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Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization

Applied Microbiology and Biotechnology volume 94, pages 799–808 (2012)Cite this article

Abstract

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 103 cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (±1.1) × 106 and 5.0 (±2.3) × 106 cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.

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Acknowledgements

The authors sincerely acknowledge the Department of Biological Sciences at Michigan Technological University for financial support; Dr. Ryan Gilbert, Department of Biomedical Engineering at Michigan Technological University for microscope and imaging facilities.

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Authors and Affiliations

  1. Department of Microbiology and Molecular Biology, NSF International, 789 N Dixboro Road, Ann Arbor, MI, 48105, USA

    Ratul Saha & Robert S. Donofrio

  2. Department of Biological Sciences, Michigan Technological University, Dow 740, 1400 Townsend Drive, Houghton, MI, 49931, USA

    Ratul Saha & Susan T. Bagley

  3. Center for Biofilm Engineering, Montana State University, 366 EPS Building, Bozeman, MT, 59717, USA

    Darla M. Goeres

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  1. Ratul Saha
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  2. Robert S. Donofrio
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  3. Darla M. Goeres
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  4. Susan T. Bagley
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Correspondence to Ratul Saha.

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Saha, R., Donofrio, R.S., Goeres, D.M. et al. Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization. Appl Microbiol Biotechnol 94, 799–808 (2012). https://doi.org/10.1007/s00253-011-3647-y

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  • DOI: https://doi.org/10.1007/s00253-011-3647-y

Keywords

  • Metalworking fluid
  • rRNA group I Pseudomonas
  • FISH
  • 16S rRNA
  • Biofilms

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