Congo Red Agar Biofilm Assay

Detection of biofilm formation in all isolates was studied by the CRA method according to the protocol described by Freeman et al [10 (link)]. CRA medium was prepared with brain heart infusion broth (Merck, Darmstadt, Germany) 37 g/L, sucrose (Merck, Darmstadt, Germany) 50 g/L, agar-agar (Pronadisa, Laboratories Conda, S.A., Madrid, Spain) 10 g/L, and Congo red dye (Merck, Darmstadt, Germany) 0.8 g/L. Prepared CRA plates were inoculated with a 0.5 McFarland turbidity standard of microorganism and incubated aerobically at 37°C for 24 hours. For color evaluation of colonies, a 5-colourimetric scale method was used. Biofilm-positive isolates appeared as dry opaque black and bright black colonies, while biofilm-negative variants developed a red, pink with or without a darkening at the center, or Bordeaux red colonies [6 (link)].