Performing a Manual Count for Sperm Concentration

1. Performing a Manual Countfor Sperm Concentration Dr. Bruce Eilts Louisiana State University

2. Diluent – Formol Buffered Saline (Water works too!)

3. A one page ‘how to’. Proceed for step by step instructions.

4. You have now created a 1:100 dilution and are ready to load the hemacytometer Click once to proceed! Raw Semen FBS or H2O 0.1 mL→1:100 0.1 mL 0.9 mL 0.1 mL 0.9 mL 0.9 mL

5. Neubauer chamber

6. Load each side of the chamber.

7. Load each side of the chamber.

9. 40x Objective Phase ideal Match 40x phase ring 40 Phase contrast makes counting much easier. If you do not have a phase contrast microscope, you will have to decrease the light by closing the diaphragm.

10. There are 9 big squares on the hemacytomter

11. 1 2 3 4 5 6 7 8 9

12. Count the middle big square bounded by triple lines.

13. There are 25 smaller squares within the middle big square bounded by triple lines. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

14. Each of the 25 smaller squares has 16 smaller squares

15. Count each cell in the 25 squares

16. Count cells on the left and top lines or on the right and bottom lines as you go to prevent from counting cells twice

17. How many cells do you count here?

18. You should get 32 – Click to follow the counting ball.

19. Count both sides of the chamber. Results should be within 10% of each other. If the results are not within 10% of each other, repeat the entire counting procedure.

20. Number of cells counted in the 25 squares = Sperm X 106/mL

21. Number of cells counted in the 25 squares = 32 in our example. Sperm concentration= 32 X 106/sperm cells/mL

22. If sperm concentration= 32 X 106/sperm cells/mL and Volume = 15 mL Total count is: 480 X 106

23. You have finished successfully!