Source: Schlafer, S., et al. Ratiometric Imaging of Extracellular pH in Dental Biofilms. J. Vis. Exp. (2016)
This video demonstrates a real-time assay for monitoring extracellular pH in dental biofilms using a pH-sensitive ratiometric probe. When an in situ-grown bacterial biofilm is exposed to glucose, a reduction in extracellular pH near the bacterial biomass protonates the probe, leading to its increased concentration inside the bacteria. The disparity in emission wavelength between the protonated and unprotonated probe is utilized to calculate the pH across the extracellular matrix of the biofilm and track pH changes over time.
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Collection of In Situ Grown Dental Biofilm Samples
2. Biofilm pH Imaging
3. Digital Image Analysis
The authors have nothing to disclose.
Zeiss LSM 510 META | Zeiss | N/A | |
C-Apochromat 63X water immersion objective | Zeiss | N/A | |
XL Incubator | PeCON | N/A | |
SNARF-4F 5-(and-6)-Carboxylic Acid | Life Technologies | S23920 | |
Dimethyl sulfoxide | Life Technologies | D12345 | |
HEPES | Life Technologies | 11344-041 | |
Costar 96-well black clear-bottom plate | Fisher Scientific | 07-200-567 | |
Custom-made glass slabs (4x4x1 mm; 1,200 grit) | Menzel | N/A | |
Alginate impression material | GC Corporation | N/A | |
Acrylic Adjusting Logic Sets/set of acrylic dental burs | Axis Dental | LS-906 | |
Orthodontic retainer containers | Spark Medical Equipment Co., Ltd | SK-WDTC01 | |
Sticky wax | Dentsply | N/A | |
Chewing paraffin wax | Ivoclar Vivadent AG | N/A | |
Dithiothreitol | Sigma Aldrich | D0632 | Used during preparation of salivary solution |
0.45 µm and 0.2 µm syringe filters | Sigma Aldrich | CLS431220; CLS431219 | |
daime | University of Vienna, Austria | http://dome.csb.univie.ac.at/daime | |
ImageJ | NIH, Bethesda, Maryland, USA | http://imagej.nih.gov/ij/ |