the ordinary yeast fungus. Used at times as an antiseptic. S. anginae. — Found in a case of angina. S. blanchardi. — Found in a jelly-like tumor mass of the abdomen. The budding cells varied from 2 to 2o/£. Probably identical with S. tumefaciens. Endomyces. — Forms spores in the interior of filaments. E. albicans. — The organism of thrush. It produces a false membrane, especially on buccal surfaces. Grows 102 STUDY AND IDENTIFICATION OF MOULDS. only in acid media. Hence propriety of alkaline treatment. Cryptococcus. — Reproduces by budding, but ascospore formation not observed. Not a well-recognized genus. The diseases caused by it are termed blastomycoses. C. Gilchristi. — The cells are about i6/z in diameter and have a thick membrane. They reproduce by budding. May invade internal organs. It is cultivable. A mould, somewhat similar, is the Coccidioides im- FIG. 40. — Thrush fungus. (Kolle and Wassermann.) mitis of Ophuls. This has a mycelial growth. The infection frequently becomes generalized. The small bodies, about 3//, in the Molluscum con- tagiosum cells are thought by some to be yeasts. They are more probably artefacts. Plimmer's bodies in cancer cells belong in this group. They also are probably other than parasites. Gymnoasci. — Belonging to the family Gymnoasci we have the genera Trichophyton, Microsporum and Achorion. The trichophytons are generally known as the large-spored ring- worms. The spores are in chains and may be inside the hair or both RINGWORM. '03 outside and inside. Many of them are of animal origin, especially from the horse and the cat. T. Tonsurans. — Gives a crater-like culture with fine marginal rays. Fungus wholly inside the hair. Causes most of the large-spored scalp ringworms and many body cases. FIG. 41. — More common fungi, i, culture of Achorion schoenleini (favus); 2 culture of Trichophyton tonsurans; 3, culture of Trichophyton sabouraudi; 4, sporangium of Aspergillus; 5, culture of Trichophyton mentagrophytes; 6, culture of Microsporum audouini; 7, mycelium and spores of Malassezia fur- fur; 8, Cryptococcus gilchristi; 9, A and B, sporangium and mycelium of Mucor corymbifer; 10, Penicilium; n, Saccharomycestumefaciens; 12, Discomyces bovis. T. Sabouraudi. — Has a heaped-up festooned sort of culture. There is a similar fungus with a violet culture. These cause some of the scalp and beard ring- worms. T. Mentagrophytes. — This is the megalosporon endo- 104 STUDY AND IDENTIFICATION OF MOULDS ectothrix of Sabouraud. The external spores are in chains or in short mycelial threads, not mosaics of spores. There are varieties from horse, cat and bird. The lesions are more inflammatory than those of the endothrix class. Most of the beard and bcdy ringworms belong to this group — very few scalp cases. The cultures are finely rayed. The so called small spored ringwrorm is the Microsporum audouini. The fungus is packed as a mcsaic of spores, chiefly on the outside of the hairs. It is the chief cause of the ringworm of the scalp of children. It gives a downy-white culture. The Achorion schoenleini is the cause of favus. The cultures are rather wrinkled. It is characterized by the scutulum or favus cup. In the suborder Carpoascus wre have to consider the family Peri- sporiaceae. In this family the asci are completely enclosed by the investing membrane, the perithecium. When this rots the spores are set free. There are two genera of interest. Penicillium and Asper- gillus. Penicillium. — While Penicillium does at times form perithecia, yet they characteristically show chains of spores. The common P. glaucum resembles a hand with terminal beads. P. Crustaceum. — Is the common blue-green mould. It has been deemed pathogenic in cases of chronic catarrh of the eustachian tube and in gastric hyperacidity. Aspergillus. — These have sterigmata carrying chains of spores. Of the pathogenic Aspergilli we have: 1. A. fumigatus. — This has been considered as the cause of pellagra. 2. A. repens. — This has been found in the auditory canal and may produce a false membrane. 3. A. flavus. — This has been found in the discharges of chronic ear diseases. 4. A. concentricus. — Tnis is the cause of an important tropical ringworm, Tinea imbricata. The scales TROPICAL SKIN DISEASES. 105 are dry, like pieces of tissue-paper. There are generally about four rings which do not heal in the center. General appearance is that of watered silk. There are no inflammatory lesion^. Com- mon in Malay peninsula. Also found in some parts of the Philippines and in China. Some authorities consider the fungus to be a Trichophyton. FIG. 42. — Tropical fungi, i, concentric rings of Aspergillus concentricus; 2, sporangium of A. concentricus; 3, Aspergillus pictor; 4, Microsporoides minu- tissimus; 5, Trichosporum giganteum; 6, black granules of Madurella mycetomi; 7, yellow grains of Discomyces madurae. 5. A pictor.— This is the cause of a skin affection of Central America. In the affection colored spots appear on the skin, chiefly on face, forearms and chest. The disease is attended with a mangy odor. Spots are of various colors; if the superficial epithelium 106 STUDY AND IDENTIFICATION OF MOULDS. is affected we have a dark violet color. Deeper involvement gives red spots. Hyphomycetes. — In this order are grouped certain genera which cannot properly be assigned to any of the other orders. Discomyces bovis. This is the well-known ray fungus, the cause of actinomycosis. In man it is at times found in chronic sup- purative conditions attended with much granulation tissue. Such pus may show7 small yellow-gray granules about the size of a pin's head. When spread out between two slides the central portion shows a net-work of mycelium with bulbous thread-like rays going to the periphery. The " clubs" at the periphery are degenerate structures and do not stain by Gram. The central mycelium is Gram positive. This mould is essentially an anaerobe and should be cultivated in a deep glucose agar stab. It may also be cultivated in bouillon. In this it grows at bottom. Growth is dry and chalky. In diagnosis look for the little granules. Curetting of the sinuses may give the "ray fungus" when they are not found free in the pus. Discomyces madurae. This is a ray fungus found in the yellow "fish- roe" granules of madura foot. It is strictly aerobic in cultures, thus differing from actinomycosis. For diagnosis proceed as for D. bovis. Madurella mycetomi. This is the cause of the black "gunpowder" granules of madura foot. It is a mycelial mass with rather oval shaped swollen segments. It is at times cultivable on potato and agar as felted masses of gray growth, which later becomes almost black. Malassezia furfur. This is the fungus of Tinea versicolor. It is common both in temperate and in tropical climates. It is characterized by dirty yellow spots about covered parts of the body. Scrapings show a profusion of mycelial threads and interspersed spores. It is very difficult to cultivate. Microsporoides minutissimus. This is generally considered as the DIAGNOSIS AND CULTIVATION OF FUNGI. IOf cause of Erythrasma or dhobies itch, a very common inter- trigo of the tropics. It is characterized by its narrow mycelium and small spores. Various fungi are found in this affection. Castellani considers the cause of dhobies itch to be a trichophyton. T. Cruris. Trichosporum giganteum. This is the cause of a disease of the hairs, known in Columbia as "Piedra," so-called from the small gritty- like masses along the length of the hair. These spores are arranged like mosaics about the hair. DIAGNOSIS OF FUNGI. The most expeditious way to examine fungi is to treat the scales or hairs with a 10% solution of caustic potash or soda. Then crush between two slides; heat moderately over the flame and examine. Tribondeau's method is to treat the scales with ether, then with alcohol and finally with water. Next put the sediment (it is convenient to use a centrifuge) in a drop of caustic soda solution. Cover with a cover-glass, and after the preparation has stood about an hour run glycerin under the cover-glass. A very satisfactory method is to scrape the scales with a small scalpel, and smear out the material so obtained in a loopful of white of egg or blood-serum on a glass slide. By scraping vigorously the serum may be obtained from the patient. After the smear has dried, treat it with alcohol and ether to get rid of the fat. It may then be stained with Wright's stain or by Gram's method. The ordinary Gram method may be used or the decolorizing may be done with aniline oil, observing the decolorization under the low power of the microscope. Yeasts are best examined in hanging drop on the plain slide with vaselin cell, as given under Blood. CULTIVATION OF FUNGI. Moulds grow well on media with an acid reaction, so that by adjusting the reaction to + 2 percent or even higher, we permit of the growth of the fungi, but inhibit bacterial development. IO8 STUDY AND IDENTIFICATION OF MOULDS. Glycerin agar, bread paste or potato media are all suitable, but the best medium is that of Sabouraud: Maltose, 4 grams. Peptone, i grams. Agar, 1.5 grams. Water, 100 c.c. Make the reaction about + 2. Before inoculating media with moulds, some recommend placing the material in 60% alcohol for one or two hours to kill the bacteria. The moulds withstand such treatment. In cultivating moulds it is best to use small Erlenmeyer flasks, containing about one-fourth of an inch of media on the bottom, for the development of the colonies. In order to separate the mould we may take the hair or scales on a sterile slide and cut them into small fragments with a sterile knife. Then moisten a platinum loop from the surface of an agar slant, touch a fragment with the loop, and when it adheres transfer it to the agar slant. Make four or five inoculations on the surface and from suitable growth, after four to seven days, inoculate the medium in the Erlenmeyer flask. Plauth recommends receiving the mould material between two sterile glass slides. Seal the edges of the slides w;Lh wax and place the preparation in a moist chamber for four to seven days. From developing fungus growth inoculate the medium in the Erlenmeyer flask. A Petri dish containing several layers of thoroughly moistened filter-paper in top and bottom, makes a satisfactory moist chamber. CHAPTER XL BACTERIOLOGY OF WATER, AIR, MILK, ETC. BACTERIOLOGICAL EXAMINATION OF WATER. WHILE in a chemical examination as to the character of a water there are certain relations between the ammonias, nitrates, chlorides, etc., which indicate the probable animal as against vegetable nature of the organic matter present, yet it is a more or less presumptive evidence. In a bacteriological examination of water the finding of the colon bacillus may from a practical stand point be considered as positive evidence of human fecal contamination. Theoretically, the possibility of organisms being present corresponding culturally to B. coli and de- rived from cereals is to be considered. Also the faeces of animals con- tain an organism which cannot be differentiated from the colon bacillus. In detecting sewage contamination in water to which varying amounts of sewage had been added, it was found that the bacterial tests were from 10 to 100 times more delicate than the chemical ones. As showing sewage contamination of water, the presence of the B. coli has been generally accepted as the most satisfactory indication. The English authorities consider sewage streptococci and the spore- bearing B. enteritidis sporogenes as of value as indicators as well as the B. coli — the presence of sewage streptococci indicating very recent sewage contamination and that of the B. enteritidis sporogenes, in the absence of streptococci and colon bacilli, as evidence of sewrage contamination at some period more or less remote. In the United States the colon bacillus alone is considered the indi- cator of sewage contamination, and all tests, presumptive or positive, are based on the presence of this organism. In collecting samples of water for bacteriological examination, the following points should be considered: i. The bottles, which should have a capacity of from 25 to 100 c.c,, 109 110 BACTERIOLOGY OF WATER, AIR, MILK, ETC. should be sterile. Sterilization may be effected by heat or by rinsing with a little sulphuric acid and subsequently washing out thoroughly with the suspected water before collection. The utmost care must be exercised that the ringers do not come in contact with the glass stopper or the neck of the bottle while filling it. If the specimen is to be sent some distance, it should be packed in ice to prevent bacterial development. Frankland states that a count of 1000 became 6000 in 6 hours and 48,000 in 48 hours. In water packed in ice for a consider- able time, however, the bacterial count may diminish. 2. If collecting from city water supplies, secure the sample direct from the mains and let the water run from the tap a few minutes be- fore collection. If the water be taken from a pond, stream or cistern, be sure that the specimen comes from at least 10 inches below the sur- face. As sedimentation is the most important method in self-purifica- tion of rivers and ponds, it will be understood that any stirring up of the mud on the bottom will enormously increase a bacterial count. Quantitative Bacteriological Examination. i. Deliver definite quantities of the water to be examined into tubes of liquefied gelatin or agar and plate out the same or into a series of Petri dishes. The water should be deposited in the center of the plate and the melted gelatin or agar poured directly on the water and then, carefully tilting to and fro, mix the water and the media. One set of plates should be of gelatin and incubated at room temperature; a similar set should be of lactose litmus agar and incubated at 38° C. If the water is highly contaminated, it is neces- sary to dilute it; thus, with river water, which may contain from 2000 to 10,000 bacteria per c.c., a dilution of i to 100 would be desirable. Ordinarily it wrill be sufficient to deliver from a sterile graduated pipette .2, .3, and .5 c.c. of the water in each of 2 sets of plates: one set for gelatin, the other for agar. When gelatin is not at hand or convenient to work with, the gelatin plates may be replaced by others of lactose litmus agar for incubation at room temperature. After 24 hours at 38° C. or 48 hours at 20° C., the count should be made. Example: Forty colonies were counted on the gelatin plate con- BACTERIOLOGY OF WATER. Ill tainkig .2 c.c. (1/5) of the water. The number of organisms would be 200 per c.c. Ten colonies were counted on the agar plate containing .2 c.c. and incubated at 38° C. Number of bacteria developing at body temperature equals 50 per c.c. There is no strict standard as to the number of bacteria a water should contain per c.c. Koch's standard of 100 colonies per c.c. is generally given. It is by the qualitative rather than the quantitative analysis that one should judge a water. If there should be very many colonies on a plate, the surface can be marked off into segments with a blue pencil. If very numerous, cut out of a piece of paper a space equal to i square centimeter. By counting the number of colonies inclosed in this space at different parts of the plate, we can strike an average for each space of i square centimeter. To find the number of such spaces contained in the plate, multiply the square of the radius of the plate by 3.1416. Then multi- ply this number by the average per square centimeter, and we have the total number of colonies on the plate. This is the principle of the Jeffers disk. The relative proportion between the bacterial count at 20° C. and that at 38° C. is of great importance from a qualitative stand-point, as will be seen later. 2. Deliver into a. series of Durham fermentation tubes containing glucose bouillon and into another series containing lactose bouillon varying definite amounts of the water to be examined. In tubes show- ing the presence of gas in both glucose and lactose bouillon the evidence is presumptive that the colon bacillus is present. For the positive demonstration plates must be made from such tubes as show gas. It is sufficient to deliver from graduated pipettes in each series quantities of water varying in amount from .1 c.c. to 10 c.c. In our laboratory we inoculate with .1 c.c., .2 c.c., .5 c.c., i c.c. and 10 c.c. of the suspected water. If the .1 c.c. tubes show gas, we have reason to assume that the water contained at least 10 colon bacilli per c.c. If only the 10 c.c. tubes showed gas — those with less amounts not having gas — we would be in a position to state that the water contained the colon bacillus in quantities of 10 c.c., but not in quantities of i c.c. or less Many authorities regard water as suspicious only when the colon 112 BACTERIOLOGY OF WATER. AIR, MILK. ETC. bacillus is present in quantities of 10 c.c. or less; waters of good quali- ties frequently showing the presence of the colon bacillus in quantities of 100 to 500 c.c. It is generally accepted that if a water shows the presence of the colon bacillus in quantities of i c.c. or less, it should be regarded as suspicious. At the present time the medium that gives the least source of error in carrying out the quantitative presumptive tests is the bile lactose. It is made by adding i%, of lactose to ox bile, and fermentation tubes of the media showing gas may be considered as very probably con- taining the colon bacillus. The percentage of error with this method is reported to be only 11%, while with glucose fermentation tubes the error is more than 50%. Gas formation is usually shown in 48 hours, but it is advisable to continue the incubation for 72 hours. Even with this method plates should be made. 3 . As the colon and sewage streptococci ferment lactose with the pro- duction of acid and hence produce pink colonies on lactose litmus agar, much information can be obtained from the proportion existing between the number of pink colonies and those not having such a color. Waters of fair degree of purity rarely give any pink colonies. Qualitative Bacteriological Examination. General Considerations. — In some countries the proportion of liquefying to nonliquefying colonies on gelatin plates is considered of importance. Certain sewage organisms belonging to the proteus and cloaca group liquefy gelatin; consequently, if the proportion of liquefy- ing to nonliquefying be greater than as i to 10, the water is considered suspicious. The test is not considered by American authorities as of any particular value. The American Public Health Association recognizes the importance of the information obtained from a comparison of the number of organisms developing at 38° C. and those developing at 20° C. Bacteria whose normal habitat is the intestinal canal naturally develop well at body temperature, while normal water bacteria prefer the average temperature of the water in rivers and lakes. Consequently when the COLON BACILLUS IN WATER ANALYSIS. 113 number of organisms developing at 38° C. at all approximates the num- ber developing at 20° C., there is a strong suspicion that sewage or- ganisms may be present. Normal waters give proportions of i to 25 or i to 50, while in sewage contaminated waters the proportion may be as i to 4 or less. In addition, the appearance of pink colonies on the lactose litmus agar is of great assistance in judging of a water. Both sewage strepto- cocci and the colon bacillus give pink colonies — those of the streptococci are smaller and more vermilion in color. Microscopic examination will differentiate the cocci from the bacilli. It is well to bear in mind that the pink colonies after 24 hours may turn blue in 48 hours from the development of ammonia and amines. Consequently the lactose litmus agar plates should be studied after 24 hours. A good water supply will rarely show a pink colony, while in a sewage contaminated one the pink colonies wrill probably predominate. The diagnostic characteristics considered important by the Ameri- can authorities in reporting the colon bacillus are: 1 . Typical morphology, nonsporing bacillus, relatively small and often quite thick. 2. Motility in young broth cultures. (This is at times unsatisfac- tory, as some strains of the colon bacillus do not show it even in young bouillon cultures.) 3. Gas formula in dextrose broth. Of about 50% of gas produced, 1/3 should be absorbed by a 2% solution of sodium hydrate (CO2). The remaining gas is hydrogen. (Later views indicate that the gas formula is exceedingly variable and should not be depended upon. To carry out this test one fills the bulb of a fermentation tube with the caustic soda solution then, holding the thumb over the opening or with a rubber stopper, the bouillon culture and the soda solution are mixed by tilting the fermentation tube to and fro. The total amount of gas is first recorded and then that remaining after the CO2 has been absorbed is reported as hydrogen.) 4. Nonliquefaction of gelatin. 5. Fermentation of lactose with gas production. 6. Indol production. 7. Reduction of nitrates to nitrites. 8 114 BACTERIOLOGY OF WATER, AIR, MILK, ETC NOTE. — The reduction of neutral red with a greenish-yellow fluorescence is very striking and has been suggested as a test for the colon bacillus. Many other organisms, especially those of the hog cholera group, have this power. It is convenient, however, to color glucose bouillon with about i% of a 1/2% solution of neutral red. Isolation of the Typhoid Bacillus from Water. This is probably the most discouraging procedure which can be taken up in a laboratory. Only the most recent reports of such isola- tion from water supplies, which have been verified by immunity reac- tions, can be accepted and of these the number of instances is exceed- ingly small. Owing to the long period of incubation, the typhoid organisms may have died out before the outbreak of an epidemic suggests the examination of the water supply. There have been various methods proposed for the detection of the B. typhosus in water. A method which wrould offer about as reasonable a chance of success as any other would be to pass 2 or 3 liters of the water through a Berkefeld filter; then to take up in a small quantity of water all the bacteria held back by the filter. Then plate oat on lactose litmus agar and examine colonies which do not show any pink coloration. The dysentery bacillus has about the same cultural characteristics as the typhoid one, so that it is important to note motility. If from such a colony you obtain an organism giving the cultural characteristics of B. typhosus, carry out agglutination and preferably bacteriolytic tests as well. Some strains of typhoid, espe- cially when recently isolated from the body, do not show agglutination. The Conradi Drigalski, the malachite-green and various caffeine containing plating media have been highly recommended. Isolation of the Cholera Spirillum from Water. The method proposed by Koch in 1893 does not seem to have been improved upon by later investigators. To 100 c.c. of the suspected water add i% of peptone and i% of salt. Incubate at 38° C., and at intervals of 8, 12 and 18 hours examine microscopically loopfuls taken from the surface of the liquid in the flask. So soon as comma-shape BACTERIOLOGICAL EXAMINATION OF MILK. 1 15 organisms are observed, plate out on agar. The colonies showing morphologically characteristic organisms should be tested as to ag- glutination and bacteriolysis. Inasmuch as the true cholera spirillum shows a marked cholera-red reaction it is well to inoculate a tube of peptone solution from such a colony and add a drop of concentrated sulphuric acid after incubating for 18 hours. The rose-pink colora- tion is given by the cholera spirillum with the acid alone — the nitroso factor in the reaction being produced by the organism. BACTERIOLOGICAL EXAMINATION OF MILK. A bacterial milk count is of comparatively little value as showing whether a milk is dangerous or not. As a matter of fact, a milk which contains several million of bacteria per c.c. might be less dangerous than one containing only a few thousand, especially if in the latter there were numerous liquefiers and gas producers present. There is, however, one point of importance in connection with the quantitative estimation of bacteria in milk, and that is the fact that in order to keep the development of the bacteria within the limits of 10,000 to 50,000 per c.c., it is necessary that the requirements of cleanliness in milking and the rapid cooling of the milk after obtaining it and the keeping of the temperature below 50° C. be rigidly observed. If a milk has a high count it shows some error in the handling of the milk. In making a quantitative bacteriological examination, the principle is the same as with water. Make a known dilution of the milk with sterile water; add definite quantities of this diluted milk to tubes of melted agar or gelatin and pour into plates. The diluted milk may also be delivered in the center of the plate and the melted agar or gelatin poured directly on it, mixing thoroughly. . Always shake the bottle well before taking sample. Example: Added i c.c. of milk to 199 c.c. of sterile water in a large flask (500 to 1000 c.c.). After shaking thoroughly, take i c.c. of this i : 200 dilution and add it to 99 c.c. of sterile water. Shak- ing thoroughly, wre have a dilution of i : 20,000. Of this we added .5 c.c. to a tube of gelatin or agar. After incubation the plate showed Il6 BACTERIOLOGY OF WATER, AIR, MILK; ETC. 75 colonies. Therefore the milk contained in each c.c. 75 x 2 x 20,000 (dilution) = 3,000,000 — the number of bacteria in each c.c. of milk. Lactose litmus gelatin or agar is to be preferred in milk-work, as the normal lactic acid bacteria produce reddish colonies which are very striking. A standard easily attained for high-grade, certified milk would be 5,000 to 10,000 per c.c. In the qualitative examination of milk, many dairies employ the fermentation tube, any organism producing gas being considered undesirable. Again liquefying organisms, as shown by the presence of such bacteria in the gelatin plates, is evidence of probable contami- nation by fecal bacteria. A question which seems difficult to decide is as to the general nature of the so-called normal lactic acid bacteria of milk. Some describe them as very short, broad bacilli with very small colonies, fermenting lactose with the formation of lactic acid. Others consider that the streptococci are the organisms which are concerned with the normal fermentative changes. In examining specimens of milk considered the best on the market, I have repeatedly found the small red colonies on lactose litmus agar to be streptococci. In connection with the organisms present in the tablets used for treat- ing milk to produce lactic acid for the treatment of intestinal disorders, and considered to be normal lactic acid bacteria, I have found both streptococci and bacilli. These have all agreed, however, in not pro- ducing gas in either lactose or glucose fermentation tubes. Another source of information as to the quality of a milk may be derived from a study of the number of leukocytes or pus cells con- tained in i c.c. of the milk. The Doane-Buckley method is probably the most accurate. In this you throw down the cellular contents of 10 c.c. of milk in a cen- trifuge revolving about 1000 times a minute for ten to twenty minutes. Then remove supernatant milk and add 0.5 c.c. of Toisson's solution to the sediment. You thus have the leukocytes of 10 c.c. contained in 0.5 c.c. (Concentrated twenty times.) Make a haemacytometer pre- paration as for blood and find the average number of cells for each square millimeter. Then multiply this by 10 to get the number of cells in a cubic millimeter. As a cubic millimeter is one thousand times smaller than a cubic centimeter, you multiply the number per BACTERIOLOGICAL EXAMINATION OF AIR. Ilj cubic millimeter by one thousand. Then, as the milk was concentrated twenty times, you divide by 20. (If it were diluted twenty times, you would multiply by 20.) Example: Found an average of 50 cells per square millimeter. This would make 500 per cubic millimeter, and 500,000 per c.c.; then 500,000 divided by 20 would give 25,000. There is no agreement as to a standard for allowable leukocytes. Even in apparently healthy animals they may exceed 100,000 per c.c. Doane has suggested 500,000 per c.c. as a preferable limit. The smear methods for determining the number of leukocytes present do not compare in accuracy with the volumetric ones. BACTERIOLOGICAL EXAMINATION OF AIR. In Paris a cubic meter of air was found to contain the following number of organisms: Suburbs.— Winter, 145 moulds, 170 bacteria. Summer, 245 moulds, 345 bacteria. City Hall. — Winter, 1345 moulds, 4305 bacteria. Summer, 2500 moulds, 9845 bacteria. Air of hospitals, especially after sweeping, may contain 50,000 bacteria per cubic meter. There does not seem to be any particular relation between the amount of carbon dioxide in air and the bacterial content. Petri's Rough Method. — Exposure of a lactose litmus agar plate (capacity 100 sq. cm.) for five minutes will give the number of organ- isms present in ten liters of air. Multiply by 100 for one cubic meter. The two groups of organisms usually found in air are (i) bacteria and (2) moulds. Moulds (spores) may be carried by currents of air; bacteria, however, are generally carried about by particles of dust or finely divided liquids (spray). On the lactose litmus agar plate staphylococci and streptococci show as bright red colonies. Sedgwick Tucker Sterile Granulated Sugar Method. — Sterilize aerobioscope and introduce granulated sugar on support. Again sterilize (not over 120° C. in dry-air sterilizer). Allow a given quan- tity of air to pass through; then shake the sugar into wide part of Il8 BACTERIOLOGY OF WATER, AIR, MILK, ETC. aerobioscope. Now pour in 10 or 15 c.c. of melted gelatin (40° C.) to dissolve sugar. Roll tubes as for Esmarch roll cultures, and incubate at room temperature. To draw air through the aerobioscope, connect the small end with a piece of rubber tubing which is attached to a tube in the stopper of an aspirating bottle. Having poured a definite quantity of wrater into the aspirating bottle, allow the water to run out. The same quantity of air will be drawn through the sugar of the aerobioscope as the amount of water passing out of the aspirating bottle. The bacteria and moulds are caught by the sugar. Example. — Passed 10 liters of air through the aerobioscope. The bacteria in this quantity of air showed 75 colonies when incubated FIG. 43. — Sedgwick-Tucker aerobioscope. (Williams.} at 20° C. The unit being one cubic meter or one thousand liters, we have only obtained the bacteria of one hundredth of the unit. Hence multiplying 75 by 100 gives 7,500 bacteria as present in one cubic meter of the air examined. In comparing the results with the aerobioscope with those obtained by exposing a plate as in Petri's method for ten instead of five minutes, it was found that the latter was sufficiently in accord to make it a satisfactory approximate quantitative method. The simplicity and ease of access of the colonies developing in it make it preferable when the air of operating-rooms or hospital wards is to be examined. CHAPTER XII. PRACTICAL METHODS IN IMMUNITY. THAT which prevents the gaining of a foothold by disease organisms in the animal body or which neutralizes their harmful products or destroys the parasites is termed immunity. In the main, the question of immunity hinges on the powers of resistance of the human body and the aggressiveness or virulence of the invading organism. It must always be kept in mind that immunity is only relative; thus the fowl, which is practically immune to tetanus, may be made to suc- cumb by reducing its resistance by refrigeration or by increasing the amount of poison introduced. The insusceptibility which the fowl has to tetanus or which man has to many diseases of animals is best termed inherent immunity, and is at present only a subject of theo- retical interest. When immunity to a given disease is obtained as a result of an attack of the disease in question or by laboratory methods of inoculation, this is termed properly an acquired immunity, and in the former case is a naturally acquired immunity or "natural im- munity" and in the second is an artificially acquired immunity or " artificial immunity." As a result of an attack of a disease or in response to the stimulus of the injection of the organism or its products, we have developed in the man so injected certain specific antagonistic properties to that organism, which are usually demonstrable in the blood serum or other body fluids, and to which we apply the terms agglutinating power, opsonic power or bacteriolytic power. The term antibody is also applied. All three powers may be present together in equal or in varying degree or one or more may be absent. By agglutinating power we mean that which causes evenly distributed organisms to come together and form clumps. By opsonic power we mean that which so alters the resistance of bacteria that the phagocytes ingest them. By bacteriolytic power we mean that which brings about 119 120 PRACTICAL METHODS IN IMMUNITY. disintegration or lysis of the specific organism. The bacterium which causes the disease or which is used in inoculation for the pro- duction of immunity is termed the specific organism. Of the different kinds of immunity only artificial immunity will be considered. This may be obtained in two ways : i . By injecting the bacteria or their products into man or animals and as the result of the activity of the cells of the animal invaded, antibodies are formed which neutralize the toxins of or destroy the specific bac- teria. These antibodies which are supposed to be thrown off (free receptors) or which may remain attached to the cell (sessile receptors) may re- main potential for months or years and so confer a more or less enduring immunity. This is termed active im- munity. 2. When we take the serum of a man or animal immunized actively and inject it with its contained anti- bodies into a second animal or man, we confer an im- munity on the second animal; but as his cells take no active part in the production of the immunity, but are only pas- sive, we term this immunity " passive immunity." If this serum which is introduced in passive immunity only neutralizes the toxic products of the infecting bacteria, we term it antitoxic passive immunity and designate the immune serum as antitoxic serum. If it destroys the organism, we call it antimicrobic serum, and the immunity, antimi- crobic passive immunity. Some immune sera are both antitoxic and antimicrobic. It is well to remember that some organisms produce a toxin which FIG. 44. — Receptors of the first order uniting with toxin. (Journal oj the American Medi- cal Association. 1905. P. 955.) a, Cell receptor; 6, toxin molecule; c, hap- tophore of the toxin molecule; d, toxophore of the toxin molecule; e, haptophore of the cell receptor. ANTITOXIC AND ANTIMICROBIC SERA. 121 is given off while the bacterium is alive; and in other instances the toxin is intracellular and is o ily given off when the bacterium disintegrates; consequently, an antimicrobic serum may cause the liberation of toxin. Diphtheria, tetanus or botulism antisera are instances of antitoxic sera, while practically all others are antimicrobic. There is but one factor to consider in an antitoxic serum and that is the protoplasmic particles which are thrown off from the cell in response to the injury incident to the attack upon the cell by the toxin particles. This free particle in the circulation represents the entire mechanism of antitoxic im- munity. It is capable of uniting with the toxin mole- cule and neutralizing its toxic power, or rather so binding its combining end (haptophore group) that it is incapable of attaching itself to a cell, so that the poisonous end of the toxin (toxophore group) cannot have access to the cell. In antimicrobic sera we have two factors to consider, the first is a protoplasmic par- ticle quite similar to the anti- toxin molecule, but which in itself has no power of injuring its specific bacterium. This particle is generally referred to as the amboceptor or immune body. It is the specific product of the activity of a specific bacterium or foreign cell against the body cells attacked. It with- stands a temperature above 56° C. and of itself is incapable of injuring the bacterium in response to whose attack it was produced. The second factor in the bacteriolysis of the specific bacterium, or the FIG. 45. — Receptors of the second order and of some substance uniting with one of them. (Journal of the American Medical Association. 1905. P. 1113.) c, Cell recepto: of the second order; d, tox- ophore or zymophore group of the receptor; e , haptophore of the receptor; /, Food substance or product of bacterial disintegration uniting with the haptophore of the cell receptor. 122 PRACTICAL METHODS IN IMMUNITY. haemolysis of the specific foreign cell, is something normally present in the serum of every animal, and which is capable of disintegrating a foreign cell or bacterium, provided it can have access to the cell or bacterium through an intermediary amboceptor (hence the ambo- ceptor is sometimes called an intermediary body). This something is called the "complement." It is by some called "alexine," by others cytase (Metchnikoff). The complement cannot act upon and destroy an invading bacterium or cell unless the amboceptor is FIG. 46. — Receptor of the third order, and of some substance uniting with one of them. (Journal 0} the American Medical Association. 1905. P 1369 ) c, Cell receptor of the third order — an amboceptor; e, one of the haptophores of the amboceptor, with which some food substance or product of bacterial disintegra- tion (/) may unite; g, the other haptophore of the amboceptor with which com- plement may unite; k, complement ; h, the haptophore; z, the zymotoxic group of complements. present to make the necessary connection. The complement is destroyed by a temperature of 56° C., so that, if we heat the serum from an immune animal to 56° C., the complement it naturally con- tains is destroyed, and the amboceptor it contains, which is not injured by such a temperature, is incapable of destroying bacteria or cells, unless we replace the complement which has been destroyed by fresh complement. This is done experimentally by adding the serum ACTIVATION OF IMMUNE SERA. I23 of a nonimmunized animal which contains the complement, but no specific immune body (amboceptor) to the heated serum. This is termed "activating," and a serum so treated is said to be "activated." When an immune serum has been heated to 56° C., it is said to have been "inactivated". FIG. 47. — i, Red cells + normal serum. No amboceptor. Nohemolysis. A. com- plement; B, normal red cell. 2. Red cells + immune serum. Complement and amboceptor. Hemolysis. C, complement; D, amboceptor; E, hemolyzed red cell. 3. Red cells + immune serum heated to 56° C. Inactivated. Complement de- strojed. No hemolysis, F, destroyed complement; G, amboceptor; H, red cells. 4. Red cells + heated immune serum + fresh serum. (Activated by contained complement). Hemolysis I, destroyed complement; J, fresh complement ; K, am- boceptor; L, hemolyzed red cell. 5, Diagram showing antitoxin production. a, toxin molecule; &, antitoxin molecule; c, neutralization of toxin by antitoxin. 6. Diagram showing bacteriolysin. d, complement; e, amboceptor; /, bacillus. When we allow a mixture of bacteria or cells to remain in contact with their specific immune serum which has been inactivated, the amboceptors attach themselves to the bacteria or cells, so that now, upon adding normal serum (complement), these bacteria or cells are so pre- 124 PRACTICAL METHODS IN IMMUNITY. pared that the complement can disintegrate them. This experiment is termed " sensitizing" and cells so treated are said to be "sensitized." METHODS FOR OBTAINING IMMUNE SERA. While a convalescent from a disease may be utilized to obtain an antitoxic, agglutinating, opsonic or bacteriolytic serum against the specific bacterium, yet this is more conveniently obtained from an animal which has been immunized against the bacterium or cell in question. The rabbit is the most convenient animal to employ for the production of immune sera where the object is to have at hand a serum for use in diagnosis. Where sera are used on an extensive scale, as in the production of curative sera, larger animals are employed. There are two application of serum diagnosis : i . Where the bacterium is known and the serum is to be diagnosed. 2. Where the serum is known and the bacterium is to be diagnosed. The first is employed by testing the agglutinating or bacteriolytic power of the serum taken from a patient upon pure cultures of the organ- ism which is suspected as the cause of the disease. The Widal test (ag- glutination) is the best instance of this procedure. This method is of practical value in the diagnosis only of typhoid, Malta fever and paratyphoid. In diseases like cholera and bacillary dysentery, the disease has run its course before agglutinating power becomes apparent in the serum. This method, however, may be used to prove that a convalescent has suffered from a suspected disease. Thus, by test- ing the agglutinating power of a serum, one or two weeks after re- covery from a suspicious case of ptomaine poisoning, we may be able to demonstrate that the case in question was cholera. The second method has wider application, and is the one in which we use the sera of animals which have been immunized with known bacteria. Or- ganisms isolated from urine, faeces or blood of patients, or those obtained from water or food supplies may be identified by testing the agglu- tinating, opsonic or bacteriolytic power of known sera against them. This has a wide range of applicability. The testing of the opsonic power of the sera in man or animals immunized against plague, and possibly cerebrospinal meningitis, seems to give more definite informa- METHODS FOR OBTAINING IMMUNE SERA. 125 tion than do agglutination or bacteriolytic tests. With the majority of other organisms, however, the agglutination test is the one almost always preferred. Even in a small laboratory there are no particular difficulties in the way of having on hand rabbits immunized against typhoid, paratyphoid Malta fever, acid producing and nonacid producing strains of dysentery, cholera, etc. Just as we inject men with vaccines prepared from various bacteria in opsonic therapy, so we inject animals to produce sera for diagnosis. We may use either a bouillon culture or the growth on agar slants taken up with salt solution as the inoculating material. This is heated for one hour at 60° C. to kill the bacteria. Where we desire to produce a serum which will disintegrate red blood cells (haemolytic serum), we inject about 4 c.c. of the defibrinated blood of the animal for which we wish to produce a specific serum. Thus for a serum for use in a medicolegal case we would inject the rabbit with human blood. The most convenient way to defibrinate blood is to break a section of glass tubing into fragments, put these fragments into a glass test-tube, sterilize tube and contents in a flame or sterilizer and, when cool, let the blood drop into the test-tube (we may use a Wright's pipette with a rubber bulb to take up the blood from a punc- ture of the finger and eject it into the tube). By shaking, the fibrin collects on the glass fragments, and we have the corpuscular emulsion to inject. Inject about 4 c.c. of the defibrinated blood or i c.c. of the killed bacterial bouillon culture into the peritoneal cavity of the rabbit. The easiest way to inject the rabbit is to hold the animal head down and plunge the needle in the median line into the abdominal cavity, forcing in the contents of the syringe. The intestines gravitate down- ward and by entering the needle below the limits of the bladder we avoid injuring any vital part. It may be more satisfactory to at first inject only about 1/2 c.c., and then if there is very little reaction, as shown by the appetite and spirits of the rabbit, to inject about 4 days latter i c.c. About 4 or 5 injections at intervals of 3 to 5 days will usually produce an immune serum. Some animals do not seem to be capable of producing antibodies, so that it may be necessary to use one or more rabbits before a satisfactory serum is obtained. The most convenient way of obtaining serum for a test is to cut across one 126 PRACTICAL METHODS IN IMMUNITY. of the marginal veins of the rabbit's ear, and collect the blood in a Wright's U-tube. Centrifugalizing, we have the serum ready for use. The immune body and agglutinin in serum remain active for weeks when kept in the refrigerator. The complement and opsonin, however, begin to deteriorate at once and have disappeared by the fifth day. Consequently, for opsonic and bacteriolytic and haemolytic experiments, fresh serum — 12 to 24 hours — must be used, or it may be activated. AGGLUTINATION TESTS. There are two methods of testing the agglutinating powers of a serum — the microscopical and the macroscopical or sedimentation method. i. For the microscopical method draw up serum to the mark .5 of the white pipette. Then draw up salt solution to the mark 1 1 . This when mixed gives a dilution of i to 20. One loopful of the diluted serum and one loopful of a bouillon culture or salt solution suspen- sion of the organism to be tested gives a dilution of i to 40. One loop- ful of the 1-20 diluted serum and 3 loopfuls of the bacterial suspension give a dilution of 1-80. These two dilutions answer in ordinary diag- nostic tests. The red pipette with a i-ioo or 1-200 dilution may be used where dilutions approaching i-iooo are desired. Having mixed the diluted serum and the bacterial suspension on a cover-glass, we invert it over a vaselined concave slide and examine with a high power, a dry objective (1/6 in.). It is simpler to make a ring of vaselin to fit the cover-glass and make the mixture of diluted serum and culture in the center of this ring or square. Then apply the cover-glass, press it down on the vaselin ring and examine as with the ordinary hanging drop. In making dilutions it is preferable to use salt solution, as the phenomenon of agglutination requires the presence of salts. Ordi- narily, 30 minutes is a sufficient time to wait before reporting the absence of agglutination. Agglutination is more rapid at body tempera- ture than at room temperature. In reporting agglutination, always give time and dilution. It is absolutely necessary that a control prepa- ration be prepared in every instance; that is, one with the bacterial culture alone or with a normal serum of the same dilution as the lowest used. Some normal sera will agglutinate in i to 10 dilution, and group MACROSCOPICAL AGGLUTINATION. 127 agglutinations (as paratyphoid with typhoid serum) may occur in i to 40 or possibly higher. It is very unusual for sera to agglutinate any other bacteria than its specific one in dilutions as high as 1-80. 