Computational protocol: Nanostructured titanium surfaces exhibit recalcitrance towards Staphylococcus epidermidis biofilm formation

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Protocol publication

[…] The titanium substrates were removed from wells with sterile forceps and gently rinsed three times with Phosphate Buffered Saline (PBS, pH = 7.4) to remove non-adherent or loosely adhered bacteria. The bacteria were then stained with a solution of the FilmTrackerTM Live/Dead® Biofilm Viability kit (Invitrogen, Life Technologies, Carlsbad, CA, USA). In this study, 1.5 µl of SYTO®9 and 1 µl of propidium iodide stain were added to 1 ml of PBS. After that, 150 µl of the staining solution was gently added to each substrate and plates were incubated for 15 minutes in the dark. Suspensions were then aspirated and the titanium substrates were transferred into a new well plate with ample PBS to fully immerse the sample. Samples were then visualized with a Nikon A1 confocal laser scanning microscope (CLSM) with a 40x water dipping lens. For the bacterial adhesion assay for 2 hours’ incubation, the surface coverage of bacteria under each field of view was determined by calculating the surface area of live and dead bacteria cells with ImageJ. For the early-stage biofilms formed after 2 days, the biomass under each field of view was determined by COMSTAT2 plugin (Lyngby, Denmark) in ImageJ. For each substrate type, three independent experiments were performed, and on each sampled surface, five equidistant evenly spaced fields (105.96 × 105.96 µm2) were selected. […]

Pipeline specifications

Software tools ImageJ, Comstat
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Bacteria
Chemicals Titanium