Activation of the oocyte (i.e. oogenesis) is followed by the incorporation of euplasmatic material and/or yolk proteins. Marking a whole mount of the mites’ ovaries with toluidine blue is a rapid method to confirm such incorporation and therefore initiation of oogenesis (Garrido et al., 2000).
1. Remove ventral shield of the mites with thin dissecting forceps under a binocular microscope.
2. Excise ovary together with spermatheca and lyrate organ.
3. Place in PBS buffer (phosphate buffered saline, pH 7.2–7.4).
4. Fix in formalin (4 %) for 30 min.
5. Wash three times with PBS buffer.
6. Incubate in toluidine blue (0.005 %) for 30 min.
The duration of incubation might need optimization, which can be tested on the coloration of activated oocytes approximately 12 h after cell sealing.
7. Rinse with PBS buffer for 15 min.
8. Repeat rinsing two more times.
9. Verify the colouring of the oocytes under a microscope at 400x magnification.
Pros: easier and faster than the alternative histological method. Detects initiation of oogenesis with high resolution.
Cons: somewhat tedious; subjective grading of oocyte colour.