Computational protocol: Srv Mediated Dispersal of Streptococcal Biofilms Through SpeB Is Observed in CovRS+ Strains

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Protocol publication

[…] Lab-tek II chambered #1.5 German borosilicate coverglass wells (Nunc) were coated in Poly-L-Lysine (Sigma), seeded with logarithmic phase cultures (3 ml), and incubated for 12, 24 or 48 h at 37°C, 5% CO2. Supernatant was removed and biofilms were washed once with 1× Dulbecco's Phosphate Buffered Saline (DPBS). Biofilms were stained with a Live/Dead BacLight viability kit (Invitrogen) before samples were visualized using a Nikon Eclipse Ti CLSM and Nikon EZ-C1 v. 3.80 software. Twelve image stacks of Z-series, each representing a different field of view, were collected for each strain at each time point. The Z-slice images were exported into MATLAB (version 5.1) using NIS Elements Imaging Software, and COMSTAT analysis was performed using the Image Processing Toolbox to calculate total biomass (µm3/µm2) and average thickness (µm) as previously described , . [...] Tissue samples were excised and fixed at 8 dpi as previously described (n = 3/strain) . Briefly, samples were fixed with fresh 1% paraformaldehyde for 24 hours at 4°C, stored in 70% ethanol at room temperature, and paraffin embedded for sectioning. Taylor's Brown-Brenn modified Gram-stain was used for Gram-staining tissue sections. A Nikon Eclipse TE300 Light Microscope (Nikon) was used to examine microcolony formation in Gram-stained sections, QImaging Retiga-EXi camera (AES) was used to capture images, and ImageJ version 1.43 software (rsbweb.nih.gov) was used to store images. […]

Pipeline specifications

Software tools Comstat, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Infection
Chemicals Cysteine