Plant tissue culture is done under aseptic conditions. For this, we require good knowledge of sterilization processes. For plant tissue culture we use glasswares, instruments, gloves, lab coats, and different articles. These articles must be free from vegetative as well as spores of microorganisms.
As we know that for aseptic conditions different strategies are used such as cleaning, disinfectant, and then sterilization.
Cleaning is removing the visible dirt, soil, or spots on the objects. For cleaning, we usually use soap or detergents.
Disinfection
By disinfection, we kill all vegetative microorganisms, but these processes do not kill all types of spores of microorganisms. So, the main principle of disinfection is to reduce microorganisms to a level so that they cannot multiply easily. Ethanol, H2O2 are used as a disinfectant. This method is used for non-living objects.
Sterilization
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| DIFFERENT METHODS OF STERILIZATION |
It is killing the microorganisms in all forms that are, in vegetative form or in spore stages.
Sterilization uses different processes. sterilization is done by the physical, and chemical methods that are widely used in plant tissue culture. The physical methods use sunlight, heat, radiation, and filtration, and chemical methods use different Chemicals to sterilize the objects.
In the plant tissue culture laboratory, we usually use heat radiation and filtration for sterilization. Heat comes under two categories dry heat and moist heat for dry heat we use red heat, flaming, incineration, hot air oven, or infrared rays.
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| STERILIZATION USING HEAT |
Dry heat
This process uses heating items to sterilize them. Instruments are sterilized by dry heat. There are different methods used such as
Red heat
It is heating the instrument to be red hot, this method is used for culture loops, blade handles, forceps, etc.
Flaming is passing the instrument through the flame. The metal instruments are sterilized by this method.
Incineration is the sterilization of the used items, here the infected items are incinerated before they are discarded.
Hot air oven
Hot air is used to sterilize instruments, apparatus, and glassware. Washed and dried instruments are sterilized using a hot air oven. Instruments are arranged in a hot air oven so that hot air can freely circulate between the instruments. Temperature is maintained at 60ºC in starting and then the instruments are sterilized at 120ºC for 60 min. It kills vegetative as well as spores of microorganisms.
Wet heat there can be three different processes. Media and sera are sterilized by this method.
Below 100ºC includes pasteurization, where the content is heated at 60ºC for 30 minutes and then cooled to 13ºC, the serum is sterilization is done by this method.
A hundred degrees and then above 100ºC, for laboratory we use above 100ºC sterilization and autoclaving.
Autoclave
The autoclave uses a temperature of 121ºC and it uses high pressure. Concentrated steam is used to sterilize the objects. It is used to sterilize non-living items. Concentrated steam at 121ºC temperature. When the steam is condensed on the surface of the object, it heats the surface to such a high temperature that kills the microorganisms and their spores.
Filtration
For filtration in the laboratory, membranes are used so that microorganisms cannot pass through them and these membrane filters are used in laminar airflow. LAF is used to maintain sterilized conditions during culture experiments.
Chemicals
Chemicals used for sterilization in the laboratory are alcohol, phenol, chlorine solutions. HgCl2 is widely used for sterilizing surface sterilizing of the living tissue.
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