Maintenance of aseptic environment:
All culture vessels, media and instruments used in handling tissues as well as the explants must be sterilized. The importance is to keep the air surface and floor free of dust. All operations are carried out in laminar air-flow, a sterile cabinet. Infection can be classified in three ways:
1. The air contains a large quantity of suspended microorganisms in the form of fungal and bacterial spores.
2. The plant tissue is covered with pathogens on its surface.
3. The human body (a skin, breathe etc) carries several microorganisms.
In general, the methods of elimination of these sources of infection can be grouped under different categories of sterilization procedures:
1. Preparation of sterile media, culture vessels and instruments (sterilization is done in autoclave)
2. Preparation of sterile plant growth regulators stocks (by filter sterilization)
3. Aseptic working condition
4. Explants (isolated tissues) are sterilized using chemical sterilents, e.g. HgCl2 and NaOCl.
Sterilization: It follows that all the articles used in the plant cell culture must be sterilized to kill the microorganisms that are present.
A. Steam or Wet sterilization (Autoclaving):
This relies on the sterilization effect of super-heated steam under pressure as in a domestic pressure cooker. The size of the equipment used can be as small as one litre or even as large as several thousand litres. Most instruments/ nutrient media are sterilized with the use of an autoclave and the autoclave has a temperature range of 115- 1350C. The standard conditions for autoclaving has a temperature of 1210C and a pressure of 15 psi (Pounds per square inch) for 15 minutes to achieve sterility. This figure is based on the conditions necessary to kill thermophilic microorganisms. The time taken for liquids to reach this temperature depends on their volume. It may also depend on the thickness of the vessel. The temperature of 1210C can only be achieved at 15 psi. The efficiency of autoclave can be checked in several ways:
The most efficient way is to use an autoclave tape. When the autoclave tape is autoclaved, a reaction causes dark diagonal strips to appear on the tape indicating that it is autoclaved.
1. Excessive autoclaving should be avoided as it will degrade some medium components, particularly sucrose and agar breakdown under prolonged heating. Especially when under pressure and in an acidic environment. A few extremely thermoduraic microorganisms exist that can survive elevated temperature for sometime. But 15-30 minutes kill even those.
2. At the bottom of the autoclave the level of water should be verified.
3. To ensure that the lid of the autoclave is properly closed.
4. To ensure that the air- exhaust is functioning normally.
5. Not to accelerate the reduction of pressure after the required time of autoclaving. If the temperature is not reduced slowly, the media begin to boil again. Also the medium in the containers might burst out from their closures because of the fast and forced release of pressure.
6. Bottles, when being autoclaved, should not be tightly screwed and their tops should be loose. After autoclaving these bottles are kept in the laminar air-flow and the tops of these bottles are tightened on cooling.
B. Filter sterilization:
Some growth regulators like amino acids and vitamins are heat labile and get destroyed on autoclaving with the rest of the nutrient medium. Therefore, it is sterilized by filtration through a sieve or a filtration assembly using filter membranes of 0.22 Âµm to 0.45Âµm size.
It can only be carried out under condition where UV radiation is available. Consequently, its use is restricted generally to purchased consumables like petridishes and pipettes. UV lights may be used to kill organisms in rooms or areas of work benches in which manipulation of cultures is carried out. It is however, dangerous and should not be turned on while any other work is in progress. UV light of some wavelengths can damage eyes and skin.
D. Laminar Airflow Cabinet:
This is the primary equipment used for aseptic manipulation. This cabinet should be used for horizontal air-flow from the back to the front, and equipped with gas corks in the presence of gas burners. Air is drawn in electric fans and passed through the coarse filter and then through the fine bacterial filter (HEPA). HEPA or High Efficiency Particulate Air Filter is an apparatus designed such that the air-flow through the working place flows in direct lines (i.e. laminar flow). Care is taken not to disturb this flow too much by vigorous movements. Before commencing any experiment it is desirable to clean the working surface with 70% alcohol. The air filters should be cleaned and changed periodically.