*2.6. Bacterial Adhesion Test*

*Streptococcus mutans* (ATCC 25175, Microbiologics, St. Cloud, MN, USA) was used to assess bacterial adhesion to control and experimental surfaces following a previously validated protocol [30,34]. *S. mutans* was grown in Brain Heart Infusion (BHI) agar (Scharlab S.L., Barcelona, Spain) supplemented with 0.2 U/mL bacitracin (Sigma Fluka, St. Louis, MO, USA) followed by incubation for 24 h at 37 ± 1 ◦C. Then, *S. mutans* was cultured in peptone water for 24 h at 37 ± 1 ◦C. The bacterial suspension was centrifuged at 5000× *g* for 15 min, supernatant was discarded and the bacterial pellet was re-suspended in peptone water at 107 CFU/mL by measuring the nephelometric turbidity unit (NTU) (based on a calibration curve of NTU vs. CFU/mL). 15 plates from control and each experimental

group were used for bacterial adhesion tests. Each plate from each group was placed at the bottom of the well of a 24-well non-treated polystyrene plate (Costar, Corning Inc., NY, USA) and 500 μL of the bacterial solution was added to cover each SS 316L plate. Polystyrene plates were incubated for 8 h at 37 ± 1 ◦C to allow bacterial adhesion. After this time, experimental and control plates were carefully rinsed three times with 0.9% saline solution (Corpaul, Medellin, Colombia) to remove non-adherent bacterial cells. Then, each sample was subjected to sonication (Qsonica 125, Newtown, CT, USA) at 50% power for 3 sec to quantify viable adherent bacteria. Sonicated solutions were serially diluted and 10 μL were cultured in BHI agar, by triplicate, following the drop plate method [35]. Culture plates were incubated for 48 h at 37 ± 1 ◦C and then Colony Forming Units (CFU) were counted.
