A Manual of Clinical Diagnosis

by James Campbell Todd

The Thoma-Zeiss instrument consists 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 thick cover-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.

FIG. 71.—Method of drawing blood into the pipet (Boston).

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 severe anemia, 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 not evenly 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. To understand 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, and vice 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.