2.3. LIVE/DEAD® BacLight™

The biofilm on the specimens was stained with the LIVE/DEAD^® BacLight™ Bacterial Viability Kit L7012 (Invitrogen, Molecular Probes, Eugene, OR, USA). That kit contains two stains targeting nucleic acid that differ in terms of spectral properties and the ability to penetrate bacteria. SYTO 9 (3.34 mM, 300-µL solution in dimethyl sulfoxide) is a green fluorescent stain that penetrates both bacteria with intact and damaged membranes. Propidium iodide (20 mM, 300-µL solution in dimethyl sulfoxide) is a red fluorescent stain that penetrates only bacteria with a damaged membrane. When both stains are present in the cell, propidium iodide displaces SYTO 9, resulting in green fluorescent bacteria with intact membranes and red fluorescent bacteria with damaged membranes. The biofilm was stained for 10 min with the staining solution, which contained 1 µL of each stain in 1000 µL of 0.9% NaCl solution. Then, specimens were investigated by fluorescence microscopy (Axio Imager.M2, Carl Zeiss Microscopy GmbH, Jena, Deutschland) using a fluorescein diacetate filter (Sigma, St. Louis, MO, USA) and an ethidium bromide filter (Roth, Mannheim, Deutschland). Of each specimen, six pictures were taken. The software ImageJ 1.52 (NIH, Bethesda, MD, USA) was used to determine the viability and the coverage of specimens with bacteria. Coverage is the area fraction of bacteria from the total area of the image. For that, bacteria were selected manually using the software, which calculated the number of pixels of the selected area. To determine the vitality, green and red bacteria were selected and evaluated separately. In addition to the number of selected areas, the brightness of pixels was taken into account. Therefore, bacteria lying on top of each other shone brighter and, thus, received a higher measured value. The software generated a single value for green and red bacteria. Vitality was determined by dividing the result of green bacteria by the sum of green and red bacteria.