6.Bacteria.—Normal urine is free from bacteria in the bladder, but becomes contaminated in passing through the urethra. Various non-pathogenic bacteria, notablyMicrococcus ureæ(Fig. 54), are always present in decomposing urine. In suppurations of the urinary tract pus-producing organisms may be found. In many infectious diseases the specific germs may be eliminated inthe urine without producing any local lesion. Typhoid bacilli have been known to persist for months and even years after the attack.
Bacteria produce a cloudiness which will not clear upon filtration. They are easily seen with the one-sixth objective in the routine microscopic examination. Ordinarily, no attempt is made to identify any but the tubercle bacillus and the gonococcus.
Tubercle bacilliare nearly always present in the urine when tuberculosis exists in any part of the urinary tract, but are often difficult to find, especially when the urine contains little or no pus.
Detection of Tubercle Bacilli in Urine.—The urine should be obtained by catheter after careful cleansing of the parts.
(1) Centrifugalize thoroughly, after dissolving any sediment of urates or phosphates by gentle heat or acetic acid. Pour off the supernatant fluid, add water, and centrifugalize again. Addition of one or two volumes of alcohol will favor centrifugalization by lowering the specific gravity.
(2) Make thin smears of the sediment, adding a little egg-albumen if necessary to make the smear adhere to the glass; dry, and fix in the usual way.
(3) Stain with carbol-fuchsin, steaming, for at least three minutes.
(4) Wash in water, and then in 20 per cent. nitric acid until only a faint pink color remains.
(5) Wash in water.
(6) Soak in alcohol fifteen minutes or longer. This decolorizes the smegma bacillus (p. 35), which is often present in the urine, and might easily be mistaken for the tubercle bacillus. It is unlikely, however, to be present in catheterized specimens. It is always safest to soak the smear in alcohol for severalhours or over night, since some strains of the smegma bacillus are very resistant.
(7) Wash in water.
(8) Apply Löffler's methylene-blue solution one-half minute.
(9) Rinse in water, dry between filter-papers, and examine with the one-twelfth objective.
When the bacilli are scarce, the following method may be tried. It is applicable also to other fluids. If the fluid is not albuminous, add a little egg-albumen. Coagulate the albumen by gentle heat and centrifugalize. The bacilli will be carried down with the albumen. Separate the albumen, mix with artificial gastric juice (for preparation of which see test for pepsin,p. 222), and set in an incubator or warm place until digested. Finally, centrifugalize and stain as described above. The bacilli do not stain so well as in the ordinary methods.
A careful search of many smears may be necessary to find the bacilli. They usually lie in clusters (see Plate V). Failure to find them in suspicious cases should be followed by inoculation of guinea-pigs; this is the court of last appeal, and must also be sometimes resorted to in order to exclude the smegma bacillus.
In gonorrhoeagonococciare sometimes found in the sediment, but more commonly in the "gonorrheal threads," or "floaters." In themselves, these threads are by no means diagnostic of gonorrhea. Detection of the gonococcus is described later (p. 264).
7.Animal parasitesare rare in the urine. Hooklets and scolices ofTænia echinococcus(Fig. 55) and embryos ofFilaria sanguinis hominishave been met. In Africa the ova, and even adults, ofDistoma hæmatobiumare common, accompanying "Egyptian hematuria."
Other parasites, most of which are described inChapter VI, may be present from contaminations. A wormwhich is especially interesting isAnguillula aceti, the "vinegar eel." This is generally present in the sediment of table vinegar, and may reach the urine through use of vinegar in vaginal douches, or through contamination of the bottle in which the urine is contained. It has been mistaken forStrongyloides intestinalisand forFilaria sanguinis hominis. It closely resembles the former inboth adult and embryo stages. The young embryos have about the same length as filaria embryos, but are nearly twice as broad and the intestinal canal is easily seen (compare Figs. 56 and107).
C. EXTRANEOUSSTRUCTURES
C. EXTRANEOUSSTRUCTURES
The laboratory worker must familiarize himself with the microscopic appearance of the more common of the numerous structures which may be present from accidental contamination (Fig. 57).
