Adherence of L. amylovorus strains to mucus
The adherence of the L. amylovorus strains to porcine gastric mucins (type II, Sigma) or to porcine small intestinal mucus was studied essentially as described earlier [35], but by using a nucleic acid binding fluorescent stain SYTO®9 (Molecular Probes, Eugene, OR) rather than tritium for bacterial labeling, and PBS as the buffer. To label the bacterial cells in the experiments, the strains were cultivated overnight, collected, washed twice with 0.85% NaCl and suspended into the original volume of 0.85% NaCl, and then 1 μl of 5 mM SYTO®9 solution was added per 1 ml of cell suspension, followed by a 15 minute incubation in the dark with vigorous shaking, after which the cells were collected and washed twice with PBS. After the adherence assay, the input (added) and output (remaining) fluorescence values were measured in a microplate reader (Victor Multilabel Plate Reader, Perkin Elmer, Waltham, MA) and the adherence was expressed as the proportion (%) of the original fluorescence remaining, after first subtracting the background fluorescence from mucus-coated wells without bacteria (for outputs) and from wells filled with PBS (for inputs).