Crystal Violet Biofilm Eradication Assay

A standard crystal violet biofilm eradication assay was performed with minor modifications as described previously.24 (link) This method gives the total amount of the biofilm material binding to the dye, including extracellular and cellular components. The 24-hour biofilms, formed in a 96-well tissue culture microtiter plate, were washed three times with 200 μL of sterile PBS solution and air-dried. One hundred microliters of antimicrobial composition dissolved in LB liquid nutrient medium (Invitrogen, Carlsbad, CA, USA) was added to each corresponding well, and the plates were incubated for 24 hours at 37°C. Then, the waste media was removed, and the plates were washed three times with 200 μL PBS solution, air-dried and stained with crystal violet 0.1% (in water) (Lenreactiv, St. Petersburg, Russia) for exactly 2 minutes. The stained biofilms were washed three times with 200 μL PBS solution, air-dried and solubilized with 200 μL of 95% ethanol for 1 hour. Then, the biofilm-associated dye was measured at OD₅₇₀ using the Epoch reader (BioTek Instruments). Each experiment was performed in two independent assays. The minimum biofilm eradication concentration (MBEC) was assessed with MBEC₉₀, which is the concentration of antimicrobials decreasing crystal violet binding in preformed biofilms by 90% compared to untreated control.