title: Monitoring of extracellular pH in young dental biofilms grown in vivo in the presence and absence of sucrose.
abstract: OBJECTIVE
  pH in dental biofilms is of central importance for the development of caries. We used the ratiometric pH-sensitive dye C-SNARF-4 in combination with digital image analysis to monitor extracellular pH in dental biofilms grown in situ with and without sucrose supply.
  
  
  METHODS
  Dental biofilms (48 h) from 10 individuals were collected on glass slabs mounted on intra-oral appliances. During growth, appliances were immersed extra-orally in either physiological saline or 4% sucrose for 2 min, eight times per day. Fluorescence emissions of C-SNARF-4 in deep layers of the biofilms were recorded ex vivo with confocal microscopy for 15 min or for 1 h after exposure to 0.4% glucose. Extracellular pH was determined ratiometrically using digital image analysis.
  
  
  RESULTS
  Extracellular pH dropped rapidly in most examined sites after addition of glucose. Distinct pH microenvironments were observed within single biofilms. The variation in pH was similar between sites within the same biofilm and sites from different individuals. pH drop patterns did not differ between biofilms exposed to sucrose-free and sucrose-rich environments.
  
  
  CONCLUSIONS
  The present study is the first of its kind to apply the combination of pH ratiometry and digital image analysis to systematically record extracellular pH in intact dental biofilms from several individuals for up to 1 h. We observed highly heterogeneous pH landscapes and the presence of acidogenic microenvironments - 'acidogenic hotspots' - within the biofilms. The data suggest that pH drops in young (48 h) dental biofilms are independent of the sucrose supply during growth.
authors.affiliation: Department of Dentistry, HEALTH, Aarhus University, Aarhus, Denmark; idige@odont.au.dk.
authors.forename: Irene
authors.initials: I
authors.lastname: Dige
authors.affiliation: Department of Dentistry, HEALTH, Aarhus University, Aarhus, Denmark.
authors.forename: Vibeke
authors.initials: V
authors.lastname: Baelum
authors.affiliation: Department of Dentistry, HEALTH, Aarhus University, Aarhus, Denmark.
authors.forename: Bente
authors.initials: B
authors.lastname: Nyvad
authors.affiliation: Department of Dentistry, HEALTH, Aarhus University, Aarhus, Denmark.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schlafer
keywords: C-SNARF-4; dental biofilms; digital image analysis; imaging; pH; ratiometric imaging; sucrose