162 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES 



a-hydroxyphenazine, and an oil that forms insoluble salts with calcium, 

 barium, and heavy metals. The last appears to be similar to what has 

 previously been described as pyocyanic acid, a substance strongly lytic 

 to V. comma. All three compounds were isolated by extraction with 

 chloroform. 



Different strains of Ps. aeruginosa may produce either pyocyanase or 

 pyocyanin or both, the production of the two not proceeding in a paral- 

 lel manner. Among the amino acids, alanine and tyrosine were found to 

 be favorable to pyocyanin production (27, 320), although the effect 

 of tyrosine is not very significant (340, 341, 452, 529). It was suggested 

 (407) that the antagonistic action of young cultures of Ps. aeruginosa 

 is due primarily to the presence of pyocyanin, whereas in older cultures 

 pyocyanase is largely concerned. 



The determination of the nature of the antibacterial substances of 

 Ps. aeruginosa can be carried out in the following manner (407) : the 

 organism is grown in bouillon for 14 daysj the cultures are heated for a 

 half hour at 75° C. to kill the living cells j they are then centrifuged, 

 the liquid is treated with chloroform which extracts the pigment, and 

 the chloroform solution is concentrated in vacuo at 50° C. j the aqueous 

 solution remaining after chloroform extraction is acidified with hydro- 

 chloric acid and again shaken five times with chloroform, thus extract- 

 ing the fatty acids. It was found that, on removing the pigment, the 

 antibacterial properties are very little diminished j however, when both 

 the pigment and the fatty acids are removed, no antibacterial action is 

 left in the culture. S. aureus is commonly used as the test bacterium. 



In most cases the broth culture of the organism is first extracted with 

 ether, giving pyocyanase, and the residue treated with chloroform, 

 yielding pyocyanin. The solution left after the removal of the blue 

 chloroform extract may be again treated with ether, giving a yellow 

 pigment, which also has some activity (501 ). This pigment is a deriva- 

 tive of pyocyanin and is often designated ( 1006) as hemipyocyanin. It 

 may also be obtained by acidifying pyocyanin with acetic acid and heat- 

 ing. The fluorescin remaining in the culture after the ether and chloro- 

 form extraction was found to be inactive. In old cultures, pyocyanin is 

 changed into a brown pigment, pyoxanthose. A fourth pigment, which 

 is yellow in transmissible light and fluorescent-green in reflected light. 



