The viability of the cells was estimated after subjecting bacteria to the aforementioned bleach concentrations and staining the cells with the LIVE/DEAD BacLight Bacterial Viability and Counting Kit (Molecular Probes) according to the manufacturer's instructions (Berney et al., 2007 (link); Singh et al., 2012 (link)). Later, cells were examined either by confocal microscopy (Zeiss LSM780) or by flow cytometry (Bio-Rad S3e Cell Sorter). In the flow cytometry analysis, the gating strategy was defined according to the LIVE/DEAD BacLight kit (Berney et al., 2007 (link)). Microspheres of 6 μm diameter were used as the standard for absolute cell quantification. The results obtained in flow cytometry were analyzed using the FlowJo software (Tree Star, Ashland, OR).
Live dead baclight bacterial viability and counting kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The LIVE/DEAD BacLight Bacterial Viability and Counting Kit is a fluorescence-based assay used to determine the proportion of live and dead bacterial cells in a sample. The kit contains two fluorescent dyes that differentially stain live and dead cells, enabling their quantification using fluorescence microscopy or flow cytometry.
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Lab products found in correlation
Lsrfortessa flow cytometer, by BD (3 mentions)
Facsdiva v8, by BD (2 mentions)
S3e cell sorter, by Bio-Rad (1 mentions)
Accuri c6, by BD (1 mentions)
S 3000n scanning electron microscope, by Hitachi (1 mentions)
Facs canto 2 instrument, by BD (1 mentions)
Streptavidin pe, by BioLegend (1 mentions)
Attune next, by Thermo Fisher Scientific (1 mentions)
Jsm 6310, by JEOL (1 mentions)
Novocyte flow cytometry, by Agilent Technologies (1 mentions)
Fluorescent microscope, by Leica (1 mentions)
Tp reader, by Thermo Fisher Scientific (1 mentions)
Attune acoustic focusing cytometer, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Nutrient agar, by BD (1 mentions)
Blood agar, by BD (1 mentions)
Mueller hinton broth and agar, by BD (1 mentions)
Nutrient broth, by Merck Group (1 mentions)
Luria bertani broth, by Thermo Fisher Scientific (1 mentions)
31 protocols using live dead baclight bacterial viability and counting kit
1
Quantifying Bacterial Viability with LIVE/DEAD Staining
2
Antibiotic Susceptibility Assay
3
Bacterial Viability in Fecal Microbiota
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Bacterial viability in fecal microbiota samples from fresh and frozen stool was measured by flow cytometry using LIVE/DEAD BacLight™ Bacterial Viability and Counting Kit (L34856, Molecular Probes) according to manufacturer instructions (Molecular Probes, Oregon, United States). In brief, 977 μL of 0.9% NaCl, 1.5 μL of SYTO9, 1.5 μL of propidium iodide (PI), and 10 μL of the diluted sample were added to the flow cytometry analysis tube. Samples were 10-fold diluted in 0.9% NaCl. The tube was incubated for 15 min in the dark at room temperature and 10 μL of the microsphere suspension (beads) was added to the stained sample. The total volume of the sample in the flow cytometry analysis tube was 1000 μL. The samples were analyzed on an LSR Fortessa flow cytometer (Becton Dickinson, New Jersey, United States) with FACS Diva v8 software (Becton Dickinson). The gating strategy was as described in our first work (Bilinski et al., 2020 (link)). Three main cell populations were observed—alive, dead, and unknown (probably alive, probably dead) with a special not-alive-not-dead group of cells (SYTO9–PI–). The number of bacteria per mL in each analyzed gate was counted according to the following formula taken from the manufacturer materials:
4
Anaerobic Co-culture Viability Assay
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LIVE/DEAD BacLight Bacterial Viability and Counting Kit (L34856R; Molecular Probes, Inc, Eugene, OR) was adopted in this assay. Both R gnavus and A muciniphila were anaerobically cultured in modified GAM broth at 37°C. For each reaction, R gnavus cells (1 × 108) were incubated with the green fluorescent SYTO 9 nucleic acid stain (20 μmol/L) at 37°C for 2 hours. After washing (centrifuged at 15,000g for 10 minutes) twice at room temperature, the bacterial pellet was resuspended in 100 μL modified GAM. R gnavus was then anaerobically co-cultured with an increasing amount (1 × 107 to 1 × 1010) of A muciniphila or the respective CM in a total volume of 1 mL modified GAM broth for overnight at 37°C. Once washed twice with 1 mL 150 mmol/L NaCl, the bacteria were stained with 40 μmol/L red fluorescent propidium iodide in 100 μL of 150 mmol/L NaCl. The live and dead bacteria were quantitated with flow cytometer using the FITC-channel for R gnavus and the V450 channel for dead cell detection.
