472 



FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 



300 or less than 30 colonies were not used in quan- 

 titative estimates except in a very few instances. 

 The exceptions were in cases where either the most 

 dilute plates contained more than 300 colonies or 

 the undiluted plate contained less than 30 colonies. 



The bacterial counts most likely represent 

 minimal concentrations, because nutritional and 

 environmental requirements of an entire bacterial 

 population cannot be satisfied with any one me- 

 dium or with a single set of incubation conditions. 

 We made no attempt to enumerate anaerobic 

 bacteria. All colonies except those with the 

 typical appearance of molds were counted, there- 

 fore, any microorganisms producing bacterialike 

 colonies were included in the counts. 



Since we could not prepare pour-plates of all 

 water samples immediately after collecting them, 

 another possible source of error in the counts 

 should be considered. Both quantitative and 

 qualitative changes in the bacterial population 

 may have occurred before some of the samples 

 were plated, particularly those plated several 

 hours or even days after collection. Immediately 

 after collection all water samples were refrigerated 

 (4° C.) until shortly before preparation of the 



plates. In most cases the storage period did not 

 exceed 6 hours; however, this period varied con- 

 siderably in some experiments, especially those 

 in which several samples were counted. Conse- 

 quently, we have recorded the extremes of the 

 storage period for each experiment. 



DINOFLAGELLATE ENUMERATION 



The concentration of G. hrevis and other dino- 

 flagellates was determined in two steps: (1) a 

 preliminary counting of 1.0-ml., 0.1-ml., and 0.01- 

 ml. aliquots from a sample mixed by gently swirling 

 the tube (vigorous shaking frequently causes many 

 of the organisms to cytolyze) to determine sample 

 size best for counting, and (2) counting 3 to 9 

 aliquots of the quantity selected in the first step. 

 The latter counts were averaged to obtain the G. 

 brevis concentrations. The counts probably repre- 

 sent minimal levels because the organism tends to 

 disintegrate when manipulated. Because of this 

 tendency, only one aliquot was withdrawn at a 

 time and it was counted immediately. A wide- 

 field stereoscopic microscope with a magnification 

 of X 54 was used in making the counts. 



EXPERIMENTS WITH UNIALGAL CULTURES OF GYMNODINIUM BREVIS AND 



OTHER DINOFLAGELLATES 



Seven experiments testing the toxicity to fish of 

 unialgal cultures of G. brevis and some other dino- 

 flagellates were performed. All of these studies, 

 even those which were preliminary, such as experi- 

 ments 1 through 3, are presented because the de- 

 tails and results vary considerably in some cases. 

 In some experiments only one test fish was used 

 per container because either the available fish 



were too few or the containers were too small to 

 accommodate more. Moreover, duplicate con- 

 tainers were iiot always used because of limita- 

 tions imposed by insufficient supply of either test 

 fish or test materials. We have taken special care 

 to record all experimental details, some of which 

 may be of no significance, since they could prove 

 of value to others in reviewing our work. 



EXPERIMENT 1.— A Simple Test of the Toxicity of Gymnodinium brevis Cultures 



This experiment was conducted to determine 

 whether unialgal G. brevis cultures would kill fish. 

 We used a 3K-week-old culture, replenished with 

 fresh medium three times weekly, that contained 

 1.8 million organisms per liter. Sea water from a 

 lagoon at the east end of Galveston Island, Texas — 

 the locality where the test fish were collected — 

 served as control material. The test materials 

 were not aerated. One rough silversides {Membras 

 vagrans), 3J^ inches long, and one sailfin molly 

 (Mollienisia latipinna), 2K to 3 inches long, were 

 placed in each of two 1 -liter beakers — one con- 



taining sea water, the other G. brevis culture. 

 The beakers were covered with polyethylene 

 sheeting. 



Membras vagrans survived only 4 minutes in 

 the G. brevis culture whereas this species survived 

 43 minutes in the sea water. M. latipinna died 

 after 85 minutes' exposure to the G. brevis culture 

 and the fish in the sea water was alive when the 

 experiment was discontinued 4 days later. Al- 

 though the lethality of G. brevis cultures to fish 

 is evident from these results, they do not neces- 

 sarily prove that a toxic substance is involved. 



