Human oral biofilm formed in vitro with a saliva inoculum and using sterile saliva as its sole source of nutrient. The 25-μm-thick biofilm was grown overnight suspended from the underside of the coverslip of a flowcell with saliva flowing through once at 0.2 ml per min. Bacterial juxtaposition and biofilm architecture were imaged by confocal scanning laser microscopy after staining the cells with Live/Dead stain (Molecular Probes, Eugene, Oreg.). The color of the cells is from the red (propidium iodide; damaged or permeable cell membrane) and green (SYTO 9; healthy cell) fluorescent stains. Colocalization of both fluorophores results in yellow staining. Confocal scanning laser microscopy acquires optical sections through the biofilm; each optical section is 0.5 μm thick. The entire biofilm is represented in six images (A to F). Panel A is the 0.5-μm optical section at the substratum and shows the biofilm footprint. Panel F is the top 0.5 μm of the biofilm where it projects into the lumen of the flowcell. The other four projection images contain eight sections per projection and show the 4-μm-thick regions from 4 to 8 μm from the substratum (B), 8 to 12 μm from the substratum (C), 12 to 16 μm from the substratum (D), and 16 to 20 μm from the substratum (E). Regions indicated by arrows are described in the text. Bar, 10 μm. Microscopic observations and image acquisition were performed on a TCS 4D system (Leica Lasertechnik GmbH, Heidelberg, Germany).
Color Climax Film Nr 1391 44
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