*3.14. Biofilms Growth and Enzymatic Treatment*

The strains of Bacillus subtilis, Bacillus licheniformis, Bacillus aegricola, Bacillus berkelogi, Pseudomonas aeruginosa, Yersinia pseudotuberculosis, and Salmonella enteritidis were used. An overnight bacterial culture was diluted with the appropriate nutrient medium and incubated in a 96-well plate at 200 μl per well for 24 h at 37 ◦C, or for 3 days at 22–24 ◦C. After incubation, loose cells were removed from the wells, and wells were washed three times with 0.85% NaCl. The biofilm was stained with 0.5% crystal violet (CV) for 20 min at room temperature. The dye was removed from the wells, and unbound CV was washed with tap water. The plates were air dried, 2% acetic acid in 95% ethanol was added to each well, and the absorbance was determined at 600 nm.

Inhibition of biofilm formation was tested using the method in [63]. Test substances at various concentrations were added to the wells. All experiments were repeated four times. The destruction of biofilms by the investigated substances was carried out as follows. After the formation of biofilms for a certain time, unattached cells were removed and the wells were washed with 0.85% NaCl. The enzyme was added to each well in the appropriate buffer and incubated under the conditions for determination of the activity of the studied enzyme. Then, the plate was processed as described above.
