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Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids

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Abstract

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.

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Acknowledgments

Work for this paper was supported through the project No III.B.05 of the second stage of the Multi-Annual Program: “Improving the Safety and Working Conditions”, funded in 2011–2013 for research and development activities by the Ministry of Science and Higher Education/National Center for Research and Development. Coordinator: Central Institute for Labour Protection—National Research Institute. We thank Agnieszka Wielechowska i Monika Uchańska for their excellent technical assistance.

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  1. Department of Microbiology, Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163, Warsaw, Poland

    Elżbieta Anna Trafny, Rafał Lewandowski, Irena Zawistowska-Marciniak & Małgorzata Stępińska

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  1. Elżbieta Anna Trafny
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  2. Rafał Lewandowski
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  3. Irena Zawistowska-Marciniak
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  4. Małgorzata Stępińska
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Correspondence to Elżbieta Anna Trafny.

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Trafny, E.A., Lewandowski, R., Zawistowska-Marciniak, I. et al. Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids. World J Microbiol Biotechnol 29, 1635–1643 (2013). https://doi.org/10.1007/s11274-013-1326-0

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  • DOI: https://doi.org/10.1007/s11274-013-1326-0

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