Hcp 2 instrument

Cells collected at 16 h in the dynamic filamentation/acquisition assay were subjected to SEM analysis. One microlitre of cells were mixed with 1 ml of PBS and 100 μl of heparin, vortexed and centrifuged at 500 rpm for 5 min at room temperature. The supernatant was collected and centrifuged for 10 min at 2000 rpm. Bacterial cells in the pellet were fixed in 1 ml of 2.5% glutaraldehyde in PBS (pH 7.4) at 4 °C for 2 h. Following a reported protocol,^{53 (link)} the bacterial cells were washed 3 times in PBS, fixed on the slides and dehydrated by ethanol. With an HCP-2 instrument (Hitachi, Japan), the bacterial cells were critically point dried using liquid CO₂. The samples were imaged with an FEI NOVA NANOSEM450 scanning electron microscope (Thermo Fisher Scientific).

For scanning electron microscopy (SEM), the biofilms on pins were fixed with 0.1 M phosphate buffer solutions (pH 7.4) containing 2% paraformaldehyde and 2.5% glutaraldehyde, and subsequently with 0.1 M phosphate buffer solutions (pH 7.4) containing 1% osmium tetroxide. The samples were solvent-exchanged with 50, 70, and 95% ethanol, ethanol, ethanol dehydrated with a molecular sieve, and then isoamyl acetate. After critical point drying using an HCP-2 instrument (Hitachi, Tokyo, Japan), the samples were mounted on substrates using silver paste and coated with osmium. The surfaces were observed with a JSM-6340F instrument (JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV.
For fluorescence microscopy, the biofilms on pins before and after the treatment with Ca(OH)₂–H₂O₂ solutions were stained using LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA). Specifically, the pins with biofilms were immersed in aqueous solutions of 4.8 µM SYTO 9 and 24 µM propidium iodide for 15 min in the dark. The samples were observed by a BZ-X800 instrument equipped with a Plan Apochromat 2x objective (Keyence, Osaka, Japan). Observations were conducted using an OP-87763 filter (excitation: 470 nm, absorption: 525 nm), an OP-87764 filter (excitation: 545 nm, absorption: 605 nm) for SYTO 9, and propidium iodide, respectively, at an exposure time of 1/6 s.