CHAPTER VII

III. ARTHROPODA

Most of these are external parasites, and the reader is referred to the standard works upon diseases of the skin for descriptions. The itch-mite (Acarus scabiei) and the louse (Pediculus capitis,corporis,vel pubis) are the more common members of the group.

A number of flies may deposit their ova in wounds or in such of the body cavities as they can reach, and the resulting maggots may cause intense irritation. Ova may be swallowed with the food and the maggots appear in the feces. Probably most important is the "screw worm," the larva ofCompsomyia macellaria, infection with which is not rare in some parts of the United States. The ova are most commonly deposited in the nasal passages, and the larvæ, which may be present in great numbers, burrow through the soft parts, cartilage, and even bone, always with serious and often with fatal results.

PUS

Pus contains much granular débris and numerous more or less degenerated cells, the great majority being polymorphonuclear leukocytes—so-called "pus-corpuscles." Eosinophilic leukocytes are common in gonorrheal pus and in asthmatic sputum. Examination of pus is directed chiefly to detection of bacteria.

When very few bacteria are present, culture methods must be resorted to, but such methods do not come within the scope of this work. When considerable numbers are present, they can be detected and often identified in cover-glass smears. Several smears should be made, dried, and fixed as described under Sputum (p. 32). One of these should be stained one-fourth to one-half minute with Löffler's methylene-blue, rinsed well with water, dried, mounted, and examined with an oil-immersion lens. This will show all bacteria except the tubercle bacillus, and often no other stain is necessary for their identification. In many cases special stains must be applied.

Gram's method(p. 40) is a very useful aid in distinguishing certain bacteria. The more important organisms react to this staining method as follows:

The most common pus-producing organisms arestaphylococciandstreptococci. They are both cocci, or spheres, their average diameter being about 1 µ. Staphylococci are commonly grouped in clusters, often compared to bunches of grapes (Fig. 113). There are several varieties, which can be distinguished only in cultures. Streptococci are arranged side by side, forming chains of variable length (Fig. 114). Sometimes there are only three or four individuals in a chain; sometimes a chainis so long as to extend across several microscopic fields. Streptococci are more virulent than staphylococci, and are less common.

Should bacteria resemblingpneumococcibe found, Buerger's method (p. 37) should be tried. When it is inconvenient to stain before the smears have dried, capsules can be shown by the method of Hiss. The dried and fixed smear is covered with a stain composed of 5 c.c. saturated alcoholic solution gentian-violet and 95 c.c. distilled water, and heated until steam rises. The preparation is then washed with 20 per cent. solution of copper sulphate, dried, and mounted in Canada balsam.

Pneumococci may give rise to inflammations in many locations. When they form short chains, demonstration of the capsule is necessary to distinguish them from streptococci.

If tuberculosis be suspected, the smears should be stained by one of the methods for thetubercle bacillus(pp.32and127), or guinea-pigs may be inoculated.The bacilli are generally difficult to find in pus, and bacteria-free pus would suggest tuberculosis.

Gonococci, when typical, can usually be identified with sufficient certainty for clinical purposes in the smear stained with Löffler's methylene-blue. They are coffee-bean-shaped cocci which lie in pairs with their flat surfaces together (Fig. 116). They lie for the most part within pus-cells, an occasional cell being filled with them, while the surrounding cells contain few or none. A few arefound outside of the cells. It is not usual to find gonococci when many other bacteria are present, even though the pus is primarily of gonorrheal origin. Whenever the identity of the organism is at all questionable, Gram's method should be tried. In rare instances it may be necessary to resort to cultures. The gonococcus is distinguished by its failure to grow upon ordinary media.

Gonococci are generally easily found in pus from untreated acute and subacute gonorrheal inflammations,—conjunctivitis, urethritis, etc.,—but are found with difficulty in pus from chronic inflammations and abscesses, and in urinary sediments.

PERITONEAL, PLEURAL, AND PERICARDIAL FLUIDS

The serous cavities contain very little fluid normally, but considerable quantities are frequently present as a result of pathologic conditions. The pathologic fluids are classed as transudates and exudates.

Transudatesare non-inflammatory in origin. They contain only a few cells, and less than 2.5 per cent. of albumin, and do not coagulate spontaneously. The specific gravity is below 1018. Micro-organisms are seldom present.

