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

Abstract

2,3,5-triphenyltetrazolium chloride (TTC) is a dye largely used for enumeration of microbial colonies in solid culture media, being a key component of the dry rehydratable film system used for microbiological analysis of food. This dye is colorless in the oxidized 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 of microorganisms in thirty four pasteurized milk samples, with the aim to verify the frequency of microorganisms that are unable to reduce TTC. Milk samples were decimally diluted in saline and pour-plated in PCA plus 0.015% TTC. Colonies were counted after 24h and 48 h of incubation at 35°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%) colonies that were colorless in 24h became red in 48h. From those that didn't reduce TTC in 48h, 233 were purified and Gram stained. 229 (98.71%) of them were Gram positive cocci and bacilli. The results show that there is a high percentage of microorganisms unable to reduce TTC in pasteurized milk, which cannot be detected by laboratory procedures based on the formation of red colonies.