2. For the macroscopical or sedimentation test, take a series of small test-tubes (3/8 x 3 in.) and deposit i c.c. of salt solution in each of the series. Now, having taken an empty test-tube, drop 4 drops of serum in it and then add 1 2 drops of salt solution. This approximately gives i c.c. of a 1-4 dilution of the serum. With a rubber-bulb capillary pipette, which has been graduated to hold 16 drops or i c.c. draw up the contents of the tube containing the i to 4 serum and add it to the next tube containing i c.c. of salt solution. This gives a dilution of i to 8. Now mix thoroughly by drawing up and forcing out with the bulb pipette, and then withdraw i c.c and add to the next tube containing i c.c. of salt solution. This gives a dilution of i to 16. Having mixed as before, again withdraw i c.c. of the mixture and add it to the i c.c. in the next tube. We now have a dilution of i to 32. Again withdrawing i c.c. and adding it to the fourth tube containing i c.c. of salt solution we have a dilution of i to 64. In tube i there is i c.c. of a dilution of the serum of i to 8; in tube 2, there is i c.c. of a dilution of i to 16; in tube 3, of i to 32. Tube 4 contains 2 c.c. of i to 64. Now adding i c.c. of a cul- ture of typhoid or any other organism, we have the dilution of the serum in each tube doubled. Tube i now contains a serum in dilution of i to 16, acting on the bacteria; tube 2 of a i to 32; tube 3 of i to 64. Now place these tubes in the incubator and after 2-5 hours or over- night, we examine for the clearing up of the supernatant fluid. If the serum in a certain dilution agglutinates, the clumps gravitate to the bottom and the upper part becomes clear. If so desired, these dilutions may be carried on to i to several hundred in the same way. It is safer to work with dead cultures instead of living ones. To prepare, in- oculate a flask of bouillon containing about 150 c.c. with typhoid or any other culture. Allow to grow for 1 8 to 24 hours and then add i c.c. of formalin. One percent of formalin is frequently used to kill the cul- tures. At the end of 24 hours the sterile cultures may be used as with the live cultures.* * A very convenient method in general use in Germany is the following: Make dilutions of serum in ordinary test-tubes (J by 6 inches) as described for the small 128 PRACTICAL METHODS IN IMMUNITY. H^MOLYTIC EXPERIMENTS. Take the blood of the animal that has been used to immunize the rabbit and receive it in a graduated centrifuge tube containing salt solution which has had i% of sodium citrate added to it. This prevents the coagulation of the blood. After mixing, centrifuge and pipette off supernatant fluid. Note the graduation reached by the sediment of red cells and make up with salt solution to 20 times its volume. If the cells reach the 1/2 c.c. mark, add 10 c.c. of salt solu- tion. This gives a 5% emulsion of red cells — the percentage usually used in hemolytic experiments. To carry out the test, simply add i c.c. of this 5% mixture of red cells to each of the series of tubes containing diluted serum, as with the macroscopic agglutination tests. Place in the incubator for 2-5 hours. The red cells settle to the bottom, and tubes showing haemolysis have a light reddish to rich haemoglobin color. Tubes not showing haemolysis remain white. BACTERIOLYTIC EXPERIMENTS. These may be carried out in the peritoneal cavity of a guinea pig, injecting mixtures of immune sera and the bacterial culture. Upon withdrawing, after 15-60 minutes, the bacteria are granular and disintegrated. This is the well-known Pfeiffer's phenomenon, and was once considered the most important test for cholera. See Cholera. There have been several accidents (death of Orgel) with this test, and it is not practicable except in a well equipped laboratory. Instead of a guinea-pig, we may simply take a fresh serum of known dilution, and mix it with an equal quantity of the bacterial emulsion in a capillary pipette; sealing off the end of the pipette, we incubate for 15 minutes. Then filing off the end we mix the culture thoroughly on a test-tubes. Then take a loopful (2 mg.) of culture from an 18 to 24 hour old agar culture and emulsify it thoroughly in the dilution in the first test-tube — repeat the process in the second tube and so on. This procedure is much oafer than when live cultures are added with a pipette. Again, the dilution is unchanged by this addition whereas it is doubled when an equal volume of culture is added to the diluted scrum. A control should always be made in normal salt solution. After incubating, observe flocculent precipitates (agglutination) by tilting the fluid in the tubes to form a thin layer and to obtain the most advantageous light and look for a fine curdy precipitate (agglutination) or a uniformly turbid emulsion (negative reaction) . COLON BACILLUS IN WATER ANALYSIS. 113 number of organisms developing at 38° C. at all approximates the num- ber developing at 20° C., there is a strong suspicion that sewage or- ganisms may be present. Normal waters give proportions of i to 25 or i to 50, while in sewage contaminated waters the proportion may be as i to 4 or less. In addition, the appearance of pink colonies on the lactose litmus agar is of great assistance in judging of a water. Both sewage strepto- cocci and the colon bacillus give pink colonies — those of the streptococci are smaller and more vermilion in color. Microscopic examination will differentiate the cocci from the bacilli. It is well to bear in mind that the pink colonies after 24 hours may turn blue in 48 hours from the development of ammonia and amines. Consequently the lactose litmus agar plates should be studied after 24 hours. A good water supply will rarely show a pink colony, while in a sewage contaminated one the pink colonies will probably predominate. The diagnostic characteristics considered important by the Ameri- can authorities in reporting the colon bacillus are: 1. Typical morphology, nonsporing bacillus, relatively small and often quite thick. 2. Motility in young broth cultures. (This is at times unsatisfac- tory, as some strains of the colon bacillus do not show it even in young bouillon cultures.) 3. Gas formula in dextrose broth. Of about 50% of gas produced, 1/3 should be absorbed by a 2% solution of sodium hydrate (CO2). The remaining gas is hydrogen. (Later views indicate that the gas formula is exceedingly variable and should not be depended upon. To carry out this test one fills the bulb of a fermentation tube with the caustic soda solution then, holding the thumb over the opening or with a rubber stopper, the bouillon culture and the soda solution are mixed by tilting the fermentation tube to and fro. The total amount of gas is first recorded and then that remaining after the CO2 has been absorbed is reported as hydrogen.) 4. Nonliquefaction of gelatin. 5. Fermentation of lactose with gas production. 6. Indol production. 7. Reduction of nitrates to nitrites. 8 114 BACTERIOLOGY OF WATER, AIR, MILK, ETC NOTE. — The reduction of neutral red with a greenish-yellow fluorescence is very striking and has been suggested as a test for the colon bacillus. Many other organisms, especially those of the hog cholera group, have this power. It is convenient, however, to color glucose bouillon with about i% of a 1/2% solution of neutral red. Isolation of the Typhoid Bacillus from Water. This is probably the most discouraging procedure which can be taken up in a laboratory. Only the most recent reports of such isola- tion from water supplies, which have been verified by immunity reac- tions, can be accepted and of these the number of instances is exceed- ingly small. Owing to the long period of incubation, the typhoid organisms may have died out before the outbreak of an epidemic suggests the examination of the water supply. There have been various methods proposed for the detection of the B. typhosus in water. A method which would offer about as reasonable a chance of success as any other would be to pass 2 or 3 liters of the water through a Berkefeld filter; then to take up in a small quantity of water all the bacteria held back by the filter. Then plate out on lactose litmus agar and examine colonies which do not show any pink coloration. The dysentery bacillus has about the same cultural characteristics as the typhoid one, so that it is important to note motility. If from such a colony you obtain an organism giving the cultural characteristics of B. typhosus, carry out agglutination and preferably bacteriolytic tests as well. Some strains of typhoid, espe- cially when recently isolated from the body, do not showr agglutination. The Conradi Drigalski, the malachite-green and various caffeine containing plating media have been highly recommended. Isolation of the Cholera Spirillum from Water. The method proposed by Koch in 1893 does not seem to have been improved upon by later investigators. To 100 c.c. of the suspected water add i% of peptone and i% of salt. Incubate at 38° C., and at intervals of 8, 12 and 18 hours examine microscopically loopfuls taken from the surface of the liquid in the flask. So soon as comma-shape BACTERIOLOGICAL EXAMINATION OF MILK. 115 organisms are observed, plate out on agar. The colonies showing morphologically characteristic organisms should be tested as to ag- glutination and bacteriolysis. Inasmuch as the true cholera spirillum shows a marked cholera-red reaction it is well to inoculate a tube of peptone solution from such a colony and add a drop of concentrated sulphuric acid after incubating for 18 hours. The rose-pink colora- tion is given by the cholera spirillum with the acid alone — the nitroso factor in the reaction being produced by the organism. BACTERIOLOGICAL EXAMINATION OF MILK. A bacterial milk count is of comparatively little value as showing whether a milk is dangerous or not. As a matter of fact, a milk which contains several million of bacteria per c.c. might be less dangerous than one containing only a few thousand, especially if in the latter there were numerous liquefiers and gas producers present. There is, however, one point of importance in connection with the quantitative estimation of bacteria in milk, and that is the fact that in order to keep the development of the bacteria within the limits of 10,000 to 50,000 per c.c., it is necessary that the requirements of cleanliness in milking and the rapid cooling of the milk after obtaining it and the keeping of the temperature below 50° C. be rigidly observed. If a milk has a high count it shows some error in the handling of the milk. In making a quantitative bacteriological examination, the principle is the same as with water. Make a known dilution of the milk with sterile water; add definite quantities of this diluted milk to tubes of melted agar or gelatin and pour into plates. The diluted milk may also be delivered in the center of the plate and the melted agar or gelatin poured directly on it, mixing thoroughly. Always shake the bottle well before taking sample. Example: Added i c.c. of milk to 199 c.c. of sterile water in a large flask (500 to 1000 c.c.). After shaking thoroughly, take i c.c. of this i : 200 dilution and add it to 99 c.c. of sterile water. Shak- ing thoroughly, we have a dilution of i : 20,000. Of this we added .5 c.c. to a tube of gelatin or agar. After incubation the plate showed Il6 BACTERIOLOGY OF WATER, AIR, MILK; ETC. 75 colonies. Therefore the milk contained in each c.c. 75 x 2 x 20,000 (dilution) = 3,000,000 — the number of bacteria in each c.c. of milk. Lactose litmus gelatin or agar is to be preferred in milk-work, as the normal lactic acid bacteria produce reddish colonies which are very striking. A standard easily attained for high-grade, certified milk would be 5,000 to 10,000 per c.c. In the qualitative examination of milk, many dairies employ the fermentation tube, any organism producing gas being considered undesirable. Again liquefying organisms, as shown by the presence of such bacteria in the gelatin plates, is evidence of probable contami- nation by fecal bacteria. A question which seems difficult to decide is as to the general nature of the so-called normal lactic acid bacteria of milk. Some describe them as very short, broad bacilli with very small colonies, fermenting lactose with the formation of lactic acid. Others consider that the streptococci are the organisms which are concerned with the normal fermentative changes. In examining specimens of milk considered the best on the market, I have repeatedly found the small red colonies on lactose litmus agar to be streptococci. In connection with the organisms present in the tablets used for treat- ing milk to produce lactic acid for the treatment of intestinal disorders, and considered to be normal lactic acid bacteria, I have found both streptococci and bacilli. These have all agreed, however, in not pro- ducing gas in either lactose or glucose fermentation tubes. Another source of information as to the quality of a milk may be derived from a study of the number of leukocytes or pus cells con- tained in i c.c. of the milk. The Doane-Buckley method is probably the most accurate. In this you throw down the cellular contents of 10 c.c. of milk in a cen- trifuge revolving about 1000 times a minute for ten to twenty minutes. Then remove supernatant milk and add 0.5 c.c. of Toisson's solution to the sediment. You thus have the leukocytes of 10 c.c. contained in 0.5 c.c. (Concentrated twenty times.) Make a haemacytometer pre- paration as for blood and find the average number of cells for each square millimeter. Then multiply this by 10 to get the number of cells in a cubic millimeter. As a cubic millimeter is one thousand times smaller than a cubic centimeter, you multiply the number per BACTERIOLOGICAL EXAMINATION OF AIR. 117 cubic millimeter by one thousand. Then, as the milk was concentrated twenty times, you divide by 20. (If it were diluted twenty times, you would multiply by 20.) Example: Found an average of 50 cells per square millimeter. This would make 500 per cubic millimeter, and 500,000 per c.c.; . then 500,000 divided by 20 would give 25,000. |There is no agreement as to a standard for allowable leukocytes. Even in apparently healthy animals they may exceed 100,000 per c.c. Doane has suggested 500,000 per c.c. as a preferable limit. The smear methods for determining the number of leukocytes present do not compare in accuracy with the volumetric ones. BACTERIOLOGICAL EXAMINATION OF AIR. In Paris a cubic meter of. air was found to contain the following number of organisms: Suburbs. — Winter, 145 moulds, 170 bacteria. Summer, 245 moulds, 345 bacteria. City Hall. — Winter, 1345 moulds, 4305 bacteria. Summer, 2500 moulds, 9845 bacteria. Air of hospitals, especially after sweeping, may contain 50,000 bacteria per cubic meter. There does not seem to be any particular relation between the amount of carbon dioxide in air and the bacterial content. Petri's Rough Method.— Exposure of a lactose litmus agar plate (capacity 100 sq. cm.) for five minutes will give the number of organ- isms present in ten liters of air. Multiply by 100 for one cubic meter. The two groups of organisms usually found in air are (i) bacteria and (2) moulds. Moulds (spores) may be carried by currents of air; bacteria, however, are generally carried about by particles of dust or finely divided liquids (spray). On the lactose litmus agar plate staphylococci and streptococci show as bright red colonies. Sedgwick Tucker Sterile Granulated Sugar Method. — Sterilize aerobioscope and introduce granulated sugar on support. Again sterilize (not over 120° C. in dry-air sterilizer). Allow a given quan- tity of air to pass through; then shake the sugar into wide part of Il8 BACTERIOLOGY OF WATER, AIR, MILK, ETC. aerobioscope. Now pour in 10 or 15 c.c. of melted gelatin (40° C.) to dissolve sugar. Roll tubes as for Esmarch roll cultures, and incubate at room temperature. To draw air through the aerobioscope, connect the small end with a piece of rubber tubing which is attached to a tube in the stopper of an aspirating bottle. Having poured a definite quantity of water into the aspirating bottle, allow the water to run out.. The same quantity of air will be drawn through the sugar of the aerobioscope as the amount of water passing out of the aspirating bottle. The bacteria and moulds are caught by the sugar. Example. — Passed 10 liters of air through the aerobioscope. The bacteria in this quantity of air showed 75 colonies when incubated FIG. 43. — Sedgwick-Tucker aerobioscope. (Williams.) at 20° C. The unit being one cubic meter or one thousand liters, we have only obtained the bacteria of one hundredth of the unit. Hence multiplying 75 by 100 gives 7,500 bacteria as present in one cubic meter of the air examined. In comparing the results with the aerobioscope with those obtained by exposing a plate as in Petri's method for ten instead of five minutes, it was found that the latter was sufficiently in accord to make it a satisfactory approximate quantitative method. The simplicity and ease of access of the colonies developing in it make it preferable when the air of operating-rooms or hospital wards is to be examined. CHAPTER XII. PRACTICAL METHODS IN IMMUNITY. THAT which prevents the gaining of a foothold by disease organisms in the animal body or which neutralizes their harmful products or destroys the parasites is termed immunity. In the main, the question of immunity hinges on the powers of resistance of the human body and the aggressiveness or virulence of the invading organism. It must always be kept in mind that immunity is only relative; thus the fowl, which is practically immune to tetanus, may be made to suc- cumb by reducing its resistance by refrigeration or by increasing the amount of poison introduced. The insusceptibility which the fowl has to tetanus or which man has to many diseases of animals is best termed inherent immunity, and is at present only a subject of theo- retical interest. When immunity to a given disease is obtained as a result of an attack of the disease in question or by laboratory methods of inoculation, this is termed properly an acquired immunity, and in the former case is a naturally acquired immunity or " natural im- munity" and in the second is an artificially acquired immunity or " artificial immunity." As a result of an attack of a disease or in response to the stimulus of the injection of the organism or its products, we have developed in the man so injected certain specific antagonistic properties to that organism, which are usually demonstrable in the blood serum or other body fluids, and to which we apply the terms agglutinating power, opsonic power or bacteriolytic power. The term antibody is also applied. All three powers may be present together in equal or in varying degree or one or more may be absent. By agglutinating power we mean that which causes evenly distributed organisms to come together and form clumps. By opsonic power we mean that which so alters the resistance of bacteria that the phagocytes ingest them. By bacteriolytic power we mean that which brings about 119 120 PRACTICAL METHODS IN IMMUNITY. disintegration or lysis of the specific organism. The bacterium which causes the disease or which is used in inoculation for the pro- duction of immunity is termed the specific organism. Of the different kinds of immunity only artificial immunity will be considered. This may be obtained in two ways : i . By injecting the bacteria or their products into man or animals and as the result of the activity of the cells of the animal invaded, antibodies are formed which neutralize the toxins of or destroy the specific bac- teria. These antibodies which are supposed to be thrown off (free receptors) or which may remain attached to the cell (sessile receptors) may re- main potential for months or years and so confer a more or less enduring immunity. This is termed active im- munity. 2. When we take the serum of a man or animal immunized actively and inject it with its contained anti- bodies into a second animal or man, we confer an im- munity on the second animal; but as his cells take no active part in the production of the immunity, but are only pas- sive, we term this immunity "passive immunity." If this serum which is introduced in passive immunity only neutralizes the toxic products of the infecting bacteria, we term it antitoxic passive immunity and designate the immune serum as antitoxic serum. If it destroys the organism, we call it antimicrobic serum, and the immunity, antimi- crobic passive immunity. Some immune sera are both antitoxic and antimicrobic. It is well to remember that some organisms produce a toxin which FIG. 44. — Receptors of the first order uniting with toxin. (Journal of the American Medi- cal Association. 1905. P. 955.) a, Cell receptor; 6; toxin molecule; c, hap- tophore of the toxin molecule; d, toxophore of the toxin molecule; e, haptophore of the cell receptor. ANTITOXIC AND ANTIMICROBIC SERA. 121 is given off while the bacterium is alive; and in other instances the toxin is intracellular and is oily given off when the bacterium disintegrates; consequently, an antimicrobic serum may cause the liberation of toxin. Diphtheria, tetanus or botulism antisera are instances of antitoxic sera, while practically all others are antimicrobic. There is but one factor to consider in an antitoxic serum and that is the protoplasmic particles which are thrown off from the cell in response to the injury incident to the attack upon the cell by the toxin particles. This free particle in the circulation represents the entire mechanism of antitoxic im- munity. It is capable of uniting with the toxin mole- cule and neutralizing ifs toxic power, or rather so binding its combining end (haptophore group) that it is incapable of attaching itself to a cell, so that the poisonous end of the toxin (toxophore group) cannot have access to the cell. In antimicrobic sera we have two factors to consider, the first is a protoplasmic par- ticle quite similar to the anti- toxin molecule, but which in itself has no power of injuring its specific bacterium. This particle is generally referred to as the amboceptor or immune body. It is the specific product of the activity of a specific bacterium or foreign cell against the body cells attacked. It with- stands a temperature above 56° C. and of itself is incapable of injuring the bacterium in response to whose attack it was produced. The second factor in the bacteriolysis of the specific bacterium, or the FIG. 45. — Receptors of the second order and of some substance uniting with one of them. (Journal oj the American Medical Association. 1905. P. 1113.) c, Cell recepto; of the second order; d, tox- ophore or zymophore group of the receptor; e, haptophore of the receptor; /, Food substance or product of bacterial disintegration uniting with the haptophore of the cell receptor. 122 PRACTICAL METHODS IN IMMUNITY. haemolysis of the specific foreign cell, is something normally present in the serum of every animal, and which is capable of disintegrating a foreign cell or bacterium, provided it can have access to the cell or bacterium through an intermediary amboceptor (hence the ambo- ceptor is sometimes called an intermediary body). This something is called the "complement." It is by some called "alexine," by others cytase (MetchnikorT). The complement cannot act upon and destroy an invading bacterium or cell unless the amboceptor is FIG. 46. — Receptor of the third order, and of some substance uniting with one of them. (Journal 0} the American Medical Association. 1905. P 1369 ) c, Cell receptor of the third order — an amboceptor; e, one of the haptophores of the amboceptor, with which some food substance or product of bacterial disintegra- tion (/) may unite; g; the other haptophore of the amboceptor with which com- plement may unite; k, complement ; h, the haptophore; z, the zymotoxic group of complements. present to make the necessary connection. The complement is destroyed by a temperature of 56° C., so that, if we heat the serum from an immune animal to 56° C., the complement it naturally con- tains is destroyed, and the amboceptor it contains, which is not injured by such a temperature, is incapable of destroying bacteria or cells, unless we replace the complement which has been destroyed by fresh complement. This is done experimentally by adding the serum ACTIVATION OF IMMUNE SERA. I23 of a non immunized animal which contains the complement, but no specific immune body (amboceptor) to the heated serum. This is termed "activating," and a serum so treated is said to be "activated." When an immune serum has been heated to 56° C., it is said to have been "inactivated". FIG. 47. — i, Red cells + normal serum. No amboceptor. Nohemolysis. A. com- plement; B, normal red eel!. 2. Red cells + immune serum. Complement and amboceptor. Hemolysis. C, complement; D, amboceptor; E, hemolyzed red cell. 3. Red cells + immune serum heated to 56° C. Inactivated. Complement de- stroyed. No hemolysis. F, destroyed complement; G, amboceptor; H, red cells. 4. Red cells + heated immune serum + fresh serum. (Activated by contained complement). Hemolysis. I, destroyed complement; J, fresh complement; K, am- boceptor; L, hemolyzed red cell. 5, Diagram showing antitoxin production. a, toxin molecule; 6, antitoxin molecule; c, neutralization of toxin by antitoxin. 6. Diagram showing bacteriolysin. d, complement; e, amboceptor; /, bacillus. When we allow a mixture of bacteria or cells to remain in contact with their specific immune serum which has been inactivated, the amboceptors attach themselves to the bacteria or cells, so that now, upon adding normal serum (complement), these bacteria or cells are so pre- 124 PRACTICAL METHODS IN IMMUNITY. pared that the complement can disintegrate them. This experiment is termed "sensitizing" and cells so treated are said to be "sensitized." METHODS FOR OBTAINING IMMUNE SERA. While a convalescent from a disease may be utilized to obtain an antitoxic, agglutinating, opsonic or bacteriolytic serum against the specific bacterium, yet this is more conveniently obtained from an animal which has been immunized against the bacterium or cell in question. The rabbit is the most convenient animal to employ for the production of immune sera where the object is to have at hand a serum for use in diagnosis. Where sera are used on an extensive scale, as in the production of curative sera, larger animals are employed. There are two application of serum diagnosis: i. Where the bacterium is known and the serum is to be diagnosed. 2. Where the serum is known and the bacterium is to be diagnosed. The first is employed by testing the agglutinating or bacteriolytic power of the serum taken from a patient upon pure cultures of the organ- ism which is suspected as the cause of the disease. The Widal test (ag- glutination) is the best instance of this procedure. This method is of practical value in the diagnosis only of typhoid, Malta fever and paratyphoid. In diseases like cholera and bacillary dysentery, the disease has run its course before agglutinating power becomes apparent in the serum. This method, however, may be used to prove that a convalescent has suffered from a suspected disease. Thus, by test- ing the agglutinating power of a serum, one or two weeks after re- covery from a suspicious case of ptomaine poisoning, we may be able to demonstrate that the case in question was cholera. The second method has wider application, and is the one in which we use the sera of animals which have been immunized with known bacteria. Or- ganisms isolated from urine, faeces or blood of patients, or those obtained from water or food supplies may be identified by testing the agglu- tinating, opsonic or bacteriolytic power of known sera against them. This has a wide range of applicability. The testing of the opsonic power of the sera in man or animals immunized against plague, and possibly cerebrospinal meningitis, seems to give more definite informa- METHODS FOR OBTAINING IMMUNE SERA. 125 tion than do agglutination or bacteriolytic tests. With the majority of other organisms, however, the agglutination test is the one almost always preferred. Even in a small laboratory there are no particular difficulties in the way of having on hand rabbits immunized against typhoid, paratyphoid Malta fever, acid producing and nonacid producing strains of dysentery, cholera, etc. Just as we inject men with vaccines prepared from various bacteria in opsonic therapy, so we inject animals to produce sera for diagnosis. We may use either a bouillon culture or the growth on agar. slants taken up with salt solution as the inoculating material. This is heated for one hour at 60° C. to kill the bacteria. Where we desire to produce a serum which will disintegrate red blood cells (haemolytic serum), we inject about 4 c.c. of the defibrinated blood of the animal for which we wish to produce a specific serum. Thus for a serum for use in a medicolegal case we would inject the rabbit with human blood. The most convenient way to defibrinate blood is to break a section of glass tubing into fragments, put these fragments into a glass test-tube, sterilize tube and contents in a flame or sterilizer and, when cool, let the blood drop into the test-tube (we may use a Wright's pipette with a rubber bulb to take up the blood from a punc- ture of the finger and eject it into the tube). By shaking, the fibrin collects on the glass fragments, and we have the corpuscular emulsion to inject. Inject about 4 c.c. of the defibrinated blood or i c.c. of the killed bacterial bouillon culture into the peritoneal cavity of the rabbit. The easiest wray to inject the rabbit is to hold the animal head down and plunge the needle in the median line into the abdominal cavity, forcing in the contents of the syringe. The intestines gravitate down- ward and by entering the needle below the limits of the bladder we avoid injuring any vital part. It may be more satisfactory to at first inject only about 1/2 c.c., and then if there is very little reaction, as shown by the appetite and spirits of the rabbit, to inject about 4 days later i c.c. About 4 or 5 injections at intervals of 3 to 5 days will usually produce an immune serum. Some animals do not seem to be capable of producing antibodies, so that it may be necessary to use one or more rabbits before a satisfactory serum is obtained. The most convenient way of obtaining serum for a test is to cut across one 126 PRACTICAL METHODS IN IMMUNITY. of the marginal veins of the rabbit's ear, and collect the blood in a Wright's U-tube. Centrifugalizing, we have the serum ready for use. The immune body and agglutinin in serum remain active for weeks when kept in the refrigerator. The complement and opsonin, however, begin to deteriorate at once and have disappeared by the fifth day. Consequently, for opsonic and bacteriolytic and haemolytic experiments, fresh serum — 12 to 24 hours — must be used, or it may be activated. AGGLUTINATION TESTS. There are two methods of testing the agglutinating powers of a serum — the microscopical and the macroscopical or sedimentation method. i. For the microscopical method draw up serum to the mark .5 of the white pipette. Then draw up salt solution to the mark n. This when mixed gives a dilution of i to 20. One loopful of the diluted serum and one loopful of a bouillon culture or salt solution suspen- sion of the organism to be tested gives a dilution of i to 40. One loop- ful of the 1-20 diluted serum and 3 loopfuls of the bacterial suspension give a dilution of 1-80. These two dilutions answer in ordinary diag- nostic tests. The red pipette with a i-ioo or 1-200 dilution may be used where dilutions approaching i-iooo are desired. Having mixed the diluted serum and the bacterial suspension on a cover-glass, we invert it over a vaselined concave slide and examine with a high power, a dry objective (1/6 in.). It is simpler to make a ring of vaselin to fit the cover-glass and make the mixture of diluted serum and culture in the center of this ring or square. Then apply the cover-glass, press it down on the vaselin ring and examine as with the ordinary hanging drop. In making dilutions it is preferable to use salt solution, as the phenomenon of agglutination requires the presence of salts. Ordi- narily, 30 minutes is a sufficient time to wait before reporting the absence of agglutination. Agglutination is more rapid at body tempera- ture than at room temperature. In reporting agglutination, always give time and dilution. It is absolutely necessary that a control prepa- ration be prepared in every instance; that is, one with the bacterial culture alone or with a normal serum of the same dilution as the lowest used. Some normal sera will agglutinate in i to 10 dilution, and group MACROSCOPICAL AGGLUTINATION. 127 agglutinations (as paratyphoid with typhoid serum) may occur in i to 40 or possibly higher. It is very unusual for sera to agglutinate any other bacteria than its specific one in dilutions as high as 1-80. 2. For the macroscopical or sedimentation test, take a series of small test-tubes (3/8 x 3 in.) and deposit i c.c. of salt solution in each of the series. Now, having taken an empty test-tube, drop 4 drops of serum in it and then add 1 2 drops of salt solution. This approximately gives i c.c. of a 1-4 dilution of the serum. With a rubber-bulb capillary pipette, which has been graduated to hold 16 drops or i c.c. draw up the contents of the tube containing the i to 4 serum and add it to the next tube containing i c.c. of salt solution. This gives a dilution of i to 8. Now mix thoroughly by drawing up and forcing out with the bulb pipette, and then withdraw i c.c and add to the next tube containing i c.c. of salt solution. This gives a dilution of i to 16. Having mixed as before, again withdraw i c.c. of the mixture and add it to the i c.c. in the next tube. We now have a dilution of i to 32. Again withdrawing i c.c. and adding it to the fourth tube containing i c.c. of salt solution we have a dilution of i to 64. In tube i there is i c.c. of a dilution of the serum of i to 8; in tube 2, there is i c.c. of a dilution of i to 16; in tube 3, of i to 32. Tube 4 contains 2 c.c. of i to 64. Now adding i c.c. of a cul- ture of typhoid or any other organism, we have the dilution of the serum in each tube doubled. Tube i now contains a serum in dilution of i to 1 6, acting on the bacteria; tube 2 of a i to 32; tube 3 of i to 64. Now place these tubes in the incubator and after 2-5 hours or over- night, we examine for the clearing up of the supernatant fluid. If the serum in a certain dilution agglutinates, the clumps gravitate to the bottom and the upper part becomes clear. If so desired, these dilutions may be carried on to i to several hundred in the same way. It is safer to work with dead cultures instead of living ones. To prepare, in- oculate a flask of bouillon containing about 150 c.c. writh typhoid or any other culture. Allow to grow for 18 to 24 hours and then add i c.c. of formalin. One percent of formalin is frequently used to kill the cul- tures. At the end of 24 hours the sterile cultures may be used as with the live cultures.* *A very convenient method in general use in Germany is the following: Make dilutions of serum in ordinary test-tubes (J by 6 inches) as described for the small 128 PRACTICAL METHODS IN IMMUNITY. H^MOLYTIC EXPERIMENTS. Take the blood of the animal that has been used to immunize the rabbit and receive it in a graduated centrifuge tube containing salt solution which has had i% of sodium citrate added to it. This prevents the coagulation of the blood. After mixing, centrifuge and pipette off supernatant fluid. Note the graduation reached by the sediment of red cells and make up with salt solution to 20 times its volume. If the cells reach the 1/2 c.c. mark, add 10 c.c. of salt solu- tion. This gives a 5% emulsion of red cells — the percentage usually used in hemolytic experiments. To carry out the test, simply add i c.c. of this 5% mixture of red cells to each of the series of tubes containing diluted serum, as with the macroscopic agglutination tests. Place in the incubator for 2-5 hours. The red cells settle to the bottom, and tubes showing haemolysis have a light reddish to rich haemoglobin color. Tubes not showing haemolysis remain white. BACTERIOLYTIC EXPERIMENTS. These may be carried out in the peritoneal cavity of a guinea-pig, injecting mixtures of immune sera and the bacterial culture. Upon withdrawing, after 15-60 minutes, the bacteria are granular and disintegrated. This is the well-known Pfeiffer's phenomenon, and was once considered the most important test for cholera. See Cholera. There have been several accidents (death of Orgel) with this test, and it is not practicable except in a well- equipped laboratory. Instead of a guinea-pig, we may simply take a fresh serum of known dilution, and mix it with an equal quantity of the bacterial emulsion in a capillary pipette; sealing off the end of the pipette, we incubate for 15 minutes. Then filing off the end we mix the culture thoroughly on a test-tubes. Then take a loopful (2 mg.) of culture from an 18 to 24 hour old agar culture and emulsify it thoroughly in the dilution in the first test-tube — repeat the process in the second tube and so on. This procedure is much bafer than when live cultures are added with a pipelte. Again, the dilution is unchanged by this addition whereas it is doubled when an equal volume of culture is added to the diluted serum. A control should always be made in normal salt solution. After incubating, observe flocculent precipitates (agglutination) by tilting the fluid in the tubes to form a thin layer and to obtain the most advantageous light and look for a fine curdy precipitate (agglutination) or a uniformly turbid emulsion (negative reaction). IMMUNITY EXPERIMENTS. I2Q sterile slide; deposit a small drop fora hanging-drop preparation and draw up the remaining mixture into the same tube and again incubate. The time and dilution with which the culture becomes nonmotile and granular (bacteriolytic disintegration) should be recorded. Controls with normal serum are always necessary. DEVIATION OF THE COMPLEMENT. It has been found that if there is not sufficient immune body in a mixture of normal serum, containing abundant complement, and bacterial emulsion, only a portion of the bacteria present will be destroyed. Increasing the amount of immune body with a constant quantity of normal serum, we reach a point where all the bacteria are destroyed. Now, if we continue to increase beyond this point the addition of immune serum, the destruction of the bacteria ceases, and the cultures will again contain myriads of living bacteria. To carry out the test, make a series of tubes containing mixtures of bacteria with the same quantity in each of normal serum. Thus, each tube contains 1/2 c.c. of bacterial emulsion and 1/2 c.c. of i-io normal serum. Now inactivate a tube of i-ioo immune serum and to each of the tubes of normal serum and bacterial emulsion add increasing drops of the inactivated i-ioo immune serum. Thus, i drop to No. i tube; 2 drops to No. 2 tube and so on. After incubating for 2 hours, we take a pipette and plate out a fraction of a drop in an agar plate. The limit at which bacteriolysis is complete is showrn by there being an absence of colonies. Beyond or below that point colonies are more or less abundant. The explanation of this phenomenon of deviation or deflection of the complement is that where we have an excess of amboceptors for available receptors on the bacterial cells, only a portion of the ambo- ceptors can attach themselves to their specific bacteria. The free amboceptors, not being able to form a union with the bacterial cell receptors (for which they have a greater affinity), combine with the complement present. Unless the complement be in excess, there will be no free complement left to join onto the amboceptors attached to the bacterial cells, and consequently bacteriolysis does not take place and the plate cultures show an abundance of colonies. 9 130 PRACTICAL METHODS IN IMMUNITY. FIXATION OR ABSORPTION OF THE COMPLEMENT. One of the controversies in connection with the nature of the complement is that regarding the question of the unity of complements or whether there exist different kinds of complements for different amboceptors (unity and multiplicity of complement). To prove that a single complement will act with varying amboceptors, Bordet and Gengou showed that the same complement would activate both haemolytic and bacteriolytic immune bodies. If to a mixture of typhoid bacteria and inactivated typhoid immune serum some guinea pig serum is added and the mixture be allowed to remain at 37° C. for 2 hours, and then sensitized red cells be added and the mix- ture again be placed in the incubator for 2 hours, no haemolysis will be found to have occurred, because the bacteria have absorbed all the guinea-pig complement through the intervening typhoid amboceptors, and there is no complement left to haemolyze the red cells through the specific blood-cell amboceptors. If, instead of immune typhoid serum, the serum of a normal person had been used, there would have been no amboceptors to unite the complement to the bacterial cells. The complement would then be at hand to unite with the sensitized red cells subsequently added and bring about their haemolysis, as shown by the ruby color of the supernatant fluid. This phenomenon of Bordet and Gengou has been utilized by Wasserman for the diagnosis of diseases where cultures are not applicable. It is in the diagnosis of syphilis that it is best known. It at present being impossible to obtain cultures of Treponema pallidum, we use emulsions of the liver of a syphilitic foetus, which have been filtered so as to be clear, instead of a culture. The syphilitic liver, as can be observed by staining according to Levaditi's method, is packed with spirochaetes. For the immune bodies we take the serum of the patient, or if a case of locomotor ataxia or general paresis, the cerebrospinal fluid. This is heated to 56° C. to destroy complement (inactivation). For the complement we use normal guinea-pig serum in a dilution of i-io. For the sensitized cells we use the cells of some animal whose defibri- nated blood has been used to immunize a second animal, as human blood injected into rabbits. Consequently, as for haemolysis experi- OPSONIC POWER. 131 ments, we should use a 5% emulsion of human red cells in salt solution, to which has been added the inactivated serum of the animal immune to human red cells. This immune serum should be capable of dis- solving red cells in a dilution of at least i to 1000. Experiment. — In a test-tube put 4 drops of the extract of syphilitic liver and the same amount of the inactivated serum of the patient. Now add i c.c. of a i-io dilution of guinea-pig serum and allow the mixture to remain in the incubator at 37° C. for i hour. Then add i c.c. of the sensitized 5% emulsion of red cells, and if haemolysis does not take place, the patient has syphilis. Controls should not only be made with serum from normal persons, but also extracts prepared from normal livers should be used as well. This is one of the most exacting of laboratory diagnostic tests. DETERMINATION OF OPSONIC POWER AND THE PREPARATION OF VACCINES. The following modification of Irishman's method takes very little time and skill and is applicable in the determination of the organism concerned in an infection, as in Wright's method. The control of vaccine treatment by taking opsonic indices from time to time does not seem to have met with much favor in this country — the sources of error being as great, if not greater, than ordinary variations in the opsonic index during the negative and positive phases. Method: Emulsify, by repeatedly drawing up and ejecting with a bulb capillary pipette, a small loopful of a young agar culture of the organism to be diagnosed in 12 to 15 drops of salt solution containing i% of sodium citrate (the citrate prevents coagulation). This may most conveniently be done in a watch glass. Now puncture the ear of the patient and draw up blood to a point marked on the capillary bulb pipette with a grease pen- cil. Draw in air to make a slight break in the column and then draw up bacterial emulsion to the same mark. Thoroughly mix the blood and emulsion on a slide by drawing up and ejecting the mixture. Then finally draw up the mixture to about 2 inches above the tip and seal the tip off in the flame. Put the pipette preparation in the in- cubator for exactly 15 minutes. Prepare a similar preparation, using the blood of a normal person. At the expiration of 15 minutes' incu- 132 PRACTICAL METHODS IN IMMUNITY. bation for the patient's blood and the same time for the control blood, file off the sealed tip, cautiously eject the contents on a slide, and after mixing, spread a film on a slide or cover-glass. Fix with burning alcohol and stain with formoi fuchsin. With Wright's stain the dark nucleus obscures some of the bacteria. Counting the phago- cytized bacteria in a given number of polymorphonuclears, we obtain an average number of bacteria phagccytized per cell. Repeating the count with the control or normal blood, we likewise have the average number of bacteria taken up per cell. Dividing the patient's average by the normal average, we have the opsonic index. If the average for 50 of the patient's cells was 8 and that of the control only 4, the patient's index would be 2, or twice the normal. The practical value of this test is where 2 or more organisms are in a body fluid we may ascertain the causative organism by noting marked variation from the normal in the patient's opsonic index for that particular organism and not for the other organism. This variation may be of the nature of a high or low opsonic index. Preparation of Vaccines. — It has been found satisfactory to make use of stock vaccines in gonorrhceal and tuberculous affections. In case of other infections, however, and preferably with gonorrhceal infections, the causative organism should be isolated from pus, sputum, urine, blood or other material (autogenous vaccine). In treatment of tuberculosis Wright prefers Koch's T. R. or Neu Tuberculin in doses of from 1/5000 to 1/800 of a milligram. Some prefer Koch's more recent Bazillen emulsion. Having isolated the organism, it is inoculated upon one or more agar slants, and after a growth of from 5 to 7 hours with streptococci and pneumococci, or with 18 hours for staphylococci and colon, the growth on these inoculated slants is taken up with salt solution, thoroughly shaken up in the diluting solu- tion and standardized. The most practical way is to gently rub off the growth on the agar in about i or 2 c.c. of salt solution with a platinum loop. Then pour the bacterial emulsion into a sterile test-tube and repeat the process with 3 to 5 agar slants, until we have from 6 to 10 c.c. of the emulsion in the sterile test-tube. By heating to melting-point in the flame a piece of glass rod and attaching it to the rim of the test-tube (also melted), PREPARATION OF VACCINES. 133 we have a handle with which to draw out the test-tube when heated about i inch from the mouth in a blowpipe flame. Drawing this out, we let it cool, and then filing the constricted portion we break it off and seal it in the flame. By shaking up and down vigorously for 5 to 15 minutes, the bacteria are distributed evenly in the salt solution. The sealed test-tube is then placed in a water-bath at 60° C. and heated at this temperature for i hour. Again shake. The constricted sealed end is again filed off and a few drops shaken out in a watch glass for standardization, and at the same time a few drops are deposited on an agar slant as a test for sterility. (Incubation for 24 hours should not show growth). Wright found that by taking a definite quantity of blood and a similar quantity of bacterial emulsion, mixing the blood and bacterial emulsion, then making a smear and staining, it was possible to de- termine the ratio of bacteria to red cells, and from this the number of bacteria per cubic centimeter could be determined. For example, if we find 3 bacteria to each red cell we should have 15,000,000 bacteria to i cubic millimeter. (There being 5,000,000 red cells to the cubic millimeter.) As one cubic centimeter is 1,000 times greater than i cubic millimeter, there would be 15,000,000,000 bacteria in each cubic centimeter of such an emulsion, or vaccine, as it is termed. The standardization may be made with a haemacytometer.