Exudatesare of inflammatory origin. They are richer in cells and albumin, and tend to coagulate upon standing. The specific gravity is above 1018. Bacteria are generally present, and often numerous. The amount of albumin is estimated by Esbach's method, after diluting the fluid. Bacteria are recognized by cultures, animal inoculation, or stained smears.

Exudates are usually classed as serous, serofibrinous, seropurulent, purulent, putrid, and hemorrhagic, whichterms require no explanation. In addition, chylous and chyloid exudates are occasionally met, particularly in the peritoneal cavity. In the chylous form the milkiness is due mainly to the presence of minute fat-droplets, and is the result of rupture of a lymph-vessel. Chyloid exudates are milky chiefly from proteids in suspension, or fine débris from broken-down cells. These exudates are most frequently seen in carcinoma and tuberculosis of the peritoneum.

Cytodiagnosis.—This consists in a differential count of the cells in a transudate or exudate, particularly one of pleural or peritoneal origin.

The fresh fluid, obtained by aspiration, is centrifugalized for at least five minutes; the supernatant liquid is poured off; and cover-glass smears are made and dried in the air. The smears are then stained with Wright's blood-stain, to which one-third its volume of pure methyl-alcohol hasbeen added. Cover the smear with this fluid for one-half minute, then dilute with eight or ten drops of water, and let stand about two minutes. Wash gently in water, anddry by holding the cover-glass between the fingers over a flame. Mount in balsam and examine with a one-twelfth objective.

Predominance of polymorphonuclear leukocytes (pus-corpuscles) points to an acute infectious process (Fig. 117).

Predominance of lymphocytes (Fig. 118) generally signifies tuberculosis. Tuberculous pleurisy due to direct extension from the lung may give excess of polymorphonuclears owing to mixed infection.

Predominance of endothelial cells, few cells of any kind being present, indicates a transudate (Fig. 119). Endothelial cells generally predominate in carcinoma, but are accompanied by considerable numbers of lymphocytes and red blood-corpuscles.

CEREBROSPINAL FLUID

Examination of the fluid obtained by lumbar puncture is of value in diagnosis of certain forms of meningitis.

Tubercle bacillican be found in the majority of cases of tuberculous meningitis. The sediment, obtained by thorough centrifugalization or by coagulation and digestion (p. 128) is spread upon slides and stained by one of the methods already given. A considerable number of smears should be examined. In doubtful cases, inoculation of guinea-pigs must be resorted to.

TheDiplococcus intracellularis meningitidisis recognized as the cause of epidemic cerebrospinal fever, and can be detected in the cerebrospinal fluid of most cases, especially those which run an acute course. Cover-glass smears from the sediment should be stained by the method for the gonococcus. The meningococcus is an intracellular diplococcus which often cannot be distinguishedfrom the gonococcus in stained smears (Fig. 120). It, also, decolorizes by Gram's method. The presence of such a diplococcus in meningeal exudates is, however, sufficient for its identification.

Various organisms have been found in other forms of meningitis—the pneumococcus most frequently. In some cases no micro-organisms can be detected even by culture methods.

ANIMAL INOCULATION

Inoculation of animals is one of the most reliable means of verifying the presence of certain micro-organisms in fluids and other material.

Clinically, it is applied almost exclusively to demonstration of the tubercle bacillus when other means havefailed or are uncertain. The guinea-pig is the most suitable animal for this purpose. When the suspected material is fluid and contains pus, it should be well centrifugalized, and one or two cubic centimeters of the sediment injected by means of a large hypodermic needle into the peritoneal cavity or underneath the loose skin of the groin. Fluids from which no sediment can be obtained must be injected directly into the peritoneal cavity, since at least 10 c.c. are required, which is too great an amount to inject hypodermically. Solid material should be placed in a pocket made by snipping the skin of the groin with scissors, and freeing it from the underlying tissues for a short distance around the opening. When the intraperitoneal method is selected, several animals must be inoculated, since some are likely to die from peritonitis caused by other organisms before the tubercle bacillus has had time to produce its characteristic lesions.

The animals should be killed at the end of six or eight weeks, if they do not die before that time, and a careful postmortem examination should be made for the characteristic pearly-gray or yellow tubercles scattered over the peritoneum and through the abdominal organs, particularly the spleen, and for caseous inguinal and retroperitoneal lymph-glands. The tubercles and portions of the caseous glands should be crushed between two slides, dried, and stained for tubercle bacilli. The bacilli are difficult to find in the caseous material.

