Copper powder was supplied by TraceXtec (TraceXtec, Pty., Ltd., South Africa). PDLLA (Mw 75−120 kDa) and PEO (Mw 200 kDa), N,N-dimethylformamide (DMF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle's medium (DMEM), Hams-F12, fetal bovine serum (FBS), hydrocortisone, insulin, cholera toxin and PenStrep were obtained from Sigma-Aldrich (St. Louis, MO, USA). The LIVE/DEAD® BaclightTM Bacterial Viability kit and FilmTracerTM SYPRO® Ruby Biofilm Matrix Stain were from Thermo Fisher Scientific (Massachusetts, USA). All reagents were of analytical grade.
Live dead baclighttm bacterial viability kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
The LIVE/DEAD® BacLight™ Bacterial Viability Kit is a fluorescence-based assay used to distinguish live and dead bacterial cells. The kit utilizes two nucleic acid stains, SYTO® 9 and propidium iodide, to provide a rapid and reliable method for assessing bacterial cell viability.
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Lab products found in correlation
Cx40 microscope, by Olympus (4 mentions)
Image pro plus, by Media Cybernetics (4 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Axiocam mrm, by Zeiss (3 mentions)
Axio imager m2 optical microscope, by Zeiss (3 mentions)
Igor pro 6.22a, by Wavemetrics (3 mentions)
Evolutiontm mp 5.1 ccd color camera, by Media Cybernetics (3 mentions)
U rfl t burner, by Olympus (3 mentions)
Filmtracertm sypro ruby biofilm matrix stain, by Thermo Fisher Scientific (2 mentions)
Axio scope a1, by Zeiss (2 mentions)
Zen 2, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Zen blue 3, by Zeiss (2 mentions)
U mwb mirror cube unit, by Olympus (2 mentions)
A1r laser scanning confocal microscope, by Nikon (2 mentions)
Nis elements ar software, by Nikon (2 mentions)
Pen strep, by Merck Group (1 mentions)
Ham s f 12, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
75 protocols using live dead baclighttm bacterial viability kit
1
Copper-Polymer Composite Biocompatibility
2
Antimicrobial Efficacy of Matrine Formulations
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CS with a low viscosity <200 mPa·S, matrine with a purity of 98%, and D-proline with a purity of 99% were purchased from Aladdin Chemistry Co. Ltd. (China). Lecithin with a purity >98% was provided by Shanghai Macklin Biochemical Co. Ltd (China). The 2216E medium powders were purchased from Qingdao Hope Bio-Technology Corporation. Different pH phosphate-buffered saline (PBS) solutions were stored after sterilization. Live/dead® BacLight TM Bacterial Viability Kit (L13152) was obtained from Thermo Fisher Scientific (America). The E. coli (ATCC 8739), S. aureus (ATCC 6538), and P. aeruginosa (MCCC 1A00099) were purchased from the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
3
Visualization of P. aeruginosa Biofilm Formation
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To study the effect of di-rhamnolipids on P. aeruginosa PAO1 adherence to glass or plastic surfaces biofilms were developed on cover slips and examined under the microscope. An overnight culture of P. aeruginosa PAO1 was diluted to 5 × 107 cfu mL-1 in LB and 2 ml was added per well of six well microtiter plate containing glass or plastic cover slips. After 24 h, non-adherent cells were removed and biofilms were washed with 0.9% NaCl and stained with 2.5 μM SYTO9 green fluorescent dye and 2.5 μM propidium iodide (PI) red fluorescent dye of Live/Dead staining kit (LIVE/DEAD® BacLightTM Bacterial Viability Kit, Thermo Fisher Scientific, Waltham, MA, United States). Cells were observed under a fluorescence microscope (Olympus BX51, Applied Imaging Corp., San Jose, CA, United States) under 100,000 × magnification (glass) or 40,000 × magnification (plastic).
4
Phytochemicals' Effect on C. jejuni Viability
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We determined the effect of phytochemicals on the viability of C. jejuni S-8 using a Live/Dead BacLightTM bacterial viability kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. Briefly, C. jejuni S-8 was cultured overnight at 42°C under microaerophilic conditions. The culture was dispensed in the 24-well plates and treated with 0.25% of the phytochemicals for 5 min, followed by the addition of SYTO-9 and propidium iodide. SYTO-9 and propidium iodide stains were used for the differential staining of live and dead cells. The cells were transferred to the microscope slide and observed under the fluorescent microscope (BZ-810, Keyence Co., IL, United States) at a ×20 objective lens.
5
Biofilm Viability Assessment by Confocal Microscopy
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Using the LIVE/DEAD® BacLightTM Bacterial Viability Kit (Thermo Fisher Scientific, Braunschweig, Germany), the plaque deposits were fluorescently stained according to the manufacturer’s protocol and fixed with 2.5% glutaraldehyde (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). A phosphate-buffered saline solution (Biochrom GmbH, Berlin, Germany) was used to store the stained samples at 4 °C. This enabled the initial intraoral biofilm formation to be assessed under close-to in vivo conditions. Three-dimensional images of the biofilm were taken by CLSM (SP2, Leica Microsystems GmbH, Wetzlar, Germany). For each test specimen, five defined positions were microscopically examined at a magnification of 10 × and 63 ×. The biofilm volume per test specimen and the live/dead distribution were quantified using the Imaris software package (Imaris x64 6.2.1, Bitplane AG, Zurich, Switzerland). In addition, a representative three-dimensional reconstruction of the biofilm was carried out. The biofilm surface coverage per test specimen was determined using the Leica LAS AF Lite and the ImageJ software (Leica LAS AF Lite, Leica Microsystems GmbH, Wetzlar, Germany; ImageJ, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/ ).
