Baclight bacterial viability kit

S. mutans biofilm model was based on previous methodology [19 (link)] with some modifications. Briefly, after being coated with sterile salivary pellicle, two samples of each experimental (GIC + tt-farnesol) and control group (GIC) were incubated with S. mutans UA 159 in BHI broth supplemented with 10% sucrose at 37 °C for 1, 3 and 5 days in a 5% supplemented CO₂ environment. The medium pH was measured daily. Then, biofilm bacterial viability (colony forming unities-CFU/mg of biofilm), biomass (dry weight-DW), and biochemical composition (insoluble-ASP and water-soluble-WSP extracellular polysaccharides) was analyzed by colorimetric methods. Biofilm model was performed in duplicate on at least four independent experiments.
The morphology of live and dead bacteria on biofilm surface was analyzed by confocal laser scanning microscopy (CLSM; Leica TCS SP1, Leica Lasertechnik GmbH, Heidelberg, Germany) using HCX APOL U-V-I 40X/0.8-numerical-aperture water immersion objective. Biofilms were stained with a live/dead BacLight bacterial viability kit (Molecular Probes. Invitrogen, Eugene, Oregon. USA) in accordance with the manufacturer. Afterwards, the samples were incubated at room temperature in the dark for 15 min and examined under a CLSM [19 (link)].

Bacteria (Nine Mile and Guyana strains of C. burnetii) were prepared as previously reported (12 (link)). Avirulent variants of Nine Mile bacteria were obtained after repeated passages in L929 cells. Bacteria were stored at −80°C, their concentration was determined by Gimenez staining, and bacterial viability was assessed using the live/dead BacLight bacterial viability kit (Molecular Probes, Life Technologies) (51 (link)). Bacteria were inactivated at 95°C for 30 min. S. aureus (ATCC 25923) bacteria were grown on blood agar plates (bioMérieux) and quantified by flow cytometry (FACS BD Fortessa).

Selected samples from each medicament group underwent confocal microscopy to evaluate the antifungal activity among the groups and observe the pattern of microbial colonization as described in a previous study [52 (link)]. The treated samples were rinsed with PBS and stained with a live/dead BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA) and subsequently examined using confocal laser scanning microscopy (CLSM) (Nikon C2¹ system, Nikon Instruments Inc., Melville, NY, USA).

S. aureus ATCC29213 (5×10⁶ CFU/mL) was cultured with 32, 64, or128μg/mL roemerine at 200 rpm and 37°C for 6h; the control group was treated without drugs under the same conditions. Bacteria were harvested, washed, stained with Syto 9 and propidium iodide (BacLight Bacterial Viability kit; Molecular Probes)[15 (link)], and then examined by H600L fluorescence microscopy (Nikon, Japan).

The structural organization of the biofilm was examined with confocal fluorescence imaging with a Leica TCS CARS SP 8X microscope (Leica Microsystems, Wetzlar, Germany), using HC PL APO CS2 20X/0.75 numerical-aperture multi immersion and HX PL APO CS2 63X/1.2 numerical-aperture water immersion objectives. The imaged biofilms were stained using a live/dead BacLight bacterial viability kit (Molecular Probes. Invitrogen, Eugene, Oregon, USA). The stains were prepared according to manufacturer directions and were left to incubate at room temperature and in the dark for 15 min prior to examination under the confocal scanning laser microscope (CSLM). Light excitation was performed with a two-laser system, a 488 nm Argon laser, and a 561 nm DPSS laser, the emission windows configured to exclude the excitation wavelength of the two lasers and to meet the emission wavelength of the live/dead fluorescence marker. The emission window for the 488-nm laser was set at 500 nm—530 nm, and for the 561-nm laser, at 620 nm—640 nm.