THE MOUTH

Micro-organisms are always present in large numbers. Among these isLeptothrix buccalis(Fig. 121), which is especially abundant in the crypts of the tonsils and thetartar of the teeth. The whitish patches ofpharyngomycosis leptothricaare largely composed of these fungi. They are slender, segmented threads, which generally, but not always, stain violet with Lugol's solution, and are readily seen with a one-sixth objective. At times they are observed in the sputum and stomach fluid. In the former they might be mistaken for elastic fibers; in the latter, for Boas-Oppler bacilli. In either case, the reaction with iodin will distinguish them.

Thrushis a disease of the mouth seen most often in children, and characterized by the presence of white patches upon the mucous membrane. It is caused by the thrush fungus,Oïdium albicans. When a bit from one of the patches is pressed out between a slide and cover and examined with a one-sixth objective, the fungus is seen to consist of a network of branching segmented hyphæ with numerous spores, both within the hyphæ and in the meshes between them (Fig. 122). The meshes also contain leukocytes, epithelial cells, and granular débris.

Acute pseudomembranous inflammations, which occur chiefly upon the tonsils and nasopharynx, are generally caused by the diphtheria bacillus, but may result from streptococcic infection. In many cases diphtheriabacilli can be demonstrated in smears made from the membrane and stained with Löffler's methylene-blue or 2 per cent. aqueous solution of methyl-green. They are straight or curved rods, which vary markedly in size and outline, and stain very irregularly (Fig. 123). A characteristic form is a palely tinted rod with several deeply stained granules (metachromatic bodies), or with one such granule at each end. They stain by Gram's method. It is generally necessary, and always safer, to make a culture upon blood-serum, incubate for twelve hours, and examine smears from the growth.

Vincent's anginais a chronic pseudomembranous and ulcerative inflammation of pharynx and tonsils. It is probably caused by two micro-organisms living in symbiosis—one a fusiform bacillus, the other a long spirillum (Fig. 124). They can readily be demonstrated in smears stained with Löffler's methylene-blue. The bacillus is spindle shaped, more or less pointed at theends, and about 6 to 12 µ long. The spirillum is a very slender, wavy thread, about 30 to 40 µ long.

Tuberculous ulcerationsof mouth and pharynx can generally be diagnosed from curetings made after careful cleansing of the surface. The curetings are well rubbed between slide and cover, and the smears thus made are dried, fixed, and stained for tubercle bacilli. Since there is much danger of contamination from tuberculous sputum, the presence of tubercle bacilli is significant only in proportion to the thoroughness with which the ulcer was cleansed. The diagnosis is certain when the bacilli are found within groups of cells which have not been disassociated in making the smears.

THE EYE

Staphylococci,pneumococci, andstreptococciare probably the most common of the bacteria to be found in nonspecific conjunctivitis and keratitis. The usual cause of acute infectious conjunctivitis, especially in cities, seems to be theKoch-Weeks bacillus. This is a minute, slender rod, which lies within and between the pus-corpuscles (Fig. 125), and is negative to Gram's stain. In smears it cannot bedistinguished from the influenza bacillus, although its length is somewhat greater. Thediplobacillus of Morax and Axenfeldgives rise to an acute or chronic blepharo-conjunctivitis without follicles or membrane, for which zinc sulphate seems to be a specific. It is widely distributed geographically, and is common in many regions. The organism is a short, thick diplobacillus, is frequently intracellular, and is Gram-negative (Fig. 126). A delicate capsule can sometimes be made out.

Early diagnosis of gonorrheal ophthalmia is extremely important, and can be made with certainty only by detection ofgonococciin the discharge. They are easily found in smears from untreated cases. After treatment is begun they soon disappear, even though the discharge continues.

Pseudomembranous conjunctivitis generally shows eitherstreptococciordiphtheria bacilli. In diagnosing diphtheric conjunctivitis, one must be on his guard against thexerosis bacillus, which is a frequent inhabitant of the conjunctival sac in healthy persons, and which is identical morphologically with the diphtheria bacillus. The clinical picture is hence more significant than the microscopic findings.

Various micro-organisms—bacteria, molds, protozoa—have been described in connection with trachoma, but the specific organism of the disease is not definitely known.

