Media Sterilization – Plant Tissue Culture Protocol

Plant tissue culture media are generally sterilized by autoclaving at 121 °C and 1.05 kg/cm2 (15-20 psi). The time required for sterilization depends upon the volume of medium in the vessel. The minimum times required for sterilization of different volumes of medium are listed below. It is advisable to dispense medium in small aliquots whenever possible as many media components are broken down on prolonged exposure to heat. There is evidence that medium exposed to temperatures in excess of 121 °C may not properly gel or may result in poor cell growth.

Minimum Autoclaving Time for Plant Tissue Culture Medium
 

Volume of Medium per Vessel (ml) Minimum Autoclaving (min)*
25 20
50 25
100 28
250 31
500 35
1000 40
2000 48
4000 63
*Minimum autoclaving time includes the time required for the liquid volume to reach the sterilizing temerature
(121 °C) and 15 min. at 121 °C (Burger, 1988). Times may vary due to differences in autoclaves. Validation with your system is recommended.

Several medium components are considered thermolabile and should not be autoclaved. Stock solutions of the heat labile components are prepared and filter sterilized through a 0.22 µm filter into a sterile container. The filtered solution is aseptically added to the culture medium, which has been autoclaved and allowed to cool to approximately 35-45 °C. The medium is then dispensed under sterile conditions. Experimentation with your system is recommended.