Glass microscope slides

Glass microscope slides (Corning Incorporated, New York, USA) were cut to obtain slides with 10 × 5 × 1 mm (Figure 1(a)). Glass slides were autoclaved, anchored vertically with metal devices, and suspended in a 24-well culture plate (Figure 1(b)).

This S. mutans biofilm model was a modified version detailed by Koo et al. [22 (link)] and Ccahuana-Vásquez and Cury [23 (link)], using culture medium and inoculum prepared as indicated by CLSI M7-A6 [21 ]. S. mutans UA159 biofilms were formed on glass microscope slides (Corning® Incorporated, New York, USA), suspended vertically in Ultrafiltered (10 kDa molecular weight cut-off membrane; Amicon) Tryptone-Yeast Extract Broth (UTYEB) [22 (link)], or cation-adjusted-Müller-Hinton broth (Sigma-Aldrich Co., St. Louis, MO, USA) with or without lysed horse blood (MHS or MHS, resp.) [21 ], all containing 1% sucrose, at 37°C, 10% CO₂ for 5 days. After 48 h one group of biofilm (baseline 48 h) was collected and the other groups were treated by 1 min, twice a day, with the respective solutions, for 3 days: group 2, 0.9% NaCl (Baseline 120 h) (n = 4); group 3, CHX 1 μg/mL (0.0001%) (n = 4); group 4, CHX 10 μg/mL (0.001%) (n = 4); group 5, CHX 120 μg/mL (0.012%) (n = 4); group 6, CHX 1.200 μg/mL (0.12%) (n = 4). The biomass and viable bacteria of biofilms were determined and the culture medium pH was determined daily, as an indicator of biofilm acidogenicity. The determination of the pH medium of groups 2 to 6 was carried out directly on the plate, with a calibrated pH meter and controlled environment.