Live dead baclight

To determine the proportion of live and dead B. burgdorferi following doxycycline treatment, Live/Dead BacLight^® (Molecular Probes) staining was performed on untreated and 50 μg/mL-treated B. burgdorferi per the manufacturer’s instructions. Briefly, 1.0 μL of 1.67 mM SYTO-9, a green nucleic acid stain, and 1.0 μL of 1.67 mM propidium iodide, which will only stain cells with damaged cell membranes red, were thawed and mixed in equal proportions. A volume of 1.5 μL of the stain mixture was then added to 500 μL of B. burgdorferi suspended in phosphate buffered saline, pH 7.4 (Gibco), incubated in the dark for 15 min, then applied to a slide and coverslip for viewing and counting. The samples were viewed and counted in live/dead ratios using a fluorescent microscope. The excitation/emission maxima for SYTO-9 is 480/500 nm, and 490/635 nm for propidium iodide. Images captured were obtained using a Nuance FX^®36 fluorescence microscope (Leica) and software, with an optimal emission filter range of 500–560 nm for SYTO-9, and 600–650 nm for propidium iodide. Results were calculated as percent viability, and reported as mean ± SD per group.

The effect of two thiazolidione derivatives on cell viability in mature biofilms (24 h) was determined using the Live/Dead Bacterial Viability method (Live/Dead BacLight, Molecular Probes, USA) with SYTO 9 and propidium iodide (PI) dyes to stain live and dead cells within mature biofilms [36 (link)]. Overnight cultures of E. faecium EF16M64 was grown in cell-culture dishes (Fluorodish, FD35–100) with TSBG medium and incubated at 37 °C for 24 h. Planktonic bacteria were then removed and discarded, and fresh TSBG containing the derivatives monotherapy (at MIC concentrations) or the derivatives combined with a final concentration of 8 × MIC of ampicillin and daptomycin were added and incubated at 37 °C for a further 72 h, exchanging the media every 24 h for fresh media containing appropriate derivatives. After staining, mature biofilms were observed under a confocal laser scanning microscope (CLSM, Leica) with oil-immersion objective. IMARIS 7.0.0 software (Bitplane) was used to edit and analyze the original images. Green and red fluorescence represented the viable and dead cells, respectively. The PI/total percentage, representing the proportion of dead cells within the mature biofilm, was estimated using ImageJ software (Rawak Software Inc., Stuttgart, Germany). This assay was performed in triplicate and similar results were obtained.

The effect of the derivatives on cell viability in mature biofilms (24 h old) was determined using the Live/Dead Bacterial Viability method (Live/Dead BacLight, Molecular Probes, USA). Individual wells of cell-culture dishes (WPI, USA) were filled with diluted culture, after incubation at 37°C for 24 h, the planktonic cells were removed and fresh TSB containing the derivatives at concentrations of 4 × MIC was added and incubated at 37°C for another 16 h. The mature biofilms were stained with SYTO 9 and propidium iodide at room temperature for 15 min, following gentle washing of the dishes with normal saline, and were observed under a Leica TCS SP5 CLSM with a 63 × 1.4-NA oil-immersion objective. Further, image analysis was performed using IMARIS 7.0.0 software (Bitplane). The fluorescence quantities of biofilm were determined using Image J software. This assay was performed in triplicate and similar results were obtained.

Nitrogen-fixation assays were performed with ten plants per condition at 14 days post-inoculation, as previously described^{9 (link)}. Plant biomass production was determined from the fresh weight of shoots that were removed from whole plants. The nitrogen and carbon content was determined on pools of three de-nodulated whole plants that were dried in an oven at 60 °C for 48 h. The dried samples were analyzed with an Isoprime element analyzer (Elementar).
For microscopy analysis, nodules were harvested, embedded with agarose (6%), and then freshly sectioned with a Leica VT1200S vibratome (Leica Microsystems GmbH) into 50 µm tissue slices. Slices were incubated for 20 min in Live/Dead BacLight (Molecular Probes) staining solution in Tris buffer (50 mM), pH 7.0, containing 0.01% CalcofluorWhiteM2R (Sigma). Sections were washed to remove excess of dye and observed using a Leica TCS SP8x confocal microscope.
Toluidine blue staining of thin sections was made as follows. Nodules were fixed in 1% glutaraldehyde/4% paraformaldehyde. After washing, nodules were dehydrated in ethanol series and embedded in Technovit 7100 resin (KulzerHistoTechnik) according to the manufacturer’s instructions. Five µm sections were obtained with a Leica RM2155 microtome and stained with toluidine blue (0.005%). Bright field microscopy was performed with an Eclipse 80i microscope (Nikon).

Biofilms were grown in a flow cell (model FC91; BioSurface Technologies, Corp., Bozeman, MT, USA) with connective tubing draining the effluent into a container as described previously (Fux et al., 2004 (link)). Overnight culture of S. mutans was resuspended in BHI–1% sucrose and a 2 ml inoculum [5 × 10⁵colony forming units (CFUs)/ml] was injected into the inoculation port containing polystyrene (PLS) blocks (VWR Scientific, CA, USA). Bacteria were allowed to attach on PLS blocks for 45 min before a continuous flow of 1 ml/min at 37°C was started. The flowthrough was collected, and the dispersed cells were harvested by centrifugation and resuspended in PBS. The amount of dispersed cells was determined by plate counting and presented as the log (CFU/ml). The remaining adherent biofilm on the PLS blocks was stained with Live/Dead BacLight (Molecular Probes, Invitrogen, Carlsbad, CA, USA) and scanned by confocal laser scanning microscope (LSM 710; Zeiss, Jena, Germany), followed with COMSTAT program analysis (Liu et al., 2012 (link)).

We used LIVE/DEAD BacLight (Molecular Probe) to assay the viability of cells following RecF overexpression. After 16h of culture in the presence of the indicated concentration of arabinose, cells were spun down, washed and resuspended in 0.85% NaCl in order to be incubated with the adequate solution allowing the differential staining of live and dead cells as described by the manufacturer (Molecular Probe). Following the incubation, imaging of cells was carried out with inverted microscope Nikon N-STORM (100x Objective in epifluorescence mode) equipped with an ORCA FLASH 4.0 camera (512 × 512 pixel, Hamamatsu). For each experiment, 2.5 μl of cells were dispersed on a cover-slide 24 × 50 mm, No. 1.5 (Azer scientific) under agar pad 1.5% agarose. For each condition a biological triplicate was imaged. Cells were imaged in the brightfield (100 ms, 4.5V), with a DsRed filter (640 nm, 100 ms, 4.5 V) to image dead cells and with a GFP filter for live cells (488 nm, 50 ms, 4.5 V). Cells were analysed with Fiji equipped with MicrobeJ. Only individual cells in focus were selected. The number of cells for each experiment was greater than 400 cells. The percentage of dead cells was calculated as followed:
For easiest differentiation of live and dead cells on the images, the LUTs of DsRed and GFP channels were respectively changed to yellow and blue before merging the three channels.