Chapter 10
Insecta (Blatta ria)
Subprotocol 1
Embryos
Materials
- Dissecting microscope
- Centrifuge and centrifuge tube
- Petri dish
- Maximov slide
- Culture vessel
- 70% ethanol
- Physiological solution
- Trypsin solution
- Culture medium (S-20; see Appendix 1)
- Cockroach adult with oothecae
Procedure
1. Collect oothecae.
2. Surface sterilize the oothecae by submersion in 70% ethanol for 5 min.
3. Rinse the oothecae with sterile distilled water.
4. Place the oothecae in physiological solution on a Maximov slide.
PROTOCOL
Equipment
Solutions
Animals
Sterilization
1. Cut the oothecae, and isolate the embryos.
Dissection
2. Collect the embryos, and wash in physiological solution.
l. Transfer the embryos into 0.01 %-0.05% trypsin solution.
Trypsinization
2. Allow digestion with trypsin for 10-15 min.
3. Add an equal amount of culture medium containing serum.
l. Dissociate the embryos by vigorous pipetting.
Culture
2. Centrifuge the resulting cell suspension at 275 g for 10 min.
set-up
3. Discard the supernatant, and suspend the cell pellet in culture medium.
4. Inoculate the cell suspension into a culture vessel.