Yeast-cellsare smooth, colorless, highly refractive, spheric or ovoid cells. They sometimes reach the size ofa leukocyte, but are generally smaller (seeFig. 88,l). They might be mistaken by the inexperienced for red blood-corpuscles, fat-droplets, or the spheric crystals of calcium oxalate, but are distinguished by the facts that they are not of uniform size; that they tend to adhere in short chains; that small buds may often be seen adhering to the larger cells; and that they do not give the hemoglobin test, are not stained by osmic acid or Sudan but are colored brown by Lugol's solution, and are insoluble in acids and alkalis. Yeast-cells multiply rapidly in diabetic urine, and may reach the bladder and multiply there.
Mold fungi(Fig. 58) are characterized by refractive, jointed, or branched rods (hyphæ), often arranged in a network, and by highly refractive, spheric or ovoid spores. They are common in urine which has stood exposed to the air.
Fibersof wool, cotton, linen, or silk, derived from towels, the clothing of the patient, or the dust in the air are present in almost every urine.Fat-dropletsare most frequently derived from unclean bottles or oiled catheters.Starch-granulesmay reach the urine from towels, theclothing, or dusting-powders. They are recognized by their concentric striations and their blue color with iodin solution.Lycopodium granules(Fig. 59) may also reach the urine from dusting-powders. They might be mistaken for the ova of parasites.Bubbles of airare often confusing to beginners, but are easily recognized after once being seen.Scratchesandflawsin the glass of slide or cover are likewise a common source of confusion to beginners.
IV. THE URINE IN DISEASE
IV. THE URINE IN DISEASE
In this section the characteristics of the urine in those diseases which produce distinctive urinary changes will be briefly reviewed.
1.Renal Hyperemia.—Active hyperemiais usually an early stage of acute nephritis, but may occur independently as a result of temporary irritation. The urine is generally decreased in quantity, highly colored, and strongly acid. Albumin is always present—usually in traces only, but sometimes in considerable amount for a day or two. The sediment contains a few hyaline and finely granular casts and an occasional red blood-cell. In very severe hyperemia the urine approaches that of acute nephritis.
Passive hyperemiaoccurs most commonly in diseases of the heart and liver and in pregnancy. The quantity of urine is somewhat low and the color high, except in pregnancy. Albumin is present in small amount only. The sediment contains a very few hyaline or finely granularcasts. In pregnancy the amount of albumin should be carefully watched, as any considerable quantity, and especially a rapid increase, strongly suggests approaching eclampsia.
2.Nephritis.—The various degenerative and inflammatory conditions grouped under the name of nephritis have certain features in common. The urine in all cases contains albumin and tube-casts, and in all well-marked cases shows a decrease of normal solids, especially of urea and the chlorids. The characteristics of the different forms are well shown in the table on opposite page, modified from Hill.
3.Renal Tuberculosis.—The urine is pale, usually cloudy. The quantity may not be affected, but is apt to be increased. In early cases the reaction is faintly acid and there are traces of albumin and a few renal cells. In advanced cases the urine is alkaline, has an offensive odor, and is irritating to the bladder. Albumin in varying amounts is always present. Pus is nearly always present, though frequently not abundant. It is generally intimately mixed with the urine, and does not settle so quickly as the pus of cystitis. Casts, though present, are rarely abundant, and are obscured by the pus. Small amounts of blood are common. Tubercle bacilli are nearly always present, although animal inoculation may be necessary to detect them.
4.Renal Calculus.—The urine is usually somewhat concentrated, with high color and strongly acid reaction. Small amounts of albumin and a few casts may be present as a result of kidney irritation. Blood is frequently present, especially in the daytime and after severe exercise. Crystals of the substance composing the calculus—uricacid, calcium oxalate, cystin—may often be found. The presence of a calculus generally produces pyelitis, and variable amounts of pus then appear, the urine remaining acid in reaction.
5.Pyelitis.—In pyelitis the urine is slightly acid, and contains a small or moderate amount of pus, together with many spindle and caudate epithelial cells. Pus-casts may appear if the process extends up into the kidney tubules (see Fig. 62). Albumin is always present, and its amount, in proportion to the amount of pus, is decidedly greater than is found in cystitis.
