Live dead baclight staining kit

The live or dead status of the tested bacteria was observed through a LIVE/DEAD BacLight staining kit (Invitrogen, CA) and visualized by confocal laser scanning microscopy (CLSM). Bacteria with intact cell membranes were stained green, whereas those with damaged membranes were stained red. Briefly, S. aureus USA300 cells in the mid-log phase growth were collected and washed twice with PBS. Then, 800 µL bacterial suspension (1 × 10⁸ CFU/mL) was added to 200 µL of LysSYL with a final concentration of 4×MIC (128 µg/mL), and incubated at 37 °C for 1 h. Subsequently, the mixture was collected, washed once with PBS, and resuspended in PBS. Next, 2 mL suspensions were added into a 35-mm-diameter glass-bottom microwell dish and dyed with a 1:1 mixture of SYTO9 and propidium iodide (PI) in the dark for 15 min. After staining, the bacteria were observed by a Zeiss LSM880 confocal microscope (Carl Zeiss, Germany). Moreover, the real-time observation of the bactericidal process was also captured by CLSM.

The permafrost ice wedge sampling site and sample sterilization method were described previously (11 (link)). Briefly, the ice block from Fox tunnel, Alaska, USA was submerged in 75% (v/v) ethanol solution for 5 s and the surface was burned to destroy any contaminating bacteria. The ice was stamped onto agar plates to confirm the absence of surface contamination. The ice block was thoroughly rinsed using 0.85% (w/v) NaCl, then melted and 10-fold serially diluted in R2A broth. Melted ice dilutions (10⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) were mixed with each amount of the Rpf protein (0, 1.25, 12.5, 125, and 1250 pM, and 12.5 pM denatured Rpf) being tested for a total of 30 treatment combinations in triplicate. The results from the denatured proteins were not significantly different from the 0 rRpf controls and, therefore, were not shown in the Results section. Mixtures were spread onto R2A agar medium containing antimicrobials (0.05 g nystatin L⁻¹ and 0.01 g cycloheximide L⁻¹) to inhibit fungal growth. Plates were incubated at 15°C for 2 weeks and colony numbers were counted daily. The presence of living cells in the melted ice was confirmed by epifluorescence microscopy after staining using the Live/Dead Baclight staining kit (Invitrogen, USA) as previously described (23 (link)).

To study the effect of oral bacteria on keratinocyte adhesion, titanium discs were seeded with 1 ml of OKF6/Tert 2 cells and these were allowed to attach for 16 hours. After this time, 1 ml of the four-species bacterial consortium containing 10⁷ cells in serum-free keratinocyte medium as described above, was added and the discs then incubated in 5% CO₂ in air at 37°C for an additional 8 hours. After a total incubation time of 24 hours, the discs were washed gently with serum-free keratinocyte medium to remove non-attached cells and the remaining adherent cells stained with Live/Dead BacLight staining kit (Molecular Probes) prior to visualization in a fluorescence microscope. To evaluate strength of attachment of the bacteria-treated cells to the substratum, the same standardised washing procedure as outlined above was used. Alternatively, titanium discs were seeded with 1 ml of the four-species bacterial consortium containing 10⁷ cells in serum-free keratinocyte medium (5% CO₂ in air at 37°C) for 2 hours and washed three times with serum-free keratinocyte medium before the addition of 1 × 10⁵ cells in 1 ml serum-free keratinocyte medium for 22 hours.

For time-kill curve analysis, planktonic cells of GBS 89, GBS 121, and S. agalactiae ATCC 13813 (1–5 × 10⁵ CFU/mL) were incubated in TSB containing MIC levels of eugenol. At determined time points (0, 1, 2, 4, 6, 8, 10, and 24 h), aliquots were aseptically transferred to TSA plus 5% sheep blood plates and the CFU counts were determined after incubation at 37°C for 24 h. The bacterial viability of eugenol-treated GBS was also evaluated, using LIVE/DEAD BacLight staining kit (Molecular Probes, Invitrogen) according to the manufactures' recommendations. This assay is based on the detection of two nucleic acid fluorescent stains. Green-fluorescent SYTO 9 labels live and dead bacteria, whereas the red fluorescent propidium iodide selectively labels bacteria with permeable (damaged) membranes. Bacterial cells were placed on a glass coverslip and analyzed by fluorescence microscopy (LEICA DM2000). All assays were carried out in triplicate on three different occasions.

Titanium discs were placed in 24-well tissue culture dishes (Becton Dickinson), seeded with either keratinocytes or gingival fibroblasts (1 × 10⁵ cells in 1 ml DF-K medium or DMEM supplemented with penicillin (50 units/ml), streptomycin (50 μg/ml) and 5% fetal calf serum, respectively) and incubated in 5% CO₂ in air at 37°C for 24 hours. This time point was chosen since a sufficient number of cells had adhered in order for the results to be reliable but little cell division had occurred. The discs were gently washed with PBS to remove non-attached cells and stained using Live/Dead BacLight staining kit (Molecular Probes). Adherent cells were visualized using a fluorescence microscope. To evaluate how well the cells were attached to the substratum, a standardised washing procedure was used to remove loosely adhered cells [23 (link)]. Discs in culture dishes containing 1 ml PBS were shaken at 100 rpm for 2 × 5 minutes (IKA Vibrax rotary shaker, GMBH & Co., Germany). Cells remaining after this procedure were counted manually after staining with Live/Dead BacLight staining kit and image capture using fluorescence microscopy. The number of cells remaining after the wash was expressed as a ratio of control (number of cells present before washing).

Lactate concentration in culture medium and protein-based biomass volume of the planktonic cells were by HPLC and a protein quantification assay, as previously reported36 (link). SigmaPlot 11.0 was used for data (mean and standard deviation) statistical analysis. CLSM was used to analyze the development and structure of the biofilms attached on anode surfaces15 (link). Before CLSM observation, biofilm samples were stained with LIVE/DEAD BacLight staining kit (Molecular Probes) which can distinguished viable (with green fluorescence) or unviable or stressed (with red fluorescence) bacterial cells. Randomly sampled view fields were observed and analyzed for each anode biofilm. To obtain three-dimensional structure information, the biofilm sample was observed under the “z-Stack” model of the Zen software (Zeiss)15 (link).

A confocal laser scanning microscope (CLSM, LSM 700, Zeiss) was used to analyze the biofilm structure and viability. Before CLSM analysis, a piece of graphite plate with biofilm (0.5 × 1 cm) was sampled from each reactor in an anaerobic glove box and was dipped in sterilized PBS to remove loosely attached planktonic cells or debris. The sample was then stained with LIVE/DEAD BacLight staining kit (Molecular Probes, Invitrogen), by which cells with damaged membrane can be stained by PI and visualized as red cells while the intact cells are stained by both PI and SYTO 9 and visualized as green cells. Randomly sampled view-fields of each biofilm were observed (n ≥ 10), pixel-based biofilm viability was analyzed and presented as described before9 (link)10 (link). A CFU count was also performed with LB agar plates. The biofilm cells were scraped into LB broth with a sterilized blade. The LB broth containing biofilm cells was then gently blended to scatter the cell clusters and observed under a microscope to avoid cell death or clustering. The blended samples were spread on the LB agar plate and the CFUs were counted after 24 and 48 hours.