Bacterial Adhesion Assay with Asialofetuin

FmlH-dependent bacterial adhesion was analyzed as described previously with minor modifications [47 (link)]. Briefly, Immulon 4HBX microtiter plates (Thermo Electron Corp.) were coated with 20 μg/mL of asialofetuin (Sigma-Aldrich) and plates were quenched with 0.2% BSA in PBS. Bacterial suspensions in PBS (OD540 = 2) were added to each well in a volume of 100 μl and plates were spun for 2 min at 750 x g. After extensive washing, bound bacteria were stained with 0.1% (vol/vol) crystal violet for 20 min at room temperature and were washed several times with water. Then 100 μl of 50% (vol/vol) ethanol was added to each well, and the absorbance at a wavelength of 600 nm was measured. For inhibition, bacterial binding was tested in the presence of 1% galactose (Sigma).