Sperm Count Protocol
Obtain sperm mass from female cloaca using glass capillary tube and place in 50 l of
PBS (be sure to remove all sperm that might be in the sides of the cloaca).
If using sperm mass (from mating) start with first step. If using expressed sperm,
skip to 2.
1) Add 3 l of 6GDU/ml bromelain to the sperm mass and PBS and tap to mix. Sperm
mass should dissolve after 3-5 minutes.
2) As soon as mass is dissolved, add 490 l of PBS, and then 10l of 4% PFA fixative to
prevent the cells from swimming.
3) Mix well by tapping.
4) Add 5 l of the solution to the hemocytometer, and place coverslip on top. Place on
microscope and allow cells to settle for ~5 min.
5) Count and record the cells (head only!) in each of the 25 center squares (if each
square is >80-90 cells, only count the top two rows)
6) If cell head is in lined area, use central line to determine which box the sperm is
counted. If >50% cell head is in 1 square, count it to that square, if ~50%, use cell
where tail is located.
7) Total volume of central 25 squares = 0.1l. Multiply total number by 5000 to get total
sperm count. Record this number in notebook.
8) To make slide from these cells, mix remaining cells by tapping, then removed 250l
into a second tube.
9) Add 150l of 4% PFA to the tube and let fix for 5 minutes. Spin down for 30-45
seconds.
10) Pipet off PBS/PFA, discard. Add 100l of ultrapure water to cells, and mix by tapping.
11) Pipet ~20l of cells onto clean slide and allow to dry completely. See staining
procedure in protocol below.
Sperm Morphology Protocol
Collect sperm manually and place it in 150 l PBS using a capillary tube.
Fixing and staining:
1) Add 50 l 4% PFA to tube, tape to mix, and allow cells to fix in solution for ~5
minutes.
2) Spin down cells in desktop centrifuge for 30-45 seconds.
3) Pipet off the PBS and PFA, and add 100 l of ultrapure H20. Tap by mixing.
4) Pipet 10 l of cells and H20 onto clean slide. Spread cells using the side of the pipet
tip across the slide to cover ~75% of surface area, and allow to dry completely (make
sure the slides are completely dry or cells will slough off during when rinsing of stain).
5) Pipet 100 l of Sperm Blue onto the slide, and spread across slide using pipet tip.
Allow to stain for 5 minutes. **Use the white ‘Thermo’ pipets for staining**
6) Drain excess stain on paper towel, and then rinse by dipping in distilled water. Allow
to dry.