* Having determined the strength of the stock vaccine, we should pre- pare a dilute vaccine for injection. This is most conveniently carried out by filling vials with 50 c.c. of salt solution, plugging with cotton, then sterilizing in the autoclave. A sterile rubber cap is now drawn over the mouth of the vial. Sterility is insured by plunging the rubber cap and neck in boiling water. If the stock vaccine showed 5,000,000,- ooo bacteria per c.c. and we desired to have a vaccine containing 200,- 000,000 bacteria per c.c., it would only be necessary to draw out 2 c.c. of the salt solution by means of a sterile syringe needle inserted through the rubber cap and replace it with 2 c.c. of the bacterial emul- sion. Example : In introducing 2 c.c. of a vaccine containing 5 billion *This is best done by drawing up the vaccine to .5 with either the red or white pipette, according to concentration, and then sucking up i to 10 dilute carbol fuchsin to 1 1 or 101. Allow the bacteria to settle on the shelf for 10 minutes before counting. Count as in making a red count. 134 PRACTICAL METHODS IN IMMUNITY. bacteria per c.c., we throw in 10 billion bacteria in a volume equal to 50 c.c. Then each c.c. of the 50 c.c. in the bottle would contain 10,000,000,000 divided by 50 or 200 million in each c.c. If we only want a vaccine containing 100 million per c.c.. we should only add i c.c. We now add 1/4% of Trikresol to the vaccine in order to insure ster- ility. (Introduced with syringe, inserting needle through rubber cap.) The syringe is best sterilized by drawing up vaselin or olive oil heated to 150° C., and the neck and rubber cap of the bottle in boiling water. We now draw up the desired dose of bacteria. If glass syringes are used, simply boiling in water suffices. The ordinary doses are: For gono- cocci, streptococci, pneumococci and colon vaccines 5 million to 50 million. For staphylococci 200 million to one billion. NOTES ON BACTERIOLOGY. NOTES ON BACTERIOLOGY. NOTES ON BACTERIOLOGY. NOTES ON BACTERIOLOGY. PART II. STUDY OF THE BLOOD. CHAPTER XIII. MICROMETRY AND BLOOD PREPARATIONS. MlCROMETRY. IN the examination of blood and faeces preparations, especially when the identification of animal parasites is in question, there is nothing that assists more than a knowledge of the measurements of the object studied. The making of such measurements microscopic- ally is termed micrometry. Micrometry is also indispensable in bacteriology and cytodiagnosis as well as in animal parasitology. The most practical way of making these measurements is with an ocular micrometer. These can be bought separately, or a glass disk (disk micrometer) with lines ruled on it can be dropped into the ocular to rest on the diaphragm inside the ocular. The ruled surface of this glass diaphragm should be placed downward. As was stated in con- nection with the microscope, the image of the object is formed at the level of the diaphragm rim inside the ocular, consequently the lines of the image cut those of the lines ruled on the glass in the ocular. Once having standardized the value of the spaces of the ocular microm- eter for each different objective, all that is necessary subsequently in measuring is to count the number of lines or spaces which the image of the object fills and then, knowing the value of each space for that objective, to multiply the number of spaces by the value of a single space. The unit in micrometry is the mikron. This is usually written /* i36 MICROMETRY AND BLOOD PREPARATIONS. and is the i/iooo part of a millimeter. There are 1000 mikronsina millimeter. To standardize: For this purpose it is necessary to have a scale of known measurements. The stage micrometers are usually ruled in spaces of .1 and .01 mm. The lines which are i/io of a millimeter FIG. 48. — Micrometry diagrams, i. Ocular micrometer with stage micrometer. 50 spaces of ocular micrometer cover two 100 micron spaces and ten 10 micron spaces; equal 300 microns. Each division on ocular micrometer equals 6 microns. 2. Ocular micrometer subtending image of whip worm egg. 9 spaces of ocular micrometer cover Whipworm egg. Each space equals 6 microns. Whipworm egg equals 54 microns. 3. Ocular micrometer with ruling of hasmacytometer. 50 spaces of ocular micrometer cover space equal to width of 6 small squares 50 x 6 = 300 microns. Each division of ocular micrometer equals 6 microns. apart are consequently separated by a distance of loomikrons; those i/ 100 of a millimeter apart are separated by a distance of 10 mikrons. The ocular micrometer is usually ruled with 50 or 100 lines or spaces, separated by longer lines into groups of 5 and 10. Having brought the lines on the stage micrometer to a focus, we MICROMETRY. 137 determine the number of spaces on the stage micrometer which the 100 divisions of the ocular micrometer cover. To distinguish the ruling of the ocular from that of the stage micrometer, revolve the ocular with the fingers. The tube length which is used at the time of standardizing must always be adhered to in subsequent measurements. Example: With a 2/3-in. objective, the 100 rulings of the ocular fill in 15 of the i/io millimeter rulings (ioo//) and 3 of the i/ioo millimeter spaces (lOfi). Consequently the 100 spaces of the ocular cover 1530 mikrons (15 x 100 = 1500; 3 x 10 = 30). Then if 100 spaces equal 1530 mikrons, one space would equal 15.3 mikrons. With the i/6-in. objective the 100 ocular spaces would cover about 3 of the i/ 10 millimeter (ioo(«) spaces of the stage micrometer. Then the 100 spaces would equal 300 mikrons and one space would equal 3 mikrons. The ruling of the slide of a Thoma Zeiss haemacytometer will answer as well as a stage micrometer. The small squares are 1/20 of a millimeter square, consequently the distance between the lines border- ing the small square is 1/20 millimeter or 50 mikrons. Now, if wifh the 1/6 in. objective, the 100 lines on the ocular fill in the spaces of 6 small squares, the length of such a space would be 50 x 6 = 300 mikrons. This divided by 100 spaces would equal 3 ,«. The most accurate instrument for measuring is the filar microm- eter. These are expensive. Measurements can also be made with the camera lucida, but it takes considerable time to make the adjust- ments necessary, so that it is not convenient. With an ocular microm- eter one can make measurements of blood-cells, amoebae, etc., in a few seconds — it only being necessary to slip in the ocular micrometer. Rule for determining the magnifying power of microscopic lenses: Measure the diameter of the lens of the objective in inches — the approximate equivalent focal distance is about twice the diameter. Dividing 10 by the equivalent focal distance gives the magnifying power of the lens. This should be multiplied by the number of times the ocular magnifies. Example: The diameter of the lens of the objective was found to measure 1/2 in., the focal distance would then be about i inch. Dividing 10 by i we have 10 as the magnifying 138 MICROMETRY AND BLOOD PREPARATIONS. power of the lens of the objective. If we were using a No. 4 ocular, the magnifying power would be approximately 40. BLOOD PREPARATIONS. To obtain blood, except for blood cultures, use either a platino- iridium hypodermic needle which can be sterilized in the flame, a small lancet or a surgical needle with cutting edge. A steel pen with one nib broken off or the glass needle of Wright may also be used. To make a glass needle, pull straight apart a piece of capillary tubing in a very small flame. Tap the fine point to break off the very delicate extremity. Scarcely any pain attends the use of such a needle. In puncturing either the tip of the finger or lobule of the ear a quick piano- touch-like stroke should be used. The ear is preferable, as it is less sensitive and there is less danger of infection. Before puncturing, the skin should be cleaned with 70% alcohol and allowed to dry. It is advisable to sterilize the needle before using it. The first drop of blocd which exudes should be taken up on the paper of the Ta'lquist haemoglobinometer, using subsequent ones for the blood pipettes and smears. If it is necessary to make a complete blood examination, it is rather difficult to draw up the blood in the pipettes, dilute it and then get material for fresh blood preparations and films without undue squeezing, which is to be avoided. Of course, fresh punctures can be made. Ordinarily, complete blood examinations are not called for. It is only a white count or a differ- ential count or an examination for malaria that is required. HEMOGLOBIN ESTIMATION. The most accurate instrument for this purpose is the Miescher modification of the v. Fleischl haemoglobinometer. The glass wedge for comparison with the diluted blood is the same in each instrument, but by the use of a diluting pipette accurate dilutions are possible in the Miescher. There are two cells provided — one 12 millimeters high, the other 15 millimeters; the idea of this being to enable one to make separate comparisons and to select the central parts of the glass-wedge scale, where comparison is more accurate than at the ends. If using a i to 200 dilution and the deeper cell, the reading of the HEMOGLOBIN ESTIMATION. 139 scale is the proper one; if the 12 millimeter cell is used, the reading is only 4/5 of what it should be. Thus a reading of 100% with the 15- millimeter cell would show with the 12-millimeter one a reading of 80% for the same blood. The apparatus is quite expensive and requires considerable time in making the estimation. Sahli's Haemometer.— A simple and ap- parently very scientific instrument which has been recently introduced is the Sahli modifi- cation of the Gower haemoglobinometer. In- stead of the tinted glass, or gelatin colored with picrocarmine to resemble a definite blood dilution, Sahli uses as a standard the same corloring matter as is present in the tube containing the blood. By acting on blood with 10 times its volume of N/io HC1, haematin hydrochlorate is produced, which gives a brownish-yellow color. In the standard tube, which is sealed, a dilution representing i% of normal blood is used. To apply this test, pour in N/ 10 HC1 to the mark 10 on the scale of the graduated tube. Add to this 20 cubic millimeters of the blood to be examined, drawn up by the capillary pipette provided. So soon as the mixture assumes a clear brown color, add water drop by drop until the color of the tubes matches. The reading of the height of the aqueous dilution on the scale gives the Hb. reading. The tubes are encased in a vulcanite frame with rectangular apertures. This gives the same optical impression as would piano -parallel glass sides. It recommended that the N/io HC1 be preserved with chloroform. Tallquist's Haemoglobin Scale.— This is a small book of specially prepared filter-paper with a color-scale plate of 10 shades of blood colors. These are so tinted as to match blood taken up on a piece of the filter-paper and are graded from 10 to 100. So soon as the FIG. 49. — Sahli's haemo- globinometer. (Greene.) is 140 MICROMETRY AND BLOOD PREPARATIONS. blood on the filter-paper has lost its humid gloss, the comparison should be made. This is best done by shifting the blood stained piece of filter-paper suddenly from one to the other of the holes cut in each shade — the piece of filter-paper being underneath the color plate. The error with this method is probably not over 10% after a little experience. If the colored plate is not kept in the dark, the tints tend to fade. The use of this method requires neither time nor trouble. To COUNT BLOOD-CORPUSCLES. The instrument almost universally used is the Thoma-Zeiss haemacytometer. The apparatus consists of two pipettes, one for leukocytes, graduated to give a dilution of i to 10 or greater; the other, for red cells to give a dilution of i to 100 or greater. The white pipette has the mark ii above the bulb and the red pipette the mark 101. In addition, there is a counting chamber. This consists of a square of glass with a round hole in the center. Occupying the center of this round hole is a circular disk of glass of less diameter, so that an encircling channel is left. The square and the FIG. 50. — Thoma-Zeiss blood circle of glass are cemented to a heavy counter, showing pipette count- lags siide The surfaces of each are mg chamber and ruled field. (Greene.} absolutely level and highly polished. That of the circular disk is ruled into squares of varying size and is exactly i/io of a millimeter below the level of the surface of the surrounding glass square. When a polished piano-parallel cover-glass rests on the shelf, as the outer square glass is termed, there is a space left between its under surface and the ruled disk of .1 millimeter. The channel around the disk is termed the moat or ditch. The most desirable rulings are those of Turck and of Zappert. In these the entire ruled surface consists of 9 large squares, each i millimeter square. These are subdivided, and in the central large square are to be found the small COUNTING RED CELLS. 141 squares used for averaging the red cells. These small squares are 1/20 of a millimeter square and are arranged in 9 groups of 16 small squares by bordering double-ruled lines. As the unit in blood counting is the cubic millimeter, if one counted all the white cells lying within one of the large squares (i millimeter square), he would have only counted the cells in a layer i/io of the required depth, so that it would be necessary to multiply the number obtained by 10. This product, multiplied by the dilution of the blood, would give the number of white cells in a cubic millimeter of undiluted blood. To make a red count : Having a fairly large drop of blood, apply the tip of the 10 1 pipette to it and, holding the pipette horizontally, care- fully and slowly draw up with suction on the rubber tube a column of blood to exactly .5 or i. The variation of 1/25 of an inch from the mark would make a difference of almost 3 percent. If the column goes above .5, it can be gently tapped down on a piece of filter-paper until the .5 line is cut. Now insert the tip of the pipette into some diluting fluid and, revolving the pipette on its long axis while filling it by suction, you continue until the mark 101 is reached. A variation of 1/25 of an inch at this mark would only give an error of about 1/30 of i%. After mixing thoroughly, by shaking for one or two minutes, the fluid in the pipette below the bulb is expelled (this, of coarse, is only diluting fluid). A drop of the diluted blood of a size just sufficient to cover the disk when the cover-glass is adjusted, is then deposited on the disk and the cover-glass applied by a sort of sliding movement, best obtained by using a forceps in one hand assisted by the thumb and index-finger of the other. Among diluting fluids Toisson's is probably the best : Sodium chloride, i gram. Sodium sulphate, 8 grams. Glycerin, 30 c.c. Distilled water, 160 c.c. Dissolve the sodium chloride and the sodium sulphate in the glycerin water and add sufficient methyl or gentian violet to give a rich violet tint. A salt solution of about 2% strength, tinged with about i drop of a 142 MICROMETRY AND BLOOD PREPARATIONS. saturated alcoholic solution of gentian violet to about 50 c.c., is a good substitute, or the salt solution alone will answer when no white count is to be made at the same time as the red one. It is important to work quickly in adjusting the cover-glass, or there will be cells settling in the center of the drop from a greater depth than the one which the apposition of the cover-glass makes (i/io millimeter deep). A good preparation should show: 1. Presence of Newton's rings. 2. Absence of air bubbles. 3. Entire surface of ruled disc covered. 4. Equal distribution of cells. Before counting, about 5 minutes should be allowed for the settling of the cells. It will be remembered that the small squares are 1/20 millimeter square. The depth of fluid from upper surface of shelf to lower sur- face of cover-glass is i/io mm. Hence each space embraced by the small square and the depth of fluid is 1/4000 of the unit used in esti- mating number of corpuscles in blood, or i cubic millimeter (1/20x1/20 x 1/10 = 1/4000). Count 100 of the small squares (this enables one to use decimals). There are 9 squares between double -ruled lines, each containing 16 small squares. Count the number of corpuscles in the 1 6 small squares contained in upper left-hand double-ruled square. Put down this count. Next count corpuscles in the adjoining 16 squares. Put down this count. Then in third, 16 squares. Put down the number. Now move down to next row of three double- ruled squares. Count the number of corpuscles in each of the three 1 6 square spaces and set down the numbers for addition. We have now counted 96 small squares (6 x 16). Count at any place 4 additional small squares and add number of blood -cells contained therein to those in the 96 small squares already counted. Divide the sum by 100 or simply point off two decimals. This gives the average for each small square. Multiply this by the dilution and then (as the small square is only 1/4000 cu. mm.) by 4000. This will give the number of cor- puscles in i cubic millimeter. Example: 100 small squares contained 655 red cells. Pointing off 6.55 equals average number of red cells per LENCOCYTE COUNTING. 143 small square. Multiply by dilution (200) and then by 4000 (the small square is 4000 times smaller than the unit: icu. mm.) — 6.55x200 = 1310 x 4000 = 5,240,000. At least 100 small squares, and preferably 200 should be counted. If the blood appears normal, one may simply count the number of red cells in 5 of the 16 small square spaces (80 small squares). Having added the numbers and multiplying by 10,000, you obtain the number of cells in i cubic millimeter. (Eighty small squares is 1/50 of the unit of i cu. mm., or 4000 small squares. The blood dilution being i to 200, we have 50 x 200 x number of cells in 80 small squares.) In counting, count corpuscles lying on the lines above and to the right. Do not count those lying on lines below and to the left. In the small squares count only corpuscles lying in the space or cutting the upper line. This prevents counting the same cell twice. To Count White Cells.— Draw up the fluid in the white pipette to the mark .5. Then, still holding the pipette as near the horizontal as possible, because the column of blood tends to fall down in the larger bore, draw up by suction a diluting fluid which will disintegrate the red cells without injuring the whites. The best fluid is .3% of glacial acetic acid in water. This makes the white cells stand out as highly refractile bodies. Some prefer to tinge the fluid with gentian violet. The .5 mark is preferred because it takes a very large drop of blood to fill the tube up to the i mark and if there is much of a leukocy- tosis a i to 10 dilution is not sufficient. In leukemic blood it is better to use the red pipette with the .3% acetic acid solution The blood having been drawn up to .5, we have a dilution of i to 20. Making a preparation, exactly as was done in the case of the red count, we count all of the white cells in one of the large squares (i sq. mm.). The cross ruling greatly facilitates this. Note the number. Then count a second and a third large square. Strike an average for the large squares counted and multiply this by 10, as the depth of the fluid gives a content equal to only i/io of a cubic millimeter. Then multiply by the dilution. Example: First large square 50; second large square 70; third large square 60. Average 60. Then 60 x 10 x 20 = 12,000, the number of leukocytes in i cubic millimeter of blood. The count may be made with a low power (2/3-in. objective) as the 144 MICROMETRY AND BLOOD PREPARATIONS. leukocytes stand out like pearls. It is better, however, to use a higher power, so that pieces of foreign material may be recognized and not enumerated as white cells. When it is desired to make a white count with the same preparation as is used for the red one, especially if the ruling is of the old style (only central ruling and not in 9 large squares as with Zappert and Turck), it is advisable to make use of the method of counting by fields. With a Leitz No. 4 ocular and a No. 6 objective, with a tube length of 120 millimeters, it will be observed that the field so obtained has a diameter of 8 small squares. Now, remembering that the area of a circle equals the square of the radius multiplied by *-, or 3.1416, we have the following calculation: The diameter being 8 small squares, the radius would be 4 small squares. Squaring the radius, we have 1 6. This multiplied by 3.1416 gives us 50. This means that every field, with the microscope adjusted as stated, contains 50 of the small squares, or 1/80 of the unit of i cubic millimeter of the diluted blood. By keeping a single red cell in view while moving the mechanical stage from right to left or from above downward, we know that a new field of 50 small squares is brought into view when the circumference of the field cuts this individual cell. Example: As 2000 small squares would ordinarily be a sufficient number to count for a white count, this would require us to count the number of leukocytes in 40 of the designated microscopic fields (this, of course, is only 1/2 the unit, hence we should multiply by 2). Counted 40 fields and noted 50 white cells. 50 x 2 = 100 x 200 (the dilution in red pipette) = 20,000. Consequently 20,000 would represent the number of leukocytes in i cubic millimeter of the blood examined. After making a blood count, the haemacytometer slide should be cleaned with soap and water and then rubbed dry, preferably with an old piece of linen. As the accuracy of the counting chamber depends upon the integrity of the cement, any reagent such as alcohol, xylol, etc., and in particular heat, will ruin the instrument. The pipettes should be cleaned by inserting the ends into the tube from a vacuum pump, as a Chapman pump. First draw water or i% sod. carbonate solution through the pipette, then alcohol, then ether, and finally FRESH BLOOD FILMS. 145 allow air to pass through to dry the interior. If the interior is stained, use i% HC1 in alcohol. If a vacuum pump is not at hand, a bicycle pump or suction by mouth will answer. PREPARATIONS FOR THE STUDY OF FRESH BLOOD. Many authorities prefer a fresh-blood specimen to a stained dried smear in the study of parasites of the blood. In malaria in particular there is so much information as to species to be obtained from a fresh specimen that the employment of this method should never be ne- glected. While waiting for the film to stain one has 5 or 6 minutes which could not be better spent than in examining the fresh specimen which only requires a moment to make. Manson's Method. — Have a perfectly clean cover-glass and slide. Touch the apex of the exuding drop of blood with the cover-glass and drop it on the center of the slide. The blood flows out in a film which exhibits an "empty zone" in the center. Surrounding this we have the "zone of scattered corpuscles, " next the "single layer zone" and the "zone of rouleaux" at the periphery. It is well to ring the prepara- tion with vaselin. When desiring to demonstrate the flagellated bodies in malaria, it is well to breathe on the cover-glass just prior to touching the drop of blood. The Method of Ross is very easy of application and gives most satisfactory preparations. Take a perfectly clean slide and make a vaselin ring or square of the size of the cover-glass. Then, having taken up the blood on the cover-glass, drop it so that its margin rests on the vaselin ring. Gently pressing down the cover-glass on the vase- lin makes beautiful preparations wrhich keep for a very long time. If it is desired to study the action of stains on living cells, this method is also applicable. A very practical way to do this is to tinge .85% salt solution containing i% sodium citrate (the same as is used in opsonic work) with methylene azur, gentian violet or methyl green. With a Wright bulb pipette, take up one part of blood, then one part of tinted salt solution. Mix them quickly on a slide and then deposit a small drop of the mixture in the center of the vaselin ring and immediately apply a cover-glass and press down the margins as before. This method will be found of great practical value. 146 MICROMETRY AND BLOOD PREPARATIONS PREPARATION AND STAINING OF DRIED FILMS. When preparations are desired for a differential count, Ehrlich's method of making films is to be preferred as the different types of leukocytes are more evenly distributed. In making smears by spread- ing, there is a tendency for the polymorphonuclears to be concentrated at the margin while lymphocytes remain in the central part of the film. FIG. 51. — Blood technic. i, 2, 3, Method for making blood smear on slide; 4, U tube for resting slides while staining; 5, slide showing grease pencil marking, marking prevents stain from overflowing; 6, method for drawing apart cover glasses in making blood smear. In Ehrlich's method we have perfectly clean dry cover-slips. Take up a small drop of blood without touching the surface of the ear or finger. Drop this cover-glass immediately on a second one and as soon as the blood runs out in a film, draw the two cover-slips apart in a plane parallel to the cover-glasses. Slide them apart. Ehrlich uses forceps to hold the cover-glasses to avoid moisture from the fingers. Of the various methods of spreading films on slides there is none BLOOD FILMS. 147 equal to that described by Daniels. In this the drop of blood is drawn along and not pushed. along. The films are even, can be made of any desired thickness by changing the angle of the drawing slide, and there is little liability of crushing pathological cells. Take a small drop of blood on the end of a clean slide. Touch a second slide about 1/2 inch from end with the drop and as soon as the blood runs out along the line of the slide end, slide it at an angle of 45° to the other end of the horizontal slide. The blood is pulled or drawn behind the ad- vancing edge of the advancing slide. An angle less than 45° makes a thinner film. One greater, a thicker film. Of the various methods of making smears by means of cigarette paper, rubber tissue, needles, etc., the best seems to be to take a piece of capillary glass tubing and use this instead of a needle in making the film. There is one advantage about the strip of cigarette paper touched to the drop of blood and drawn out along the slide or cover glass and that is that it is almost impossible not to make a working preparation by this method. In the making of smears the chief points are to make the smear as soon after taking the blood as possible and to have slides and cover- glasses scrupulously clean. It is well to flame all slides and cover- glasses which are to be used for blood-work. This is the best method of getting rid of grease. Fixation of Film. — In Wright's, Irishman's and other similar stains the methyl-alcohol solvent causes the fixation. In staining with Giemsa's stain, Ehrlich's tri-acid, haematoxylin and eosin, Smith's formol fuchsin, and with thionin, separate fixation is necessary. For Giemsa and thionin, either alcohol and ether (15 minutes), ab- solute alcohol (10 to 15 minutes) or methyl alcohol (2 to 5 minutes) answer well. Formalin vapor, for 5 to 10 seconds, is also used for fixation. For Ehrlich's tri-acid, haematoxylin and eosin and formol fuchsin, heat gives the best results. The best method is to place the films in an oven provided with a thermometer. Raise the temperature of the oven to 135° C. and then remove the burner. After the oven has cooled, take out the fixed slides or slips. Some prefer to place a crystal of urea on the slide, then hold it over 148 MICROMETRY AND BLOOD PREPARATIONS. the flame until the urea melts. This shows that a temperature be- tween 130° and 135° C. has been reached. One of the handiest methods is to drop a few drops of 95% alcohol on the slide or cover glass. Allow this to flow over the entire surface; then get rid of the excess of alcohol by touching the edge to a piece o£. filter paper for a second or two. Then light the remaining alcohol film from the flame and allow the burning alcohol to burn itself out. A chemical fixation which gives good fixation for haematoxylin and tri-acid stains (not equal to heat) is a modification of Zenker's fluid (Whitney). To Muller's fluid, which is potassium bichromate 2 gms., sodium sulphate i gm. and water 100 c.c., add 5 gms. of bichloride of mercury and 5 c.c. of nitric acid (C. P.). Fixation is obtained in 5 seconds. Staining Blood-films. — As separate staining with eosin and methylene blue rarely gives good preparations and as the modifications of the Romanowsky stain recommended are easy to make and employ, and give much greater information, the separate method of staining is not recommended. The most satisfactory single stain is thionin. Rees' Thionin Solution. — Take of thionin 1.5 gms., alcohol 10 c.c., aqueous solution of carbolic acid (5%) 100 c.c. Keep this as a stock solution. It should be at least two weeks old before using. For use, filter off 5 c.c. and make up to 20 c.c. with water. 1. Fix films (a) by heat, (b) by alcohol and ether, or (c) preferably by i% formalin in 95% alcohol for i minute. 2. Stain for from 10 to 20 minutes. Wash and mount. Malarial parasites are stained purplish; nuclei of leukocytes, blue; red cells, faint greenish-blue. Ehrlich's Tri-acid or Triple Stain. — There are required : 1. Sat. aq. sol. orange G. (Dissolve 3 grams in 50 c.c. water.) 2. Sat. aq. sol. acid fuchsin. (Dissolve 10 grams in 50 c.c. water.) 3. Sat. aq. sol. methyl green. (Dissolve 10 grams in 50 c.c. water.) These three solutions may be kept as stock solutions. They keep well in the dark. To make the stain: Add 9 c.c. of No. 2 (acid fuchsin) to 18 c.c. of No. i (orange G.). After they are mixed thoroughly, add 20 c.c. of No. 3 (methyl green). Then, after the first ROMANOWSKY METHODS OF STAINING. 149 three ingredients are well mixed, add 5 c.c. of glycerin. Mix, then add 15 c.c. of alcohol; again mix, and finally add 30 c.c. of distilled water. Keep the mixed stain about i week before using. The best fixatives are heat and Whitney's modified Zenker. To use: Stain films from 2 to 5 minutes. Then wash and mount. The tri-acid stain is a good tissue stain. The objections to the triacid stain are that it does not stain malarial parasites or mast cells and that failure to obtain good results is of frequent occurrence. Wright's Method. — The stain is made by adding r gram of methylene blue (Grubler) to 100 c.c. of a 1/2% solution of sodium bicarbonate in water. This mixture is heated for i hour in an Arnold sterilizer. When cool, add to the methylene-blue solution 500 c.c. of a i to 1000 eosin solution (yellow eosin, water soluble). Add the eosin solution slowly, stirring constantly until the blue color is lost and the mixture becomes purple with a yellow metallic luster on the surface and there is formed a finely granular black precipitate. Collect this precipitate on a filter-paper and when thoroughly dry (dry in the incubator at 38° C.) dissolve .3 gm. in 100 c.c. of pure methyl alcohol (acetone free). This constitutes the stock solution. For use filter off 20 c.c. and add to the filtrate 5 c.c. of methyl alcohol. A modification by Batch is very satisfactory. In this method in- stead of polychroming the methylene blue with sodium bicarbonate and heat, the method of Borrel is used. Dissolve i gm. of methylene blue in 100 c.c. of distilled water. Next dissolve .5 gm. of silver nitrate in 50 c.c. of distilled water. To the silver solution add a 2 to 5% caustic soda solution until the silver oxide is completely precipi- tated. Wrash the precipitated silver oxide several times with distilled water. This is best accomplished by pouring the wash-water on the heavy black precipitate in the flask, agitating, then decanting and again pouring on water. After removing all excess of alkali by repeated washings, add the methylene-blue solution to. the precipitated silver oxide in the flask. Allow to stand about 10 days, occasionally shaking until a purplish color develops. The process may be hastened in an incubator. When polychroming is complete, filter off and add to the filtrate the i to 1000 eosin solution and proceed exactly as with Wright's stain. 150 MICROMETRY AND BLOOD PREPARATIONS In Leishman's method the polychroming is accomplished by adding i gm. of methylene blue to 100 c.c. of a 1/2% solution of sodium car- bonate. This is kept at 65° C. for 12 hours and allowed to stand at room temperature for 10 days before the eosin solution is added. The succeeding steps are as for Wright's stain. The modification of the Romanowsky stain, which is used in the laboratory of the U. S. Naval Medical School and which can be rec- ommended as giving good results with the least expenditure of time in making and which by the addition of either acid or alkaline alcohol can be made to give the staining effect desired, is that of Hospital Steward R. W. King, U. S. Navy. The preparation of the stain is as follows : Dissolve i gram of Grubler's methylene blue and 0.5 gram of sodium, bicarbonate in 100 c.c. of distilled water. Transfer to porce- lain dish and evaportate to dryness over Bunsen burner or alcohol flame. The fluid may be allowed to boil gently until about half of the water has escaped, when the heat should be somewhat reduced. Evaporation is now facilitated by causing the fluid to flow upon the sides of the dish by tilting. This also overcomes the tendency to spluttering. The heat should be continued until the last trace of moisture has disappeared. The absolutely dry stain is then removed from the dish and preserved in small well-stoppered vials. (If larger quantities than the above are to be made, separate dishes must be used.) Stock solution: Dissolve 0.3 gram of the polychromatic blue and 0.175 gram of Grubler's eosin in 100 c.c. of pure methyl alcohol. Allow to stand for three hours and filter. Add 25 drops of 3% hydro- chloric acid alcohol (used in T. B. staining). For use, take 25 c.c. of the stock solution and add eight drops of the alkaline alcohol (5 c.c. 10% sol. caustic soda to 100 c.c. 95% alcohol), and test by staining a section of a fresh blood film. If the blue over- stains, add one drop of the acid alcohol and test again. If the eosin overstains (nuclei stain poorly), add two or three drops of the alkaline alcohol. In this way it will only require one or two trials to adjust the staining properties of the fluid, after which it will keep unchanged for some time. At any time, however, if it is found not to give good ROMANOWSKY STAINING METHODS. 151 results, the addition of a drop or so of either the acid or alkaline alcohol, as indicated, will restore its original staining properties. The acid and alkaline alcohols may be used in the same way and with about the same results with Wright's staining fluid. In all Romanowsky methods distilled water should be used. If not obtainable, the best substitute is rain-water collected in the open and not from a roof. Method of staining: 1. Make films and air dry. 2. Cover dry film preparation with the methyl-alcohol stain for i minute (to fix). 3. Add water to the stain on the cover-glass or slide, drop by drop, until a yellow metallic scum begins to form. It is advisable to add the drops of water rapidly in order to eliminate precipitates on the stained film. Practically, we may add i drop of water for every drop of stain used. 4. Wash thoroughly in water until the film has a pinkish tint. 5. Dry with filter-paper and mount. Red cells are stained orange to pink; nuclei shades of violet; eosinophile granules, red; neutrophile granules, yellow to lilac; blood platelets, purplish; malarial parasites, blue; chromatin, metallic red to rose-pink. Giemsa's Modification of the Romanowsky Method. — This is one of the most perfect of the modifications. The objection is that greater time in staining films is required than with the Wright or Leishman method and the stain is very expensive. Take of Azur II eosin 0.3 gm. Azur II 0.08 gm. Dissolve this amount of dry powder in 25 c.c. of glycerin at 60° C. Then add 25 c.c. of methyl alcohol at the same temperature. Allow the glycerin methyl- alcohol solution to stand over night and then filter. This is the stock stain. To use: Dilute i c.c. with 10 to 15 c.c. of water. If i to 1000 potassium carbonate solution is used instead of water it stains more deeply. Having fixed the smear with methyl alcohol for 5 minutes, pour on the diluted stain and after 15 to 30 min. wash off and continue washing with distilled water until the film has a slight pink tinge. For Treponema pallidum stain from 2 to 12 hours. I$2 MICROMETRY AND BLOOD PREPARATIONS. While the Romanowsky methods are more satisfactory for differential counts and for the demonstration of the malarial parasites, and es- pecially for differentiating species, yet by reason of the liability to deterioration in the tropics of methylene blue the haematoxylin methods may be preferable. Many workers in blood-work and cytodiagnosis prefer the haematoxylin. 1. Fix the film either by heat or with Whitney's fixative. Heat is to be preferred. 2. Stain with Meyer's hemalum or Delafield's haematoxylin for from 5 to 15 minutes according to the stain. Fre- quently 3 minutes will be found sufficient. To make the hemalum, dissolve .5 gm. of haematin in 25 c.c. of 95% alcohol. Next dissolve 25 gm. of ammonia alum in 500 c.c. of distilled water. Mix the two solutions and allow to ripen for a few days. The stain should be satisfactory in 2 or 3 days. To make Delafield's haematoxylin, dissolve i gm. of haematoxylin crystals in 6 c.c. of 95% alcohol. Add this to 100 c.c. of saturated aqueous solution of ammonia alum. After exposure to light for a week, the color changes to a deep blue-purple. Add to this ripened stain 25 c.c. of glycerin and 25 c.c. of methyl alcohol and, after it has stood for about two days, filter. The stain should be filtered from time to time as a sediment forms. This makes a stock solution which should be diluted 10 to 15 times with water when staining. 3. Wash for 2 to 5 minutes in tap water to develop the haematoxylin color. 4. Stain either with a i to 1000 aqueous solution of eosin or with a 1/2 of i% eosin solution in 70% alcohol. The eosin staining only requires 15 to 30 seconds. 5. Wash and examine. IODOPHILIA. This reaction is supposed to be due to the presence of glycogen, especially in the polymorphonuclears, in suppurative conditions. IODOPHILIA REACTION. 153 Make blood-smears on cover-glasses as usual, and after they dry, but without fixation, mount them in a drop of the following solution: Iodine, i part. Potassium iodide, 3 parts. Gum arabic, 50 parts. Water, 100 parts. Small brown masses in the polymorphonuclears or lying extra- cellular indicate a positive iodophilia. CHAPTER XIV. NORMAL AND PATHOLOGICAL BLOOD. In considering what may be termed normal blood, it must be borne in mind that the normal varies for men, women and children: Hb. Red cells. Leukocytes. Men, 90 to 110%, 5 to 5 1/2 million, 7500. Women, 80 to 100%, 4 1/2 to 5 million, 7500. Children, 70 to 80%, 4 1/2 to 5 million, 9000. COLOR-INDEX. This is obtained by dividing the percentage of the haemoglobin by the percentage of red cells, five million red cells being considered as 100%. To obtain the percentage of red cells it is only necessary to multiply the two extreme figures to the left by two. Thus if a count showed the presence of 1,700,000 red cells, the percentage would be 34. (17x2 = 34.) If the Hb. percentage in this case were 50; then the color index would be 50 -H 34, or 1.4. In normal blood the color-index is, approximately, i. In anaemias we have three types of color-index: (i) The perni- cious anaemia type, which is above i. Here we have a greater re- duction in red cells than we have of the haemoglobin content of each cell. (2) The normal type, when both red cells and haemoglobin are proportionally decreased, as in anaemia following haemorrhage. (3) The chlorotic type. Here there is a great decrease in haemoglobin percentage, but only a moderate decrease in the number of red cells. Hence the color-index is only a fraction of i. For example, in a case of chlorosis we have 40% of haemoglobin and 90% of red cells, 40 -j- 90 = .4 RED CELLS. In considering the corpuscular richness of a specimen of blood, it must be remembered that this does not necessarily bear any relation to 154 NUCLEATED RED CELLS. 155 the quantity of blood in the body. Thus, a more or less bloodless- looking individual, the total quantity of whose blood is greatly reduced, may notwithstanding give a normal red count. In examining a specimen of peripheral blood we get a qualitative, not a quantitative result. Normally, we have an increase in red cells in those living at high altitudes. An altitude of two thousand feet increases the red count about one million, and a height of six thousand feet about two million. Profuse sweats and diarrhoeas also increase the red count. Pathologi- cally, in chronic polycythemia with cyanosis and splenic enlargement, we have a red count of about ten million. In cyanosis from heart disease, etc., and in Addison's disease there is also an increase in red cells. The normal red cell or erythrocyte measures about 7.5/4 in diameter. It is nonnucleated and normally stains with acid dyes, taking the pink of eosin or the orange of orange G. If larger, 10 to 20^, it is called a macrocyte; if smaller, 3 to 6/j, a microcyte. Macrocytes are rather indicative of severe forms of anaemia, the microcytes, of less grave types. When the red cell is distorted in shape, it is called a poikilocyte. Care must be exercised that distorted shapes are not due to faulty technic. Crenation and vacuolation of red cells are marked in poorly prepared specimens. In addition to variation in size and shape, we also have pathological variation in staining affinities. Poly chromatophilia.— This shows itself by red cells taking a brownish to a dirty blue tint, as is frequently seen in immature red cells, especially nucleated ones. Granular basophilic degeneration (also termed punctate baso- philia and stippling) refers to the presence of blue dots in the pink back-ground of stained red cells. It is found in many severe anaemias, as pernicious anaemia, the leukaemias, malarial cachexia, etc. It is very characteristic of lead poisoning. The nucleated red cell, while normal for the marrow, is always pathological for the blood of the peripheral circulation. Normoblasts have the diameter of a normal red cell. The nucleus is round and stains intensely with basic dyes, often appearing almost black. 156 NORMAL AND PATHOLOGICAL BLOOD. Another characteristic is that it frequently appears as does the setting in a ring. Some give the term microblast to smaller nucleated forms. In normoblasts the red cell proper stains normally. The megaloblasts not only have a greater diameter than the normoblast, but the nucleus is poor in chromatin, stains less intensely and is less distinctly out- lined. Instead of being round, the nucleus is irregular and may be trefoil in shape. The cytoplasm surrounding the nucleus shows polychromatophilia. This contrasted with the pure blue of the lymphocytes should differentiate. Normoblasts are found in secondary anaemias, and especially in myelogenous leukaemia. Megaloblasts are peculiarly characteristic of pernicious anaemia. Enormous megaloblasts are sometimes termed gigantoblasts. In aplastic anaemia (a severe type of pernicious anaemia), in con- trast to ordinary pernicious anaemia, nucleated reds are very rarely found. There is also very little poikilocytosis, and the color-index is about normal. It is a rare, rapidly fatal anaemia, particularly of young women. WHITE CELLS. Owing to the conflicting views as to origin, nature and functions of the various leukocytes, their classification is in a state of confusion. As regards the appearance of the cells, this of course varies as "the stain used, and it requires considerable experience for a single individual to be able to positively recognize the difference between a lymphocyte and a large mononuclear when one specimen is stained with a Roman- owsky stain, another with Ehrlich's triacid, and a third with haema- toxylin and eosin. This, of course, is intensified when different persons adhere to the method of staining which they prefer and are at a loss to appreciate differences which are brought out by some other stain used by some other person. Even with the same stain used with different speci- mens of blood we find the staining characteristics of various leukocytes imperceptibly merging, the one into the other, so that at times it is impossible for one, even with his own standard of differentiation, to be sure whether he is dealing with a lymphocyte or a large mononuclear. The difficulty is even greater when wre deal with Turck's irritation forms and with myelocytes. LYMPHOCYTES. 157 Without going into the various granule stainings so thoroughly brought out by Ehrlich, we shall immediately take up the question of a practical classification for use in making a differential count. As the Romanowsky method of staining (Wright, Leishman or Giemsa) gives us information not yielded by either haematoxylin and eosin or the triacid, the points of differentiation to be referred to in that which follows is with blood so stained. In considering the staining affinities of different parts of the leukocytes, it is convenient to divide such into basic ones, acid ones and those which may be said to be on the border line betwreen these — the so-called neutrophilic affinities. With Wright's stain we have the eosinophile cr oxyphile affinity of the granules of eosinophiles for acid dyes, in this case eosin. The nuclei and basophile granules have affinities in greater or less degree for basic stains (the blue and the violet shading resulting from methylene blue as modified by polychroming). With the granules in the cyto- plasm of the polymorphonuclears and neutrophilic myelocytes, and to a less extent in the transitional, we have a staining which merges into a yellowish-red on the one extreme and into a lilac on the other. As a standard, neutrophilic granules should be a mean of these extremes. Not only by reason of the authority of Ehrlich, but because such a division gives all variations, which can then be combined by one preferring a simpler classification, it would seem proper to divide the normal leukocytes into: 1. Small lymphocytes. These are small round cells about the size of a red corpuscle with a large centrally-placed, deeply violet stain- ing nucleus and a narrow zone of cytoplasm. This cytoplasm may not be more than a mere crescentic fringe. This is the type of lymph- ocytes which makes up the greater proportion of the leukocytes in chronic lymphatic leukaemia. At times these cells seem to be com- posed of nucleus alone. 2. Large lymphocytes. These are of the same type as small lymphocytes, but possessing more cytoplasm. The nucleus, while round and taking a fairly deep rich violet stain, does not stain so deeply as the nucleus of the small lymphocytes. The cytoplasm is a clear translucent 158 NORMAL AND PATHOLOGICAL BLOOD. pure blue. It may contain pinkish granules known as azur granules, but these are of rather large size and do not mar the glass-like appearance. They are from 9 to 15/1 in diameter and are com- mon in children. In the acute lymphatic leukaemias they at times predominate. 3 Large mononuclears. These are large round or oval cells with a nucleus which has lost the richness of violet staining of the lympho- cyte nucleus. The nucleus is furthermore frequently irregular in outline or may show the commencing indentation of the transitional nucleus. There is not that sharp distinction between nucleus and cytoplasm that exists in the lymphocytes. The cytoplasm of the large mononu- clear gives the impression of opacity, as if it were frosted glass instead of clear glass. The neutrophile mottling which begins to appear causes a disappearance of the pure blue character of the cytoplasm of the lymphocyte. It is principally by the washed-out staining of the nucleus and the opaque lilac of the cytoplasm, that we differentiate them from the lymphocytes. They greatly resemble Turck's irritation forms or plasma cells and may be confused with myelocytes. 