5.5.3. Procedure

  1. Make Sybr 14 working solution:
    a. Add 20 µl Sybr 14 (from Invitrogen kit) to 980 µl DMSO
    b. Store at -20°C in the dark.
  2. Make sufficient volume of sperm diluent of HEPES, NaCl, and BSA (HNB). See Table 4
  3. For spermathecae
    a. Transfer diluent with sperm (from step 5.4.1.) to a labelled 2 ml glass vial.
    b. After all spermathecae have been dissected, add 10 µl Sybr 14 in DMSO to each vial.
    c. Incubate 5-10 min at 36°C.
    d. Add 5 µl propidium iodide.
    e. Incubate 5-10 min at 36°C.
  4. For seminal vesicles
    Gently disrupt seminal membrane to release sperm in diluent.
    a. Discard seminal membrane and pour sperm solution into a 2 ml glass vial.
    b. Stain seminal vesicles:
    i. Add 10 µl Sybr 14/DMSO.
    ii. Incubate 15 min at 36°C.
    iii. Add 6 µl propidium iodide.
    iv. Incubate another 15 min at 36°C.
  5. Turn on fluorescent lamp and camera, and open photo software on computer.
  6. Use microdispenser to load haemocytometer with 10 µl sperm solution across both chambers.
  7. Place haemocytometer on microscope table and examine under low magnification to centre view on grid (Fig. 26).
  8. Switch to high magnification (200x then 400x).
  9. Turn off visible light and open fluorescence. Focus using FITC filter, then take an image using the camera. This will be a picture of the dead sperm in the field.
  10. Without moving the haemocycometer, switch to Rhodamine filter and take another image. This will be a picture of the live sperm in the same field.
  11. Move to a new field of view and repeat; a total of five replicates should be minimum, preferably greater particularly if there is large variation among fields.
  12. Save pictures using descriptive names indicating sample number, live or dead sperm, and picture number.
  13. Switch back to low-resolution and close fluorescent lamp aperture.
  14. Clean haemocytometer and cover slip with a kimwipe.
  15. Repeat for each new sample.