Laboratory Requirements for Tissue Cultur

General Organization
Localize each portion of the tissue culture procedure in a specified place in the laboratory. An
assembly-line arrangement of work areas (such as, media preparation, glassware washing,
sterilization, microscopy, and aseptic transfers) facilitates all operations and enhances cleanliness.
Media (tissue culture and nutrient agar) are available from Carolina Biological Supply Co.,
Burlington, NC. Laminar flow hoods are available from several suppliers.

Glassware
Use glassware that has only been used for tissue culture and not other experiments. Toxic metal
ions absorbed on glassware can be especially troublesome. Wash glassware with laboratory
detergent, then rinse several times with tap water and, finally, rinse with purified water.

High-purity Water
Use only high-purity water in tissue culture procedures. Double glass distilled water or
deionized water from an ion-exchanger are acceptable. Water should not be stored, but used
immediately. Regular maintenance and monitoring of water purification equipment are necessary.
Purified water for tissue culture can also be purchased.

Plant Material
Plants used in tissue culture need to be healthy and actively growing. Stressed plants,
particularly water-stressed plants, usually do not grow as tissue cultures. Insect and disease-free
greenhouse plants are rendered aseptic more readily, so contamination rate is lower when these
plants are used in tissue culture procedures. Seeds that can be easily surface sterilized usually
produce contamination-free plants that can be grown under clean greenhouse conditions for later
experimental use.

Aseptic Technique
The essence of aseptic technique is the exclusion of invading microorganisms during
experimental procedures. If sterile tissues are available, then the exclusion of microorganisms is
accomplished by using sterile instruments and culture media concurrently with standard
bacteriological transfer procedures to avoid extraneous contamination.
Media and apparatus are rendered sterile by autoclaving at 15 lbs/inch2 (121°C) for 15 minutes.
The use of disposable sterile plasticware reduces the need for some autoclaving. Alternative
sterilization techniques such as filter sterilization must be employed for heat-labile substances like
cytokinins.
Aseptic transfers can be made on the laboratory bench top by using standard bacteriological
techniques (i.e., flaming instruments prior to use and flaming the opening of receiving vessels prior
to transfer). 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).
If experimental tissues are not aseptic, then surface sterilization procedures specific to the
tissues are employed. Common sterilants are ethyl alcohol and/or chlorox with an added surfactant.
Concentration of sterilants and exposure time are determined empirically.