4. Transitionals. These appear as but a later stage in the decay of the large mononuclears; the nucleus is more indented, frequently horse-shoe shaped, and has a washed out violet shade of less intensity than that of the large mononuclears. These are the cells so often disrupted in smears. These four kinds of cells are frequently referred to as the lymphocyte series, and although many authorities consider that the small lympho- cyte represents a more mature cell than the others of this class, yet it is thought by others that the age of the cell increases as we go from small lymphocytes to large lymphocytes, thence to the large mononu- clear; and then in the transitional we have the decrepit stage which precedes dissolution. The old view that the transitional was the precursor of the polymorphonuclear has few advocates at the present time. While it is convenient to consider. these hyaline cells as representing different stages in development, yet from a stand-point of immunity this is untenable. The large mononuclears and transitionals are the GRANULAR LEUKOCYTES. 159 cells in which we find certain animal cells and pigment phagocytized, as is the case in malaria. These cells are the macrophages of Metch- nikoff and are probably derived from the bone marrow. The lymphocytes take origin from the lymphoid tissue, and very probably the large lymphocyte is a younger, more immature cell than the small lymphocyte. Consequently, we probably have marrow lymphocytes and gland lymphocytes. In addition to the series of leukocytes just considered we have present normally in the blood three types of granular cells distinguished according to the staining affinity of their granules. These are : 1 . Polymorphonuclear leukocytes. This cell normally constitutes the greater proportion of the leukocytes. It is an amoeboid, actively phagocytic cell, about 10 or i2/x in diameter, and is the microphage of Metchnikoff. Bacteria are actively phagocytized by this cell, and it is the cell concerned in determining the opsonic power of blood to various bacteria. It has fine lilac granules which are termed neutrophilic. (epsilon granules). The single nucleus is rich in character and is lobose like the kernel of an English walnut; frequently it resembles the letter z. These cells are derived from the neutrophilic myelocytes of the bone marrow. It is in these cells that the glycogen, or iodophil, granules appear in certain suppurative conditions. 2. Eosinophile Leukocytes. — These are very striking cells with coarse granules staining brilliantly pink, the eosinophile, oxyphile or acidophile granules (alpha granules of Ehrlich). The cells are a little larger than the polymorphonuclears. The normal eosinophile is to be distinguished from the eosinophilic myelocyte by its possessing two distinct lobes in the nucleus. The nucleus of the myelocyte is round. The eosinophile is the cell so frequently increased in infections by intestinal animal parasites. 3. Mast Cells. — These also have coarse granules, but they stain a deep violet blue. Hence they are basophile granules (gamma granules). In fresh blood these granules do not show up very well, thus they can be distinguished from the highly refractile granules of the eosinophile. The-tri-lobed nucleus stains less intensely than the granules. As a rule, the mast cell is about the size of a pol\ morphonuclear. l6o NORMAL AND PATHOLOGICAL BLOOD. In a differential count of normal blood we find about the following percentages. Polymorphonuclears, 65 to 70%, About 5000 per c. mm. Small lymphocytes, 20 to 25%, About 1500 per c. mm. Large lymphocytes, 5 to 10%, About 500 per c. mm. Large mononuclears, i to 2%, About 100 per c. mm. Transitionals, 2 to 4%, About 200 per c. mm. Eosinophiles, i to 2%, About 100 per c. mm. Mast cells, 1/4 to 1/2%, About 25 per c. mm. The leukocytes which are found in the peripheral circulation only in pathological conditions are: 1. Neutrophilic Myelocytes. — The common type is a large cell with a large centrally-placed, feebly-staining nucleus. This may be recognized by the difficulty of distinguishing the nucleus from the cytoplasm, there being no sharp line separating these parts of the cell. They imperceptibly merge into one another. They differ from a large mononuclear in that the cytoplasm is distinctly dotted with neutrophile granules, and that we cannot make out a distinct line of separation of a slightly irregular or indented nucleus from the sur- rounding slightly neutrophilic cytoplasm. Cornil has described a very large myelocyte with eccentrically-placed nucleus and neutrophilic granules. Myelocytes are at times found with both basophilic and neutrophilic granules, and may rarely be seen to have all three kinds of granules on a single myelocyte, acidophile, basophile and neutrophile. 2. Eosinophilic Myelocytes. — These can be distinguished from normal eosinophiles by their possessing a single round nucleus, not bilobed. These myelocytes may be as large as a normal eosinophile, but frequently are no larger than a red cell. The neutrophile myelocyte is characteristic of spleno-myelogenous leuka&mia, the eosinophile one of myelogenic leukaemia. The oc- currence of an occasional myelocyte is frequently noted in conditions having a Jeukocytosis. In diphtheria their presence in numbers is of bad prognostic import. Myelocytes are of diagnostic importance in metastases of malignant tumors. BLOOD PLATELETS AND HEMOKONIA. l6l 3. The Irritation Cell of Turck, or Plasma Cell.— This cell has a faintly-staining, eccentrically-placed nucleus, and a dark opaque blue, frequently vacuolated, cytoplasm. They are usually recorded as large mononuclears. BLOOD PLATELETS. These are normally present in blood in the number of about 350,000 per cubic millimeter. They disintegrate very quickly after the blood is withdrawn. Wright has demonstrated that they are pinched-off projections of giant cells of the bone marrow. They consist only of protoplasm, no nuclear material. They do not contain haemoglobin. In conditions where giant cells are less abundant, as in pernicious anaemia, the blood platelets are less abundant. In myelogenous leukemia they are very abundant. They vary in size from 2 to 5/1 according as a larger or smaller pseudopod of a giant cell has been broken off. Stained with Wright's stain, they are more purplish than blue and show thread-light projections. They are often mistaken for the protozoal causes of various diseases. Especially are they confused with malarial parasites when lying on a red cell. The blood plate has no brick-red chromatic material; it is purplish rather than blue, and has no pigment grains. It is advisable to compare these isolated blood- plates writh the larger or smaller aggregations scattered about the smears. In this way their true character is apparent. In addition to blood platelets, which in fresh blood can only be observed when a fixative is used, we have other confusing bodies. The hemokonia of Muller are small, highly refractile bodies showing active oscillatory movement. They are supposed to be cast-off granules of eosinophiles or other leukocytes. Pinched off fragments of red cells may also appear as possible protozoal bodies. LEUKOPENIA. This is a term used to designate a reduction in the normal number of leukocytes. A leukocyte count of 5000 would represent a slight leukopenia; one of 2000, a marked leukopenia. In the later stages of typhoid, and in acute miliary tuberculosis, we expect a moderate leukopenia. Chronic alcoholism and chronic arsenic poisoning cause 162 NORMAL AND PATHOLOGICAL BLOOD a reduction in the number of the white cells. Pernicious anaemia shows a marked leukopenia, as is also the case with Banti's disease. Two tropical diseases, Kala-azar and dengue, show a marked leuko- penia, the counts often being below 2500. During the apyrexial period of malaria we may have a white count of 5000. EOSINOPHILIA. Where the eosinophiles are increased to 5%, we have a moderate eosinophilia. In some cases of infection with intestinal parasites, especially hook-worms, but also from other parasites, as round and whip-worms, we may have an eosinophilia of 30 to 50%. In Guam, among the natives, it is difficult to find an eosinophile count under 15%. The eosinophilia of trichinosis is best known, and a combination of this blood finding with fever and marked pains of muscles, would justify the excision of a piece of muscle for examination for encysted embryos. In true asthma eosinophilia is marked, and its absence is of value in indicating other causes for the condition. Certain skin diseases, especially pemphigus, also show eosinophilia. LEUKOCYTOSIS. It is to an increase in the polymorphonuclears that this term is usually applied, the term lymphocytosis or eosinophilia being em- ployed where white cells of eosinophile or lymphocyte nature are increased. We have physiological leukocytosis in the latter weeks of pregnancy, also in the new-born, and in connection with digestion. Pathological Leukocytosis. — Pneumonia. In this disease we have a leukocytosis of 20,000 to 30,000 or higher. The eosinophiles are almost absent. A normal leukocyte count in pneumonia makes a prognosis unfavorable. Septic processes. The leukocyte count is of great value, especially when we obtain a leukocytosis with 80 to 90% of polymorphonuclears, as in appendicitis, cholecystitis or other suppurative conditions. According to Cabot, leukocytosis varies in infections as follows: i. Severe infection — good resistance; early marked and persistent leukocytosis. LEUKOCYTOSIS. 163 2. Slight infection — slight resistance; leukocytosis present, but not marked. 3. In fulminating infections \ve may have no increase in whites, but a higher percentage of polymorphonuclears. 4. Slight infection and good resistance may not be pro- ductive of leukocytosis. Spirochaeta fevers, as relapsing fever, may give a leukocytosis of from 25,000 to 50,000. Small-pox, especially at time of pustulation, plague, scarlet fever and liver abscess give a leukocytosis of from 12,000 to 15,000. FIG. 52. — Leukocytosis (40,000); sixteen polymorphonuclears in field. (Cabot.) Erysipelas and epidemic cerebrospinal meningitis also give a leukocytosis of from 15,000 to 20,000. In malignant diseases we some- times have a moderate leukocytosis. Rogers states that in liver abscess, with a leukocytosis of 15,000 to 20,000, we have onlyabout 75 to 77% of polymorphonuclears — there being also a moderate increase in the percentage of large mononuclears. LYMPHOCYTOSIS. Of course, the disease in which we have the most marked lymphocy- tosis is lymphatic leukaemia. Whooping-cough may give a lymphocytosis of 20,000 to 30,000. 164 NORMAL AND PATHOLOGICAL BLOOD. Young children have normally an excessive proportion of lympho- cytes. This is apt to be particularly marked in hereditary syphilis. Enlarged tonsils may give rise to a lymphocytosis of 10,000 to 15,000, when more than 50% of the white cells will be lymphocytes. Rickets and scurvy give a lymphocytosis. DISEASES IN WHICH THERE is A NORMAL LEUKOCYTE COUNT. Uncomplicated tuberculosis, influenza, Malta fever, measles, trypanosomiasis, malaria, syphilis and chlorosis. In malaria we have a leukocytosis at the time of the rigor, while during the apyrexial period there is a moderate leukopenia. In malaria we have a marked increase in the percentage of the large mononuclears and transitionals. These may form from 25% to 35% of the leukocytes. When bearing particles of pigment they are known as melaniferous leukocytes — macrophages which have ingested malarial material. In dengue, at the time of the terminal rash, we may have as great a percentage of large mononuclears. In this disease, however, we have a great dimi- nution of polymorphonuclears from the start (25 to 40%). Instead of a large mononuclear we have at the onset a lymphocytic increase. There is an increase of large mononuclears in trypanosomiasis. THE PRIMARY ANEMIAS. Chlorosis. — In chlorosis it is the reduction of haemoglobin with the slight numerical variation from normal of the red cells that makes for a diagnosis. The color-index is very low. There is nothing abnormal about the leukocytes. Microcytes may be present, and very occasion- ally a normoblast. Macrocytes and megaloblasts are always absent. Blood of chlorotics is very pale and very fluid and coagulates rapidly, hence frequency of thrombosis. Simple Primary Anaemia. — This condition is not recognized by many authors, but is a convenient term under which to group anaemias which are neither chlorosis nor pernicious anaemia and for which no assignable cause can be designated. It is a secondary anaemia with- out a cause. In it color-index is about normal, there is no change in the leukocytes and cases go on to recovery. Pernicious Anaemia. — In pernicious anaemia we obtain a very PERNICIOUS ANAEMIA. 165 fluid, but normally-colored drop of blood upon puncture. The yellow marrow of the long bones is transformed into a soft, bright red lymphoid tissue, smears from which show great numbers of megaloblasts. Areas of fatty degeneration are characteristic, especially the tiger-lily spots in the heart muscle. Iron-containing pigment (hemosiderin) is found in the liver, spleen and kidneys. Areas of degeneration in the spinal cord may account for nervous symptoms. The red cells fre- quently fall below 2,000,000 with patients going about. Cases have been reported with counts under 200,000. The color-index is high. Megaloblasts are the most characteristic qualitative change in the red FIG. 53. — Pernicious anaemia. M.m, Megaloblasts; n, normoblast; s, stippling (punctate basophilia). (Cabot.) cells. Megaloblastic crises may at certain times show enormous numbers of 'megaloblasts. Cases often present remissions in which no megaloblasts can be found. In such cases the presence of many macrocytes should prevent an examiner reporting against a pernicious anaemia previously diagnosed. Poikilocytosis, polychromatophilia and stippling are also features of the disease. Normoblasts are far less frequent than megaloblasts and there is usually a moderate lymphocytosis. Myelocytes may be present. Cases of pernicious anaemia show remissions during which the patient is apparently on the road to recovery. Such improvements are only temporary. The remissions may last from two months to 1 66 NORMAL AND PATHOLOGICAL BLOOD. possibly three or four years. Especially in the anaemia of Dibothrio- cephalus latus do we have a picture of pernicious anaemia. It is supposed to be due to a toxin present in the heads of these tape- worms. \ SECONDARY ANEMIAS. These are the anaemias which can be definitely traced to some disease not of the haemopoietic system. In some secondary anaemias, as in syphilis, carcinoma and tuberculosis, we have a chlorotic color- index (chloro anaemias). In secondary anaemias polychromatophilia, poikilocytosis and punctate basophilia (stippling) may be present. This latter is very marked in lead poisoning, but in certain cases of malarial cachexia it may be equally prominent. The only form of nucleated red cell seen is the normoblast, in very small numbers, or it may not be present, Megaloblasts are practically never seen, except in some of the very severe parasitic anaemias, as the broad Russian tape-worm infection. The red cells generally number between 2,000,000 and 4,000,000, thus differentiating chlorosis. The leukocytes are frequently increased to 15,000. In the anaemia of splenic anaemia there is a marked leuko- penia. In anaemias from malignant tumors the color-index is usually of the chlorotic type — the haemoglobin content of the red cells being more affected than the number. Normoblasts are usually present, and this finding may differentiate gastric cancer from ulcer. In bone marrow metastases megaloblasts may be expected. Myelocytes and so-called tumor cells (large cells with faintly-staining vacuolated nuclei and but little cytoplasm) may also be found. As a rule, there is a moderate leukocytosis in malignant disease. Eosinophiles may be largely increased in sarcoma. THE LEUKAEMIAS. It is in the leukaemias that we have the greatest increase in the num- ber of white cells. These cases show more or less anaemia, but we may have cases of myelogenous leukaemia showing 250,000 leukocytes per cubic millimeter without particular change in the red cells. The more marked the red-cell change the more severe the condition. There are two well-defined types of leukaemia, the lymphatic and the MYELOGENOUS LEUKEMIA. 167 splenomyelogenous. It must be borne in mind, however, that while a greater change in the lymphatic glands may produce the lymphatic type, yet even in such cases we expect to find alteration in bone marrow and spleen; that is, there is a general involvement of the haemopoietic system in all leukaemias, the activity being most marked in spleen and bone marrow in certain cases and in lymphatic glands in others. Myelogenous leukaemia is a very rare disease, about five times as rare as pernicious anaemia. Lymphoid leukaemia is still more rare. Splenomyelogenous Leukaemia (myeloid leukaemia). — The differentiation of the blood picture of this disease from leukocytosis FIG. 54. — Myelogenous leukaemia, m, Myelocyte; p, polymorphonuclear; /, mast cell; n, normoblast. (Cabot.) does not depend on the number of leukocytes, but on the presence and large proportion of myelocytes. We expect both neutrophilic and eosinophilic myelocytes in myeloid leukaemia — the proportion of these varies, but, as a rule, the neutrophilic one is the common one. The blood in advanced cases is milky and shows a most marked buffy coat. The marrow is largely replaced by a yellow pyoid material. The spleen may weigh ten pounds. The leukocyte count is on the average from 200,000 to 500,000. Cases are recorded of more than one million white cells. The neutrophilic myelocytes make up about thirty to forty per cent, of these and, about equal in number, are found the 1 68 NORMAL AND PATHOLOGICAL BLOOD. polymorphonuclears, while the percentage of the lymphocytes is decreased (2 to 5%) and normal eosinophiles, eosinophilic myelocytes, and large mononuclears make up the remaining percentages. We usually have great numbers of normoblasts. Megaloblasts may be rarely found. The red count is usually about 2,500,000 and the color- index low. Lymphatic Leukaemia. — In this we have glandular enlargements, but not such large masses as in Hodgkin's disease. The red cells are usually reduced about one-half and the color-index is a little below normal. Normoblasts are rarely found. Myelocytes, as a rule, are FIG. 55. — Lymphatic leukaemia, p, polymorphonuclear; m, megaloblast; e, eosinopm'le. Twenty-one lymphocytes in this field. (Cabot.} absent, but may amount to 5% of the leukocytes. The predominating leukocyte (75 to 98%) is the small lymphocyte. In acute lymphatic leukaemia the large lymphocytes predominate. The leukocyte count is never so great as in myeloid leukaemia, rarely exceeding 125,000. Hodgkin's disease is usually considered as a disease with marked glandular enlargements, but with a negative blood picture. Undoubt- edly the view that so-called lymphosarcomata, lymphatic leukaemia and Hodgkin's disease merge into one another and that they represent a malignant cell formation in the haemopoietic system is the conservative one to take. NOTES ON BLOOD WORK. NOTES ON BLOOD WORK. NOTES ON BLOOD WORK. NOTES ON BLOOD WORK. PART III. ANIMAL PARASITOLOGY. CHAPTER XV. GENERAL CONSIDERATIONS OF CLASSIFICATION AND METHODS. ANIMALS that are in all respects alike we term a Species. Of course the male and female of a species may be very unlike, but as a result of mating they produce young having characteristics similar to the parents. Now, if, as in the case of the mosquitoes causing yellow fever, we find some with straight silvery lines and others uniformly showing crescentic silvery bands about thorax, yet resembling each other closely in the respect of being dark, brilliantly-marked mosquitoes, we should consider them as being separate species with a certain relationship to which the term Genus is applied. In naming a species we always first write the name of the genus, which has a Greek or Latin name, com- mencing with a capital, and follow with the specific term, which latter commences with a small letter. Thus we designate the dark silver- marked mosquitoes as belonging to the genus Stegomyia; those showing the characteristics of curved silver bands on dorsal surface of thorax we designate as Stegomyia calopus ; the species with the straight silver lines we call Stegomyia scutellaris. Again, certain genera show resemblances which enable us to make broader groupings to which we apply the name Subfamily. Thus the genus Stegomyia and the genus Culex have the similar characteristics of palpi in the female being shorter than the straight proboscis ; we there- fore classify all species of Stegomyia and all species of Culex under the designation Culicinae. The name of a subfamily ends in "inae." Now, 12 169 170 CONSIDERATIONS OF CLASSIFICATION AND METHODS again, certain insects are different from others in having scales on the wings. We find that not only do the Culicinae have such character- istics, but the same is observed with the Anophelinae and other similar scale-wing insects. All of these we term a Family and we speak of the Culicidae, meaning the family of mosquitoes. The name of a family ends in "idae." Many families are not subdivided into subfamilies, but are directly separated into genera. Again, a genus may have only a single species. When there are a number of families agreeing closely in some striking characteristic, we group them together into an Order; thus, the family of mosquitoes closely resembling many other families of insects in possessing a pair of well-developed wings are grouped in the order Diptera; all of which resemble certain other animals in the possession of a distinct head, thorax and abdomen with 3 pairs of legs projecting from the thorax. This collection of animals we call a Class; thus, we speak of the class Insecta. It will be observed that the insects have no internal skeleton, but instead a chitinous cuticle, the exoskeleton. Spiders, ticks, etc., resemble them in this respect, and we now apply to all such animals the wider designation, Branch or Phylum. Inasmuch as the animal kingdom is divided into the branches Protozoa, Porifera, Ccelenterata, Echinodermata, Vermes, Arthropoda, Mollusca and Chordata, we see that the branch is the largest grouping we employ. To descend in the scale we have belonging to the branch, the classes; to the class, the orders; to the order, the families; to the family, the subfamilies; to the subfamily, the genera; to the genus, the species. Occasionally a species is further divided into subspecies. There are certain terms employed in animal parasitology which it is necessary to understand. Among these we shall refer to the follow- ing: i. True Parasitism. By this is understood the condition where the parasite does harm to the host, deriving all the benefit of the association. A good example of this would be the hook-worm infecting man or animals. 2.!*Mutualism. In such an association there is mutual benefit to each party of the association. An instance of this would be the presence of colon bacilli in the intestines. The bacillus is furnished ZOOLOGICAL -NOMENCLATURE. I 7 1 a suitable habitat and in return protects its host against strictly pathogenic bacteria. 3. Commensalism. Here there is benefit to the parasite, but no injury to the host. An example of this kind would be furnished in the case of the Trichomonas vaginalis which lives in the vaginal mucus, but, so far as known, does no injury to the host. 4. Nomenclature. When the thousands of different species, genera, etc., of animals is considered, it will be readily perceived that, unless some system existed for their designation, indescribable con- fusion would prevail. To avoid this, the International Code, based on the rules of Linnaeus (1751), requires Latin or Latinized names. There are certain rules governing the naming of animals. Of these, the law of priority provides that the oldest name, under the code, of any genus or species is its proper zoological name. The history of the naming of the organism of syphilis illustrates this well. Schaudinn gave this organism in 1905 the name of Spirochaeta pallida. Ehren- burg, in 1838, had used the name Spirochaeta for animals of a different character, so that this designation of the genus was not permissible under the code. Villemin, a little later, proposed the name of Spiro- nema. This term, however, was found to have been used in 1864. Consequently it was not available. Stiles then proposed the name Microspironema but as Schaudinn only about two weeks before had offered the designation Treponema, the name Treponema pallidum had to be accepted as the proper zoological name for the organism of syphilis. Another point is that names are not definitions, consequently the fact of lack of appropriateness of any name is no objection to its con- tinuation. This will appeal to anyone as a wise provision, because if a different name were substituted each time a designation more descrip- tive or applicable were invented it would be utterly destructive to system. When it is considered that some of our parasites have approxi- mately 50 different designations, for the most part given by medical observers, it will be appreciated how much the zoologist has aided us in trying to eliminate all but the single proper zoological name. The objections so frequently heard among physicians in connection with adopting new names for old ones are not well founded. Wher- 172 CONSIDERATIONS OF CLASSIFICATION AND METHODS. ever confusion has reigned, the establishment of order always results in temporary greater confusion. There is no doubt that the student taking up this subject a few years hence will have the satisfaction, thanks to the zoolo'gist, of only having to burden his mind with one name^for one parasite. 5. Terminology. This applies to appropriate designations for different organs, symptoms, etc., and is not subject to any rule other than that of good usage. Class CHAPTER XVI. THE PROTOZOA. CLASSIFICATION OF PROTOZOA Order Gmnamceba- Genus Species • f E. coli Entamceba j E. histolyticax [ E. buccalis Leydenia L. gemmipara *• S. recurrentis S. vincenti Spirochaeta S. duttoni S. carteri S. refringens Treponema Vv/V T. pallidum T. pertenue *, Trypanosoma l^a* ' Tnchomonas . T. gambiense~ f T. vaginalis \ T. intestinalis Lamblia L. intestinalis Babesia B. bigemina Leishmania . ^-fKcWg, f L. donovani \ L. tropica Balantidium B. coli E. stiedae Isospora I. bigemina (P. vivax Plasmodium P. malaria? -J, P. falciparum X Rhkopoda (Sarcodina) These throw out proto- plasmic projections called pseudopodia. Flagella'ta (Mastigophora) These move by means of undulating membranes or flagella. Infusoria Heterotricha (Ciliata) These have contractile vacuoles and numerous fine cilia which are shorter than flagella and have a sweeping stroke. Spopozoa Coccidiaria These have no motile organs. They live para- sitically in the cells or tissues of other animals. Haemosporidia Reproduction by spores. THE PROTOZOA. RmzopoDA (SARCODINA). In this class of protozoa the pseudopodia serve the double purpose of nutrition and locomotion. These protoplasmic extensions may be 173 174 THE PROTOZOA quite broad or very narrow. As a rule, the thicker the pseudopod the more rapid the movement. Some rhizopods have hard shell-like coverings which are secreted in or on the ectosarc. These skeletons have openings through which the pseudopods project. The pseudopo- dia may be made up only of ectoplasm or both ectoplasm and endoplasm may take part. Amoeboid movement always starts in the ectoplasm. In FIG. 56. — Various protozoa. i, Entamoeba coli; 2, Entamoeba histolytica; 3, Leydenia gemmipara; 4, Trichomonas vaginalis; 5, Trichomonas intestinalis; 6, Lamblia intestinalis; 7, flagellated Leishmania donovani; 8, Leishmania donovani in phagocyte; 9, Eimeria stiedae; 10, Isospora bigemina; n, Trypanosoma gam- biense; 12, Balantidium coli. addition to the nucleus, which the so-called chromatin staining methods bring out as reddish areas, we frequently observe smaller aggregations of chromatin staining material in the cytoplasm. This extranuclear chromatin is supposed to play a part in the more intricate divisions which such protozoa undergo. Food vacuoles and contractile vacuoles are present in many rhizopods. INTESTINAL AMCEB^. 175 Entamoeba coli. — This is considered by Schaudinn to be a harm- less inhabitant of the intestines and its presence in the faeces is not considered of importance. Musgrave and Clegg do not recognize a distinction between a nonpathogenic and a pathogenic amoeba, but consider that the presence of amoebae, in the absence of symptoms, is to be explained by the nonestablishment of a satisfactory symbiosis with some bacterium or other parasite. They state, that as a result of extended observation, persons harboring amoebae will sooner or later develop dysentery. As regards the morphological points of distinc- tion, they state that even in pure cultures, descended from a single amoeba, the same variations in size motility, etc., may be observed. They also consider that amoebae having all the characteristics of the harmless commensal may cause marked pathological change. Craig claims that the E. coli cannot be cultivated and, that several years since noting E. coli in stools of healthy persons, these persons have remained free of any dysenteric symptoms. The only safe way in recognizing amoebae in stools is to note amoeboid movement. The encysted amoebae can scarcely be differ- entiated from many vegetable cells and especially from large phago- cvtic cells, of probable endothelial origin. By the use of neutral red in very dilute solution the granular endoplasm will be observed to take up the brick-red stain. E. coli varies greatly in size (8 to 40/1). There is no well-marked distinction between a granular interior and a more compact, hyaline exterior. The nucleus is centrally situated, is distinct and on staining with Wright's stain shows the chromatin coloration. It is sluggishly motile and is of a grayish-white color. When stained it does not show a distinction between endoplasm and ectosarc. The infecting stage is an encysted form with 8 amcebulae. Entamceba histolytica. — This is considered the pathogenic amceba. Schaudinn considers that it is by the possession of its tough, tenacious, glassy and highly refractile ectoplasm that it is able to bore its way into the submucosa of the large intestine and bring about those gelatinous like necroses, which, by undermining, even- tually result in dysenteric ulcerations. It is also the species found in tropical liver abscess. As described 176 THE PROTOZOA by Schaudinn, it has a marked differentiation between the glassy ectoplasm and the granular endoplasm. The nucleus is indistinct, eccentric and stains feebly. The movement is more active and the color more greenish-yellow than E. coli. Craig notes the character- istic staining of the E. histolytica, this being a dark blue ectoplasm encircling a lighter blue endoplasm. In dividing, there is a process of budding. These little spore-like bodies form at the periphery of the encysted amoeba and form the infecting stage. Cold or other inimical influences tend to make amoebae encyst, hence faeces should be ex- amined as soon as possible after the stool is passed. A particle of mucus pressed down with a cover-glass makes a satisfactory prepara- tion. If necessary to dilute, use blood-warm salt solution — not plain water. Entamoeba buccalis. — Obtained from the mouths of persons with dental caries. It does not appear to have pathogenic character- istics. Castellani has reported an intestinal amoeba with an undulatory membrane. He has given it the name of E. undulans. Leydenia gemmipara. — It is a question whether these bodies were animal parasites or simply body cells showing amoeboid movement. They were found in the ascitic fluid of 2 cases of carcinomatosis. They varied in size from 3 to 36 //. FLAGELLATA (MASTIGOPHORA). In this class of protozoa the adults have flagella for the purposes of locomotion and the obtaining of food. Some flagellates more or less resemble rhizopods in being amoeboid and in having an ectoplasm and an endoplasm. The body is fre- quently covered by a cuticle. Some flagellates have a definite mouth part, the cytostome, which leads to a blind oesophagus ; others absorb food by absorption through the body wall. In addition to flagella, some flagellates possess an undulating membrane. All flagellates possess a nucleus and some have contractile vacuoles. The flagellum may arise directly from the nucleus or from a small kinetic nucleus, the blepharoplast. The most important flagellates of man are the haemoflagellates. RELAPSING FEVER. 177 Among these we may include the blood spirochaetes and the organism of syphilis, which have many resemblances to the spiral form of bac- teria, together with the three genera in which protozoal characteristics are marked, namely, Leishmania, Trypanosoma and Trypanoplasma. In addition we have flagellates in the intestinal canal and in the vaginal secretion. Some authors place the genus Piroplasma with the flagel- lates and there has been controversy concerning the nature of certain projections from these bodies. It would seem preferably however, to consider them under the sporozoa. FIG. 57. — Spirilla of relapsing fever from blood of a man. (Kolle and Wassermann.) Spirochaeta. The generic term Spirochaeta is applied to flagellates having a spiral shape, an undulating membrane and no flagella. This genus is one about which there are two views: one, that the members belong to the bacteria; the other, that they are protozoa. The absence of demonstrable nucleus and blepharoplast make them apparently vegetable in nature while the variations in thickness, the fact of trans- mission by an arthropod and indications of a longitudinal, rather than a transverse division, would indicate protozoal affinities. S. recurrentis.— This is the organism of relapsing fever. It was 178 THE PROTOZOA. formerly considered a bacterium and was termed the Spirochaeta obermeieri. It is present in the blood of persons suffering from the disease during the pyrexia. During the apyrexia they are not found in the peripheral circulation. At this time they are present in great numbers in the spleen where they are actively phagocytized. The disease is supposed to be transmitted by bed-bugs or lice. Monkeys are susceptible and, after passage through monkeys, rats can be inoculated. S. duttoni. — This is the cause of South African tick fever or Tete fever. The disease is similar to relapsing fever, but there are generally four or five febrile paroxysms with apyrexial intervals. The disease is readily transmitted to ordinary laboratory animals, especially the rat. Rats which have recovered from S. recurrentis can be infected by S. duttoni and vice versa. The disease is transmitted by the bite either of the adult or larval Ornithodoros moubata. Koch found spirochaetes in the eggs of the ovaries of ticks which had fed on persons with the disease. It is thus an instance of hereditary transmission. Other spirochaetes that have been considered as pathogenic for the type of relapsing fever in India and that of America are the S. carteri and the S. novyi. S. vincenti. — This is a very delicate spiral-shaped organism which has been found in conjunction with a fusiform bacillus in a throat inflammation, usually termed Vincent's angina. S. refringens. — This Spirochaeta is frequently associated with the Treponema pallidum and is common in genital ulcerations. It is thicker, has less regular and more flattened curves and stains more readily. Treponema. The genus Treponema has no undulating membrane and has a flagellum at each end. Treponema pallidum. — This is the cause of syphilis. It is char- acterized by its very geometric regularity in the spirals, which are deeply cut, and in focussing up and down continue in focus (like a corkscrew). They require from one to two hours to stain distinctly with Giemsa's stain and the attenuated ends or flagella should always YAWS. 179 be noted before reporting their presence. To stain them in section Levaditi's method is the best. T. pertenue. — An organism of similar morphology was first reported by Castellani as present in yaws. It is found in smears and sections as with T. pallidum. FIG. 58. — The spirochaeta refringens is the larger and more darkly stained organism, while the lightly stained and more delicate parasites is the Spirochaeta pallida. (Treponema pallidum). From a chancre stained with Wright's blood stain. (Hirsch — by Rosenberger.) Trypanosoma. The genus Trypanosoma has a more or less spindle-shaped body, along one border of which runs an undulating membrane. There is one flagellum bordering the membrane and projecting like a whip l8o THE PROTOZOA. posteriorly. There is a nucleus (macronucleus) and a blepharoplast (micronucleus — centrosome), the latter being located anteriorly as a chromatin staining dot or rod. From this blepharoplast the flagellum proceeds posteriorily bordering the undulating membrane and pro- jecting freely beyond the posterior end. The nucleus is larger, nearer the posterior end, and does not stain so intensely as the blepharoplast. T. gambiense. — This is the trypanosome causing human trypano- somiasis, the latter stage of which is known as sleeping sickness. It is from 17 to 28/1 long, and from 1.5 to 2/z wide. It is very difficult to distinguish the human trypanosome from some of the other pathogenic ones by staining methods. The immunity test is the most reliable. An animal recovered from an infection by a certain trypanosome does not possess immunity for other pathogenic ones. Novy and McNeal cultivated T. lewisi in water of condensation on blood agar, but up to the present T. gambiense has not been culti- vated. It is present in the blood, usually in exceedingly small numbers, and in the lymphatic glands of patients. It is by puncture of the glands that we have the best means of finding the parasites. It is also found in the cerebrospinal fluid in sleeping sickness. The parasite stains readily with Wright's stain. The transmitting agent is the Glossina palpalis. It is not known whether this occurs by direct or indirect transmission. At any rate, there must be some peculiarity about the tsetse fly, either in the reaction of its salivary secretion or otherwise, to make it the only well-recognized agent in the spread of the disease. No tsetse fly, no trypanosomiasis. Koch observed certain developmental forms in the bulb of the proboscis, but whether they represented a developmental cycle or not is not settled. Other authorities think it possible that trypanosomes may encyst in the digestive tract and so the flies transmit the disease along with their faeces. This does not seem to be possible in connection with human infections. Koch found several cases where infection had taken place by coitus. This is the method of infection in T. equiperdum, a trypanosome disease of horses. Of the more important trypanosome diseases of animals may be mentioned : KALA AZAR. l8l 1. Nagana. Pathogenic for domestic animals. T. brucei. 2. Surra. Pathogenic for horses in India and Philippines. T. evansi. 3. Dourine. Transmitted by coitus in horses. T. equiper- dum. 4. Mai de caderas. Affects horses in South America. T. equinum. A harmless infection, especially in sewer rats, is due to T. lewisi. There are many trypanosomes in birds, fish, frogs, etc. Trypanoplasma. The genus Trypanoplasma has a rather large blepharoplast, from which arise two flagella. One extends forward as a free anterior flagellum, while the other projects posteriorly, running along the border of the undulating membrane. This Genus is not known for man. Leishmania. The genus Leishmania includes two species: one, the L. donovani, the parasite of kala azar, and the other the L. tropica, the parasite of oriental sore. These are undoubtedly different species, inasmuch as in sections of India, where tropical ulcer was common, there was no kala azar, and in Assam where kala azar prevailed there were no Leishman-Donoyan bodies to be found in smears from the tropical ulceration there present, except rarely in cases of general infection. L. tropica has not been cultivated. These parasites are typically intracellular, being within either polymorphonuclears, which contain only one or two of the bodies, or in large mononuclears, in which there may be as many as six. They may be packed, however, in phagocytic endothelial cells. The parasites occur in the peripheral circulation in about 80% of the cases. They abound in the liver and spleen. The parasite is oval and about 2x3^. There are two distinct chromatin staining masses. The larger nu- cleus is more or less spherical and stains faintly, while the smaller chromatin mass is generally rod-shaped and stains intensely. It has been recently recommended that instead of liver or splenic puncture 1 82 THE PROTOZOA. for the demonstration of these bodies, a blister be raised and a smear from that containing many polymorphonuclears might show these bodies. The affection is characterized by a leucopaenia so that it is very difficult to demonstrate the parasites in ordinary blood smears. By cultivating the parasites obtained from splenic puncture in acidified sodium citrate solution at room temperature, Rogers suc- ceeded in obtaining flagellated forms similar to Herpetomonas. ' An anterior flagellum proceeds directly from the blepharoplast. The bed-bug is supposed to be the intermediary host. Trichomonas. Trichomonas vaginalis. — This parasite has a fusiform body and is about 18 x 10/1. It has three flagella arising from the anterior end and an undulating membrane. It lives in vaginal mucus which has an acid reaction. A change of reaction, as at menstrutation, causes them to disappear. Forms similar to the T. vaginalis have been found in the intestine and in sputum from putrid bronchitis. These flagellates are generally considered harmless,, although doubt as to this is expressed by some authors. Lamblia. Lamblia intestinalis. — These parasites are about 10 x 15^ and have a pear shaped body with a depression at the blunt anterior end. This depression enables the flagellate to attach itself to the summit of an epithelial cell. Around the depression are three pairs of flagella which are constantly in motion. Another pair of flagella project from either side of the blunt little tail-like projection. When stained, the parasites have a pyriform shape with two chromatin staining areas on either side of the anterior end. When encysted, they assume a circular shape. This parasite is generally considered as of little importance, but inasmuch as, when in great numbers in the csecum and appendix, they may give rise to symptoms resembling appendicitis and as they are responsible for a chronic and intractable diarrhoea associated with mental and physical depression, this is undoubtedly an affection only minor in importance to amoebic infection. It is a very common infection in the tropics. SPOROZOA. 183 INFUSORIA (CILIATA.) The bodies of Infusoria are oval and may be free or attached to a stalk-like contractile pedicle, as with Vorticella, or they may be sessile. The cilia, which are characteristic, may be markedly developed around the cystostome (mouth) and serve the purpose of directing food into the interior, while others act as locomotor organs. The body is enveloped by a cuticle which may only have one opening or slit, to serve as mouth ; or it may have a second one, a cytopyge or anus. Usually the fecal matter is ejected through a pore which may be visible only when in use. They usually have a large nucleus and a small one. Infusoria tend to encyst when conditions are unfavorable. Balantidium coli. — This is the only ciliate of importance in man. It is a common parasite of hogs. It is from 60 to loofj. long by 50 to 70 ,« broad, and has a peristome at its anterior end which becomes narrow as it passes backward. It has an anus. The ectosarc and the endorsarc are distinctly marked. These parasites cause an affection similar to dysentery and may bring about a fatal termination. It is almost impossible to escape noticing the actively moving bodies if a fecal examination is made. When encysted they are round. Another ciliate, the Nyctotherus faba, has a kidney -shaped body and is about 25 by 15;*. It has a large contractile vacuole at the posterior end. It has a large nucleus in the center with a small fusi- form micronucleus lying close to it. It has only been reported once for man. SPOROZOA. This class of protozoa gets its name from the method of reproduc- tion— sporulation. These parasites rarely show binary fission. While the sporozoa are found within cells, in the tissues and in internal cavities, as intestine and bile ducts, yet it is as inhabitants of the blood that they have their greatest importance for man — these are known as Haemosporidia. A sporozoon may be either naked or amoeboid or be covered with a distinct cuticle. Coccidiaria. The parasites of the order Coccidiaria are almost exclusively found in the intestines and in the organs connected with it. In the vegetative DESCRIPTION OF PLATE I. (Kolle and Wassermann.) Malarial Parasites. 1. Two tertian parasites about thirty-six hours old, attacked blood-cor- puscles magnified. 2. Tertian parasite about thirty-six hours old; stained by Romanowsky's method. The black granule in the parasite is not pigment but chromatin. Next to it and to the left is a large lymphocyte, and under it the black spot is a blood plate. 3. Tertian parasite, division form nearby is a polynuclear leukocyte. 4. Quartan parasite, ribbon form. 5. Quartan parasite, undergoing division. 6. Tropical fever parasite. (^Estivo-autumnal.) In one blood -corpuscle may be seen a smaller, medium, and large tropical fever-ring parasite. 7. Tropical fever parasite. Gametes half moon spherical form. Smear from bone marrow. 8. Tropical fever parasite which is preparing for division heaped up in the blood capillaries of the brain. Asexual Forms. 9. Smaller tertian ring about twelve hours old. 10. Tertian parasite about thirty-six hours old, so-called ameboid form. 11. Tertian parasite still showing ring fever, forty-two hours old. 12. Tertian parasite, two hours before febrile attack. The pigment is beginning to arrange itself in streaks or lines. 13. Tertian parasite further advanced in division. Pigment collected in large quantities. 14. Further advanced in the division. (Tertian parasite.) PLATE I. MALARIA. 185 stage it lives within an epithelial cell, which it destroys. Afterward it falls into the lumen lined by this epithelial cell and sporulates, either by the method of schizogony or sporogony. Eimeria stiedae. — This sporozoon is usually known as the Coccidium cuniculi or C. oviforme. It is most frequently found in the epithelium of the bile ducts. It has very rarely been reported for man. The parasite is about 40 x 2o//, and is oval in shape with a double outlined integument. The sporozoites, which form inside, are falci- form in shape. These escape and enter fresh epithelial cells, and thus the process of schizogony goes on. The parasites of the liver are larger than those found in the intestines, these latter being only about 30 x i5//. In the faeces the form most often found is the oocyst, about 40 x 25//. Isospora bigemina. — This parasite, formerly called the Coccidium bigeminum, lives in the intestinal villi of dogs and cats. It is about 12 x 8fjL, and shows a highly refractile envelope containing two biscuit-shaped spores. It has been reported for man. Haemosporidia. Of the sporozoa found in the blood (Haemosporidia), the malarial parasites are the only ones connected with disease in man. In addition to man, infections with parasites of a similar nature are found in monkeys (Plasmodium kochi; the sexual forms alone seem to be present), in birds (PL praecox; this infection is usually desig- nated Proteosoma. An infection of crows and pigeons of like nature is Halteridium). Numerous haemosporidia have been reported for bats, various other mammals, tortoises, lizards, etc. The life history of the malarial parasite is one of the most interesting chapters in medicine. Laveran discovered the parasite in 1880. In 1885, Golgi noted that sporulation occurred simultaneously at time of malarial paroxysm. Koch, Golgi and Celli demonstrated existence of different species for different types of fever. King and Laveran (1884) considered possibility of mosquito transmission. Manson (1894) formulated hypothesis that gametes were destined to undergo develop- ment in the mosquito from observing that flagellated bodies only ap- peared some time after the blood was withdrawn. DESCRIPTION OF PLATE II. (Kolle and Wassermann.) Malarial Parasites. 