Optimisation of the fluorescein diacetate antibacterial assay

Stephanus Wanandy, Nynke Brouwer, Qian Liu, Andrew Mahon, Stephen Cork, Peter Karuso, Subramanyam Vemulpad, Joanne Jamie

    Research output: Contribution to journalArticlepeer-review

    25 Citations (Scopus)

    Abstract

    The fluorescein diacetate (FDA) antibacterial assay relies on the cleavage of fluorescein diacetate by metabolically active bacteria. The recent finding that microbiological media can lead to significant levels of cleavage has reduced the reliability of the assay. Using the nucleophilic scavengers N-ethylmaleimide and maleic anhydride, we have demonstrated that this abiotic cleavage is most likely due to nucleophiles such as cysteine and histidine commonly present in the media. To increase the reliability of the assay we have modified the original assay conditions to include use of dilute medium (peptone 0.2% w/v, yeast extract 0.1% w/v and NaCl 0.1% w/v) in a non-nucleophilic buffer and overnight incubation of the medium after addition of antibacterial agents. The optimised fluorescein diacetate assay has been used to determine the MIC of gentamicin, tetracycline and chloramphenicol for Escherichia coli, Staphyloccocus aureus and Pseudomonas aeruginosa and gave quantitative results that were reproducible and consistent with published data.

    Original languageEnglish
    Pages (from-to)21-30
    Number of pages10
    JournalJournal of Microbiological Methods
    Volume60
    Issue number1
    DOIs
    Publication statusPublished - Jan 2005

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