184
Methods for Setting Up Primary Cultures Specific to Animal Groups
fortified with amino acids and hemolymph of marine molluscs. The culture was maintained at 15°C, and the cells survived for 4 months. During
cultivation, mitotic division was observed.
Subprotocol 13
Gills of the Hard Clams (Bivalvia)
The following procedure is based mainly on the methods of Chen et al.
(1989) and Auzoux et al. (1993) for the culture of gill tissues of a hard
clam.
Materials
Equipment - Surgical knife
- Forceps
- Scissors
- Beaker
- 25-cm 2 plastic flask (Falcon, Becton-Dickinson)
- Pasteur pipette
Solutions - Leibovitz's L-15 medium (double strength)
- Clam hemolymph (see Comments)
- Clorox (Clorox Clorox Co.)
- Trypsin-EDTA solution
- Antibiotic mixture (10 IU Iml bacitracin, 400 IU Iml polymyxin B, 20 j..lgl
ml ampicillin, 300 IU/ml penicillin G, 300 j..lg/ml streptomycin, 50 j..lgl
ml amphotericin B, 50 IU/ml nystatin, 125 j..lg/ml flag, and 200 j..lg/ml
solnicol)
- 0.025% potassium permanganate
Animals - Hard clam Meretrix lusoria (approximately 10 g weight) or butterfish
clam Ruditapes decussatus
Preliminary
sterilization
Dissection
Sterilization
Procedure
1. Wipe the surface of the clams with 70% ethanol-saturated absorbent
cotton, and dry.
2. Maintain the clams for 1-2 days in a saline solution containing 200 IVI
ml penicillin, 200 j..lg/ml streptomycin, and 50 IVlml nystatin.
1. Open the shell by cutting the adductor muscle.
2. Remove the gill tissues (cf. Fig. 4).
1. Wash the gill tissues in double-strength L-15 medium containing the
antibiotic mixture by changing the medium three times.
Methods for Setting Up Primary Cultures Specific to Animal Groups
fortified with amino acids and hemolymph of marine molluscs. The culture was maintained at 15°C, and the cells survived for 4 months. During
cultivation, mitotic division was observed.
Subprotocol 13
Gills of the Hard Clams (Bivalvia)
The following procedure is based mainly on the methods of Chen et al.
(1989) and Auzoux et al. (1993) for the culture of gill tissues of a hard
clam.
Materials
Equipment - Surgical knife
- Forceps
- Scissors
- Beaker
- 25-cm 2 plastic flask (Falcon, Becton-Dickinson)
- Pasteur pipette
Solutions - Leibovitz's L-15 medium (double strength)
- Clam hemolymph (see Comments)
- Clorox (Clorox Clorox Co.)
- Trypsin-EDTA solution
- Antibiotic mixture (10 IU Iml bacitracin, 400 IU Iml polymyxin B, 20 j..lgl
ml ampicillin, 300 IU/ml penicillin G, 300 j..lg/ml streptomycin, 50 j..lgl
ml amphotericin B, 50 IU/ml nystatin, 125 j..lg/ml flag, and 200 j..lg/ml
solnicol)
- 0.025% potassium permanganate
Animals - Hard clam Meretrix lusoria (approximately 10 g weight) or butterfish
clam Ruditapes decussatus
Preliminary
sterilization
Dissection
Sterilization
Procedure
1. Wipe the surface of the clams with 70% ethanol-saturated absorbent
cotton, and dry.
2. Maintain the clams for 1-2 days in a saline solution containing 200 IVI
ml penicillin, 200 j..lg/ml streptomycin, and 50 IVlml nystatin.
1. Open the shell by cutting the adductor muscle.
2. Remove the gill tissues (cf. Fig. 4).
1. Wash the gill tissues in double-strength L-15 medium containing the
antibiotic mixture by changing the medium three times.