UNIALGAL AXD BACTERIA-FREE CULTURES OF G. BREVIS 



475 



mum of 8/3 hours to a maximum of 24 hours — 

 the (hu-atioii of tlie experiment (table 3). The 

 bacterial counts of botli the G. brevis culture 

 (bottle 1) and the G. brenti-iree bacterial culture 

 (bottle 2) had decreased since experiment 4 was 

 conducted. Just as in experiment 4, however, 

 the G. brevis culture had a much higher count — 

 2.9 to 3.4 million bacteria per ml. in contrast 

 with 0.20 to 0.23 million per ml. for the G. brems- 

 free culture. 



One of the containers of G. brevis culture (6) 

 used in experiment 5 was employed in a supple- 

 mentary study to determine whether adding 

 several fish to the same culture would affect its 

 toxicity. Another phase of this study was to 

 check the response of fish transferred to sea water 

 after being subjected to G. brevis culture. Im- 

 mediately after the fish in container 6 died (29 

 minutes after beginning of experiment 5) it was 

 removed and the first of five additional M. cephalus 

 were placed in this container. This fish suc- 

 cumbed after 21 minutes' exposure. After re- 

 moving the dead Hsh, the second one was allowed 

 to remain in container 6 for 15 minutes It was 

 then transferred to sea water (container 1) where 

 it died 12 minutes later. Exposures of 15 minutes 

 and 7 minutes, respectively, in container 6, were 

 required to kill the third and fourth fish. Each 

 fish was removed from the container after it died. 

 After 3 minutes' exposure in container 6, the 



Table 3. — Experiment 5: Effects on MiiKil cephalus of 

 nnialgal cultures of Gymnodiniirn brevis ami Proro- 

 centru'ii sp. and Gymnodiniu-n brevis-/ree culture pre- 

 sumed to contain bacteria associated with this organism in 

 uniatgal cultures 



1 Time (hr.rmin.) of cessation of opercular movement. 

 ' Death did not occur during the 24-hour test period. 



fifth fish was removed to container 1 where it 

 survived for 2% liours. 



Experiments 4 and 5 (tables 2 and 3) were in- 

 adequately controlled with regard to quantities of 

 bacteria. Experiment 6 was performed in an 

 attempt to correct this shortcoming. 



EXPERIMENT 6. — Comparison of Toxicity of Unialgal Cultures of Gymnodinium brevis and Prorocentrum sp., 

 and Effects of Heating and Filtration on Toxicity 



Besides attempting to ascertain the source of 

 the toxic substance in unialgal G. brevis cultures, 

 this experiment included a study of the effects of 

 heating and filtration on the toxicity of such 

 cultures. 



One month prior to conducting experiment 6 

 the remaining portion of the G. brevis-iree bac- 

 terial culture (bottle 2) received an inoculum of 

 unialgal Prorocentrum sp., which had proved non- 

 toxic to M. cephalus in experiment 5 (table 3). 

 This step was taken in an attempt to increase the 

 bacterial concentration in bottle 2 to a level com- 

 parable to that in the G. brevis culture (bottle 1). 

 Centrifuged sea water was used in addition to 

 these two bottles of material, which were 4% 

 montiis old at this time. These three materials 

 were tested in duplicate (containers 1 through 6). 



The test materials in all of these containers, except 

 one container (2) of sea water, were sampled for 

 bacterial counts just before the fish were added. 

 These samples were refrigerated 20 minutes to 2}i 

 hours before the plates were poured. 



Five containers (7 through 11) of the test ma- 

 terial were used to test the effects of heating and 

 filtration on the toxicity of unialgal G. brevis cul- 

 tures. Bacterial counts were not made for these 

 materials because such information was not 

 needed. A filtrate, which was prepared by passing 

 G. brevis culture througli filter paper (Whatman 

 No. 42), was tested in duplicate. A single con- 

 tainer of another test material consisted of the 

 residues retained by the two filter-paper discs 

 eluted in 2 liters of sea water. Two liters of 0. 

 brevis culture were passed tlirough each disc. 



