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Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Diluted seawater (diluted with distilled water to 650 mOsmollkg osmolarity and filter sterilized)
- Filtered seawater
- 2.6% NaCl
- Antibiotic solution 1 (penicillin G 500 IU, streptomycin sulfate 50 Ilg,
amphotericin B 5 Ilg, glucose 3 Ilg/ml; prepared with diluted seawater)
- Antibiotic solution 2 (2.6% NaCl containing 400 IU/ml penicillin,
400 Ilg/ml streptomycin, and 0.25 Ilg/ml amphotericin B)
- 0.25% Trypsin-EDTA in 2.6% NaCl containing antibiotic solution 2
- Cecil's medium (see Appendix 1)
Animals - Adult clams Spisula solidissima
Miscellaneous - Gauze
- Rubber bands
Procedure
Preliminary 1. Scrub the exterior surface of the shells with a stiff brush in running
sterilization
seawater.
2. Place 50 clams in 41 diluted seawater.
3. Change the diluted seawater four times at hourly intervals.
4. Immerse the clams in 70% ethanol for 30 s, and dry them in a laminar
flow cabinet.
Dissection 1. Cut the adductor muscles carefully and open the shell.
2. Dissect the mantle from the shell, care being taken to avoid damaging
the pericardial membrane.
3. Rinse the pericardial area with a strong spray of 70% ethanol.
4. Remove the pericardial membrane with forceps (Fig. 11).
5. Remove the heart carefully, and place it in a 50-ml beaker containing
20 ml antibiotic solution 1.
6. Collect 20 hearts in a beaker.
Sterilization 1. Change the antibiotic solution every 15 min for a total of six times.
2. Place a single layer of gauze over the top of the beaker, and hold the
gauze in place with a rubber band.
3. Wash the cardiac tissues for 10 min in running filtered seawater.
4. Transfer the cardiac tissues in 2.6% NaCI to a petri dish.
5. Wash the tissues with four changes of antibiotic solution 2.
Dissection 1. Mince the cardiac tissues finely.
2. Transfer the tissues to 30 ml trypsin-EDTA solution.
3. Place the flask on a magnetic stirrer and digest the tissues for 4 h at
20°C.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Diluted seawater (diluted with distilled water to 650 mOsmollkg osmolarity and filter sterilized)
- Filtered seawater
- 2.6% NaCl
- Antibiotic solution 1 (penicillin G 500 IU, streptomycin sulfate 50 Ilg,
amphotericin B 5 Ilg, glucose 3 Ilg/ml; prepared with diluted seawater)
- Antibiotic solution 2 (2.6% NaCl containing 400 IU/ml penicillin,
400 Ilg/ml streptomycin, and 0.25 Ilg/ml amphotericin B)
- 0.25% Trypsin-EDTA in 2.6% NaCl containing antibiotic solution 2
- Cecil's medium (see Appendix 1)
Animals - Adult clams Spisula solidissima
Miscellaneous - Gauze
- Rubber bands
Procedure
Preliminary 1. Scrub the exterior surface of the shells with a stiff brush in running
sterilization
seawater.
2. Place 50 clams in 41 diluted seawater.
3. Change the diluted seawater four times at hourly intervals.
4. Immerse the clams in 70% ethanol for 30 s, and dry them in a laminar
flow cabinet.
Dissection 1. Cut the adductor muscles carefully and open the shell.
2. Dissect the mantle from the shell, care being taken to avoid damaging
the pericardial membrane.
3. Rinse the pericardial area with a strong spray of 70% ethanol.
4. Remove the pericardial membrane with forceps (Fig. 11).
5. Remove the heart carefully, and place it in a 50-ml beaker containing
20 ml antibiotic solution 1.
6. Collect 20 hearts in a beaker.
Sterilization 1. Change the antibiotic solution every 15 min for a total of six times.
2. Place a single layer of gauze over the top of the beaker, and hold the
gauze in place with a rubber band.
3. Wash the cardiac tissues for 10 min in running filtered seawater.
4. Transfer the cardiac tissues in 2.6% NaCI to a petri dish.
5. Wash the tissues with four changes of antibiotic solution 2.
Dissection 1. Mince the cardiac tissues finely.
2. Transfer the tissues to 30 ml trypsin-EDTA solution.
3. Place the flask on a magnetic stirrer and digest the tissues for 4 h at
20°C.