The following points highlight the five main steps for aseptic manipulation. The steps are: 1. Sterilisation of the Culture Vessels and Instruments 2. Sterilisation of Nutrient Media 3. Sterilisation of Culture Rooms and Transfer-Area 4. Sterilisation of Explant 5. Aseptic Transfer of Explant/Sub-Culturing.
Step # 1. Sterilisation of the Culture Vessels and Instruments:
For sterilisation of culture vessels, glassware’s, instruments, etc. it is better to use steam sterilisation techniques i.e., by autoclaving at high pressure (15 pound/inch2) and high temperature (121°C) for 15-20 mins. All the items should be properly covered with aluminium foil before sterilisation. Glassware’s, metal instruments can also be sterilised by exposure to dry hot air oven (160°-180°C) for 2-4 hrs.
Plastic labware of special types, cotton plugs, gauze, filters can be sterilised using steam sterilisation technique. The autoclaved instruments like forceps, scalpels, needles, spatula are further sterilised by flame sterilisation technique before use by dipping in 95% alcohol, and followed by flaming and cooling.
Step # 2. Sterilisation of Nutrient Media:
The nutrient media used in tissue culture are commonly sterilised by autoclaving and filter sterilisation. Macro-, micronutrients and other stable compound mixtures are autoclaved, whereas the thermolabile compounds are filter-sterilised separately and mixed with the media whenever necessary. Culture media containing high conc. of sugars when autoclaved the carbohydrates get decomposed under high pressure.
Some vitamins, amino acids, plant extracts and hormones are thermolabile, they require filter sterilisation. The solutions are passed through a bacterial membrane filter under pressure (pore size 0.2 µm), the sterilised solution is then mixed separately with autoclaved media.
Step # 3. Sterilisation of Culture Rooms and Transfer-Area:
The floor and walls of culture rooms are cleaned by gently washing with detergent then by wiping them with 2% sodium hypochlorite solution and then using ethanol. Commercially available disinfectants like Lysol can be used for this purpose. The process of surface sterilisation of culture rooms should be done at regular intervals.
The transfer-area is also sterilised once or twice a month by washing with common brand of antifungal agent. Transfer rooms or areas are further sterilised by UV light. UV radiation is harmful for eyes, so UV radiation should be done before operation.
Where laminar air flow cabinet is used for transfer, the surface is cleaned by ethanol and chamber is sterilised by UV light before work in progress. Then the sterile air is flowed through HEPA filter during works.
Step # 4. Sterilisation of Explant:
All different kinds of plant materials or explants should be surface sterilised by a variety of chemicals before using in tissue culture (Fig. 16.3). It is the eradication of surface microorganisms with the help of different chemicals. The type and concentration of different chemical sterilant to be used for sterilisation of different types of explants and exposure time must be decided experimentally.
Sometimes the sterilisation procedure may lead to lethality to plant tissue, so the use of disinfectants should be tested. Here are few disinfectants which are used commonly for surface sterilisation of different explants (Table 16.2).
(a) 1% solution of Sodium hypochlorite (NaClO), commercial bleach having 5% active chlorine can be used.
(b) 4%-l0% solution of Calcium hypochlorite [Ca(ClO)2] can be used, it enters within the plant tissue slower than sodium hypochlorite.
(c) 1% solution of Bromine-water.
(d) 0.01-0.1% solution of Mercuric Chloride (HgCl2) which is an extremely toxic substance for plant tissue, repeated rinsing with water is very much essential.
(e) 70% Ethyl alcohol is used for sterilisation of plant material dipping them for 30 sec-2 mins.
(f) 10% Hydrogen peroxide (H2O2) solution is effective for end surface sterilisation.
All these sterilants should be washed out properly before using the explant as the retention of these chemical substances may affect the establishment of successful tissue culture. But in most of the cases it becomes difficult to determine the optimal conditions for each kind of tissue.
So to avoid this problem the explants can be taken from aseptically grown plants developed from the surface sterilised seeds as these seeds are more resistant to chemicals due to presence of seed coat.
For this purpose, the seeds are surface sterilised and then cultured aseptically in basal nutrient media. These give rise to aseptic seedlings from which the different explants can be used. Explants from such seedlings need no further sterilisation (Fig. 16.4).
But for another culture and shoot tip culture the explants are collected from outside grown plant. For these kinds of explants addition of few drops of surfactant (Trito-X or Tween-20) to the solution or treating the plant material in a solution of Cetavlon for 2 min. before exposing to sterilant may enhance sterilisation efficiency.
Step # 5. Aseptic Transfer of Explant/Sub-Culturing:
Control of contamination largely depends upon the operator’s technique while transferring the sterilised explant/sub-culturing into the sterile culture vial containing nutrient media under aseptic condition. Dust from the surface, hair, hands and clothes are the potential sources of contamination.
Before starting the transfer procedure the surface of transfer area and hands should wiped with 95% ethanol; sterile clothes (aprons) should be used. All the metallic equipment’s used for transfer (inoculating needle, forceps, scalpel) should be dipped into 95% ethanol and then flamed and cooled. The tissue material should not touch the edge of culture vessel during transfer.