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Optimization of Two Immunofluorescent Antibodies for the Detection of Escherichia coli Using Immunofluorescent Microscopy and Flow Cytometry

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Abstract

Two commercially available fluorescein isothiocyanate (FITC) -conjugated anti-Escherichia coli antibodies, tested for immunofluorescence were assessed for their suitability in screening E. coli using flow cytometry. Staining efficacy was initially tested using immunofluorescent microscopy; and further optimization was carried out using flow cytometry. Initially, an acetone fixation step was utilized; however, it was determined statistically that the step could be omitted without impacting the assay and thus reduce the time involved. There was no statistical difference between the staining proficiency of the two antibodies employed. The percentage staining was quite low, approximately 10% for the two antibodies, which indicated that both were equally sensitive but ultimately, more specific antibodies are required for the detection of E. coli. Known proportions of target-E. coli (105, 106, and 107 cells/ml) were mixed with large quantities of non-target bacteria; there was a significant correlation among all the antibodies at the different bacterial cell concentrations. Therefore, despite the low staining percentage achieved on the bacterial cultures, there is a representative and comparative level of staining occurring, between samples and between bacterial strains.

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Acknowledgments

We would like to acknowledge the Marine Institute Advanced Technologies (Strategic Marine RTDI) Programme and the EPA as part of the Science, Technology, Research and Innovation for the Environment (STRIVE) Programme 2007–2013. The Irish Government under the National Development Plan 2007–2013 financed the research. The authors owe their thanks and appreciation to Maurice O’Donoghue, Department of Microbiology, University College Cork for his overall assistance in the laboratory.

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Authors and Affiliations

  1. Department of Zoology, Ecology and Plant Science, University College Cork, The Cooperage, Distillery Fields, North Mall, Cork, Ireland

    Moira McCarthy

  2. Department of Zoology, Ecology and Plant Science, University College Cork, Cork Enterprise Centre, Distillery Fields, North Mall, Cork, Ireland

    Sarah C. Culloty

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  1. Moira McCarthy
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  2. Sarah C. Culloty
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Correspondence to Moira McCarthy.

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McCarthy, M., Culloty, S.C. Optimization of Two Immunofluorescent Antibodies for the Detection of Escherichia coli Using Immunofluorescent Microscopy and Flow Cytometry. Curr Microbiol 62, 402–408 (2011). https://doi.org/10.1007/s00284-010-9721-3

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  • DOI: https://doi.org/10.1007/s00284-010-9721-3

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