6.Cystitis.—-Inacuteandsubacutecases the urine is acid and contains a variable amount of pus, with many epithelial cells from the bladder—chiefly large round, pyriform, and rounded squamous cells. Red blood-corpuscles are often numerous.
Inchroniccases the urine is generally alkaline. It ispale and cloudy from the presence of pus, which is abundant and settles readily into a viscid sediment. The sediment usually contains abundant amorphous phosphates and crystals of triple phosphate and ammonium urate. Vesical epithelium is common. Numerous bacteria are always present (see Fig. 63).
7.Vesical Calculus, Tumors, and Tuberculosis.—These conditions produce a chronic cystitis, with its characteristic urine. Blood, however, is more frequently present and more abundant than in ordinary cystitis. With neoplasms, especially, considerable hemorrhages are apt to occur. Particles of the tumor are sometimes passed with the urine. No diagnosis can be made from the presence of isolated tumor cells. In tuberculosis tubercle bacilli can generally be detected.
8.Diabetes Insipidus.—Characteristic of this disease is the continued excretion of very large quantities of pale, watery urine, containing neither albumin nor sugar. Thespecific gravity varies between 1.001 and 1.005. The daily output of solids, especially urea, is increased.
9.Diabetes Mellitus.—The quantity of urine is very large. The color is generally pale, while the specific gravity is nearly always high—1.030 to 1.050, very rarely below 1.020. The presence of glucose is the essential feature of the disease. The amount of glucose is often very great, sometimes exceeding 8 per cent., while the total elimination may exceed 500 gm. in twenty-four hours. It may be absent temporarily. Acetone is generally present in advanced cases. Diacetic acid may be present, and usually warrants an unfavorable prognosis.
Preliminary Considerations.—The blood consists of a fluid of complicated and variable composition, the plasma, in which are suspended great numbers of microscopic structures: viz., red corpuscles, white corpuscles, blood-platelets, and blood-dust.
Red corpuscles, orerythrocytes, are biconcave discs, red when viewed by reflected light or in thick layer, and straw-colored when viewed by transmitted light or in thin layer. They give the blood its red color. They are cells which have been highly differentiated for the purpose of carrying oxygen from the lungs to the tissues. This is accomplished by means of an iron-bearing proteid, hemoglobin, which they contain. In the lungs hemoglobin forms a loose combination with oxygen, which it readily gives up when it reaches the tissues. Normal erythrocytes do not contain nuclei. They are formed from preëxisting nucleated cells in the bone-marrow.
White corpuscles, orleukocytes, are less highly differentiated cells. There are several varieties. They all contain nuclei, and most of them contain granules which vary in size and staining properties. They are formed in the bone-marrow and lymphoid tissues.
Blood-platelets, orblood-plaques, are colorless or slightly bluish, spheric or ovoid bodies, about one-third or one-half the diameter of an erythrocyte. Their structure, nature, and origin have not been definitely determined.
Theblood-dust of Müllerconsists of fine granules which have vibratory motion. Little is known of them. It has been suggested that they are granules from disintegrated leukocytes.
Coagulationconsists essentially in the transformation of fibrinogen, one of the proteins of the blood, into fibrin by means of a ferment derived from disintegration of the leukocytes. The resulting coagulum is made up of a meshwork of fibrin fibrils with entangled corpuscles and plaques. The clear, straw-colored fluid which is left after separation of the coagulum is calledblood-serum. Normally, coagulation takes place in two to eight minutes after the blood leaves the vessels. It is frequently desirable to determine the coagulation time. The simplest method is to place a drop of blood upon a perfectly clean slide, and to draw a needle through it at half-minute intervals. When the clot is dragged along by the needle, coagulation has taken place. This method is probably sufficient for ordinary clinical work. For very accurate results the method of Russell and Brodie, for which the reader is referred to the larger text-books, is recommended. Coagulation is notably delayed in hemophilia and icterus and after administration of citric acid. It is hastened by administration of calcium chlorid.
