470 FISHERY BULLETIN OF THE FISH AND WILDLIFE SERVICE 



PROCEDURES FOR TESTING STERILITY AND ENUMERATING ORGANISMS 



Bacteria-free cultures were grown in the same 

 medium prescribed by Wilson and Collier (1955) 

 and were carried through several subcultures. 

 After 10 months they showed no apparent dimi- 

 nution in vigor. Several media were used to 

 establish sterihty. All G. brevis cultures origi- 

 nated from a culture obtained from a sample 

 collected in a bloom that occurred near the coast 

 of Florida in September 1953. Details of the 

 procedures for culturing G. breds, and the methods 

 used to obtain bacteria-free cultures will be 

 presented in another paper. 



STERILITY-TESTING MEDIA 



Cultiu-es of G. breds used for transferring were 

 tested for sterility in media prepared according 

 to Spencer (1952): (1) peptone sea water (0.5%, 

 bacto-peptone, 0.01%, FePOi dissolved in 75% 

 aged sea water) and (2) peptone sea-water agar 

 (peptone sea water plus 1 .5% bacto-agar) . These 

 media as well as all other sterility-testing media 

 subsequently described were dispensed in screw- 

 cap tubes and autoclaved at 121° C. for 15 

 minutes. 



We frequently used four other media similar to 

 those employed by Droop (1954) for routine 

 sterility testing. These media included (1) dis- 

 tilled-water liquid, (2) distilled-water agar, (3) sea- 

 water liquid, and (4) sea-water agar. Our media 

 contained the following substances: 0.5% dex- 

 trose, 0.1% Difco neopeptone, 0.4% bacto-beef 

 extract, 0.5% bacto-yeast extract, 0.015% sodium 

 acetate (NaC2H302.3H20), and soil extract (2.0 

 ml./lOO ml.). These substances (dextrose was 

 often excluded) with and without bacto-agar 

 (1.5%) were dissolved in both distilled water and 

 75% aged sea water to give the four combinations. 

 Droop (1954) listed the substances used, but not 

 the quantities. A personal communication (1956) , 

 however, revealed that his formula contained the 

 organic substances in concentrations which were 

 roughly 10 to 15 times less than the quantities we 

 used. Furthermore, he included bacto-tryptone, 

 which was not listed in his paper, whereas we used 

 Difco neopeptone. Subsequent to the comple- 

 tion of the present studies, the absence of bacteria 

 from several G. breds cultures was confirmed with 

 media of Droop's formulation and also with these 

 media diluted to 10 percent. 



Other media used to supplement the routine 



tests included (1) the sterility-test medium used 

 by Sweeney (1954) containing 0.05% bacto- 

 peptone, 0.0136% sodium acetate (NaCsHjOz. 

 3H2O), 0.0202% KNO3, 0.00356% K2HPO4, 

 0.00016% FeCl3.6H20, and 0.000012% MnC^. 

 4H2O dissolved in 75% aged sea water with and 

 without bacto-agar (1.5%); (2) Spencer's peptone 

 sea-water media supplemented with 0.1% bacto- 

 yeast extract as employed in medium 2116E 

 (Morita and ZoBell, 1955); (3) semisolid medium 

 composed of 0.075% trypticase (Baltimore Bio- 

 logical Laboratory), 0.075%, bacto-peptone, 

 0.075% bacto-yeast extract, 0.01% sodium acetate 

 (NaC2H302.3H20), and 0.2%o Difco special (Noble) 

 agar dissolved in aged sea water; (4) 1% bacto- 

 peptone in aged sea water with and without 

 bacto-agar (1.5%), the medium used by Bein 

 (1954) to isolate and cultivate certain chromo- 

 genic bacteria found in Florida waters; and 

 (5) Spencer's (1952) casein sea-water agar com- 

 posed of 0.05% bacto-peptone, 0.05% bacto- 

 isoelectric casein, 0.05% soluble starch, 0.1% 

 (v/v) glycerol, 0.02% K2HPO4, and 1.5% bacto- 

 agar dissolved in 75% sea water. 



Sterility tests for anaerobic bacteria were con- 

 ducted occasionally with three difi'erent media: 

 (1) Bacto-fluid thioglycollate medium rehydrated 

 with both distilled water and 75% aged sea 

 water; (2) the general anaerobic medium (slightly 

 modified) used for marine bacteria by Morita and 

 ZoBell (1955) containing 0.5%o bacto-peptone, 

 0.1%, bacto-yeast extract, 0.01% FePO^, 0.1% 

 sodium formaldehydesulphoxylate, and 0.0001% 

 resazurin dissolved in 75% aged sea water with 

 and without bacto-agar (1.5%,); and (3) an anaer- 

 obic 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. 

 before addition of the test culture which was 

 mixed by swirling the tube before the agar solidi- 

 fied. After adding the test culture, sterile melted 

 vaspar (50% vaseline and 50% paraffin) was 

 poured into each tube of anaerobic medium, 

 except the fluid thioglycollate medium, to exclude 



oxvgen. 



INOCULATION AND INCUBATION 



All sterility tests, unless otherwise indicated, 

 were made with 1.0 ml. of test cvilture in 10.0 ml. 



