used. Similar figures were obtained for the 

 dinoflagellate Prorocentrum micans by Kain and 

 Fogg (I960) with fluorescent light, but satu- 

 ration occurred at only 300 ft. -c. with tungsten 

 illumination. Our values for saturation of 

 Gymnodinium growth are much less than those 

 observed for dinoflagellate photosynthesis by 

 Ryther (1956). He observed saturation at 2,500 

 ft.-c. There may be a difference between 

 optimum light requirements for growth, and 

 those for photosynthesis. 



The remaining work on Gymnodinium con- 

 cerned its chemical nutrient requirements. 

 Various artificial sea waters have been tried 

 as substitutes for sea-water media. The basic 

 medium tried with Gymnodinium was Provasoli's 

 (1957) ASP- 2, which contains all the major 

 salts and minor nutrients present in sea water , 

 plus vitamin B12 ^'^'^ other vitamins known 

 to stimulate growth. This mediunri was also 

 modified by substituting nitrilotriacetic acid 

 for EDTA as a chelating agent, changing the 

 trace metal content, and by adding soil extract. 

 Some growth in ASP- 2 occurred when soil 

 extract was added, but this was nnuch less 

 than in enriched sea-water controls; other- 

 wise, no growth occurred in ASP-2 or various 

 modifications thereof. Toxicity of artificial 

 sea water was shown by the loss of cellular 

 motility, cell enlargement, and cell wall thick- 

 ening. Some growth occurred in another arti- 

 ficial mediunn, ASP-6. However, none of the 

 various modifications resulted in any in- 

 crease of growth over a sea-water medium. 

 These experin-ients indicate that some unknown 

 component in sea water is necessary for 

 maxinnum growth of Gymnodinium. 



The nitrate requirements of the Dome iso- 

 late were studied in separate cultures con- 

 taining sea-water nnedia prepared with con- 

 centrations of added nitrate ranging fronn 

 0-2,000 fi. g.-at./l. These were incubated at 

 800 foot-candles and 25° C. The growth rate 

 was identical in all cultures. This suggested 

 that Gymnodinium could effectively utilize con- 

 centrations of nitrate as low as those supplied 

 by the sea water and soil extract in these 

 media. 



This was confirmed by growing the alga 

 (Dome isolate) in a mass culture containing 

 sea water enriched with phosphate, EDTA, soil 

 extract and 50 |x g.-at./l. of added nitrate. 

 Samples were taken at intervals of 12-16 

 hours for cell counting and for analyses of the 

 nitrate concentration of the mediunn. In two 

 such experiments, the growth rate decreased 

 when a concentration of 3 to 7 fj, g.-at./l. of 

 nitrate remained. Nitrate was shown to be a 

 limiting factor by adding nitrate to separate 

 portions of the cell suspension after the rate 

 had decreased. The original rate was nearly 

 restored by this addition. When growth in the 



naass culture ceased, the nitrate concentration 

 in the medium was 1 (i g.-at./l. 



Calculations of the amount of nitrogen re- 

 quired per Gymnodinium cell were made on a 

 rate basis by dividing the increments of change 

 of cell nunnbers by the incrennents of change 

 of nitrate between sampling times. They were 

 also made by dividing the final population by 

 the amount supplied. The imean of these deter- 

 n-iinations, with both methods, was 1.1 

 fJ.g.-at.N/cell; on a cell volume basis it was 

 0.49 /xg.-at.N/mm^. Kain and Fogg (I960) 

 give values for various phytoplankton which 

 range from 0. 1 9 to 1 .6 ji. g.-at.N/mm.^ 



Comparison of the nitrate concentration in 

 surface water of the eastern tropical Pacific 

 Ocean with the rate-limiting concentration 

 of 3 to 7 (1 g.-at./l. shows that the latter is 

 fivefold to tenfold greater than the surface 

 concentration except at Dome stations where 

 the two are equal (table 3). Thus theCymno(/Jnium 

 should grow at its maximum rate in the 

 Dome area, where it was originally obtained, 

 provided that factors other than nitrate are 

 also not limiting. 



Experiments on other nitrogenous nutrients 

 and on phosphate requirements are planned. 



Results with ^annocA^oris .- -This alga was ob- 

 tained from station 19 of cruise TO-58-2, off 

 the Gulf of Tehuantepec. It is a tiny green rod, 

 about 2-3 (J, in length and 1 \j. wide. Dr. J. B. 

 Lackey tentatively identified it as a species 

 of Chloretla. However, it reproduces without the 

 release of autospores, and therefore is prob- 

 ably a species of Nannochloria. 



The light and temperature requirements of 

 this alga have not yet been studied in detail. 

 However, stock and experim.ental cultures 

 have been grown at 500 ft.-c. and 21° C. 



Nannochloria grows just as well in ASP-2 

 (artificial sea water) as in the enriched 

 sea-water medium. However, if vitamin B12 

 is omitted from ASP-2, the alga grows poorly 

 and does not survive a second transfer. If 

 this result can be obtained consistently the 

 organisnn could serve as a useful bioassay 

 organism for B12 i^i sea water: it has a short 

 generation time (8-10 hours), its growth could 

 be measured easily by turbidimetric nnethods, 

 and internal stores of B12 could be reduced 

 by growing it in ASP-2 without Bj2 before the 

 assay. 



Nitrate and phosphate requirements of this 

 alga have been not yet established with any 

 precision. Batch cultures in ASP-2 medium 

 containing to 100 |i.g.-at./l. of nitrogen and 

 0-100 |i g.-at./l. of phosphorus showed that 

 enough nutrients could be carried over in the 



27 



