Confocal laser scanning microscopy

A live-dead BacLight bacterial viability and counting kit {"type":"entrez-nucleotide","attrs":{"text":"L34856","term_id":"515727","term_text":"L34856"}}L34856 (Thermofisher Scientific, Ireland) was used. This included SYTO 9, a green fluorescent nuclear and chromosome counterstain, and propidium iodide (PI), a red-fluorescent stain which is not permeant to live cells, and thus was used to detect dead cells. The staining procedure was based on the instructions from the kit manufacturer with slight modifications. The working solution of fluorescent stains was prepared by adding 1.5 μL of SYTO 9 stain and 1.5 μL of propidium iodide stain to 1 mL of sterile 0.85% sodium chloride (NaCl). This working solution was prepared and used the same day. Each untreated and treated biofilm slide was covered with 200 µL of prepared staining solution and incubated at room temperature in dark for 15 min. The biofilm slides were carefully washed with 1 mL of NaCl solution twice to remove any dye residues. The biofilm samples were then placed on a 35 mm diameter glass-bottomed dish (ibidi GmbH, Martinsried, Germany) containing one drop of the mounting solution obtained from the live/dead BacLight bacterial viability kit L7007 (Thermofisher Scientific, Ireland). The slides were directly examined on a confocal laser scanning microscope (Olympus Fluoview FV1000) equipped with a 60x / 1.35 NA oil immersion objective. At least 3 randomly chosen microscopic fields were examined for each sample. A noise reduction filter was applied to the images in the Olympus Fluoview FV1000 software.