The membrane integrity of the cells was evaluated as fully described by Barros et al. [4 (link)]. Briefly, the cell suspension was properly diluted, stained for 7 min with the nucleic acids SYTO9TM and propidium iodide (PI), available from the LIVE/DEAD® BaclightTM kit (Invitrogen/Molecular Probes, Eugene, OR, USA), and filtered through a 0.2 μm Nucleopore® (Whatman, Middlesex, UK) black polycarbonate membrane. Afterwards, the membranes were prepared for microscope observation (LEICA DMLB2; Leica Microsystems Ltd., Heerbrugg, Switzerland). The biofilm cells were observed under a 100× immersion oil objective of a CCD camera coupled with a mercury lamp HBO/100W/3 (λexcitation = 480–500 nm and λemission = 485 nm; Chroma 61000-V2 DAPI/FITC/TRITC). The cell numbers were estimated from the counts of a minimum of 15 fields of view (6.03 × 10−5 cm2) and the results were presented as the SYTO 9 and PI uptake percentages.
Live dead baclighttm kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LIVE/DEAD BacLight™ kit is a fluorescence-based assay used to distinguish viable and non-viable bacterial cells. It employs two nucleic acid-binding stains, SYTO 9 and propidium iodide, to label bacterial cells. The kit provides a rapid and reliable method for the assessment of bacterial cell viability.
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8 protocols using live dead baclighttm kit
1
Evaluating Membrane Integrity of Cells
2
Bacterial Viability Analysis by Flow Cytometry
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All data were obtained using a FACSCanto flow cytometer (Becton, Dickinson and Company, Erembodegem, Belgium), and acquired and processed using FacsDiva software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.
Bacterial viability was assessed using the LIVE/DEAD BacLightTM kit (Molecular Probes Eugene, OR, USA) as described by the manufacturer. This bacterial viability kit is widely used in flow cytometry and consists of two nucleic acid stains: green fluorescent SYTO 9 is cell-permeable and freely enters all tested bacteria, either live or dead, while red fluorescent propidium iodide (PI) can only enter membrane-comprised cells [30 ]. In our set-up, 977 μl of sterile saline cell suspension was added to 20 μl of the treated bacteria. These samples were immediately stained with 3 μl of a mixture of SYTO 9 (5 μM final concentration) and PI (30 μM final concentration) and incubated for 15 minutes in the dark at room temperature. FCM measurements were performed immediately thereafter.
Bacterial viability was assessed using the LIVE/DEAD BacLightTM kit (Molecular Probes Eugene, OR, USA) as described by the manufacturer. This bacterial viability kit is widely used in flow cytometry and consists of two nucleic acid stains: green fluorescent SYTO 9 is cell-permeable and freely enters all tested bacteria, either live or dead, while red fluorescent propidium iodide (PI) can only enter membrane-comprised cells [30 ]. In our set-up, 977 μl of sterile saline cell suspension was added to 20 μl of the treated bacteria. These samples were immediately stained with 3 μl of a mixture of SYTO 9 (5 μM final concentration) and PI (30 μM final concentration) and incubated for 15 minutes in the dark at room temperature. FCM measurements were performed immediately thereafter.
3
Detection of MRSA VBNC Cells
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The developed CPA assays were utilized to detect the VBNC cells. The experimental strains were cultured to the exponential growth period. To induce the VBNC state of MRSA, the culture was diluted to the final concentration at 108 CFU/ml with food homogenate (Cantonese pastry, steamed bread, rice flour; Guangzhou Restaurant, Guangzhou, China). Then, they were stored at −20°C to induce the VBNC state for further use of PMA-CPA. The trend of the number of culturable bacteria was used to make sure that the cells enter into culturable state. And culturable and viable cell enumerations were preformed every 3 days by traditional culture method. The LIVE/DEAD BacLightTM kit (Thermo Fisher Scientific, United States) was performed under fluorescence microscope after the culturable colonies no longer form on agar medium. After confirmation of the VBNC state, PMA-CPA has been applied for detection.
4
Membrane Integrity Analysis of NDM-1 E. coli Cells
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Membrane integrity of the NDM-1 E. coli cells after treatment with plant extracts was analyzed as described by Kennedy et al. (2011) (link) using the LIVE/DEAD® BacLightTM Kit (Thermo Scientific, United States). Flow cytometry was optimized by preparing a standard by mixing live and dead cells in phosphate buffer. Bacterial cell density in the mid-exponential growth phase was adjusted to 1 × 108 cfu/ml, and treated with 2× MIC of plant extracts at 37°C for 1 h. The treated bacterial cells were incubated with 5 μM SYTO 9 in the dark for 15 min, and propidium iodide (PI) was added to a final concentration of 30 μM. Colistin (4 μg/ml) and DMSO (4%) were used as positive and negative controls, respectively. The cells were analyzed in a flow cytometer and the signals were captured using FL1 and FL3 channels (BD FACS Calibur, United States).
