2.  Establishing aseptic cultures

Plant tissue culture media contain sugar and so support the growth of many microorganisms (bacteria and fungi). When these microorganisms reach a medium, they generally grow much faster than the cultured plant materials. Their growth and toxic metabolites will affect, and may even kill, the tissue cultures. It is, therefore, essential to maintain a completely aseptic environment inside the culture vessels.

There are several possible sources of contamination of the medium:

• •  the culture vessel

•  the medium itself

•  the explant (plant tissue)

•  the environment of the transfer area

•  the instruments used to handle plant material during establishment and subculture

•  the environment of the culture room.

Autoclaving media will eliminate contamination from the culture vessel or the medium. In some cases, substances such as gibberellic acid, abscisic acid (ABA), urea and certain vitamins are thermolabile and break down upon autoclaving. These chemicals can be sterilized by membrane filtration using microfilters of pore size 0.22-0.45 µm which is suitable enough to exclude pathogens. Later the filter sterilized compound can be added to autoclaved medium cooled to around 40°C.

To prevent the environment of the culture room from being the source of contamination, keep the culture room as dust- free as possible and remove contaminated cultures from the area as soon as they are detected. Ideally, the culture room should be clean, filtered air which has passed through high efficiency particulate air (HEPA) filters.

The transfer area in most laboratories is within a laminar air-flow cabinet. A laminar air-flow cabinet has a small fan which blows air through a coarse filter to remove large dust particles and then through a fine HEPA filter to remove microbes, their spores and other particles larger than 0.3 µm. The velocity of the air coming out of the fine filter is about 27 ± 3 m/min, which keeps airborne microorganisms out of the working area. The working area is swabbed with 70% alcohol (or equivalent) and instruments dipped in 70% alcohol, flamed and cooled before use.

Caution : Prolonged contact with alcohol can cause skin irritation, and other health problems can result from the inhalation of fumes. Use ethanol rather than methanol, and surgical gloves when handling. Take care with ultraviolet light as it can permanently damage eyes and promote skin cancer. Laminar flow cabinets equipped with ultraviolet light for surface sterilization should be fitted with safety doors which can be closed when ultraviolet light is used.

Plant surfaces carry a wide range of microorganisms. The tissue must be thoroughly surface-sterilized before being placed on the nutrient medium. Discard cultures with fungal or bacterial contamination. Solutions of sodium or calcium hypochlorite are usually effective in disinfecting plant tissues. Placing tissues in a 0.5 to 1% solution of sodium hypochlorite for 10 to 15 minutes will disinfect most tissues. Surface sterilants are toxic to plant tissues. Choose the concentration of the sterilizing agent and the length of time to minimize tissue damage, which shows up as white, bleached areas. Other techniques for surface sterilisation include dipping plant material for a few seconds in 90% ethanol or placing in running water for 30 minutes and 2 hours before disinfection.