Ool^ tacto-yeast extract, 0.01^ FePOy, 0,1% sodium formaldehyde - 

 sulphoxylate, and 0.0001^ resaziorin dissolved in 75^ aged sea 

 water with and without bacto-agar (1.5^); and (3) an anaerobic 

 medium prepared by adding 0.01^ sodium thioglycollate to the 

 semisolid medium described in the previous paragraph „ The melted 

 general anaerobic agar medium was cooled to /40-42° C, prior to adding 

 the test culture which was mixed by swirling the tube before the 

 agar solidified. After adding the test culture, sterile melted 

 "vaspar" (50^ vaseline and 50% paraffin) was poured into each 

 tube of anaerobic medium, except fluid thioglycollate medium, to 

 exclude oxygen. 



Inocnlation and Incubation Procedures 



All sterility tests, unless otherwise indicated, were made 

 with 1.0 ml of test culture in 10.0 ml of medium. The agar media 



were inoculated in the following ways: (l) pour -plate mixing 



test culture in a sterile Petri dish with melted medium cooled to 



4-0--42 C, (2) streak-plate streaking 0.1 ml of test culture on 



a freshly prepared plate, (3) stab 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 (/^) slant cul- 

 tures 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 

 desication and mold contamination while incubating. Semisolid 

 medium was inoculated by stabbing to the bottom with a micro- 

 pipette and then gradually releasing the inoculum as the pipette 

 was slowly withdrawn. 



We incubated the sterility-test cultures in the dark at 

 28-30° Cfor a minimum of 6 weeks before discarding them as 

 sterile. This temperature level was selected since some of the 

 bacteria isolated from the unialgal cultures of G. 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 

 for inoculating agar cultures were used. One set was incubated 

 with illumination (175-300 ft-ca.) and temperature (24-25° C^ 

 the same as used for G. brevis cultures; the other set was incu- 

 bated 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 



