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1. Using an alcohol-flamed spatula, carefully place two heart explants in Culture
each of the eight wells.
set-up
2. Replace the lid and carefully tape it in order to seal the periphery of
the cluster plate.
3. Incubate at 15°C.
4. Replace one-half of the culture medium every 3 days. This partial
change of culture medium is suggested in order to retain any benefit
obtained by using a conditioned medium. The pH of the medium
should remain at approximately 7.2-7.4.
Results
According to the report of Stephens and Hetrick (1979), migration of
amoebocytes from the explant started within 5 min, and the migrated
amoebocytes attached to the surface of the vessels. These cells exhibited
pseudopodia extensions of various lengths. Mitosis was not observed. In
addition, hyalinocytes and fibrocytes were observed to migrate from the
explants.
Subprotocol 11
Cells from Larval Tissues of Bivalves (Bivalvia)
Materials
- Centrifuge and centrifuge tubes
- Water bath
- Water pump
- Glass homogenizer
- Pasteur pipette
- Petri dish
- T-25 culture flask
- Sterilized seawater
- MMBSS (see Appendix 1)
- Culture medium Pf-35 (see Appendix 1)
Equipment
Solutions
- Veliger larvae (Fig. 17) of the pearl oyster P. fucata and the black lip Animals
pearl oyster P. margaritifera
Procedure
1. Rinse veliger larvae in sterilized running seawater.
Sterilization
2. Wash the larvae with MMBSS containing 2,000 Ilg/ml penicillin and
500 Ilg/ml kanamycin.