An assessment of the metabolic activity of starved and vegetative bacteria using two redox dyes

The abilities of two redox dyes, 2-(p-iodophenyl)-3-(p-nitrophenyl)5-phenyltetrazolium chloride (INT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to determine the metabolic activity of vegetative and starved cultures of Pseudomonas putida and a thermophilic sulphate reducing bacterium (t-SRB) Ex296, were compared. INT detected higher levels of metabolic activity with starved cells of both cultures, INT and CTC could both be used to stain vegetative anaerobic cultures without attendant problems of background reduction. CTC is reduced to an orange fluorescent formazan by metabolically active cells. During development of the CTC method for use with anaerobic cultures a novel property of this tetrazolium salt was observed. When CTC was added to glutaraldehyde-fixed (i.e. dead) cells they fluoresced green under epifluorescence microscopy. This allowed the total cell population to be counted, offering a new alternative to the traditional method of staining total populations with acridine orange.