Fill both sides of the hemocytometer. Focus on the large center square, using either 200X or 400X magnification. The counting area consists of 25 small squares within the large center square. The squares usually counted are the four corner squares and the center square, all of which are marked R in the images on the right. A minimum of 200 sperm should be counted on each side of the hemocytometer. If this number is not achieved by counting the five small squares, the entire center square (all 25 small squares) should be counted on both sides of the hemocytometer. If a minimum of 200 sperm are not counted within the center square, additional ruled areas should be counted. It may also be necessary to repeat the procedure using a smaller dilution.
Count both sides of the hemocytometer and take the average of the two counts to calculate the actual count per mL, if the replicate counts are acceptably close. If the counts are not within the acceptable limits according to your laboratory's requirements, begin the procedure again; thoroughly mixing the original sample, loading, and counting both sides of a clean hemocytometer.
- Phase contrast microscopy can enhance the appearance of sperm and allow differentiation from round cells and other elements.
- Count only whole spermatozoa that have heads and tails.
- Count the sperm if the head lies within the square counted.
- If the head lies on a boundary line between two squares, the sperm should be counted if it is on the upper boundary line or the left-hand side boundary line (see red lines on lower image on the right), but not if it is the lower boundary line or the right-hand side boundary line. (Some laboratories may instead determine the boundaries to be counted as the lower boundary line and the left-hand side boundary line; the important point is to be consistent).