Improved pH-ratiometry for the three-dimensional mapping of pH microenvironments in biofilms under flow conditions

Confocal microscopy-based monitoring of pH in biofilms is gaining increasing interest, as it allows for a quick assessment of horizontal pH gradients without mechanically perturbing the biofilm. Ratiometric monitoring of pH with the fluorescent dye C-SNARF-4 has been used to reliably map extracellular pH in the basal layers of biofilms, but only under static conditions. Here, we expand this methodology to measurements of vertical gradients in multispecies in vitro-grown and in situ-grown dental biofilms of different age, and to pH measurements in in vitro-grown biofilms under flow conditions. After static incubation with glucose, young in vitro-grown biofilms (30h) were more acidogenic than older biofilms (120h). However, under dynamic conditions mimicking the oral salivary flow, low pH was only preserved in older biofilms. As both types of biofilm were of similar thickness (~20 μm), these findings highlight the importance of cell density and biofilm matrix maturation for pH developments. In both in vitro-grown and in in situ-grown biofilms, horizontal and vertical pH gradients were observed. Under static conditions, the surface layer of the biofilms tended to be more acidic, whereas the bottom layer became more acidic under dynamic conditions. Compared to in vitro-grown biofilms, 120 h in situ-grown biofilms showed higher acidogenicity during static incubation. This study shows that pH ratiometry with C-SNARF-4 is well-suited to monitor extracellular pH in thin biofilms in all three dimensions. The different pH dynamics observed under static and dynamic conditions argue for the implementation of flow during real-time assessment of biofilm pH.