plates were exposed by necessary manipulations. The position 

 of the colonies as well as the appearance of similar colonies on 

 some control plates (uninoculated agar plates), which were treated 

 in the same manner as the test cultures, supports this conclusion. 

 We rarely encountered accidental contamination of sterility-test 

 culture contained in screw-cap tubes. 



Miscellaneous Checks for Sterility 



We consider that the medium used for culturing G, brevis 

 is unlikely to be suitable for the growth of photosynthetic bacteria. 

 Nevertheless, a few cultures were checked for such organisms. 

 The checks were made with a medium developed by Dr, T. J, Starr 

 of this laboratory for the isolation of marine non-sulfur purple 

 bacteria. This medium is composed of: 0.2^ sodium acetate 

 (NaC2H302.3H20), 0,05^' Na2S03.7H20, 0,01^ MgSO/ =7H20, 0.05^ K2HPO/, 

 0.1MNH^)2S0/, 0.0001^ FeCl3.6H20, 2.5^ NaCl, 0.01% bacto-yeast 

 extract , 0.01^ sodium thioglycollate, and 1.5^ Difco special 

 (Noble) agar dissolved in double distilled water. Just before 

 inoculatlGn the medium was melted, and sterile NaHCOo solution 

 was added aseptically to each tube to give a 0.1^ concentration 

 and a final pH of 8.0. This medium was inoculated and treated 

 in the same manner as previously described for the general anaerobic 

 medium. These sterility-test cultures, which were incubated under 

 the same light and temperature conditions used for G, brevis, 

 showed no evidence of growth after 6 weeks. 



Phase-contrast microscopic examination (970X) of wet prepara- 

 tions of a few G4. brevis cultures which were determined to be pure 

 by cultural methods did not reveal any contaminating organisms. 

 These examinations, conducted several months after the initial 

 establishment of bacteria-free cultures, were performed to check 

 for possible contaminants which may have maintained themselves 

 in G. brevis culture medium after repeated subculturing, and yet 

 not have grown in any of the sterility- test media employed. 



Procedure for Enumerating Bacteria 



Aerobic bacterial counts presented in the experiments to follow 

 were estimated by plating serial dilutions of the test sample. 

 The dilution water blanks (75^ aged sea water) dispensed in 9-ml 

 amounts in screw-cap tubes were autoclaved at 121 "Co for 15 minutes. 

 Serial dilutions of 1:10; 1:100; 1:1,000; 1:10,000; and 1:100,000 

 were prepared of each sample to be counted o One ml of each dilution 

 and 10.0 ml of melted Spencer agar, cooled to 4O-4.2 C, were mixed 

 in a sterile Petri dish by gentle swirling before the agar hardened. 

 Because of the possibility of low counts a plate was also prepared 

 with 1.0 ml of undiluted sample. After the agar hardened, the 

 plates were sealed with masking tape and incubated in an inverted 

 position for 4 to 7 days at 28-30 C, Colonies were eniomerated 



