Live dead baclight bacterial viability kit

After the experimental abutments and retaining screws were removed at the end of the experiments, six abutment and retaining screw combinations (unmodified titanium) from group A and eight from group B were immediately used for fluorescent staining of the attached biofilm. After fixation with 2.5% glutardialdehyde (Carl Roth GmbH, Karlsruhe, Germany) in PBS, SYTO®9 (from the LIVE/DEAD® BacLight™ Bacterial Viability Kit, Life Technologies, Darmstadt, Germany) was applied at a 1:1000 dilution in PBS. Subsequent microscopic examination and quantification of the complete volume of the attached biofilm was done using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Mannheim, Germany). The SYTO®9 dye was excited at 488 nm and the emission was measured from 500 to 545 nm. Microscopic examination and quantification of the complete volume of the attached biofilm was done by a researcher blinded to sample identity. Prominent features of the geometry of the retaining screw were used to allow a reproducible scanning procedure. The surface marked in green in Fig. 4A was scanned at a tenfold magnification during microscopic examination with a z-step size of 3 µm. The biofilm volume attached to the titanium surface was later quantified by analyzing the obtained z-stacks with the software IMARIS (Version 8.4, release 2016, Oxford Instruments, Abington, UK).

Fluorescent propidium iodide (PI) stain was used with the SYTO^® 9 (Live/Dead^® BacLight™ Bacterial Viability Kit, Life Technologies GmbH, Darmstadt, Germany) to determine the number of viable and dead bacterial cells [53 (link)]. Intact cells and those with disrupted membranes can be penetrated by the green fluorescence stain SYTO^® 9, whereas the red-fluorescent PI can only penetrate disrupted cell membranes. Hence, viable and active bacterial cells fluoresce green and non-intact cells fluoresce red. The PI and SYTO^® 9 were diluted in 0.9% NaCl to achieve a final concentration of 0.1 nmol/mL. The different materials covered with the initial bacterial adhesion were then transferred to multiwell plates and stained with 1 mL SYTO^® 9/PI solution in 0.9% NaCl per well, for 15 min at room temperature, in a dark chamber. The stained materials were subsequently placed with the contaminated side on a drop of 0.9% NaCl solution in an 8-chambered cover glass (µ Slide 8 well, ibidi GmbH, Munich, Germany), and analyzed using an inverse epifluorescence microscope (ApoTome.2, Axio Observer.Z1, ZEISS, Oberkochen, Germany) with a 63 × oil immersion objective (Plan-Apochromat 63x/1.4 Oil DIC, ZEISS, Oberkochen, Germany).

P. gingivalis strain W50 (ATCC 53978) was obtained from the culture collection of the Oral Health Cooperative Research Centre at the Melbourne Dental School. P. gingivalis W50 was grown and harvested as described (O'Brien-Simpson et al., 2000 (link); Lam et al., 2016 (link)). Growth conditions of batch cultures were monitored at 650 nm using a spectrophotometer (model 295E, Perkin-Elmer, Germany). As before (Cecil et al., 2016 (link)), cells were harvested during late exponential growth by centrifugation (7,000 g, 20 min at 4°C) and enumerated (number/ml) by flow cytometry using a Cell Lab Quanta SC flow cytometer (Beckman Coulter, Australia) and a LIVE/DEAD BacLight™ Bacterial Viability Kit (Life Technologies, Australia). Bacteria were resuspended in 0.01 M phosphate buffered saline (PBS, Sigma-Aldrich), pH 7.4, before incubation with macrophages. Aliquots of P. gingivalis were heat-killed at 70°C for 1 h, as described (Palm et al., 2013 (link)).

For biofilm antibiotic susceptibility and RNA extraction, biofilms were grown in a continuous-flow reactor system with size 13 (1 m in length) Masterflex silicone tubing (Cole Parmer) at a flow rate of 0.1 ml/min, as previously described using 20-fold-diluted LB medium (3 (link)– (link)5 ). For protein extraction, biofilms were grown in a continuous-flow reactor system with size 14 (1 m in length) Masterflex silicone tubing (Cole Parmer) at a flow rate of 0.2 ml/min using 20-fold-diluted LB medium. Carbenicillin at 10 μg/ml was added to the growth medium for plasmid maintenance. To visualize and quantify biofilm formation, biofilms were grown in 24-well plates in 5-fold-diluted LB medium containing 10 μg/ml carbenicillin, with the growth medium being exchanged every 12 h as previously described (23 (link)). Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Inc., Wetzlar, Germany) and the LIVE/DEAD BacLight bacterial viability kit (Life Technologies, Inc.). Quantitative analysis of the confocal laser scanning microscope images of 24-well plate-grown biofilms was performed using COMSTAT (54 (link)).

Bacterial viability after EPL treatment was assessed by fluorescence emitted by EPL-treated compared to non-treated bacterial cells. Initially log-phase bacterial cultures were adjusted to OD_600nm 0.1, and centrifuged at 10,000×g for 15 min. Supernatant was removed and pellets suspended in EPL solutions at MIC or distilled water control. Cells were incubated at 28 °C for one hour when 1 µl of SYTO 9 from the Live/Dead BacLight bacterial viability kit (Life Technologies) was added to 1 ml of the bacterial suspensions. The samples were then incubated in the dark for 15 min and fluorescence measured using flat bottom black plates and fluorimeter (PerkinElmer). Samples were excited at 470 nm and emission spectrum (490–700 nm) recorded. Three biological replicas were performed, and two technical replica readings of each sample were taken. Controls included water-only treatment (non EPL-treated) and EPL + fluorophores (without bacterial cells). Fluorescence microscopy was used to visualize viable and non-viable cells stained with 3 µL of a 1:1 mixture of SYTO 9 and propidium iodide from the Live/Dead BacLight kit components. Cells were prepared as described above and imaged on an Evos FL fluorescence microscope (ThermoFisher), in which viable cells fluoresce in green and non-viable cells in red.