Single Cell Culture
Single cell culture is a method of growing an isolated cell aseptically on a nutrient medium under controlled conditions.
The technique was pioneered by Hildebrandt who attempted to culture single cells of a tumourous hybrid tobacco plant (Nicotiana tabacum with N. glutinosa).
The basic principle involved is the isolation of a large number of intact living cells and culturing them on a suitable nutrient medium.
Single cells can be isolated, either mechanically or enzymatically, from a number of tissues and organs of plant as well as from callus tissue and cell suspension.
An Explant refers to the part of the plant that has been taken for cell culture(tissue culture in general). Mechanical isolation involves tearing or chopping the surface sterilized explants to expose the cells, followed by scraping of the cells with a fine scalpel to liberate the single cell. Gentle grinding of surface sterilized explants in a sterilized mortar-pestle, followed by cleaning the cells by filtration and centrifugation is now widely used for the large-scale mechanical isolation of viable cells.
In large-scale isolation of free cells from the surface sterilized plants frst the the pectin is dissolved by pectinase or macerozyme treatment. Using enzymes to isolate a cell has made it possible to obtain pure preparation of viable cells with less effort and time.
Single cells are traditionally isolated from established friable callus tissue and cell suspension culture. Mechanically, single cells are carefully isolated from cell suspension or friable callus with a needle or fine glass capillary. Sometimes, the friable tissue is transferred to liquid medium and the medium is continuously agitated by a shaker. Agitation of liquid medium prevents cell aggregation and helps in separation of aggregated cells, if any. Thus, a cell suspension is formed. The cell suspension is first filtered to remove cell clumps and the filtrate is then centrifuged to collect the single cells from the pellet.
The isolated single cell can be cultured either in liquid medium or on solid medium. 5 basic methods exist for culturing single cells: paper raft nurse technique, the Petri dish plating technique, the micro chamber technique, the micro droplet technique, the plating with nurse tissue technique.
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