Sterilization

Sterilization is the technique used to eliminate microbes such as bacteria and fungi from the culture medium, the culture vessels, and the explant itself, since even a single contaminating cell can quickly overrun and destroy a culture. Four things must each be sterilized in turn. The general aseptic environment is maintained by sterilizing glassware, forceps, scalpels and other accessories either by wet-steam autoclaving at 15 psi (121 degC) for 15 to 30 minutes, or by dipping them in 70% ethanol followed by flaming and cooling. The culture room itself is kept sterile by washing its floor and walls first with detergent and then with 2% sodium hypochlorite or 95% ethanol, while the laminar air-flow cabinet's work surface is wiped with 95% ethanol and then exposed to UV radiation for 15 minutes. Nutrient media are sterilized by dispensing them into glass containers, plugging or sealing the containers, and then autoclaving at the same 15 psi (121 degC) for 15 to 30 minutes - though heat-sensitive components such as plant extracts, vitamins, amino acids and hormones are instead sterilized separately by passing them through a Millipore filter with a 0.2 mm pore diameter before being added to the already-sterilized medium inside the laminar air-flow chamber. Finally, the explant material itself is surface-s …