Sterilization of the media and material is generally done at 121 °C but sometimes it is required to do at low temperature (115 °C) because of heat sensitivity of the material. This sterilization below 121 °C is called Low-Temperature Sterilization.
Reinforced Medium, Rappaport Vassiliadis Medium, Wilson and Blair's BBS Agar, GN Broth, Triple Sugar Iron Agar, Bismuth Sulphite Agar Medium etc. are sterilized at 115°C and 10 psi pressure. These media are sterilized separately at this temperature.
These culture media contains some heat sensitive ingredients that can degrade at 121°C losing their nutritive quality. This media shall not support the growth of specified microorganisms. When these media are sterilized at high temperature, the microorganism can grow but shall not develop the characteristic colonies due to the degradation of the heat sensitive ingredients.
121 °C sterilization cycle is run for 15 minutes but when the temperature is decreased to 115 °C, time of sterilization is increased for effective killing of the microorganisms. It is recommended to run the 115 °C sterilization cycle for 30 minutes or it can be calculated by the D value. Suppose D value at 115 °C is 3.75 and we want 8 log reduction then we have to run the sterilization cycle 3.75x8=30 minutes.
All sterilization processes must be validated. Low-temperature sterilization validation is done by using Bacillus subtilis (Bacillus atrophaeus) ATCC 5230. Strips and ampoules containing the spores of B. subtilis are sterilized with the media load similarly as the normal autoclave validation. Every strip contains 1x106 spores.
Spore strips are incubated in soybean casein digest medium at 35°C for seven days. Growth should not be observed for a valid sterilization process. The growth of the biological indicator is indicated by the turbidity.
Turbidity – Improper sterilization
No turbidity – Proper sterilization
If ampoules of Bacillus subtilis are used as a biological indicator then media should be released from the bulb for the growth of the spores. Ampoules are also incubated at 35°C for seven days and observed for the change of color of the ampoule.
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