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Home >> Plant Tissue Culture >> Sterilization Methods Used in Tissue Culture Laboratory
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Sterilization Methods Used in Tissue Culture Laboratory - 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:
(i) dry heat treatment,
(ii) flame sterilization,
(iii) autoclaving,
(iv) filter sterilization,
(v) wiping with 70% ethanol, and
(vi) surface sterilization.

Dry Heat
Glassware and Teflon plasticware (empty vessels), and instruments may be sterilized by dry heat in an oven at 160-180°C for 3 hr. But most workers prefer to autoclave glass and plasticware, 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.

Flame Sterilization
Instruments like forceps, scalpels, needles, etc. are ordinarily 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 mouths of culture vessels prior to inoculation/subculture.

Autoclaving
Culture vessels, etc. (both empty and containing media) are generally sterilized by heating in an autoclave or a pressure cooker to 121°C at 15 p.s.i. (pounds per square inch; 1.06 kg/cm2) for 15 (20-50 ml medium) to 40 (21 medium) minutes.

Sterilization during autoclaving depends mainly on temperature. Certain types of plasticware and some instrument, e.g., micropipettes, etc., are also autoclavable. Care should be taken to properly stopper all the vessels and to open the autoclave only when its pressure gauge indicates zero pressure.

 
 
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