5
Bacterial Viability Quantification by Flow Cytometry
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Bacterial viability in samples was measured by flow cytometry using the LIVE/DEAD BacLight™ Bacterial Viability and Counting Kit (L34856, Molecular Probes) according to manufacturer instructions (Molecular Probes) [20 ]. Briefly, 977 µL of 0.9% NaCl, 1.5 µL of SYTO9, 1.5 µL of propidium iodide (PI) and 10 µL of diluted sample were added to a flow cytometry analysis tube. Samples were 10-fold diluted in 0.9% NaCl. The tube was incubated for 15 min in a dark at room temperature. A quantity of 10 µL of the microsphere suspension (beads) was added to the stained sample. The total volume of the sample in the flow cytometry analysis tube was 1000 µL. The samples were analyzed on a LSR Fortessa flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with FACS Diva v8 software (Becton Dickinson). The gating strategy is shown in Figure 1 and shows three main cell populations—alive, dead and unknown (probably alive, probably dead) with a special “double negative” group of cells (SYTO9−PI−). The number of bacteria per mL in each analyzed gate was counted according to the following formula taken from the manufacturer materials:
6
Quantifying H. pylori Viability
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H. pylori was standardized to an
OD600 of 0.4 in rich media and incubated for 4 days in
the absence of S-glycoside or presence of 2 mM BacSBn 4, 1 mM DATSBn
5, or 2 mM FucSBn 6. Cells were analyzed prior to incubation and after
4 days of incubation using the LIVE/DEAD BacLight Bacterial Viability
and Counting Kit (Invitrogen) according to manufacturer’s instructions.
Following staining with propidium iodide and SYTO 9 dyes included
in the kit, cells were analyzed by flow cytometry using a BD Accuri
C6+ (BD Biosciences, San Jose, California) instrument,
with 10,000 live cells gated for each replicate. The number of live
and dead H. pylori cells were counted
using FlowJo software to determine the percentage of live H. pylori (% live = 100*[(# live cells)/(# live cells
+ # dead cells)]).
OD600 of 0.4 in rich media and incubated for 4 days in
the absence of S-glycoside or presence of 2 mM BacSBn 4, 1 mM DATSBn
5, or 2 mM FucSBn 6. Cells were analyzed prior to incubation and after
4 days of incubation using the LIVE/DEAD BacLight Bacterial Viability
and Counting Kit (Invitrogen) according to manufacturer’s instructions.
Following staining with propidium iodide and SYTO 9 dyes included
in the kit, cells were analyzed by flow cytometry using a BD Accuri
C6+ (BD Biosciences, San Jose, California) instrument,
with 10,000 live cells gated for each replicate. The number of live
and dead H. pylori cells were counted
using FlowJo software to determine the percentage of live H. pylori (% live = 100*[(# live cells)/(# live cells
+ # dead cells)]).
7
Analyzing Biofilm Formation in M. synoviae
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Confocal microscopy was used to analyze the biofilm of M. synoviae as previously described with some modifications (Yi et al., 2020 (link)). Briefly, biofilms were formed with or without magnolol on round coverslips in a 12-well plate. After a 72-h incubation, the coverslips were gently washed three times with PBS to remove poorly attached cells and stained with LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit according to the manufacturer’s protocol (ABI L34856; Invitrogen, USA). SYTO 9 was used to label the live bacteria, which fluoresced green (488 nm). Propidium iodide was used to label the dead bacteria, which fluoresced red (561 nm). The sample was subsequently incubated at room temperature for 10 min, washed three times with PBS, and imaged by confocal microscopy.