THE EAR

By far the most frequent exciting causes of acute otitis media are the pneumococcus and the streptococcus. The finding of other bacteria in the discharge generally indicates a secondary infection, except in cases complicating infectious diseases, such as typhoid fever, diphtheria, and influenza. Discharges which have continued for some time are practically always contaminated with the staphylococcus. The presence of the streptococcus should be a cause of uneasiness, since it much more frequently leads to mastoid disease and meningitis than does the pneumococcus. The staphylococcus, bacillus of Friedländer, colon bacillus, and Bacillus pyocyaneus may be met in chronic middle-ear disease.

In tuberculous disease the tubercle bacillus is present in the discharge, but its detection offers some difficulties. It is rarely easy to find, and precautions must always be taken to exclude the smegma and other acid-fast bacilli (p. 35), which are especially liable to be present in the ear. Rather striking is the tendency of old squamous cells to retain the red stain, and fragments of such cells may mislead the unwary.

PARASITIC DISEASES OF THE SKIN

Favus, tinea versicolor, and the various forms of ring-worm are caused by members of the fungus group. To demonstrate them, a crust or a hair from the affected area is softened with a few drops of 20 per cent. caustic soda solution, pressed out between a slide and cover, and examined with a one-sixth objective. They consist of a more or less dense network of hyphæ and numerous round or oval refractive spores. The cuts in standard works upon diseases of the skin will aid in differentiating the members of the group.

MILK

A large number of analyses of human and cow's milk are averaged by Holt as follows, Jersey milk being excluded because of its excessive fat:

The reaction of human milk is slightly alkaline; of cow's, neutral or slightly acid. The specific gravity of each is about 1.028 to 1.032. Human milk is sterile when secreted, but derives a few bacteria from the lacteal ducts. Cow's milk, as usually sold, contains large numbers of bacteria. Microscopically, human milk is a fairly homogeneous emulsion of fat, and is practically destitute of cellular elements.

Chemic examination of milk is of great value in solvingthe problems of infant feeding. The sample examined should be the middle milk, or the entire quantity from one breast. The fat and proteid can be estimated roughly, but accurately enough for many clinical purposes, by means of Holt's apparatus, which consists of a 10 c.c. cream gage and a small hydrometer (Fig. 127). The cream gage is filled to the 0 mark with milk, allowed to stand for twenty-four hours at room temperature, and the percentage of cream then read off. The percentage offat is three-fifths that of the cream. The proteid is then approximated from a consideration of the specific gravity and the percentage of fat. The salts and sugar very seldom vary sufficiently to affect the specific gravity, hence a high specific gravity must be due to either an increase of proteid or decrease of fat, or both, and vice versâ. With normal specific gravity the proteid is high when the fat is high, and vice versâ. The method is not accurate with cow's milk.

For more accurate work the following methods, applicable to either human or cow's milk, are simple and satisfactory.

Fat.—Leffmann-Beam Method.—This is essentially the widely used Babcock method, modified for the small quantities of milk obtainable from the human mammary gland. The apparatus consists of a special tube which fits the aluminum shield of the medical centrifuge (Fig. 128) and a 5 c.c. pipet. Owing to its narrow stem, the tube is difficult to fill and to clean. Exactly 5 c.c. of the milk are introduced into the tube by means of the pipet, and 1 c.c. of a mixture of equal parts of concentrated hydrochloric acid and amyl-alcohol is added and well mixed. The tube is filled to the 0 mark with concentrated sulphuric acid, adding a few drops at a time and agitating constantly. This is revolved in the centrifuge at 1000 revolutions a minute for three minutes, or until the fat has separated. The percentage is then read off upon the stem, each small division representing 0.2 per cent. of fat.

Proteids.—T. R. Boggs' Modification of the Esbach Method.—This is applied as for urinary albumin (p. 74), substituting Boggs' reagent for Esbach's. The reagent is prepared as follows:

When the phosphotungstic acid is completely dissolved, mix the two solutions. This reagent is quite stable if kept in a dark glass bottle.

Before examination, the milk should be diluted according to the probable amount of proteid, and allowance made in the subsequent reading. For human milk the optimum dilution is 1:10; for cow's milk, 1:20. Dilution must be accurate.

