Initiation of Aseptic Cultures

The nutrient medium (basal medium) used for plant tissue culture would promote luxuriant growth of micro-organisms such as bacteria and fungi. Widely used basal medium for tissue and culture studies is that of Murashige and Skoog (1962). Reaching the medium, these microbes grow much faster than the plant cells cover the tissue surfaces impeding its growth and finally killing it. The microbes may be present in the medium right from the beginning. To destroy them the culture vessels containing the medium are properly plugged and autoclaved (steam heating under pressure) at 121? C for 15-20 minutes.

Also you can use pressure cooker for sterilizing small volumes of media. If you use presterilised, non autoclavable culture vessels then the medium is autoclaved in 100-1000 ml. coming or pyrex flasks or bottles and the sterilised medium poured into the culture vessels under aseptic conditions. You should know that most of the media constituents are heat stable and can be added to the medium before autoclaving. Growth regulators such as Gibberellic acid, Abscisic acid, Zeatin and some vitamins are thermolabile i.e. they are rapidly degraded by elevated temperatures. The solutions of such compounds are sterilised by passing them through a bacteria proof filter membrane (Pore size 0.2 μm-0.45 μm) and then added to the autoclaved medium cooled to 60° C.