THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 133 



should be plated for isolation of pure cultures. It should be remembered 

 that infections from such wounds are commonly polymicrobic in origin. 



Preliminary Purification Procedures 



It is often difficult to isolate anaerobic bacteria from enrichments which 

 also contain aerobic bacteria. It would be presumed that aerobic bac- 

 teria 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, sporeforming aerobes. 

 In fecal and perhaps other samples the contamination may include non- 

 sporeforming anaerobes. If the nonsporeforming anaerobe is wanted, 

 then anaerobic plating and picking of isolated colonies should be com- 

 bined 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 ensure 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- 

 sporeforming types is as follows : Heat subcultures from the contaminated 

 enrichment, retaining the original tube, of course, unheated. Heat the 

 newly inoculated tubes 20 min at 80°C or a shorter time a# higher tem- 

 peratures. Take care to ensure 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 sporeforming aerobes the above 

 procedure may not be satisfactory, owing to the heat resistance of the 

 aerobic spores. In this case, one may employ dyes as bacteriostatic 

 agents. Nearly all, if not all, aerobic sporeformers are inhibited by 

 crystal violet, and most of the anaerobic types are relatively resistant. 

 Two or three serial transfers may therefore be made in a medium contain- 

 ing this dye (approximately 1 : 100,000 final concentration) to eliminate 

 the aerobe. The exact concentration of the dye to be used may vary 

 with the medium and the conditions at hand. If the dye is used in some 

 of the complex media, its effectiveness may be reduced during sterilization ; 

 therefore, the dye should be added to such media after sterilization. 

 Either liquid or solid media may be used. 



