Practical bacteriology, blood work and animal parasitology, including bacteriological keys, zoological tables and explanatory clinical notes

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Preface To The Fifth Edition

Although the fourth edition of this laboratory manual of internal medicine was issued less than two years ago it has been found necessary to revise it very extensively in view of the important advances made as a result of the medical work of the present war.

In this connection much new material has been added to the section on agglutination as applied to grouping meningococci and pneumococci. The Dreyer method for interpretation of agglutination reactions of typhoid and paratyphoid infections has also been given in the section on "Immunity."

A new chapter dealing with the medical importance of poisonous arthropods and fishes has been inserted.

In the section on "Disinfectants and Insecticides" much new material has been added in particular a practical method of preparing Dakin's solution and recent investigations as to delousing methods. In view of its practical value a brief presentation of Mosenthal's nephritic test diet method of studying renal functioning will be found under " Urine. " A table giving data as to the most important communicable diseases, based upon the report of a Conunittee of the American Public Health Association, will be found in the front linings.

A large number of the old illustrations have been replaced by new and better ones. In particular the four plates of the malarial parasites contained in former editions have been replaced by a single and more instructive one.

Notwithstanding the utmost effort at condensation it has been found necessary to increase the size of the present edition by sixty-two pages.

Preface To The Fourth Edition

As noted in previous prefaces, this manual represents the notes of a course along the laboratory side of internal medicine which has seemed to the author practical and concise.

The exhaustion of a large edition of this laboratory guide in a little more than two years would indicate a demand for a book of this character. By combining notes on point*? of clinical importance with laboratory details such a presentation of the subject would appear of more value to the student of internal medicine than the bare laboratory technic.

In view of the enormous advances in internal medicine since the appearance of the last edition it has been very difficult to incorporate new material and at the same time retain the pocket manual feature. This has been done, however, by the greater use of paragraphs of smaller type separating those printed in larger t)rpe, a plan which adds to the accessibility of the contents as well as lessening the number of pages. Notwithstanding such utilization of space it has been necessary to increase the Contents of the book by almost one hundred pages.

To further facilitate quick reference numerous additional bold type headings of paragraphs have been introduced.

It has been a difficult matter to write concise discussions of such subjects as acidosis, anaphylaxis, deficiencies of renal functioning, etc., where space was so limited.

Practically every chapter in the book has been carefully revised and new material added to each. In some of the sections the changes in our views during the past two or three years have been so marked that it has been necessary to entirely rewrite large portions of such chapters.

A new chapter, dealing with diseases of doubtful or only recently determined etiology has been added to Part IV. In this will be found discussions of the vitamine theory in beriberi and pellagra as well as recent findings in connection with such diseases as typhus fever, Oroya fever, verruga, rat bite fever, spotted fever of the Rocky Mountains and sprue.

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VIU PREFACE

In the appendix a new section has been added on the chemical blood examinations, and in that on insecticides the recent views as to the best methods of destroying lice to control the spread of typhus fever and relapsing fever have been incorporated.

There has also been added to the appendix a section dealing with anatomical and physiological normals to furnish ready reference for work in the pathological or chemical laboratory.

Among the tests more recently accepted as of practical value and incorporated in this edition may be mentioned the following: Schick test for diphtheria immunity, tests for recognition of acidosis, tests for efficiency in renal functioning, Petroff 's method for culturing tubercle bacilli, Wolff and Junghans' test for gastric carcinoma, Bronfenbrenner's modification of Aberhalden's technic, tests as applied to the duodenal fluid, Lange's colloidal gold test for general paresis, Fontana's spirochete staining technic, Gluzinski's gastric carcinoma test, and many others.

There have also been many new illustrations of animal parasites substituted for those in the third edition which did not appear to have sufficient teaching value.

Preface To The First Edition

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 himdred graduates of the various medical schools of the country from the standpoint 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 successful candidates, while under instruction at the Nav^ 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 possibiUties 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 employed to eliminate as far af*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 organisms, yet when it was required of them to outline the procedure by which they would differentiate members of the typhoid-colon groups when encoimtered in a plate made from faeces, the problem appeared to them impossible. They possessed the information, but did not know how to apply it.

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X PREFACE

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 sahent 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 Parasites."

In the chapter on Media Making, it is believed that anyone after reading this section and following the instructions will be able to satisfactorily 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 important 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 cUmates, unless by reason of such measures of diagnosis being indispensable in the tropics, it has, however, been my endeavor to treat every tropical question, T«diether in blood work, bacteriology, or animal parasitology, in a more complete way than is usual in manuals of this character. Therefore it is beUeved that this Uttle 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 Naval Medical School that I feel justified in presenting a concise outline 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..

