NUTRITIONAL AND ENVIRONMENTAL CONDITIONS 29 



charcoal or these resins. Weak anion (IR 4B » and weak cation I IRC 50) 

 (lid not remove the activity from the spent medium. 



When solvent extraction methods were used, the activity could he ex- 

 tracted from the lyophilized spent medium by means of acidic ethanol or 

 acidic aqueous acetone (80'^ acetone). Neutral ethanol seemed to destroy 

 the activity, inasmuch as, after such extraction, neither soluble nor insolu- 

 ble fractions were active, either individually or in combination. 



The sporogenic activity of the whole culture filtrates of PA 3679-h was 

 stable on storage in either acid or neutral conditions but, when stored under 

 alkaline conditions in the refrigerator, the activity was lost. Purified active 

 fractions diminished in activity when stored under refrigeration at pH 8.5. 



A partially defined medium (Medium Q), containing 2% acid-hydro- 

 lyzed casein (NBC, "vitamin-free and salt-free"), biotin, thiamine, niacin, 

 and pyridoxine, plus minerals and 0.1% sodium thioglycollate, supported 

 growth of the organism. The spent medium from this growth appeared to 

 be sporogenically active when refortified with trypticase at a 2% level, but 

 was not active when refortified with the dry ingredient of the growth med- 

 ium. It seems, therefore, that the organism needs something from the tryp- 

 ticase as well as something from the spent Medium Q. The fresh Medium 

 Q to which trypticase was added at the same level yielded sporulation which 

 was inferior to that of 4% trypticase broth. However, 2% trypticase broth, 

 using either the sporogenic or nonsporogenic lots of trypticase and 2% 

 vitamin-free acid-hydrolyzed casein with added thiamine, was superior to 

 4% trypticase broth, and equal to the trypticase refortified spent Medium Q. 



Microbiological amino acid assays and two-dimensional paper chromatog- 

 raphy showed that growth of PA 3679-h in medium Q exhausted the me- 

 dium of phenylalanine, tyrosine, proline, serine, threonine, methionine, 

 and arginine. Leucine, isoleucine, and valine were diminished. Glutamic 

 acid, aspartic acid, and tryptophane were present in the spent medium in 

 about the original concentration. Histidine, lysine, a-aminobutyric, y-amino- 

 butyric acids, and an unidentified ninhydrin positive material were synthe- 

 sized during growth. 



Displacement chromatography using Dowex 50 cation exchange resin, 

 NaCl, CaClo, and NH4OH as displacers showed that the activity of the spent 

 medium was resident in those fractions at the end of the neutral amino acids 

 and ahead of the fractions containing the basic amino acids. The active 

 fractions contained the amino acids leucine, isoleucine and y-aminobutyric 

 acid. These fractions also contained an unidentified fluorescent material. On 

 one 'occasion sporogenic activity extended beyond those fractions contain- 

 ing neutral ninhydrin positive materials. These active fractions also con- 

 tained no basic amino acids. 



