Bacterial staining for confocal laser scanning microscopy (CLSM) and fluorescence Live/Dead assay

Biofilms before and after treatment by AAP and AAU were stained using the Live/Dead BacLight Bacterial Viability kit L7007 (Thermofisher Scientific, Dublin, Ireland) according to the manufacturer’s protocols.

For confocal analysis, a fresh working solution was prepared by adding 1.5 μl of each SYTO 9 and PI stain to 1 ml of 0.85% sterile sodium chloride (NaCl) solution. One hundred microliters of staining solution was added onto each biofilm coupon and incubated at room temperature in the dark for 15 min. The coupons were washed gently with NaCl twice to remove excess stain and its residues. The stained samples were placed in 35 mm diameter glass-bottomed µ-dish (ibidi GmbH, Martinsried, Germany) with a drop of mounting solution from the staining kit. Biofilms were visualised and imaged using a confocal laser scanning microscope (Olympus Fluoview FV1000) equipped with a 60×/1.35 NA oil immersion objective.

For microplate reader analysis, bacteria were re-suspended from the coupons’ surface in 10 mL of sterile water using glass beads as described previously. Samples were stained by adding 100 μl freshly prepared stain solution (3 μl SYTO 9 + 3 μl PI working + 1 ml 0.85% NaCl solution) to 100 μl bacterial suspension per well in a 96-well fluorescence micro-plate (Costar Multiple Well Cell Culture Plates, Fisher Scientific Ireland Ltd). The stained samples were incubated at room temperature in the dark for 15 min. Fluorescence intensity was measured with the Spark multimode microplate reader (Tecan UK Ltd. UK) using a 488 nm excitation filter (for both SYTO9 and PI) and a 530 nm (for SYTO 9) and 630 nm (for PI) emission filter.