PREFACE XI

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 immediately 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 parasites 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 haye been: Albutt'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; Daniel's Laboratory Studies in Tropical Medicine; Manson's Tropical Diseases; Gedoelst's Les Champignons Parasites; Neveu-Lemaire Parasitologie Humaine; Chester's Determinative Bacteriology; Lehmann and Neumann's Bacteriology.

E. R. S.

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CHAPTER II.— Culture Media

Nutrient bouillon, 22 — Standardization of reaction, 23 — Sugar-free bouillon, 26 — Glycerine bouillon, 27 — Peptone solution, 27 — ^Nutrient agar, 28 — Glycerine £^ar egg medium, 30 — Gelatine, 30-— Litmus milk, 31 — Potato, 32 — Blood serum, 33 — Blood agar, 34 — Bile and faeces media, 36-39 — Culture media for protozoa, 40.

CHAPTER XI. — Bacteriology of Water, Air, and Milk. CHAPTER Xll. — Practical Methods of Immunity

Methods of obtaining immune sera, 182 — Agglutination tests, 184 — Precipitins, 191 — Deviation of the Complement, 194 — Fixation of the Complement, 195 — The Wassermann reaction, 196 — Opsonic power and preparation of vaccines, 209 — Anaphylaxis, 215.

CHAPTER XIII. — Micrometry and Blood Preparations

Micrometry, 221 — Haemoglobin estimation, 224 — Counting blood, 227 — Study of fresh blood, 232 — Blood films, 234 — Staining blood films, 238 — lodophilia, 242 — Occult blood, 246 — Acidosis, 248.

CHAPTER XIV. — Normal and Pathological Blood

Color index, 252 — Red cells, 252 — ^White cells, 255 — ^Eosinophilia, 264 — ^Leukocytosis, 265 — ^Lymphocytosis, 266 — Diseases with a normal leukocyte count, 267 — ^The primary anaemias, 268 — Secondary anaemias, 270 — The leukemias, 271.

CHAPTER XVII.— The Plat Worms

Flukes, 324 — Liver flukes, 327 — Intestinal flukes, 329 — ^Lung flukes, 330 — Blood flukes, 332 — Cestodes, 334 — Somatic taeniasis, 341.

CHAPTER XVIIL— The Round Worms.

Filariidae, 347 — Key to filarial larvae, 353 — Trichinosis, 356 — Hookworms, 359 — ^Ascaridae, 365 — ^Leeches, 367.

Part IV. Clinical Bacteriology And Animal Parasitology Op The Various Body

FLUIDS AND ORGANS

CHAPTER XXIV. — Diagnosis of Infections of the Ocular Region, 427. CHAPTER XXV. — Diagnosis of Infections of the Nasal Cavities, 430. CHAPTER XXVI: — Examination of Buccal and Pharyngeal Material,

432.

CHAPTER XXVII.— Examination of Sputum, 435.

CHAPTER XXVIII.— The Urine, 440— Renal Functioning, 449. CHAPTER XXIX.— The FiECES, 454.

CHAPTER XXX. — Blood Cultures and Blood Parasites, 461. CHAPTER XXXI. — The Stomach Contents, 465 — Duodenal Fluid, 466. CHAPTER XXXIL— Examination of Pus, 468.

CHAPTER XXXIIL— Skin Infections, 471.

CHAPTER XXXIV.— Cytodiagnosis, 473— Spinal Fluid, 475. CHAPTER XXXV. — Rabies, Vaccinia and Smallpox, 480 — Filterable

Viruses, 484.

CHAPTER XXXVL— Diseases of Unknown Origin, 486.

Preparation of Tissues for Examination in Microscopic Sections, 495.

Mounting and Preparation of Animal Parasites, 504.

Preparation of Normal Solutions, 506.

Chemical Examination of Blood, 507.

Chemical Examination of the Urine, 513. ,

Chemical Examination of the Gastric Contents, 527.

Chemical Tests of Duodenal Fluid, 529.

Disinfectants and Insecticides, 530.

Anatomical and Physiological Normals, 540.

BACTERIOLOGY, BL00D-W1DRK AND ANIMAL PARASITOLOGY

CHAPTER I APPARATUS The Microscope

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The most important piece of apparatus for the laboratory worker is the microscope. Very satisfactory microscopes can be purchased in this country. It is impossible to do good microscopical work unless the microscope gives and continues to give good definition and the working parts remain firm.

