Sugar challenge with planktonic NCTC 10449 cells
This protocol is extracted from research article:
Fluorescent nanosensors reveal dynamic pH gradients during biofilm formation
NPJ Biofilms Microbiomes, Jun 17, 2021; DOI: 10.1038/s41522-021-00221-8

An overnight culture of S. mutans NCTC 10449 in BHI was spun down and re-suspended in PBS for 30 min as a starvation step. The cells were spun down again and re-suspended in saline before being mixed with positively charged nanosensors in saline to obtain an OD600 of 1 and a nanosensor concentration of 1 mg mL−1. The suspension was aliquoted into a Greiner 24-well, PS, flat glass bottom, black walled microplate using 400 µm per well. Imaging was performed using widefield fluorescence microscopy with the ×40/0.6 lens (×40 objective with ×1.5 additional magnification). An image was taken (brightfield, OG-FAM excitation = 460 nm, TAMRA excitation = 550 nm) for time point 0 min before 20 µL of either glucose, sucrose, xylose, xylitol (all 1% final conc.) or saline was added to their respective wells. Images were taken every 5 min for 30 min. Additional controls with glucose, sucrose, xylose, xylitol and saline solution added to 1 mg mL−1 positively charged nanosensors in saline were also imaged to confirm the absence of any pH change brought on by the solutions themselves.

The calibration was performed using a Greiner 24-well microplate where pH buffers from pH 8 to pH 2.5 were mixed with positively charged polyacrylamide nanosensors for a final concentration of 1 mg mL−1. Images were taken from each pH using the same exposure settings as the experiment. The fluorescence intensity ratio from each pH was plotted and the linear regression calculated to determine the pH values from the fluorescence intensities generated during the experiment.

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