Determining Biofilm Antimicrobial Sensitivity

The effect of antimicrobial agents on the viability of biofilm bacteria was assessed using a standard trimethyl tetrazolium chloride (TTC) test identical to that described previously.24 (link) The 24-hour biofilms, formed in a 96-well tissue culture microtiter plate, were washed three times with 200 μL of sterile physiological buffered saline (PBS) to remove unattached bacteria and then air-dried. An antimicrobial agent dissolved in 100 μL LB liquid nutrient medium (Thermo Fisher Scientific) was added to each corresponding well, and the plates were incubated for 24 hours at 37°C in a humidified incubator. Then, 11 μL of 0.2% TTC (Lenreactiv, St. Petersburg, Russia) was added to a final concentration of 0.02%. After 1 hour of incubation at 37°C, the OD₅₄₀ was measured on the Epoch microplate reader (BioTek Instruments, Winooski, VT, USA). The mean OD₅₄₀ value of 48-hour biofilm cells without antimicrobial agent treatment was set as the control. Each experiment was performed in two independent assays. The minimum biofilm-inhibiting concentration (MBIC) was assessed with MBIC₉₀, the concentration decreasing TTC staining by 90% compared to the control.