Washing and Sterilization for Plant Tissue Culture, Glassware, Distilled Water, Agar medium, Microbial Contaminents

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Washing and sterilization
New glassware is always washed using detergents especially designed for the purpose of removing all traces of acids, etc. Finally the glassware is rinsed in tap water and then in distilled water. Sometimes, the glassware may need to be autoclaved before washing to remove agar medium and to destroy microbial contaminants
The culture medium and the glassware are autoclaved at 100°C for 20 min. If autoclave is not available, a pressure cooker may be used. Growth factors, such as GA3, Zeatin, ABA, urea, etc. (which are Thermolabile) should not be autoclaved, but sterilized separately by membrance filtration. Instruments, such as forcepts, scalpels, needles, spatula are sterilized by dipping in 96% ethanol followed by flaming and cooling

Plant material is surface sterilized using sodium or calcium hypochlorite solution (0.3 – 0.6%) for 15-30 min. for ovule, embryo and endosperm, culture, only the seeds are surface sterilized and the ovule or embryo or endosperm is dissected under aseptic conditions (usually in a laminar air flow cabinet). Similarly for shoot apices, the shoot buds are surface sterilized and the apices are dissected under aseptic conditions. After surface sterilization, plant material is rinsed 3-4 times in sterile distilled water

All operations including transfer of plant materials are carried out under aseptic conditions, preferably under the hood of a laminar air flow cabinet, which is commercially available for tissue culture laboratories. These cabinets have now replaced UV – sterilization wooden cabinets earlier utilized for this purpose

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