Biofilm Formation Assay (Crystal Violet-Based Method)

All isolates were screened for their biofilm formation ability in 96-well plates using the crystal violet-based assay.¹⁸ Briefly, 18 h trypticase soya broth (TSB) cultures of tested staphylococci isolates were normalized to an optical density (OD) of one at 600 nm, and then diluted 1:100 with fresh TSB. Volumes of 200 μL of the diluted cultures were used to fill the wells of a 96-well flat-bottomed plates which were incubated for 24 h at 37 ◦C. Non-inoculated TSB was included as a blank (negative control) and S. aureus strains US300 was used as a biofilm forming positive control.¹⁹ The biofilms were washed three times with PBS and left to dry overnight after discarding the growth medium and the unattached cells. Crystal violet (0.4% w/v) was used to stain the adherent cells at room temperature for 15 min. Then, wells were washed, 150 μL of absolute ethanol was added, and absorbance at OD₅₉₅ was recorded.¹⁸ An isolate was classified as strong biofilm former if the OD₅₉₅ was more than 0.24, moderate (0.12–0.24) and weak (non-biofilm forming) if OD₅₉₅ was less than 0.12.^20,21