136 MANUAL OF MICROBIOLOGICAL METHODS 



the new tube of medium, for it is this portion of the medium which will 

 stay reduced the longest. Although it is possible to initiate growth from 

 a small number of cells, in routine studies use a more adequate inoculum. 

 To facilitate the placing of the inoculum in the bottom of the tube with 

 liquid and semisolid media, substitute a Wright or Pasteur pipet (used 

 with small rubber bulbs) for the inoculation needle. By this means trans- 

 fer a small drop (0.1 or 0.2 ml) of the culture to the new tube. Use the 

 pipet also in the isolation of subsurface colonies particularly from media 

 in which the concentration of agar is reduced. Prepare these pipets from 

 6- to 8-in. lengths of sterile 8- to 9-mm soft glass tubing (with cotton plug 

 in each end) by applying heat to the center of the glass and pulling to form 

 two capillary pipets. 



In general, use a culture from 16-20 hr old. With the pathogenic types 

 this time may be extended a few hours with no harm. 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 suit- 

 able 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 min) 

 after inoculation. 



Generally, if an anaerobic sporeforming culture is desired in an experi- 

 ment, inoculate a tube of a favorable medium from a stock culture which 

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

 ment rather than the inoculation of the latter tube or flask directly from 

 the spore-containing culture. Maintain the stock culture in the spore 

 state, follow the above transfer routine rather than carry the anaerobe in a 

 serial passage, and use such cultures for sources of inoculum for experi- 

 mental flasks or tubes. This is particularly true with the actively 

 fermentative types, where serial passage may yield a culture of undesir- 

 able 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 Clostridium 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 last perhaps being the best. With the butyric-butyl 

 group, use plain corn mash or potato infusion. Prepare the plain corn 



