The hemacytometer can be used to count the number of spermatozoa per mL semen.
Diluent: (100 mL distilled water) + (1 mL neutral buffered formalin) + (5 grams sodium bicarbonate)
Procedure:
1) Make a 1:20 dilution with 0.5 mL of semen diluted to 10 mL. Mix.
2) Charge both sides of the hemacytometer and wait 2 minutes for the immobilized sperm (fixed by formalin) to settle.
3) Count the central chamber (square #5) on both sides of a hemocytometer and add the numbers together. Each square is 1 mm square with a depth of 0.1 mm, for a volume of 0.1 cubic mm. According to convention, elements are counted if they lie on the topmost and leftmost lines, but not the bottommost or rightmost lines.
4) Repeat the count on a fresh sample.
5) Average the two counts.
6) Multiply the average by 100,000, to get total spermatozoa per mL.
where:
• 100,000 (from step 6) = ((dilution factor 20) / ((number of squares 2) * (volume 0.1 cubic mm))) * (1000 cubic mm/mL)]
Limitations:
• The test shows considerable intra- and inter-technologist variation.
