Tetrazolium reduction as an aid for streptococcal growth detection with agar dilution susceptibility testing

A dye reduction method for determining a definitive endpoint with agar dilution susceptibility testing has been developed. Bacterial growth was determined by applying to the inoculum spot a dye solution containing 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride and phenazine methosulfate. Viable colonies reduced the tetrazolium salt to a visible red color within 3 to 5 min. The minimum inhibitory concentrations of six antimicrobial agents tested against 167 clinical streptococcal isolates were recorded before and after the addition of the tetrazolium-phenazine methosulfate solution. A total of 252 discrepancies (25%) were observed, and of these, 30 (12%) differed by more than one tested antibiotic concentration. Endpoint reproducibility of the dye procedure was assessed by four technologists in a double-blind study. A 2.7-fold reduction in disagreement was observed when the dye was used. Use of the tetrazolium-phenazine methosulfate solution involves little deviation from standard antimicrobial susceptibility test procedures and yields more accurate, as well as reproducible, susceptibility results.