particular batch of sterile medium possibly was lower than 5,9- 

 The pH of the medium in container 13 (duplicate for container L4) 

 was 6.6 after 2L, hours. The pH values, after 2-4 hours, for the 

 millip®re 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, brevis cultures used in experiment 8 varied from 7.6 to 7.7, 

 No pH values were ascertained initially in experiment 9, excepting 

 the pH value of 7.8 for the week-old sterile culture medium used 

 for eluting the millipore and filter paper residue. 



The pH of culture medium either with or without G, brevis 

 customarily drops after fish are introduced; this decrease probably 

 results from increased bacterial growth and accumulation of fish 

 waste products. Excepting two containers of culture medium (13 

 and L4) and a container of filter paper residues of a bacteria-free 

 G. brevis culture eluted in sterile culture medium (/4) of experiment 

 9, a minimal pH value of 7.0 was recorded after test periods as long 

 as 2^ to 30 hours in the two experiments (8 and 9) conducted with 

 initially bacteria-free G„ brevis 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 'iSO low. It is our surmise that 

 the abnormally low pH resulted from failure to rinse the culture 

 flask after hot nitric acid (7^) was used in the cleaning process. 

 By actual test we found that failing to rinse the flask after the 

 nitric acid treatment did signficantly lower the pH of 2,0 liters 

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

 autoclaved medium stabilized at approximately 6,4., 



A flask of culture medium, companion to one with the low pH 

 (containers 13 and L4) of experiment 9, which was inoculated with 

 G. brevis failed to support growth of this organism after incubating 

 one month. Unfortunately, the medium in this flask was discarded 

 without checking the pH. Since the two flasks of mediimn were 

 prepared at the same time, we consider that G, brevis possibly 

 failed to grow because the pH was unfavorable. We have experienced 

 thus far only this one failure of bacteria-free G. brevis to grow 

 in low-form culture flasks (3-liter). Growth of G, brevis has not 

 occured 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 unsatisfactory for this organism. 



The results of experiment 9a (Table 9) show that the culture 

 medium in container I4 was no longer toxic to small mullet when 

 retested about 2L, hours after experiment 9 was begun. The pH 

 of this medium was 6,4 about one hour after beginning experiment 9a. 



34 