8
Visualizing Acinetobacter sp. DW-1 Cells
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Acinetobacter sp. DW-1 cells immobilized on polyhedron hollow polypropylene balls were examined using a S-3000N scanning electron microscope (Hitachi, Tokyo, Japan). In preparations, cells were fixed in 0.1 M phosphate buffer solution (PBS) containing 3% (v/v) glutaraldehyde at 4 °C for 5 h. The samples were then washed with PBS (pH 7.0) six times (20 min each). The samples were then gradually dehydrated with ethanol. Dehydrated cells were filtered through a 0.2 µm polycarbonate filter, dried with a CO2-critical point dryer, coated with gold and subsequently observed by SEM at 20 kV. Confocal laser scanning microscopy (CLSM) (Zeiss, Berlin, Germany) analysis was performed using a LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit (Invitrogen, Carlsbad, USA) with an appropriate mixture of the SYTO 9 and propidium iodide (PI).
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Acinetobacter sp. DW-1 cells immobilized on polyhedron hollow polypropylene balls were examined using a S-3000N scanning electron microscope (Hitachi, Tokyo, Japan). In preparations, cells were fixed in 0.1 M phosphate buffer solution (PBS) containing 3% (v/v) glutaraldehyde at 4 °C for 5 h. The samples were then washed with PBS (pH 7.0) six times (20 min each). The samples were then gradually dehydrated with ethanol. Dehydrated cells were filtered through a 0.2 µm polycarbonate filter, dried with a CO2-critical point dryer, coated with gold and subsequently observed by SEM at 20 kV. Confocal laser scanning microscopy (CLSM) (Zeiss, Berlin, Germany) analysis was performed using a LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit (Invitrogen, Carlsbad, USA) with an appropriate mixture of the SYTO 9 and propidium iodide (PI).
9
Bacterial Binding Assay with GP2
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Fecal bacterial samples were analyzed by spectrophotometer and 1 × 107 cells (assuming OD 600 1.0 = 5 × 108 cell/mL) were fixed by 4% paraformaldehyde at 4 °C for 3 h. After centrifuge and discard supernatants, bacteria were resuspended and incubated with 10 μg rGP2 or PBS at 4 °C overnight. GP2-bound bacteria were detected by using a combination of anti-mGP2 antibody (MBL, 2F11-C3, #D278-3, D278-5, D278-6, 1:100) and PE-conjugated anti-rat IgG2a antibody (BioLegend, # 407508, 1:100) or PE-Streptavidin (Biolegend, #405203 or BD, # 554061) diluted 1:100. For human GP2, PE-conjugated anti-human GP2 (MBL, #D277-5, 1:100) were used. Live bacteria were stained with LIVE/DEAD BacLight, Bacterial Viability and Counting Kit (Invitrogen, #L34856) in accordance with the manufacturer’s instructions. Flow cytometry analysis was performed with a FACSCanto II instrument (BD Biosciences) or ATTUNE Next (Thermo Fisher Scientific). Gating strategies for the bacterial flow cytometry were shown in Supplementary Fig. 19
10
Biofilm Formation and Characterization
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An overnight culture of E. coli AB1157 was diluted 300-fold in NB, and thereafter the cells were grown on glass slides for 24 h at 30 °C, leading to the formation of biofilms. Then, the supernatant excess solution was removed from the slides and a fresh NB solution was added and supplemented with AuHCl4. The bacterial cells were further grown for 24 h. Biofilms were analyzed by using crystal violet as described above. Bacterial viability in biofilms was assessed with Live/Dead BacLight Bacterial Viability and Counting Kit (Invitrogen, Molecular Probes) via scanning confocal laser microscopy (Zeiss LSM 510 laser module, Carl Zeiss GmbH, Jena, Germany) with an excitation wavelength at 488 nm.
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