15. Complete division of the parasite. Typical mulberry form. 16. To the left is the completed division form, an almost developed gamete, which is to be recognized by its dispersed pigment. 17. A tertian ring parasite, small size broken up. 18. Three-fold infection with tertian parasite. The oval black granules are the chromatin granules. 19. To the left, tertian parasite with large, sharply demarked, and deeply colored chromatin granules. To the right, tertian parasite. Both thirty-six hours old. Both probably gametes. 20. Tertian parasite thirty-six hours old, ring form. 21. Tertian parasite with beginning chromatin division, with eight chroma- tin segments. 22. Tertian parasite chromatin division farther advanced with twelve chromatin granules, in part triangular in form. 23. Completed division figure of a tertian parasite. Twenty-two chroma- tin granules. 24. The young tertian parasites separating themselves from each other. The pigment remains behind in the middle. 25. Quartan ring parasite, which is hard to differentiate from large tropical ring or small tertian ring. 26. Quartan ring lengthening itself. 27. Small quartan ribbon form. 28. The quartan ribbon increases in width. The dark places consist al- most entirely of pigment. PLATE II. MALARIA. 187 Ross (1895) demonstrated that flagellation takes place in the stomach of the mosquito. McCallum (1897) saw fertilization of macrogametes by microgametes of Halteridium. Opie recognized differences in sexual charactersistics. Ross (1898) demonstrated life cycle of bird malaria (Proteosoma), showing formation of zygotes and presence of sporozoites in salivary glands. Grassi and Bignami proved the cycle for Anophelinae for human malaria. In 1900 (Sambon and Low), infected mosquitoes from Italy were sent to London, where, by biting, they infected two persons. Life History. — When man is at first infected by sporozoites we have starting up a non-sexual cycle which is completed in from forty-eight to seventy-two hours, according to the species of parasite. The falciform sporozoite bores into a red cell, assumes a round shape and continues to enlarge (schizont). Approaching maturity, it shows division into a varying number of spore-like bodies. At this stage the parasite is termed a merocyte. When the merocyte ruptures, these spore-like bodies or merozoites enter a fresh red cell and develop as before. At the time that the merocyte ruptures it is supposed that a toxin is given off which causes the malarial paroxysm. The cycle goes on by geometric progression from the first introduction of the sporozoite, but it is usually about two weeks before a sufficient number of merocytes rupture simultaneously to produce sufficient toxin for symptoms (period of incubation). This cycle is termed schizogony. After a varying time, whether by reason of necessity for renewal of vigor of the parasite by a respite from sporulation, or whether from a stand-point of survival of the species, sexual forms (gametes) develop. Some think that sporozoites of sexual and nonsexual characteristics are injected at the same time. It is usually considered, however, that sexual forms develop from preexisting nonsexual parasites. These gametes show two types: the one which contains more pig- ment, has less chromatin and stains more deeply blue is the female — a macrogamete; the other with more chromatin, less pigment and stain- ing grayish-green rather than blue is the male — a microgametocyte. When the gametes are taken into the stomach of the Anophelinae, the male cell throws off spermatozoa -like projections, which have an active DESCRIPTION OF PLATE III. (Kolle and Wassermann). Malarial Parasite. 29>3°>3I- The quartan ribbon increases in width. The dark places con- sist almost entirely of pigment. 32. Beginning division of the quartan parasite and the black spot in the middle is the collected pigment. 33- Quartan ring. 34. Double infection with quartan parasites. 35. Wide quartan band. The fine black stippling in the upper half of the parasite is pigment. 36. Beginning division of the quartan parasite. The chromatin (black fleck) is split into four parts. 37. Division advanced, quartan parasites. 38. Typical division figure of the quartan parasite. 39. Finished division of the quartan parasite. Ten young parasites, pig- ment in the middle. 40. Young parasites separated from one another. 41. Small and medium tropical ring, the latter in a transition stage to a large tropical ring. 42. Small, medium and large tropical ring, together in one corpuscle. PLATE III, MALARIA. 189 iashing movement and break off from the now useless cell carrier and are thereafter termed microgametes. These fertilize the macrogametes and this body now becomes a zygote. By a boring-like movement the zygote goes through the walls of the mosquito's stomach, stopping just under the outer epithelial layer of the stomach or mid-gut. It continues to enlarge until about the end of .7-0% . FIG. 59. — Sexual and non- sexual cycle of malaria, i, Schizonts; 2, merocyte; 3, merozoites; 4, macrogamete; 5, microgametocyte; 6 and 7, gametes in stomach of mosquito; 8, microgametocyte throwing off microgametes; 9, microgamete fer- tilizing macrogamete; 10, vermiculus or zygote; n and 12, Zygotes; 13, Zygote distended with sporozoites; 14, sporozoites. one week it has grown to be about 60 /i in diameter and has become packed with hundreds of delicate falciform bodies. Zygotes of benign tertian show little rod-like particles of yellowish pigment — those of malignant tertian black clumps, which, however, are not so coarse as those of quartan. The mature zygote now ruptures and the sporozoites are thrown off into the body cavity. They make their way to the salivary glands and DESCRIPTION OF PLATE IV. (Kolle and Wassermann.) Malarial Parasite. 43. To the left a young (spore) tropical parasite. To the right a medium and large tropical parasite. 44. An almost fully developed tropical parasite. The black granules are pigment heaps. 45. Young parasites separated from one another. Broken up division . forms twenty-one new parasites. 46. To the left a red blood-corpuscle with basophilic, karyochromatophilic granules. Prototype of malarial parasite. On the right a red blood -cor- puscle with remains of nucleus. Sexual Forms or Gametes. 47. An earlier quartan gamete (macrogamete in sphere form), female. 48. An earlier quartan gamete (microgametocyte), male. 49. Tertian gamete, male form (microgametocyte). 50. Tertian gamete, female (macrogamete). 51. Tertian gamete (microgametocyte) still within a red blood-corpuscle. 52. Macrogamete tertian within a red blood-corpuscle. 53. Tropical fever, (^stivo-autumnal) gamete, half moon (crescent) still lying in a red blood-corpuscle. In the middle is the pigment. The concave side of the crescent is spanned by the border of the red blood-corpuscle. 54. Gamete, tropical fever parasite. 55. Gamete of tropical fever parasite heavily pigmented. 56. Gamete of the tropical fever parasite (flagellated form), microgameto- cyte sending out microgametes (flagella or spermatozoa). PLATE IV. MALARIA. 191 thence, by way of the veneno-salivary duct in the hypopharynx, they are introduced into the circulation of the person bitten by the mosquito, and start a nonsexual cycle. As the sexual life takes place in the mosquito, this insect is the definitive host — man is only the interme- diary host. There are three species of malarial parasites: (i) the Plasmodium vivax, that of benign tertian — cycle, forty-eight hours; (2) the Plasmo- dium malariae, that of quartan — cycle, seventy-two hours; and (3) the Plasmodium falciparum, that of aestivo-autumnal or malignant tertian- cycle of forty-eight hours. Variations in cycles may be produced by infected mosquitoes biting on successive nights, so that one crop will mature and sporulate twenty- four hours before the second. This would give a quotidian type of fever. In aestivo-autumnal infections anticipation and retardation in the sporulation causes a very protracted paroxysm, lasting eighteen to thirty-six hours; this tends to give a continued fever instead of the characteristic type. UNSTAINED SPECIMEN (FRESH BLOOD). P. vivax. (Benign tertian.) P. malariae. (Quartan.) P. falciparum. (Malignant tertian) (iEstivo-autumnal.) Character of the Swollen and light About the size and Tendency to distor- infected red : in color. color of a normal tion of red cell cell. red cell. rather than cre- . nation. Shriveled appearance. (Brassy color.) Character of Amoeboid outline. Has band or ribbon- Small, distinc 1 1 y young schizont Rarely more than like appearance. round, crater like one in r.c. dots not more than one-sixth diameter of red cell. Two to foui parasites in one red cell common. Amoeboid with fine Rather oval in Only seen in over- Character of ma- yellow-biown pig- shape. Sluggish whelming infec- ture schizont ment which shows movement of per- tion. Have scanty active swarming ipherally placed fine black pig- movement. coarse black pig- ment clumped ment. together. 192 THE PROTOZOA. STAINED SPECIMEN. P. vivax. (Benign tertian ) P. malariae. (Quartan.) P. falciparum. (Malignant tertian) ti vo-autumnal . ) Character of in- Larger and lighter About normal size Shows distortion fected red cell. pink than normal and staining. and some poly- red cell. Shows chromatophilia "Schiiffner's and stip p 1 i n g. dots." Rarely we have coarse cleft-like reddish dots — Maurer's spots. Character of Chromatin mass Round rings with Very small sharp young schizont. usually single and the chromatin band-like rings, situated inside mass almost cen- with a chromatin the ring of the trally situated. mass piotruding irregularly out - outside the ring. lined blue para- Often appears on site. periphery of red cell. Frequently two chromatin dots. Character of ma- Fine pigment rather Coarse pigment Very rarely seen in ture schizont evenly distribu- rather peripher- peripheral circu- ted in irregularly ally arranged in lation in ordinary outlined parasite. an oval parasite. infection. P i g - ment clumps Character of me- Irregular division Rather regular di- early. rocyte. into fifteen or vision into 8 or Sporulation occurs more spore-like 10 merozoites — in spleen, brain, chromatin dot Daisy. etc. Rarely in segments. peripheral circu- lation. 8 to 10 chromatin stain- ing merozoites. Character of gam- Round. Round. Crescent. etes. In the diagnosis of malaria one should always examine both a fresh specimen and a stained one, as each method gives valuable information in differentiating species. When time will not permit the examination by both methods, always use the smear stained by Wright's stain, as the small peripherally situated rings of aestivo-autumnal fever may escape notice in a fresh specimen. SARCOSPORIDIA. Sarcosporidia are sporozoa found in the striped muscles of various SARCOSPORIDIA. 193 mammals and birds. They are common in the pig and mouse and have been reported for man. They are known also as Miescher's tubes when in muscle fibers. They are divided into three genera: Miescheria, when parasitic in muscle fiber; Balbiania, when parasitic in the intervening connective tissue of the muscles, and Sarcocystis. In addition to the protozoa previously referred to, there are certain infections which are considered by certain authors to be protozoal in nature. FIG. 60. — Diagram showing development of different species of malarial parasites. Cytorrhyctes vaccinae. — These parasites develop within the epithelial cells of stratified epithelium. In vaccinia, Councilman and his colleagues consider that the development only takes place in the cytoplasm of the cell. In variola, however, the developmental cycle affects the nucleus. Cytorrhyctes luis, reported as the cause of syphilis, sporulates in the blood-vessels and in the connective tissue, not in epithelial cells. Cytorrhyctes scarlatinae was reported by Mallory to have been found in the skin in four cases of scarlet fever. CHAPTER XVII. FLAT WORMS. CLASSIFICATION or THE PLATYHELMINTHES (FLAT WORMS). Class Trematoda Family Fasciolklae 0 Cestoda Paramphistomidae \r^\ Schislocomklae Dibothriocephalidae Taeniidae Genus Species Fasciola F. hepatica Fasciolopsis F. buski Dicrocoelium D. lanceatum Pajagonimus P. westermani Qpisthorchis O. felineus Clonorchis G. sinensis , C. endemicus V Heterophyes Cladorchis H. heterophyes C. watsoni \ Gastrodiscus G hominis ' S. haematobium Schistosomum S. japonicum S. mansoni ae Dibothriocephalus D. latus f Dipylidium D. caninum Hymeode.pis H. nana H. diminuta T ' T. solium 1 asnia T. saginata Davainea D. madagascanensis. NOTE. — Two larval Taeniidae are found in man (Cysticercus cellulosae and Echinococcus polymorphus) . Also two larval Dibothriocephalidae (Sparganum mansoni and Sparganum prolifer). TREMATODES OR FLUKES. Flukes are generally leaf-like in outline, exhibiting marked variation in size and shape. The largest human fluke, F. buski, is from 2 to 3 inches in length, while the H. heterophyes is less than 1/12 of an inch in length. The most important fluke, the liver fluke, Clonorchis endemicus, is flat and almost transparent, while the almost equally important lung fluke, the Paragonimus westermani, is oval, almost round and reddish-brown in color. With the exception of the Schis- tosomidce, all flukes are hermaphrodites, and, with the exception of this 194 DISTOMIASIS. 195 family, all flukes have operculated eggs. The only other operculated (with a lid) eggs we meet with in man are those of the Dibothrioceph- alidae. Flukes have two suckers which, except in the Paramphistomidae, are quite near each other — one is termed the oral sucker and the other the ventral sucker or acetabulum. The intestinal tract consists of a pharynx, proceeding from the oral sucker, which bifurcates and terminates in blind intestinal caeca. The life history of the important human flukes is unknown. It is supposed that this, in a measure, may resemble that of the common liver-fluke disease of sheep (sheep rot). In this the eggs containing a ciliated embryo (Miracidium) pass out in the faeces. This embryo is hatched out and, gaining the water, swims about actively until it reaches some suitable mollusk or crustacean (Limnaea trunca'tula). By means of a pointed end, it bores its way into the body of the gastropod and becomes either a bag-like structure (the sporocyst) or develops into a creature with an alimentary canal (redia). From the sporocyst or redia minute little worms resembling adult flukes in possessing suckers, but differing in the possession of a tail, develop (Cercaria). Having reached maturity, these cercariae leave the sporocyst or redia, and, as in case of F. hepatica, become encysted on blades of grass, to be eaten by sheep and again com- mence the cycle. The encysted cercariae develop into adult liver flukes. It is probable that with many flukes the cercariae enter some host, as mollusk, insect or fish, and that it is by eating such animals as food that man becomes infected. Looss thinks it possible that the miracidium of Schistosum haematobium may bore its way directly into man, as do the larvae of the hook-worm. Manson also suggests that the reporting by Musgrave of 100 mature lung flukes in a psoas abscess makes it very probable that these parasites entered the body as miracidia. The idea in China is that the infection with the com- mon liver fluke of man is brought about by eating fish. Fluke disease is generally known as distomatosis or distomiasis. LIVER FLUKES. Fasciola hepatica. — This fluke, while of enormous economic importance by reason of destruction of sheep, has only been reported 196 FLAT WORMS. 23 times in man, and in these instances does not seem to have occa- sioned marked symptoms. There is, however, a possible importance of F. hepatica in connection with a peculiar affection known as "halzoun." This results from the eating of raw goats' liver, and it is supposed that the 'flukes crawl up from the stomach and, entering the 5. FIG. 61. — Trematodes of man, natural size, i, Oonorchis endemicus (Opisthor- chis sinensis) ; 2, Gastrodiscus hominis; 3, Dicrocoelium lanceatum; 4, Hetcrophyes heterophyes; 5, Schistosomum haematobium; 6, Fasciola hepatica; 7, Paragonimus westermani; 8, Fasciolopsis buski; 9, Opisthorchis felineus; 10, anatomy of C. en- demicus (enlarged). G. P., genital pore; V. S., ventral sucker; V. G. vitelline glands; R. S., receptaculum seminis; T., branched testicles. larynx or attaching themselves about the glottis, produce the asphyxia characteristic of the disease. Dicrocoelium lanceatum. — This has only been reported 7 times in man. The symptoms are unimportant; the fluke is about 1/3 of an inch long. Clonorchis endemicus. — This fluke and the C. sinensis are the most important of the human liver flukes. Until recently these flukes DISTOMIASIS. 197 were known as Opisthorcis sinensis. This fluke is very common in China and Japan — in certain sections of Japan, 20% of the population being infected. This fluke is about 3/4 of an inch long and 1/6 of an inch broad. When squeezed out of the thickened bile ducts it is so transparent and glairy as almost to resemble glairy mucus. This fluke is supposed to produce most serious symptoms, as indigestion, swelling and tenderness of liver, ascites, oedema, and a fatal cachexia. As a matter of fact, many physicians in China attribute very little pathogenic importance to it. The disease is diagnosed by the presence of the ova in the stools. Opisthorchis felineus. — This fluke is smaller than the C. endemi- cus, and is a common parasite of the gall bladder and bile ducts of cats. In certain parts of Siberia the parasite is found in more than 6% of the human autopsies. Intestinal Flukes. Cladorchis watsoni. — This fluke is about 1/3 of an inch long, and has an indistinct oral sucker and a large sucker at the other end. This parasite has only been once reported. Gastrodiscus hominis. — This fluke is about 1/4 of an inch long and has a disk about 1/6 of an inch in diameter from which proceeds a teat-like projection, bearing an oral sucker. While it has only been reported twice for man, indications are that it is probably fairly common in India and Assam. Fasciolopsis buski. — This is probably a rather common parasite in India, as Dobson found the eggs in i% of the stools of more than 1000 coolies. The fluke is from 2 to 3 inches in length and about 1/2 of an inch in breadth. It is thick, brown in color and has a very large acetabulum. These parasites cause dyspeptic symptoms and an irregular diarrhcea. Heterophyes heterophyes. — This exceedingly small fluke, which can be recognized by its small size (less than 1/12 of an inch long) and large, prominent acetabulum, was formerly supposed to be rare. Looss, however, has shown that it is quite common in Egypt, he having found it twice in Alexandria in 9 autopsies. The parasites occupy the ileum. It is common in dogs. Tp8 FLAT WORMS. LUNG FLUKES. Paragonimus westermani. — In certain parts of Japan and For- mosa it is estimated that as many as 10% of the inhabitants may harbor this parasite. It is also common in China, and recently many cases have been reported in the Philippines. Dr. Stiles states that around Cincinnati there is quite a heavy infection among the hogs, so that it may be that certain cases diagnosed in man as pulmonary tuberculosis may be due to this disease. It is popularly known as endemic haemoptysis on account of the accompanying symptoms of chronic cough and expectoration of a rusty-brown sputum. After violent exertion, and at times without manifest reason, attacks of haemoptysis of varying degrees of severity come on. The characteristic ova are constant in the sputum and establish the diagnosis. The fluke itself is a little more than 1/3 of an inch long and is almost round on transverse section. It is rather flesh- like in appearance. The flukes are usually found in tunnels in the lungs, the walls of which are of thickened connective tissue. There may be also cysts formed from the breaking down of adjacent tunnel walls. In addition to lung infection with this fluke, brain, liver and intestinal infections may be found. Musgrave was the first one to call attention to the frequency of general infection with this parasite (paragonomiasis) in the Philippines. He found it in 1 7 cases in one year. The life history, beyond the stage of miracidium, is unknown. As stated previously, Manson suggests that the miracidium may enter directly by the skin. BLOOD FLUKES. Schistosomun haematobium. — Flukes of the circulatory system. These flukes are of great importance in Egypt, Japan and the West Indies. The disease is named bilharziasis after Bilharz who first as- sociated the parasite and the disease. It seems probable that there are at least three human species, differentiated principally by the character of the egg. In the blood-fluke disease of Egypt, S. haematobium, the parasite chiefly infects the bladder and the egg has a terminal spine. BILHARZIASIS. 1 99 The lateral-spined ovum is also found in the faeces. In the West Indies, as shown by the reports of Surgeon Holcomb from Porto Rico, rectal bilharziasis is rather common. In these cases the egg is prac- tically always lateral-spined. The adults of this species, the S. mansoni, are scarcely, if at all, to be distinguished from the S. haema- tobium. With the S. japonicum, the name of the Eastern species, there is not only the difference in that the eggs are without spines, but, in addition, the skin of the adult parasite is nottuberculated,as is the case with the other two species. Catto considers that the S. japonicum may live in both arteries and veins. The other two species only live in branches of the portal vein. The blood flukes are about 1/2 inch long. All of these flukes are hermaphroditic, but live separately until maturity. At this time the female enters what is known as the gynaeco- phoric canal of the male; this canal is formed by the infolding of the sides of the flat male fluke, thus giving a rounded appearance to the male. The female is longer than the male (about 5/6 of an inch long), and is thread-like. Her two extremities project from the canal of the male in which she lives. The most prominent symptoms of the Bilharz disease are haema- turia and bladder irritation; later on calculus formation. In rectal bilharziasis the symptoms are more those of bleeding piles or of a mild dysentery. In the Japanese infection the symptoms point more to liver and spleen, there being ascites, cachexia and a bloody diarrhoea. The life history is not known of any of these flukes. Looss con- jectures that it is probable that the miracidium enters the skin, not re- quiring an intermediary host. Frequent experiments have failed to show any mollusk, etc., which attracted the embryo. Evidence seems to show that those who are constantly wading about in the water of the pools or the mud of the fields are the ones most subject to infection. If urine containing eggs is diluted with water the miracidium breaks out of the shell and swims about as if in search of some desired object. The views are also entertained that the miracidium may gain access to the body through the drinking water; there is much evidence against this. However access to the body is gained, it is known that the larval 200 FLAT WORMS. forms make their way to the liver where they develop. Arriving at maturity, the males and females become united and proceed to the terminal branches of the portal vein^ where the irritating eggs, given off by the female, give rise to the symptoms. CESTODE OR TAPE- WORM INFECTIONS. The cestodes and trematodes constitute the two great divisions of the flat-worms. Anatomically, a tape-worm may be considered as a series of individual flukes united in one ribbon-like colony. The cestode segments, or proglottides, differ, however, from the flukes in not having an alimentary canal and in having a cellular instead of a chitinous external covering. A tape-worm is divided, into the segment-producing controlling head and the series of segments or proglottides together known as the strobila. The head and neck together form the scolex. Tape-worm heads are provided with suctorial or hook-like suckers, or both, to enable them to hold on to the intestinal mucosa. The importance of the head is generally recognized by the well-known fact that the per- manent evacuation of one of these parasites is only arrived at when the head as well as the segments is expelled. Otherwise, additional segments will be produced. Even in tape-worms twenty-five to thirty feet in length, the head is no larger than a small shot. It carries the suckers or booklets which best enable us to differentiate the different species. The segments adjacent to the head are im- mature— the sexually -mature ones being found from the middle of the body onward. The sexually-mature segment possesses a varying number of testicles: 3 in H. nana and as many as 2000 in T. saginata. As with the flukes, they also have ovaries, yolk glands, uterus, genital pore, etc. The location of the genital pore and the character of the branching of the uterus are of the greatest importance in differentia- tion. The sexually-mature proglottides may either expe their ova, when these would be found in the faeces or, as is common, they break off and pass out themselves in the faeces They then either expel the eggs or may be eaten by some animal and in this way effect an entrance for their ova. The "hexacanth" or 6-hooked embryo, also called the onchosphere, is the essential part of the egg. The egg shell is dis- * TAPE WORMS. 201 solved off in the alimentary canal of the animal ingesting it, and the onchosphere bores its way through the gut to later become encysted in various tissues. In some tape-worms a ciliated embryo is liberated from the egg shell and swims about actively to enter some fish or other animal. When the 6-hooked embryo reaches its proper tissue, the hooklets are discarded and a scolex similar to the parent one is devel- to. C. FIG. 62. — Tape worms. A. i, 2 and 3, Scolex, proglottides and ovum of Taenia solium; B. 4, 5, 6 and 7, Scolex, proglottides and ovum of Dibothriocephalus latus; C, 8, 9 and 10, Scolex, proglottides and ovum of Taeenia saginata. oped. At this time we have a bladderlike structure with the scolex inverted in it. This little cyst with its scolex when ingested by another animal is digested, and the scolex establishing itself in the intestine, develops a series of segments. The ciliated embryo of the D. latus does not form a cyst, but instead a worm-like creature similar to the adult. This is termed a Plerocercoid. Small laminated calcareous corpuscles are characteristic of cestode tissue. 14 202 FLAT WORMS. INFECTIONS. Taenia saginata. — This very widely distributed tape-worm is often termed the unarmed tape-worm, to distinguish it from the T. solium or armed tape-worm. It is from 10 to 25 feet long and has several hun- dred proglottides. The small pear-shaped head has 4 pigmented elliptical suckers and no hooklets. The segments are plumper than those of T. solium, hence the name saginata. The single lateral genital pore projects markedly and in a series of segments presents, as a rule, first on one side, and then on the opposite side of the next seg- ment (alternating). The best way to distinguish a segment of the T. saginata from the T. solium is by counting the number of lateral uterine branches; these number 15 to 30 and branch dichotomously. The lateral divisions of the uterus of the T. solium are tree-like in their blanching and only number 5 to 12 on each side. The ox is the in- termediate host. The 6-hooked embryo, having worked its way from the alimentary canal to the muscles or liver of the ox, become encysted (Cysticercus bovis). This little bladder-like structure is about 1/4 by i / 3 inches. Being ingested by man's eating raw or imperfectly cooked meat, the adult stage becomes established in his alimentary canal. In Abyssinia the infection is said to be universal, and a man without a tape worm to be a freak. An important point is the fact that the larval stage never appears in man. It is this fact which makes it a so much less dangerous parasite than the T. solium, which readily es- tablishes a larval existence in man if the ova are introduced into the human stomach. Cooking meat always destroys the Cysticercus. A period of about 2 months elapses after the ingestion of the Cysticer- cus before the mature segments pass out of the rectum. These not only make their exit with the faeces, but are also capable of wandering out at other times. In this they differ from the segments of T. solium. T. saginata next to H. nana is the common tape-worm of the United States. Dr. Stiles has examined several hundred tape-worms during the past few years and has found only one T. solium. Taenia solium. — The measly -pork tape-worm is smaller than the T. saginata and differs from it in having a globular head, with a rostel- lum, which is crowned by 26 to 28 hooklets. The segments have only 5 to 10 branches and are expelled only at the time of defecation. IAIM. WORMS. 203 The segments or the ova having been ingested by a hog, the 6-hooked embryo is liberated and becomes encysted in the muscles of the hog, as an invaginated scolex. Pork containing this Cysticercus (Cysticercus cellulosae) is known as measly pork. If one by chance should carry the eggs on his fingers to his mouth, as the result of examining mature segments, the larval stage may be established in man. If this infection is not heavy, very few symptoms may be observed. The Cysticercus, however, tends to invade the brain, next in frequency the eye, and so causes convulsions, death or blindness. Instead of only being the size of a pea, these cysts when forming in the brain may be the size of a walnut or larger. T. solium is comparatively common in North Germany, but is exceedingly rare in England and the United States. Taenia africana. — This is an unarmed tape-worm, only about 5 feet long. It was found in a native soldier in German East Africa. Hymenolepis nana. — This is generally known as the dwarf tape worm — it is the smallest of the human tape-worms. It is from 1/4 of an inch to 1/2 inch in length, and is less than 1/25 of an inch in breadth. The genus Hymenolepis has lateral g. pores, all of which are on the same side. The head has 4 suckers and a rostellum, which is usually invaginated. The rostellum has 24 to 30 booklets encircling it. The eggs of this species are quite characteristic, there being 2 distinct membranes. The inner one has 2 distinct knobs, from which thread- like filaments proceed. The eggs of the H. diminuta have a thicker, striated outer membrane and there are no filaments. The eggs of the Dipylidium caninum are similar, but are found in the faeces in aggre- gations— several eggs in a packet. The dwarf tape-worm has been found to be the most common tape-worm in the United States. Dr. Stiles found it in about 5% of children in a Washington orphanage. It has been estimated that in certain parts of Italy 10% of the children may be infected. The symptoms, especially nervous ones, may be marked in this infection. Although very small, yet the number of parasites may be very great, even more than 1000. A form found in rats, which may be identical with H. nana, does not require an intermediate host. The 6-hooked embryo bores into the intestinal villus and there delveops a Cercocystis (larva of small dimensions with but little fluid). When fully developed, it 204 FLAT WORMS. drops into the lumen of the gut, and a new parasite is added to the already existing number of parasites. This explains the heavy infection. H. diminuta and H. lanceolata have also been reported for man a few times. Dipylidium caninum. — This is a common parasite of dogs and cats. The larval stage is passed in lice and fleas. The cases of human infection have been principally in children, probably from getting dog lice or fleas in their mouths. The head has 4 suckers and a rostellum. which has 3 or 4 rows of encircling hooklets. The segments have the shape of melon seeds and have bilateral genital pores. Davainea madagascariensis. — This tape-worm has been found in Siam and Mauritius. It is about 10 inches long. The head has 4 suckers and a rostellum with 90 hooklets. The genital pores are unilateral. The cockroach is supposed to be the intermediate host. DIBOTHRIOCEPHALID.E INFECTIONS. Dibothriocephalus latus. — This is frequently termed the broad Russian tape-worm. It has a small olive-shaped head with 2 deep winding suctorial grooves on each side; it has neither rostellum nor hooklets. The segments are quite broad, being about. 1/2 by 1/5 in. At the end of the strobile they are more nearly square. The segments are very numerous, 3000 or more. The fully developed worm is about 30 feet long. The uterus in each segment is rosette-shaped and the genital pore is ventrally situated. The eggs of this species have an operculum and a ciliated embryo. This ciliated embryo swims around and either enters some fish, especially pike, directly or through an as yet unknown intermediary. This parasite produces an intense anaemia similar to pernicious anaemia. It is a frequent parasite in Switzerland, Bavaria, Japan, Scandinavia and Russia. Recently several cases have been reported from our Northwest, and some of the fish of the waters of that region are said to be infected. The larva is a plerocercoid and is about i inch long. It is said that salting, smoking or other ordinary methods of preserving fish will not kill it. A tape-worm, Diplogonoporus grandis has been reported from Japan. In this there are 2 complete sets of genital organs to each segment. HYDATID DISEASE. 205 SOMATIC T^NIASIS. While rarely we may have the larval stage of T. solium present in man, and while certain bothriocephalid larvae (Sparagnum mansoni and Sparganum proliferum) infect man, yet they are unimportant as compared with the larval stage of the Taenia echinococcus. The adult stage of this parasite is passed in dogs. It is one of the smallest tape-worms known, being only about 1/6 in. long. It has a head with 4 FIG, 63. — Tape worms, i, 2 and 3, head, melon-shaped segments and egg packet of Dipylidium caninum; 4, 5, 6 and 10, entire worm magnified, head, larval stage in intestinal villus and ovum of Hymenolepis nana; 7, echinococcus cyst; A, mother cyst; D, daughter cyst; E, granddaughter cyst; C, scolex in brood capsule; B, brood capsule; G, parenchymatous layer; F, laminated layer; 8 and 9, Taenia echinococcus; 9, natural size. suckers and a rostellum encircled with hooks. There are only 3 to 4 segments. The larval stage, on the contrary, gives oneof the largest of larval cestodes. The larval stage is also found in hogs and sheep, and it is probably by reason of the dog eating the echinococcus cyst of such animals at the abattoir that we owe the increase in this serious infection. 206 FLAT WORMS. Man contracts the infection from association with dogs. The disease is peculiarly prevalent in Iceland. As stated above, the adult stage is passed in the intestine of the dog. Should the egg-bearing segments passed by the dog contaminate the hands of man and a single egg be ingested, we may have hundreds of Taenia larvae produced. The 6-hooked embryo, leaving its shell, bores its way through the walls of the alimentary tract and especially seeks the liver, just as the onchosphere of the T. solium seeks the brain and eye. In the development of the cyst, after the onchosphere has come to rest at some point in the liver, we have formed at first an indistinctly laminated external envelope with coarsely granular fluid contents. Later on the contents become transparent, and 2 distinct layers can be observed: (i) The external, markedly laminated one, and (2) the internal one, made up of small cells externally and large cells and cal- careous corpuscles internally. This internal lining membrane is known as the parenchymatous or germinal layer. When the external layer is incised it curls up by reason of its elasticity. This is character- istic of such a cyst. In addition, we have an enveloping connective- tissue capsule formed by the surrounding liver substance. From the germinal layer arise the brood capsules and the scolices. In these brood capsules we have the cellular layer external — just the reverse of the mother cyst. Scolices may develop either on the outside or inside of these brood capsules. It is interesting to note that one onchosphere may develop hundreds of scolices. From the parenchymatous layer of the mother cyst, daughter cysts are formed; these have an external stratified layer and an internal parenchymatous one; within them a varying number of scolices may develop. From these daughter cysts, granddaughter cysts may arise — all within the mother cyst- — and hence are termed endogenous. At times the daughter cysts work their way external to the mother cyst and proceed to develop in a manner similar to the endogenous formation. The exogenous development is rare in man, but common in hogs. Hydatids containing no scolices are called sterile. These cysts may be as large as a child's head, but are usually smaller. The fluid of these cysts contains about i% of NaCl, also a trace of sugar; in addition there is a toxin which produces urticaria and acts as a cardiac depress- LARVAL TAPE WORM INFECTIONS. 20' ant. If any quantity should escape into the peritoneal cavity at operation, it may cause death. Hydatids develop very slowly, and the duration of the disease is usually from 2 to 8 years. Sparganum mansoni. — This is a larval bothriocephalid which is FIG. 64. — Ova of intestinal parasites with measurements in mikrons. i Fasciola hepatica (150x80); 2, Taenia saginata (25); 3, Ascaris lumbncoides (65x50); 4, Fasciolopsis buski (125x75); 5, Necator americanus (70x40); 6, Dibothrio- cephalus latus (70x45); 7, Schistosomum haematobium (100x50); 8, Trichoceph- alus trichiurus (55x25); 9, Strongyloides stercoralis (60x30); 10. Schistosomum mansoni (110x60); n, Hymenolepis nana (56x53); 12, Echinorhynchus gigas (100x80); 13 and 15, Oxyuris vermicularis (50x20); 14, Heterophyes heterophyes (28x16); 16, Clonorchis endemicus (28x16). about 5 to 10 inches long and has been reported 10 times in Japan. It has been found in various parts of the body. Sparganum prolifer. — This has been reported from Japan as a larval form in the subcutaneous tissue. Stiles has found these larval forms in skin lesions in Florida. They show themselves as bizarre grub-like forms. CHAPTER XVIII. THE ROUND WORMS. CLASSIFICATION OF THE NEMATHELMINTHES (ROUND WORMS). Class. /O Nematoda, Family. Genus. Species. Angiostomidae, Strongyloides, S. stercoralis. (Dracunculus, D. medinensis. F. bancrofti. F. loa. Filariidae, 1 Filaria, F. perstans. I F. demarquayi. F. ozzardi. F. philippinensis Trichotrachelidae, Trichocephalus, Trichinella, T. trichiurus. T. spiralis. Eustrongylus, E. gigas. Strongylidae, TrichostrongyluS; Agchylostoma, T. instabilis. A. duodenale. Necator, N. americanus. Ascaridae, Ascaris, A. lumbricoides. A. canis. Oxyuris, O. vermicularis. Gigantorhynchus G. gigas. {Hirudo, H. medicinalis. Limnatis, L. nilotica. Haemadipsa, H. ceylonica. Acanthocephala, Hirudinea, NOTE. — The Strongyloides stercoralis was formerly described under two desig- nations: (i) Anguillula intestinalis, a parasitic generation and (2) Anguillula ster- coralis, a free living generation. ROUND WORMS OR NEMATODES. All nematodes are covered by a cuticle which varies in thickness. The sexes are, as a rule, separate. The male can usually be recognized by its small size, its curved or curled posterior end, and at times exhib- iting an umbrella -like expansion — the copula tory bursa. The spicules, chitinous copulatory structures, may be observed drawn up in the worm or projected out of the cloaca. Certain papillae in the region of the anus are valuable in differen- tiation. In the female the vulva is usually situated about the middle of the ventral surface. 208 COCHIN-CHINA DIARRHCEA. 2OQ ANGIOSTOMID^:. Strongyloides stercoralis. — This parasite was formerly thought to be the cause of Cochin-China diarrhoea. It presents two genera- tions: i. Parasitical or intestinal form. 2. The free living or fecal form. i. The intestinal form (also known as Anguillula intestinalis) is represented only by females. These are about 1/12 of an inch long and reproduce parthenogenetically. The embryos escape from the eggs while still in the intestine, so that in the faeces we only find actively motile embryos. The eggs, which are strung out in a chain, never appear in the faeces except during purgation. As they greatly re- semble hook-worm eggs, this is a point of great practical importance. In fresh faeces we find hook-worm eggs and Strongyloides embryos. If the temperature is low, these rhabditiform embryos develop into filariform embryos, which being ingested form the infecting stage. If the temperature is warm, 25° to 35° C., these embryos develop into: (2) The free living form. In this we have males and females; these copulate and we have produced rhabditiform larvae, which later change to filariform ones. These being ingested, start up the parasiti- cal generation. The embryos are rather common in stools in the tropics. These embryos have pointed tails and are about 250 x i3/£. They have a double cesophageal bulb. This family is of the greatest importance to man. It is also one about which much confusion exists.as to the adult type; hence anyone finding adult filariae should fix them in hot 5% glycerin alcohol (alcohol 70%), and subsequently mount in glycerin gelatin. Formalin is not to be used. These worms are most likely to be seen as writhing thread- like worms, especially in the lymphatic glands and connective tissue, about body cavities. Fil'a'f ift medinensis. — The Guinea or Medina worm, of which until recently only the female was known, is of great importance in parts of India, Africa and Arabia. The female is a thread-like worm, about 20 to 30^ inches long. The habitat is the subcutaneous and 210 THE ROUND WORMS. intermuscular connective tissue, especially of the lower extremity. The mouth is terminal and the body uniformly cylindrical. The uterus is a continuous tube filled with sharp-tailed, transversely striated embryos, 650 x 17/4 and constitutes the greater part of the body, the alimentary canal being pressed to one side. The genital organs 13. /-OB. FIG. 65. — Round worms (Filariidae). i, Hooked posterior extremity and anterior extremity of Dracunculus medinensis; 2, cross section of uterus filled with embryos, D. medinensis; 3 and 4, free embryo and embryos of D. medinensis in intermediate host (Cyclops); 5, natural size of female" Filar'a bancrofti; 6, embryo of F. ban- crofti in blood; 7, tail of male F. bancrofti; 8, male and female of F. loa (natural size); 9, tuberculated integument and posterior end of male F. loa; 10, posterior end of male F. perstans; n, male of F. bancrofti (natural size); 12, blunt tailed embryo of F. perstans; 13, sharp tailed embryo of F. demarquayi. probably discharge through the oesophagus. The body when being extracted is rather transparent. The tip of the tail is bent, form- ing a sort of anchoring hook. Recently Leiper fed monkeys on bananas containing infected Cyclops, and at the autopsy six months later obtained both male and female forms. FILARIASIS. 211 As regards the life history, Fedschenko showed that the embryos when liberated swam around in water and finally entered the bodies of species of the genus Cyclops. The female tends to come to the surface in the lower extremities, and experiments show that if on the blister-like point of emergence some water be squeezed out from a sponge, the uterus will eject a milky-looking fluid containing myriads of embryos. This would indicate that the worm selects the lower extremity so that the embryos may gain access to the Cyclops when the host is wading through the water. Leiper showed that a strength of HC1 equal to that of gastric juice killed the Cyclops, but made the Dracunculus embryos very active. From this he judged that infection must probably take place from drinking water containing infected Cyclops. The disease is known as "Dracontiasis." Filaria loa. — This is a thread-like worm, about i to 2 inches long. The cuticle is characterized by distinct wart-like structures. The males are smaller than the females and have 3 preanal papillae and 2 postanal ones. There are 2 short unequal spicules. The life history is not satisfactorily established. The young are born ovoviviparously, and it has been suggested that the localized cedemas, known as Calabar swelling, may be due to the irritation produced by these eggs. The embryos almost exactly resemble those of F. bancrofti. They have a diurnal periodicity, however, appearing in the blood about 8 A. M., increasing to noon and disappearing about 9 P. M. The intermediate host is unknown. The adult worms have a tendency to wrander about in the subcutaneous connective tissue, especially about the region of the orbit or even under the conjunctiva. Filaria bancrofti. — This is the most important of the filarial worms. The embryos have been carried in medical books as Filaria sanguinis hominis. This species is the cause of the common mani- festations of filariasis, such as elephantiasis, varicose groin glands, chyluria, lymph scrotum, etc. F. bancrofti is transversely striated, and lives in lymphatics of trunk and extremities. The sexes are usually found together. The females are about 3 inches long and the males less than 2 inches. The tails of both sexes are incurved, but that of the male is more so. The 212 THE ROUND WORMS. head is club-shaped. The sheathed embryos are supposed to be born viviparously and Manson supposes that as a result of injury to the parent worm and resulting extrusion of eggs, that the blocking of lymph channels occurs. These embryos show a nocturnal periodicity. Dur- ing the day they remain in the lungs. The disease is transmitted especially by Culex fatigans. The sheathed embryos, getting into stomach of mosquito, wriggle out of the sheath, they then bore their way through walls of stomach and enter into a sort of passive stage, during which further development takes place. They finally become distributed in the muscles of the thorax and make their way along the fleshy labium, to enter the wound in a person bitten by a mosquito, by way of Button's membrane. Filaria perstans. — The adults are found in connective tissue and deeper fat, especially about the mesentery and abdominal aorta. The female is about 3 inches long; the male is rarely found and is less than 2 inches long. These worms are characterized by incurved tails, the extremity of which has two triangular appendages giving a bifid appearance. The embryos do not possess a sheath and have a blunt tail. The life history is unknown. Both mosquito and tick have been incriminated. The embryos are always present in the peripheral circulation — hence perstans. There does not seem to be any symptomatology. Filaria demarquayi. — The habitat of this filarial worm is the West Indies. The embryo has no sheath and has a sharp tail. Other filarial species which have been reported are F. magalhaesi, F. ozzardi, F. volvulus, F. powelli and F. philippinensis. A species called F. gigas is now considered to have been only the hair of the leg of a fly. The embryos have usually been given such names as F. nocturna, F. diurna, etc. Of course the embryos and the parent should have the same name. It has been proposed to designate these embryos the same as the parent, but with the use of the term Microfilaria instead of Filaria. The points usually noted in the description of filarial embryos are : 1. Presence or absence of periodicity of embryos in peripheral circulation. 