2,3,5-Triphenyltetrazolium chloride as a novel tool in germicide dynamics

A novel, colorimetric method using 2,3,5-triphenyltetrazolium chloride (TTC) in tandem with membrane filtration is described for the determination of death rates of Escherichia coli (E. coli) due to microbiocides. This method enables results to be obtained on the same day in contrast to the 18 h required by the accepted aerobic plate count method. The microbiocides investigated were the preservatives 2-bromo-2-nitro-1,3-propanediol (Bronopol), N-(hydroxymethyl)-N-(1,3-dihydroxymethyl-2,5- dioxo-4-imidazolidinyl)-N'-(hydroxymethyl)-urea (Germall II), phenethyl alcohol, and benzyl alcohol. D values (time required per log reduction of E coli) were determined by this method, and equations relating the D values to preservative concentrations were derived [i.e., eta values (the logarithmic values relating changes in rates of kill for specified changes in concentration) and A values (extrapolated D values at 1% concentration) were determined]. these equations are compared with those previously published using the accepted aerobic plate count method. The potential advantages of this method are that it has a broad range of application as TTC is reduced by a wide variety of microbes; the test is easily done; results can be achieved in one day; dead cells do not cause interference; test sensitivity can be increased by increasing the length of incubation time or by using membrane filtration in tandem with TTC reduction; and preservative inactivation may be achieved by filtration and flushing with an inactivator, or by adding neutralizers to the TTC broth.