Elisa reader

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.

For biofilm biomass quantification, after 24 h + 24 h of incubation in an anaerobic chamber, exposed and unexposed discs were treated based on our previous studies [9 (link),20 (link)]. The discs were washed from dead cells, air-dried, and stained with 0.1% Crystal Violet. Then, the discs were resuspended in 200 μL ethanol. After the removal of the disc, elution was measured at OD₅₇₀ using an ELISA reader (SAFAS, Munich, Germany).

The effects of composite disks on the growth in the biofilm supernatant was determined. The planktonic phase, coming from the S. mutans CH02 biofilm formation, was removed from each well and transferred to wells of a new 96-well polystyrene flat-bottomed microtiter plates to evaluate the total mass amount by determining the OD_600nm with 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.