Hexidium iodide

Streptococcus mutans binding to type IV collagen and fibrinogen was also assessed using confocal laser scanning microscopy as described previously^{52 (link)} with some modifications. Type IV collagen or fibrinogen were added to chambered coverglass wells (CultureWell; Grace Bio Labs, Bend, OR, USA) and incubated overnight at 4 °C. The coated wells were washed three times with PBS, blocked for 1.5 h with 5% BSA in PBS at 37 °C, and washed again with PBS containing 0.01% Tween 20. S. mutans cells were collected, stained with hexidium iodide (Molecular Probes), and added to the coated wells (2 × 10⁹ CFU/well) in PBS. The cells were cultured anaerobically at 37 °C for 18 h in the dark. Non-attached S. mutans cells were removed by washing with PBS, and the adherent cells were observed using a confocal laser scanning microscope (LSM510; Carl Zeiss, Oberkochem, Germany) with a 63 × oil immersion objective.

Octanol, heptanol, carbenoxolone, oleamide, LaCl₃, Etd⁺, procion orange MX2R, brilliant blue G (BBG), Evans blue, propidium iodide, 4-Bromo calcium ionophore A23187 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The 9-amino-6-Chloro-2-Methoxyacridine (ACMA), DAPI⁺², hexidium iodide, nuclear yellow, BOBO 1-iodide, 7-aminoactinomycin D (7-ADD), YOYO 1-iodide, TOTO 1-iodide and ethidium homodimer–2–bromide obtained from Invitrogen (Massachusetts, USA), indocyanine green from Pulsion medical systems, (Feldkirchen, Germany) methylene blue from Euromed (Santiago, Chile).

Observation of S. mutans strains binding to type I collagen using confocal laser scanning microscopy was performed by a method described previously^{43 (link)}, with some modifications. The collagen-binding assay described above was performed using a chambered cover glass system (CultureWell™, Grace Bio Labs, Bend, OR, USA) instead of a 96-well plate. After binding the bacteria to collagen for 3 h, bacterial cells were stained with 5 µl of 10 mM hexidium iodide (Invitrogen, Carlsbad, CA, USA) in 1 ml of Hanks’ balanced salt solution (Lonza, Walkersville, MD, USA) for 15 min at room temperature in the dark. Stained bacteria were observed by confocal scanning laser microscopy using a TCS-SP5 microscope (Leica Microsystems GmbH, Wetzlar, Germany) with reflected laser light at 543 nm, as well as a DMI6000 B fluorescence microscope (Leica Microsystems GmbH) and a 63 × oil immersion objective.

The bacterial biofilm formed on the monofilament was analysed using confocal laser scanning microscopy as described previously^{31 (link)}, with some modifications. The S. mutans MT8148 adhering to the monofilaments were resuspended in 1 ml of sterile distilled water including 5 µl of 10 mM Hexidium Iodide (Invitrogen, Carlsbad, CA, USA) and incubated in the dark for 15 min at room temperature. The monofilament was washed with Hanks’ Balanced Salt Solution (Lonza, Walkersville, MD, USA), fixed with 4% paraformaldehyde, and mounted on a slide glass. The bacterial mass of S. mutans on the monofilament was observed by confocal scanning laser microscopy using a TCS-SP5 Microscope (Leica Microsystems GmbH, Wetzlar, Germany) with reflected laser light at 543 nm, as well as a DMI6000 B Fluorescence Microscope (Leica) and a 63 × oil immersion objective.

Bacterial growth and biofilm formation were analyzed using confocal laser scanning microscopy, as described previously^{14 (link), 49 (link)}, with some modifications. In the bacterial growth assay, 500 μL of the cultured bacteria was collected by centrifugation, resuspended in 500 μL of PBS including 2.5 µl of 10 mM hexidium iodide (Invitrogen, Carlsbad, CA, USA), and incubated in the dark for 15 min at room temperature. Then, 20 μL of the bacterial suspension applied to the cover glass was fixed with 3% paraformaldehyde (FUJIFULM Wako Pure Chemical Corporation). For the biofilm assay, the biofilm formed on a chambered cover glass system (CultureWell™, Grace Bio Labs, Bend, OR, USA) or S. mutans cultured on the enamel test piece was stained with 5 µl of 10 mM hexidium iodide in 1 ml of Hanks’ balanced salt solution (Lonza, Walkersville, MD, USA) for 15 min at room temperature in the dark. The plates were then washed with PBS and fixed with 3% paraformaldehyde. Bacterial growth and biofilm formation were observed by confocal scanning laser microscopy using a LSM510 (Carl Zeiss, Oberkochem, Germany) with reflected laser light at 543 nm, as well as a DMI6000 B fluorescence microscope (Leica Microsystems GmbH) and a 63 × oil immersion objective.

Structural analysis of biofilms by confocal laser scanning microscopy was performed as previously described, with some modifications [36 (link),37 (link)]. Briefly, cultured bacteria (final concentration 1.0 × 10⁷ CFU/mL) were added to BHI broth containing 1% (w/v) sucrose, with or without shikonin dispersion (0, 0.2, 2, 20, or 200 μM shikonin). Then, 200 µL of the bacterial suspension was added to a chambered coverglass system (CultureWell™, Grace Bio Labs, Bend, OR, USA) and incubated at 37 °C for 18 h in the dark. The bacterial cells were then stained with 5 µL of 10 mM hexidium iodide (Invitrogen, Carlsbad, CA, USA) in 1 mL of Hanks’ balanced salt solution (Lonza, Walkersville, MD, USA) for 15 min at room temperature in the dark, and the chambered coverglass systems were washed with PBS. Biofilms were observed by confocal scanning laser microscopy using an LSM510 (Carl Zeiss, Oberkochem, Germany) (https://www.zeiss.co.jp/corporate/home.html, accessed on 1 November 2022) with a reflective laser beam at 543 nm.