Journal of Microbiological Methods, Volume 54, Issue 3 , September 2003, Pages 315-323
S. Gaines (a), T. C. Jamesa (b), M. Folan (c), A. W. Baird (d) and C. O'Farrelly (a)
(a) Education and Research Centre, St. Vincent's University Hospital, Elm Park, Dublin 4, Ireland
(b) The Moyne Institute for Preventive Medicine, Trinity College, Dublin 2, Ireland
(c) Westgate Biological Ltd., Roebuck Castle, University College Dublin, Dublin 4, Ireland
(d) Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University College Dublin, Dublin 4, Ireland
Received 18 September 2002; revised 17 December 2002; accepted 20 December 2002. ; Available online 24 May 2003.
Abstract
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 M 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¥107 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.
Author Keywords: Adhesion; Dental caries; Fluorescence; Hydroxylapatite; Streptococcus mutans
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