Biotechnology and Bioinformatics (1+1)

3.1.6. Preparation of fish for explants

3.1.6.1.Tissues

• External tissues : Fin, skin, barbels, cornea and caudal and trunk portions should be washed in cold chlorinated tap water and rinsed in sterile BSS.
• Antibiotic s such as polymyxin-B (2000 IU/ml), streptomycin (100-1500 m g/ml) and penicillin can be used via bath treatment for gill purification, followed by treatment with BSS.

• Immersion of eggs for 1 sec in 95% ethanol and then transferring to sterile water should be employed for aseptic removal of embryos.
• Gambusia in gravid condition is immersed momentarily in methiolate and washed twice in 70% alcohol. The fish is dried with sterile cotton and aseptically the embryos are removed.

Internal tissues

• Sterile embryos may be obtained by surface sterilization of either eggs or gravid females.
• Unless an animal is infected and with the exception of the digestive tract, internal tissues of fishes are sterile and their aseptic removal is simple.
• Prior to opening the fish, the area of incision or when feasible the entire fish is topically disinfected or sterilized.
• It is advantageous to remove scales from heavily scales fish. Isopropanol (70%),
• Ethanol (70%) and 500 ppm available chlorine solution are used to disinfect the external surfaces.

3.1.6.2.Seeding density for primary monolayer culture

• The cells to be cultured are harvested by centrifugation. It is gene rally agreed that 200 g for 10 min is both adequate and safe.
• Cells from many fishes readily tolerate centrifugation at 20^oC or even higher but frictional heating coupled with high ambient temperature may injure cells from cold water fishes.
• The cell density varies from 1 to 3x10⁵ cells/ml.

3.1.6.3. Seeding density for cell lines

The seeding density for subcultures of cell lines will vary with the cell, the medium and the particular need. The usual density various from 10⁴ to 10⁵ cells/ml but 10³–10⁴ cells/ml can be adequate under good conditions.

3.1.6.4. Choice of explant in the order of decreasing importance

Embryo, gonad, swim bladder, fin, mesentery, cornea, gill, heart and skin.