4.3. Treatment of Biofilms and pH Measurement

The biofilms were treated 72, 78, and 96 h after the beginning of the biofilm formation, for 1 min [30,31]. The two first treatments were performed in order to achieve and verify the cumulative effect of the compounds, similarly to what happens in in vivo conditions after brushing with a fluoridated dentifrice. Regarding the last treatment, it was performed in order to evaluate the effect of the compounds on the biofilms right after the treatment [30,31].

The treatments were performed with HMP solutions of 0.25% (0.25HMP), 0.5% (0.5HMP), 1% (1HMP), 0.25HMP + 500 ppm F (0.25HMP/F), 0.5 HMP + 500 ppm F (0.5HMP/F), and 1HMP + 500 ppm F (1HMP/F), prepared by diluting the compounds in deionized water [12]. Solutions containing 500 and 1100 ppm F and pure AS (without F or HMP) were tested as controls. After the last treatment, the biofilms were gently washed with 1 mL AS (for 10 s) [12]. The biofilms were then scraped with a cell scraper and transferred, with the assistance of a pipette, to microtubes. For pH measurement, it was performed using a pH electrode (PHR-146 Micro Combination pH Electrode, Fisher Scientific, California, USA), previously calibrated with 7.0 and 4.0 pH standards [29]. The biofilm pH was measured in the total biofilm, before the separation of the biofilm fluid and biomass. Given that the biofilm pH should be measured immediately after the last treatment or the sucrose exposure (for those biofilms exposed to sucrose), the biofilm pH was determined before the separation of the solid and fluid phases.

In another set of experiments, after the last treatment, AS was removed from the wells, and the biofilms were submitted to a cariogenic challenge by their exposure to a 20% sucrose solution for 3 min [29]. The sucrose solution was then removed, and the biofilms were scraped from the wells and transferred to microtubes (within 1 min after removal of the sucrose solution). The pH analysis was performed as described above [29]. The sucrose challenge above was based on a previous study [29], which involved the evaluation of sucrose solutions at different concentrations administered at different duration periods in order to resemble clinical conditions. It was validated that a mixed biofilm of S. mutans and C. albicans exposed to a 20% sucrose solution for 3 min exhibited a pattern of pH change similar to that observed in vivo.

All the parameters analysed in this study (i.e., biofilm pH, and F, Ca, and P from the biofilm fluid and biomass) have been analysed in biofilms exposed or not to the sucrose challenge, in separate experiments.