Application of a tetrazolium dye as an indicator of viability in anaerobicbacteria

The use of the redox dye 5-cyano-2,3,-ditolyl tetrazolium chloride (CTC) fo r evaluating the metabolic activity of aerobic bacteria has gained wide app lication in recent years. In this study, we examined the utility of CTC in capturing the metabolic activity of anaerobic bacteria. In addition, the fa ctors contributing to abiotic reduction of CTC were also examined. CTC was used ill conjunction with the fluorochrome 5-(4,6-dichlorotriazinyl) aminof luorescein (DTAF), that targets bacterial cell wall proteins. to quantitate the active fraction of total bacterial numbers. Facultative anaerobic bact eria, including Escherichia coli grown fermentatively, and Pseudomonas chlo rophis, P. fluorescens, P. stutzeri, and P. pseudoalcalegenes subsp. pseudo alcalegenes grown under nitrate-reducing conditions, actively reduced CTC d uring all phases of growth. Greater than 95% of these cells accumulated int racellular CTC-formazan crystals during the exponential phase. Obligate ana erobic bacteria, including Syntrophus aciditrophicus grown fermentatively, Geobacter sulfurreducens grown with fumarate as the electron acceptor, Desu lfovibrio desulfuricans subsp. desulfuricans and D. halophilus grown under sulfate-reducing conditions, Methanobacterium formicicum grown on formate, H-2 and CO2, and Methanobacterium thermoautotrophicum grown autotrophically on H-2 and CO2 all reduced CTC to intracellular CTC-formazan crystals. Thr optimal CTC concentration for all organisms examined was 5 mM. Anaerobic C TC incubations were not required for quantification of anaerobically grown cells. CTC-formazan production by all cultures examined was proportional to biomass production, and CTC reduction was observed even in the absence of added nutrients. CTC was reduced by culture fluids containing: ferric citra te as electron acceptor following growth of either G. metallireducens or G. sulfurreducens, Abiotic reduction of CTC was observed in the presence of a scorbic acid, cysteine hydrochloride, dithiothreitol, NADH, NADPH, Fe(II)CI ,, sodium thioglycolic acid and sodium sulfide. These results suggest that while CTC can be used to capture the metabolic activity of anaerobic bacter ia, care must be taken to avoid abiotic reduction of CTC. (C) 1999 Elsevier Science B.V. kll rights reserved.