Bacterial localization and viability assessment in human ex vivo dentinal tubules by fluorescence confocal laser scanning microscopy

Parmar D, Hauman CHJ, Leichter JW, McNaughton A, Tompkins GR. Bacterial localization and viability assessment in human ex vivo dentinal tubules by fluorescence confocal laser scanning microscopy. International Endodontic Journal, 44, 644–651, 2011. Abstract Aim  To develop a convenient method for the localization and quantification of live and dead bacteria in human ex vivo mineralized dentinal tubules. Methodology  The roots from human single-rooted teeth (n = 12) were infected with Enterococcus faecalis V583 and either treated with calcium hydroxide paste or left untreated; six control roots were uninoculated and untreated. Following further incubation, roots were stained with fluorescent DNA-binding reagents, washed thoroughly, sectioned and examined by confocal laser scanning microscopy. Computer-assisted determinations of fluorescence (bacterial viability) were compared statistically. Results  Bacteria were distributed in the tubules throughout the length of the roots but tubule penetration distance was slightly reduced in the apical sections. There was no significant difference in bacterial tubule penetration between roots from different teeth and small standard deviations indicated reproducibility appropriate for experimental application. Following treatment with calcium hydroxide paste, live and dead bacteria were readily distinguishable by contrasting green and red fluorescence. Bacterial viability determinations amongst roots treated in the same way were not significantly different, and the small standard deviation is commensurate with experimental application. Conclusions  Fluorescent viability staining is a convenient, accurate and reproducible method for localizing and quantifying live and dead bacteria in human ex vivo mineralized dentinal tubules.