Semen must be diluted prior to counting sperm if a hemocytometer is used. In additon to preventing overlapping of sperm cells, the diluting fluid immobilizes the sperm in the chamber to further facilitate counting.
Following liquefaction (at about 30 minutes), mix the sample manually by swirling the container several times. Thorough mixing is essential for accurate counting. Calibrated automatic pipettes are used to prepare a dilution. Some laboratories may require two separate dilutions, as recommended by WHO 5th edition. Because of the viscosity of semen, the semen should be added to the diluent using a positive pressure pipettor.
The dilution often used for routine sperm counts is 1:20 but the actual dilution factor will vary depending on the sperm concentration that was noted during the initial microscopic evaluation. If a high concentration of sperm are noted, a greater dilution will be necessary. For low concentrations, a minimally diluted (eg, 1:2) specimen may be required. The appropriate dilution is determined by estimating the concentration needed to achieve a count of at least 200 spermatozoa in the area that is counted on each side of the hemocytometer.