An efficient method to obtain axenic cultures of Alexandrium tamarense—a PSP-producing dinoflagellate

The fact that species of harmful algae maintained in the laboratory harbor a complex bacterial flora increases the difficulties involved in the study of the relationship between bacteria and algae. An efficient method to remove bacteria from a laboratory culture of the marine dinoflagellate Alexandrium tamarense is presented in this paper. The alga was subjected to repeated washing, lysozyme/SDS and antibiotic treatment with a mixture of gentamycin, streptomycin, cephalothin and rifampicin. Axenic status was confirmed after subculturing three times in sterile f/2 medium without antibiotics. Bacteria could not be detected in various media, both solid and liquid, nor by epifluorescence microscopy and PCR amplification of 16S rDNA of both eubacteria and archaea. Bacterial presence was monitored throughout a full growth cycle and, following subculture, no bacteria were detected using the above methods. This method is more efficient and less time-consuming than other methods and the resultant axenic A. tamarense cultures would provide a simpler system for further study of bacteria–alga interactions.