Labeling reagent

The planktonic growth was placed in a 96-well flat bottom microtiter plate and incubated with 20 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) labeling reagent (Sigma–Aldrich Chemical Co., St. Louis, MO, USA) at concentration of 5 mg/mL for 2 h, as indicated by the manufacturer. After incubation, 100 μL of Dimethyl Sulfoxide (DMSO, Sigma–Aldrich Chemical Co., St. Louis, MO, USA) was added at each well and incubated for 10 min at the dark. Medium incubated with composite disks served as negative control. The assay is based on the metabolic conversion of water soluble MTT compound to a colored insoluble formazan derivate. Viable cells with active metabolism convert MTT into formazan; however, dead cells lose this ability [30 (link)]. The optical density reading was measured spectrophotometrically at 570 nm by ELISA reader (SAFAS, Munich, Germany). For the detection, the planktonic phases coming from 39 disks (10 tests and 3 negative controls for each different CR material) in triplicate, for a total of 117 disks, were analyzed.

To evaluate the metabolic activity of the biofilm formed by S. mutans CH02 on composite surfaces, the disks were incubated with 100 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) labeling reagent (Sigma–Aldrich Chemical Co. St. Louis, MO, USA) at the concentration of 5 mg/mL for 2 h, as indicated by the manufacturer. A total of 100 µL of supernatant from each composite disk was transferred to a 96-well flat bottom microtiter plate, and the optical density reading of formazan derivative was read at 570 nm using an ELISA reader spectrophotometer (SAFAS, Munich, Germany). Disks incubated with media alone served as negative control. For this detection, 39 disks (10 tests and 3 negative controls for each different CR material) were analyzed in triplicate, for a total of 117 disks.