Maintenance of aseptic condition, in plant tissue culture

Introduction
Aseptic technique
Sterilizing the culture vessels and instruments
Sterilization of culture media
Sterilizing Transfer area
Sterilizing culture rooms
Sterilizing Plant material
Transfer of the explants
Conclusions
References

1. plant tissue culture Maintenance of aseptic condition By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. Synopsis Introduction Aseptic technique Sterilizing the culture vessels and instruments Sterilization of culture media Sterilizing Transfer area Sterilizing culture rooms Sterilizing Plant material Transfer of the explants Conclusions References

3. INTRODUCTION Aseptic technique is absolutely necessary for the successful establishment and maintenance of plant cell, tissue and organ cultures. The in vitro environment in which the plant material is grown is also ideal for the proliferation of microorganisms. In most cases the microorganisms outgrow the plant tissues, resulting in their death . Contamination can also spread from culture to culture. The purpose of aseptic technique is minimize the possibility that microorganisms remain in or enter the cultures.

4. Aseptic technique Aseptic condition means free from all micro-organism. Propagation of plant tissues in culture requires aseptic techniques to ensure equipment is free of contaminants such as bacteria, fungi, or algae, that may otherwise quickly overgrow the culture and destroy it. Aseptic techniques combine protocols to remove contaminants from material (sterilization, disinfection) and procedures to maintain sterility during manipulation of material.

5. Glassware and plastic ware washing Detergents especially designed for washing laboratory glassware and plastic ware are available. After soaking in detergent solution for a suitable period (preferably overnight) the apparatus is thoroughly rinsed first in tap water and then in distilled water.

6. Sterilizing the culture vessels and Instruments Hot air oven Glass culture vials, metal instruments and aluminium foil can all be sterilized by exposure to hot dry air (160°-180 °C) for 2-4 hr in a hot air oven. Autoclaving Autoclaving is the method of sterilizing with water vapour under high pressure. Nearly all microbes are killed on exposure to the superheated steam of an autoclave. In order to be sterilized, the item must be held at 121°C, 15 psi, for at least 15 minutes.

7. Flame sterilization The instruments used for aseptic manipulations, such as forceps, scalpels, needles, and spatula, are normally sterilized by dipping in 75% ethanol followed by flaming and cooling. This technique is called flame sterilization . This is done at the start of the transfer work and several times during the operation. Ethylene Oxide Gas Plastic containers that cannot be heated are sterilized commercially by ethylene oxide gas. These items are sold already sterile . Examples of such items are plastic petri dishes, plastic centrifuge tubes etc .

8. UV Radiation It is possible to use germicidal lamps to sterilize items in the transfer hood when no one is working there. We do not do this. UV lamps should not be used when people are present because the light is damaging to eyes and skin. Plants left under UV lamps will die.

9. Sterilization of culture media Two methods are commonly used to sterilize culture media. 1.autoclaving The Culture media, distilled water, and other heat stable mixtures can be autoclaved in glass containers that are sealed with cotton plugs, aluminum foil, or plastic closures. And the medium is autoclaved (steam heating under pressure) at 121°C for 15-40 min from the time.

10. 2.membrane filtration Sterilization Organic compounds such as some growth regulators, amino acids, and vitamins may be degraded during autoclaving. These compounds require filter sterilization through a 0.22 μm membrane. Several manufacturers make nitrocellulose membranes that can be sterilized by autoclaving. Fig. - ' Swinnex ' Millipore filter assembly for sterilizing small volumes of liquids.

11. Sterilizing Transfer area The transference room, where the laminar flow chambers are, is a place which must be kept very clean, since it is next to the culture room. In this area, great care must be taken in the laminar flow chamber, the internal walls must be cleaned with alcohol (70%). The filters and pre-filters ( Hepa ) within the chambers must be continuously revised to avoid contamination. During propagation, hands and table surface will be continuously cleaned with a piece of cotton soaked in alcohol. Aseptic transfers are more easily performed in a transfer chamber such as a laminar flow hood, which is also preferably equipped with a Bunsen burner ( Bottino , 1981).

12. Laminar flow hood

13. Sterilizing culture rooms The disinfection of floors and shelves must be continuous. The tube racks must be cleaned with a fungicide solution before they are put into the culture room for the first time. The inside doors and walls must be frequently examined to avoid the preserve of fungi. The air-conditioner must be regularly checked .

14. culture rooms

15. Sterilizing Plant material Surfaces of plant parts carry a wide range of microbial contaminants. To avoid this source of infection the tissue must be thoroughly surface sterilized before planting it on the nutrient medium. surface sterilization by treatment with a disinfectant solution at a suitable concentrations for a specified period.

16. The procedure is as follows:- The explants are washed with tap water. They are washed thoroughly by keeping them under running tap water for an hour. After washing, the material is treated with freshly prepared saturated chlorine water for 2 minutes. It is then rinsed with sterile distilled water. The material is surface-sterilized with freshly prepared 0.1% mercuric chloride solution for 1 minute, followed by 4–5 times with sterilized distilled water to remove all traces of contamination.

17. Transfer of the explants The surface-sterilized explants are transferred to an inoculation chamber where aseptic conditions are maintained. During this process dust, hair, hands and clothes are potential sources of contamination, against which it is essential to take precaution.

18. Conclusions The maintenance of sterility is achieved by providing a workspace that minimizes the chance of introducing contaminants into cultures. This is impotent for commercial tissue culture for which the contamination could be hazardous.

19. References Plant Tissue Culture, a Revised Edition – S. S. Bhojwani and M. K. Razdan (1990 Elsevier Science B.V .) Plant Tissue Culture – K. K. Day