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 journalArticleResearchpeer-review

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.

LanguageEnglish
Pages21-30
Number of pages10
JournalJournal of Microbiological Methods
Volume60
Issue number1
DOIs
Publication statusPublished - Jan 2005

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Maleic Anhydrides
Peptones
Ethylmaleimide
Chloramphenicol
Gentamicins
Tetracycline
Histidine
Pseudomonas aeruginosa
Cysteine
Buffers
Yeasts
Escherichia coli
Anti-Bacterial Agents
Bacteria
3',6'-diacetylfluorescein

Cite this

Wanandy, Stephanus ; Brouwer, Nynke ; Liu, Qian ; Mahon, Andrew ; Cork, Stephen ; Karuso, Peter ; Vemulpad, Subramanyam ; Jamie, Joanne. / Optimisation of the fluorescein diacetate antibacterial assay. In: Journal of Microbiological Methods. 2005 ; Vol. 60, No. 1. pp. 21-30.
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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.",
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Optimisation of the fluorescein diacetate antibacterial assay. / Wanandy, Stephanus; Brouwer, Nynke; Liu, Qian; Mahon, Andrew; Cork, Stephen; Karuso, Peter; Vemulpad, Subramanyam; Jamie, Joanne.

In: Journal of Microbiological Methods, Vol. 60, No. 1, 01.2005, p. 21-30.

Research output: Contribution to journalArticleResearchpeer-review

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