For most clinical examinations only one drop of blood is required. This may be obtained from the lobe of the ear, the palmar surface of the tip of the finger, or, in the case of infants, the plantar surface of the great toe. With nervous children the lobe of the ear is preferable, as it prevents their seeing what is being done. An edematous or congested part should be avoided. The site should be well rubbed with alcohol to remove dirt and epithelialdébris and to increase the amount of blood in the part. After allowing sufficient time for the circulation to equalize, the skin is punctured with a blood lancet (of which there are several patterns upon the market) or some substitute, as a Hagedorn needle, aspirating needle, trocar, or a pen with one of its nibs broken off. Nothing is more unsatisfactory than an ordinary sewing-needle. The lancet should be cleaned with alcohol before and after using, but need not be sterilized. If the puncture be made with afirm, quick stroke, it is practically painless. The first drop of blood which appears should be wiped away, and the second used for examination. The blood should not be pressed out, since this dilutes it with serum from the tissues; but moderate pressure some distance above the puncture is allowable.
When a larger amount of blood is required, it may be obtained with a sterile hypodermic syringe from one of the veins at the elbow.
Clinical study of the blood may be discussed under the following heads: I. Hemoglobin. II. Enumeration of erythrocytes. III. Color index. IV. Enumeration of leukocytes. V. Enumeration of plaques. VI. Study of stained blood. VII. Blood parasites. VIII. Serum reactions. IX. Tests for recognition of blood. X. Special blood pathology.
I. HEMOGLOBIN
I. HEMOGLOBIN
Hemoglobin is an iron-bearing proteid. It is found only within the red corpuscles, and constitutes about 90 per cent. of their weight. The actual amount of hemoglobin is never estimated clinically: it is the relation which the amount present bears to the normal which is determined. Thus the expression, "50 per cent. hemoglobin," when used clinically, means that the blood contains 50 per cent. of the normal. Theoretically, the normal would be 100 per cent., but with the methods of estimation in general use the blood of healthy persons ranges from 85 to 105 per cent.; these figures may, therefore, be taken as normal.
Increase of hemoglobin, orhyperchromemia, is uncommon, and is probably more apparent than real. It accompanies an increase in number of erythrocytes, and may be noted in change of residence from a lower to a higher altitude; in poorly compensated heart disease with cyanosis; in concentration of the blood from any cause, as the severe diarrhea of cholera; and in "idiopathic polycythemia."
Decrease of hemoglobin, oroligochromemia, is very common and important. It is the most striking feature of the secondary anemias (p. 204). Here the hemoglobin loss may be slight or very great. In mild cases a slight decrease of hemoglobin is the only blood change noted. In very severe cases, especially in repeated hemorrhages, malignant disease, and infection by the worms uncinaria and bothriocephalus latus, hemoglobin may fall to 15 per cent. Hemoglobin is always diminished, and usually very greatly, in chlorosis, pernicious anemia, and leukemia.
Estimation of Hemoglobin.—There are many methods, but none is entirely satisfactory. Those which are most widely used are here described.
(1)Von Fleischl Method.—The apparatus consists of a stand somewhat like the base and stage of a microscope (Fig. 65). Under the stage is a movable bar of colored glass, shading from pale pink at one end to deep red at the other. The frame in which this bar is held is marked with a scale of hemoglobin percentages corresponding to the different shades of red. By means of a rack and pinion, the color-bar can be moved from end to end beneath a round opening in the center of the stage. A small metal cylinder, which has a glass bottom and which is divided vertically into two equal compartments, can be placed over the opening in the stage so that one of itscompartments lies directly over the color-bar. Accompanying the instrument are a number of short capillary tubes in metal handles.
Having punctured the finger-tip or lobe of the ear as already described, wipe off the first drop of blood, and from the second fill one of the capillary tubes. Hold the tube horizontally, and touch its tip to the drop of blood, which will readily flow into it if it be clean and dry. Avoid getting any blood upon its outer surface. With a medicine-dropper, rinse the blood from the tube into one of the compartments of the cylinder, using distilled water, and mix well. Fill both compartments level full with distilled water, and place the cylinder over the opening in the stage so that the compartment which contains only water lies directly over the bar of colored glass.
In a dark room, with the light from a candle reflected up through the cylinder, move the color-bar along with a jerking motion until both compartments have the same depth of color. The number upon the scale corresponding to the portion of the color-bar which is now under the cylinder gives the percentage of hemoglobin. While comparing the two colors, place the instrument so that they will fall upon the right and left halves of the retina, rather than upon the upper and lower halves; and protect the eye from the light with a cylinder of paper or pasteboard. After use, clean the metal cylinder with water, and wash the capillary tube with water, alcohol, and ether, successively. Results with this instrument are accurate to within about 5 per cent.
