Following liquefaction (20-30 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. 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 total sperm count. For high concentration specimens a greater dilution will be necessary. For low concentrations an undiluted or minimally diluted specimen may be required. The appropriate dilution is determined by estimating the concentration needed to do a count of at least 100 cells per side of the loaded hemacytometer.

The diluent that may be used for sperm counts on a hemacytometer can be as follows: 5 gm of sodium bicarbonate in 100 ml of distilled water, plus 1ml of formalin (neutral).