Folding microscope stands are now made which are perfectly satisfactory, such instruments, however, have only the advantage of occupying less space in a case so that unless the question of compactness is involved, as in an outfit for the military services or for a microscopist who travels about a great deal, the ordinary rigid horseshoe base is to be preferred.

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. For the examination of colonies growing in Petri dishes we also use the stage unencumbered with the mechanical stage. A triple or quadruple nose-piece, according to the number of objectives used, is also indispensable.

One should always use a magnifying glass or better a lens in a tripod or an aplanatic triplet in the study of microscopic objects, prior to examination with. the microscope. A dissecting microscope is better for this purpose and is very useful in dissecting mosquitoes, etc. In fact the dissecting microscope is almost essential in the examination of helminthological and entomological specimens. Of particular value is a stage forceps for orienting insects, especially mosquitoes, when examined on the stage of either a compound or simple microscope.

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2 APPAIIATUS

The following precautions should be observed to prevent injury to the microscope;

I. II the fineadjustment works through the ann of the microscope, always grasp the instrument by the pillar which supports the stage. In those microscopes, however, which are not constructed in this way the arm is made to serve in lifting the instrument.

2. Always keep the microscope in its case or covered with a bell jar when not in use in order to keep away the dust. A piece of black cloth supported on a wire lamp-shade frame makes a most convenient protecting covering.

3. Alcohol ruins the lacquer of an instrument and care should be observed to keepallpartsof the microscope from coming in contact with acids, alkalies, chloroform or lylol as well as alcohol.

4. Always use Japanese lens paper in wiping off the dust from dry objectives or the immersion oil from the 2-mm. one.

Should one neglect to wipe off the oil from the oil-immersion objective, the dried oil can be removed by wiping with a drop of xylol on lens paper, but the cleaning should be done as rapidly as possible, with a final wiping oS with dry lens paper, to avoid damage by the xylol to the setting of the lenses. Throw lens paper away after using

Fig. I.— Partsofmicroscopf Eyepiece. D, Drawtube. fi, Rack.

P.H, Pinion head. T. Body M.H, Micrometer head. R.N. Revolving nose-piece. O. Objective!

M.S. Mechanical stage. 5. Stag.

H, Handle, S.S, Sub-stage. /. It. elination joint. P. Pillar, if, Mil ror. B, Base.

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5. Lenses are very liable to deteriorate in the tropics. One should be careful to protect his instrument from the direct light of the tropical sun.

6. li any oil is used on the mechanical should be wiped off to avoid the catching of dust parts for lubrication, all e: or gritty particles.

Objectives. — To meet the demands of clinical microscopy there should be three objectives, preferably a i6-mm, {%-ihch), a 4-mm. (J^-inch) and a 2-mm. {J-f 2-inch) homogeneous oil immersion one.

The Zeiss AA is a 17-mm. objective, and the Leitz No. 3, an i8-mm. or>i-inch one. The Zeiss D is about 4.3-mm. and the Leitz No. 6, 4.4-mm. or ^-inch. A dustproof quadruple nose-piece with four objectives will be found a great convea-

OBJECTIVES 3

ience (in addition to the ?^-inch and J^2-uich objectives, a J^-inch for urine and blood counting, with a J^-inch for examining hanging-drop preparations and for quick eicamination 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.

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 K-inch objective may have to be approached to the object so that the distance intervening may be only J^ inch or even less. This explains the frequent inability to focus an object when a high-power dry objective (J^-inch or J^-inch) 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.

Oil Immersion. — ^In using the oil-immersion objective always dip the - ' lens in the oil and practically touch the cover-glass — the eye being at a level with the stage — before beginning to focus. With the coarse adjustment one can feel the contact with the cover-glass, which is impossible with the fine adjustment. It saves time and disappointment to make a preHminary examination of a preparation requiring the high dry or immersion lens with a low power (%-inch) before employing the higher power; in this way we locate or center a suitable field for study.

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 standpoint 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 3^-inch of high numerical aperture there may not be sufl&cient working distance 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. The skill of the optician, however, can obviate this defect in an objective of high numerical aperture so that it may combine the qualities of perfect definition with sufficient working distance.

Oculars. — ^As regards oculars (eye-pieces) a Leitz No. i and a No. 4 w will best meet the requirements. For high magnification a No. 8 may y be of service. 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 their equivalent focal distance and are referred to as 3^ -inch, i-inch and 2-inch oculars.

A i-inch or 25-mm. ocular magnifies the magnification produced by the objective about 10 times while a 2-inch or 50-mm. one increases the magnification of the objective four times.

