Optimisation of the fluorescein diacetate antibacterial assay.
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.
Wanandy
Stephanus
S
Department of Chemistry, Macquarie University, Sydney NSW 2109, Australia.
Brouwer
Nynke
N
Liu
Qian
Q
Mahon
Andrew
A
Cork
Stephen
S
Karuso
Peter
P
Vemulpad
Subramanyam
S
Jamie
Joanne
J
D000596:Amino Acids / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002021:Buffers; D002701:Chloramphenicol / Q000494:pharmacology; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D005033:Ethylmaleimide / Q000737:chemistry; D005452:Fluoresceins / Q000378:metabolism*; D005839:Gentamicins / Q000494:pharmacology; D007202:Indicators and Reagents / Q000378:metabolism; D008299:Maleic Anhydrides / Q000737:chemistry; D008826:Microbial Sensitivity Tests / Q000379:methods; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000254:growth & development / Q000378:metabolism*; D013752:Tetracycline / Q000494:pharmacology
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D002021:Buffers; D005452:Fluoresceins; D005839:Gentamicins; D007202:Indicators and Reagents; D008299:Maleic Anhydrides; D002701:Chloramphenicol; D013752:Tetracycline; D005033:Ethylmaleimide; C018506:diacetylfluorescein