Uv vis spectroscopy microplate reader

Osteoblast (hFOB) cell viability was evaluated using MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St.
Louis, MO) assay. To prepare MTT solution, 5 mg of MTT is dissolved in 1 ml
of sterile filtered PBS. 100 μl of MTT solution was then added to
each sample in 24-well plates followed by addition of 900 μl of cell
medium. The samples were incubated for 2 h. 1 ml of MTT solubilizer is
prepared using 10% Triton X-100, 0.1N HCl and isopropanol. After
incubation, 600 μl of MTT solubilizer was added to dissolve the
formazan crystals. Thereafter, 100 μl of that solution was
transferred into a 96-well plate, and read by UV–Vis spectroscopy
microplate reader (BioTek) at 570 nm. To ensure reproducibility, all samples
were used in triplicate. Data are presented as mean ± standard
deviation. Student’s t-test was used to perform statistical analyses
and P values < 0.05 and <0.0001 are considered
significant and extremely significant.

The antibacterial efficacy against S. aureus (Carolina biological supply company, Burlington, NC) was performed as per the modified ISO 22196: 2011 Standard [24 (link)]. Optical densities of different serial dilutions of bacterial suspension were measured at 625 nm using UV–Vis spectroscopy microplate reader (BioTek) and compared with the McFarland standard. Based on the bacteria count, 10⁶ CFU of bacteria was seeded on top of each sterilized sample (in 24 well plates) with the addition of 1 mL broth media. The bacteria added well plates were kept at 37°C, and 90% humidity, inside the oven. After 36 h of sample-bacteria interaction, the bacteria containing samples were taken out in glass vials and filled with 1 mL phosphate buffer solution (PBS). The vials were vortexed for 15 s followed by plating of 10 μL of suspension on the agar plate by streaking method. The agar plates were kept inside an oven (37°C, and 90% humidity) for 24 h. After that, the bacterial colony formation was analyzed from the photographed plates.

The antibacterial efficacy of the coatings was tested against S. aureus, according to the modified ISO 22196: 2011 Standard [14 ]. Optical densities of the activated bacterial suspensions were measured at 625 nm using a UV–Vis spectroscopy microplate reader (BioTek) after a serial dilution and compared with the McFarland standard. Based on this quantification, each sterilized sample in a 24 well plate was loaded with 10⁶ CFU of bacteria with 1 mL broth media. The well plates were incubated at 37 °C and 90% humidity. After 24, 48, and 72 h of sample – bacteria interaction, the samples were moved to a glass vial containing 1 mL phosphate buffer solution (PBS). Vortexing of each vial was performed for 15 s, and 10 μL of bacterial suspension was plated on the agar plates, followed by 24 h incubation at 37 °C. The bacterial colony formation was checked from the photographed plates after 24 h incubation. The following equations were used to calculate each treatment’s antibacterial potential (R %).
$$\text{R}\%=\left({\text{N}}_{\text{control}}-{\text{N}}_{\text{treatment}}\right)/{\text{N}}_{\text{control}}$$
$$\text{Bacterial}\text{cell}\text{viability}(\%)={\text{N}}_{\text{treatment}}/{\text{N}}_{\text{control}}\times 100\%$$
where N_control and N_treatment represent the number of colonies formed in the control and treatment sample agar plates, respectively, at each time point.