106 Marine Microbiology 



Richard E. Nonis who purified it, was abundant in a pond in 

 Berkeley Aquatic Park, California. Amphidinium rhynchoceph- 

 alum Anassimowa, from a sample of water of the inland Salton 

 Sea, and A. carter ii'' from a unialgal culture kindly supplied by 

 Dr. Robert Guillard (isolated from Great Pond, Falmouth Mass.) 

 were both purified in 1955 by transfer and growth for fifteen days 

 in a medium containing 600 units/ml of penicillin followed by a 

 few micropipette washings. 



All the strains of dinoflagellates, except A. carterii and 

 Exuviella cassuhica, were lost because of repeated failures of the 

 refrigerator-incubators. Knowledge of their nutrition is therefore 

 sketchy. Cultures were grown in screw-capped tubes, incubated 

 at 15 to 20 C under 200 to 400 foot-candles of "cool white" 

 fluorescent light. 



GROWTH IN ARTIFICIAL MEDIA 



Construction of reproducible, non-precipitating marine me- 

 dia was approached in two convergent ways: a) by studying the 

 nutrition of single marine species in the hope of finding common 

 requirements; b) by finding ways to simplify sea water-like solu- 

 tions, to avoid precipitation during autoclaving, and to buffer 

 the artificial media. 



G. calif orniciim, as the first marine organism obtained by 

 us in bacteria-free culture, was a major test organism in devising 

 good media (15, pp. 402-10). An early medium for G. calif orni- 

 ciim ( 13 ) shows how clumsy were the first attempts ( Table 1 ) . 

 When we began with marine organisms it was known that 

 Gymnodinitim splendens grew in aged seawater enriched with 

 N, P, and soil extract (17). Soil extract for freshwater algae 

 could be replaced by B-vitamins and trace metals (13). Soil 

 extract was similarly replaced for G. californicmn. But, to avoid 

 precipitates, we employed a high concentration (20 mg5^) of 

 ethylenediamine tetraacetic acid (EDTA). Such concentrations of 

 EDTA created difficulties. In calcium-rich media (and in solu- 

 tions similar to seawater) EDTA forms the dicalcium salt which 

 is poorly soluble; also the excess EDTA had to be compensated 



* Formerly designated as A. klebsii. 



