5.4 Sterile Technique
5.4.1 Clean Equipment
Successful tissue culture requires the maintenance of a sterile environment. All tissue
culture work is done in a laminar flow hood. The laminar flow hood filters air with a
dust filter and a high-efficiency particulate air (HEPA) filter (Fig. 5.3). It is important to
keep the hood clean, which can be done by wiping it with 70% alcohol. The instruments
used should also be dipped in 70% ethanol and sterilized using flame or glass beads.
Hands should be disinfected with ethanol before handling cultures in order to avoid con-
tamination. It is imperitive to maintain axenic conditions throughout the life of cultures:
from explant to the production of whole plants. Entire experiments have been lost
because of an episode of fungal or bacterial contamination at any stage of culture (see
Fig. 5.4). Especially problematic are fungal contaminants that are propagated by spores
that might blow into a hood from an environmental source. Therefore, it is important to
work away from the unsterile edge of a laminar flow hood. Culture rooms or chambers
must be maintained as clean as possible to control any airborne contaminants.
5.4.2 Surface Sterilization of Explants
Plant tissues inherently have various bacteria and fungi on their surfaces. It is important that
the explant be devoid of any surface contaminants prior to tissue culture since contaminants
can grow in the culture medium, rendering the culture nonsterile. In addition, they compete
with the plant tissue for nutrition, thus depriving the plant tissue of nutrients. Bacteria and
especially fungi can rapidly overtake plant tissues and kill them (Fig. 5.4). The surface ster-
ilants chosen for an experiment typically depend on the type of explant and also plant
species. Explants are commonly surface-sterilized using sodium hypochlorite (household
bleach), ethanol, and fungicides when using field-grown tissues. The time of sterilization
is dependent on the type of tissue; for example, leaf tissue will require a shorter sterilization
time than will seeds with a tough seed coat. Wetting agents such as Tween added to the
Figure 5.3.Researcher working in the laminar flow hood.118 TISSUE CULTURE: THE MANIPULATION OF PLANT DEVELOPMENT