0.4 to 40 [xg.at/l of phosphorus if the 

 calcium content was between 140 and 

 400 mg/l. Media with phosphorus con- 

 centrations as high as 400 |ig. at/1 sup- 

 ported good growth of G^. breve if the 

 calcium concentration was between 140 

 amd 250 per liter. On the other hand, 

 media with calcium concentration as 

 high as 1,000 mg per liter supported 

 good growth if the phosphorus content 

 was between 0.04 and 4.0 |j. g. at/1. The 

 above values for calcium and phosphorus 

 should be revised slightly upward (less 

 than 0.01 percent) because of the occur- 

 rence of these elements as contaminants 

 in the other components of the medium. 



[p. 72.] 



" Vitamins . Three water soluble vita- 

 mins, thiamine (Bj), biotin (B3) and 

 cobalamin (B12 )> have been used con- 

 sistently in artificial sea water media 

 for bacteria-free G. breve . Results of 

 earlier experiments indicated that 

 G. breve would not grow in these media 

 unless these vitamins were added. In 

 addition, thiamine was the most effec- 

 tive of the three vitannins, but a com- 

 bination of thiamine and biotin supported 

 better growth than thiamine alone. Dur- 

 ing the past year, a series of nine 

 experiments was conducted to define 

 more clearly the role of these vitamins 

 in the growth of G. breve, [p. 72-73.] 



"Results of these experiments are 

 sinnilar in many ways to the results 

 of previous work in that thiamine was 

 the most active of the three vitamins 

 and a connbination of thiamine and biotin 

 was better thain thiamine alone. How- 

 ever, these experiments indicate that 

 the inclusion of cobalamin along with 

 the thiamine and biotin further im- 

 proved growth. In the earlier experi- 

 ments, we had omitted cobalamin in ten 

 serial subcultures without diminution 

 of growth. In recent experiments, omis- 

 sion of cobalamin has resulted in lesser 

 growth. To ascertain the source of the 

 apparent discrepancy in these results 

 we will initiate growth experiments 

 using cobalamin Eind the other vitamins 

 from more tham one supply house, 

 [p. 73.] 



"The need for thiamine is pronounced 

 in the experiments conducted to date. 

 There is a possibility that similar or- 

 ganic compounds will substitute for 

 thiannine and we plan to conduct experi- 

 ments to determine if such is the case. 

 If not, assays of this vitamin in the 

 field may be valuable for forecasting 

 red tides, [p. 73-74,] 



" Trace elennents . In earlier experi- 

 ments we were unable to grow G. breve 

 in artificial sea water nnedia unless a 



Effect of some vitajnins and a group of trace 



elements on the growth of G, breve in artificial 

 sea water medium (Numbers represent percent of 

 cultures with each addition in each growth 

 category) 



■■■ Bi (Thiamine), B3 (Biotin), 

 T (Trace element group) 



B12 (Cobalamin), 



group of trace elements was added. We 

 have attempted to determine the ele- 

 ments of this group that are necessary 

 for growth, but to date the results are 

 inconclusive. Some elements, notably 

 zinc, titanium, zirconium, and man- 

 ganese and boron may be required by 

 G. breve . The results of experiments 

 using these as additives indicate that 

 they nnay improve growth. Media con- 

 taining copper, nickel, rubidium, molyb- 

 denum and barium have on occasions 

 supported better growth tham media to 

 which they were not added. The addition 

 of iron has not improved growth regard- 

 less of the concentration or form in 

 which it was added. This does not mean 

 that iron is not needed by the orgamism 

 because it is a common contaminaint 

 of the major salts used in preparing the 

 medium. 



"Aged sea water collected from am 

 area of G. breve bloom will substitute 

 for the group of trace elements. The 

 addition of twenty milliliters of this 

 water per liter of medium will support 

 growth of G. breve when usedto replace 

 the trace elennents. These results indi- 

 cate that this water contains a rela- 

 tively high concentration of the required 

 group. 



"These elements, with the possible 

 exception of zirconium, are normally 

 present in sea water, and their absence 

 would probably not be a limiting factor 

 for red tides. The form amd higher 

 concentrations of these elements may 

 be a limiting factor of red tides. We 

 must add metal chelators or metal 



74 



