Live dead baclight stain

A staining procedure was undertaken to determine the viability of biofilm bacteria prior to and after exposure to the test dressings. Using both the simple biofilm and the simulated wound polymicrobial biofilm models, biofilm controls (at the start and end of each experiment) and residual biofilm samples (after dressing removal) were exposed to Live/Dead® BacLight stain (Molecular Probes, Invitrogen) for 10 minutes at room temperature in darkness prior to analysis. Calcofluor White (Fluka® Analytical) staining at room temperature in darkness was used to establish the effect of the dressings on the biofilm extracellular polymeric substances (EPS). In the case of the simulated wound polymicrobial biofilm model, peptide nucleic acid fluorescence in situ hybridisation (PNA FISH, AdvanDx Inc.) was used to differentiate the two organisms (S. aureus and K. pneumoniae) using fluorescent labels to enable visualisation of the polymicrobial biofilm population. PNA FISH was conducted according to the manufacturer's instructions, with the exception of a 90-minute hybridisation step at 55 ± 1°C, and omission of a water rinse step. Stained samples were examined immediately using confocal laser scanning microscopy (CLSM; Leica TCS SP2, Leica Microsystems) and images were captured for later analysis.

A LEICA model DM IRE2 microscope was used to collect confocal images further processed using imaris ×64 (Bitplane, Concord, MA, USA) software. Briefly, hydroxyapatite‐coated CBD pegs containing biofilms were aseptically removed from the lid using alcohol‐flamed pliers. The collected pegs were rinsed twice with 200 μl of 0.9% NaCl solution in a microtiter plate and stained using the Live/Dead^® BacLight™ stain (Molecular Probes, Ontario, Canada) for 30 minutes prior to visualization (Harrison et al., 2006; Cerca et al., 2012). This kit contains SYTO 9 green‐fluorescent nucleic acid stain and propidium iodide, which is a red‐fluorescent dye (Dailey, 2006). These stains have diverse spectral characteristics, and they are able to differentially penetrate bacterial cells. SYTO 9 generally labels all bacterial cells in population, while propidium iodide penetrates only bacterial cells with damaged membranes (Dailey, 2006). The thickness of the biofilms has been estimated through obtaining high‐resolution optical images with depth selectivity, allowing 3D images of complex biological samples (Dailey, 2006).

Mutant or wild-type strains, with the pGLOW reporter, were grown in EVCC + Aviguard. Oxyrase (Sigma-Aldrich; St. Louis, MO) was added to the medium (1:20 final dilution) and overlaid with mineral oil to create low oxygen conditions. Salmonella starting cell density was 10⁵ CFU/mL. The lyophilized Aviguard was reconstituted as recommended by the manufacturer (Lallemand Animal Nutrition) in sterile saline with Oxyrase and used to inoculate EVCC cecal medium (1:20 dilution) (7.99 Log₁₀ viable cells/mL). The viability of the rehydrated Aviguard was determined microscopically using LIVE/DEAD BacLight stain (Molecular Probes). Sterile 96-well, black-walled polystyrene microtiter plates with a clear bottom (Fisher Scientific) were used to monitor Salmonella fluorescence with a BioTek Synergy HT 96-well fluorescence microtiter plate reader at 37°C. A filter was added to the BioTek Synergy HT fluorescent plate reader (BioTek; Winooski, VT) to record pGLOW fluorescence based on its excitation frequency (Drepper et al., 2007 (link)). Fluorescence was recorded every 30 min. Strains were run in triplicate.