A recent modification of the von Fleischl apparatus by Miescher gives an error which need not exceed 1 per cent. It is, however, better adapted to laboratory use than to the needs of the practitioner.
(2)The Sahli hemoglobinometer(Fig. 66) is an improved form of the well known Gowers instrument. It consists of a hermetically sealed comparison tube containing a 1 per cent. solution of acid hematin, a graduated test-tube of the samediameter, and a pipet of 20 c.mm. capacity. The two tubes are held in a black frame with a white ground-glass back.
Place a few drops of decinormal hydrochloric acid solution in the graduated tube. Obtain a drop of blood and draw it into the pipet to the 20 c.mm. mark. Wipe off the tip of the pipet, blow its contents into the hydrochloric acid solution in the tube, and rinse well. In a few minutes the hemoglobin is changed to acid hematin. Place the two tubes in the compartments of the frame, and dilute the fluid with water drop by drop, mixing after each addition, until it has exactly the same color as the comparison tube. The graduation corresponding to the surface of the fluid then indicates thepercentage of hemoglobin. Decinormal hydrochloric acid solution may be prepared with sufficient accuracy for this purpose by adding 15 c.c. of the concentrated acid to 985 c.c. distilled water. A little chloroform should be added as a preservative.
This method is very satisfactory in practice, and is accurate to within 5 per cent. The comparison tube is said to keep its color indefinitely, but, unfortunately, not all the instruments upon the market are well standardized.
(3)Dare's hemoglobinometer(Fig. 67) differs from the others in using undiluted blood. The blood is allowed to flow by capillarity into the slit between two small plates of glass. It is then placed in the instrument and compared with different portions of a circular disc of colored glass. The reading must be made quickly, before clotting takes place. This instrument is easy to use, and is one of the most accurate.
(4)Hammerschlag Method.—This is an indirect method which depends upon the fact that the percentage of hemoglobin varies directly with the specific gravity of the blood. It yields fairly accurate results except in leukemia, where the large number of leukocytes disturbs the relation.
Mix chloroform and benzol in a urinometer tube so thatthe specific gravity of the mixture is near the probable specific gravity of the blood. Add a drop of blood by means of a pipet of small caliber. If the drop floats near the surface, add a little benzol; if it sinks to the bottom, add a little chloroform. When it remains stationary near the middle, the mixture has the same specific gravity as the blood. Take the specific gravity with a urinometer, and obtain the corresponding percentage of hemoglobin from the following table:
For accurate results with this method, care and patience are demanded. The following precautions must be observed:
(a) The two fluids must be well mixed after each addition of chloroform or benzol. Close the tube with the thumb and invert several times. Should this cause the drop of blood to break up into very small ones, adjust the specific gravity as accurately as possible with these, and test it with a fresh drop.
(b) The drop of blood must not be too large; it must not contain an air-bubble, it must not adhere to the side of the tube, and it must not remain long in the fluid.
(c) The urinometer must be standardized for the chloroform-benzol mixture. Most urinometers give a reading two or three degrees too high, owing to the low surface tension. Make a mixture such that a drop of distilled water will remain suspended in it (i.e., with a specific gravity of 1.000) and correct the urinometer by this.
(5)Tallquist Method.—The popular Tallquist hemoglobinometer consists simply of a book of small sheets of absorbent paper and a carefully printed scale of colors (Fig. 68).
Take up a large drop of blood with the absorbent paper,and when the humid gloss is leaving, compare the stain with the color scale. The color which it matches gives the percentage of hemoglobin. Except in practised hands, this method is accurate only to within 10 or 20 per cent.
Of the methods given, the physician should select the one which best meets his needs. With any method, practice is essential to accuracy. The von Fleischl has long been the standard instrument, but has lately fallen into some disfavor. For accurate work the best instruments are the von Fleischl-Miescher and the Dare. They are, however, expensive, and it is doubtful whether they are enough more accurate than the Sahli instrument to justify the difference in cost. The latter is probably the most satisfactory for the practitioner, provided awell-standardized color-tube is obtained. The specific gravity method is very useful when special instruments are not at hand. The Tallquist scale is so inexpensive and so convenient that it should be used by every physician at the bedside and in hurried office work; but it should not supersede the more accurate methods.
