Computational protocol: How to Study Biofilms after Microbial Colonization of Materials Used in Orthopaedic Implants

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Protocol publication

[…] Stained biofilms were examined with a confocal laser microscope (Leica model TCS SP5; Leica Microsystems CMS GmbH, Mannheim, Germany) using a 20× dry objective (HC PL FLUOTAR 20.0 × 0.50 DRY) plus a 2× electronic zoom. In order to minimize the air contact and maintain constant sample moisture condition, a coverslip was used on the specimen.A 488 nm laser line was used to excite SYTO® 9, while the fluorescent emission was detected from 500 to 540 nm. PI was excited with 561 nm laser line and its fluorescent emission was detected from 600 to 695 nm. In order to avoid false co-localization of fluorescence due to microbial movement, we opted for a simultaneous acquisition mode of the two channels, in which the laser beam scanned the visual field at a frequency of 700 Hz. Moreover, using a third laser line (633 nm) in reflection mode, it was possible to determine with high accuracy both titanium disc (starting acquisition point) and coverslip (ending acquisition point) reflecting surfaces. Images from at least four randomly selected areas were acquired for each disk. For each of them, sequential optical sections of 2 µm were collected along the z axis over the complete thickness of the sample to be subsequently analyzed, quantified by Fiji software (Fiji, ImageJ, Wayne Rasband National Institutes of Health) and rendered into 3D mode by Imaris software (Imaris, Bitplane AG). […]

Pipeline specifications

Software tools ImageJ, Imaris
Application Microscopic phenotype analysis
Chemicals Titanium