UNIALGAL AND BACTERIA-FREE CULTURES OF G. BREVIS 



487 



heavy bacterial growth, the initial pH of this 

 particular hatch of sterile medium possibly was 

 lower than 5.9. The pH of the medium in con- 

 tainer 13 (duplicate for container 14) was 6.6 

 after 24 hours. The pH values, after 24 hours, 

 for the millipore and paper filtrates of the other 

 batch of sterile culture medium used in experiment 

 9 were 7.2 and 7.0, respectively. The initial pH 

 of month-old sterile culture medium and bacteria- 

 free G. hreris cultures used in experiment 8 varied 

 from 7.6 to 7.7. No pH values were ascertained 

 initially in experiment 9, excepting a pH of 7.8 

 for the week-old sterile culture medium used for 

 eluting the millipore and filter-paper residues. 



The pH of culture medium either with or with- 

 out G. hrevis customarily drops after fish are 

 introduced; this decrease probably results from 

 increased bacterial growth and accumulation of 

 fish waste products. Excepting two containers 

 (13 and 14) of culture medium and a container 

 (4) of filter-paper residues of a bacteria-free G. 

 breris culture eluted in sterile culture medium in 

 experiment 9, a minimal pH value ol 7.0 was 

 recorded after test periods as long as 24 to 30 

 hours in the two experiments, 8 and 9, conducted 

 with initially bacteria-free G. hrevis cultures and 

 sterile culture medium. Small mullet survived in 

 culture medium with pH values as low as 7.0 and 

 7.1 in experiments 8 and 9. 



We do not know why the pH of one particular 

 flask of culture medium used in experiment 9 was 

 so low. It is our surmise that the abnormally 

 low pH resulted from failure to rinse the culture 

 flask after 7-percent, hot nitric acid was used in 

 the cleaning process. By actual test we found that 

 failing to rinse the flask after the nitric-acid treat- 

 ment did significantly lower the pH of 2.0 liters 

 of sea-water-base medium. In this particular test 

 the pH of the autoclaved mediiun stabilized at 

 approximately 6.4. 



A flask of culture medium, companion to one 

 with the low pH (containers 13 and 14) of experi- 

 ment 9, that was inoculated with G. hrevis failed 

 to support growth of this organism after incubat- 

 ing for 1 month. Unfortunately, the medium in 

 this flask was discarded without checking the pH. 

 Since the two flasks of mediimi were prepared at 

 the same time, we consider that G. hrevis possibly 

 failed to grow because the pH was unfavorable. 

 We have experienced thus far only this one failure 

 of bacteria-free G. hrevis to grow in low-form 



culture flasks (3-liter). Growth of G. hrevis has not 

 occurred in sea-water-base medium with an initial 

 pH of less than 7.0. Furthermore, preliminary 

 studies indicate that a pH below 7.4 is unsatis- 

 factory for this organism. 



The results of experiment 9a (table 9) show 

 that the culture medium in container 14 was no 

 longer toxic to small mullet when retested about 

 24 hours after experiment 9 began. The pH of 

 this medium was 6.4 about 1 hour after start of 

 experiment 9a. All four test fish were alive after 

 24 hours; two of them were alive after 5 days. 

 The pH of the medium in container 14 was 6.9 

 at this time. 



The effects of millipore and paper filtration on 

 the toxicity of bacteria-free G. hrevis cultures 

 were the same as observed when unialgal cultures 

 were so treated. The more toxic portion of the 

 culture passes through the millipore membrane, 

 whereas filter paper retains the more toxic fraction. 



Bacteria-free G. hrevis cultures proved just as 

 toxic as the unialgal ones in simultaneous tests 

 with comparable concentrations of this organism. 

 For example, in experiment 8 (table 6) the death 

 times for M. cephalus in initially bacteria-free 

 cultures varied from a minimum of 15 minutes to 

 a maximum of 4 hours and 44 minutes. Seven of 

 the 16 M. cephalus died in less time than was 

 required (1 hour and 13 minutes) to kill the two 

 M. cephalus subjected to a unialgal culture. The 

 C. variegatus succumbed in the bacteria-free cul- 

 tiu-es in periods varying from a minimum of 3 

 hours and 18 minutes to a maximum of 32 hours; 

 13 of the 16 test fish died within 8 hours. Nine 

 of the 16 C. variegatus in bacteria-free cultures died 

 in less than the minimum time (6% hours) required 

 to kill the two C. variegatus in a unialgal culture. 

 Further data for comparison of the effects of 

 bacteria-free and unialgal cultures are available 

 from experiment 9 (table 8). Two large M. 

 cephalus subjected to unialgal cultures died in 10 

 and 14 minutes. The four large mullet in the 

 bacteria-free cultures died within 23 to 41 minutes. 

 The death times for six small M. cephalus in the 

 unialgal cultures varied from 15 minutes to 2% 

 hours. The extremes of death times for the 12 

 small mullet in bacteria-free cultures were quite 

 similar — 25 minutes to 2 hours and 28 minutes. 



Despite the evidence that bacteria are not 

 directly responsible for the toxic effects of G. hrevis 

 cultures, the possibility that bacteria play a 



