Filmtracer live dead biofilm viability kit

Biofilms were grown under various conditions in the chamber slide method described above. Before confocal microscopy, biofilms were stained using the Filmtracer Live/Dead biofilm viability kit (Life Technologies, Burlington, ON, Canada). The medium was gently removed from the chambers, and 200 μl total of the Live/Dead stain was added to the chambers. After 45 min of incubation, the stain was removed, and fresh medium was placed in the wells. These chamber slides were used for confocal imaging. Confocal images were acquired using a Quorum WaveFX spinning disk confocal system (Quorum Technologies Inc., Guelph, Canada). All images were acquired using a 25× water objective (total magnification, ×250) on a Zeiss AxioVert 200 M Microscope. Spectral Borealis lasers (green, 491 nm; red, 561 nm) were used for excitation. Emission filter sets of 515/40 and 624/40 were used to visualize the SYTO9 and propidium iodide stains, respectively. For each experiment, each isolate tested was performed in duplicate technical replicate (two chambers per condition) and four images per chamber were captured for analysis. Each experiment had three biological replicates (three independent experiments).

Pseudomonas aeruginosa overnight cultures were diluted to 1 × 10⁶ CFU/mL. An aliquot of 125 µL of bacterial suspension was cultured with 125 µL of LL-37 and RP557 solution at a quarter MIC in an eight-well chambered glass (LAB TEK, USA). The chambers were cultured at 37°C for 48 h to form mature biofilms. After incubation, the bacterial suspension was discarded, and the biofilm was washed with 0.85% saline. Biofilm was stained with the FilmTracer LIVE/DEAD Biofilm Viability Kit (Life Technologies Corporation, Eugene, USA) for 20 min in the dark. Then, the dye was discarded, and each well was rinsed. Biofilms were examined by confocal laser scanning microscopy on ZEISS LSM 800 (ZEISS, Germany) using a 20× objective. Laser excitation was performed at 488 nm for SYTO9 and 561 nm for PI. Emission was collected from 510 nm to 550 nm for SYTO 9 and 630 nm to 670 nm for PI. Cell viability was determined by the intensity of red and green fluorescence using ImageJ (NIH, MD, USA).

A. pleuropneumoniae 81750 and K17 biofilm-like structures were pre-formed overnight (THB-NAD, 37°C, anaerobic chamber) in the wells of a 96-well clear bottom black wall microplate (Greiner Bio-One North America, Monroe, NC, USA). After removing planktonic and loosely-attached bacteria by aspiration, the biofilm-like structures were exposed (4 h, 37°C, anaerobic chamber) to either H₂O₂ (0.57, 1.15, 2.3, and 4.6 mM) or a culture supernatant of S. pluranimalium 2N12 prepared as described above. Biofilm-like structure viability was assessed using the FilmTracer LIVE/DEAD Biofilm Viability kit (Life Technologies Corporation, Eugene, OR, USA) according to the manufacturer's protocol. Assays were performed in triplicate, and the means ± SD were calculated.

The mature P. aeruginosa PAO1 biofilm was formed in six-well plates (Multiwell Cell Culture Plate, Becton Dickinson, NJ, USA) for 24 h at 37 °C in a TSB medium. Following the incubation, planktonic cells were removed by washing with saline using a shaker at 100 rpm for 10 min. Polyethylene terephthalate (PET) membrane with a pore diameter of 1 µm (as an element of the Cell Culture Insert, Becton Dickinson, NJ, USA) containing the agarose-immobilized phages was placed in the culture well and wells were supplemented with 3 ml of fresh TSB medium. Eradication of bacterial cells and biofilm was monitored for the next 24 h. The P. aeruginosa PAO1 cells in the biofilm were stained with Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s instruction and visualized using a fluorescence microscope (Zeiss Axio Scope.A1, Carl Zeiss Canada Ltd. Toronto, Canada). Each assay was repeated at least in triplicate. The representative image of biofilm structure with the highest similarity (the level of aggregation with the distribution and proportion of green (alive) and dead (red) bacterial cells) from 4 images in each repeat (3) was presented.

For this part of the study, all strains of L. monocytogenes (Table 1) were co-cultured with B52 strain and collected after 48 h, as described above. Then, the coupons were stained with FilmTracer™ LIVE/DEAD^® Biofilm Viability Kit (Life Technologies, Eugene, OR, United States) following manufacturer instructions to observe the final biofilm morphology and the distribution of live (green-emitting) cells and damaged/dead (red-emitting) cells. Biofilms were visualized under a Leica DM6000 epifluorescence microscope (Leica, Wetzlar, Germany) using a 40× dry objective and 10× ocular lenses. Representative images of each sample were acquired with a Leica DFC365 FX camera and the Metamorph MMAF software (Molecular Devices, Sunnyvale, CA, United States).

To visualize viable bacteria biofilms, LIVE/DEAD staining followed by confocal laser scanning microscopy was performed. Biofilms were grown on 3D printed samples as described in the scanning electron microscopy (SEM) section. After an incubation period of 24h, the samples were washed three times in physiological saline (0.9% NaCl). The biofilm formation was analyzed using the FilmTracer™ LIVE/DEAD Biofilm Viability Kit (Molecular Probes, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The SYTO 9 green fluorescent nucleic acid stain measured at 482 nm excitation and 500 nm emission was used to visualize live bacteria with an intact cell membrane. Bacteria with a compromised membrane, considered dead or dying, were stained with propidium iodide (red), measured at 490 nm excitation and 635 nm emission. To obtain a percentage of live and dead cells, five images of the same magnification were evaluated with FIJI (ImageJ) software for each material using the following routine: First, the images were converted to binary images. Then the amount of white and black pixels for each image was calculated with the FIJI selection tool. The last step was to compare the number of pixels of the living and dead cells to the background pixel values.

Biofilms were formed on polystyrene Chamber Slides (n° 177,445; Thermo Scientific, Ottawa, Canada). For this purpose, overnight cultures of R^Rϕ1+, R^Sϕ1-, and R^W strains grown in LB medium were diluted to a final concentration of 0.001 and seeded into a chamber slide at 37 °C for 36 h to assess biofilm thickness and cell viability. The biofilm cell viability was determined with the FilmTracer™ LIVE/DEAD® Biofilm Viability Kit (Molecular Probes, Invitrogen, Carlsbad, California, USA) following the manufacturer’s instructions. Microscopic observations and image acquisitions were performed as described [41 (link)].