Biofilm Formation Assay Protocol

Biofilm formation was performed in LB medium (Biocorp), Christensen broth (Cullen et al. 2015 (link)) and in Artificial Urine (Stankowska et al. 2012 (link)) at 37 °C in 96-well microtiter plates (Nunclon). Overnight culture was diluted (1:100) and transferred into wells in volume 200 μl and incubated overnight at 37 °C with a cover. The broth was removed, biofilm was triple-washed with 0.9 % NaCl solution (200 μl). The 200 μl of 0.01 % (w/v) crystal violet solution was added, incubated at room temperature (RT) for 15 min. and washed five times with 0.9 % NaCl solution. The washed wells were filled with 200 μl of 95 % ethanol at RT for 15 min. Crystal violet absorbance was measured at the λ = 595 nm with an Infinite M200PRO microplate reader (Tecan). Assay was performed in at least 3 independent repeats. An average value of the absorbance was presented on the graph with standard deviation (SD) as error bars.