Q.How will you avoid the growing of microbes in nutrient medium during culture process? What are the techniques used to remove the microbes?

Explant sterilization removes surface contamination through washing, ethanol treatment, a sterilant, and sterile rinsing under aseptic conditions.

In plant tissue culture, the explant (the piece of plant tissue used to start the culture) must be completely free of surface microbial contamination -- bacteria and fungi -- before it is placed on the nutrient medium, since any contaminant would rapidly overgrow the culture. The standard sterilization procedure involves the following steps:

1. Washing: the explant is first washed thoroughly under running tap water, sometimes with a mild detergent, to remove adhering dust, soil and surface debris.
2. Ethanol treatment: it is then briefly dipped in 70% ethanol for a short period (a few seconds to about a minute), which acts as a quick surface disinfectant.
3. Treatment with a sterilant: the explant is next treated with a suitable chemical sterilant for a controlled duration (typically a few minutes) -- most commonly a dilute solution of sodium hypochlorite (NaOCl), such as diluted commercial bleach, or mercuric chloride (HgCl2). These agents kill surface-adhering bacteria and fungal spores without excessively damaging the plant tissue if the concentration and exposure time are properly controlled. …

Sterilization eliminates all microbial contaminants; a tissue-culture room is sterilized using UV light, chemical fumigation, alcohol wiping and a laminar air-flow cabinet.

Sterilization is defined as the process by which all living microorganisms -- including bacteria, fungi, viruses and their resistant spores -- are completely killed or removed from an object, culture medium, instrument or an entire working area, so that no contaminating organism remains. This is critical in plant tissue culture because even a single contaminating microbe can outcompete and destroy an in-vitro plant culture, since the nutrient medium used is equally favourable for microbial growth.

Sterilizing a plant tissue-culture room typically involves:

1. UV (ultraviolet) light exposure: the culture/transfer room and laminar air-flow cabinet are exposed to UV light (usually for about 20-30 minutes before use) to kill airborne and surface microorganisms.
2. Chemical fumigation: the room is periodically fumigated using formaldehyde or glutaraldehyde vapour, which are effective disinfectants against a broad range of microorganisms.
3. Surface disinfection: work surfaces, the interior of the laminar flow hood, and instruments are wiped down with 70% ethyl or isopropyl alcohol (or another suitable disinfectant) before and during use. …