Inlab micro

First, root dentin samples were predemineralized in 1 L of a demineralizing solution (pH 5.0, 37 °C) containing 50 mM acetic acid, 3 mM CaCl₂ × H₂O, 3 mM KH₂PO₂ and 6 mM methyl-hydroxydiphosphonate for 2 days [31 (link)]. The pH of the solution was monitored daily (InLab micro, Mettler-Toledo, Giessen, Germany) and if necessary adjusted using HCl (Roth, Karlsruhe, Germany) or 10 M KOH. Samples were then incubated with overnight cultures of LGG in MRS-S for 24 h to induce bacterially contaminated lesions. Biofilms were removed from the surface areas of each specimen with a sterile scalpel. To allow enumeration of baseline bacterial load of induced lesions, dentin from one quarter of the lesion was now excavated using sterile rose-head burs and the net weight of excavated dentin assessed (Analytical Plus, Ohaus, Nänikon, Switzerland). Dentin was dissolved in 1 mL NaCl (0.9%), vortex mixed, and plated on MRS agar in various dilutions (10²–10⁴). Agar plates were cultured at 37 °C and 5% CO₂ (CO₂ Incubator, Heraeus Kulzer, Hanau, Germany). After 48 h, colony forming units per µg dentin (CFU/µg) were enumerated.

The determination of the intracellular pH was performed after Scholes and Mitchell (1970) (link) via permeabilization of the membrane with 5% (vol/vol) butanol. Cells were harvested by centrifugation (150 ml culture, 10.000 × g, 10 min, 4°C, Beckman J2-HS), the supernatant was discarded and the pellet was resuspended in 5 ml of non-buffered solution (0.3 M NaCl, pH 7). The measurement was performed in a 5 ml glass tube with an OD₄₃₆ of 20. For the determination of the pH_i under anoxic conditions the glass tube was closed with a rubber stopper and flushed with N₂ for 2 h. A pH electrode (type Inlab Micro, Mettler Toledo) was immersed and the suspension was stirred. The data were logged (AD converter ADC-16, pico Technology) with the software MPwin (version 2008.08.25, Cypionka, 2005 ). For permeabilization 250 μl of butanol were added into the stirred suspension. From the pH changes after butanol addition at different outer pH values, the pH_i could be determined.

Each sample was immersed only from CEJ to apex in 300 μl of deionized water in a separate vial and stored at 37°C with 100% humidity. pH of the immersion solutions was measured using the pH meter (420A pH meter; Thermo Orion, Inc., Beverly, MA, USA) with microelectrode (InLab® Micro; Mettler Toledo, Greifensee, Switzerland) at 7, 14, and 28 days. After each measurement, the immersion solutions were replaced with fresh 300 μl of deionized water in each vial.