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Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Chernin's BSS (see Appendix 1)
- Trypsin solution (a 0.25% (w/v) solution of trypsin (Difco; 1 : 250) -
in Chernin's BSS containing penicillin (100 units/ml) and streptomycin; (100 f.1g/ml) pH 7.6-7.8)
- Chernin's medium (see Appendix 1)
Miscellaneous - Gauze
- Watercress
- Romaine lettuce
Animals - Aquatic snail Australorbis glabratus, with a 13-17 mm shell diameter
(reared at 26°C on watercress and romaine lettuce)
Procedure
Sterilization 1. Remove excess water from the snails with the gauze.
2. Wipe the snails with 70% ethanol and air-dry them.
Dissection l. Place a snail on its side between two glass slides in a petri dish.
2. Break the shell by gentle crushing.
3. Clear the shell fragments from the pericardial area using forceps.
4. Expose the heart by using the 30-G syringe needles to tear open the
pericardial membrane.
5. Slip a hooked needle under the heart at the auriculoventricular junction, and lift the heart slightly (Fig. 12).
6. Cut the stretched auricle anteriorly with iris scissors.
7. Cut the ventricle at its base.
8. Collect five hearts in a 50-mm petri dish containing 5 ml trypsin solution.
Dissection l. Agitate the solution gently every 10-15 min.
2. After 50-60 min, transfer the hearts to a tube containing 5 ml nutrient
medium, and leave them for 3 min.
Culture l. Replace the medium, and leave the hearts for another 3 min.
set-up 2. Finally, transfer the hearts into a culture flask with l.5-2.0 ml culture
medium.
3. Leave the flask undisturbed for approximately 40 min, then place it in
an incubator at 26-28°C.
Results
According to Chernin (1963), amoebocytes and epithelial cell-like cells
migrate from the explanted hearts (Fig. 13). The former cells appeared
within 24 h near the margins of the explants. The amoebocytes were variable with regard to shape and size, and sometimes contained small par-
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Chernin's BSS (see Appendix 1)
- Trypsin solution (a 0.25% (w/v) solution of trypsin (Difco; 1 : 250) -
in Chernin's BSS containing penicillin (100 units/ml) and streptomycin; (100 f.1g/ml) pH 7.6-7.8)
- Chernin's medium (see Appendix 1)
Miscellaneous - Gauze
- Watercress
- Romaine lettuce
Animals - Aquatic snail Australorbis glabratus, with a 13-17 mm shell diameter
(reared at 26°C on watercress and romaine lettuce)
Procedure
Sterilization 1. Remove excess water from the snails with the gauze.
2. Wipe the snails with 70% ethanol and air-dry them.
Dissection l. Place a snail on its side between two glass slides in a petri dish.
2. Break the shell by gentle crushing.
3. Clear the shell fragments from the pericardial area using forceps.
4. Expose the heart by using the 30-G syringe needles to tear open the
pericardial membrane.
5. Slip a hooked needle under the heart at the auriculoventricular junction, and lift the heart slightly (Fig. 12).
6. Cut the stretched auricle anteriorly with iris scissors.
7. Cut the ventricle at its base.
8. Collect five hearts in a 50-mm petri dish containing 5 ml trypsin solution.
Dissection l. Agitate the solution gently every 10-15 min.
2. After 50-60 min, transfer the hearts to a tube containing 5 ml nutrient
medium, and leave them for 3 min.
Culture l. Replace the medium, and leave the hearts for another 3 min.
set-up 2. Finally, transfer the hearts into a culture flask with l.5-2.0 ml culture
medium.
3. Leave the flask undisturbed for approximately 40 min, then place it in
an incubator at 26-28°C.
Results
According to Chernin (1963), amoebocytes and epithelial cell-like cells
migrate from the explanted hearts (Fig. 13). The former cells appeared
within 24 h near the margins of the explants. The amoebocytes were variable with regard to shape and size, and sometimes contained small par-