The depth of fluid over the ruled surface is 1/10 mm., hence each of these small squares is 1/10 × 1/20 × 1/20 = 1/4000 of a c.mm., so that it takes 4000 such spaces to equal the unit for blood counting (1 c.mm.). My practice in making red counts is to count the red cells in five of the groups of 16 small squares. This in normal blood is about 100 for the 16 squares. After counting 5 groups of 16 we have counted the red cells of 80 small squares which is 1/50 of 4000 (the number in the 1 c.mm. unit). For this reason 50 × 200 (the blood dilution) = 10,000, so that it is only necessary to multiply the number of red cells found in 5 groups of 16 small squares by 10,000 in order to obtain the number of red cells per c.mm. For more accurate determination the process can be repeated with a second or third drop of the diluted blood, which would give an average from 160 or from 240 small squares.
To Count White Cells.—Draw up the blood in the white pipette to the 0.5 line. Then, still holding the pipette as near the horizontal as possible, because the column of blood tends to fall down in the larger bore, draw up by suction a diluting fluid which will disintegrate the red cells without injuring the whites. The best fluid is 0.5% of glacial acetic acid in water. This makes the white cells stand out as highly refractile bodies. Some prefer to tinge the fluid with neutral red or gentian violet. The 0.5 mark is preferred because it takes a very large drop of blood to fill the tube up to the 1 mark and if there is much of a leucocytosis a 1 to 10 dilution is not sufficient.
The blood having been drawn up to 0.5, we have a dilution of 1 to 20.
Making a preparation, exactly as was done in the case of the red count, we count all of the white cells in one of the large squares (1 sq. mm.). The cross ruling greatly facilitates this. Note the number. Then count a second and a third square. Strike an average of the large squares counted and multiply this by 10, as the depth of the fluid gives a content equal to only 1/10 of a c.mm. Then multiply by the dilution.
Example.—First large square 50; second large square 70; third large square 60. Average 60. Then 60 × 10 × 20 = 12,000, the number of leucocytes in 1 c.mm. of blood. In order to save time the count is preferably made with a low power (⅔-inch objective) as the leucocytes stand out like pearls. It is more accurate, however, to use a higher power, so that pieces of foreign material may be recognized and not enumerated as white cells.
If one will accustom himself to comparing the distribution of the leucocytes in a well-made stained dried-blood film, prepared according to Ehrlich’s cover-glass method, with that in a haemacytometer preparation, he can readily acquire an experience which will enable him to determine with considerable accuracy the degree of leucocytosis by the examination of a stained, cover-glass preparation alone.
After making a blood count, the haemacytometer slide should be cleaned with soap and water and then rubbed dry, preferably with an old piece of linen. As the accuracy of the counting chamber depends upon the integrity of the cement, any reagent such as alcohol, xylol, etc., and in particular, heat, will ruin the instrument. The pipettes should be cleaned by inserting the ends into the tube from a vacuum pump, as a Chapman pump. First draw water or 1% sod. carbonate solution through the pipette, then alcohol, then ether, and finally allow air to pass through to dry the interior. If the interior is stained, used 1% HCL in alcohol. If a vacuum pump is not at hand, a bicycle pump or suction by mouth will answer.
Preparations for the Study of Fresh Blood
Many authorities prefer a fresh blood specimen to a stained dried smear in the study of parasites of the blood. In malaria in particular there is so much information as to species to be obtained from a fresh specimen that the employment of this method should never be neglected. While waiting for the film to stain one has five or six minutes which could not be better spent than in examining the fresh specimen which only requires a moment to make.
Manson’s Method.—Have a perfectly clean cover-glass and slide. Touch the apex of the exuding drop of blood with the cover-glass and drop it on the center of the slide. The blood flows out in a film which exhibits an “empty zone” in the center. Surrounding this we have the “zone of scattered corpuscles,” next the “single layer zone” and the “zone of rouleaux” at the periphery. It is well to ring the preparation with vaseline. When desiring to demonstrate the flagellated bodies in malaria, it is well to breathe on the cover-glass just prior to touching the drop of blood.