2. Presence or absence of a sac sheath around the embryo. THE WHIP WORM. 213 3. Accurate measurements. 4. Shape and description of head and tail ends. 5. Character of movement. Key to Filarial Larvae. A. Sheath present. 1. No periodicity. F. philippinensis. Tightly-fitting sheath; not flattened out beyond extremi- ties. Tail is pointed and abruptly attenuated. Lashing progression move- ment. 320x6.5/4. 2. Periodicity exhibited. a. Nocturnal periodicity. F. bancrofti (F. nocturna). Pointed tail; loose sheath; lashing movement. 300x7.5/4. b. Diurnal periodicity. F. loa (F. diurna). Pointed tail; loose sheath; cannot be distinguished from F. nocturna except by periodicity. 300x7. 5/4. B. Abscence of sheath. None of these exhibit a periodicity, being continuously present. 1. Blunt tail — F. perstans. 200x4.5/4. 2. Sharply-pointed tail: a. F. demarquayi. 210x5/4. b. F. ozzardi. 215x5/4. NOTE. — A filarial embryo, F. powelli, reported once. It has a sheath, nocturnal periodicity, and is about 130x5/4. TRICHOTRACHELID^:. These have a long thin neck and a thicker terminal portion. The oesophagus is of the single row of cells type. The anus is terminal; there is only one ovary. Trichocephalus trichiurus. — This is usually called the whip- worm — the thickened body representing the handle and the narrow neck the lash. It is one of the most common parasites in both temper- ate and tropical climates. The egg is very characteristic in having an oval shape with knobs at either extremity. It resembles a platter with handles. The male is almost 2 inches long, and has the terminal portion curled up in a spiral. It has a single terminal spicule. The female is a Jittle longer than the male, and has the terminal part in the shape of a comma instead of being coiled. The neck only contains the cesophagus. The great powers of resistance of the ova may account for their general distribution; they may live for months under conditions of freezing and so forth. There is no intermediate 214 THE ROUND WORMS. host. The worm arrives at sexual maturity in about one month after ingestion. The whip-worm prefers the caecum, but also lives in the lower end of the ileum and the appendix. The neck burrows into the mucosa, and much importance has been attributed by the French to the possibility of this paving a way for the FIG. 66. — Round worms, i, Encysted embryo of Trichinella spiralis; 2, male and female of T. spiralis; 3, male and female of Trichocephalus trichiurus; 4, egg of T. trichiurus; 5 and 6. head and male and female of Ascaris canis; 7, 8 and 9, head, egg and male and female of Oxyuris vermicularis; 10, n and 12, head, egg and tail of Ascaris lumbricoides; 13 and 14, head and egg of Echinorhynchusgigas; 15, 1 6 and 17, parthenogenetic female and rhabditiform and filariform embryos of Strongyloides stercoralis. entrance of pathogenic bacteria. They do not seem to produce serious symptoms. Trichinella spiralis. — The cause of trichinosis is usually termed Trichina spiralis in medical works. The adults live in the duodenum and jejunum; the males are about 1/20 of an inch long and the female TRICHINOSIS. 215 about 1/6 of an inch. The female gives off embryos from the vulva which is near the mouth end (viviparous). After fertilization of the females the males die, and the females then begin to produce embryos to the number of more than 1000 each. These pass out of the intestine and wander about to the striated muscle; it being about 10 days be- fore they reach the muscle. In the muscle they become encysted as the oval areas containing coiled-up embryos that everyone is familiar with. These oval areas are |about 450x250/4. The encysted larvae may remain alive as long as 10 to 20 years; finally, however, the cyst undergoes calcareous infiltration and the embryo dies. Among FIG. 67. — Trichina spiralis (Zlegler). cannibals it would be easy to keep the cycle going by eating improperly cooked or raw human meat, the parasite being thus transmitted. As this would not explain the transmission among civilized men, the following is the life history: Man obtains his infection from eating raw pork, the embryos encysted in the muscle of the hog being liberated in the stomach, and the males and females developing in the intestine as above described. The hog may gain his infection by eating the meat of other hogs or rats. These rats eat scraps of pork at slaughter houses and become infected. In man, while the adults are breeding in the intestine, we have gastrointestinal symptoms. About 10 to 20 days after infection the embryos begin to wander and we have the acute 2l6 THE ROUND WORMS. muscle pains. In the diagnosis we should try to obtain specimens of the pork which has caused the trouble in order to examine for encysted Trichinae. During the diarrhceal stage we may examine the stools for adult worms. In particular examine the blood for eosinophilia. STRONG YLID.E. In this family the male has a caudal bursa, a prehensile sort of expansion at the posterior end for copulatory purposes. Eustrongylus gigas. — This is the largest round worm infecting man; it is usually found in the pelvis of the kidney. There seem to be 12 authentic cases of infection in man. The females are about 40 inches long and about 1/3 of an inch in breadth. The copulatory bursa of the male distinguishes it from Ascaris. The source of in- fection is unknown. The very characteristic ova, with gouged-out oval depressions, may be found in the urine. Trichostrongylus instabilis. — This is a small strongyle formerly known as Strongylus subtilis. The male is about 1/6 of an inch long, and the female about 1/4 of an inch. It has been found in the upper part of the small intestine of inhabitants of Egypt and Japan. It does not appear to produce symptoms. Agchylostoma duodenale. — The hook-worm, so called for the hook-like projections of the head dorsally, is probably the most impor- tant of the animal parasites. This specimen in Europe and Africa and the Necator americanus in the new world cause an immense amount of invaliding. The Egyptian anaemia and the Porto Rican anaemia are caused by this parasite. Hook-worms may be found in the small intestine of man in enormous numbers. They either produce their effects by feeding on the mucosa or by causing loss of blood. The males are little than more 1/3 of an inch long and the females little more than 1/2 inch in length. The male can readily be distinguished by his umbrella-like expansion or copulatory bursa. The tail of the female is pointed. The mouth of the Old World hook-worm has 4 claw- like teeth on the ventral side of the buccal cavity and 2 on the dorsal aspect. In N. americanus the ventral teeth are replaced by chitinous plates. The copulatory bursa of the N. americanus is also different, HOOK WORMS. 217 being bipartite in the division of dorsal ray rather than tripartite, as with the A. duodenale. The delicate-shelled eggs pass out in the faeces and in i or 2 days a rhabditiform embryo (200 x 14/4) is produced. After moulting twice, it remains rather quiescent; it is at this stage that it burrows into the skin of man, producing the so-called " ground itch" at the site of 7. FIG. 68. — Hookworm anatomy, i, Rhabditiform embryo of Strongyloides ster- coralis emerging from egg; 2 and 3, egg and male and female of hookworm; 4 and 5, head and copulatory bursa of male Ancylostoma duodenale; 6 and 7, head and copulatory bursa of male Necator americanus. Dorsal ray, N, america- nus. shows deep cleavage and bipartite tips. entrance. Having gained access to the lymphatics and veins, they eventually reach the lungs. Here they get into the bronchioles and undergo a third moulting. They then wrork their way up the trachea to the glottis and are swallowed to then become adults in the intestine. Dr. Stiles, \vhile accepting this theory of the life history, thinks it 15 2l8 THE ROUND WORMS. probable that infection is also brought about by swallowing directly some infecting stage. Necator americanus.— This is the species of hook-worm found in the Southern States and the West Indies. It is very prevalent in Guam, L. I. It was found by Looss in pigmies from Central Africa, so that this parasite was undoubtedly brought to this part of the world by slaves. The eggs of N. americanus are larger than those of A. duode- nale. In hook-worm disease we have ground itch, tibial ulcer, anaemia, interference with physical and mental development and, in bad cases, dirt eating. ASCARID^:. These have 3 papillae around oral cavity. The male has 2 equal- length spicules. An intermediary host is not needed in the life history of this family. Ascaris lumbricoides. — The male worm is from 5 to 8 inches long and the female from 7 to 15 inches in length. They are from 1/7 to 1/4 of an inch in diameter. The body of the worm resembles the ordinary earth-worm, but is more grayish than red. The ova are very characteristic with a rough mammillated exterior. This at times is shelled off and we have a smooth egg which may be mistaken for eggs of other parasites. The eggs leave the body in the faeces and after a long time — a few weeks to several months, according to temperature- develop an embryo which remains in the shell until swallowed by some man or animal. It is stated that they will remain alive for years. On being swallowed, the embryo leaves the egg and we have males and females developing in the intestine. In countries where such parasites abound, as in Guam and the Philippines, the possibility of their getting into the peritoneal cavity through operative measures on the intestine must always be thought of. Ascaris canis. — This is a parasite of the dog and cat, but is oc- casionally found in children. It is much smaller than the A. lum- bricoides— male is 2 to 3 inches long, female 4 to 5 inches in length. The parasites are characterized by the presence of wing-like pro- jections from the anterior end (arrow-like head). Oxyuris vermicularis.— This parasite is also known as the pin- THREAD WORMS. 219 worm, seat-worm or thread-worm. The male is about 1/6 of an inch long and the female a little less than 1/2 an inch in length. The male has a single spicule and the female a long tapering tail. The eggs are thin-shelled, plano-convex and show a coiled-up embryo. After ingestion of eggs, the adults develop in the small intestine where copu- lation takes place; the males then die. The fertilized females go to the caecum and colon where they remain until they reach maturity. At this time the females wander to the rectum where they either expel their ova or themselves, working their way out of the anus. This usually occurs at night, and the scratching induced by the itching causes the eggs to be widely spread about the region of the anus. The worms may also wander into the vagina, urethra or under prepuce. It will be seen that as a result of the scratching, the fingers become contaminated with ova which may be carried to the mouth and so cause a fresh in- fection. A knowledge of the life history — the early location in the small intestine, and later on in the large — shows that treatment should be dual in its direction. Enemata for the gravid female in the rectum and santonin and calomel for the young adults in the small intestine. ACANTHOCEPHALA. These are called thorn-headed worms on account of a proboscis which projects like a little peg. It has several rows of hooks pro- jecting backward which enable it to attach itself firmly to the intestinal wall. The worm absorbs nourishment through the general body wall, there being no alimentary canal or mouth. These worms are common in hogs. The 3 shelled eggs are very striking and the intermediate stage is in June bugs. The Echinorhynchus or Gigantorhynchus gigas — E. hominis E. and moniliformis — have also been reported for man. HIRUDINEA (LEECHES). Hirudino medicinalis. — This is the leech used medicinally for the abstraction of blood. They have a secretion which prevents coagula- tion of the blood so that when removed the wound still continues to bleed. Hirudo nilotica. — This species has been found in many parts of 22O THE ROUND WORMS. Northern Africa and, gaining access to the stomach through drinking water, it wanders to the pharynx, nares and even trachea. Manson refers, to a case of obstinate epistaxis and headache caused by a leech in the nostril. Haemadipsa ceylonica. — These are land leeches found in India, Philippines, Australia and South America. They are only about i inch long and are slender. They leave the damp earth to climb shrubs and from there to drop on animals or man passing through the forest. The bites are painless, but may be followed by ulcers. They may get into the nostrils. CHAPTER XIX. THE ARACHNOIDS. Order. Acarina, CLASSIFICATION OF THE ARACHNOIDEA. Family. Subfamily. ' Genus. Species. Trombidiidae, Trombidium, T. holosericeum. Gamasidae, Dermanyssus, D. gallinae. Tyroglyphidae, TV™!™!,,,* / T- farinae> T. longior. Sarcoptidae, Demodicidae, Ixodidae, Linguatulida, Sarcoptes, Demodex, Argas, Ornithodoros, Ixodes, Hyalomma, Rhipicephalus, Dermacentor, Margaropus, Amblyomma, Haemaphysalis, f Linguatula, \ Porocephalus, THE ARACHNOIDEA. Argasinae, Ixodinae, S. scabiei. D. folliculorum, A. persicus. A. miniatus. O. saviguyi. I. ricinus. H. aegyptium, R. bursa. f D. reticulatus. \ D. andersoni. M. annulatus. A. hebraeum. H. leachi. L. rhinaria. P. constrictus. The Archnoidea differ from the Insecta in having the head and thorax fused together. They also have 4 pairs of ambulatory ap- pendages, while the insects only have 3 pairs. The Arachnoidea never have compound eyes — these when present being simple. Of the 2 orders of Archnoidea of interest medically the Acarina is far more important than the Linguatulida. ACARINA. Of the acarines we are chiefly interested in the mites and the ticks. The acarines do not show any separation of the abdomen from the cephalo-thorax. A hexapod larva develops from the egg; this is succeeded by an octopod nymph which differs from the adult in not having sexual organs. 221 222 THE ARACHNOIDS. The Trombidiidae. These generally have a soft integument and are often brightly colored. A very common and annoying member of this family is the hexapod larva of the Trombidium holosericeum. It is usually designated Leptus autumnalis. Popularly it is termed "harvest mite," "red bug" or jig- ger. They are found in the fields in the autumn and attack both man and animals. The itching and redness produced is at times called autumnal erythema. There is a Trombidium in Mexico which has a predilection for the skin of the eye-lids, prepuce and navel. The Ked- ani mite is believed by the Japanese authorities to bring about infection with Japanese river fever or Tsutsugamushi, as the result of trans- mitting either a bacterium or protozoon by its bite. The disease some- what resembles typhus, although an eschar at the site of the bite and lymphatic involvement is present. Of the Gamasidae, which generally have a hard body, only the Dermanyssus gallinae is of interest. This coleopterous mite infests chicken-houses and sucks the blood of the inmates. They will also attack man. Poultrymen may be troubled writh a sort of eczema on the backs of the hands and forearms, similar to scabies, resulting from bites by these mites. They measure 350 x 650^. They have no eyes. Tyroglyphidae. Mites of this family live on cheese, flour, dried fruits, etc. They are chiefly of importance because of their being occasionally found in urine, faeces, etc., and being striking objects, the question of pathogen- icity arises. The T. longior has been associated with intestinal trouble (probably a coincidence, patient having eaten cheese containing these mites). Glyciphagi are found in sugar and are the cause of what is known as "grocers' itch." Rhizoglyphus parasiticus is reported to be the cause of an itch-like affection of the feet of coolies on tea plantations. To distinguish: the dorsum of Glyciphagus is hairy or plumose; that of Tyroglyphus has both claws and suckers on tarsi, while Rhizoglyphus has only claws. ITCH MITES. 223 Sarcoptidae. These are small eyeless mites with a transversely striated cuticle. They live on the epidermis of man and various animals. It is the female that makes the tunnels in the skin between the ringers, on penis, flexor surface of forearm, etc. The male dies off after copulation. The female passes through 4 stages: (i) larva; (2) nymph; resembles 7-oe, FIG. 69. — Mites, i, Demodex folliculorum; 2, Kedani mite; 3, Trombidium holosericeum; 4 and 6, Female and male Sarcoptes scabiei; 5, Tyroglyphus farinae; 7, Rhizoglyphus parasiticus. adult, but no sexual organs; (3) the pubescent female; (4) the egg- bearing female. A pair of itch mites may produce 1,500,000 descend- ants in 3 months. Transference of eggs, larvae or pubescent females do not seem to transmit scabies. It is the egg-laden female only. The human itch mite, Sarcoptes scabiei, is an oval mite, the male is 250 x 150/4; the female is about 400 x 300/4. Besides- the difference in size, the male maybe distinguished from the female by the fact that the third and 224 THE ARACHNOIDS. fourth pairs of legs in the female have bristles, but in the male, the fourth pair has suckers. The tunnels made by the female have the egg-bearing female at the blind end; scattered all along are faeces, eggs, larvae; the eggs being next the mother and the more mature young at the entrance to the gallery. A diagnosis can be made from the finding of either eggs or larvae. The eggs are 140/4 long and hatch out in 4 to 5 days. A female becomes mature in about 3 weeks. Different animals have different species of itch mites. Demodicidae (Hair follicle mites). Demodex folliculorum — This is a vermiform acarine about 400/4 long; the eggs are about 75/4 long; they chiefly live in the sebaceous glands of nose and forehead. Ixodidae. This family of the Arachnoidea is one of great medical interest and of growing importance. While only proven the intermediary hosts in the case of the organism of African tick fever and the as yet undis- covered cause of spotted fever of the Rocky Mountains, there is con- siderable speculation as to the possibility of blackwater fever being due to a Babesia (piroplasma). Piroplasmata of animals seem to be invariably transmitted by ticks. Very important diseases due to these small pear-shaped organisms within red cells are known for various animals, the best known being that of cattle in Texas and known as Texas fever. Other diseases are Rhodesian fever (cattle), heart water (sheep) and malignant jaundice of dogs. In these diseases there are pathological features which resemble blackwater fever of man. It is of interest to note that it was with the transmission of Texas fever through an intermediate host (the tick) that Smith and Kilborne (1889-1893) established the zoologi- cal principle of transmission of disease through arthropod interme- diary hosts. This led up to the work on malaria, yellow fever, etc. Ticks differ from insects in having 4 pairs of legs, only 2 pairs of mouth parts and no antennae. They differ from other acarines in having a median probe-shaped puncturing organ, the hypostome, which is beset with numerous teeth projecting backward. The head, TICKS. 225 or capitulum, or rostrum, is the part which projects anteriorly from the body. This carries the piercing parts which are the single hypostome and a pair of piercing chitinous structures, the chelicerae. A pair of palpi consisting of segments are on either side of the biting parts. Very important structures are the stigmal plates. These are striking mosaic-like areas which are located just posterior to each hind leg in the Ixodinae and between the third and fourth legs in the Argasinae. As the greatest confusion exists as to the classification of ticks, Dr. Charles W. Stiles has now in hand a system of classifying ticks according to the appearance of these plates as seen under the high power of a micro- scope. There is great variation in the outline and general picture of these stigmal plates in the different species. The stigmal orifice, the opening of the tracheal system, is in the center. The Ixodinae have a scutum or shield like chitinous structure on the dorsal surface. It covers almost the entire back of the tick in the male and only a small portion in the female. The genital opening is toward the anterior part of the ventral surface. The anus, with anterior or posterior anal grooves, is near the posterior third of the venter. Life History of Ticks. — This varies greatly according to the sub- family, genus and species. The female Ornithodoros savignyi lays about 140 eggs. The larva does not leave the egg, but moults inside, and finally emerges as an 8-legged nymph. It lives in the dust in the cracks of the native huts and comes out at night to feed on the sleeping natives. As the possibility for destruction are not so great as with many Ixodinae the necessity for thousands of eggs is not imperative for the continuation of the species as with the Ixodinae. With some of the Ixcdinae the females lay from 5000 to 20,000 eggs during several days or weeks and then die. The egg is preferably deposited near grass. The egg stage lasts from 2 to 6 months, when the 6-legged larva emerges. Crawling up the grass a passing animal is gotten upon. After feeding, the larva drops to the ground, and changes to the pupal stage which has 4 pairs of legs. The pupa crawls up a blade of grass and gets on a passing animal (the second one). Feeding it falls to the ground where it remains 8 to 10 weeks. It moults and develops into an adult tick. These males and females gain access to another animal — the males fecundate the females, after which the 226 THE ARACHNOIDS. female gorges herself with blood; afterward dropping off the animal and laying eggs. With some ticks fewer hosts suffice. Classification of Ixodidae. Subfamily Argosinae. — Head concealed by bcdy when viewed dorsally. No scutum. Stigmal plates between third and fourth legs. Adults have no suckers beneath claws. FIG. 70. — Ticks, i, Dorsal aspect of female Ixodinae; 2, ventral aspect of same; 3, dorsal aspect of male Ixodinas showing larger scutum or shield; 4, ventral aspect of same; 5 and 6, ventral and dorsal surface of Ornithodoros moubata (Argasinae); 7, mouth parts of tick; 8, Stigmal plate of tick. Genus Argas. — Body narrow in front. Margins thin and acute. No eyes. The A. persicus (Miana bug) of Persia has been supposed to be concerned in the transmission of a serious disease. Genus Ornithodoros. — Margins of bcdy rounded. Skin has many irregular tubercles. O. savignyi has 2 pairs of eyes near base of TICKS. 227 mouth parts. It is the intermediate host of Sp. duttoni. (South African tick fever.) Subfamily Ixodinae. — Mouth parts project in front of body when viewed dorsally. Scutum present. Stigmal plates posterior to fourth pair of legs. Adults have suckers on claws. Section Ixodce. — Transverse recurved preanal groove in female. Genus Ixodes. Section Rhipicephalus . — No preanal, but postanal groove in female. In the genera Aponomma and Hvalomma the palpi are long and slender. The genus Aponomma has eyes. Amblyomma has no eyes. Palpi are short in the genera Haemaphysalis, Dermacentor and Margaropus. Haemaphysalis has no eyes; Dermacentor and Margaropus have eyes. In Dermacentor the head is transversely oblong; in Margaropus it is hexagonal. Dermacentor andersonii transmits spotted fever of the Rocky Mountains. LlNGUATULIDA. These are vermiform acarines more or less distinctly annulated. They have hooks at either side of the mouth. Linguatula rhinaria. — This has been observed in man both in larval and adult stages. The female lays eggs which, gaining freedom through the nasal mucus, are swallowed by various animals. A larva develops which bores its way through the gut and encysts in the liver or mesenteric glands. After several moultings, they work their way again to the intestines and so get out of the body of their host; or they may wander to lungs and trachea and either escape or take up their position in the nostrils and produce eggs. Consequently, one animal may act as intermediate and definitive host or these cycles may take place in distinct animal hosts. Porocephalus constrictus.— The adult form lives in snakes and the eggs are probably ingested by drinking water. These eggs de- velop into a curled-up, ringed larva, which is encysted especially in the liver or lungs. These escape and are swallowed by the snakes, their definitive hosts. CHAPTER XX. THE INSECTS. CLASSIFICATION OF THE CLASS INSECTA. Order. Family. Subfamily Genus. Species. Rhynchota (Hemip- tera), Pediculidae, Acanthiidae, J Pediculus, I Phthirius, Acanthia, f P. capitis. \ P. vestimenti. P. pubis. A. lectularia. Reduviidae, Conorrhinus, C. sanguisuga. Pulicii iae' f Pnlex f P. irritans. j JT UlcXj |P. cheopis. Pulicidae, ] Ceratophyllus, [ Ctneocephalus, C. fasciatus. C. serraticeps. Sarco] )syl- Sarcopsylla, S. penetrans. linae > Simulidae, Simulium, S. damnosum. (buffalo gnats), (mbwa). Psychodidse, Phlebotomus, P. minutus. (sand flies), Chironomidae, Ceratopogon, C. pulicaris. (midges) f Culicinae, Culex, C. fatigans. Diptera, Culicidae, -I Anophe- Anopheles, A. maculipennis. [ linae, Tabanus, ' T. bovinus. Haematopota, H. pluvialis. Pangonia, P. beckeri. Tabanidae, Chrysops, C. dispar. (horseflies) Glossina, G. palpalis. Musca, M. domestica. Auchmeromyia, A. luteola. Muscidae, Calliphora, C. vomitoria. Lucilia, L. caesar. [ Chrysomia, C. macellaria. (screw- worm) . Sarcophagidae, f Sarcophaga, \ Ochromyia, S. carnaria. O. anthropophaga (Estridae, f Dermatobia, \ Hypoderma, D. cyaniventris. H. diana. INSECTA. The class Insecta belongs to the branch Arthropoda. The species belonging to it far outnumber those of any other branch of the animal 228 LICE. 229 kindgom. Besides the classes Insecta and Arachnoidea, we have the Crustacea (crabs, lobsters) and the Myriapoda (centipedes, etc.). The two latter are of very little importance medically. The Arthropoda have segmented bodies, but they differ from the worms in having jointed appendages for the purposes of taking in food and moving from place to place. They also have an exoskeleton which is more or less unyielding from the deposit of chitin in the cuticle. The class Insecta have one pair of antennae, 3 pairs of mouth parts (the fused labium being considered as one pair), and 3 pairs of legs. They have 3 divisions of the body — head, thorax and abdomen. The air is supplied by means of tracheae — branching breathing tubes. Insects have 2 pairs of wings, the second pair of which is frequently rudimentary and shows simply as knob-like projections. These are termed halteres or balancers. In some insects both pairs of wings are rudimentary, as in the Aphaniptera. Of the class Insecta only the Rhynchota (Hemiptera) and the Diptera are of special importance. RHYNCHOTA. The Rhynchota are sucking insects in which the lower lip forms a long thin tube or rostrum which can be bent under the head or thorax. Inside this tube are biting parts — mandibles and maxillae. The Pediculidae. In this family there are no wings and there is no metamorphosis. The acorn-shaped eggs (nits) are deposited on hairs of the host. Pediculus capitis. — The female is about 1/12 of an inch long; the male smaller. They vary in color according to the color of the hair of the host. The eggs are deposited on the hairs of the head. The thorax is as broa'd as the abdomen. There seems to be a marked preference exhibited by lice for their own peculiar racial host. It has recently been suggested that this might account for certain peculiari- ties in infection where different races were living together and under similar conditions as to food and environment, and yet only one race contracts the disease. (Beriberi.) Pediculus vestimenti. — This louse lives about the neck and trunk 230 THE INSECTS. and deposits its eggs in the clothing. It is almost twice the size of the P. capitis and the abdominal segment is broader than the thorax. Phthirius pubis. — This louse is popularly known as the crab- louse. The female is little more than 1/25 of an inch in length, and the male a trifle less. They are almost square. They have a pref- erence for the white race and live about the pubic region. The female lays about a dozen eggs, which hatch out in about a wreek. FIG. 71. — Important Rhynchota. i, Head louse; 2, body louse; 3 crab louse; 4, bed bug; 5, mexican bed bug (Conorhinus sanguisuga). The Acanthiidae. These have a flattened body, a 3 -jointed rostrum and 4-jointed antennae. Their wings are atrophied. Acanthia lectularia. — This is the cosmopolitan bed-bug. It measures about 1/5 by i/ 8 of an inch. It is of a brownish-red color. The bed^bug^lives in cracks and crevices, especially about beds. It is said they can migrate from house to house. At any rate, they are BED-BUGS. 231 frequently transferred with wash clothes. They have a penetrating odor. The eggs are deposited in cracks and in 10 days they hatch out into larvae which pass insensibly into adults by a series of moultings (5). The bed-bug is very probably the intermediate host in kala azar, and it has been incriminated in connection with typhus fever and relapsing fever. Reduviidae. Conorrhinus sanguisuga. — This is known as the Texas or Mexican bed bug, and was formerly the foe of the common bed-bug, but having gottten a taste for human blood through the Cimex or Ancathia, it now prefers man. It is extending toward the North. It has wings. The bites are much more severe than those of the com- mon bed bug. It is of a dark brown color, nearly an inch in length, with a long, flat, narrow head and a short thick rostrum. They can run as well as fly. They bite at night. DIPTERA. The order Diptera is of great importance medically in a variety of ways, either by the direct irritation of their bites, by their transmitting disease directly, as does the common housefly typhoid fever, or by acting as intermediate hosts for various parasites. They are character- ized by mouth parts formed for puncturing, sucking or licking. They present a complete metamorphosis, larva, pupa and imago. As a rule, the diptera have a distinct pair of wings, the second pair being rudimentary. With the Aphaniptera the wings are practically absent. Under the Aphaniptera, \ve have to consider the Pulicidae or flea family. Pulicidae. This family is divided into 2 subfamilies— the Pulicinae and the Sarcopsyllinae. In the former the female remains practically un- changed after fecundation, in the latter the abdomen becomes enormously distended with eggs, and the female remains stationary after her impregnation in the burrow which she has made under the skin. 232 THE INSECTS. Pulicinae. — Formerly, with the exception of infection with D. canium, the fleas were only under suspicion as carriers of disease; ideas having been entertained as to their being possible transmitters of relapsing fever, typhus fever and kala-azar. As a result of the con- vincing experiments of the British Plague Commission, their role in the transmission of plague has been absolutely established. It is by the bite of the Pulex cheopis that plague is chiefly transmitted from rat to rat, and in bubonic and septicaemic plague it is apparently the in- termediary in human infection. The puncturing apparatus of the flea consists of a pointed epipharynx and 2 mandibles. By the ap- position of the mandibles to the epipharynx a tube is formed through which the blood is sucked up. The antennae are inconspicuous and are in close apposition to the sides of the head, behind the eyes, and can only be well made out with a lens. Fleas have 3 pairs of legs, and the male can be distinguished from the female by its smaller size and the conspicuous coiled-up penis within the abdomen. The body of the flea is flattened laterally. They may or may not have eyes, and certain conspicuous structures called combs are of importance in classification. In the metamorphosis of the flea the eggs are hatched out in dust of crevices, etc., into bristled larvae in about i week. The larva forms a cocoon and develops into" a nymph which has 3 pairs of legs. The nymphs emerge from the cocoon as adult fleas in about 3 weeks after the larva forms it. Key to the Fleas. A. No eyes, or eyes indistinct. 1. Densely spinose. Combs on some abdominal segments. Hystrichopsylla. 2. Combs on head and prothorax, none on abdomen. Typh- lopsylla. B. Eyes present. 1. Combs along inferior border of head and posterior border of prothorax. Ctenocephalus. 2. No combs along inferior border of head, but on posterior border of prothorax. Ceratophyllus. 3. No combs. Pulex. FLfcAS. 233 The common house flea of Europe is the Pulex irritans; that of the Tinted States the Ctenocephalus serraticeps or dog flea. The flea that is implicated with plague is the Pulex cheopis. It resembles P. irritans, but is more yellow than brown in color. It also has a greater number of bristles on the head. The ocular bristle runs above and in front of FIG. 72. — Various pulicidae. i, Ceratophyllus fasciatus (rat flea); 2, Pulex- cheopis (plague transmitting flea); 3, Ctenocephalus serraticeps (dog flea); 4, Sar- copsylla penetrans (chigoe); 5, head of flea showing palps; 6, female of S. pene- trans distended with eggs after burrowing under skin. the eye; that of P. irritans below. It is principally the flea of the M. decumanus, the sewer rat; but the house rat, M. rattus, becomes in- fected from coming in contact \vith the sewer rat in the basement. Sarcopsyllinae. Belonging to the subfamily Sarcopsyllinae, the Sarcopsylla penetrans is of great importance in tropical countries. It is known as 16 234 THE INSECTS the chigoe, nigua or jigger. The male is unimportant. The female, which when unimpregnated is only about 1/24 of an inch long, when impregnated bores its way into the skin of man, especially about the toes, soles of the feet or finger nails, and in the chosen site develops enormously, becoming as large as a small pea. A small black spot in the center of a tense rather pale area is characteristic. The abdomen of the female is filled with eggs. The metamorphosis is similar to that of the flea. Sarcopsylla can be differentiated from the flea by the pro- portionately larger head to the body, and especially by the fact that the head is the shape of the head of a fish, distinctly pointed. With the fleas the lower border of the head comes out in a straight line to join the curve of the upper part. In the Sarcopsylla lower and upper border of head are both curved. Tabanidae. This is the family of horseflies or gadflies. It is the most numer ous family of the Diptera — there being more than 1000 species. The females are blood suckers; the males live on flowers. They are large and stockily built. The eyes are usually very brilliant in color, and in the male make up the greater part of the head. The eggs are laid on leaves; the larva is carniverous; the pupa free. Tabanus autumnalis. — Is about 3/4 of an inch long; it is dark in color, and has 4 longitudinal bands on the thorax. • The last joint of the antennae has a crescentic notch. The wings do not overlap. No spurs at tip of hind tibia. Haematopota pluvialis.— In the Haematopota there is no crescentic antennal notch, and the wings overlap. The abdomen is narrower than in Tabanus. They also have spurs on hind tibia. The brimp, one of the Haematopota, bites man severely. Pangonia beckeri. — The genus Pangonia is characterized by a very long, slender and more or less horizontal proboscis. Chrysops dispar. — Chrysops and Pangonia have spurs at tip of hind tibiae. The wings are widely separated and spotted. The antennae of Chrysops are especially long and slender. Chrysops and Haematopota produce the greatest amount of pain from their bites. BITING FLIES. 235 The Tabanidae are not implicated as intermediate hosts in the trans- mission of disease. By their bites, however, they may transmit disease directly, as with anthrax. Muscidae. The common housefly, M. domestica, is the best example of this family. This fly is incapable of biting, but may transmit disease \ 7- oft. FIG. 73. — Insects in which imago stage is important, i, Stomoxys calcitrans; 2, arista of Glossina palpalis; 3, Glossina palpalis (tsetse fly). 4, Tabanus autum nalis. directly, carrying infectious material from the source, as in faeces, to the food about to be ingested. Their role in typhoid fever is one of im- mense importance. • In the Muscidae the antennae hang down in front of the head in 3 segments, and have a plumose arista. There are no bristles on ab- domen except at tip. 236 THE INSECTS. Stomoxys calcitrans. — These greatly resemble the common housefly in size and shape. They can be easily distinguished by the black, piercing proboscis extending beyond the head. There are longitudinal stripes on the thorax and spots on the abdomen. The proboscis on examination will be seen to be bent at an angle near its base. The palps are short and slender. The wings diverge widely. The genus Stomoxys includes vicious biters. This is the fly which comes into houses before a rain, and which has given the common housefly the reputation of biting before a rain. Stomoxys may be implicated in transmitting surra (Trypanosoma evansi). The horsefly (Haematobia irritans) rarely bites man. In these the palpi are much longer than in Stomoxys. Glossina palpalis. — This is the tsetse fly that is responsible for the transmission of human trypanosomiasis and the later stage of the disease, sleeping sickness. The tsetse fly is a small brownish fly about 1/3 of an inch long. The proboscis extends vertically and has a bulb at its base. The arista is plumose only on the upper side. The wings are carried flat, closed over one another, like scissor blades. The most characteristic feature of the tsetse fly is the way the fourth longitudinal vein bends up abruptly to meet the oblique transverse vein. In Stomoxys, the wings separate; in Hsematopota they just meet, and in Glossina they cross. The tsetse fly does not lay eggs, but gives birth to a single full-grown larva which immediately becomes a pupa. Glos- sina morsitans transmits the cattle trypanosomiasis disease, nagana. Auchmeromyia luteola. — This is an African fly, the larva of which is known as the " Congo floor maggot," and is a blood sucker. The larva is of a dirty -white color and about 2/3 of an inch long. It crawls out at night and feeds on the sleeping natives. This is the only known instance of a blood-sucking larva. Calliphora vomitoria and Lucilia caesar. — These are flies with brilliant metallic-colored abdomens, commonly called blue-bottle flies. They deposit their eggs on tainted meat and in wounds. Many cases of obscure abdominal trouble are probably due to the larvae of these flies. Intestinal myiasis is undoubtedly of greater importance than has been thought. The larvae, with hook-like projections anteriorly and a ringed body, can easily be recognized in the faeces. They have been MYIASIS. 237 mistaken for flukes. They also have a tendency to be attracted by those with ozena and the larvae may develop in the nostrils. Chrysomyiamacellaria. — This is known as the screw-worm when in the larval stage. The adult fly resembles the blue-bottle flies. The thorax is striped. The eggs, which number 250 or more, when deposited in the nostrils or in wounds, develop into the screw-worm larvae, which may, by going up into the frontal sinus, cause death. FIG. 74. — Insects in which larval stage is important, i and 2, Insect and larval forms of Dermatobia cyaniventris; A, ver macaque, B, Torcel or Berne; 4 and 6, Insect and larva of Calliphora vomitoria; 5 and 7, insect and larva of Compso- myia macellaria (screw worm). Sarcophagidae. These are known as "flesh flies." Sarcophaga carnaria. — This is a grayish fly with 3 stripes on thorax and black spots on each segment of the abdomen. It is vivi- parous. The larvae gain access to nasal and other cavities and there 238 THE INSECTS. develop. Cases of death have been reported. Naturally, the fly deposits its larvae on decaying flesh. In times of war all of these flies become important by reason of "maggots" in the wound. Ochromyia anthropophaga. — This is an African fly whose larvae develop under the skin of man and animals. It is known as the Ver de Cayor. The larva resembles the Ver Macaque. (Estridae. Dermatobia cyaniventris. — These are large, thick-set flies, with prominent head and eyes, small antennae and a marked narrowing at the junction of thorax and abdomen. The abdomen is a metallic blue. The larvae are deposited under the skin in various parts of the body. When the larvae move they cause considerable pain. At first the larva is club-shaped, but later on it becomes oval. The former is called Ver Macaque, the latter Torcel. Hypoderma diana. — The larval form of this fly has been reported 3 times for man. CHAPTER XXI. THE MOSQUITOES. MOSQUITOES (Culicidae) are of the greatest importance medically, not only from their influence upon health in general by reason of inter- ference with sleep and possibly from direct transmission of disease, but, more specifically, they are the only means by which it at present appears possible to bring about infection with such diseases as yellow fever, malaria, filariasis and possibly dengue. In addition, many diseases of animals are transmitted by mosquitoes. The Culicidae differ from all other Diptera in having scales on their wings and generally on head, thorax or abdomen. To identify a mosquito, examine a wing and note the scales; also note the presence of two distinct fork cells and, in addition, that the costal vein passes completely around the border of the wing, making a sort of fringe with its scales. Mosquitoes undergo a complete metamorphosis, there developing from the egg a voracious, rapidly- growing larva; next, a nongrowing, nonf ceding stage — the pupa or nymph. There the head and thorax are combined in an oval body, from the back of which projects the syphon tubes; and, tucked in ventrally is a small tail-like appendage. The fully developed insect emerges from the pupa. The principal mosquito -like, blood-sucking Diptera which are frequently mistaken for mosquitoes — none of which have scales on their wings — are the following: i. Chironomidae or Midges. — The blood-sucking species of Chironomidae, which are found in most parts of the wrorld, belong chiefly to the genus "Ceratopogon." These midges are of very small size, about 1/12 of an inch long, are able to get through netting and, usually being in swarms, they are exceedingly troublesome. One of the 239 240 THE MOSQUITOES. midges, the "jejen" of Cuba, is a great scourge, its small size enabling it to enter eyes and nostrils. The larva of Chironomus is a red worm -like creature; the pupa has a tufted head. 2. Simulidae or Buffalo Gnats. — These are small blood- thirsty insects only about 1/8 of an inch in length. The thorax is humped, the legs are short and the proboscis short and inconspicuous. One species, the S. damnosum, known by the natives of Uganda as "Mbwa," is greatly dreaded; its bites causing swellings and sores. 3. Psychodidae. — These are small, hairy, slender midges, with long legs and a long proboscis. They are only about if 1 2 of an inch in length. Mosquitoes have 3 main parts of the body — the head, the thorax and the abdomen. On the head, the space behind the two compound eyes is called the frons, in front, and the occiput posteriorly. The nape is back of the occiput. The bulbous prolongation of the frons which projects over the attachment of the proboscis is. the clypeus. The clypeus is hairy in the Culex; scaly in Stegomyia. The proboscis is straight in all mosquitoes of importance medically. It consists of a fleshy, scaled, gutter-shaped portion beneath, known as the labium, which terminates in two hinge-joint processes — the labella. At the end of the labium is a thin membrane (Button's membrane). It is through this that filarial embryos are supposed to pass on their way from the interior of the labium to enter the person bitten. The labium may be considered as the sheath of a knife, holding and protecting the slender, blade-like penetrating organs. Lying in this groove we have, from above downward, the horseshoe-shaped labrum — epipharynx, the under surface of which is open. This when closed by the under- lying hypopharynx forms a tube through which the blood is sucked up by the mosquito. In the hypopharynx, which somewhat resembles a hypodermic needle, is a channel, the veneno-salivary duct. It is down this channel that the malarial sporozoite passes. There are 2 pairs of mandibles and 2 pairs of maxillae on either side of the hypopharynx — MOSQUITOES. 241 the mandibles above and the maxillae below. The serrations of the maxillae are coarser than those of the mandibles. The sensory organs, the palps, lie on either side of and slightly above the proboscis. These are of the utmost importance in differentiating mosquitoes and must not be confused with the antennae, which are attached above the palpi and at the sides of the clypeus. These antennae are of importance in dis- tinguishing the sex of the mosquito. FIG. 75. — Anatomy of mosquito, i, Dorsal view of mosquito; 2, wing of mosquito; A, costal vein; B, mid cross vein; C, posterior cross vein; D, first fork- cell; E, second fork-cell; 3, various types of scales; a, flat head scales; b and c, Mansonia wing scales; d, upright forked head scales; e f , g and h, various shapes of thoracic scales. The thorax is largely made up of the mesothorax, at the posterior margin of which is a small, sharply -defined piece, the scutellum; this may be smooth or trilobed. Underneath and posterior to the scutellum is the metanotum; the metanotum is bare in Culicinae, has hairs in Dendromyinae and scales in Joblotinae. 242 THE MOSQUITOES. There is a pair of wings attached to the posterior part of the mesothorax and, more posteriorly still, a pair of rudimentary wings (halteres) attached to the metanotum. The 3 pairs of legs are at- tached to the thorax. There are 9 segments in the abdomen. The genitalia arise from the terminal segment as bilobed processes. In the male there is a pair of hook-like appendages or claspers, between which, and ventrally situated, are the harpes, also a pair of chitinous processes. In considering the question of the possible danger which might arise from the introduction of a case of yellow fever, malaria or filariasis, it would give the greatest information if the ova were at hand so that we could by watching the development from egg to larva, pupa and insect, have all the points from which to decide as to the genera developing in the given locality. It is generally a very easy matter to dip out large numbers of larvae from the pools and having noted the characteristics of the larvae, to do the same when the pupae develop; so that we have only to verify our identification when the insect emerges from the pupa. THE OVA. The egg raft of Culex, containing about 250 ova, is quite perceptible on the surface of the water as a black, scooped-out mass, about 1/5 of an inch in length. The eggs are set vertically in the raft. The eggs of the Stegomyia are laid singly and have a pearl-necklace-like fringe around them. The Anophelinae eggs are oval in shape with air-cell projections from either side. They are laid in triangle and ribbon patterns. The markings of these air cells vary and have been used for differentiation. The length of time of the egg stage varies according to temperature and other conditions — i to 3 days for Stegomyia and 2 to 4 days for Anophelinae. The Anophelinae are more difficult to raise than Culex or Stegomyia. LARV.E. There are two great classes of larvae — the siphonate and the asiphonate. The latter are always Anophelinae. The Culicinae larvae have a projecting breathing tube at the MOSQUITOES. 