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Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Balanced salt solution for oyster (BSSO; see Appendix 1)
- Chloride solution (105 m150°C chloride water (Societe Langlois), 15 g
NaHC0 3 , 365 g caster sugar, distilled water to 1,420 ml)
- Trypsin-EDTA (2 g trypsin 1 : 250, 0.4 g EDTA, 8 g NaCl, 2.77 g
Na z HP0 4 ·12H z O, 0.2 g KHzPOill distilled water; pH 7.4)
- Seawater-Tween (0.5 ml Tween-20 is added to 500 ml autoclaved seawater)
- Leibovitz's L-15 medium
- Modified L-15 medium (L-15 medium (3x) and seawater in a ratio of
1 : 1, further supplemented with 10% FBS and 5% Crassotrea gigas
hemolymph obtained from the pericardial cavity)
- Scherer's medium (Difco Laboratories)
- Medium A-12 (see Appendix 1).
Animals - American oyster Crassostrea virginica, or the Pacific oyster C. gigas
Procedure
Explant culture
Sterilization l. Wash and scrub the valve exterior under tap water.
2. Bathe the oyster in 70% ethanol for 30 s.
3. Let the oysters dry under a sterile hood.
Dissection 1. Break the hinge ligament.
2. Cut the adductor muscle carefully.
3. Open the oysters without damaging the pericardial membrane
(Fig. 14).
4. Wash the animals and the inner shells with seawater-Tween.
5. Rinse the oysters with autoclaved seawater.
6. Make an incision in the pericardial membrane and cut off the heart
ventricle, leaving the black-colored auricle.
7. Chop the ventricle into pieces of approximately 1 mm z •
Culture l. Wash the fragments for 20 min with three changes of sterile BSSO conset-up
taining 100 IV/ml penicillin, and 100 j.lg/ml streptomycin.
2. Place 10-15 fragments in each plastic flask.
3. Remove any excess fluid.
4. Keep the cultures at 20°C overnight (18-20 h).
5. Add 3.0 ml culture medium (medium A-12 or a mixture of Scherer's
medium, BSSO, and chicken serum in a ratio of 45: 45: 10).
6. Incubate the culture at 20°C.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Balanced salt solution for oyster (BSSO; see Appendix 1)
- Chloride solution (105 m150°C chloride water (Societe Langlois), 15 g
NaHC0 3 , 365 g caster sugar, distilled water to 1,420 ml)
- Trypsin-EDTA (2 g trypsin 1 : 250, 0.4 g EDTA, 8 g NaCl, 2.77 g
Na z HP0 4 ·12H z O, 0.2 g KHzPOill distilled water; pH 7.4)
- Seawater-Tween (0.5 ml Tween-20 is added to 500 ml autoclaved seawater)
- Leibovitz's L-15 medium
- Modified L-15 medium (L-15 medium (3x) and seawater in a ratio of
1 : 1, further supplemented with 10% FBS and 5% Crassotrea gigas
hemolymph obtained from the pericardial cavity)
- Scherer's medium (Difco Laboratories)
- Medium A-12 (see Appendix 1).
Animals - American oyster Crassostrea virginica, or the Pacific oyster C. gigas
Procedure
Explant culture
Sterilization l. Wash and scrub the valve exterior under tap water.
2. Bathe the oyster in 70% ethanol for 30 s.
3. Let the oysters dry under a sterile hood.
Dissection 1. Break the hinge ligament.
2. Cut the adductor muscle carefully.
3. Open the oysters without damaging the pericardial membrane
(Fig. 14).
4. Wash the animals and the inner shells with seawater-Tween.
5. Rinse the oysters with autoclaved seawater.
6. Make an incision in the pericardial membrane and cut off the heart
ventricle, leaving the black-colored auricle.
7. Chop the ventricle into pieces of approximately 1 mm z •
Culture l. Wash the fragments for 20 min with three changes of sterile BSSO conset-up
taining 100 IV/ml penicillin, and 100 j.lg/ml streptomycin.
2. Place 10-15 fragments in each plastic flask.
3. Remove any excess fluid.
4. Keep the cultures at 20°C overnight (18-20 h).
5. Add 3.0 ml culture medium (medium A-12 or a mixture of Scherer's
medium, BSSO, and chicken serum in a ratio of 45: 45: 10).
6. Incubate the culture at 20°C.