Sterilization:-
All the materials, e.g., vessels, instruments, medium, plant material, etc., used in culture work must be freed from microbes. This is achieved by one of the following approaches:
1. Dry heat,
2. Flame sterilization,
3. Autoclaving,
4. Filter sterilization,
5. Wiping with 70% ethanol,
6. Surface sterilization,
1.Dry Heat
Glassware and Teflon plastic ware, and instruments may be sterilized by dry heat an oven at 160-180°C for 3 hours. But most workers prefer to autoclave glassware and plastic ware etc. And flame sterilize instruments like forceps, etc. More recently, glass bead sterilizers (300°C) are being employed for the sterilization of forceps, scalpels, etc. these devices use dry heat.
2.Flame Sterilization
Instrumental like forceps, scalpels, needles, etc. Are ordinary flame sterilized by dipping them in 95% alcohol followed by flaming. These instruments are repeatedly sterilized during the operation to avoid contamination. It is customary to flame the mouth of culture vessels prior to inoculation.
3.Autoclaving
Culture vessels, etc. Are generally sterilized by heating in an autoclave or a pressure cooker to 121°C at 15p.s.i. (pounds per square inch 1.06kg/cm2) for 15 to 40 minutes, The time being longer for larger medium volumes. Sterilization during autoclaving depends mainly on temperature. Certain types of plastic ware and some instruments, e.g., micropipettes, etc. Are also autoclave. Care should be taken to properly stopper all the vessels and to open the autoclave only its pressure gauge indicates zero pressure.
4. Filter Sterilization
Some growth regulators, e.g., GA3, zeatin, ABA (abscisic acid), urea, certain vitamins, and enzymes are heat labile. Such compounds are filter sterilized by passing their solution through a membrane filter of 0.45μ or lower pore size. The membrane filter is held in a suitable assembly, the assembly together with the filter is sterilized by autoclaving before use. Filter sterilized heat labile compounds are added to autoclaved and cooled media, in case of agar medium, they are added when the medium has cooled to about 40°C and is still liquefied.
Laminar flow cabinets are used to create an aseptic working space by blowing filter-sterilized air through an enclosed space. The air is first filtered through a coarse profiteer to remove large particles, it is then passed through a HEPA (high efficiency particulate air) filter, which filters out all particles larger than 0.3μm. This sterilized air blows through the cabinet at 1.8 km/hr. which is sufficient to keep the enclosed working area aseptic.
The surfaces that can not be sterilized by other techniques, e.g., platform of the laminar flow cabinet, hands of the operator, are sterilized by wiping them thoroughly with 70% ethyl alcohol is allowed to dry.
6. Surface Sterilization
All the plant materials to be used for culture are treated with an appropriate sterilizing agent to inactive the microbes present on their surface, This is called surface sterilization. Surface sterilization protocol will depend mainly on the source and the type of tissue of the explant is the excised piece of tissue or organ used for culture.
The sterilizing agent used for surface disinfection are calcium hypochlorite (9-10%), sodium hypochlorite (2%), mercuric chloride (0.1-1%), silver nitrate (1%), Bromine water (1-2%), H2O2 (10-12%) and antibiotics (4-50 mg/l). Of these, calcium or sodium hypochlorite and HgCl2 (satisfactory results) are the most commonly used. The duration of treatment varies form 15-30min. Since these agents are also toxic to plant tissues, the duration and the concentration used should be such as to cause minimum tissue death, and the rinsing after treatment should remove them as completely as possible.
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