Biofilm formation on surfaces

All experimental procedures or materials, devices and equipment were aseptically performed or maintained; a lack of cross-contamination was confirmed using empty plates. The primary culture was diluted with fresh broth media to achieve an optical density (OD) at 600 nm (OD₆₀₀) value of 0.9 to 1.0 (DeNovix DS-C Spectrophotometer) for the biofilm formation test on PS. For this test, we used 14-mL round-bottom tubes (40114; SPL Life Sciences, Korea) made of PS which is commonly used in biofilm-forming experiments^22,25. A 1-mL diluted bacterial suspension was dispensed in a 14-mL round-bottom tube that was sterilized by gamma irradiation, followed by incubation at 37 °C with shaking at 50 rpm⁵⁴ for 24, 48 and 72 h. Then the non-adherent planktonic cells and the culture tubes were separately collected.

Rectangular TiAl6V4 plates (Grade 23: 90% Titanium, 60% Aluminum, 4% Vanadium, 20 width × 16 height mm; Jeil Medical Corporation, Korea) were prepared for the biofilm formation test on Ti by submerging them in acetone and rinsing with ultrapure water, followed by autoclaving at 121 °C for 20 min with a pressure of 15 psi. After drying in a dry oven at 60 °C, the autoclaved rectangular Ti plate was placed on the bottom of a 50-mL conical tube. After diluting the primary culture 100-fold (OD₆₀₀ = approximately 0.01) with fresh broth, a 5-mL aliquot of the diluted bacterial suspension was added to the Ti-containing tubes; the Ti plate was halfway submerged to form a biofilm at the air–liquid interface in the middle. The caps were tightly closed during incubation at 37 °C with shaking at 50 rpm⁵⁵ for three days. Finally, the Ti plates were separately retrieved from the non-adherent planktonic cells and culture tubes.