Each resin disk with 3-day biofilm was washed with cysteine peptone water (CPW) to remove the non-adherent bacteria. Live/dead bacterial kit (Molecular Probes, Eugene, OR) was used following the manufacturer’s instructions. A mixture of 2.5 μM SYTO 9 and 2.5 μM propidium iodide was used to stain each sample for 15 min in the dark room. Live bacteria were stained with SYTO 9 to emit a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to emit a red fluorescence. Biofilms were imaged with an inverted epifluorescence microscope (TE2000-S, Nikon, Melville, NY). Three resin disks were tested for each bonding agent with each biofilm, using a total of 15 disks for multispecies biofilm and 24 disks for four single-species biofilms for live/dead staining. Five random images were taken for each disk, yielding 15 images for each bonding agent with each type of single or multi-species biofilm.
Live dead bacterial kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead Bacterial Kit is a fluorescence-based assay that enables the differentiation and quantification of live and dead bacterial cells. The kit utilizes two nucleic acid stains with distinct fluorescent properties to label cells, allowing for the visualization and enumeration of viable and non-viable bacterial populations.
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6 protocols using live dead bacterial kit
1
Quantifying Live/Dead Biofilm on Dental Resins
2
Visualizing Live and Dead Bacteria in Biofilms
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The 2-day biofilm specimens were rinsed with phosphate-buffered saline (PBS) to eradicate non-adherent bacteria. Next, adhesive samples were stained via a live/dead bacterial kit (Molecular Probes, Eugene, OR, USA). A measurement of 2.5 µM of each SYTO 9 and propidium iodide was mixed and applied to the samples for 15 min. Propidium iodide stained the dead bacteria and emitted red fluorescence, and SYTO 9 stained live bacteria and emitted green fluorescence [30 (link)]. An inverted fluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY, USA) was used to image three stained disks per group, and five randomly picked views were taken for each sample yielding a total of 15 images per group.
3
Bacterial Cell Viability Assay
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SYTO9 and propidium iodide (PI) were prepared according to the manufacturer’s instructions [Live/Dead Bacterial Kit, Molecular Probes, Gothenburg, Sweden (Brantl, 2007 (link))]. Cells in 50 μl of culture were stained with 0.1 mM SYTO9/PI mix for 15 min in dark at 25°C, washed with PBS solution, and images were taken with excitation and emission of 483/503 nm for SYTO9 and 485/630 nm for PI.
4
Biofilm Formation and Live/Dead Staining
5
Magnetic Nanoparticle-Mediated Antimicrobial Treatment
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All the chemical agents were analytical reagents (AR). Iron oxide (II, III) magnetic nanoparticles solution was obtained from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). Dopamine hydrochloride, Tris (hydroxymethyl) aminomethane, Tris (hydroxymethyl) aminomethane hydrochloride, and minocycline hydrochloride (Mino) were provided by Aladdin Biochemical Technology (Shanghai, China). Brain heart infusion (BHI) broth was supplied by Qingdao Hope Bio-Technology (Qingdao, China). Hemin was obtained from Hefei BASF Bio-Technology (Hefei, China). The Cell Counting Kit-8 (CCK-8) and AO/EB double stain Kit were supplied by KeyGen Biotechnology (Nanjing, China). The MTT staining kit was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). LIVE/DEAD bacterial kit was provided by Molecular Probes (Eugene, USA). The antibodies against IL-6, IL-1β, and TNF-α were sourced from Affinity Biosciences Co., Ltd (Changzhou, China).
6
Quantifying P. gingivalis Colonization on MAG Microbeads
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To determine the concentration of MAG, four kinds of microbeads with final MAG concentrations of 0 μM, 10 μM, 100 μM, and 1000 μM were fabricated. Prior to P. gingivalis inoculation, bacteria were washed three times with PBS and re-suspended in fresh WC anaerobic broth. Bacterial cell counts were determined using a spectrophotometer, where the optical density of 1.0 at 600 nm corresponded to 1 × 109 CFU/mL [36 (link)]. The microbeads were placed in a 96-well plate, one per well. MAG microbeads were incubated with 3 × 104 CFU/mL and 3 × 106 CFU/mL density of P. gingivalis in bacterial broth for 24 h. After 24 h of incubation, P. gingivalis colonization on the surface of the microbeads was detected. Microbeads were washed with PBS to remove the non-adherent bacteria. For the detection of bacteria viability, a live/dead bacterial kit (Molecular Probes, Eugene, OR, USA) was used according to the manufacturer’s instructions. Live bacteria were SYTO 9-positive (green fluorescence), whereas bacteria with compromised membranes were propidium-iodide-positive (red fluorescence). Fluorescent labeling was detected by an inverted epifluorescence microscope (TE2000-S, Nikon, Tokyo, Japan).
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