490 



FISHERY BULLETIN OF THE FrSH AND WILDLIFE SERVICE 



24-hour test cultures, we could not duplicate his 

 inoculation procedures. 



In our experiment, 24-hour cultures were ob- 

 tained by inoculating two loops of culture removed 

 from a 24-hour slant culture (1-percent peptone 

 agar) into duplicate Erlenmeyer flasks containing 

 approximately 150 ml. of sterile 0.1 -percent pep- 

 tone solution in aged sea water. One flask was 

 incubated at 30° C. and the other at 25°. The 

 culture incubated at 30° was deep orange after 24 

 hours and the one incubated at the lower tempera- 

 ture was yellow. The more-intensely pigmented 

 culture was used because it probably contained 

 the greater concentration of bacteria. The ratio 

 of bacterial-culture volume to sea-water volume 

 varied from a maximum of 33.0 ml. of 24-hour 

 culture to 1.0 liter of sea water, which is about the 

 ratio used by Bein, to a minimum of one-hun- 

 dredth of this ratio. Sea water and sterile 0.1- 

 percent peptone solution were used as control 

 materials. Each experimental container (2-liter 

 beaker) received 1 .0 liter of sea water, which was 

 aerated continuously. Two mullet (3 to 4 in. 

 long) were placed in each container. The test 

 fish, which had been maintained in the laboratory 

 for several days, were acclimated in the experi- 

 mental containers for about 24 hours before the 

 beginning of the study. The water, which became 

 cloudy in all containers by the end of the acclima- 

 tion period, was replaced with fresh sea water 

 shortly before the test materials were added. 

 Samples were collected for bacterial counts about 

 30 minutes after the experiment began. These 



samples were refrigerated % hour to 2 hours before 

 preparation of the pour-plates. 



.A.11 fish were alive when the experiment was 

 discontinued 5 days later and had shown no evi- 

 dence of distress. A second set of bacterial- 

 count samples was taken at this time. The 

 samples were plated after being refrigerated ^ to 

 3 hours. All bacterial plates were prepared with 

 Bein's agar medium, and they were counted 

 after 6, 12, 15, and 21 days' incubation. The 

 number of pigmented subsurface colonies as well 

 as the pigment intensity of such colonies increased 

 after prolonged incubation. After 21 days, how- 

 ever, some colonies began to lose pigment. Bac- 

 terial counts of the test materials used in this ex- 

 periment are listed in table 10. 



RESULTS OF STUDIES WITH BACTERIA 



The results of the preliminary experiment with 

 some of the bacteria isolated from unialgal 

 Gymnodinium brevis cultures suggest that cultures 

 of the two dominant colony types, both nonpig- 

 mented, and a sparsely occurring pigmented form 

 are not toxic to fish. 



On the contrary, a "red bacterium" isolated 

 from 6. brevis-miested water off the west coast 

 of Florida appears to be toxic to fish. Neverthe- 

 less, until this bacterium is found more abun- 

 dantly or its association with fish kills is estab- 

 lished, we shall not consider it of importance as 

 a cause of fish mortality occurring off the west 

 coast of Florida. Further toxicity studies with 

 this organism have been discontinued until such 



Table 10. — Initial and final counts of bacteria in materials used in experiment testing effect of Flavobacterium 



piscicida on Mugil cephalus 



' The number of F. piscicida based on the observed number of deep-orange 

 colonies. 



2 Since some of the chromogenic colonies varied from either yellow to orange 

 or orange to yellow at various intervals during the 21 days of incubation, 

 orange and yellow colonies were combined to indicate the maximum ob- 

 Berved number of bacteria in this color range. There were no definitely 



orange colonies in samples from containers 3 and 8; about one-third of the 

 pigmented colonies in the sample from container 6 and more than one-half of 

 those in samples from all other containers were definitely orange. 



3 Approximately one-sixteenth of the bacteria produced yellow pigment. 



* Approximately one-tenth of the bacteria produced yellow to light-orange 

 pigment. 



