16 ROBERT E. COLLIER 



improved after a few transfers in the butyl alcohol fermentation and why 

 you get more spores. 



Orin Halvorson: We have assumed that, if we continue to grow these 

 cultures in the vegetative state, we favor the growth of non-sporeforming 

 mutants, and therefore we want to avoid too many transfers in the vegeta- 

 tive state. 



SUSSMAN : The observation by Mr. Collier that cells only partially spor- 

 ulated will complete the process in distilled water needs, I think, a little 

 clarification. Is it possible that in distilled water you are getting lysis which 

 would provide nutrients for this process? 



Collier: I made total counts in all of these studies and found that the 

 lysis of vegetative cells without spores was neghgible, but nutrients could 

 have come from the sporangia that lysed. 



RoBiNOW: Faced with the problem of having cultures in the early stages 

 of spore formation for classwork at 3 :15 in the afternoon, I found quite 

 some time ago the following procedure useful. When Bacillus megaterium 

 is grown overnight at 16°C. on potato extract agar, confluent growths are 

 produced. When these cultures, 18 hours later, are transferred to 37^, rapid 

 and simultaneous spore formation occurs three hours later. Five hours later 

 approximately two-thirds of the cells are in the advanced stages of spore 

 formation which is useful for cytological and teaching purposes. 



Campbell: Dr. Brown, what concentration of lysozyme did you use in 

 cleaning your spores? 



Brown: We used a concentration of about 0.5 mg per ml. We realize this 

 is high, but we had a very heavy suspension and we used this routinely to 

 separate the spores and it worked well with PA 3679. Mr. Titus has used 

 it successfully with B. ster other mophilus. 



LuND: In regard to using lysozyme with PA 3679, we have also done 

 that and we used about the same concentration, but we adjusted the pH to 

 11 where there seems to be an automatic, almost immediate dissolution of all 

 vegetative cells. We thought at first that this gave us clean suspensions; 

 however, upon examing the cells under the phase microscope, we thought 

 we saw shadows around those spores that were the shadows of the original 

 sporangia. We tried staining these by various methods and finally found 

 that hematoxylin gave a stainable outline of the original cell. 



Powell: We've recently had a great gleam of hope about the prepara- 

 tion of clean spore suspensions and we find that when a laboratory strain 

 of Cereus sporulates, it builds up a very strong lytic system which will 

 attack vegetative cell walls. Now, my colleague, Mr. Strange, has done 

 more work on this enzyme than I have, so perhaps it is a little premature. 



