Question

Sterilization methods in tissue culture and applications

Answer 1

General Techniques:

Preparations of nutrient medium, steriliza­tion, aseptic manipulation, maintenance of cul­ture are the general techniques.

Technique # 1. Preparation of Culture Medium:tissues and organs get their appropriate nutrient and growth require­ments from the intact plant body for their organised growth and development. Isolated cell, tissues and organs also need nutrients for their in vitro growth and development. So, nutrients are supplied artificially according to the medium formulated by several workers. The main objec­tive of medium preparation is to culture the cell, tissue and organ in vitro.

Pour filtered sucrose solution and salt, vi­tamins

(v) Add 5% to 8% agar to the liquid medium to make solid medium. Heat to 60°C to dis­solve the agar completely. Otherwise, with­out adding agar, liquid medium can be used for culture.

Technique # 2. Sterilization Procedure:

Principle:

The culture medium, especially when it contains sugar, will also support the growth of micro-organisms like bacteria, fungi etc. So if they come in contact with medium either in cel­lular form or in spore form, the micro-organisms grow faster than the higher plant tissue due to their brief life cycle and will kill the tissue. The micro-organisms may come from glass vials, in­struments, nutrient medium used for culture and even from plant material itself. Therefore, the surface of plant tissue and all non-living articles including nutrient medium should be sterilized.

Procedure:

(i) Sterilization of non-living Articles:

The routine sterilization procedure of non-living articles such as nutrient medium, glass goods, autoclaving un­der steam at a pressure of 15 lb/in2 and a temperature of 120°C for 15 minutes.

Thermolabile compounds are often added in the medium and such medium is sterilized ei­ther at room temperature or in cold by passing through bacterial filter.

An alternative method of sterilizing glass goods and instruments is by heating in an oven at 150°C for 3-4 hrs.

It should be noted that when autoclaving screw capped glass vials, care should be taken to ensure that the caps are not closed too tightly so that gases can expand without the risk of explo­sion.

(ii) Sterilization of Plant Material:

Plant ma­terial which is to be cultured, should be sur­face sterilized to remove the surface borne microorganisms. This procedure is done in front of a laminar air flow or inside the in­oculation chamber before the plant mate­rial is inoculated onto the culture medium.

(1) Thoroughly washed plant material or ex- plant in tap water is immersed in 5% v/v solution of liquid detergent such as ‘Teepol’ for 10-15 minutes. Then wash the mate­rial thoroughly in tap water and finally in distilled water. This step can be done in the general laboratory. Subsequent steps are done in front of a laminar air flow or the pre-sterilized inoculation chamber.

(2) Dip the explants in 70% ethyl alcohol for 60 seconds.

(3) Immediately transfer the material into an autoclaved jaw bottle and pour 0.1% mer­curic chloride (HgCl2) 5-10% Sodium hypo­chlorite (v/v) solution. Keep them for 10- 15 minutes. During that period, the bottle is frequently swirled for shaking so that all surfaces of plant material come equally in contact with sterilant.

(4) After 10-15 minutes, decant the sterilant and wash the explants thoroughly with several changes of autoclaved distilled water to remove all traces of sterilant.

(5) Then the explants are ready for culture.

Technique # 3. Preparation of Aseptic Plants:

Principle:

Surface sterilization of plant tissue may cause some deleterious effect because most of the sterilants are toxic chemicals. Seeds can more or less resist such deleterious effect due to the presence of its seed coat. So to avoid the sur­face sterilization of plant tissue, seeds are sur­face sterilized and are cultured on simple basal nutrient medium.

Seeds in culture germinate and give rise to an aseptic seedling. Explants from such seedlings grown under aseptic and con­trolled conditions ar

(1) Wash the dry seeds thoroughly with tap wa­ter.

(2) Dip the seeds in 5% Teepol solution (v/v) for 10-15 minutes. Decant the Teepol so­lution and wash the seeds again with tap water and finally with distilled water.

(3) Rinse the seeds with 70% ethyl alcohol for 1

(4) Decant the sterilant and wash 3-4 times with autoclaved distilled water.

(5) Transfer the seeds from bottle to autoclaved petridish with the aid of sterile forceps.

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