PBS prior to the addition of either 100 μg/ml of DNase I (final concentration
in PBS; Sigma-Aldrich), 10 μg/ml of Proteinase K (Qiagen) or PBS
(as a control) and incubated at 30 °C for 1 h. After the treatment, the
biofilms were washed three times to remove enzymes prior to biofilm
quantification by both the CV assay and cell enumeration.
The influence of Proteinase K on the initial attachmentwas tested by
addition of Proteinase K (final concentration of 10 μg/ml) alongwith the
inoculated BHIMnG and after 1 or 2.5 h incubation at 30 °C, themedium
was removed and the wells were washed three times with 2 ml of PBS
to remove the unattached cells. Attached cells were enumerated on
MRS agar.