Computational protocol: Quaternary ammonium-induced multidrug tolerant Streptococcus mutans persisters elevate cariogenic virulence in vitro

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Protocol publication

[…] For the confocal laser scanning microscopic imaging, biofilms were developed as described above except that they were grown on sterilized glass slides.For live/dead staining, biofilms on the glass slides were stained using the BacLight live/dead bacterial viability kit (Molecular Probes, Eugene, OR, USA). Briefly, live bacteria were stained with SYTO9 to produce green fluorescence, whereas dead bacteria with compromised membranes were stained with propidium iodide (PI) to produce red fluorescence. Biofilm images were captured using a DMIRE2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with a 60 × oil immersion objective lens.EPS staining was conducted according to previous studies.– The polysaccharides were stained by adding 2.5 μmol·mL−1 Alexa Fluor 647-labeled dextran conjugate (Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA) into the biofilm formation medium at the beginning of the experiment, and then, the culture was incubated at 37 °C. The bacteria of the biofilm were stained with 2.5 μmol·mL−1 SYTO9 (Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA) for 15 min. The biofilms were imaged with a Leica DMIRE2 confocal laser scanning microscope equipped with a 60 × oil immersion objective lens.Each biofilm was scanned at five randomly selected positions. All three-dimensional reconstructions of the biofilms were rendered by Imaris 7.0 software (Bitplane, Zürich, Switzerland), and representative pictures are shown. The quantification of live/dead and EPS/bacteria volume ratios was performed with Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA) and COMSTAT, respectively. […]

Pipeline specifications

Software tools Imaris, Comstat
Application Laser scanning microscopy