THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA iii4,-17 



infection and therefore more than one colony type from a single sample is not to be un- 

 expected. With slight modifications their suggestions are as follows: 



(1) Inoculate heavily tubes of beef heart medium. Use these subsequently only 

 if the primary plating fails. 



(2) Introduce swabs or fragments of tissue into 8 ml. amounts of thioglycollate 

 broth, mix well, and make 1:10, 1:100, and 1:1000 dilutions in the same medium (not 

 saline) . 



(3) From each dilution prepare surface plates on clear peptone-thioglycollate 

 agar and pour plates in semisolid agar. As an additional or alternate medium, use blood 

 agar; in which case hemolysis, if present, is an additional helpful characteristic. Incu- 

 bate the plates at 37° C. in a Brewer or Mcintosh and Fildes jar. Place a petri dish 

 lid containing granular CaCL at the bottom of the stack of plates, and another at the 

 top, to absorb the moisture which forms in the jars. Use the Brewer or Spray plate 

 if an anaerobic jar is not available. 



PRELIMINARY PURIFICATION PROCEDURES 



It is often difficult to isolate anaerobic bacteria from enrichments 

 which also contain aerobic bacteria. It would be presumed that 

 aerobic bacteria could ordinarily be eliminated merely by the 

 anaerobic environment when this is introduced. Often in practice 

 this is not the case, and other procedures must be instituted. It is 

 of value frequently to attempt partial or complete elimination of 

 the contaminants in tube culture using a liquid medium before 

 plating is done. Materials derived from human or animal sources, 

 other than feces, are usually contaminated with non-sporulating 

 aerobic rods and cocci. Cultures derived from milk, soil, water, 

 grains, feces, etc., contain, in addition, spore-forming aerobes. In 

 fecal and perhaps other samples the contamination may include 

 non-spore-forming anaerobes. If the non-spore-forming anaerobe 

 is wanted, then anaerobic plating, and picking of isolated colonies, 

 should be combined with optimum temperature and selective medium 

 to secure the culture. In all cases the original enrichment tube 

 should be preserved in the refrigerator, after growth is evident, 

 until the purification routine is successfully completed. This will 

 insure a supply of starting material should something go wrong with 

 the purification. 



Generally one of the easiest practices to be followed to get rid of 

 non-spore-forming types is as follows: Heat subcultures from the 

 contaminated enrichment, retaining the original tube, of course, 

 unheated. Heat the newly inoculated tubes 20 minutes at 80° C. 

 or a shorter time at higher temperatures. Take care to insure the 

 presence of the spores of the anaerobe. Use old cultures in a sugar- 

 free medium as the best source of material to be heated, although 

 other cultures may be satisfactory in special situations. 



For enrichments contaminated with spore-forming aerobes the 

 above procedure may not be satisfactory, due to the heat resistance 

 of the aerobic spores. In this case, one may employ dyes as bacterio- 

 static agents. Nearly all, if not all, aerobic spore-formers are 

 inhibited by crystal violet, and most of the anaerobic types are 

 relatively resistant. Two or three serial transfers may, therefore. 



