UNIALGAL AXD BACTERIA-FREE CULTURES OF G. BREVIS 



485 



and unialgal G. hrems cultures. Howovor, large 

 M. cephalus survived considerably longer in the two 

 containers of sterile culture medium than in the 

 G. hrevifi cultures. One large fisli survived the 24- 

 hour test period and the other lived at least 8K 

 hours. In contrast, the death time for the six large 

 midlet in three different unfiltered G. hrei-is 

 cultures, two of which were bacteria-free, varied 

 from 10 to 41 minutes. All fish in the sea water 

 lived for at least 1% hours and two of them survived 

 the test period. 



The early deaths of the small mullet in con- 

 tainers 13 and 14 were probably due to the 

 abnormally low pH of the culture medium. The 

 pH of the material in container 14 was 5.9 about 

 14 hours after beginning of experiment and in- 

 creased to 6.4 after 25 hours. A pH value of 6.6 

 was obtained for the culture medium in container 

 13 at the end of the test period. 



The control fish survived much better in the 

 batch of sterile culture medium (containers 5, 6, 

 7, and 8) used in the filtration phase of this ex- 

 periment than they did in the batch of medium 

 (containers 13 and 14) used in the nonfiltration 

 phase. All (4) of the large mullet and two-thirds 

 (8) of the small mullet subjected to filtrates and 

 residues of culture medium survived the 24-hour 

 test period. On the contrary, none of the fish (4 

 large mullet and 12 small mullet) exposed to 

 filtrates and residues of the G. brevls culture sur- 

 vived tlie test period. The difference between the 

 effects of the two methods of filtration on the 

 toxicity of the G. brevis culture was marked. 



The millipore filtrate was much more toxic to 

 the fish than the residues; in the filtrate the death 

 times varied from 14 to 104 minutes in contrast 

 with a variation of 5 to 13)2 hours in the residues. 

 The toxicity of the filter-paper residues was 

 greater than the filtrate; the test fish lived from 

 39 to 42 minutes in the residues, whereas they 

 survived from 44 minutes to about 8 hours in the 

 filtrate. 



The attempt to reduce the initial bacterial con- 

 tamination of the bacteria-free G. brevis cultures 

 and the sterile culture medium by the previously 

 mentioned precautions was successful. The counts 

 for these materials varied from to 100 bacteria 

 per ml. The bacterial counts in the initially 

 bacteria-free G. breris cultures varied from 4,400 

 to 30,000 per ml. at the time the last fish died in 

 the container (% hour to 2]^ hours later). The 

 initial count in the unialgal culture was 1.3 million 

 bacteria per ml.; when the last fish died 2^2 hours 

 later the count was 1.4 million bacteria per ml. 

 At the end of the test period the bacterial concen- 

 tration was in excess of 80 million per ml. in one 

 container (13) of initially sterile culture medium. 

 After about 14 hours the count was 25 to 50 

 million bacteria per ml. in the other container (14) 

 of culture medium and in excess of 80 million per 

 ml. in one container of sea water. 



The pH of the initially sterile culture medium 

 in container 14 increased from 5.9 to 6.4 during 

 the 11 -hour period after the last fish died. Addi- 

 tional fish were subjected to this material to test 

 its toxicity at the higher pH. 



EXPERIMENT 9a. — Supplementary Toxicity Tests of Some Test Materials Previously Used in Experiment 9 



Near the close of experiment 9, we conducted a 

 supplementary study to determine whether the 

 initially sterile culture medium in container 14 

 was still toxic to small M. cephalus. Five other 

 containers originally a part of experiment 9 — one 

 container (15) of 85-percent, autoclaved sea water 

 and the four containers (9, 10, 11, and 12) of 

 initially bacteria-free G. brevis culture — were in- 

 cluded in this study. The other containers of 

 culture medium (13) and sea water (16) were not 

 used because this part of experiment 9 was still in 

 progress, as they contained one or more live fish. 



Small mullet from the group used in experiment 

 9 served as test fish. Four of these fish, which 

 were maintained in 85-percent, autoclaved sea 



water for 24 hours, were placed in each container. 

 Only two of the four containers of G. breiis culture 

 were aerated in an attempt to determine the 

 effects of agitation and aeration on the toxicity 

 of G. brevis. The culture medium (container 14) 

 and sea water (container 15) were not aerated. 

 The results (table 9) show that G. brevis cultures 

 were still toxic to small mullet, but the fish sur- 

 vived well in the previously toxic culture medium. 

 The 16 fish in the G. brevis cultures died in from 8 

 to 125 minutes; 10 of them survived less than 

 1 hour. In the control materials (containers 14 

 and 15), however, only 1 of the 8 fish failed to 

 survive the 24-hour test period; 1 fish in the sea 

 water succumbed after 5^4 hours. 



