tJNIALGAX, AXD BACTERIA-FREE CUinTRES OF G. BREVIS 



471 



of medium. The agar media were inoculated in 

 tlie following ways: (1) Pour-plate — mixing test 

 culture in a sterile Petri disli with melted medium 

 cooled to 40°^2° C, (2) streak-plate— streaking 

 0.1 ml. of test culture on a freslily prepared plate, 

 (3) stah culture — placing 0.1 ml. of test culture into 

 medium in screw-cap tubes (20 mm. x 125 mm.), 

 and then stabbing an inoculating needle to the 

 bottom, and (4) slant cultures — placing the test 

 culture on freshly slanted medium in screw-cap 

 tubes. Slant cultures were generally prepared for 

 most routine tests. The agar plates were sealed 

 with masking tape to prevent desiccation and mold 

 contamination during incubation. Semisolid me- 

 dium was inoculated by stabbing to the bottom 

 with a micropipette and then gradually releasing 

 the inoculum as the pipette was slowly withdrawn. 



We incubated tlie sterility-test cultures in the 

 dark at 28°-30° C. for a minimum of 6 weeks 

 before discarding them as sterile. This tempera- 

 ture level was selected since some of the bacteria 

 isolated from the unialgal cultures of 6. brevis 

 appear to grow more slowly at 24°-25° C 



On one occasion the sterility of several cultures 

 was tested in duplicate in various liquid and agar 

 media; the four methods tor inoculating agar cul- 

 tures were used. One set was incubated with 

 illumination (175-300 ft.-c.) and temperatui'e 

 (24°-25° C.) the same as used tor G. brevis cul- 

 tures; the other set was incubated in the dark at 

 28°-30° C. After 6 weeks none of the cultures 

 showed either visible colonies or cloudiness of any 

 sort except an occasional mold or bacterial colony 

 on the surface of a few streak- and pour-plates. 



We attribute the occasional appearance of mold 

 or bacterial colonies in our test cultures, especially 

 on the surface at the periphery of streak- and 

 pour-plates, to contamination while the plates 

 were exposed by necessary manipulations. The 

 position of the colonies as well as the appearance 

 of similar colonies on some control plates (unin- 

 oculated agar plates), which were treated in tlie 

 same manner as the test cultures, supports this 

 conclusion. We rarely encountered accidental 

 contamination of sterility-test cultures contained 

 in screw-cap tubes. 



MISCELLANEOUS CHECKS FOR STERILITY 



We consider that the medium used for cultiuing 

 G. brevis is unlikely to be suitable for the growth of 

 photosjmthetic 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 nonsulfur purple bacteria. Tiiis medium 

 is composed of 0.2% sodium acetate (XaC2H302. 

 3H.,0), 0.05% \a2SO3.7HjO,0.01% MgS04.7H20, 

 0.05% K2HPO4, 0.1%(NH4)2SO4, 0.0001% FeCl,. 

 6H2O, 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 inoculation the medium was 

 melted, and sterile NaHCOa solution was added 

 aseptically to each tube to give a 0.1% concentra- 

 tion 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 (X 970) 

 of wet preparations of a few G. brevis cultures that 

 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 cul- 

 tures, were performed to check for possible con- 

 taminants which might have maintained themselves 

 in G. brevis culture medium after repeated sub- 

 culturing, and yet had not grown in any of the 

 sterility-test media employed. 



BACTERIA ENUMERATION 



Aerobic bacterial counts presented in the experi- 

 ments 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° C. 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. One ml. 

 of each dilution and 10.0 ml. of melted Spencer 

 agar, cooled to 40°-42° C, were mixed in a sterile 

 Petri dish by gentle swirling before the agar 

 hardened. Because of tiu> 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 enumerated with the aid of a Quebec 

 colonv counter. Plates with either more than 



