CHAPTER 2 Standard procedures
49
Note: This value can only be a rough estimate because so few spermatozoa are counted and volumes may be inaccurate.
2.11.1.1 Procedure 1. Dilute two aliquots of the semen sample 1 + 1 (1:2) with fixative, as above. 2. Fill each chamber of the haemocytometer with the replicate dilutions, one replicate per chamber. 3. Store the haemocytometer horizontally for at least 4 minutes at room temperature in a humid chamber (e.g. on water-saturated filter paper in a covered Petri dish) to prevent drying out. The immobilized cells will sediment onto the grid during this time. 4. Examine the haemocytometer with phase-contrast optics at ×200 or ×400 magnification. 5. Count at least 200 spermatozoa in each replicate, in order to achieve an acceptably low sampling error (see Box 2.7 and Table 2.2). 6. Examine one chamber grid by grid, and continue counting until at least 200 spermatozoa have been observed and a complete grid has been examined. Counting must be done by complete grids; do not stop in the middle of a grid. 7. Make a note of the number of grids assessed to reach at least 200 spermatozoa. The same number of grids will be counted from the other chamber of the haemocytometer. 8. Tally the number of spermatozoa and grids with the aid of a laboratory counter. 9. Switch to the second chamber of the haemocytometer and perform the replicate count on the same number of grids (the same volume) as the first replicate, even if this yields fewer than 200 spermatozoa. 10. Calculate the sum and difference of the two numbers. 11. Determine the acceptability of the difference from Table 2.5 (which extends Table 2.4 to lower sperm numbers) or Fig. A7.1, Appendix 7. (Each shows the maximum difference between two counts that is expected to occur in 95% of samples because of sampling error alone). 12. If the difference is acceptable, calculate the concentration (see Section 2.11.1.2). If the difference is too high, make two new preparations as described above and repeat replicate counts (see Box 2.10). 13. Report the average sperm concentration to two significant figures. 14. Calculate the total number of spermatozoa per ejaculate (see Section 2.11.1.5).