Chapter 14
PROTOCOL
Echinodermata
Subprotocol 1
Starfish (Asteroidea) Embryos
The following protocol is based on the methods of Kaneko et al. (1995).
Materials
- Hand centrifuge
Equipment
- Centrifuge and centrifuge tube (10 ml)
- Incubator
- Petri dishes (4 and 6 cm diameter)
- Pasteur pipettes
- Artificial seawater (ASW; Jamarin U; Jamarin Laboratory)
Solutions
- SSW (filter-sterilized seawater 100 ml, penicillin G potassium 5,000 IU,
streptomycin sulfate 5 mg)
- Culture medium (SSW 100 ml, newborn calf serum 4 ml, pH 8.1)
- Dissociation medium (glycine 9.0 g, distilled water 100 ml, SSW 1 ml,
FBS 6 ml, pH 6.6)
- Treatment medium (glycine 4.5 g, half-strength Ca 2 + - and Mg2+ -free
ASW 100 mI, pH 6.7)
Matured eggs of the starfish Asterias amurensis, prepared as follows:
Animals
- Dissect the ovaries from the starfish (Fig. 1)
Cut the ovaries into several fragments
Wash the fragments twice with seawater
Place the fragments in a small petri dish
Add 4 ml of 1O- 6 M L-methyladenine, and leave for 15 min
Prepare the spermatozoa by making several cuts in a testis on a dry
petri dish. The spermatozoa can be kept in a refrigerator, and used
within 2 days
Inseminate the eggs with the spermatozoa within 1 h after beginning the L-methyladenine treatment
Allow the embryos to develop in sterile seawater at 18°C
PROTOCOL
Echinodermata
Subprotocol 1
Starfish (Asteroidea) Embryos
The following protocol is based on the methods of Kaneko et al. (1995).
Materials
- Hand centrifuge
Equipment
- Centrifuge and centrifuge tube (10 ml)
- Incubator
- Petri dishes (4 and 6 cm diameter)
- Pasteur pipettes
- Artificial seawater (ASW; Jamarin U; Jamarin Laboratory)
Solutions
- SSW (filter-sterilized seawater 100 ml, penicillin G potassium 5,000 IU,
streptomycin sulfate 5 mg)
- Culture medium (SSW 100 ml, newborn calf serum 4 ml, pH 8.1)
- Dissociation medium (glycine 9.0 g, distilled water 100 ml, SSW 1 ml,
FBS 6 ml, pH 6.6)
- Treatment medium (glycine 4.5 g, half-strength Ca 2 + - and Mg2+ -free
ASW 100 mI, pH 6.7)
Matured eggs of the starfish Asterias amurensis, prepared as follows:
Animals
- Dissect the ovaries from the starfish (Fig. 1)
Cut the ovaries into several fragments
Wash the fragments twice with seawater
Place the fragments in a small petri dish
Add 4 ml of 1O- 6 M L-methyladenine, and leave for 15 min
Prepare the spermatozoa by making several cuts in a testis on a dry
petri dish. The spermatozoa can be kept in a refrigerator, and used
within 2 days
Inseminate the eggs with the spermatozoa within 1 h after beginning the L-methyladenine treatment
Allow the embryos to develop in sterile seawater at 18°C