Bhi agar

After 24 hours, the membranes were removed from the wells and washed 3 times with PBS (Sigma-Aldrich) to remove non-adherent biofilm cells. To determine the antibiofilm effect of BNCs, the membranes were transferred to plastic containers containing 2 mL of PBS (Sigma-Aldrich). The biofilms were removed from the substrates by sonication for 15 minutes at an amplitude of 40 W, and the resulting suspensions were serially diluted (1:100). Aliquots of 100 µL were plated on BHI agar (KASVI). The plates were incubated under aerobic conditions at 37°C, for 24 hours to 48 hours. The number of CFU/mL was then determined. The experiment was carried out in triplicate.
For the qualitative assessment of biofilm formation, 2 samples from each group were observed using a scanning electron microscope (SEM). For processing, the membranes were fixed in 2.5% glutaraldehyde, dehydrated in an alcohol ascending chain, mounted in stubs, and covered with gold. With the microscope operating at 10 kW, photomicrographs of significant areas were taken at magnifications ranging from ×1,000 to ×10,000.

The overnight cultures were diluted in BHI broth (KASVI) to obtain a suspension of approximately 5 × 10⁸ colony-forming units (CFU)/mL (DO₆₀₀ ≈ 0.5) for each cell line. Next, 100 µL of each suspension was individually plated on BHI agar (KASVI) and spread with a sterile swab in 3 directions. Membranes (G1 to G4, Ø = 6 mm) were placed at equidistant points. Filter paper discs (Ø = 6 mm) (Unifill, Curitiba, PR, Brazil) moistened with 10 µL of 0.12% CHX comprised the positive control group. The plates were incubated at 37°C for 48 hours, under aerobic conditions. The diameter of the bacterial growth inhibition zones formed around each material was measured in millimeters with the aid of a digital caliper and optical microscope. Three plates were used per bacterial strain in each replicate. The experiment was carried out in triplicate.