ii,,-16 MANUAL OF METHODS FOR PURE CULTURE STUDY 



deep columns in flasks or bottles, and sterilized 20 minutes at 15 

 lbs. pressure. Upon cooling, if methylene blue is present, a greenish 

 blue color should develop at the surface and sometimes to some distance 

 below the surface if the medium is disturbed; upon standing a short 

 time, however, the usual amber color indicative of anaerobiosis will 

 return. The medium may be stored {at room temperature, not in a 

 refrigerator) for several days, or even a few weeks, and used without 

 the heating, required by most media, to expel absorbed oxygen. 



Use: Recommended as the medium of choice in the enrichment of 

 the pathogenic anaerobes. Particularly useful in hospital labora- 

 tories where small amounts may be made as needed from the dry 

 powder. Not recommended for isolation of the butyric-butyl group. 

 Since it is claimed that the thioglycollate not only maintains a low 

 0/R potential, but also combines with and inactivates most of the 

 mercurials, (Daily and Blubaugh, 1941; Blubaugh and Reed, 1943; 

 Nungester et at., 1943), this medium is suggested for use in the routine 

 sterility testing of biological materials including vaccines, serums, 

 catgut, etc. (Marshal et al., 1940; Federal Register, 1942). 



Beef Heart {or beef tissue) Infusion Medium. Several different 

 formulae are available for this medium; although there seems to be 

 little reason to choose any particular one, in preference to another, 

 the following is satisfactory: Allow 500 g. of beef heart (or lean beef 

 meat) to stand overnight in refrigerator in 1,000 ml. of tap water. 

 Trim fat from the meat, and mince or grind before adding to the 

 water. Remove from icebox and boil over free flame for 15 minutes 

 or steam in Arnold sterilizer for 30 minutes. Separate tissue from 

 liquid by passing through two layers of cheese cloth in a fluted glass 

 funnel, and save both portions. Add 10 g. peptone and 5 g. NaCl to 

 the liquid after restoring to volume. If necessary, heat briefly to dis- 

 solve peptone. Adjust to pH 7.6 with 1 N NaOH and boil for 15-20 

 minutes or heat in Arnold sterilizer for 30 minutes. Filter through 

 paper. If needed immediately, tube broth over a 2 cm. column of 

 tissue, and sterilize 45 minutes at 15 pounds pressure. If not needed 

 immediately, sterilize broth in screw-capped bottles, and rapidly dry 

 tissue in incubator with forced circulation. These may be used at 

 any later time. Check the sterility of the medium before use by in- 

 cubation for at least 24 hours at 37° C. 



Use: For enrichment or general cultivation of pathogenic anaerobes; 

 not suitable for the butyric-butyl group of the thermophilic anaerobes. 

 Has some diagnostic value as certain species produce a reddening of 

 the tissue. (Strongly proteolytic organisms cause a disintegration of 

 the meat tissue with the release of offensive odors.) Suitable for 

 stock cultures of most of the pathogenic types, as in most instances 

 (exception C. perfringens) spore production may be detected after 

 48 hours. Certain proteolytic species deposit crystals of tyrosine in 

 this medium upon extended incubation. 



