Frequency of 2,3,5-triphenyltetrazolium chloride (TTC) non-reducing bacteria in pasteurized milk

ABSTRACT2,3,5-triphenyltetrazolium chloride (TTC) is a dye largely used for enumeration ofmicrobial colonies in solid culture media, being a key component of the dry rehydratablefilm system used for microbiological analysis of food. This dye is colorless in theoxidized form and red when reduced by microorganisms, due to formation of formazan.In this study, TTC was added to Plate Count Agar (PCA) for enumeration ofmicroorganisms in thirty four pasteurized milk samples, with the aim to verify thefrequency of microorganisms that are unable to reduce TTC. Milk samples weredecimally diluted in saline and pour-plated in PCA plus 0.015% TTC. Colonies werecounted after 24h and 48 h of incubation at 35 o C. From a total of 50,574 colonies,19,665 (38.88%) did not reduce TTC in 48h. It was observed that 571 (6.36%) coloniesthat were colorless in 24h became red in 48h. From those that didn™t reduce TTC in48h, 233 were purified and Gram stained. 229 (98.71%) of them were Gram positivecocci and bacilli. The results show that there is a high percentage of microorganismsunable to reduce TTC in pasteurized milk, which cannot be detected by laboratoryprocedures based on the formation of red colonies.Key words: 2,3,5-triphenyltetrazolium chloride, TTC, pasteurized milk, microbial counts