Crystal violet staining for biofilm analysis

To test whether 2-MA and AHA inhibited biofilm activity, biofilms were prepared as follows. P. mirabilis was subcultured (1:1000 into LB, total volume of 1 mL), each subculture was added to a 12-well micro-plate (Corning, New York, USA) thus the starting inoculum was 10³ CFU/mL. Microplates were incubated statically with varying concentrations of compound (1, 2.5, 5, 7.5, 10 and 15 mM) for 24 h at 37 °C. To quantify the biofilms crystal violet staining was used, whereby biofilms are washed 3 × with milliQ water removing planktonic cells. The adhered cells were stained with crystal violet solution (0.1% (w/v), 500 μL) and incubated statically for 15 min at RT. The stain was removed with 3 × exhaustive washes with milliQ water and dried at RT for 30 min. Quantification of adhered cells occurred by adding decolorizing solution (33% (v/v) acetic acid, 500 μL) to each well and the absorbance measured at 595 nm. Positive control: absence of compound, negative control: absence of bacteria.