To extract RNA or evaluate dispersion, biofilms were grown for 5 days under continuous flow conditions in biofilm tube reactors (1 m long, size 14 silicone tubing, Masterflex, Cole Parmer, Inc.) with an inner surface area of (25 cm2 at a flow rate of 0.2 mL/min), using 5-fold diluted VBMM medium (4 (link), 17 (link)). For plasmid maintenance, 8 μg/mL carbenicillin and 2 μg/mL gentamicin were added. Where indicated, the growth medium was supplemented with 0.1% arabinose to induce the expression of genes of interest. For the visualization of the biofilm architecture, the biofilms were grown in flow cells (glass surface, BioSurface Technologies) at a flow rate of 0.2 mL/min. Following 5 days of growth, the biofilms were viewed via confocal laser scanning microscopy (CLSM), using a Leica TCS SP5 confocal microscope. Prior to confocal microscopy, biofilms were stained using the BacLight LIVE/DEAD viability stain (Life Technologies) at a 1/1,000 dilution in the growth medium. The CLSM images were processed using LAS AF software v2.4.1. The quantitative analysis of the images was performed using the COMSTAT software package (92 (link)).
Baclight live dead viability stain
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BacLight LIVE/DEAD viability stain is a fluorescent dye-based solution used to evaluate the viability of bacterial cells. It consists of two nucleic acid-binding stains, one that labels live cells and the other that labels dead cells. The stain can be used to quickly and easily differentiate between viable and non-viable cells in a sample.
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Lab products found in correlation
Tcs sp5 confocal microscope, by Leica (2 mentions)
Masterflex, by Cole-Parmer (1 mentions)
Uplnfln, by Olympus (1 mentions)
Bx60 microscope, by Olympus (1 mentions)
Progres cf camera, by Jenoptik (1 mentions)
Sensoplate microplate, by Greiner (1 mentions)
Dm lb2, by Leica (1 mentions)
510 meta laser scanning confocal microscope, by Zeiss (1 mentions)
μ slide vi0, by Ibidi (1 mentions)
8 protocols using baclight live dead viability stain
1
Characterizing Biofilm Architecture via CLSM
2
Biofilm Architecture Analysis via CLSM
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Architecture of biofilms grown in flow cells or in microtiter plates was assessed via confocal laser scanning microscopy (CLSM). CSLM was carried out using a Leica TCS SP5 confocal microscope. Prior to confocal microscopy, biofilms were stained using the BacLight LIVE/DEAD viability stain (Life Technologies) at a 1/1000 dilution in the growth medium. The CLSM images were processed using LAS AF software v2.4.1. Quantitative analysis of the images was performed using the COMSTAT96 (link). For brightfield visualization of biofilm architecture, the samples were viewed by transmitted light using an Olympus BX60 microscope and Olympus UPLNFLN 20× and 40× objectives. Images were captured using a ProgRes CF camera (Jenoptik, Jena, Thuringia) and processed using ProgRes CapturePro 2.7.7 software.
3
Biofilm Development Under Chloride Stress
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LAHCl was added to CFS to final concentrations between 500 μM and 500 mM. The supplemented or non-supplemented CFS was used as the medium source for the inoculated Bioflux flowing biofilm system and was added 40 min after initial inoculation with CCS. After 20 h incubation (37°C), the pH of the spent supplemented and non-supplemented CFS was checked and the developed biofilms were washed in PBS (pH 7.4; 20 min, 0.2 dyn/cm2) and stained using BacLight LIVE/DEAD viability stain according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA) for 45 min at room temperature. Excess stain was removed by flowing 100 μL of PBS (pH 7.4) at 0.2 dyn/cm2 over biofilms for 20 min at room temperature.
4
Developing Multispecies Biofilms with L-Arginine
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Static oral multi-species biofilms were developed in 24-well glass-bottom Sensoplate microplates (Greiner Bio-One, Monroe, NC) using CFS as the sole nutrient source and CCS as the inoculum. When required, L-arginine HCl (LAHCl) was added to CFS at final concentrations between 50 μM–500 mM (10-fold increments). 1.5 mL of CFS (negative control) and supplemented CFS of different LAHCl concentrations were added to each well. Wells were inoculated with 15 μL of CCS. After incubation at 37°C for 22 h, wells were treated with BacLight LIVE/DEAD viability stain (3.34 μM Syto 9 and 20 μM propidium iodide) (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After 30 min, the stained biofilms were washed with 1 mL of phosphate-buffered saline (PBS; pH: 7.4) three times, and examined microscopically.
5
Visualizing Live and Dead Cells via Confocal Microscopy
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All manipulations except centrifugation and microscopy were carried out in the anaerobic chamber, and all liquids were equilibrated in the chamber overnight. For initial observation of cells from culture, washed cells were resuspended in BacLight™ Live/Dead viability stain (Invitrogen, Waltham, MA, USA) prepared in anaerobic phosphate-buffered saline (PBS) and examined by confocal microscopy (Leica SP8, Leica Microsystems, Wetzlar, Germany). Excitation wavelength was 488 nm, and emission was collected simultaneously at 505–530 nm (Syto9™—“live”) and 600–630 nm (propidium iodide—“dead”) using a 100× (1.4 NA) oil-immersion lens. Optical section thickness and z-step settings were those recommended by the system software. Transmitted white-light micrographs of wet-mount preparations were obtained using a 100× (1.4 NA) oil-immersion lens on a Leica DM LB2 (Deerfield, IL, USA) microscope with a Hamamatsu (Bridgewater, NJ, USA) Orca camera.
6
Bacterial Acid Tolerance Assay
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Bacterial suspensions (120 μL, log phase OD 0.8 at 600 nm) in MM4 medium were added directly to lanes of channel slides and incubated for 2 h at 37 °C in nitrogen under a 5% CO2 atmosphere, followed by removal of non-adherent cells by two rinses with pH 7.5 MM4 medium.40 (link) For acid adaptation, MM4 medium adjusted to sublethal pH 5.5 was added to the lanes and incubated for 2 h prior to exposure to pH 3.5 MM4 medium for an additional 2-h incubation. For acid tolerance without adaptation, pH 3.5 MM4 medium was added directly to the lanes and incubated for 2 h. Cells incubated in pH 7.5 MM4 medium were used as control. Dead and viable cells were visualised by addition of 60 μL LIVE/DEAD BacLight viability stain (Invitrogen, L7007) and counted as described above.
7
Anti-Serum Treatment of NTHI Biofilms
8
Biofilm Formation on Saliva-Coated Surfaces
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Biofilm formation was achieved on saliva-coated channel slides (μ-Slide VI 0.4, Ibidi, 80,606).39 (link) Parotid saliva (100 μL) pooled from several individuals and diluted 1:1 in PBS with 0.3 M CaCl2 was added to each lane of the channel slide and incubated overnight. The lanes were rinsed in 100 μL PBS three times to remove excess saliva and then incubated with 120 μL per lane of bacterial suspensions in log phase (OD 0.8 at 600 nm) in minimal medium MM4 containing 40 mM phosphate/citrate buffer (pH 7.5) and 20 mM glucose for 2 h at 37 °C in nitrogen in a 5% CO2 atmosphere. After the incubation, the channels were rinsed three times with MM4 medium, and adhered cells were stained with 60 μL LIVE/DEAD BacLight viability stain (Invitrogen, L7007) per lane to visualise the initial biofilm formation under a fluorescence microscope (confocal laser scanning microscopy).
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