ni„-18 MANUAL OF METHODS FOR PURE CULTURE STUDY 



be made in a medium containing this dye (approximately 1-100,000 

 final concentration) to eliminate the aerobe. The exact concen- 

 tration of the dye to be used may vary with the medium and the 

 conditions at hand. If used in some of the complex media the effec- 

 tiveness of the dye may be reduced during sterilization; therefore, 

 the dye should be added to such media after sterilization. Either 

 liquid or solid media may be used. 



Another method for elimination of aerobic spore-formers utilizes 

 the fact that while growth of the aerobe may take place in an anaero- 

 bic environment the conditions for sporulation are unfavorable. 

 Under such conditions the anaerobe will be expected to sporulate 

 freely. Thus liquid cultures in tubes or plate cultures taken from 

 an anaerobic jar are chosen for material for heating as in the case 

 of the non-spore-forming contaminants. 



ISOLATION PROCEDURES 



From a purely theoretical viewpoint, microscopic single cell 

 methods of isolation are ideal, but the low percentage of successes 

 with these procedures excludes them from any uses except research. 

 Several reports are in the literature indicating success with anaerobes 

 using the Chambers micromanipulator, or similar instruments, 

 and wherever there is great need for strains of single cell origin, 

 the technic should be attempted. Due to the sensitivity of the vege- 

 tative cells toward oxygen, it is recommended that spores be picked 

 rather than vegetative cells. One should use freshly exhausted 

 media showing highly reducing activity for the subcultures and 

 naturally the medium should be suited to the organism being purified. 

 If growth is not evident within the first 48 hours, the tubes may be 

 protected from evaporation and incubated indefinitely. Reputable 

 workers have reported dormancy of spores for six months or longer 

 duration. 



In routine problems either plating or deep agar tube methods are 

 available for purification of cultures from the original enrichment 

 tubes. As stated above, the usual procedure in the isolation of 

 anaerobes from samples in which contamination is excessive is best 

 done by attempting partial purification in tube culture. This, 

 however, need not be the case if the population of the sample is 

 dominated by one species. In these the plating routine may be 

 started without the preliminary enrichment procedure. Perhaps 

 a few words should be included concerning details of technic. Since 

 some of the anaerobes tend to spread rapidly over the surface of the 

 agar, in many instances it will be found that "poured" agar plates 

 are to be preferred to plates inoculated by streaking the surface. 

 Two common methods are available for preparing these: (1) melt 

 tubes of the plating medium, cool, and inoculate before pouring; 

 (2) place a small amount of sterile tap water in the culture dish, 

 inoculate, and pour the agar into the dish immediately. If condi- 

 tions warrant, use crystal violet in the agar. Place the plates in 

 the anaerobic environment as soon as possible. (The size of inoculum 

 to be used will vary so that some practice may be necessary to give 



