title: Bacterial localization and viability assessment in human ex vivo dentinal tubules by fluorescence confocal laser scanning microscopy.
abstract: OBJECTIVE
  To develop a convenient method for the localization and quantification of live and dead bacteria in human ex vivo mineralized dentinal tubules.
  
  
  METHODS
  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.
authors.affiliation: Sir John Walsh Research Institute, School of Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: D
authors.initials: D
authors.lastname: Parmar
authors.forename: C H J
authors.initials: CH
authors.lastname: Hauman
authors.forename: J W
authors.initials: JW
authors.lastname: Leichter
authors.forename: A
authors.initials: A
authors.lastname: McNaughton
authors.forename: G R
authors.initials: GR
authors.lastname: Tompkins
mesh_terms: D058491:Bacterial Load; D002126:Calcium Hydroxide / Q000494:pharmacology; D003786:Dental Pulp Cavity / Q000382:microbiology*; D003804:Dentin / Q000382:microbiology*; D013293:Enterococcus faecalis / Q000187:drug effects / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D050296:Microbial Viability / Q000187:drug effects; D018613:Microscopy, Confocal; D009930:Organic Chemicals; D011419:Propidium; D012388:Root Canal Irrigants / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D019227:Tooth Apex / Q000382:microbiology
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; D012388:Root Canal Irrigants; C103389:SYTO 9; D011419:Propidium; D002126:Calcium Hydroxide