II. ENUMERATION OF ERYTHROCYTES
II. ENUMERATION OF ERYTHROCYTES
In health there are about 5,000,000 red corpuscles per cubic millimeter of blood. Normal variations are slight. The number is generally a little less—about 4,500,000—in women.
Increase of red corpuscles, orpolycythemia, is unimportant. There is a decided increase following change of residence from a lower to a higher altitude, averaging about 50,000 corpuscles for each 1000 feet, but frequently much greater. The increase, however, is not permanent. In a few months the erythrocytes return to nearly their original number. Three views are offered in explanation: (a) Concentration of the blood, owing to increased evaporation from the skin; (b) stagnation of corpuscles in the peripheral vessels, because of lowered blood-pressure; (c) new-formation of corpuscles, this giving a compensatory increase of aëration surface.
Pathologically, polycythemia is uncommon. It may occur in: (a) concentration of the blood from severe watery diarrhea; (b) chronic heart disease, especially the congenital variety, with poor compensation and cyanosis; and (c) idiopathic polycythemia, which is considered to be an independent disease, and is characterized by cyanosis, blood counts of 7,000,000 to 10,000,000, hemoglobin 120 to 150 per cent., and a normal number of leukocytes.
Decrease of red corpuscles, oroligocythemia. Red corpuscles and hemoglobin are commonly decreased together, although usually not to the same extent.
Oligocythemia occurs in all but the mildest symptomatic anemias. The blood count varies from near the normal in moderate cases down to 1,500,000 in very severe cases. There is always a decrease of red cells in chlorosis, but it is often slight, and is relatively less than the decrease of hemoglobin. Leukemia gives a decided oligocythemia, the average count being about 3,000,000. The greatest loss of red cells occurs in pernicious anemia, where counts below 1,000,000 are not uncommon.
The most widely used and most satisfactory instrument for counting the corpuscles is that of Thoma-Zeiss. The hematocrit is not to be recommended for accuracy, since in anemia, where blood counts are most important, the red cells vary greatly in size and probably also in elasticity.The hematocrit is, however, useful in determining the relative volume of corpuscles and plasma, and seems to be gaining in favor.
TheThoma-Zeiss instrumentconsists of two pipets for diluting the blood and a counting chamber (Fig. 69). The counting chamber is a glass slide with a square platform in the middle. In the center of the platform is a circular opening, in which is set a small circular disc in such a manner that it is surrounded by a "ditch," and that its surface is exactly one-tenth of a millimeter below the surface of the square platform. Upon this disc is ruled a square millimeter, subdivided into 400 small squares. Each fifth row of small squares has double ruling for convenience in counting (Fig. 70). A thickcover-glass, ground perfectly plane, accompanies the counting chamber. Ordinary cover-glasses are of uneven surface, and should not be used with this instrument.
It is evident that, when the cover-glass is in place upon the platform, there is a space exactly one-tenth of a millimeter thick between it and the disc; and that, therefore, the square millimeter ruled upon the disc forms the base of a space holding exactly one-tenth of a cubic millimeter.
Technic.—To count the red corpuscles, use the pipet with 101 engraved above the bulb. It must be clean and dry. Obtain a drop of blood as already described. Suck blood into the pipet to the mark 0.5 or 1. Should the blood go beyond the mark, draw it back by touching the tip of the pipet to a moistened handkerchief. Quickly wipe off the blood adhering to the tip, plunge it into the diluting fluid, and suck the fluid up to the mark 101, slightly rotating the pipet meanwhile. This dilutes the blood 1:200 or 1:100, according to the amount of blood taken. Except in cases of severeanemia, a dilution of 1:200 is preferable. Close the ends of the pipet with the fingers, and shake vigorously until the blood and diluting fluid are well mixed.
