Protocol of Suspension Culture
1. Take 150/250 ml conical flask containing autoclaved 40/60 ml liquid medium.
2. Transfer 3-4 pieces of pre-established callus tissue from culture tube using the spoon headed spatula to conical flask.
3. Flame the neck of conical flask, close the mouth of conical flask, with piece of aluminium foil or a cotton plug. Cover the closure with piece of brown paper.
4. Place the flasks within the clamps of a rotary shaker moving at the 80-120rpm.
5. After the filtrate to settle for 10-15 minutes or centrifuge the filtrate at 500 to 1000 rpm and finally pour off the supernant.
6. Resuspend the residue cells in a requisite volume of fresh liquid medium and despise the cell suspension equally in several sterilized flasks. Place the flasks on shaker and allow the free cells and cell aggregates to grow.
7. Resuspend the residue cells in a requisite volume of fresh liquid medium and despense the cell suspension equally in several sterilized flasks ( 150/250 ml). Place the flasks on shaker and allow the free cells and cell aggregates to grow.
8. At the next subculture, repeat the preveious steps but take only one fifth of the residual cells as the inoculum and despense equally in flasks and again place them on shaker.
9. After 3-4 subculture, transfer 10ml of cell suspension from each flask into new flask containing 30 ml fresh liquid medium.
10. To prepare a growth curve of cells in suspension, transfers a definite number of cells measured accurately by a haemocytometer to a definite volume of liquid medium and incubate on shaker. Pipette out very little aliquot of cell suspension at short intervals of times and count the cell number. Plot the cell count data of a passage on a graph paper and the curve will indicate the growth pattern of suspension culture.