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To develop a colorimetric colony-screening assay to facilitate the isolation of micro-organisms capable of defluorination.A metal-dye chelate, zirconium-xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1:2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l−1 respectively), the assay could detect a fluoride application spot (5 mmol l−1) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0·1 g l−1 and some proteins digest to between 1 and 5 g l−1. A microbial enrichment culture growing on solidified medium containing 20 mmol l−1 fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated.A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro-organisms growing on solidified medium.This method can be used to facilitate the isolation of micro-organisms capable of defluorination.