Isolation of Single Cells, Sucrose, Musin cloth, Centrifugation, Cleaned Leaf pieces, Epidermis, Potassium Dextran

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	Isolation of Single Cells - The most suitable material for the isolation of single cells is the leaf tissue since it has a more or less homogeneous population of cells which are good candidates for raising defined and controlled large scale cell cultures. Cells can be isolated using mechanical or enzymatic methods. The mechanical procedure involves mild maceration of 10 g leaves in 40 ml of the grinding medium (20 μ mol sucrose, 10 μ mol MgCl2, 20 μ mol tris-HCl buffer, pH 7.8) with a mortar and pestle.
The homogenate is passed through two layers of muslin cloth and the cells thus released are washed by centrifugation at low speed using the same medium. In the enzymatic method 2 g cleaned leaf pieces (with the epidermis removed) are immersed in 20 ml filter sterilized enzyme macerozyme (0.5%) solution, 0.8% mannitol and 1 % potassium dextran sulphate (M.W. 560, S content 17.3%) in a flask. Flasks are incubated after evacuating at 25° C for 2 h on a reciprocating shaker. Cultured tissues are also widely used to obtain single cells.