Crystal violet biofilm formation assay

Selected clinical isolates were subjected to a crystal violet biofilm formation assay with slight modifications according to a previously published method.⁷⁴ Briefly, overnight cultures were adjusted to an OD₆₀₀ of 0.02 and 100 µl of the inoculum were added to each well of a flexible, non-treated U-bottom PVC 96-well plate (Corning Inc., NY, USA). Plates were sealed with an air-permeable membrane and incubated at 37 °C in humid atmosphere for 24 h. Planktonic bacteria were removed and wells were washed with water prior to the addition of 150 µl of the crystal violet staining solution (0.1% w/v in water). After 30 min incubation, the staining solution was removed and wells were again washed and air-dried. For de-staining, 200 µl of 96% ethanol was added to each well and the plate was incubated for 30 min at room temperature (RT). 125 μl of the solution was transferred to a fresh 96-well flat bottom plate before absorbance was measured at 540 nm. Each clinical isolate was tested in two independent experiments with eight technical replicates each time.