UNIALGAL AND BACTERIA-FREE CULTURES OF G. BREVIS 



473 



EXPERIMENTS 2 and 3. — Comparison of Effects of Gymnodinium brevis and Gymnodinium splendens Cultures 



fish were alive at the close of the stiulv 19 davs 



The mere presence of mimeioiis dinoflagcllates 

 may have been responsible for the toxicity of the 

 unialgal culture used in experiment 1. To test 

 this possibility, fish were subjected to unialgal G. 

 breris and 6^. splendfus cultures in experiments 2 

 and 3. Experiment 2 was conducted under the 

 same conditions as experiment 1. A 4-week-old 

 unialgal culture of G. brevis and a 10-week-old 

 unialgal culture of G. splendens that contained 2.1 

 and 2.8 mdlion organisms per liter, respectively, 

 were tested for toxicity to Mollienina latipinna 

 (2}^ to 3 in. long). Both cultures were replenished 

 with fresh medium three times weekly during the 

 incubation period. Sea water from which the test 

 fish were taken was used as control. One fish was 

 placed in each of three test materials. The fish 

 in the G. brevis culture died after 47 minutes; the 



later in the G. splendens culture and in sea water. 



Experiment 3 duplicated experiment 2 in most 

 respects, except that the cultures were a week 

 older. At this time there were 2.0 million G. brevis 

 and 2.6 million G. splendens per liter in the cul- 

 tures. M. latipinna (2^ to 3 in. long) lived only 

 68 minutes in the G. brevis culture, but they were 

 alive in the G. splendens culture and in sea water 

 3 days later when the experiment was discontinued. 



The excellent survival of the fish in G. splendens 

 cultures, in contrast with the lethality of G. brevis 

 cultures, indicates that the latter cultures con- 

 tained a toxic substance(s). Since the cultures of 

 G. brevis were not pure, the toxic substance could 

 have been produced by G. brevis, associated bac- 

 teria, or both. 



EXPERIMENT 4. — Effects of Unialgal Gymnodinium brevis Cultures and Associated Bacteria 



A series of experiments (4, 5, 6, and 7) was 

 designed mainlj' to determine whether G. brevis or 

 its associated bacterial flora is responsible for the 

 toxic effects of unialgal cultures to fish. If the 

 bacteria prove nontoxic under the same cultural 

 conditions, one could reasonably assume that G. 

 brevis produces the toxic substance. Much of the 

 value with regard to the original purpose for con- 

 ducting these experiments has been lost subse- 

 quent to the development of mass bacteria-free 

 cultures of G. brevis. Bacteria-free cultures made 

 it possible to demonstrate experimentally that G. 

 brevis produces a fish-killing substance. The de- 

 tails are presented later in this paper. 



To obtain some of the test materials used in 

 these experiments (4, 5, 6, and 7), 20 liters of cul- 

 ture medium were prepared, 5 liters of which were 

 placed in each of two P3Tex bottles {2}^ gal.) ; 

 another Pyrex bottle (5 gal.) received the remain- 

 ing 10 liters. Each bottle of medium was heated 

 to 75° C. (5 to 6 hours' heating required) on three 

 successive days to reduce the bacterial load. One 

 of the 2K-gallon bottles (No. 1) was inoculated 

 with 10.0 ml. of a 6-week-old unialgal G. breds 

 culture with a bacterial count of 8.1 million per 

 ml. The otlier 2K-galloT) bottle (No. 2) was 

 seeded with 10.0 nd. of G. brevis-hee inoculum in 

 an attempt to culture the associated bacteria. 

 This inoculum, having a bacterial count of 10.3 

 million per ml., was obtained by heating between 

 37°-39° C. for 30 minutes a portion of the same 



culture used to inoculate bottle 1. The 5-gallon 

 bottle (No. 3) containing uninoculated medium 

 was arranged so that bottles 1 and 2 could be 

 replenished from this reservoir when culture 

 materials were removed for toxicity tests and 

 chemical analyses. 



Samples were taken from the three bottles at 

 irregular intervals during the first 25 days of in- 

 cubation to follow the bacterial growth. The 

 bacterial counts (table 1) of samples taken at 1-, 

 4-, 14-, and 25-day intervals from the unialgal G. 

 brevis culture (bottle 1) and the G. brevis-iree bac- 

 terial culture (bottle 2) were comparable except 

 for the 4-day samples. The 4-da3^ sample from 

 the G. brevis culture had a bacterial count of 2.7 

 million per ml. — about 50 percent greater than 

 that of the G. bi-ems-hee bacterial culture. Some 

 bacterial counts are questionable because of pro- 

 longed refrigeration of samples before preparation 

 of the plates. They indicate, however, the rela- 

 tive number of bacteria in the three bottles at the 

 various sampling intervals. Although bottles 1 

 and 2 apparently were inoculated with the same 

 bacterial flora, we can only presume that the 

 floras that subsequently developed in these bottles 

 were qualitatively comparable. 



Approximately 6 weeks after bottles 1 and 2 

 were inoculated, materials from these bottles and 

 the reservoir (bottle 3), in addition to an 11- 

 month-old unialgal G. brevis culture and centri- 

 fuged sea water, were used to conduct experiment 



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