A novel spectrofluorometric microassay for Streptococcus mutans adherence to hydroxylapatite

Adherence to the tooth surface by Streptococcus mutans is an important step in initiation of dental caries. Current in vitro methods used to study bacterial adherence are time-consuming and may involve the use of radiolabels. The aim of this study was to develop a more convenient, high-throughput, microtitre-plate assay of bacterial adherence to hydroxylapatite. S. mutans was labelled with the fluorescent indicator BCECF/AM and fluorescence measured using a spectrofluorometer. Fluorescence microscopy confirmed label uptake. Optimal labelling occurred at 120 min with 50 μM BCECF/AM in DMSO. Viability was similar in control untreated bacterial cells, bacteria treated with DMSO alone or with the label for up to 4 h. Preliminary adherence experiments were performed using four commercially available types of hydroxylapatite. Fluorescence from pre-labelled bacteria was measured for bound cells. The assay was then optimised with respect to time and bacterial concentration using Fluka crude hydroxylapatite. Time course studies demonstrated that adherence reached saturation by 30 min incubation when using 1×10 7 cfu/ml labelled bacteria to 1 mg hydroxylapatite, coated with PBS or saliva. The fluorescence-based adherence assay was highly reproducible in repeated analyses and was useful in demonstrating interference with adherence. In conclusion, this microtitre-plate assay offers a more convenient approach to examine streptococcal adherence and could be used to screen for potential anti-adhesive agents.