Assessing Plaque Acid Tolerance

Plaque acid tolerance was evaluated using a previously validated method (Neilands et al., 2012 (link); Senneby et al., 2017 (link)). Briefly, 25 µl plaque sample was mixed with 75 µl TYE medium (1.7% tryptone, 0.3% yeast) containing 20 mM glucose and 40 mM phosphate/citrate buffer adjusted to pH 3.5 and incubated aerobically at 37°C for 2 hours. Following incubation, the cells were stained with LIVE/DEAD^® BacLight™ Fluorescent Stain (Molecular Probes) and transferred into an Ibidi mini flow cells (Ibidi GmbH). Flow-cells were then viewed with confocal laser scanning microscopy (CLSM) using a Nikon Eclipse TE2000 microscope (Nikon Corp.) with an Ar laser (488 nm laser excitation). Images were acquired with a Photometrics Prime 95B camera using Nikon NIS-Elements software. Ten randomly selected images from each sample were saved for further analysis. All confocal images were examined by an experienced oral microbiologist and given a score 1-5 as described by Senneby et al. (2017) (link). This method has been shown to have high intra-rater agreement and the scoring corresponding well to the percentage obtained by manually counting the cells (Senneby et al., 2017 (link)).