6
Bacterial Membrane Permeability Assay
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Membrane permeability was measured by fluorescence staining using the Live/Dead® BacLight TM Bacterial Viability Kit (Thermo) [20 (link)]. Briefly, 150 μL of rToBPI1/LBP or rToBPI2/LBP protein (100 μg/mL) was incubated for 2 h with equal volumes of E. coli (2.2 × 108 CFU/mL), V. alginolyticus (2.1 × 108 CFU/mL) and S. agalactiae (1.5 × 108 CFU/mL), respectively. Then 1 μL of the fluorescent staining mixture (STYO9 0.5 μL and PI 0.5 μL) was added and incubated for 30 min away from light. The samples were observed and photographed at the excitation wavelengths of 485 nm and 535 nm and the emission wavelengths of 498 nm and 617 nm. PBS mixed with bacteria was used as a control.
7
Fluorescence Microscopy of Antibiotic-Stressed Cells
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Cultures were grown as described above and treated with OFX (5 μg/ml), MMC (20 μg/ml), or Kan (50 μg/ml) at an OD600nm of 0.3. Ten microliters of cultures were collected at indicated time points, spotted on 1% agarose MOPS 0.4% glucose pads, and imaged by fluorescence microscopy. For PI staining, cells were collected at indicated time points, washed in NaCl solution (0.85%), and stained for 15 min using the LIVE/DEAD BacLightTM Bacterial Viability Kit (Thermo Fisher Scientific). Cells were then washed with the saline solution and transferred on a 1% agarose MOPS 0.4% glucose pad. For membrane potential assay, DiBAC4(3) (Thermo Fisher Scientific) was dissolved in 100% dimethyl sulfoxide (DMSO). At indicated time points, 1 ml of culture was collected and 1 µl of DiBAC4(3) (10 μg/ml) was added to the sample, followed by incubation for 15 min in the dark. Cells were then washed and transferred on 1% agarose MOPS 0.4% glucose pads. Negative control corresponds to untreated cells and positive control to heat-shocked cells at 72°C for 1 hour. For H2O2 detection after OFX treatment using H2DCFDA (Sigma-Aldrich), H2DCFDA was dissolved in 100% DMSO and, at indicated time points, 1 ml of cell culture was collected and incubated for 40 min with H2DCFDA (10 μg/ml). Cells were then washed and transferred on 1% agarose MOPS 0.4% glucose pads.
8
Visualizing Bacterial Biofilm Viability
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Immediately after washing the cells with sterile distilled water, biofilms were carefully removed from the wells with a sterile pipette tip and transferred to 100 μl sterile PBS (pH 7.3). The biofilms were stained with the LIVE/DEAD® BaclightTM Bacterial Viability kit (Thermo Fisher Scientific, Massachusetts, USA) and FilmTracerTM SYPRO® Ruby Biofilm Matrix Stain (Thermo Fisher Scientific), as recommended by the suppliers. A Carl Zeiss LSM780 confocal microscope (Carl Zeiss, Göttingen, Germany) was used to study the stained biofilms. Images were processed by using the ZEN 2011 imaging software (Carl Zeiss).
9
Bacterial Viability Assay on Enamel
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The samples were stained using a fluorescent bacterial viability kit (LIVE/DEAD® BacLightTM Bacterial Viability Kit; Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature in the dark [38 (link),39 (link)]. The specimens were embedded in 4% carboxymethyl cellulose sodium salt (Section Lab Co., Ltd., Hiroshima, Japan). The adhesive-film method was used to create a frozen section of 8 µm in thickness vertically along the enamel [40 (link)]. Fluorescent tomographic imaging was performed using a CLSM (FV-300; Olympus, Tokyo, Japan) as well as Ar 488 nm and He-Ne 543 nm lasers. Filters of 510–530 nm and ≥610 nm were used to detect SYTO 9 and propidium iodide, respectively [38 (link),39 (link)]. A 100× oil immersion objective lens was used. The assay was performed with three replicates per material and a control group.
10
Culturability and Viability Assessment of S. aureus
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To determine the culturability of S. aureus cells, the plate counting method was applied to identify the culturable cell number. The induction culture was serially diluted with 0.9% NaCl and inoculated on TSA followed by incubation at 37°C for 24 h. When culturable, the cell number was < 1 CFU/mL for 3 days, and the cells were considered to be non-culturable (Deng et al., 2015 (link)). In addition, the LIVE/DEAD® BacLightTM bacterial viability kit (Thermo Fisher Scientific, China) combined with fluorescence microscopy was used to determine whether the non-culturable cells were in the VBNC state following the manufacturer’s instructions.
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