The microculture chamber technique was first developed by Jones et al. (1960) and later was used by Vasil and Hildebrandt (1965) after some modifications. This method consists of culturing 30-50μ/ of medium containing one or more protoplasts on a microscope slide enclosed by a cover glass resting on two other cover glasses placed on either side of the drop. The cultures are sealed with sterile paraffin oil and incubated in light at 23-25º C.

Another approach to culturing protoplasts at low density is the feeder layer technique. In this technique a feeder cell layer is prepared by exposing protoplasts to irradiation with x-ray which inhibits division of cells but allows them to remain metabolically active.
The irradiated protoplasts are then plated in soft agar medium and serves as the feeder layer for the nonirradiated protoplasts which are placed above this. Coculturing of two types of protoplasts (e.g. fast growing and slow growing) is also done to aid in regeneration of cell wall and cell division by cross feeding.