A Leitz No. o is a 50-mm. ocular and magnifies four times. The Nos. i, 2, 3, 4 and 5 are 40, 35, 25, and 20 mm. respectively and give eye -piece magnifications of 5, 6, 8, 10 and 12 times.

The oculars in common use are known as negative or Huyghenian 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 within the ocular. When using a disc micrometer, it is supported by this diaphragm, and the outlines of the image are cut by the ruUngs on the glass disc, and so we are enabled to measure the size of the object 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 magnif 3dng the image, the image being formed entirely by the objective and located below the ocular. By fixing one end of a hair on the rim of the diaphragm inside the ocular with a minute drop of balsam one has a satisfactory pointer to locate any particular cell in the microscopic field.

Tube Length. — The tube length is measured from the eye lens of the ocular to the end of the tube into which the objective is screwed. If a triple nosepiece is used the tube length must be shortened that much. As a rule objectives are corrected to use with a tube length of 160 mm.

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 the No. 2, but with a little practice in cleaning cover-glasses this is negligible. Immersion lenses are less affected than dry lenses by the question of a certain thickness of cover-glasses for a certain tube length.

One of the most fruitful causes of the crushing of microscopical objects and the overling cover-glass or, what is far more important, the breaking of the cover-glass of a hanging-drop preparation and consequent risk of infection is the attempt to focus with the fine adjustment.

MICROSCOPICAL TECHNIQUE 5

Focussing. — 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 pinign) 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.

Practical Points in the Use of the Microscope. — ^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 3^2-ii^ch or a high-power dry lens.

It is well, however, in a bacterial or blood preparation to first examine the smear with the %-inch objective in order to determine suitable areas for examination with the oil-immersion objective. With tissue sections 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 glass, before using the microscope, will give a surprising amount of information.

Position. — While some workers prefer to use the microscope with the body tube inclined by the inclination joint yet one gets just as good results by keeping the tube perpendicular and it is better to accustom one's self to such a position, because it is necessitated when we work with fluid mounts.

Care of the Oil-immersion. — After using the oil-immersion objective the lens should be wiped clean of oil with a strip of Japanese lens paper or with a silk handkerchief. If the oil should dry on the surface of the lens it may be removed with a drop of xylol on a piece of lens paper. Immediately afterward the lens should be dried. Dried oil on a lens often causes the lens to be considered defective. Accidental contact of the dry objectives with oil is not uncommon and should always be thought of when satisfactory optical effects are not obtainable. In depositing the drop of immersion oil on the slide bubbles are at times formed which make it almost impossible to use the }-{ 2-inch objective. Under such circumstances I either prick the bubbles or wipe off the oil and deposit a drop anew.

It is advisable to cultivate the use of both eyes in doing microscopical work. When using one eye the other should be kept open witli accommodation relaxed. It is this squinting of the unemployed eye which so often fatigues. A strip of cardboard 4 or 5 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.

Warm Stages. — A warm stage for the study of living protozoa may be extemporized by taking a piece of copp)er about the size of the stage and with a strip projecting out anteriorly for 5 or 6 inches. The under surface of the plate is covered with flannel and a hole about i inch in diameter cut out of the center. The proper amount of heat is applied by a flame impinging on the tongue-like projection of the copper plate.

At present there are electrically heated warm stages, connected with the desk socket by a wire and plug, which are most convenient. In fact they are almost as satisfactory as the more expensive and less convenient warm chamber or microscope oven surrounding the microscope.

^ niumination. — Direct sunlight or excessively bright light is to be avoided. If such conditions must exist a white shade or muslin curtain drawn across the window is a necessity. Light from the north and from a white cloud is the most desirable. South of the equator a southern Hght. In the tropics a piece of plate glass fitted into the lower part' of a wire screen frame gives good lighting, keeps out dust, and does not interfere greatly with the circulation of the air.

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 by the iris diaphragm so as to give a contour picture. In examining a stained blood or bacterial preparation, the Abb6 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 Abb6 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. It is best to use a slide about i .5 mm. thick. Always use the plane mirror when examining stained bacterial or blood films, as a color image is desired. If artificial light is used the concave mirror gives better results as such rays are not parallel. By using a 6-inch globe filled with a very dilute solution of copper sulphate and a few drops of ammonia the rays are rendered parallel and then the plane mirror should be used. Ordinarily in examining tissue sections, the Ahh6 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. '

For microscopical work in a laboratory not properly supplied with windows or for night work the frosted incandescent bulb is very satisfactory.

An objection to artificial light is that one working almost entirely with sunlight forms standards and when using a different Ught is somewhat confused in interpretation of the microscopical picture.