million G. brevis and 2.6 million G. splendens per liter in the 

 cultures. The 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 sea water 3 days later when the experiment was discontinued. 



The excellent survival of the fish in G. splendens cultures in 

 contrast to the lethality of G. brevis cultures indicates that the 

 latter cultures contained a toxic substance(s) . Since the cultiores 

 of G. brevis were not pure, the toxic substance could have been produced 

 by G. brevis , the associated bacteria, or both. 



Experiment 4. Effects of Unialgal Gymnodinium brevis Cultures and 



the Associated Bacterial Flora 



A series of experiments (4> 5, 6, and 7) was designed mainly 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 non-toxic 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 conducting 

 these experiments has been lost subsequent to the development of 

 mass bacteria-free cultures of G. brevis . Bacteria-free cultiires 

 made it possible to demonstrate experimentally that G. brevis produces 

 a fish-killing substance. The details 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 culture medium were prepared, 5 liters 

 of which were placed in each of two Pyrex bottles (2-|--gallon) ; another 

 Pyrex bottle (5-gallon) received the remaining 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 22- 

 gallon bottles (No. 1) was inoculated with 10,0 ml of a 6-week-old 

 unialgal G. brevis cult\ire with a bacterial count of 8,1 million per 

 ml. The other 2i-gallon bottle (No. 2) was seeded with 10.0 ml of 

 G. brevis -free 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° G. 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 incubation to follow the bacterial growth. 

 The bacterial counts (Table 1) of samples taken at 1-, L,-, 14-j and 

 25-day intervals from the unialgal G. brevis culture (bottle 1) and 

 the G. brevis -free bacterial culture (bottle 2) were comparable 

 except for the 4-day samples. The 4-day sample from the G. brevis 



8 