243 posterior extremity which is called a respiratory syphon. This pro- jects off at an angle from the axis of the body, the true end of which terminates in four flap-like paddles. If you divide the length of the syphon by the breadth, you get what is known as the syphon index. In Culex the syphon is long and slender; in Stegomyia it is short and barrel-shaped. When at the surface the Culex larva has his syphon almost vertical and the body at an angle of about 45°. A-H.Ebfclinpf ?-«>« FIG. 76. — Metamorphosis of mosquitoes, i, 2, 3, 4 and 5, Eggs, larva, pupa and heads of male and female Culex; 6, 7, 8, 9 and 10, eggs, larva, pupa and heads of male and female Anopheles; n, 12, 13 14 and 15. Eggs, larva, pupa and heads of male and female Stegomyia. The Stegomyia larva hangs more vertically. As a rule, the hairs proceeding from the sides of Culex larvae are straight and the head relatively large. There are also no palmate hairs along the sides. The Anophelinae larvae have a small head which is capable of being twisted around with lightning-like rapidity. They are darker in color and have no syphon; float parallel to the surface of the water; 244 THE MOSQUITOES. have long lateral branching hairs, and on the sides of each of the 5 or 6 middle abdominal segments they have a pair of palmate hairs. These palmate hairs are supposed to aid them in keeping their position on the surface of the water. The larvae are usually called "wigglers." The duration of the larval stage is from i to 2 weeks, according to the temperature. THE PUP^:. These have a bloated-looking cephalo-thorax and a shrimp-like tail — the latter the abdomen. Very important in examining them with a lens is to note the characteristics of the syphon tubes which project from the dorsal surface. These syphons are long and slender in Culex and project from the posterior portion of the head end. In Anophelinae they are broadly funnel-shaped and arise from the middle of the head end. The syphon of the Stegomyia is triangular. The bulbous end of the Culex nymph is more vertical than the horizontally-placed cephalo-thorax of Anopheles. The duration of pupal life is short — only 1-3 days. At the end of this time the pupa comes to the surface and straightens out. The integument then splits dorsally and the perfect insect emerges. It dries its wings for a time on its raft-like pupal skin and then flies away. From the above it will be seen that the stages in the metamorphosis of the mosquito takes about 2 weeks: 1-3 days for egg stage; 7-10 days for larval stage and 2-3 days for pupal stage. DISSECTION OF THE MOSQUITO. The easiest way to secure a mosquito for dissection is to use an ordinary plugged test-tube. Slipping the open end of the test-tube over the resting mosquito; by a slight movement, the insect will fly toward the bottom. Then quickly insert the plug. If it is not desired to study the scales, the best way to kill the mosquito is by striking the tube sharply against the thigh. If it is also desired to study the scale characteristics it is better to put a drop or so of chloroform on the lower part of the cotton plug. The vapor falls to the bottom of the tube and kills the mosquito. Take the mosquito out, pull off legs and wings and MOSQUITOES. 245 then place the body in a drop of salt solution on a slide. Then hold the anterior end of the thorax by pressure of a needle. With a needle in the other hand, gently crush the chitinous connection between the sixth and seventh segments of the abdomen. Then holding the thorax firm, steadily and gently pull the last segments in the opposite direction. If this is done properly, a delicate gelatinous white mass will slowly float out in the salt solution. One should be able to secure the aliment- ary canal as far up as the proventriculus, which is just anterior to the FIG. 77. — Anatomy of mosquito, i, Cross section of proboscis of mosquito; 2. anatomy of mosquito longitudinal section; 3, tip of proboscis of mosquito; a, labrum-epipharynx; b, hypopharynx; c, mandible; d, maxilla. stomach, the part in which the malarial zygotes develop. Proceeding from before backward, we have the proventriculus, which is a sort of muscular ring at the opening of the stomach or mid-gut. Leading from the stomach we have the hind gut, which ends in the rectum. Taking origin at the posterior end of the stomach and festooning the hind gut are 5 longitudinal tubes— the Malpighian tubules. These are characterized by large granular-like cells with a prominent refractile nucleus. They are regarded as the renal structures. It is in these tubules that the embryo of the Filaria immitis of the dog develops. 246 THE MOSQUITOES. In the female mosquito, the parts withdrawn may seem to be largely made up of the white oval ovaries. These are connected with the spermathecae, in which the spermatozoa are stored after fecundation by the male. In the male the testicles are quite distinct. Next to the examination of the stomach for zygotes, which appear as wart-like excrescences on its outer surface, the most important structures are the salivary glands, where the malarial sporozoites are found. The easiest way to dissect out the salivary glands is to press down firmly, but gently, on the anterior part of the thorax, and then with the shaft of a second needle, pressing on the head, to gently draw the head away from the thorax, so that by this expression and traction movement you extract them with the head segment. They are very minute and are to be told by their exceedingly highly refractile appearance. To stain for spcrozoites, pick up the head end, and with forceps draw the severed neck along a clean dry slide, trying at the same time to smear out the adherent salivary glands. After drying, stain with Wright's stain. The sporozoites are narrow falciform bodies about 12 p in length, with a central chromatin dot. A matter about which there is dispute is as to whether the salivary glands communicate with the alimentary canal. Theobald states that there is no connection between them. DIFFERENTIATION OF CULICIN^: AND ANOPHELIN^:. It is impossible even for an entomologist to differentiate mos- quitoes without recourse to elaborate keys and tables. It is a com- paratively easy matter, however, to decide as to whether the mosquito is a probable malaria transmitter or not. While certain characteristics of the male are used to separate the ^dinae from other subfamilies, yet it is only with the female that we •concern ourselves in differentiating the Culicinae from the Anophelinac. Therefore, it is first necessary to distinguish the male from the female. If the antennae have not been torn off, this can be decided by the highly-adorned plumose antennae of the male, those of the female being sparsely decorated with short hairs. The palpi of the Anophelinae tend to be clubbed, while those of the Culex are straight. If the antennae have been broken off, look for the claspers at the end of the abdomen. MOSQUITOES. 247 Having determined that the insect is a female, we then proceed to place it either in the subfamily Culicinae or Anophelinae by a study of the relative length of the palpi to the proboscis. If the palpi are shorter than the proboscis, it belongs to the Culicinae; if as long or longer, to the Anophelinae. The palpi of the female Megarhininae are also long, but the proboscis is curved. Having settled on the subfamily, we separate the genera by con- sidering such points as character and distribution of scales on back of head, wings, thorax and abdomen; banding of proboscis, legs, ab- domen and thorax, shape of scales on wings and location of cross-veins. In the resting position Culex allows the abdomen to droop, so that FIG. 78. — Anopheles. FIG. 79. — Culex. Resting positions of anopheles and culex insects. (Drawn by C. O. Waterhouse.} it is parallel to the wall. The angle formed by the abdomen with head and proboscis gives a hunchback appearance. Anopheles when resting on a wall goes out in a straight line at an angle of about 45°. It resembles a bradawl. Classification. There are 4 subfamilies of Culicidae, differentiated according to the palpi: i. Palpi as long or longer than proboscis in male. 1. Palpi as long as proboscis in female; proboscis straight. Anophelina. 2. Palpi as long or shorter than proboscis; probos- cis curved. Megarrhinince. 3. Palpi shorter than proboscis. Cuticince. 2. Palpi shorter than proboscis in male and female. JEdince. 248 THE MOSQUITOES. The important ones from a medical stand-point are the Anophelin 33 and Culicinae. i. Scales on head only; hairs on thorax and abdomen. 2. Scales on head and thorax (narrow curved scales.) Abdomen with hairs. Anophelinae. 1. Scales on wings, large and lanceolate. Ano- pheles. 2. Wing scales small and narrow and lanceolate. Myzomyia. 3. Large inflated wing scales. Cyclolcppteron. i. Wing scales small and lanceolate. Pyretophorus. i. Abdominal scales only on ventral surface. Thoracic scales like hairs. M yzorhynchus . 3. Scales on head and thorax and abdomen. Palpi covered with thick scales. 2. Abdominal scales narrow, curved or spindle- shaped. Abdominal scales as tufts and dorsal patches. Nyssorhynchus. 3. Abdomen almost completely covered with scales and also having lateral tufts. Cellia. 4. Abdomen completely scaled. Aldrichia. NOTE. — Of the above genera only Cycloleppteron and Aldrichia are unproven malarial transmitters. The Megarhininae are of no importance medically. The genus Megarhinus has the following characteristics : 1. Large mosquitoes with brilliant metallic coloring. (Ele- phant mosquitoes.) 2. Long, curved proboscis. 3. Caudal tufts of hairs on each side of abdomen. The JEd'msd are not known to play any role in transmission of diseases. This subfamily is characterized by having the maxillary palpi much shorter in both males and females than the proboscis. One genus Sabethes is very characteristic, owing to dense paddle- like scale structures on two or more legs. MOSQUITOES. 249 Differentiation of Culicinae Genera. i. Posterior cross- i. Proboscis curved in female. Psorophora. vein nearer the 2. Proboscis straight in female, base of the wing A. Palps with 3 segments in the female. than the midcross- vein. a. Third segment somewhat longer than the first two. Culex. b. The 3 segments equal in length. Stegomyia. B. Palps with 4 segments in the female. a. Palps shorter than the third of the proboscis. Spotted wings. Theobaldia. b. Palps longer than the third of the proboscis. Irregular scales on wings. Mansonia. C. Palps with 5 segments in the female. Tcenior- hynchus. 2. Posterior cross-vein in line with midcross-vein. Joblotina. 3. Posterior cross-vein further from base of wing than midcross-vein. Mucidus. Of the Culicinae the genus Stegomyia is of importance on account of yellow fever. The totally efficient hosts for filariasis (filarial embryos found in thorax and proboscis) are chiefly among the genus Culex. The genera Mansonia and Taeniorhynchus may also trans- mit filariasis. Some think the Anophelinae genera "Cellia" and "Myzomyia" may transmit filariasis as well as malaria. The genus Culex is implicated in dengue. Stegomyia. — This is the most important culicine genus. These are mosquitoes with silver markings. The head, entirely covered with flat scales, has also some upright forked scales. Scutellum has dense flat scales. S. Calopus is deep blackish-brown with two thoracic parallel lines with curved silver-white lines outside. Banding of thorax, abdomen and legs. Culex. — Male palpi long and acuminate. Head has narrow curved and upright forked scales. Laterally, flat scales. C. fatigans supposed to carry dengue. Theobaldia. — These Culicinae have spotted wings resembling Anophelinae. These spots are due to aggregations of scales, not to dark scales. Male palps are clubbed (like Anopheles). Mucidus. — This genus has a mouldy look from long twisted gray scales. Mansonia. — This genus is characterized by broad flat asym- metrical wing scales. 250 THE MOSQUITOES. Grabhamia. — Wings have pepper-and-salt appearance with short fork cells. Tceniorhynchus. — This genus is characterized by dense wing scales, which are broadly elongated with truncated apex. Acartomyia. — Much like Grabhamia, but scales of head give ragged appearance. A. zammittii was supposed to be concerned in Malta fever (not proven). NOTES ON ANIMAL PARASITOLOGY. NOTES ON ANIMAL PARASITOLOGY. NOTES ON ANIMAL PARASITOLOGY. NOTES ON ANIMAL PARASITOLOGY. PART IV. CLINICAL BACTERIOLOGY AND ANIMAL PARASITOL- OGY OF THE VARIOUS BODY FLUIDS AND ORGANS. CHAPTER XXII. Diagnosis of Infections of the Ocular Region. IT is advisable before taking material for cultures or smears to cleanse the nasal area of the eye-lids, and especially about the carun- cles, with sterile salt solution. Then, by gently pressing on the lids, we may be able to get pure cultures of the organism causing the infection. Normally, we may find in the region of the caruncles various skin organisms, especially staphylococci, giving white colonies. A small particle of sterile cotton, wound on a toothpick, with the aid of a sterile forceps, makes an excellent swab for obtaining material for smears; the same may first be drawn over an agar surface in a Petri dish in a series of parallel lines of inoculation before making the smears on slide or cover-glass. When there is considerable discharge, a capillary pipette, with a rubber bulb, may be used to draw up sufficient material for cultures and smear. Be sure to round off the end of the pipette in the flame and not to use a very fine capillary tube. In conjunctival tultures, plates of glycerin agar or agar plates smeared with blood are to be preferred, as the gonococcus and Koch- Weeks bacillus will only grow on blood or hydrocele agar. The diphtheria and xerosis bacilli grow well on glycerin agar. In addition to the white staphylococcus, the streptococcus may be present when inflammation of the nasal duct exists. The pneumococcus is a fairly common cause of serpiginous corneal ulcerations. Active treatment is necessary. 251 252 DIAGNOSIS OF INFECTIONS OF THE OCULAR REGION. The B. xerosis is possibly a harmless organism and must not be accepted as explaining an infection unless other factors have been eliminated. Leprosy and tubercle bacilli may be found in corneal ulcerations. The true diphtheria bacillus, which the xerosis so much resembles, may cause a pseudomembranous inflammation. The gonococcus and the Koch-Weeks bacillus are usually re- sponsible for the very acute cases of conjunctivitis. Both these organisms are characteristically intracellular and are Gram negative. The diplobacillus of Morax and Axenfeld is more common in chronic, rather dry affections of the conjunctiva. In cases of ozena with involvement of the nasal ducts Fried- lander's bacillus may be found. Certain fungi of the genus Microsporum have been thought to be the cause of trachoma, as have also certain bacillary forms. One should be very conservative about reporting fungi in smears or cultures of external surfaces. The larval stage of Taenia solium (Cysticercus cellulosae) has a predilection for eye as well as brain. It is usually situated beneath the retina. The adult Filaria loa tends at times to appear under the conjunc- tiva or in the subcutaneous tissue of the eye-lids. Fly larvae have been reported from the conjunctival sacs in the helpless sick CHAPTER XXIII. DIAGNOSIS OF INFECTIONS OF THE NASAL CAVITIES. IN taking material from the nasal cavities, for the bacteriological examination, it is well to wash about the alae with sterile water and then have the patient blow his nose on a piece of sterile gauze and take the material for culture or smear from this. If the material is purulent and located at some ulcerating spot, it is best to use a specu- lum, and either touch the spot with a sterile swab or use a capillary bulb pipette with a slight bend at the end. Normally, we find only white staphylococcus colonies and colonies of short-chain streptococci. The M. tetragenus, B. xerosis and Hoff- man's bacillus are also occasionally found. In some cases of ozena we may find an organism of the Fried- lander type in pure culture. Biscuit-shaped diplococci, both Gram negative and positive, are to be found either normally or in cases of coryza. M. catarrhalis has probably been frequently reported as the meningococcus. Still, the meningococcus has been found in the nasal secretion of patients with cerebrospinal meningitis. Diphtheria involving the nasal cavity must always be kept in mind, and in quarantine investigations the examination of the nasal secretion culturally should be a part of the routine. The tubercle bacillus may be found in nasal ulcerations ; it is, how- ever, only present in exceedingly small numbers. On the other hand, one of the best diagnosistic procedures in leprosy is to examine smears from nasal mucous membrane for the B. leprae. In such ulcerations the bacilli are found in the greatest profusion. •. ' M The gonococcus has been reported for the nose. Various fungi have been reported from the nose, but in such a region the strictest conservatism in reporting should be observed. Recently sporozoa have been reported in a case of nasal polyp. 253 254 DIAGNOSIS OF INFECTIONS OF THE NASAL CAVITIES. So many degenerative changes in epithelial cells resemble protozoal forms that such findings require ample confirmation. The larval form of the Linguatula rhinaria is a rare parasite of the nasal cavities. Various fly larvae are far more common, and the "screw-worm," the larva of the Chrysomyia macellaria, is common in certain parts of tropical America, and may by its burrowing effects cause fatal results. CHAPTER XXIV. EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL. IN a preparation made from material taken by a sterile swab from the region of the normal buccal and pharyngeal cavities and stained by Gram's method, we are struck by the variety of organisms present. Gram positive and Gram negative staphylococci are present, as are also streptococci, pneumococci, leptothrix forms and very probably yeasts and sarcinae types with many Gram negative bacilli. If pseudodiphtheria organisms are present, we have these showing a FIG. 80. — Vincent's angina. Spirochaeta vincenti. (Coplin.) Gram positive reaction. If this material is smeared on agar plates and cultured at 37° C., we are struck by the fact that the colonies on the plates may be exclusively staphylococcal and streptococcal. It is very difficult, if not impossible, to distinguish a pneumo- coccus colony from a streptococcus one on a plate culture. The presence or absence, however, of the pneumococcus is distinctly shown in the 255 256 EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL. Gram stained smear, either by its lance-shaped morphology or the presence of a capsule. In has been my experience that smears from about 15% of normal individuals show capsulated pneumococci. In diphtheria examinations we rely chiefly on the cultural findings on Loffler's serum. Where the process is streptococcal or due to the organisms associated with Vincent's angina, the immediate examina- tion of a smear from the suspected spot or area gives greater diagnostic information. The streptococcus being so abundant in cultures from normal throats, it is difficult to determine its significance in a culture ; abundance of streptococci in a smear from an ulceration or bit of membrane, however, is of etiological import. By staining with Neisser's method it is possible to make an im- mediate diagnosis of diphtheria from a smear from a piece of mem- brane in about 25 percent of cases. It is well, however, to always culture such material. Material from the throat is ordinarily best obtained with a sterile copper wire cotton pledget swab. The platinum loop usually bends too easily. A sterile forceps may be more convenient for obtaining particles of membrane. It is believed that ulcerative conditions of the throat, associated with the presence of the large fusiform bacillus and delicate spirillum, which make the picture of Vincent's angina, are more common than is usually so considered. As a rule, only cultures on serum are made and very rarely direct smears. If a smear were always made and stained by Gram's method (with a contrast stain of dilute carbol fuchsin) at the same time the culture was made, it is probable that much information of value would be obtained. Direct smears are the procedure of choice in streptococcal and pneumococcal anginas as well as in Vincent's angina. Unless very familiar with the morphology of Treponema pallidum and using Giemsa's staining procedure, we should be very conserva- tive in reporting such an organism from suspected syphilitic ulcera- tions of the throat. The thrush fungus (Endomyces albicans) may be easily demon- strated in a Gram stained specimen as violet mycelial structures. Yeasts due to food particles are not infrequently observed in smears and cultures from the mouth. ANIMAL PARASITES OF THE THROAT. 257 Amoebae and flagellates have been reported from the mouth. Also in the remarkable disease "halzoun," flukes have been found to be the cause of the asphyxia. In the tropics, round worms may be vomited up and, lodging in the pharynx, may have to be extracted. CHAPTER XXV. EXAMINATION OF SPUTUM. FREQUENTLY the material submitted for examination as sputum is simply buccal or pharyngeal secretion, or more probably secretion from the nasopharynx, which has been secured by hawking. It should always be insisted upon that the sputum be raised by a true pulmonary coughing act, and not expelled with the hacking cough so frequently associated with an elongated uvula. When there is an effort to deceive, some information may be obtained from the watery, stringy, mucoid character of the buccopharyngeal material and also, from the presence of mosaic-like groups of flat epithelial cells (often packed with bacteria). The pulmonary secretion is either frothy mucus or mucopurulent material, and if the cells are alveolar they greatly resemble the plasma cells. At times these cells may contain blood pigment granules (heart disease cells). In the microscopic examination a small, cheesy particle, the size of a pin head, should be selected. This should be flattened out in a thin layer between the slide and cover-glass and should be examined for elastic tissue, heart-disease cells, eggs of animal parasites, amoebae and fungi. Echinococcus booklets, Curschman spirals besprinkled with Charcot-Leyden crystals, and haematoidin and fatty acid crystals may also be observed. It may facilitate the examination of the sputum for elastic tissue and actinomycosis and other fungi to add 10% sodium hydrate to the preparation. To make smears for staining, the sputum should be poured on a flat surface, preferably a Petri dish, and a bit of mucopurulent material selected with forceps. A dark back-ground facilitates picking out the particle. A toothpick is well adapted to smearing out such material on a slide. After using, it can be burned. When dry, the smear is best fixed by pouring on a few drops of alcohol, allowing this 258 TUBERCLE BACILLI IN SPUTUM. 259 to run over the surface, and then, after dashing off the excess of alcohol, to ignite that remaining on the film in the flame and allow to burn out. A mark with a grease pencil, about one-half inch from the end, gives a convenient surface to hold with the forceps and also prevents the stain subsequently used from running over the entire surface. Sputum should as a routine measure be stained by the Ziehl-Neelson method and by Gram's method. In examining for tubercle bacilli it may be necessary to employ some method for concentrating the bacterial content of the sputum prior to making the smear. A very satisfactory method is that of Muhlhauser-Czaplewski. Shake up the sputum with four to eight times its volume of 1/4% solution of sodium hydrate in a stoppered bottle. When the mixture has become a smooth, mucilaginous -looking fluid, add a few drops of phenolphthalein solution and bring the pink mixture to a boil. Then add drop by drop a 2% solution of acetic acid, stirring con- stantly, until the pink color is just discharged. If the least excess of acid is added over that just sufficient to cause the pink color to disap- pear, mucin wrill be precipitated. Now pour this mixture into a centri- fuge tube and smear the sediment on a slide and stain for tubercle bacilli. Sputum smears stained by some Romanowsky method or by the haematoxylin eosin stain are best adapted for the study of various cells, and in particular of the eosinophile cells so characteristic of bronchial asthma. In sputum from cancer of the lungs the large vacuolated cells may be found. When examining the sputum of the bronchopneumonia of in- fluenza the formol fuchsin gives the best results. The influenza bacilli are found in little masses, frequently grouped about small collections of M. tetragenus. The cocci stain a rich purplish-red, while the small influenza bacilli take on a light pink color. Red cells show up well in specimens stained by the Romanowsky method; if rouleaux formation is marked, it may indicate pulmonary infarction. In culturing sputum a mucopurulent mass should be washed in 260 EXAMINATION OF SPUTUM. sterile water and should then be dropped into a tube of sterile bouillon. With a sterile swab it should be emulsified and successive streaks made along the surface of an agar or a glycerin agar plate. In obtaining cultures from influenza sputum, first smear the material thoroughly over a blood-serum slant; then inoculate, by thorough smearing over the surface of successive blood-streaked agar slants, the material on the surface of the blood- serum slant. The platinum loop should be transferred from one slant to another without recharging. The in- fluenza bacillus seems to grow better if the blood-streaked agar slants are prepared just before inoculating with the sputum. All that is necessary is to sterilize an ear, puncture and take up the exuding blood with a large loop. Cultures for tubercle bacilli are impracti- cable. A guinea-pig should be inoculated. The blood-stained watery sputum of plague pneumonia should be cultured on plates of plain agar and 3% salt agar at the same time. An ordinary smear stained with carbol thionin, however, practically makes a diagnosis. Pneumococci, M. catarrhalis, and Friedlander's bacillus in sputum are best demonstrated by Gram's method of staining. Moulds, especially Aspergilli, may be found in sputum. Species of Mucor, Cryptococcus and Endomyces have also been reported. Amoebae from liver abscess rupturing into the lung may be found. Very important pulmonary infections are those with Paragonimus westermani. This is recognized by the presence of operculated eggs in the sputum. Hydatid cysts, either of the lung or of the liver, rupturing into the lung, may be recognized by the presence of echinococcus hooklets. The material is bile-stained if from the liver. CHAPTER XXVI. THE URINE. MATERIAL for staining is best obtained by centrifuging the urine, then pouring off the supernatant urine, to replace it with a i% aqueous solution of formalin. Shaking for a few seconds we again centrifuge, pour off the fluid from the sediment and make smears from the sedi- ment. The smear may be stained directly by Wright's method or after fixing by heat with Gram's stain, T. B. stain or haematoxylin and eosin. The latter is the best for the staining of epithelial cells and animal parasites; the Gram method for bacteria. It is frequently difficult to distinguish the spores of moulds from red blood-cells except by measurement and staining reactions. Spores of moulds rarely exceed five mikrons. It is difficult to determine the presence of blood in urine in higher dilution than i to 300 with the spectroscope. The occult blood-test will show it in much higher dilution. To secure urine for bacteriological examination catheterization is rarely necessary. The glans penis and meatus should be thoroughly washed with soap and water, after which dilute alcohol (50%) should be used. The greater part of the urine first passed should be rejected and only the last portion passed should be caught in a sterile recep- tacle. This may be either streaked over the surface of an agar or a lactose litmus agar plate. This lattter medium is very useful in distinguishing typhoid or paratyphoid colonies (blue) from colon, and streptococcus or staphylococcus colonies (pink). The urine may be added to tubes of melted agar and then poured. Cystitis from a colon infection gives an acid urine; that caused by Proteus vulgaris an alkaline urine. The bacilli of plague and Malta fever are also found in the urine. \Yhile the smegma bacillus in urine may be differentiated from the tubercle bacillus by the former losing its red color, by prolonged decolorization with acid alcohol, yet it is chiefly by the subcutaneous inoculation of the guinea-pig that we should diagnose genitourinary 18 261 262 THE URINE. tuberculosis. Inject the sediment after centrifuging. Gonococci are reported from Gram stained smears. Yeasts and moulds frequently contaminate urine, especially diabetic urine, after it has been passed. Amoebae and flagellates (Trichomo- nas vaginalis in females) may be found in urine. Also the itch mite is not rarely found in the urine of those having scabies of the penis. Eggs of Schistomum haematobium (bilharziosis) are important 0 fl-H-EV^ ti .9 - FIG. 81. — Starches and fibres found in urine. diagnostic findings; these are terminal-spined. Those of rectal bilharziosis are, as a rule, lateral-spined. In chylous urine the filarial embryos may be found. This examina- tion is facilitated by centrifugalization. The eggs of the E. gigas may be recognized in urinary sediment by their pitted appearance. The vinegar eel may be found in the urine of females who have used vaginal douches of vinegar. CHAPTER XXVII. THE FAECES. IF the fecal examination is to be made for the diagnosis of amoebae, in a case where the characteristic mucous stools are not present, or to verify the existence of flagellates, it is best to give a dose of salts early in the morning and examine the liquid stools which follow such treat- ment. This treatment is satisfactory for examination for intestinal parasites and ova. The blood-flecked mucus of bacillary dysentery or a piece of mucus from a typical amoebic dysentery stool are best suited for cultural examination. If the purpose of the examination is to determine the digestive power of the alimentary tract for proteids, carbohydrates or fats, it is best to use a test diet, as that of Schmidt and Strasburger. Prior to using this test diet, one should familiarize himself with the microscopic appearances resulting from such a diet in a normal person; information is then at hand to judge of variations from the normal. The examination of the faeces 7)f persons, on ordinary and specifically undetermined articles of diet, is very unsatisfactory when the state of digestion of muscle fibers and the question of fat digestion is at issue. In examining the faeces of the normal person and likewise with the patient, wait until the second or third day so that the faeces of previous diets may have passed out. Diet: breakfast, 7 A. M., bowl of oatmeal gruel (40 grams oatmeal, 10 grams butter, 200 c.c. milk, 300 c.c. water). Also one very soft- boiled egg (i min.) and 50 grams zwieback. In the forenoon, 500 c.c. of milk. For dinner, 2 o'clock, chopped beef broiled very rare (125 grams with 20 grams butter poured over it.) Also a potato puree (200 grams 263 264 THE FAECES. mashed potato, 50 grams milk, 10 grams butter). Also one-half liter of milk and 50 grams zwieback. For supper, 7 o'clock, the same articles as for breakfast. Having familiarized one's self with the degree of digestion of muscle, starch and fat in a normal person, we are in a position to judge of the state of assimilation in a patient. We judge of muscle digestion by the intactness of the striations. FIG. 82. — Familiar objects in feces. i, muscle fibres; 2, soaps; 3, vegetable hairs; 4, fatty acid crystals projecting from neutral fat globule; 5, soap crystals with leukocytes; 6, stone cells; 7, vegetable spirals; 8, pallisade cells from bean; 9, parenchyma of vegetable tissue; 10, u, 12 and 14, vegetable cells; 13, pollen. If a muscle remnant is only a homogeneous yellowish particle, it shows satisfactory digestion. If it is rectangular, with well-defined cross striations, it shows poor digestion for meat. A loopful of faeces should be smeared into a drop of Lugol's solution for starch-digestion determi- nation. Normally there should be no blue-staining starch granules CULT I RINC. FAECES. 265 Soaps are gnarled bodies everted like the pinna of an ear, while soap crystals are comparatively coarse and do not melt on application of gentle heat as do the more delicate fatty acid crystals. Neutral fat is in round or irregular globules. The best stain for fat is Sudan III (saturated solution of Sudan III in equal parts of 70% alcohol and acetone). Mix up the faeces with dilute alcohol (50 to 70%) and then add a drop of the above solution and apply a cover-glass quickly. The fat globules show as orange or golden-yellow bodies. Much connective tissue debris shows defect in gastric digestion, as only the stomach digests connective tissue. In examining a liquid stool after salts, it is well to color the drop of faeces, which is to be covered with the cover-glass, with a small loopful of 1/2% solution of neutral red. If diluting fluid is used, it should be salt solution, and not water. The neutral red tinges the granules of the endoplasm of amoebae and flagellates a very striking brown-red color, thus differentiating them from vegetable cells or body cells. Whether examining the thin faeces or the mucus particle, it is well to reserve report on amoebae or flagellates until motion is observed. Encysted protozoa are difficult to diagnose. When a smear preparation is desired, we may smear out a fragment of mucus and stain by Romanowsky's or Gram's method. The charac- ter of the bacteria present appears to be of diagnostic value — especially in the case of infants and young children. Beautiful preparations may be made by mixing the faeces with water, then centrifuging for one minute. This throws down vegetable debris and crystals. Now decant the supernatant fluid, which holds the bacteria in suspension, and add an equal amount of alcohol. Again centrifuge, decant, and smear out and examine the bacterial sediment. Gram's method, with dilute carbol fuchsin counterstaining, gives the best picture. To culture for typhoid, dysentery, cholera or other bacteria, take up the material in a tube of sterile bouillon and smear it out with a swab over a lactose litmus agar plate or an Endo or Conradi-Drigal- ski plate. Before streaking the plates they should be very dry on the surface. This can be best done by pouring into a plate with a circular piece of filter-paper in the lid and placing in the incubator for one-half 266 THE F^CES. hour to dry. The filter-paper absorbs the moisture. Then inoculate the surface of the plate with the fecal material. Muller's method for pancreatic functioning determination is to give a colomel purge two hours after a meal. A little of the liquid stool is smeared on the surface of blood-serum and the tube incubated at 60° C. (paraffin oven). If the surface is smooth, no trypsin was present; if dotted with spots of digestion liquefaction, it shows that the pancreatic secretion is present. This should be tried with the Cam- midge reaction of the urine. Epithelial cells are generally more or less disintegrated. In the mucus of bacillary dysenteric stools, however, large intact phagocytic cells are frequent, which may be mistaken for encysted amcebae. Triple phosphate crystals are frequently observed, as may also be crystals of various calcium salts. Charcot-Leyden crystals are rather indicative of helminthiases. Various flagellates, and in particular Lamblia, may be responsible for diarrhceal conditions which may cause rather serious symptoms. Balantidium coli has been reported several times as the cause of dysenteric conditions. Coccidiadea are found in the faeces. In the Philippines the Entamceba histolytica is the most important of the animal infections. Besides examining for it in a cover-glass preparation, we should attempt to make cultures. A diluted bouillon, i : 10, containing i 1/2% agar, with a reaction about — 1.5, is a satisfac- tory medium. The most important points in success seem to be proper symbiosis and proper reaction. In a series of four tubes containing the same media, but with — 2.5, — 2, — 1.5, and — i, the amcebae may only grow in one of the tubes. Walker recommends smearing a cover- glass with suitable agar, then inoculating the surface, we invert it over the hollow of a concave slide, sealing the margins with vaselin. This enables us to study development with a high power of the microscope. It is in the faeces we examine either for the parasites or for their ova in connection with practically all the flukes, except the lung fluke and the bladder fluke; for intestinal tseniases and for practically all the round worms, except the filarial ones. In the tropics, the examination of the faeces vastly exceeds in value F^CES. 267 that of the urine and is possibly more important than blood examina- tions. The larvae of various insects may at times be detected in the stools, as well as certain acarines (cheese mites, etc.) . The test for occult blood is indicated in helminthiases as well as in the conditions for which it is usually tested. CHAPTER XXVIII. BLOOD CULTURES AND BLOOD PARASITES. CLINICALLY, the most important examination of the blood for para- sites is for the presence of various bacterial infections and for certain blood protozoa and also nlarial embryos. The modern method of culturing blood, especially for the detection of typhoid or paratyphoid bacilli, is by the use of the- bile media of Conradi. Test-tubes are filled with 7 to 10 c.c. of i% peptone ox bile, or ox bile alone, and the medium is sterilized in the autoclave. It is good practice to place the syringe in a plugged test-tube containing salt solution, with the needle unscrewed. After autoclaving, the sterile syringe can be taken to the bedside in the test-tube. Using a wide test-tube, a forceps can be sterilized at the same time and used to attach the needle to the barrel of the syringe. The skin should be scrubbed gently with green-soap solution and water for about three minutes. The skin of the area to be punctured should then be sterilized by the gentle application of Harrington's solution (riot scrubbed) for one-half minute, and should then be washed with sterile water. It appears to be safe to simply scrub the area with 70% alcohol for one or two minutes. A tourniquet is now applied to distend the vein, and the needle is inserted in the direction of the venous flow. Withdrawing 5 to 10 c.c. of blood, we loosen the tourniquet, then withdraw the needle (otherwise the blood may flow from the puncture), and force out about 1/2 c.c. into the first bile tube, about i c.c. into the second, and 2 or 3 c.c. into the third. It is well to reserve some of the blood for Widal tests. The bile tubes are now incubated for 10 to 12 hours and then transfers are made to bouillon tubes. These bouillon tubes can be used in six to eight hours for testing the organism against known typhoid or paratyphoid sera. Some prefer to streak plates of lactose litmus agar with material 268 BLOOD CULTURE. 269 from the bile tubes instead of inoculating the bouillon tubes. Con- tamination with staphylococci or the presence of staphylococci, streptococci or plague bacilli in septicaemic conditions show easily accessible colonies. Schotmuller adds i to 3 c.c. of blood to liquefied agar at 45° C., and after mixing pours into plates. The standard method for- merly was to add the blood to an excess of bouillon (i to 5 c.c. of blood to 100 c.c. or more of bouillon). By using the bile media, we can take the blood from the ear in typhoid cases, if preferred. Then if chance staphylococcic contamination occurs, such colonies are readily differen- tiated from typhoid ones by the pink color on lactose litmus agar. In culturing blood in septicaemic conditions, the blood should always be drawn from the vein. Typhoid cultures are best obtained in the first week of the disease, after that time the Widal is the test of preference. If a paratyphoid serum is not at hand for testing, it may suffice to inoculate a glucose bouillon tube; gas production indicates para- typhoid. This test should be applied when a very motile organism does not show agglutination with a known typhoid serum. Anthrax and glanders should be considered in blood cultures. In Malta fever it must be remembered that colonies do not show themselves for several days. Addition of blood to melted agar is a good procedure. The examination of the blood for the parasites of malaria, filariases, kala-azar and spirillum fevers has been discussed under their respective headings. Trypanosomes from human trypanosomiasis have not as yet been cultured, and smears from gland juice or cerebrospinal fluid, seem more satisfactorv to examine than blood smears. CHAPTER XXIX. THE STOMACH CONTENTS. FROM a microscopical stand-point there is comparatively little that is of value in the examination of the gastric contents ; there is nothing very specific about the findings. A test meal is not a necessity as in the chemical examination, but either vomitus or material withdrawn with a stomach-tube two or more hours after an ordinary meal suffice. The microscopical diagnostic points in connections with distin- guishing cancer of the stomach from nonmalignant dilatation are: (i) Fragments of cancer tissue. These are very rarely found and are most difficult to diagnose. (2) The presence of flagellates in the early stages of cancer (the so-called anacid stage preceding the development of lactic acid). As flagellates prefer an alkaline medium, they disap- pear after the acidity due to lactic acid comes on. (3) The presence of the Boas-Oppler bacillus. There are probably several organisms so designated. They are lactic acid producers and are characterized by being very large bacilli (7x1/1) and arranged in long chains which stretch across the field of the microscope. They are Gram positive and do not form spores. They can be cultivated on media rich in blood and are aerobic. They should only be reported when present in great abundance and in long chains. (4) The absence of sarcinae and yeasts. The presence of these cocci and fungi in vomitus is indicative of a simple dilatation. 270 CHAPTER XXX. EXAMINATION OF PUS. Pus may be collected for examination either (i) with a platinum loop, (2) with a sterile swab, (3) with a bacteriological pipette or (4) with a hypodermic syringe. It is always well to make a smear and stain it by Gram's method at the same time that cultures are made. The Gram stain gives informa- tion as to the abundance of organisms in the pus and as to the probable findings in the culture. Pneumococci and streptococci are differen- tiated from the staphylococci in this way without the necessity of more or less extended cultural methods. When autogenous vaccines are to be made, the isolation of the exciting organism is necessary. This is best done by streaking the pus, taken up with a sterile swab and emulsified in a tube of bouillon, over the surface of an agar plate. Practically as convenient and provid- ing a more nutritious medium is to smear the material on a loop or swab over the surface of a blood-serum slant, then to inoculate a second tube from the first without recharging the loop or swab, and so on until three or four tubes are inoculated. Isolated colonies should be ob- tained in the third or fourth tube. In examining blood serum slants inoculated with purulent material, always examine the water of condensation for streptococci. A bacteriological pipette is very useful when pus is to be sent to a laboratory; the tip can be sealed in a flame and the cotton plug at the other end insures the noncontamination of the contents. The material may be drawn up either with the mouth or with a rubber bulb. The hypodermic syringe is very useful in puncturing buboes, etc., especially in plague. A small pledget of cotton on a toothpick dipped into pure carbolic acid and touched to a spot over the bubo, which after about thirty seconds is soaked with alcohol, makes a sterile anaesthetic spot at which to introduce the needle of the syringe. It 271 272 EXAMINATION OF PUS. must be remembered that when plague buboes begin to soften, the plague bacilli may be replaced by ordinary pus organisms. It is remarkable how frequently we get pure cultures from abscess material. In purulent material from abdominal abscesses we are apt to obtain mixed cultures, especially the colon bacillus and B. pyocy- aneus, in addition to ordinary pus organisms. When it is a question between streptococci and pneumococci, it is well to inoculate a mouse; the capsulated pneumococci at the autopsy make the diagnosis. Animal inoculation is also necessary in plague and glanders, and possibly anthrax. When tetanus is suspected, it should be examined for as described under Tetanus. Tuberculosis should also be identified by inoculating a guinea-pig, as well as by acid-fast staining and culture, if there be any doubt as to the nature of the material. The black or yellow granules of madura foot, as well as those of actinomycosis, should be examined as recommended in the section on fungi. Amoebae, coccidia and larval echinococci may be found in purulent material, as may also various other animal parasites, as fly larvae, sarcopsyllae, etc. CHAPTER XXXI. SKIN INFECTIONS. CULTURAL methods are to be preferred in the bacteriological examination of the skin. This is best done by washing the surface to be examined with soap and water, in order to eliminate chance organisms which may have settled on the surface of the skin in dust or as a result of contact with material containing them. Scrapings are then made with a sterile dull scalpel, and this material is emulsified in a drop of sterile water in the center of a Petri dish. A tube of melted agar at 42° C. is then poured on the inoculated drop and, by mixing, the bacterial flora is distributed over the entire surface of the plate. Of the colonies developing on such plates probably 80% will be found to be staphylococci, and of these the greater proportion will be staphylococci showing white colonies. Occasionally the aureus or citreus may be isolated. Streptococci and colon bacilli are rarely found. The Staphylococcus pyogenes aureus is the organism usually isolated from furuncles, circumscribed abscesses and carbuncles. Streptococci are the organisms to be expected in phlegmonous in- fections. Cold abscesses, which are frequently due to tuberculous infection, are, as a rule, sterile. Acne pustules may show staphylococci or the microbacillus of acne may be present. The bottle bacillus, which morphologically resembles a yeast, is considered to be the cause of dry pityriasis capitis. It may also be found in the comedones of children. In the tropics, an organism which at times produces lesions similar to impetigo and again pemphigoid eruptions and at other times wide- spreading erysipelatous conditions gives cultural characteristics 273 274 SKIN INFECTIONS. similar to S. pyogenes aureus. It is probably only a virulent aureus. It has been described under the name of Diplococcus pemphigi con- tagiosi. The Staphylococcus epidermidis albus, or stitch abscess coccus, is considered by Sabouraud to be the cause of eczema seborrhoicum. It is in scrapings from the skin of lepromata that we find acid-fast organisms in the greatest profusion. In tuberculosis of the skin the tubercle bacilli are exceedingly scarce. Inoculation of a guinea-pig will probably give positive results with the tubercle bacillus. • The leprosy bacillus is noncultivable and noninoculable for experimental animals. Anthrax and glanders cause skin lesions which can only be surely diagnosed culturally or by animal inoculation. Plague bacilli may be isolated from the primary vesicles appearing at the site of the flea bite. Tropical phagedaena is thought by some to be due to a sort of diphtheroid organism. The skin diseases due to fungi are discussed under that section. Of the skin affections caused by animal parasites, ground itch is the most important. This is a form of dermatitis due to the irritation set up by the hook-worm larvae penetrating the skin of the foot and leg. The Sarcopsylla penetrans or jigger (sand flea) is an important agent in ulcerations about the foot. Certain acarines cause skin lesions, as is also the case with the larvae of certain flies. The itch mite (Sarcoptes scabiei) is an important animal parasite of the skin. The various lice, fleas and bed-bugs are well understood as causes of skin irritation. Filarial infections are also important. CHAPTER XXXII. CYTODIAGNOSIS. THIS method of diagnosis is chiefly employed in the examination of cellular sediments of pleural, ascitic and cerebrospinal fluid. For pleural fluids we should receive the material in centrifuge tubes about one-fourth filled with 2% sodium citrate salt solution. This prevents clotting. Having thrown down the sediment, the superna- tant fluid is poured off, and in its place a i% aqueous solution of formalin is added. After mixing and allowing to stand for about five minutes, centrifugalization is again repeated and, pouring off the supernatant formalin solution, we make smears from the sediment. This is either stained by a Romanowsky method or, after fixing with heat (burning alcohol), the smear is stained with haematoxylin and eosin. At the time of securing fluid for cytodiagnosis, cultures should be made on blood-serum for various pyoqjenic bacteria and, if tuberculosis is suspected, inoculation of a guinea-pig is indicated. The interpretation of cellular sediments is more difficult than many books would indicate, there being many factors which tend to com- plicate the findings. The following are the leading differentiations: 1. A smear showing almost entirely lymphocytes with a few red cells and very rarely a polymorphonuclear indicates a tubercular process. 