Crystal Violet Biofilm Assay

The potential to form biofilms was compared in parent, P10 and X10 bacteria using a crystal violet assay. Overnight bacterial cultures were adjusted to an optical density of 0.8 and then diluted 1:100 in Mueller Hinton broth. Aliquots (150 μl) of diluted bacterial culture were transferred to the wells of a sterile 96-well microtiter plate (Corning Ltd., Weisbaden, Germany) and were incubated aerobically for 48 h at 37°C. After 48 h, the liquid in the wells was removed by inversion of the microtiter plate, and the wells were washed twice using 200 μl of sterile phosphate-buffered saline (PBS). The wells were stained with 250 μl of 1% (w/v) crystal violet solution for 1 min, rinsed twice with PBS and left to air dry at room temperature. To solubilise the attached crystal violet, 300 μl of absolute ethanol was added to each well (10 min) before measuring the absorbance (OD₆₀₀) using a PowerWave^TM XS plate reader (BioTek, Swindon, United Kingdom). Data were presented as biofilm units calculated by dividing the absorbance of the crystal violet bound biofilm by a corresponding planktonic OD₆₀₀ in order to adjust for planktonic mass. All data points were plotted and analysed using GraphPad Prism version 7.0 (GraphPad Software, California, United States) and are presented as means of biologically duplicated experiments, each comprising six technical repeats. Differences between parent and passaged bacteria (P0 vs. P10; P0 vs. X10) were determined using a Mann-Whitney test.