An Assay for Real-Time Monitoring of Extracellular pH in Dental Biofilms
Transcription
Take a multi-well plate containing a neutral pH salivary solution supplemented with glucose.
Add a dual-emission ratiometric, pH-sensitive dye.
Place the plate on a confocal microscope stage.
Introduce a glass slab with an adhered dental biofilm into the well facing down.
Bacteria in the biofilm ferment glucose, generating organic acids and lowering pH in the vicinity.
The acidic pH protonates the dye, causing its internalization and up-concentration within bacteria, staining them, while the deprotonated dye remains extracellular.
Over time, the pH becomes acidic in other areas of the biofilm.
Upon laser excitation, the protonated dye exhibits a shift in fluorescence emission compared to its deprotonated form.
Capture fluorescent images at specified intervals.
Using appropriate software, eliminate bacterial-derived fluorescence signals.
Calculate the ratio of fluorescent emission intensities from the extracellular matrix at two specific wavelengths, correlating to pH values.
Determine the decrease in extracellular biofilm pH following glucose metabolism by bacteria.
