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Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm

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Abstract

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.

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Acknowledgments

This work was supported by the Consejería de Educación of the Junta de Andalucía and the Ministerio de Ciencia y Tecnología in the Programa General del Conocimiento. The authors wish to thank T. MacFarlane for his helpful suggestions while translating the manuscript.

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Correspondence to Alberto Mariscal.

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Mariscal, A., Lopez-Gigosos, R.M., Carnero-Varo, M. et al. Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm. Appl Microbiol Biotechnol 82, 773–783 (2009). https://doi.org/10.1007/s00253-009-1879-x

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Keywords

  • Biofilm
  • Resazurin
  • Disinfectant
  • Fluorescence