Appendix 3 Example of a procedure for the culturing of algae

The purpose of culturing on the basis of the following procedure is to obtain algal cultures for toxicity tests.

Suitable methods must be used to ensure that the algal cultures are not infected with bacteria. Axenic cultures may be desirable but unialgal cultures must be established and used.

All operations must be carried out under sterile conditions in order to avoid contamination with bacteria and other algae.

See under Testing Method: Apparatus.

All nutrient salts of the medium are prepared as concentrated stock solutions and stored dark and cold. These solutions are sterilised by filtration or by autoclaving.

The medium is prepared by adding the correct amount of stock solution to sterile distilled water, taking care that no infections occur. For solid medium 0,8 % of agar is added.

The stock cultures are small algal cultures that are regularly transferred to fresh medium to act as initial test material. If the cultures are not used regularly, they are streaked out on sloped agar tubes. These are transferred to fresh medium at least once every two months.

The stock cultures are grown in conical flasks containing the appropriate medium (volume about 100 ml). When the algae are incubated at 20 °C with continuous illumination, a weekly transfer is required.

During transfer an amount of ‘old’ culture is transferred with sterile pipettes into a flask of fresh medium, so that with the fast-growing species the initial concentration is about 100 times smaller than in the old culture.

The growth rate of a species can be determined from the growth curve. If this is known, it is possible to estimate the density at which the culture should be transferred to new medium. This must be done before the culture reaches the death phase.

The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture is incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2 to 4 days. When the algal cultures contain deformed or abnormal cells, they must be discarded.