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Methods for Setting Up Primary Cultures Specific to Animal Groups
Results
According to Moulton (1978), attachment of cells to the substrate is a
prerequisite for the proliferation of cells. In the case of culture of R.
sanguineus cells, the resulting cells were spindle-shaped with cytoplasmic processes (Fig. 5). These cells formed incomplete monolayers, and
survived for up to 45 days. Cultures of cells from D. andersoni had clusters of cells within a few days, and these cells formed monolayers within
3 weeks (Fig. 6). These monolayers of cells from D. andersoni survived
for 6 months. Cultures of cells from A. maculatum showed slow but consistent growth, with cells forming clusters and incomplete monolayers.
These cells could be subcultured and continued to multiply for over 8
months.
Subprotocol 6
Shrimp (Crustacea) Hepatopancreas
Materials
Equipment - Centrifuge and centrifuge tubes
- Scissors
- Knife
- Forceps
- Dissecting tray
- Pasteur pipettes
- Beaker
- Magnetic stirrer and magnetic bar
- Nylon mesh (100 flm mesh)
Solutions - Ahearn et al.'s saline (see Appendix 1)
- Culture medium {medium 199 adapted for marine invertebrates by
the addition of the following (in mM): Na 2 S0 4 8.3; KCI 10.3; MgCl 2
12.9; NaC1293; NaHC0 3 4.1; CaCI 2 33). The medium is enriched with
20% low-protein serum replacement containing growth factors and
other growth-promoting agents. The osmolarity of the medium is 1,100
m Osmol/kg, and the pH is 7.4. The medium contains streptomycin
(100 flg/ml), penicillin (100 IU/ml), and amphotericin B (2.5 flg/ml).
- 2% Iodine
- 75% Ethyl alcohol
- lOmMEDTA
- 0.1 mM Diisopropyl fluorophosphate
Animals - Shrimp Palaemon serratus