Live dead baclight bacteria viability kit

To monitor cell viability, cells retained on surfaces were treated using a live/dead stain (LIVE/DEAD™ Baclight™ bacteria viability kit, Invitrogen, Scotland). After diluting the stains in dimethyl sulfoxide (Sigma, UK) according to the manufacturer’s instructions, the propidium iodide and Syto 9 dye components were individually diluted in a 1:10 solution in sterile distilled water in eppendorf tubes. Five microliters of each dilution was then mixed together, and 10 μL was spread across the sample and allowed to air dry in the dark in a microbiological Class II safety hood. Following drying samples were stored in the dark at 4 °C. Potentially viable and damaged cells were distinguished under the fluorescence microscope (Nikon Eclipse E600, Surrey, UK) since it is assumed that viable cells appeared green, whilst non-viable or membrane compromised cells appeared red. The microscope was mounted with an F-View II black and white digital camera (Soft Imaging System Ltd., Helperby, UK, supplied by Olympus, Hertfordshire, UK). This system used a Cell F Image Analysis package (Olympus, Hertfordshire, UK). The percentage coverage of the live and dead cells retained on the surface was measured using separate selective UV filters across the same field of view. Twenty fields of view using each UV filter were taken per surface.

Bacterial suspensions were stained with the LIVE/DEAD BacLight Bacteria Viability Kit (L7007, Invitrogen, Waltham, MA, USA). The BacLight consists of SYTO9, a membrane-permeant dye penetrating all cells, and PI, which is cell-impermeant and only enters damaged or dead cells. The staining reagent was prepared according to the manufacturer’s instructions. Briefly, component A (1.67 mM SYTO9/1.67 mM PI) and component B (1.67 mM SYTO9/18.3 mM PI) were mixed 1:1 in a microtube. Five microliters of the mixture was added to 1 mL of each bacterial suspension (5 µL/mL final concentration). The suspensions were subsequently mixed thoroughly and incubated in the dark for 25 min before measurement at RT. Microbeads (100 µL) (Invitrogen, USA) were added to the suspensions for cell quantification. Samples containing ASM without bacteria were prepared and stained to verify background noise. The autofluorescence of the bacteria was assessed using unstained cells, and positive controls were generated by pre-treating the cells with propanol (70% (v/v)) to cause membrane damage, maximizing PI penetration. Therefore, the membrane-damaged or dead cells were simply detected with the high intensity of PI.

The bacterial adhesion and biofilms formation on Smooth Ti and four groups of NT-H were also observed using CLSM. Similarly, the specimens were removed at four different time points and followed by gently washed three times with PBS. The samples were stained with 300 μL of combination dye (LIVE/DEAD BacLight bacteria viability kits, Molecular Probes, L13152) for 15 min in the dark and then were analyzed with a confocal laser scanning microscope (Leica TCS SP2; Leica Microsystems, Heidelberg, Germany). The viable bacteria with intact cell membranes appear fluorescent green, whereas nonviable bacteria with damaged membranes appear fluorescent red. The images were acquired from random positions on the surfaces of the samples. We used ATCC 43300 for the CLSM and SEM observation assays in this study.

Using bacterial live/dead staining, bacteria were stained with green fluorescent SYTO 9 and red fluorescent propidium iodide. A volume of 500 μL of the suspension with 10⁶ CFUs/mL bacteria was added to wells that contained scaffold specimens and incubated at 37 °C in a humidified atmosphere. After coincubation for 24 and 48 h with bacteria, the specimens were stained with 300 μL of combination dye (Live/Dead BacLight bacteria viability kits; Molecular Probes Life Technologies, Carlsbad, CA, USA) and observed with CLSM (Leica TCS SP2, Heidelberg, Germany). Live bacteria appear as fluorescent green, while the dead bacteria appear as fluorescent red.

After 6, 24, and 48 hours, the specimens were stained with 300 μL of a combination dye (Live/Dead BacLight bacteria viability kits; Molecular Probes Life Technologies, Carlsbad, CA, USA) and observed with a CLSM (Leica TCS SP2, Heidelberg, Germany). Bacteria were stained with green fluorescent SYTO 9 and red fluorescent propidium iodide.

After 24 h incubation, the TSB medium were removed carefully from each well, and the discs were transferred into another 48-well plate and gently washed with PBS three times. Then the wells containing the titanium discs were added with 500 μl combination dye (LIVE/DEAD Baclight bacteria viability kits, L13152; Molecular Probes, Eugene, OR, USA) in a dark environment at room temperature for 15 min and were subsequently analyzed with a CLSM (Leica TCS SP2; Leica Microsystems, Heidelberg, Germany). The live/dead kit containing two kinds of fluorescence dyes were used to distinguish the viable and non-viable cells under the fluorescence microscope, because dye SYTO 9 can make the viable bacteria with intact cell membranes display green fluorescence, whereas dye PI (Propidium Iodide) can make non-viable bacteria with damaged membranes display red fluorescence. The Leica confocal software was used to analyze the biofilm images.