infested waters which contained numerous other organisms. In addi- 

 tion, raw samples were probably held under conditions unsuitable 

 for the survival of Go brevis . 



The next approach to this problem was to obtain bacteria- 

 free or pure cultures of G, brevis . This isolation is necessary 

 to determine whether a cause and effect relationship exists between 

 G, brevis and the catastrophic fish kills. Furthermore, studies 

 of such problems as the nutritional requirements of G„ brevis , 

 nature of the toxic substance, role of associated organisms, and 

 effects of physical and chemical factors may be facilitated 

 with the use of bacteria-free cultures since the uncertainty re- 

 garding the effects of associated bacteria would be eliminated. 



The laboratory studies mentioned above, in coordination with 

 field studies, will provide a better understanding of why these 

 mass mortalities occur. Such knowledge will be helpful in predicting 

 when and where outbreaks may be expected and in determining the 

 feasibility of control measures. 



We report^herein the results of studies on the effects of 

 unialgal and bacteria-free cultures of G. brevis on fish as well 

 as the effects of some bacteria isolated from unialgal cultures 

 of this organism and from waters- off the west coast of southern 

 Florida, Based upon the results of studies with bacteria-free 

 cultures, we conclude that G, brevis produces the toxic substance(s) 

 responsible for the mass mortality of marine animals associated 

 with "blooms" of this organism in the Gulf of Mexico. 



PROCEDURES FOR TESTING STERILITY MD EMIMERATING ORGANISMS 



Bacteria-free cultures were grown in the same medium prescribed 

 by Wilson and Collier (1955) and have been carried through several 

 subcultures. After 10 months they have shown no apparent diminution 

 in vigor. Several media were used to establish sterility. All 

 G. brevis cultures originated from a culture obtained from a sample 

 collected in a "bloom" which occurred near the coast of Florida 

 in September, 1953= The details of procedures for culturing G. brevis, 

 and methods used for obtaining bacteria-free cultures will be presented 

 in another paper. 



Sterilty Testing Media 



Cultures of G. brevis used for transferring were tested for 

 sterility in media prepared according to Spencer (1952): (l) pep- 

 tone sea water (0.5^, bacto-peptone, 0,01^, FePO/ dissolved in 



2/ We are indebted to Mr, K. T. Marvin, Mrs. Alice Kitchel, and Miss 

 Jean Gates for assistance in performing the experiments reported 

 herein and Messrs. E. A. Arnold and R. S. Wheeler for identifying 

 the test fish. 



