134 MANUAL OF MICROBIOLOGICAL METHODS 



Sodium azide (0.02 per cent final concentration) together with chloral 

 hydrate (0.01 per cent final concentration) may be used, singly or 

 together, to inhibit gram-negative aerobic spreading organisms. Sorbic 

 acid (York and Vaughn, 1944) in a final concentration of 0.12-0.15 per 

 cent in thioglycollate broth will greatly aid in eliminating aerobic spore- 

 forming bacilli, staphylococci, and many gram-negative contaminants. 

 Initial enrichment broths are subcultured to sorbic acid thioglycollate 

 broth, incubated 24 hr, then plated to an isolation plate. In the event 

 that cultures are contaminated by Pseudomonas strains, polymyxin B 

 may be added to the sorbic acid thioglycollate broth; a concentration of 

 10 /ig per ml will usually inhibit these organisms and permit recovery of 

 Clostridia present (Lindberg, Mason, and Cutchins, 1954). 



Another method for elimination of aerobic sporeformers utilizes the 

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

 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 nonsporeforming 

 contaminants. 



Isolation Procedures 



From a purely theoretical viewpoint, microscopic single-cell methods of 

 isolation are ideal, but the low percentage of successes with these pro- 

 cedures 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. 

 Because of the sensitivity of the vegetative 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 hr, 

 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 avail- 

 able 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 



