Chapter 15
PROTOCOL
Mollusca
Subprotocol 1
Blastomeres of I/yanassa obsoleta (Gastropoda)
Materials
- Steel needles
Equipment
- Scissors
- Forceps
- 35-mm plastic tissue culture dish (Falcon, Becton-Dickinson)
- Pasteur pipettes
- Culture medium
Solutions
- 5% Eagle's basal medium modified for HeLa cells (Difco Laboratories)
- 1 % Horse serum
- 94% Filter-sterilized seawater
- Penicillin G
Chemicals
- Streptomycin sulfate
- Eggs of I. obsoleta at late trefoil or early two-cell stage
Animals
Procedure
1. Sterilize egg capsules by dipping them in 70% ethanol for 2-3 s.
Sterilization
1. Open the capsule, and remove the eggs into sterilized seawater.
Dissection
2. Puncture one blastomere of each egg with a steel needle leaving the
other intact.
1. Transfer 15-25 AB or CD blastomeres (Fig. 1) to 2 ml culture medium Culture
in a 35-mm plastic tissue culture dish containing 50 IU/ml penicillin set-up
G and 50 Ilglml streptomycin sulfate.
2. Keep the cultures at ambient temperature (approximately 24°C) in a
humidified chamber.