Ratiometric Imaging of Extracellular pH in Dental Biofilms Using C-SNARF-4

Objectives: pH in dental biofilms plays a central role for the development of caries lesions. For decades, pH measurements in biofilms have been limited to recording pH with electrodes/microelectrodes that do not permit monitoring horizontal pH gradients in biofilms in real-time. Quantitative fluorescent microscopy can overcome these problems. The aim of this demonstration study was to monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4 in in-situ-grown dental biofilms.Methods: Using confocal microscopy, the dye C-SNARF-4 was employed both as pH-sensitive ratiometric dye and as a bacterial stain. We tested the method on natural 48-h in-situ-grown dental biofilms from two individuals. Four biofilms per person were collected on standardized glass slabs mounted in intra-oral appliances. Digital image analysis was employed to remove the bacterial biomass from the microscopic images in order to exclusively determine extracellular pH. We monitored the pH drop at the biofilm-substratum interface in six microscopic fields of view per biofilm for 1h after exposure to 0.4% glucose.Results: Extracellular pH dropped rapidly in all specimens. In both individuals, analysis of variance showed that terminal-pH varied more between specimens (F=6.71, p=0.001) than between microscopic fields of view within single specimens (F=1.93, p=0.112). The rate of the pH drop differed significantly between the two individuals (F=6.25, p=0.017).Conclusions: The combination of pH ratiometry with C-SNARF-4 and digital image analysis allows monitoring of extracellular pH in in-situ biofilms without destroying the complex three-dimensional biofilm architecture. Within the limitations of this study using young biofilms and only two individuals the data suggest that biofilm pH differs more between individual specimens than between different microenvironments within one biofilm. Dent Res 94 (Spec Iss A), abstract number 4072, 2015