Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

Harriet Whiley, Michael Taylor, Richard Bentham

    Research output: Contribution to journalArticle

    9 Citations (Scopus)

    Abstract

    This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.

    Original languageEnglish
    Pages (from-to)304-309
    Number of pages6
    JournalJournal of Microbiological Methods
    Volume86
    Issue number3
    DOIs
    Publication statusPublished - Sep 2011

    Keywords

    • Fluorescent in situ Hybridisation (FISH)
    • Legionella longbeachae
    • Legionella spp.
    • Potting mix

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