Adherence of L. amylovorus strains to IPEC-1 cells
L. amylovorus strains were cultivated overnight in MRS-broth containing 10 μCi 3H-thymidine/ml for metabolic labeling, collected and washed twice with PBS. IPEC-1 cells grown on Thincert™ wells were washed once with PBS, and 125 μl of the labeled bacterial suspensions in DMEM/Ham’s F-12 [1:1] medium at A600nm values of 0.25, 0.5 or 1 were added per well. The plates were incubated for one hour at +37°C, 5% CO2 followed by five washes with PBS. The cells were lysed by adding 250 μl of 1% SDS in 0.1 M NaOH per well and by incubating overnight at +37°C, and the radioactivity of the lysed samples (output) was measured by liquid scintillation counting. The input radioactivity values were determined by liquid scintillation counting of the cell suspensions in DMEM/Ham’s F-12 [1:1] (A600nm = 0.25, 0.5 or 1), first treated with an equal volume of 1% SDS in 0.1 M NaOH overnight at +37°C. The adherence was expressed as the proportion (%) of the original radioactivity remaining, after first subtracting the background radioactivity from IPEC-1 cells incubated without bacteria (for outputs) and from DMEM/Ham’s F-12 [1:1] medium (for inputs).