Lactose.—The proteid should first be removed by acidifying with acetic acid, boiling, and filtering. Purdy's method may then be used as for glucose in the urine (p. 79); but it must be borne in mind that lactose reduces copper more slowly than glucose, and longer heating is, therefore, required; and that 35 c.c. of Purdy's solution is equivalent to 0.0268 gm. lactose (as compared with 0.02 gm. glucose).

It is frequently desirable to detect formalin, which is the most common preservative added to cow's milk. Add a few drops of dilute phenol solution to a few cubic centimeters of the milk, and run the mixture gently upon the surface of some strong sulphuric acid in a test-tube. If formaldehyd be present, a bright-red ring will appearat the line of contact of the fluids. This is not a specific test for formaldehyd, but nothing else likely to be added to the milk will give it.

SYPHILITIC MATERIAL

In 1905 Schaudinn and Hoffmann described the occurrence of a very slender, spiral micro-organism in the lesions of syphilis. This they namedSpirochæte pallida, because of its low refractive power and the difficulty with which it takes up staining reagents. Its etiologicrelation to syphilis is now almost universally admitted. It is not found in tertiary lesions.

Spirochæte pallidais an extremely slender, spiral, motile thread, with pointed ends. It varies considerably in length, the average being about 7 µ, or the diameter of a red blood-corpuscle; and it exhibits three to twelve, sometimes more, spiral curves, which are sharp and regular and resemble the curves of a corkscrew (Fig. 129). It is so delicate that it is difficult to see even in well-stained preparations; a high magnification and careful focusing are, therefore, required. Upon ulcerated surfaces it is often mingled with other spiral micro-organisms, which adds to the difficulty of its detection. The most notable of these isSpirochæte refringens, which is distinguished by being coarser and having fewer curves of wider and less sharp contour (Fig. 130).

Spirochæte pallidais most easily demonstrated in chancres and mucous patches, although the skin lesions—papules, pustules, roseolous areas—often contain large numbers. Tissue-juice from the deeper portions of the lesions is the most favorable material for examination, because the organisms are commonly more abundant than upon ulcerated surfaces and are rarely accompanied by other micro-organisms. After cleansing the surface a superficial incision with a scalpel or sharp needle is made at the edge of a lesion, or the surface is gently scraped away with a curet, and a drop of blood and serum is expressed. The less blood the better, because thecorpuscles may hide the spirochæte. Very thin cover-glass smears are then made.

Goldhorn's stain gives very good results. It can be purchased ready prepared from E. Leitz, New York. The unfixed smear is covered with the stain for four or five seconds. The excess of stain is poured off, and the preparation introducedslowly, with the film side down, into distilled water. It is held in this position for four or five seconds, and is then washed by shaking about in the water. By this method theSpirochæte pallidaappears of a violet color, which can be changed to bluish black by flooding with Gram's iodin solution for fifteen or twenty seconds. The preparation is then washed, dried, and mounted.

SEMEN

Absence of spermatozoa is a more common cause of sterility than is generally recognized. In some cases they are present, but lose their motility immediately after ejaculation.

Semen must be kept warm until examined. When it must be transported any considerable distance, the method suggested by Boston is convenient. The fresh semen is placed in a small bottle to the neck of which a string is attached. This is then suspended from a button on the trousers so that the bottle rests against the skin of the inguinal region. It may be carried in this way for hours. When ready to examine, place a small quantity upon a warmed slide and apply a cover. The spermatozoa are readily seen with a one-sixth objective (Fig. 53). Normally, they are abundant and in active motion.

Detection of semen in stains upon clothing, etc., isoften important. The finding of spermatozoa, after soaking the stain for an hour in normal salt solution or dilute alcohol and teasing in the same fluid, is absolute proof that the stain in question is semen, although it is not possible to distinguish human semen from that of the lower animals in this way.

Florence's Reaction.—The suspected material is softened with water, placed upon a slide with a few drops of the reagent, and examined at once with a medium power of the microscope. If the material be semen, there will be found dark-brown crystals (Fig. 131) in the form of rhombic platelets resembling hemin crystals, or of needles often grouped in clusters. These crystals canalso be obtained from crushed insects, watery extracts of various internal organs, and certain other substances, so that they are not absolute proof of the presence of semen. Negative results, upon the other hand, are conclusive, even when the semen is many years old.

The reagent consists of iodin, 2.54 gm.; potassium iodid, 1.65 gm.; and distilled water, 30 c.c.

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