Quantifying S. aureus Biofilm Formation

Biofilm formation assays were performed with S. aureus isolates (n = 30) following the protocol that was described in our previous work [19 (link)]. Briefly, the bacteria were grown overnight at 37 °C in 5 mL of tryptic soy broth (TSB; Oxoid) for 18 h. Following 18 h of incubation, the bacteria were diluted in TSB that was supplemented with 20% glucose to reach the final concentration of 10⁷ CFU/mL. Aliquots of bacteria (1 mL, 10⁷ CFU/mL) were placed in a 12-well cell culture plate and incubated at 37 °C for 48 h. After incubation, the non-adherent cells were removed and the produced biofilms on the surface of the wells were fixed at 60 °C for 1 h. The biofilm was stained with 0.1% crystal violet solution and rinsed with PBS. The OD was measured at 550 mm using a spectrophotometer. Each assay was performed in triplicate. S. aureus 15 AL was used as positive control.