In these the entire ruled surface consists of nine large squares, each 1 mm. square. These are subdivided, and in the central large square are to be found the small squares used for averaging the red cells. These small squares are 1/20 mm. square and are arranged in nine groups of 16 small squares by bordering triple-ruled lines. As the unit in blood counting is the cubic millimeter, if one counted all the white cells lying within one of the large squares (1 mm. square), he would have only counted the cells in a layer one-tenth of the required depth, so that it would be necessary to multiply the number obtained by 10. This product, multiplied by the dilution of the blood, would give the number of white cells in a cubic millimeter of undiluted blood. The Neubauer ruling is the most satisfactory.
Fig. 148.—Thoma-Levy counting chamber, Bürker double type with two Neubauer rulings.
Bürker Haemacytometer.—Some workers prefer the Bürker haemacytometer. In this there are two ruled wedge-shaped pieces of glass, separated at their bases, which take the place of the ruled disc of the Thoma apparatus. Two oblong pieces of glass are on either side of the ruled wedges and are 0.1 mm. higher, thus taking the place of the shelf. Clamps fix a cover-glass on these shelves giving a space 1/10 mm. over the ruled surfaces. The blood is run in by capillarity from the mixing pipette. I gave up this type of counter because the clamps made manipulation awkward.
Thoma-Levy Chamber.—In the Thoma-Levy modification of the Bürker apparatus the central portion of the slide is cut away and in this depression is cemented a rectangular strip of glass, divided by a central channel. Each half of this strip of glass has a Neubauer ruling on it so that one can make a white count on one side and a red one on the other, simply touching the tip of the red pipette to the space separating the under surface of the cover-glass from the ruled rectangular slips on one side and then with the white pipette repeating the same on the other side. An advantage of the Thoma-Levy is that the original thickness of the slide makes the shelf on which the cover-glass rests instead of the support being strips on either side of the ruled surfaces and cemented to the slide. The Neubauer ruling is undoubtedly the most satisfactory of the haemacytometer rulings, its rulings being simpler than those of the Türck system. The unit square in all these haemacytometers is the small square for counting red blood cells, 1/20 mm. square.
Fig. 149.—The Türck ruling. Thoma-Zeiss Haemacytometer.
To Make a Red Count.—Having a fairly large drop of blood, apply the tip of the 101 pipette to it and, holding the pipette horizontal, carefully and slowly draw up with suction on the rubber tube a column of blood to exactly 0.5. The variation of 1/25 of an inch from the mark would make a difference of almost 3%. If the column goes above 0.5, it can be gently tapped down on a piece of filter-paper until the 0.5 line is cut. Now insert the tip of the pipette into some diluting fluid, and revolving the pipette on its long axis while filling it by suction, you continue until the mark 101 is reached.
A variation of 1/25 of an inch at this mark would only give an error of about 1/30 of 1%. This gives a 1-200 dilution. After mixing thoroughly by shaking for one or two minutes, the fluid in the pipette below the bulb is expelled (this of course is only diluting fluid). A drop of the diluted blood of a size just sufficient to cover the disc when the cover-glass is adjusted, is then deposited on the disc and the cover-glass applied by a sort of sliding movement, best obtained by using forceps in one hand assisted by the thumb and index-finger of the other.
In red counts we use exclusively the small 1/20 mm. squares which are in groups of 16 bounded by triple-ruled lines.