insriALGAL AND BACTERIA-FREE CULTURES OF G. BREVIS 



491 



time that evidence is obtained to implicate it as 

 a fish-killing agent in nature. 



A bacterium producing red pigment was isolated 

 b}- Bein (1954) from Indian River on the east 

 coast of Florida at the time of an outbreak of dis- 

 colored water during August 1951. Although the 

 water was discolored when the isolation was made, 

 no fish either dead or alive were observed. Bein 

 found that this bacterium was nontoxic to several 

 species of fish. Howell (1953), studying the same 

 outbreak of discolored water, reported that no 

 great quantity of fish was killed; and that the dis- 

 coloration was caused by a dinoflagellate, which 

 he described as a new species, Gonyaulax monilata. 



The attempt to determine the minimal lethal 

 concentration of Flavobacterium piscicida failed, 

 since none of the fish died during the experi- 

 mental period. The reason for this lack of toxic 

 effect is not known. Although we followed as 

 nearly as possible the procedures used by Bein 

 (1954), the experimental conditions employed 

 were unfavorable for the bacterium, especially 

 with regard to toxin and pigment production. 

 The bacterial counts given in table 10 indicate 

 that high concentrations of F. piscicida (orange- 

 pigment-producing bacteria) were present ini- 

 tially. The second counts, which were made 

 from samples collected at the end of the 5-day 

 experimental period, however, showed that either 

 this bacterium or its chromogenic characteristic 

 decreased greatly during the intervening period. 

 A similar disappearance of chromogenic bacteria 

 occurred during the toxicity study with yellow 

 bacteria isolated from the unialgal G. brevis 

 cultures. The experimental conditions may have 

 affected the toxicogenic and chromogenic char- 

 acteristics of this bacterium in several possible 

 ways: (1) By killing the organism, (2) by in- 

 hibiting the growth of the organism, or (3) by 

 altering the toxicogenic and chromogenic prop- 

 erties of the organism. 



With only initial and terminal counts available, 

 the question as to when the decline of F. piscicida 

 occurred — either early or gradually during the 

 experimental period — can not be answered. At 

 no time during the 5-day period did the contents 

 of any container show the bright orange-yellow 

 discoloration observed by Bein after 24 hours. 

 The contents of container 3, which received the 

 maximum amount of F. piscicida culture, ex- 

 hibited a tinge of orange after 24 hours. This 



slight discoloration gradually became less notice- 

 able and completely disappeared by the fifth day. 

 The numbers of F. piscicida recorded in table 

 10 represent minimal counts, which may be 

 considerably lower than the actual concentrations, 

 because several of the white subsurface colonies 

 produced the characteristic orange pigment after 

 being transferred to agar slants (1 -percent pep- 

 tone) . All deep-orange colonies in plates prepared 

 with samples that contained F. piscicida were 

 considered to have been produced by this species. 

 A few orange colonies appeared in plates prepared 

 from some of the control samples, particularly in 

 those taken after 5 days. We made no allowance 

 for orange colonies which possibly were not 

 produced by F. piscicida, since tlie counts were 

 considered low even with the inclusion of such 

 colonies. An examination of the initial counts 

 (table 10) obtained for the control containers 

 (1 and 2) and the two containers (7 and 8) receiving 

 the smallest volumes of bacterial cultures suggests 

 that counts of bacteria, exclusive of F. piscicida, 

 varied in the order of 0.3 to 0.8 million per ml. 

 From these values we presume that the actual 

 concentration of F. piscicida was approximately 

 twice as great as the values listed. For example, 

 the initial number of F. piscicida in container 3 

 was probably about 4 million if one assumes that 

 only about 1 million of the bacteria per ml. were 

 other than F. piscicida,. The initial counts of this 

 bacterium were in relatively good agreement with 

 the various dilutions employed. For example, 

 the material in container 3, which received the 

 greatest volume of bacterial culture, yielded a 

 count of about 2 million chromogenic bacteria 

 per ml., whereas the material in container 8, 

 which received only one-hundredth as much as 

 container 3, gave a count of 20,000 chromogens 

 per ml. 



Since Bein gave only the ratio of F. piscicida 

 L'ulture and sea water employed, the concentrations 

 of bacteria used in his studies cannot be directly 

 compared with those we used. If the amount of 

 water discoloration is proportional to the abundance 

 of F. piscicida, we presume that the water in 

 Bein's aquariums probably contained in excess 

 of 4 million F. piscicida per ml. at the end of the 

 24-hour experimental period. This presumption 

 is based on the observation that the sea water in 

 container 3 of our experiment was not appreciably 



