Appendix 2 Maintenance of stock culture

Stock cultures can be maintained under lower temperatures (4-10 °C) for longer times without needing to be re-established. The Lemna growth medium may be the same as that used for testing but other nutrient rich media can be used for stock cultures.

Periodically, a number of young, light-green plants are removed to new culture vessels containing fresh medium using an aseptic technique. Under the cooler conditions suggested here, sub-culturing may be conducted at intervals of up to three months.

Chemically clean (acid-washed) and sterile glass culture vessels should be used and aseptic handling techniques employed. In the event of contamination of the stock culture e.g. by algae or fungi, steps are necessary to eliminate the contaminating organisms. In the case of algae and most other contaminating organisms, this can be achieved by surface sterilisation. A sample of the contaminated plant material is taken and the roots cut off. The material is then shaken vigorously in clean water, followed by immersion in a 0,5 % (v/v) sodium hypochlorite solution for between 30 seconds and 5 minutes. The plant material is then rinsed with sterile water and transferred, as a number of batches, into culture vessels containing fresh growth medium. Many fronds will die as a result of this treatment, especially if longer exposure periods are used, but some of those surviving will usually be free of contamination. These can then be used to re-inoculate new cultures.