Lb liquid nutrient medium

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.

The preparation of 24-hour-old biofilms corresponded to the previously described procedure ensuring the formation of a dense biofilm by the strains used in experiments.24 (link) The biofilm formation was confirmed by the staining of 24-hour biofilm with crystal violet dye. The bacteria were cultured in 5 mL of LB liquid nutrient medium (Thermo Fisher Scientific, Waltham, MA, USA) for 18–20 hours at 37°C. Overnight cultures were adjusted to 5 × 10⁵ CFU/mL measured by optical density (OD). One-hundred-microliter aliquots of the diluted bacterial suspension were inoculated into each well of a 96-well flat-bottomed polystyrene plate (Sarstedt AG & Co., Nümbrecht, Germany) and incubated in a humidified incubator for 24 hours at 37°C. The negative control was an LB liquid nutrient medium.

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.