2. Where a pyogenic process is engrafted on a tuberculous one, we have still the red cells, some degenerated lymphocytes and in particular polymorphonuclears showing fragmentation of their nuclei. 3. When a hydrothorax results from chronic heart or kidney disease, the characteristic cell is the endothelial cell, which greatly resembles a large mononuclear. 4. Some authorities consider that the cancer cell can be recognized by its occurring in masses and having a markedly vacuolated cytoplasm. 275 276 CYTODIAGNOSIS. It has been claimed that they contain glycogen by which means we can distinguish them from endothelial cells which they so much resemble. Jousset introduced ionoscopy as a means of diagnosing tuberculo- sis. The fluid was allowed to coagulate and was then digested with an artificial gastric juice. The digested material was then centrifuged and the sediment examined for tubercle bacilli. This process does not seem to have met with much favor in this country. (Using sodium citrate obviates the necessity for digesting the coagulum.) The same points will hold for ascitic fluid as for pleural fluid. In taking cerebrospinal fluid for culture and cytodiagnosis we use a stout antitoxin needle without attaching a syringe. Aspiration is responsible for many of the ill effects of lumbar puncture. The needle should be about four inches long for an adult. Sterilize the skin and needle as described for blood cultures from a vein. To make a lumbar puncture, place patient on left side with knees drawn up. A line at the level of the iliac crests passes between the third and fourth lumbar vertebrae. Select a point midway between the spinous proc- esses of these lumbar vertebrae and enter the needle 2/5 of an inch to the right of this point, pushing the needle inward and upward. Collect the material in a sterile test-tube. Make cultures on blood-serum and then centrifugalize and examine the sediment as for pleural fluids. In general terms it may be stated that: 1. A lymphocytosis indicates a tuberculous process. 2. An abundance of polymorphonuclear and eosinophilic leuko- cytes indicates a meningococcic or pneumococcic infection. A method of examination considered by neurologists as of differen- tial diagnostic value is to count the number of cells in a cubic milli- meter of the cerebrospinal fluid. The technic is to use a gentian-violet- tinged 3% solution of acetic acid. This is drawn up to the mark 0.5, and the cerebrospinal fluid is then sucked up to n. After mixing, the cell count is made with the haemocytometer. Normally we have only one or two cells per cubic millimeter, but in tabes or general paresis this is increased to 50 or 100 cells. Trypanosomiasis gives a cellular increase very similar to syphilis. CHAPTER XXXIII. RABIES. THIS is a disease of dogs and wolves, but is communicable to man and domesticated animals. The virus, whatever it may be, resides in the saliva and nervous structures. It is destroyed by a temperature of 50° C. In man the period of incubation is usually from three weeks to three months, but may be shorter or may extend over one year. Bites about the face and those with marked lacerations are particu- larly serious. Bites of rabid wolves give about four times as great a mortality as those of dogs. In the dog there are two types of the disease — dumb rabies and furious rabies. By inoculating rabbits subdurally with an emulsion of the brain or spinal cord of a rabid animal, and successively the medulla of this rabbit subdurally into other rabbits, we finally so increase the virulence of the infection that rabbits die in six days. Beyond this it is im- possible to increase the virulence and it is termed "fixed virus." To attenuate this virus the spinal cord of the rabbit is removed and is dried over caustic potash. The cord is divided into segments about one inch in length. Drying for about fifteen days seems to entirely de- stroy the virus. To prepare the material for prophylactic injections a small portion of the cord is emulsified and injected subcutaneously. The German method is to commence with a cord that has been dessiccated only eight days. At first injections are given daily, and it is possible to inject three days' cords by the sixth day. The treatment lasts for about twenty days. In the diagnosis of rabies in dogs it is preferable to preserve the animal so that the develop- ment of the symptoms may be observed. In case the dog has been killed, it may be possible to make a diagnosis by means of the Negri bodies. These are round or oval bodies from i to 2o// in diameter, which may be found in the nerve-cells, especially those of the cornu 19 277 278 RABIES. . ammonis (Hippocampus major). They may be demonstrated by staining smears of brain substance by some Romanowsky method, especially by the Giemsa stain. As their relation to the nerve-cell is more or less disturbed by such a method, it is preferable to fix brain tissue about the region of the cornu ammonis in Zenker's fluid, then to imbed in paraffin and make sections. These are stained with Giemsa 's stain and the Negri bodies are brought out as lilac-red bodies in the blue cytoplasm of the nerve-cells. It is necessary to differentiate in 95% alcohol. In addition to examining for the Negri bodies, a rabbit may be inoculated subdurally with a sterile salt-solution emulsion of the medulla of the dead dog. If the brain or cord of the dog are to be sent to a laboratory for examination they should be packed in ice or placed in glycerin. Take of glycerin one part and one part water. Sterilize the diluted glycerin by boiling, allow to cool, and drop the pieces of brain tissue into this. This does not kill the virus. It is supposed by some that Negri bodies are protozoal in nature notwithstanding the fact that the virus will pass through a coarse Berkefeld filter. Antirabic serum has been prepared by injecting sheep with emul- sions of rabid rabbits' cord and brain — at first intravenously, then subcutaneously. MISCELLANEOUS NOTES. MISCELLANEOUS NOTES. MISCELLANEOUS NOTES. MISCELLANEOUS NOTES. APPENDIX. A— PREPARATION OF TISSUES FOR EXAMINATION IN MICROSCOPIC SECTIONS. i. Fixation: a. It is most important that the tissues to be examined be placed in the fixing fluid as soon after death or operation as possible. Degenerative changes are in this way avoided. b. The piece of tissue to be fixed must not be too large. Using a sharp scalpel, or preferably a razor, a slab of tissue about one-half an inch square and not more than one-fifth of an inch thick should be dropped into the bottle containing the fixa- tive. The bottom of this bottle should have a thin layer of cotton with a piece of filter-paper covering it. There should be at least twenty times as great a volume of fixing fluid as of tissue to be fixed. Delicate tissues, as pieces of gut, should be attached to pieces of glass, wood or cardboard. c. The most convenient fixative for the average medical man is a 10% solution of ordinary commercial formalin (4% of formic aldehyde gas), either in water or, preferably, in normal salt solution. Fixation is complete in from 12 to 24 hours. By placing in the incubator, at 37° C., 2 to 12 hours in the formalin solution suffices. If fixed in the paraffin oven (56° C.), fixation is accomplished in about one-half hour. Formalin once used for fixation must be thrown away. The fixative which probably gives the best histological pictures and with which we obtain the most satisfactory haematoxylin staining is Zenker's fluid. This is Muller's fluid containing 5% of corrosive sublimate. It also contains 5% of glacial acetic acid, which latter is only added just before we are ready to fix the piece of tissue. Muller's fluid is: Pot. bichromate, 2.5 grams. Sod. sulphate, i.o grams. Water, t 100.0 c.c. Zenker's fluid fixes in about 24 hours. After all corrosive sublimate fixatives we should wash the tissues in running water for 12 to 24 hours. The precipitate of mercury in the tissues is best gotten rid of by treating the section on the slide with Lugol's solution, rather than the tissue in bulk with iodine alcohol. In Orth's fluid we add 10% of formalin to Muller's fluid (recommended for nerve tissue). A saturated corrosive sublimate solution in salt solution with the addition of 5% of glacial acetic acid may be used as a substitute for Zenker's fluid. 279 280 APPENDIX. 2. Dehydration. — After washing for twelve to twenty-four hours in running water, following corrosive sublimate fixation, or simply "washing for a few minutes after formalin, the tissues should be placed in 70% alcohol. They may be kept- in this indefinitely. If they are to be sent to a laboratory for sectioning, it is advisable lo moisten a pledget of cotton in 70% alcohol and fill in the bottom of the bottle with it. Then drop in the tissues and pack in gently over them sufficient 70% alcohol saturated cotton to fill up the bottle. All the alcohol should be absorbed by the cotton so that if the bottle should break in transit there would be no damage from the alcohol. The stopper of the bottle should be paraffined or sealed with wax. Tissues may be left in the 70% alcohol 1 2 to 24 hours and should then be trans- ferred to 95% alcohol for an equal time. They are then transferred to absolute alcohol, where they remain from 2 to 12 hours and are then placed in xylol. The time in xylol should be as short as possible. So soon as the tissue looks clear it should be removed — 30 minutes to two hours. 3. Imbedding. — The tissue is now transferred to melted paraffin. Paraffin melting at 48° C. for winter work, and that melting at 54° C. for summer. The time in the paraffin should not he prolonged. Two hours will ordinarily suffice. Some leave in the paraffin for 12 to 24 hours. Next take a paper box (made of stiff writing-paper folded over a square of wood) and fill with the melted paraffin. As quickly as possible drop in the piece of tissue taken out of the paraffin bath with heated forceps and, so soon as the paraffin begins to solidify on the surface, place the paper box in ice water. When paraffin is rapidly cooled, crystallization is less. The Acetone Method. — Take the tissues out of the 70% alcohol and place in acetone. After remaining in acetone for one to two hours, the tissues should be transferred to fresh acetone for an equal length of time. They should then be placed in xylol for about one-half hour and then imbedded in paraffin as directed above. The Chloroform Method. — The procedure may be the same as in the method of passing through alcohols to xylol, substituting chloroform for xylol and then trans- ferring to paraffin. Where absolute alcohol is not obtainable, very satisfactory results may be obtained by transferring to a mixture of 95% alcohol and chloroform after immersion in 95% alcohol. Then going from the alcohol-chloroform mixture to pure chloroform thence to paraffin. When a piece of tissue is not more than one-fourth inch square and one-eighth inch thick, it is very easy to run it through in three to six hours. Thus: 10% Formalin (in 37° C. incubator), i hour. 70% Alcohol (in 37° C. incubator), i hour. 95% Alcohol (in 37° C. incubator), i hour. Absolute Alcohol (in 37° C. incubator), 1/2 hour. Xylol (in 37° C. incubator), 1/2 hour. Paraffin (in 55° C. incubator), 1/2 to 2 hours. APPENDIX. 28l It is preferable to have a good microtome. The best is that of Minot. Very satisfactory sections can be cut with the various types of student microtomes, costing from $12 to $20. (In using a hand microtome, a razor with a flat edge is necessary. After experience, sections thin enough for histological but not for bacteriological examination can be made.) If the piece of tissue is properly dehydrated and imbedded, thin sections (3 to IOM) should be easily obtained, provided the knife be sharp. One advantage about the paraffin method is that it is only necessary to have a small part of the blade in proper condition. With celloidin the entire cutting edge must be perfect. Having cut the sections, they should be dropped on the surface of a bowl of warm water (45° C.). This causes the section to flatten out evenly. Decalcification. — This is best accomplished by fixing in 10% formalin for 24 hours, then placing a small piece of the bone (not exceeding one-half inch square and one-fifth of an inch thick) in concentrated sulphurous acid. This decalcifies in about 24 hours. Wash thoroughly in alkaline water and then in tap water. Pass through alcohols and xylol and imbed and section as before described. To Stain Sections. — It is first necessary to affix the section to a slide or cover- glass. To attach the section firmly to the slide, so that it will not become detached in subsequent treatment, pick up a section on a strip of cigarette paper. A sheet of cigarette paper is cut into about five pieces (1/2 x i 1/2 ins.). Inserting the strip of cigarette paper under the section, it is easily lifted up out of the water. Then apply the slip of cigarette paper, section downward, to a perfectly clean slide. Blot with a piece of filter-paper, then strip off the piece of filter-paper leaving the section smoothly applied to the slide. Next place in the 37° C. incubator for twelve to twenty-four hours and the section will be found to be so firmly attached that it will not be dislodged by subsequent treatment. For Immediate Diagnosis. — Take a loopful of albumin fixative (white of fresh egg, 50 c.c.; glycerin, 50 c.c.; sodium salicylate, i gram) and deposit it on a cover- glass. Now take up a loopful of 30% alcohol (i drop of 95% alcohol and two drops of water) and applying it over the albumin fixative, smear out the mixture uniformly over the cover-glass. 2. Pick up a section on a strip of cigarette paper and apply it to the prepared sur- face on the cover-glass. Blot with gentle pressure with a piece of filter-paper over the strip of cigarette paper, and strip off this latter, leaving the section attached to the cover-glass. 3. Now, turning the flame of theBunsen burner down very low or with a small alcohol flame, we hold the cover-glass in a Stewart's forceps, section side up, over the flame and slowly lower it until the paraffin is observed to melt. This shows a temperature of about 50° C. The section is fixed by the coagulation of the albumin at about 70° C. To obtain this temperature lower the cover-glass still more, and the 282 APPENDIX. moment vapor is seen to rise from the section it indicates the attachment of the section to the cover-glass. 4. Flood section on cover-glass or slide with xylol; this dissolves out the paraffin. It is better to pour off the first xylol and drop on fresh xylol (one minute). 5. Remove xylol with two applications of absolute alcohol (one minute). 6. Treat specimen with two or three applications of 95% alcohol (one to two minutes). 7. Next wash in water (one to two minutes). 8. Flood specimen with haemalum of Delafield's haematoxylin (three to seven minutes). 9. Wash in tap water for about two to five minutes until a purplish tinge is developed in the section. The alkali in ordinary tap water develops this color. 10. Apply i to 1000 eosin for thirty seconds to one minute. 11. Wash in water; then in 95% alcohol; then in absolute alcohol. 1 2. Apply a few drops of xylol and as soon as the section is perfectly transparent mount in balsam. The staining by haematoxylin and eosin is the best for the study of the histology of a section. It only requires about ten minutes to run a preparation through for diagnosis by this method. The reagents are best kept in dropping-bottles. The staining of sections on slides is exactly as for those on cover-glasses. Cop- lin's staining jars are very convenient for use in staining slides. Where the cover-glass method is used, staining by Gram's method, acid-fast stain- ing, capsule staining, etc., may be carried out as for bacterial preparations. For staining Gram positive bacteria in sections, the Gram method as for bacterial preparations, using dilute carbol fuchsin as a counter stain, gives good results. For Gram negative bacteria stain with thionin as for blood preparations (10 to 20 minutes). Then differentiate in i to 500 acetic acid solution for ten to twenty seconds, wash with water, then with 95% alcohol, and quickly through absolute alcohol and xylol. Nicolle's Method. — i. Stain with Loffler's methylen blue ten to fifteen minutes. 2. Differentiate in i to 500 acetic acid ten to twenty seconds. 3. Place in i% solution of tannin for a few seconds (fixes color). 4. Wash in water, then into 95% alcohol, absolute alcohol, xylol and balsam. Van Giesen's Stain.— Take of one percent aqueous solution acid fushsin from 5 to 15 c.c. Saturated aqueous solution picric acid 100 c.c. The method of using is to first stain with hsematoxylin in the usual way. Then pour on the picro-acid fuch- sin solution and allow to stain for one to five minutes. Wash, pass through alcohols, and xylol and mount in balsam. Connective-tissue fibres, axis cylinders and ganglion cells are stained a bright garnet red. Myelin, muscle fibers and cells generally are stained yellow. Nuclear staining is that of haematoxylin. The stronger stain is used for nerve tissue; the weaker for demonstrating connective tissue in tumors. APPENDIX. 283 Romanowsky. — Staining sections with Romanowsky stains is not very satis- factory. The differential staining seems to fade out in passing through the alco- hols. This may be avoided by blotting the section after staining and differentiation and then applying the xylol to the blotted section. After staining with Giemsa's stain for 10 to 15 minutes, differentiate with i to 500 acetic acid. When the section has a pinkish tinge, wash in water, dry, clear in xylol and mount. A very satisfactory Giemsa may be made by taking i gram of methylene blue, 1/2 gram of sodium bicarbonate and 100 c.c. of water and polychroming as for King's stain. Remove the dried stain from the porcelain dish and put it into a bottle. Then rinse out the dish with methyl alcohol and pour into the bottle. There should be 100 c.c. for this amount of stain. Let stand 3 to 5 hours, then filter. To the 100 c.c. of methyl-alcohol solution add 100 c.c. of glycerin containing 1/2 gram of yellow eosin. B— MOUNTING AND PRESERVATION OF ANIMAL PARASITES. To Mount Small Round Worms. — Wash the hook, whip or filarial worm in salt solution, then drop in 70% alcohol containing 5% of glycerin; the glycerin- alcohol mixture being at a temperature of 60° C. When cool, pour into Petri dishes and allow the alcohol to evaporate in the 37° C. incubator. Mount in glycerin jelly, preferably in a concave slide, and ring the preparation with gold size. The following is the formula for Kaiser's glycerin jelly: Soak one part of gelatin in 6 parts of distilled water for two hours. Then add 7 parts of glycerin. To the mixture add i% of carbolic acid, warm for 15 minutes, with constant stirring, and then filter through cotton. To Prepare Tape-worms. — Wash in salt solution. Wrap around a piece of glass as a glass slide and fix in salt solution containing 2 to 5% of formalin. Then keep the preparation permanently in 70% alcohol. If preferred, the specimen may be run through alcohols and xylol and mounted in balsam. Larvae. — Mosquito larvae may either be prepared as for small round worms or they may be dropped into 70% alcohol at 60° C. and then passed through alcohols and cleared in xylol and mounted in balsam. Flukes and insects may require treatment with hot (60° to 70° C.) solution of 10 to 20% sodium-hydrate solution. Then wash thoroughly in water and subsequently pass through alcohols to xylol and mount in balsam. Clove oil or cedar oil clears more slowly, but makes specimens less brittle than does xylol. Another satisfactory method is to drop insects or larvae into acetone at 60° C. and after being in this from i to 12 hours to clear in xylol or clove oil and mount in balsam. Looss has a method of first washing a small nematode or delicate fluke in salt solution. Then pouring this first salt solution out of the test-tube in which the wash- ing was carried out, to add fresh salt solution, and then an equal amount of saturated aqueous solution of bichloride of mercury, The shaking is easily carried on in the test-tube. After washing in water the worm is passed through alcohols, one strength of which should contain iodine. Clear in xylol and mount in balsam. 284 APPENDIX. To Prepare Flies or Mosquitoes for Transmission Through the Mails. — Wrap the insect carefully in a piece of tissue-paper (toilet-paper answers). Impreg- nate sawdust with 5% carbolic acid solution and fill around the folded insects in the box containing them. (Barely moisten.) C— PREPARATION OF NORMAL SOLUTIONS. A normal solution is one which contains the hydrogen equivalent of an element, expressed in grams, dissolved in sufficient distilled water to make 1000 c.c. The hydrogen equivalent is the atomic weight of any element divided by its valence. In a base, salt or acid we use the molecular weight in grams divided by valence. What may be considered as the valence of a base is shown by the number of hydroxyls combined with it; that of an acid by the number of replaceable hydrogen atoms which it contains. To make a normal solution, dissolve in distilled water a weight in grams equal to the sum of the atomic weights of the substance, divided by its valence, and make up the volume to exactly 1000 c.c. NaOH is univalent. Na = 23. O = 16. H = i. Dissolve 40 grams NaOH in water and make up to exactly 1000 c.c. Oxalic acid is COOH — COOH + 2 H2O which gives it a molecular weight of 126. As it contains two carboxyl groups it is dibasic, and it is necessary to divide the molecular weight by 2, so that for a normal solution of oxalic acid we dissolve 63 grams in a volume of distilled water made up to 1000 c.c. If a chemical laboratory is not accessible one may prepare normal solutions with an error so slight as to be unimportant in clinical work in the following way: Sodium hydrate being very hygroscopic, it is impossible to accurately prepare a normal solution by directly weighing out the substance. Instead, select perfect crystals of oxalic acid, such as can be obtained in a drug store, and weigh out on the most accurate apothecary scales obtainable exactly 6.3 grams of the most per- fect crystals in the bottle. Put these preferably in a volumetric flask and make up with distilled water to 1000 c.c. Less accurate is the use of a measuring cylinder. If care is used this should give N/io solution of oxalic acid in which the error is less than i%. Having N/io acid at hand, we may prepare N/io NaOH in the following way: Weigh out an excess of sodium hydrate (5 grams of stick caustic soda) and dissolve in 1 100 c.c. of distilled water. Take up xoc.c. of this solution with a pipette and let it run into a beaker. Add six drops of phenolphthalein solution. This gives a violet-pink color. Fill the burette with the N/io oxalic-acid solution and let it run into the sodium-hydrate solution in the beaker until the pink is just discharged. Reading off the number of c.c. of the N/io acid used, we know the strength of the sodium-hydrate solution. It is well to repeat the titration and take an average. If 10.5 c.c. of the oxalic-acid solution were required it would show that the sodium-hydrate solution was stronger than N/io, as only 10 c.c. would have been APPENDIX. 285 necessary if the NaOH solution had been N/io. It is therefore necessary to dilute the sodium-hydrate solution in the proportion of 10 to 10.5. Measure exactly 1000 c.c. of the too concentrated sodium-hydrate solution and add to it 50 c.c. of distilled water, mix thoroughly, and we have 1050 c.c. of N/io solution of NaOH. 1000 x 10.5 = 10.500. 10.500-7-10=1.050. As Acidum hydrochloricum U. S. P. is about two-thirds water (68.1%) to make N/io HC1, which would require 3.65 in 1600 c.c., it would be necessary to take about three times this amount of U. S. P. acid. Take 12 c.c. of the acid and add distilled water to make 1 100 c.c. Put 10 c.c. of this dilute solution in a beaker. Add phenol- phthalein solution and titrate. If n c.c. of N/io NaOH were required it would be necessary to add 100 c.c. of water to a volume of 1000 c.c. of the diluted hydrochloric acid. looo x n = nooo-f- 10 = noo. Other acid and alkali solutions can be made as for N/io HC1 and N/io NaOH. D— DISEASES OF UNKNOWN OR NOT DEFINITELY DETERMINED ETIOLOGY. OF TEMPERATE CLIMATES. Acute Articular Rheumatism. — Various bacteria have been reported as cause. Foot-and-mouth Disease. — Probably due to an ultramicroscopic organism. Measles. — Cause entirely unknown. Hektoen has shown that blood contains .the virus. Mumps. — Herb has implicated a diplococcus. Inoculations into Steno's duct of monkey successful. Rabies. — Probably the Negri bodies. Roetheln (German Measles). — Nothing known. Scarlet Fever. — Streptococci seem most probable cause (S. anginosus). Mallory has implicated epithelial protozoa. Small-p3X and Vaccinia. — Guarnieri and Councilman have implicated epithe- lial protozoa. Spotted Fever of the Rocky Mountains. — Supposed to be due to an unknown protozoon transmitted by a tick. Typhus Fever. — It has been suggested that the cause may be a protozoon trans- mitted by vermin. Varicella. — Entirely unknown. Whooping Cough. — Influenza-like bacilli have been implicated. OF TROPICAL CLIMATES. Ainhum. — (A disease characterized by a constricting fibrous ring, especially of little toe, often leading to spontaneous amputation.) Beriberi. — Various microorganisms and food factors suggested. Blackwater Fever. — Considered as a malarial disease, but thought by some to be possibly caused by a protozoon — a Babesia (Piroplasma) Dengue. — Supposed to be due to a protozoon transmitted by Culex fatigans 286 APPENDIX. Goundou. — (Symmetrical bony tumors of nasal processes of superior maxillary bones.) Sprue. — (A form of chronic diarrhoea characterized by diaphanous thinning of gut and ulcerations of buccal cavity.) Tsutsugamushi. — (A disease of Japan somewhat resembling typhus fever.) Supposed to be due to a protozoon transmitted by the Kedani mite. Yellow Fever. — Supposed to be due to a protozoon transmitted by the Stego- myia calopus. E— MINK'S MODIFICATION OF UNNA'S H^EMATOXYLIN. Hsematoxylin, . i gram. Alum, 8 grams. Sulphur (sublimed), i gram. Glycerin, 30 c.c. Alcohol, 50 c.c. Water, 100 c.c. Dissolve the haematoxylin in the glycerin in a mortar. Dissolve the alum in the water and add it to the glycerin baematoxylin in the mortar. Then add the sulphur and the alcohol. The solution ripens in about 3 to 4 days. Allow the sediment to remain in the bottom of the bottle containing the stain and filter off small quantities as needed. INDEX. Abbe condenser, 4 Abscess, bacteria in, 272 Acanthia lectularia, 228, 230 Acarina, 221 Acartomyia, 250 Acetone, for sections (see tissue), 280 Acid-fast bacteria, 66 Acid-fast staining, 30 Actinomycosis (see Discomyces), 106, 272 ^Edinae, 248 Agar, egg, 22 glucose, 22 glycerine, 22 nutrient, 21 plating, 34 Agchylostoma duodenale, 208, 216 Agglutination, macroscopical, 127 microscopical, 126 Ainhum, 286 Air, bacteriological examination of, 117 Aldrichia, 248 Alexin, 122 Amboceptor, 121 Amoebae, 175 Anaemia, aplastic, 156, pernicious, 154, 164 primary, 164 secondary, 166 Anaerobes, 53', 56 Buchner method, 58 cultivation of, 57 Liborious method, 58 Vignal method, 59 Wright method, 59 Anginas, 256 Animal parasites, general classification, 169 mounting of, 283 nomenclature in, 171 preservation of, 283 Ankylostoma, 208 Anophelinae, 246, 248 Anthrax, 53, 54 vaccination, 56 Antitoxin botulism, 61 diphtheria, 75 pyocyaneus, 92 tetanus, 63 Arachnoidea, 221 Argas, 221, 226 Ascaris, canis, 208, 218 lumbricoides, 208, 218 Ascitic fluid (cytodiagnosis in), 275 Aspergillus, concentricus, 104 flavus, 104 fumigatus, 104 pictor, 105 repens, 104 Auchmeromyia luteola, 228, 236 Bacillus, acidi lactici, 116 aerogenes capsulat., 53, 64 anthracis, 53, 54 anthracis symptomat., 53 botulinus, 53, 60 cloacae, 71 coli, 78, 91, 113 diphtheriae, 66, 73, 251, 253, 256 dysenteriae, 78, 89 enteritidis (Gartner), 78, 89 enteritidis sporogenes, 53 fecalis alkaligines, 85 icteroides, 85 influenzae, 78, 79 lactis aerogenes, 91 lepiae, 66, 70, 252, 253 mallei, 66. 72 mycoides, 53 of avian tuberculosis, 66 of bovine tuberculosis, 66 of chancroid, 78, 81 of Hofman, 76 of Koch- Weeks, 78, 80, 251 of malignant cedema, 53, 59 of Morax, 78, 81 287 288 INDEX. Bacillus of smegma, 66, 70 of timothy grass, 66, 67 of trachoma (Muller), 78 paratyphosus (A. and B.), 88 pestis, 78, 8 1 pneumoniae (Friedlander), 79, 81 prodigiosus, 93 proteus, 89 psittacosis, 85 pyocyaneus, 92 subtilis, 53 tetani, 53, 54, 62 tuberculosis, 66, 68 typhosus, 79, 86, 114 violaceus, 92 vulgatus, 53 xerosis, 77, 252 Bacterium, 38 Bacteriolytic experiments, 128 Balantidium coli, 183 Beriberi, 286 Bile media, 25, 268 Bilharziasis, 199 Blackwater fever, 224, 286 Blood, color index of, 154 counting red cells, 140 counting white cells, 143 counting with microscopic field, 144 cultures of, 268 differential count (normal), 160 dried films, 146 fixation of, 147 fresh preparations, 145 making preparations, 138 normal count, 154 red cells of, 154 staining of, 148 white cells of, 156 Blood platelets, 161 Blood serum, coagulating apparatus, 10 preparation of, 23 Bordet and Gengou phenomenon, 130 Bottle bacillus, 273 Bouillon, glycerine, 20 Liebig's extract in, 19 standardizing reaction of, 16, 17 sterilization of, 19 sugar, 20, 40 sugar free, 20 Broth media, 15 Buccal secretions, 255 Calliphora vomitoria, 228, 236 Capsule staining, 31 Carbol-fuchsin stain, 28 Cellia, 248 Cells, in blood, 154, 156 in cytodiagnosis, 275 Cerebrospinal fluid, 51 puncture for, 276 Cestoda, 194, 200 Chlorosis, 154, 164 Cholera, 94 in water, 114 Chironomidse, 228, 239 Chromatin stains, 151, 152 Chromogens, 92 Chrysomyia macellaria, 228, 237 Chrysops, 228, 234 Chyluria, 262 Cladorchis watsoni, 194, 197 Classification, animal kingdom, 169 arachnoidea, 221 bacilli, branching, 66 bacilli, gram negative, 78 bacilli, spore bearing, 53 bacteria, 36 cocci, 41 flat worms, 194 fungi, 99 insects, 228 mosquitoes, 247, 248, 249 protozoa, 173 round worms, 208 spirilla, 94 Clonorchis endemicus, 194, 196 Clonorchis sinensis, 194 Coccidiaria, 183 Coccidium (See Eimeria and Isospora), 185 Coley's fluid, 93 Colon bacillus, 78, 91 in water, 113 Colonies, isolation of, 34 Color index, 154 Commensalism, 171 Complement, 122 absorption of, 130 deviation of, 129 Conjunctival infections, 251 Conradi-Drigalski medium, 26 Cover glasses, 2 Cover-glass preparations, 27 Cryptococcus gilchristi, 102 Culicinae, 246 Culture media, agar, 21 bile media, 25 blood agar, 24 INDEX. 289 Culture, blood serum, 24 bouillon, 15 faeces media, 25 gelatin, 22 litmus milk, 23 peptone solution, 21 potato, 23 sterilization of, 14 sugar bouillon, 20 Cycloleppteron, 248 Cystitis, 261 Cytorrhyctes, luis, 193 scarlatinae, 193 vaccinae, 193 Cytodiagnosis, 275 Davainea madagascariensis, 204 Demodex folliculorum, 221, 224 Dengue, 249, 286 Dermacentor andersoni, 227 Dermatobia cyaniventris, 228, 238 Desk-microscopic, 10 Dhobies itch, 106 Dibothriocephalus latus, 204 Dicrocoelium lanceatum, 194, 196 Differential leukocyte count, 160 Diphtheria, 66, 73, 256 diagnosis of, 75 diphtheria-like bacilli, 76 media for growing, 74 Neisser's stain, 31 toxin of, 75 Diplococcus, crassus, 47 intracellular meningitidis, 49 lanceolat, 46 Diplognoporus grandis, 204 Diptera, 228, 231 Dipylidium caninum, 194, 204 Distomiasis, 195 Double boiler, n Dum dum fever, 181 Dunham's solution, 21 Dysentery, amoebae in, 175, 265 bacilli, 89 bacilli in faeces, 265 Eberth group, 85 Exhinococcus cysts, 205 P^chinorhynchus gigas, 219 Ehrlich, blood film method, 146 granule staining, 159 tri-acid stain, 148 Eimeria stiedae, 173, 185 Endo medium. 25 19 Endomyces albicans, 101 Endothelial cell in cytodiagnosis, 275 Entamoeba, buccalis, 173, 176 coli, 173, 175 histolytica, 173, 175, 266 Eosinophiles, 159 Eosinophilia, 162 Escherich group, 85 Eustrongylus gigas, 208, 216 Eye-piece (see ocular), 2 Eye-strain, 3 Eye infections, 251 Faeces, 263 amoebae in, 266 culturing, 265 diet for examination of, 263 fats in, 265 pancreatic test, 266 plating media, 25 soaps in, 265 Fasciola hepatica, 194, 195 Fasciolopsis buski, 194, 197 Fauces, 255 Favus, 104 Fermentation tubes, 5, 10 Films (blood), 146 Filter pump, 12 Filaria, bancrofti, 208, 2 1 1 demarquayi, 208, 212 embryos, key to, 213 loa, 208, 211, 252 medinensis, 209 ozzardi, 213 perstans, 208, 212 philippinensis, 213 powelli, 213 Fixation, blood films, 147 tissues, 279 Flagella staining, 32 Flagellata, 176 Flat worms, 194 Fleas, 232 key to, 232 Flukes, 194 of blood, 198 of intestines, 197 of liver, 195 of lungs, 198 Focus, microscopical, 3, 4 Foot and mouth disease, 285 Friedlander group, 79, 81, 252 Fungi, Achorion, 104 Ascomycetes, 100 2 go INDEX. Fungi, Aspergillus, 104 classification of, 99 Crytococcus, 102 cultivation of, 107 diagnosis of. 107 Discomyces bovis, 106 Discomyces madurae, 106 Hyphomycetes, 106 Madurella mycetomi, 106 Malassezia furfur, 106 Microsporoides, 106 Microsporum audouini, 106 Mucor, 100 Penicillium, 104 Saccharomycetes, 101 Trichophyton, 103 Trichosporum giganteum, 107 Gartner group, 85 Gastric contents, 270 Gastrodiscus hominis, 194, 197 Gelatin, 22 liquefaction of, 39 General paralysis (spinal fluid in), 276 Giemsa's stain, 151 Glanders, 66, 72 Glassware, cleaning of, 8 Glossina palpalis, 228, 236 Gonococcus, 41, 48, 251 Gonorrhoea, 48 Goundou, 286 Grabhamia, 250 Gram method, 28, 38 negative bacteria, 29 positive bacteria, 29 solution, 29 Granular degeneration (red cells,) 155 Granules (white cells,) 157 Haemacytometer, 136, 137, 140 Haemadipsa ceylonica, 220 Haematopota, 228, 234 Haematoxylin stain, 152, 286 Haemoglobin estimation, 138 Haemoglobinometers, Miescher's 138 Sahli's, 139 Tallquist, 139 Haemolytic experiments, 128 Haemosporidia, 153, 185 Haffkine, cholera vaccine, 97 plague prophylactic, 84 Halzoun, 196, 237 Hemokonia, 161 Herpetomonas, 182 Heterophyes heterophyes, 194, 197 Hirudo, medicinalis, 208, 219 nilotica, 208, 219 Hodgkin's disease, 168 Hook worms, 216 Hydatid disease, 205 Hydrocele agar, 24 Hymenolepis, nana, 194, 203 diminuta, 203 Hyphomycetes, 106 Hypoderma diana, 228, 238 Immersion objectives, 3 Immune sera, antimicrobic, 120 antitoxic, 120 diphtheria, 15 in diagnosis, 124 preparation, 124, 125 tetanus, 63 Immunity, active, 120 artificial, 119 natural, 119 passive, 120 Incubators, body temperature, n electrical, n, 12 petroleum lamp, n room temperature, 12 Indol, test for, 21 Influenza, 79 Infusoria, 183 Inoculation animals (tuberculosis), 67 animals (plague), 84 of media, 36 lodophilia, 152 Insecta, 228 Isospora bigemina, 173, 185 Itch mite, 223 Ixodidae, 221, 224, 226 Japanese river fever, 202, 286 Joints, gonococcus in, 49 Kala azar, 181 Kedani mite, 222, 286 Key to branching, curving bacilli, 66 to cocci, 41 to filarial embryos, 213 to fleas, 232 to Gram negative bacilli, 78 to spirilla, 94 to spore-bearing bacilli, 53 Lactic-acid bacteria, 116 Lamblia intestinalis, 173, 182 INDEX. 2QI Leeches, 219 Leishmania, 181 donovani, 173, 181 tropica, 173, 181 Leprosy, 70 diagnosis of, 71 in rats, 71 Leukaemia, 166 lymphatic, 168 splenomyelogenous, 167 Leukocytosis, 162 Leukopenia, 161 Leydenia gemmipara, 173, 176 Light in microscopical work, Linguatula rhinaria, 221, 227, 254 Liquefaction of gelatine, 39 Litmus, 23 Liver abscess, 163 Loeffler serum, 24, 256 stain, 28 Lumbar puncture, 276 Lymphocytosis, 163 Lymphocytes, large, 157 small, 157 Madura foot, 106 Magnifying power, 137 of oculars, 2 Malaria, 185 diagnosis of, 192 differential tables, 191, 192 life cycle, 189 life history, 187 Romanowsky stain in, 152 Mallein, 72 Malta fever, 51, 261, 269 Mansonia, 249 Mast cells, 159 Measles, 285 Megarhininae, 248 Meat poisoning, 60, 61 toxin of, 6 1 Malignant pustule, 55 Mechanical stage, i Media (see culture media), 25 Megaloblast, 156 Melaniferous leukocytes, 164 Meningococcus, 49, 276 Micrococcus, 41 catarrhalis, 41, 51, 253 melitensis, 41, 51 tetragenus, 41, 44, 253 Mi( rometer disk, 2, 135 standardization of, 136 Micrometer screw, 3 Micrometry, 2, 135 Microscope, i Microscopical sections (see tissue), 281 quick diagnostic method, 281 Milk, bacteriological examination of, "5 lactic-acid bacteria in, 116 leukocytes in, 115 Mites, 221, 222, 223 Mosquitoes, anatomy of, 240 classification of, 247, 248, 249 dissection of, 244 larvae of, 242 ova of, 242 i pupae of, 244 Moulds, (see fungi), 99 Mononuclear leukocytes, 158 Motility, 36 Brownian, 37 current, 37 Mucidus, 249 Mumps, 285 Musca domestica, 228, 235 Mutualism, 170 Myelocytes, 160 Myzomyia, 248J Myzorhynchus, 248 Nasal infections, diphtheria in, 253 leprosy in, 253 Necator americanus, 208, 218 Negri bodies, 277 Neisser's stain, 31, 256 Nematoda, 208 Nomenclature, in animal parasitology, 171 law of priority in, 171 Normob lasts, 156 Normal solutions, 284 Notes, blank, bacteriology, 134, 135 blood work, 168, 169 parasitology, animal, 250, 251 Numerical aperture, 3 Nyssorhynchus, 248 Ocular infections, 251 animal parasites in, 252 bacilli in, 252 gnococcus in, 252 M. catarrhalis in, 51 pneumococcus in, 251 Objectives, i, 2 Oculars, i, 2 2Q2 INDEX. Opisthorchis, felineus, 194, 197 sinensis, 197 Opsonic power, 119 apparatus in, 13 determination of, 131 Ornithodoros, 221, 226 Oxyuris vermicularis, 208, 219 Pangonia, 228, 234 Paragonimus westermani, 194, 198 Parasitism, 170 Pasteurelloses, 82 Pediculus capitis, 228, 229 Pediculus vestimenti, 228, 229 Pellagra, 104, Penicillium crustaceum, 104 Petri dishes, 7 Pfeiffer's phenomenon, 97, 128 Pharyngeal secretions, 255 Phthirjus pubis, 228, 230 Piedra, 107 Pinta, 105 Pipettes, bacteriological, 7, 12, 13 capillary bulb, 12 Piroplasmata, 224 Plague, 8 1 diagnosis of, 84 flea in, 84, 233 pneumonia, 260 prophylaxis, 260 Platinum wire, n Pleural fluids (cytodiagnosis), 276 Pneumococcus, 41, 46, 251 Poikilocytes, 155, 165 Polymorphonuclear leukocytes, 159 Porocephalus constrictus, 227 Protozoa, 173 Psychodidae, 228, 240 Pulex, cheopis, 228, 233 irritans, 228, 233 Pulicidae, 228, 231 Pus cultures from, 271 tetanus in, 272 Pyretophorus, 248 Rabies, 277, 285 preservation of dog in, 278 Reaction of media, 37 standardization of, 16, 17, 18, 284 Red blood-cells, counting of, 140 normal, 155 nucleated red cells, 155 polychromatophilia, 155 punctate basophilia, 155 Relapsing fever, 177 Rheumatism (acute), 285 Rhizoglyphus parasiticus, 222 Rhizopoda, 173 Rhynchota, 228, 229 Rice cooker, 10, 14, 15 Ring- worms, 103 Rocky mountain spotted fever, 224, 285 Roetheln, 285 Romanowsky stains, 151, 152 Round worms, 208 Sabouraud's medium for moulds, 1 08 Saccharomyces, anginosse, 101 blanchardi, 101 cerevisiae, 101 Sarcina lutea, 41, 43 Sarcoptes scabiei, 221, 223 Sarcophaga carnaria, 228, 237 Sarcopsylla penetrans, 228, 233 Sarcosporidia, 192 Scarlet fever, 285 Schistosomum haematobium, 198 japonicum, 199 mansoni, 199 Screw worm, 23 7> 254 Sections, making and staining, 282 Serum (see immune serum), 120 Sewage, in water, 109 Shiga's bacillus, 90 Simulidae, 228, 240 Skin infections, 273 itch mite, 274 leprosy in, 274 pus cocci in, 273 sarcopsylla in, 274 Slides, cleaning, 9 concave, 9 Smallpox, 193, 285 Sparganum, mansoni, 207 prolifer, 207 Spirillum cholerae asiaticae, 94, 114 metschnikovi, 94 of Finkler, Prior, 94 tyrogenum, 94 Spirochaeta, 177 duttoni, 173, 178, 227 recurrentis, 173, 177 refringens, 173, 178 Vincenti, 173, 178, 256 Spores, spore-bearing bacilli, 53 staining, 32 INDEX. 293 Sporozoa, 183 Sprue, 286 Sputum, 258 amoeba in, 260 centrifugalization for T. B., 259 culturing, 259 fixing smears, 258 Paragonimus eggs in, 260 plague pneumonia, 260 Staining methods, 27 Stains, acid fast, 30 Balch's, 149 capsule, 31 carbol fuchsin, 28 flagella, 32 Giemsa's, 151, 283 Gram's method, 28 haematoxylin, 152, 286 King's, 150 Leishman's, 150 Loffler's methylene blue, 28 Neisser's, 31 Nicolle's, 282 Smith's formal fuchsin, 30 spore, 32 tri-acid, 148 Van Giesen's, 282 Wright's, 149 Staphylococcus, 44 epidermidis albus, 41, 45 pyogenes albus, 41, 45 pyogenes aureus, 41, 45 Stegomyia, 249 Sterilization, Arnold, 5, 15 autoclave, 5, 6, 14 glass ware, 4 hot air, 5 pathogenic bacteria, 8 Stomach contents, 270 Boas-Oppler bacillus, 270 cancer cells in, 270 Stomoxys, 228, 236 Streptococcus, 41, 42 anginosus, 41 capsulatus, 47 coli gracilis, 41 fecal is, 41 pyogenes, 41, 43 salivarius, 41 Strong, cholera prophylactic, 97 plague vaccine, 85 Strongylidae, 216 Strongyloides stercoralis, 208, 209 Swabs,' 7 Syphilis, fixation of complement in di- agnosis, 130, 178 Giemsa's stain in, 256 Tabanus, 228, 234 Tables, insects, 228 mosquitoes, 246, 248, 249 of arachnoids, 221 of flat worms, 194 of fungi, 99 of protozoa, 173 of round worms, 194 pressure and temperature, 7 Taenia, africana, 203 echinococcus, 205 saginata, 194, 202 solium, 194, 202, 252 Taeniorhynchus, 249 Tape worms, adult, 200 somatic or larval, 205 Terminology, 172 Test-tubes, 8 Tetanus, 53, 62 antitoxin, 63 diagnosis of, 64 toxin, 63 Theobaldia, 249 Thionin, 148 Thorn-headed worm, 219 Throat examination, 236 Thrush, 101, 256 Ticks, 224 classification of, 226 life history, 225 Tinea, 103 cruris, 107 imbricata, 104 versicolor, 106 Tissue, acetone method, 280 chloroform method, 280 dehydration of, 280 fixation of, 279 imbedding, 280 preparation of for sections, 279 Titration of media, 17, 284 Toisson's solution, 141 Toxin, 121 diphtheria, 75 tetanus, 63 Transitional leukocyte, 158 Trematoda, 194 Treponema, 151, 178 pallidum, 173, 178 pertenue, 173, 179 294 INDEX. Trichomonas intestinalis, 173 vaginalis, 173, 182 Trichinella spiralis, 208, 214 Trichinosis, 214 Trichophyton mentagrophytes, 103 Sabouraudi, 103 tonsurans, 103 Trichocephalus trichiurus, 208, 213 Trichostrongylus instabilis, 216 Trombidiidae, 222 Trombidium holosericeum, 221, 222 Trypanoplasma, 181 Trypanosoma, brucei, 181 equinum, 181 equiperdum, 181 evansi, 181 gambiense, 173 and 180, 236 lewisi, 180, 181 Trypanosomiasis, 180, 164, 276 Tsetse flies, 236 Tuberculin, 69 Tuberculosis, bacillus of, 68 diagnosis of, 70 in guinea pigs, 57 Tubes, fermentation, 10 test-tubes for agglutination, 127 Wright's U-tube, 13 Turck, irritation cells, 161 ruling on haemacytometer, 140 Typhoid, agglutination in, 87 blood cultures, 269 carriers, 88 gall stones in, 87 in water, 114 serum for, 87 vaccination in, 87 Typhus fever, 285 Tyroglyphus longior, 221, 222 Urine, 261 chylous, 262 moulds in, 261 'schistosomum eggs in, 262 smegma bacillus in, 261 Vaccines, 132 preparation of, 132 standardizing of, 133 Varicella, 285 Vincent's angina, 178, 256 Water, bacteriological examination, 109 cholera spirillum in, 114 colon bacillus in, 113 qualitative bacteriological examina tion, 112 quantitative bacteriological examin- ation, no typhoid bacillus in, 114 Whip worms, 213 White blood-cells, 156 counting of, 143 normal count in disease, 164 Whooping cough, 80, 285 Widal tests, 126 Woolsorter's disease, 55 Working distance, 3 Wright's blood stain, 149, 157 method for anaerobes, 59 method for standardizing vaccines, 133 Yaws, 179 Yersin's serum, 85 Yellow fever, 249, 286 11.5 110 it5 I 10 too 75 70 t»0 4-0 57 1* IX 2,1 Xo xSo rf, 14-0 j~x"- »- — 75 ' vi v- , I — G9.S — / Q r^ — 1 ' f — -^•0- — •f — Equivalent Fahrenheit and Centigrade tables for the tem- peratures in common use in laboratories : 1. Those employed in pre- serving biological products and post mortem material; also in centrifuging experiments to separate complement and am- bcceptor (freezing tempera- tures) . 2. Those for culturing gela- tin (melting-point, 25° C.) as in water work (room tempera- tures) . 3. Those fcr growing im- portant pathogenic organisms (body temperatures). 4. Those for pasteurization and sterilization of bacterial vaccines. Also for paraffin bath (pasteurizing tempera- tures) . 5. Those for sterilization of dressings and media. Also for certain disinfection of spore- bearing bacterial contamina- tion (autoclave temperatures). UNITS IN COMMON USE IN LABORATORIES. Cubic Meter. — Unit of space for the number of organisms in air. It contains 1000 litres. It is equal to 1-308 cubic yards or 35.316 cubic feet. One thousand cubic feet, the unit of space in disinfection, is equal to 28.3 + cubic meters. Litre. — Unit of space for normal volumetric solutions. It contains 1000 cubic centimeters. It is equal to 1.0567 quarts or 33.8 + ounces. A liter of distilled water weighs i kilogram. Cubic Centimeter.— Unit of space for organisms in water, milk, vaccines, etc., ic.c.=o.27 fl. dr. There are, approximately, 16 drops in i c.c. Cubic Millimeter. — Unit of space for blood-cells. There are 1000 cubic millimeters in i cubic centimeter and i million cubic milli- meters in i litre. In water analysis, as there are i million milli- grams in one litre, parts in the million and milligrammes per litre are the same. i Meter = 3 9.3 7 inches. i Centimeter = .3937 inch. Approximately, 2/5 inch. i Millimeter = .0393 inch. Approximately, 1/25 inches. i Kilogram = 2.2+ pounds av. i Gram = 15. 432 grains. i Milligram = 0.0154 grain. Approximately, 1/64 grain. A pound avoirdupois is equal to 453.59 gm. One hundred cubic centimeters of a saturated solution contains: Water Alcohol Methylene blue, 6.68 0.66 grams. Gentian violet, i-75 4.42 grams. Basic fuchsia, 0.66 2.92 grams. YC 88491 GRAM'S METHOD OF STAINING. 1. Prepare even thin film and fix. 2. Aniline gentian violet, -2 to 5 minutes. 3. Gram's iodine solution, i to 2 minutes. (Tea-leaf color.) 4. Wash in water and decolorize with 95% alcohol. 5. Wash in water and counterstain with Bismarck brown or with dilute carbol fuchsin. 6. Wash, dry and mount. Practically all pathogenic cocci are Gram positive, except the Gonococcus, the Meningococcus, the M. catarrhalis and the M. melitensis. Practically all pathogenic bacilli are Gram negative, except the e pore-bearing ones (exception B. malig. cedemat.), the acid-fast ones and diphtheria and diphtheroid organisms.