Use of triphenyltetrazolium chloride in preservative efficacy testing

Abstract

Preservative efficacy testing without counting colonies was done by determining growth in dilutions of inoculated product following enrichment in Letheen broth with 0.001% triphenyltetrazolium chloride (TTC) in 96-well microtiter plates. Bacterial growth was indicated by the development of a red/pink color in the enrichment broth. The method was used to determine log reductions of bacteria at specified times after inoculation, and D-values were calculated using the reciprocal of the highest dilution showing growth (pink color) as the log CFU/ml bacteria at each time point. The method using TTC was validated by demonstrating that D-values for Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, and Escherichia coli in 44 aqueous cosmetic and OTC-drug products were virtually identical to those obtained when using Alamar Blue in the miniaturized system. Plotting D-values obtained using TTC as a function of D-values obtained using Alamar Blue gave a line with a slope of 0.98, which shows excellent agreement of results obtained by the two methods. This miniaturized assay system has been used for more than three years for preservative efficacy testing of several hundred cosmetic and OTC-drug product samples in our laboratory. It is recommended for laboratories that conduct large numbers of preservative efficacy tests.