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Fig. 1 | BMC Microbiology

Fig. 1

From: A novel high-content screening approach for the elucidation of C. jejuni biofilm composition and integrity

Fig. 1

Workflow of high-content screening approach to investigate the effect of a range of inhibitors on the structure, composition and integrity of Campylobacter biofilm under aeration. (1) Bacterial overnight cultures of NCTC11168 (Induced with 10μg/mL novobiocin) were equalised to an OD600 of 0.1 in MH broth (supplemented with 10μg/mL novobiocin) and 200μL were seeded into all of the wells of an optical 96-well plate. The cultures were incubated for 72h at 37°C aerobically (21% O2) to induce adherent biofilm formation. (2) Each row of the 96-well plate was selected for addition of an inhibitor to be tested for its effect on biofilm integrity. After 72h incubation, the adherent biofilms in all 96 wells were washed once with PBS in aseptic conditions followed by addition of 100μL MH broth (+10μg/mL novobiocin) into all wells except for the first well of each row. For each inhibitor, 200μL of the highest concentration was dissolved in MH broth (+10ug/mL novobiocin) and was added into the first well of each row. The inhibitors used in this study and their highest concentrations were DNAseI (25U/mL, Sigma), sodium (meta)periodate (4mg/mL, Sigma), proteinase K (20mg/mL, Promega), trypsin (0.05%, Gibco), H2O2 (30% pure, Sigma) and sodium deoxycholate (10%, Sigma). Using a multiwell pipettor, 100μL of the medium in the first well of each row was diluted in a 1:2 serial dilution along the row to a final volume of 200μL. The adherent biofilms were then incubated at 37°C for 1h in the presence of inhibitors. (3) The metabolically active bacteria were stained for 30 mins with PBS containing 40μg/mL 5-TAMRA-SE followed by counterstaining of dead bacteria and extracellular DNA structures with PBS containing 10μg/mL SytoX green dye for a further 30 minutes. (4) Automated confocal microscopy was carried out using an Opera Phenix (PerkinElmer) high-content screening microscope using a 5x/0.16 NA air objective. Images were acquired for channels using laser channels 561nm (TAMRA) and 488nm (SytoX). (5) A high-throughput image analysis approach for quantification of TAMRA and SytoX intensity and biofilm area was developed using the Columbus image data and analysis system. Heatmaps of mean biofilm area intensities across three biological replicates were generated to provide a rapid readout of biofilm inhibition

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