SUBSTANCES PRODUCED BY BACTERIA 179 



rather interesting history. Emmerich believed that it is an enzyme 

 (233, 236). Later it was found (766) that all the active substance 

 could be extracted with lipid solvents j the extraction of the cells of Ps. 

 aeruginosa with alcohol also gave active antibacterial preparations. A 

 crystalline product was finally obtained (448) 5 it was soluble in organic 

 solvents and had a bactericidal effect upon B. anthracisy S. albus, C. 

 difhtheriae, and a number of other organisms. 



In the course of time it was recognized that all the antibacterial ac- 

 tivity of the lipoid extracted from the medium was due to the presence 

 of fatty acids, so that the term pyocyanase is now used to designate the 

 antibiotic lipid, found in the medium and containing unsaturated fatty 

 acids. Certain well-defined compounds have recently been isolated, such 

 as pyolipic acid (50a). 



Schoenthal (843) obtained three compounds that possessed antibac- 

 terial properties, namely, pyocyanin, oxyphenazine, and an active oil 

 that formed insoluble salts with calcium, barium, and heavy metals. 

 The last appeared to be similar to what had previously been described 

 as pyocyanic acid, a substance highly active against 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 (346), although the effect of 

 tyrosine is not very significant (346, 461, 573). 



The determination of the nature of the antibacterial substances of 

 Ps. aeruginosa can be carried out in the following manner (418) : 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 the antibacterial properties are 

 yery little diminished by removal of the pigment 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. 