When it is not convenient to count the corpuscles at once, place a heavy rubber band around the pipet so as to close the ends, inserting a small piece of rubber-cloth or other tough, non-absorbent material if necessary to prevent the tip from punching through the rubber. It may be kept thus for twenty-four hours or longer.
When ready to make the count, mix again thoroughly by shaking; blow two or three drops of the fluid from the pipet, wipe off its tip, and then place a small drop (the proper size can be learned only by experience) upon the disc of the counting chamber. Adjust the cover immediately. Hold it by diagonal corners above the drop of fluid so that a third corner touches the slide and rests upon the edge of the platform. Place a finger upon this corner, and, by raising the finger, allow the cover to fall quickly into place. If the cover be properly adjusted, faint concentric lines of the prismatic colors—Newton's rings—can be seen between it and the platform when the slide is viewed obliquely. They indicate that the two surfaces are in close apposition. If they do not appear at once, slight pressure upon the cover may bring them out. Failure to obtain them is usually due to dirty slide or cover—both must be perfectly clean and free from dust. The drop placed upon the disc must be of such size that, when the cover is adjusted, it nearly or quite covers the disc, and that none of it runs over into the "ditch." There should be no bubbles upon the ruled area.
Allow the corpuscles to settle for a few minutes, and then examine with a low power to see that they are evenly distributed. If they are notevenly distributed over the whole disc, the counting chamber must be cleaned and a new drop placed in it.
Probably the most satisfactory objective for counting is the special one-sixth for blood work already mentioned. Tounderstand the principle of counting, it is necessary to remember that the large square (400 small squares) represents a capacity of one-tenth of a cubic millimeter. Find the number of corpuscles in the large square, multiply by 10 to find the number in 1 c.mm. of the diluted blood, and finally, by the dilution, to find the number in 1 c.mm. of undiluted blood. Instead of actually counting all the corpuscles, it is customary to count those in only a limited number of small squares, and from this to calculate the number in the large square.
In practice a convenient procedure is as follows:With a dilution of 1:200, count the cells in 80 small squares, and to the sum add 4 ciphers; with dilution of 1:100, count 40 small squares and add 4 ciphers.Thus, if with 1:200 dilution, 450 corpuscles were counted, the total count would be 4,500,000 per c.mm. This method is sufficiently accurate for all clinical purposes, provided the corpuscles are evenly distributed and two drops from the pipet be counted. It is convenient to count a block of 20 small squares in each corner of the large square. It distribution be even, the difference between the number of cells in any two such blocks should not exceed twenty. Corpuscles which touch the upper and left sides should be counted as if within the squares, those touching the lower and right sides, as outside, andvice versâ.
Diluting Fluids.—The most widely used are Hayem's and Toisson's. Both of these have high specific gravities, so that, when well mixed, the corpuscles do not separate quickly. Toisson's fluid is probably the better for beginners, because it is colored and can be easily seen as it is drawn into the pipet. It stains the nuclei of leukocytes blue, but this is no real advantage. It must be filtered frequently.
Sources of Error.—The most common sources of error in making a blood count are:
(a) Inaccurate dilution, either from faulty technic or inaccurately graduated pipets. The instruments made by Zeiss can be relied upon.
(b) Too slow manipulation, allowing a little of the blood to coagulate and remain in the capillary portion of the pipet.
(c) Inaccuracy in depth of counting chamber, which sometimes results from softening of the cement by alcohol or heat. The slide should not be cleaned with alcohol nor left to lie in the warm sunshine.
(d) Uneven distribution of the corpuscles. This results when the blood is not thoroughly mixed with the diluting fluid, or when the cover-glass is not applied soon enough after the drop is placed upon the disc.
Cleaning the Instrument.—The instrument should be cleaned immediately after using, and the counting chamber and cover must be cleaned again just before use.
Draw through the pipet, successively, water, alcohol, ether, and air. This can be done with the mouth, but it is much better to use a rubber bulb. When the mouth is used, the moisture of the breath will condense upon the interior of the pipet unless the fluids be shaken and not blown out. If blood has coagulated in the pipet—which happens when the work is done too slowly—dislodge the clot with a horse-hair, and clean with strong sulphuric acid, or let the pipet stand over night in a test-tube of the acid. Even if the pipet does not become clogged, it should be occasionally cleaned in this way.