5
Fluorescent Microscopy of Bacterial Membrane Interactions
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MRSA (ATCC 43300) and P. aeruginosa were cultured overnight separately in MHB. Following subculture, the fresh bacteria culture (10 8 CFU/mL) was centrifuged and gently washed with 0.9% (w/v) NaCl twice; then, they were incubated with different tested peptide concentrations for 1h at 37℃. The mixtures were subsequently stained with the Live/Dead BacLight TM kit (SYTO9 and propidium iodide (PI); Thermo Fisher Scientific Inc., Waltham, MA, USA). Fluorescence emission was detected by the SP8 Lighting confocal microscope (Leica, Germany). The localization of samples on the bacteria cell membranes was detected by the fluorescent confocal microscope. Mid-logarithmic phase P. aeruginosa culture was centrifuged and washed with 0.9% (w/v) NaCl. The bacterial culture (10 7 CFU/mL) was incubated with FITC-labeled DMS-PS2 at a concentration of 32 μg/mL. After the incubation of 30 min, samples were counterstained with DAPI for 10 min and observed by the fluorescent confocal laser microscope with a 63×magnification objective.
6
Quantifying Bacterial Viability and Cell Size
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The total number of bacteria (TNB) was determined according to the procedure described by Hobbie et al. [31 (link)]. Viable bacteria, estimated as bacteria with intact cytoplasmic membranes (MEMB+), were counted with Live/Dead BacLightTM kit (Thermo Fisher Scientific Inc., Madrid, Spain) as described by Joux et al. [32 (link)]. The number of culturable bacteria (CFU) was determined by spreading cell suspensions on marine agar (MA, PanReac AppliChem, Barcelona, Spain) followed by incubation for 24 h at 26 °C and cell counting.
The length variations of V. harveyi cells during their survival at 20 °C were estimated via image analysis of epifluorescence preparations [33 ] by using an image analysis system, which included a video camera of high resolution (Hamamatsu 2400, Hamamatsu Photonics, Hamamatsu City, Japan). Digitized images of microscopic fields were analyzed by Scion Image 1.62a software. In total, 200 cells were measured in each sample. The mean value (x) and the corresponding standard deviation (SD), which defined the size of the cells in the initial inoculate, were used to establish three arbitrary ranges of cell size (≤x-SD, >x-SD and ≤ x+SD, >x+SD) subsequently used to present the time-dependent changes of cell size in V. harveyi populations [5 (link)].
The length variations of V. harveyi cells during their survival at 20 °C were estimated via image analysis of epifluorescence preparations [33 ] by using an image analysis system, which included a video camera of high resolution (Hamamatsu 2400, Hamamatsu Photonics, Hamamatsu City, Japan). Digitized images of microscopic fields were analyzed by Scion Image 1.62a software. In total, 200 cells were measured in each sample. The mean value (x) and the corresponding standard deviation (SD), which defined the size of the cells in the initial inoculate, were used to establish three arbitrary ranges of cell size (≤x-SD, >x-SD and ≤ x+SD, >x+SD) subsequently used to present the time-dependent changes of cell size in V. harveyi populations [5 (link)].
7
Phage Biofilm Viability Assay
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24 hour-old biofilms were formed on 2-well μ-slides with a glass bottom (ibidi, USA) in the presence of phage phiIPLA-RODI or SM (negative control). After removing the planktonic phase, wells were washed with PBS and stained with Live/Dead® BacLightTM kit (Invitrogen AG, Basel, Switzerland). Samples were observed with a confocal scanning laser microscope (DMi8, Leica Microsystems) using a 100 × oil objective.
8
Biofilm Formation and Live/Dead Staining
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Each well of a 2-well μ-slide with a glass bottom (ibidi, United States) was inoculated with 1 ml of a cell suspension containing 106 cfu/ml in TSBG. Biofilms were then allowed to form for 24 h at 37°C. After biofilm development, the planktonic phase was removed and wells were washed with PBS prior to staining all cells (live and dead) with SYTO 9 from the LIVE/DEAD® BacLightTM Kit (Invitrogen AG, Basel, Switzerland) as indicated by the manufacturer. The biofilm samples were then observed under a DMi8 confocal laser scanning microscope (Leica Microsystems) using a 63× oil objective.
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