Posted Date: 30-Jan-2008
? Sterilization is a procedure used for the elimination of microorganisms. The maintenance of aseptic or sterile conditions is essential for the successful tissue culture procedures.
? The need for asepsis require that all culture vessels, media and instruments used in handling tissue as well as explants itself be sterilized. All the operations should be carried out in laminar airflow cabinets.
? An obvious precaution is not to share these cabinets and areas where tissue culture works being carried out with other microbiologists and plant pathologists.
? If tissue culture work itself requires screening against the pathogens or isolation of the toxins etc. then pathological work must be separated. In general different sterilization procedures can be groped into three categories.
? Preparation of sterile media, containers and small instruments
? Maintenance of aseptic conditions
? Preparation of sterilized explants material.
Preparation Of Sterile Media Containers And Small Instruments
The most popular method for sterilizing culture media and small apparatus is by autoclaving the material using steam/ dry sterling.
? The nutrient media are generally sterilized by using an autoclave. The standard autoclaving conditions are 121 0C temperatures at a pressure of 15 psi for 20min. These conditions are followed for test tubes or other containers containing between 20-50 ml of nutrient media.
? Good sterilization relies on the time, pressure, temperature and volume of object to be sterilized. It must be realized that heat penetration is very important in an autoclave so larger volume must be sterilized for longer as heat will take longer time to penetrate than with smaller volume.
? The advantage of an autoclave are speed, simplicity and destruction of viruses, while disadvantages are change in pH by 0.3-0.5 units, component can separate out and chemical reaction can occur resulting in loss of the activity of media.
? The following guidelines must be followed.
? Test tubes/ flasks containing between 20-50ml nutrient media: 20 min at 121 0C and 15psi
? Flasks containing between 50-500ml nutrient media: 25 min at 121 0C and 15psi
? Flasks containing between 500-5000mlnutrient media: 35 min at 121 0C and 15psi
? The sterilization of glassware and metallic instruments can be carried out in dry heat at 160 0C -180 0C for 3hr.
? An exposure of 160 0C dry heat for 2hr is regarded as equivalent to the moist heat sterilization at 121 0C for 15 min.
? Dry goods can either be wrapped in aluminum foil, brown paper or sealed metal containers to maintain the sterility.
? Some growth factors, amino acids, vitamins and toxins are heat labile and get destroyed during the autoclaving with rest of the medium. These chemicals are therefore sterilized through a sieve or filter assembly using filter membrane of 0.45 to 0.22µm. Most filters are of cellulose acetate or cellulose nitrate and are available in pre-sterilized plastic disposable units.
? During filter sterilization the basic medium without substance X is autoclaved in a flask. While medium is sill liquid (45-50 0C), the substance X is injected through a filter assembly. Now medium containing X is stirred and poured into pre-sterilized containers. The whole procedure is carried out in the laminar airflow cabinet.
? Pre-sterilized disposable plastic ware is generally sterilized by ultraviolet radiations. The sterilized autoclaved medium is then poured in to these containers in a laminar air flow cabinet. The laminar airflow cabinets are generally also fitted with UV lamp to sterilize the cabinet before carrying any operation in these.
Maintenance of aseptic conditions
The following procedures/techniques are employed during aseptic manipulations.
? It is essential that workers hands be relatively aseptic during manipulations work. A wash with an antibacterial detergent followed by spraying 70% ethanol on hands is quite effective. The laminar air flow should also be sprayed with70-90% ethanol before use.
? This method is used for the sterilization of the instruments during aseptic manipulations. The instruments are soaked in 70-80% alcohol followed by flaming on a burner in laminar air flow hood.
? This is essential even if instruments have been subjected to dry or steam sterilization and is done frequently during manipulations.
? A glass beads sterilizer is often used in commercial units instead of flame sterilization. It is an electric appliance in which an electric element is used to heat the small glass beads kept in a cylindrical space. The tissue culture manipulating instruments are kept in these beads while they are not in use during aseptic manipulations.
Sterilization of Explants
? Plant material can be surface sterilized by variety of chemicals. Some of the commonly used chemicals and their effectiveness are shown in comparative form in table below.
? It is frequently seen that overzealous sterilization leads not only to complete removal of microorganisms but also lethal to the plant tissue. So it is therefore important to determine the optimal conditions for each tissue.
S. No. Sterilizing Agent Conc. Ease of Removal Treatment time Remarks
1 Sodium hypochlorite 1-1.4% +++ 5-30 min Very effective
2 Calcium hypochlorite 9-10% +++ 5-30 min Very effective
3 Hydrogen peroxide 10-12% +++++ 5-15 min Effective
4 Bromine water 1-2% +++ 2-10 min Very Effective
5 Silver nitrate 1.0% + 5-30 min Effective
6 Mercuric chloride 0.1-1.0% + 2-10 min Satisfactory
7 Antibiotics 4-50mg/l ++ 30-60 min Effective
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Responses to "Sterilization Techniques"
Guest Author: Venice Maron 13 Jan 2013
This article on Sterilization Techniques helps little bit, specially on surface sterilization techniques for plants tissue culture and other basic sterlization technique. Very informative, regards
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