iii«-20 MANUAL OF METHODS FOR PURE CULTURE STUDY 



With the butyl-butyric types, however, which sporulate readily 

 in many media, there is a critical period in which the culture is not 

 very satisfactory for transfer purposes. As the culture goes into 

 the spore stage it is less and less suitable until sufficient time elapses 

 for the spores to mature. When spores are present in the inoculum, 

 with these cultures and perhaps others as well, the new tube should 

 be given a heat treatment (80° C. for 20 minutes) after inoculation. 

 Generally, if an anaerobic spore-forming culture is desired in an 

 experiment, inoculate a tube of a favorable medium from a stock 

 culture which contains spores, heat-shock it, and use the resulting 

 culture for the experiment rather than the inoculation of the latter 

 tube or flask directly from the spore containing culture. Maintain 

 the stock culture in the spore state and follow the above transfer 

 routine, rather than carry the anaerobe in a serial passage, and 

 use such cultures for sources of inoculum for experimental flasks or 

 tubes. This is particularly true with the actively fermentative types, 

 where serial passage may yield a culture of undesirable characters — 

 even though it is descended in pure state from a culture that was 

 satisfactory. 



Other Methods of Value 

 stock culture methods 



The anaerobes are susceptible to freezing-drying technic as a 

 means of preservation of cultures over a long period of time as shown 

 by Roe (1940). This technic is unnecessary, however, as species of 

 Clustridium are usually viable in spore state over a long period of 

 time. For the pathogenic group, one should use beef heart infusion, 

 alkaline egg medium, and brain mash, with the latter perhaps being 

 the best. With the butyric-butyl group, use plain corn mash or 

 potato infusion. Prepare the plain corn mash in a manner similar 

 to the method given for corn-liver medium with the exception that 

 the liver powder is omitted. Brain medium may be suitable also. 

 (See also Leaflet II, 9th Edition.) 



In any medium after all gassing has subsided and spores have been 

 demonstrated microscopically, the tube should be sealed in the flame 

 or the stopper covered to protect the medium from evaporation, 

 and the tube placed in a cool room or refrigerator. Viable sub- 

 cultures may be obtained from such tubes for months or even years 

 in some instances. Another method which has been used with suc- 

 cess is worthy of mention. This involves the storage of cultures 

 on sterile soil: Dry fresh garden soil and sift through a fine mesh 

 screen; add 5% of CaCOs to neutralize any acidity of the culture. 

 Place soil in tubes in 2 inch columns and autoclave overnight. Test 

 each tube for sterility using both aerobic and anaerobic media. If 

 sterile, add 2 or 3 ml. of a well sporulated culture with a sterile 

 pipette and dry the tube (preferably in a vacuum desiccator). To 

 obtain an active culture from this stock (which may be stored at 

 room temperature) transfer a small amount of the soil to an enrich- 

 ment medium and heat shock. By the soil stock method a relatively 

 permanent source is available from which cultures may be revived 

 as needed without destroying the stock culture. 



