Generation of free radicals from phenazine methosulfate, streptonigrin, and riboflavin in bacterial suspensions.

The existence of an EPR signal in a biological sample provides direct evidence for the presence of free radicals in the sample.' If hyperfine structure is discernible in the signal, the free radicals may be identifiable by comparison with known spectra.In a signal which is due to a known radical, the quality of the hyperfine structure can indicate the extent of binding of the radical to macromolecules.' 3 We report here that an EPR signal develops when any one of the inhibitors- phenazine methosulfate, streptonigrin, or rubiflavin-is added to a suspension of bacteria.The existence and structure of these signals may prove important to understanding the mode of action of the inhibitors.Materials and Methods.-Bacteria:Aerated cultures of Escherichia coli strain B, growing exponentially in a tris-buffered minimal medium4 or in trypticase soy broth (Difco), were harvested, washed twice with 0.15 M NaCl, and resuspended in minimal medium (without glucose) at a concentration of 1010 cells/ml.Bacillus megaterium was harvested while in exponential growth in trypticase soy broth, washed as in the case of E. coli, and resuspended at a concentration of 108 cells/ml.Bacterial suspensions were kept at 00C until used and gave satisfactory results