UNIALGAL AND BACTERIA-PREE CtTLTURES OF G. BREVIS 



483 



into the containers passed through a nonabsorbent 

 cotton filter installed in the main air line from 

 the pump. The third safeguard against excessive 

 bacterial contamination was to place the test fish 

 in autoclaved 85-percent aged sea water for 

 several minutes before their transfer to the experi- 

 mental containers. This concentration of sea 

 water is about the same as that in the culture 

 media to which the fish were exposed. 



Three different, month-old bacteria-free 6. hrevis 

 cultures, two batches of sterile culture medium of 

 the same age, a 10-week-old unialgal G. brevis 

 culture, and autoclaved 85-percent aged sea water 

 constituted the test materials. One liter of test 

 material was placed in each of the 18 containers. 

 Since the test materials were aerated more vigor- 

 ously in this study, the increased air flow made 

 equalizing the degree of aeration in each container 

 difl!icult and the agitation of the test materials 

 probably varied more. 



One portion of the experiment (containers 1 

 through 8) was designed to compare the effects of 

 millipore (HA membrane) and paper (Whatman 

 No. 40) filtration on the toxicity of one of the 

 bacteria-free G. brevis cultures. One batch of the 

 sterile culture medium treated in the same manner 

 as the G. brevis culture served as control material. 

 The residues of millipore and paper filtration of 

 both the G. brevis culture and the sterile culture 

 medium were each eluted in 1 liter of sterile culture 

 medium to obtain four of the eight test materials 

 used in this phase of the study. The test materials 

 for the remaining portion of this experiment (con- 

 tainers 9 through 18) consisted of duplicate 

 containers of two different bacteria-free G. brevis 

 cultures, sterile culture medium, autoclaved 85- 

 percent aged sea water, and a unialgal G. brevis 

 culture. The distribution of the test materials 

 and numerical designation of the containers are 

 presented in table 8. 



Samples were taken for bacterial and G. brevis 

 counts immediately after the test materials were 

 dispensed. All containers of unfiltered bacteria- 

 free G. brevis culture (9, 10, 11, and 12) and of 

 unfiltered culture medium (13 and 14), in addition 

 to a container of sea water (15) and a container of 

 unialgal G. brevis culture (17), were sampled for 

 bacterial counts. The containers of filtrates or 

 residues were not sampled because such counts 

 were not needed. These samples were refrigerated 

 4)j to 6}^ hours before being plated. Samples for 



G. brevis counts were obtained from the containers 

 with filtrates of G. brevis culture (1 and 2) and 

 bacteria-free G. brevis (9, 10, 11, and 12). The G. 

 brevis concentration of the unialgal culture used in 

 containers 17 and 18 was ascertained by withdraw- 

 ing a sample from the culture before dispensing it. 

 The G. brevis samples were counted between l}i 

 to 2% hours after collection. 



Each container received four fish: three small 

 mullet (1 to XYi in. long) and one large mullet 

 {4}i to 5K in. long). The small mullet were main- 

 tained in aerated aquariums overnight and the 

 large mullet were used within a few hours after 

 they were collected. Since the volume (1 liter) 

 of test material was somewhat small for the large 

 mullet, they thrashed about vigorously and pos- 

 sibly injured some of the smaller test fish. 



Excepting the filtrates of G. brevis cultures 

 (containers 1 and 2), the bacteria and G. brevis 

 were again enumerated for those containers for 

 which such counts were made initially. The second 

 set of bacterial samples from the five containers 

 with G. brevis (9, 10, 11, 12, and 17) was plated 

 after 4% to 5% hours' refrigeration whereas those 

 samples from the containers with culture medium 

 (13 and 14) and sea water (15) were stored only 

 Yz hour to IK hours. The second set of G. brevis 

 counts was completed between 1 hour and 2 hours 

 after the samples were collected. In addition, 

 samples for pH and salinity determinations were 

 taken from all 18 containers. Bacterial, G. brevis, 

 pH, and salinity samples were taken either shortly 

 after the death of the last fish in the container or 

 at the end of the test period (24 hours) provided 

 that one fish in the container survived, except for 

 some pH and salinity samples as noted under 

 Remarks in table 8. The room temperature 

 varied from 22° to 25° C. during the 24-houT test 

 period. 



The fish subjected to bacteria-free cidtures as 

 well as filtrates and residues of such cidtures died 

 more rapidly, for the most part, than those exposed 

 to the control materials (table 8). The survival 

 rate among the control fish, especially small M. 

 cephalus, was not good. Fish of this size group 

 died quickly in one batch of sterile culture medium 

 (containers 13 and 14). The death times, varying 

 from 25 minutes to 2% hours, for the small mullet 

 in these two containers were comparable to the 

 death times, varying from 15 minutes to 2% hours, 

 for the same-sized fish in unfiltered bacteria-free 



