Biotechnology and Bioinformatics (1+1)

3.1.8. Flow chart for primary cell culture from shrimp

Shrimp (8-15 cm)

¯

Anesthetize (cold water 4^oC/40 min or dip in 10% hypochloride)

¯

Rinse in 7% tincture of iodine to sterilize the external surfaces of shrimp and wash with Leibowitz’s solution to remove tincture of iodine.

¯

Dissect under dissection microscope aseptically to get isolated tissues of the required parts.

¯

The sterile tissues are collected in a pistri dish

¯

Trypsinize each tissue separately with 0.1% Trypsin at 37^oC for 20 min.

¯

Trypsinization yields isolated /single cells from the  tissue mass by enzymatic action

¯

Wash the trypsinized cells with Leibowitz’s medium to completely remove the trypsin. Repeat the process 2-3 times

 Collect the cells by centrifuging at 800-1000 g for 5-10 min

¯

Suspend the washed cell pellet in 5-10 ml medium in a 25cm2 tissue culture flask (medium – Eagles minimum essential medium or Grace insect medium or

Leileowitz’s L-15 medium)

¯

Incubate at 25^oC for 24-48 hrs

¯

Watch under a microscope for the formation of a monolayer

Secondary culture

•  The cell culture is called a primary culture until it is subcultured for the first time, after which it becomes a secondary culture.
• The subsequent cell cultures are known as cell lines.
• Since the primary cell culture is heterogenous, we go for selection or cloning of cells for obtaining particular cells.