Elementary plant Biochemistry and Biotechnology (2+1)

Subculture Methods

• Basically there are three types of suspension cultures: batch culture, continuous culture and immobilized culture.
 
Batch culture

• These are used for initiating single cell cultures. Cell suspensions are grown in 100-250 ml flasks each containing 20-75 ml of culture medium. The cultures are continuously propagated by routinely taking a small aliquot of the suspension and transferring it to a fresh medium (ca. 5X dilution). Batch cultures are not ideal for studies of cell growth and metabolism because there is a constant change in cell density and nutritional status of the medium. In these cultures the exponential growth with constant cell doubling time may be achieved for a short time but there is no period of steady-state growth in which the relative cell concentrations of metabolites and enzymes are constant. To a certain extent these problems are overcome by continuous cultures.

Continuous culture

• In a continuous culture, the cell population is maintained in a ~fixed number by regularly replacing a portion of the used or spent medium by fresh medium. Such culture systems are of either closed or open type. In a closed continuous culture, cells are separated from the used medium taken out for replacement and added back to the culture so that cell biomass keeps on increasing. Whereas in an open continuous culture, both cells and the used medium are taken out and replaced by equal volume of fresh medium and cells. The replacement volume is so adjusted that cultures remain at sub-maximal growth indefinitely.


• The open cultures are of either turbidostat or chemostat types. In a turbidostat, cells are allowed to grow up to a preselected turbidity (usually measured as OD) when a predetermined volume of the culture is replaced by fresh normal culture medium. But in a Chemostat, a chosen nutrient is kept in a concentration so that it is depleted very rapidly to become growth limiting, while other nutrients are still in concentrations higher than required. In such a situation, any addition of the growth limiting nutrient is reflected in cell growth. Chemostats are ideal for the determination of effects of individual nutrients on cell growth and differentiation.

Immobilized cell cultures

• Plant cells and cell groups are encapsulated in a suitable compound such as agarose, calcium alginate cells etc. or entrapped in membranes or stainless steel screens. The gel beads containing cells may be packed in a suitable column or alternatively, cells may be packed in a column of a membrane or wire cloth. Liquid medium is continuously run through the column to provide nutrients and aeration to cells. Immobilization of cells changes their cellular physiology in comparison to suspension culture cells. This offers several advantages for their use in biochemical production, but they are generally not used for other studies.