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3.1.8. Flow chart for primary cell culture from shrimp
Shrimp (8-15 cm)
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Anesthetize (cold water 4oC/40 min or dip in 10% hypochloride)
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Rinse in 7% tincture of iodine to sterilize the external surfaces of shrimp and wash with Leibowitz’s solution to remove tincture of iodine.
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Dissect under dissection microscope aseptically to get isolated tissues of the required parts.
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The sterile tissues are collected in a pistri dish
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Trypsinize each tissue separately with 0.1% Trypsin at 37oC for 20 min.
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Trypsinization yields isolated /single cells from the tissue mass by enzymatic action
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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
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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)
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Incubate at 25oC for 24-48 hrs
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Watch under a microscope for the formation of a monolayer
Secondary culture
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The cell culture is called a primary culture until it is subcultured for the first time, after which it becomes a secondary culture.
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The subsequent cell cultures are known as cell lines.
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Since the primary cell culture is heterogenous, we go for selection or cloning of cells for obtaining particular cells.
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