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An Evaluation of Three New-Generation Tetrazolium Salts for the Measurement of Respiratory Activity in Activated Sludge Microorganisms

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Abstract

XTT (3′-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate), MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt), and WST–1 (4-(3-4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzenedisulfonate) are tetrazolium salts that have become commercially available only in relatively recent years; they differ from earlier such compounds in that their reduction gives rise to a formazan product that is water soluble. We have established the sites in the prokaryotic respiratory chain at which each of the dyes is reduced to its corresponding formazan and have evaluated the suitability of each for the colorimetric estimation of electron transport system activity in populations of activated sludge microorganisms. Reduction of all three tetrazolium salts was shown to be proportional to cell biomass and oxygen uptake and to be susceptible to low levels of the reference toxicant 3,5-dichlorophenol. XTT, which was not inhibitory at concentrations of up to 2 mM and was reduced by 91% of isolates from a sample of culturable activated sludge bacteria, was chosen for further assay development. XTT-formazan production was found to be stimulated by the availability of an exogenous carbon and energy source, and by the presence of the electron-coupling agent phenazine methosulfate. Less than 3% of XTT reduction by an activated sludge sample was abiotic. An assay based on this compound could be a valuable and simple tool for the routine monitoring of the performance of wastewater treatment systems.

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Acknowledgments

The work was supported by the Queen’s University Environmental Science and Technology Research Centre (QUESTOR); a Strategic Research Infrastructure (SRIF) grant for Environmental Engineering and Biotechnology at the Queen’s University Belfast; and the Invest Northern Ireland RTD Centres of Excellence Programme.

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Authors and Affiliations

  1. School of Biology and Biochemistry and QUESTOR Centre, The Queen’s University of Belfast, Northern Ireland

    C. McCluskey, J.P. Quinn & J.W. McGrath

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  1. C. McCluskey
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  2. J.P. Quinn
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  3. J.W. McGrath
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Correspondence to J.W. McGrath.

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McCluskey, C., Quinn, J. & McGrath, J. An Evaluation of Three New-Generation Tetrazolium Salts for the Measurement of Respiratory Activity in Activated Sludge Microorganisms. Microb Ecol 49, 379–387 (2005). https://doi.org/10.1007/s00248-004-0012-z

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