Bergmann's Method of Cell Plating
In this method free cells are suspended in a liquid medium at a density twice the finally desired plating density. Melted agar containing medium of otherwise the same composition as the liquid medium is maintained at 35?c in water bath. Equal volumes of the two media are mixed and rapidly spread out in petri dishes in such a manner that the cells are evenly distributed and fixed in a thin layer (about 1 mm thick) of the medium after it has cooled and solidified.
The dishes are sealed with parafilm. The cells to be followed are marked on the outside of the plate and before the colonies derived from individual cells grow large enough to merge with each other. They are transferred to separate plates. Another popular method for single cell culture is the microchamber technique, developed by Jones et al. (1960). In this method mechanically isolated single cells are cultured in separate droplets of liquid medium. While Jones et al. used sterile microslides and three coverglasses to make microchamber, it is now possible to buy pre-sterilised plastic plates with several microwells. Individual cells are cultured in separate wells each containing 0.25 ml of the liquid medium. The culture requirement of single cells increases with decrease in the plating cell density, and the cell cultured in complete isolation require a very complex culture medium. A simple medium conditioned by growing cell suspension for some time also fulfils the requirements of single cell culture at low density.