The live dead staining of bacterial cells was done using standard protocol,29 (link) with variations. Briefly, the glass cover slip with pre-formed S. aureus biofilm was washed extensively with double distilled water, followed by washing with PBS, pH 7.0. The staining for viable cells on the cover slips was performed by the addition and 5 μM of Syto9 (ThermoFischer Scientific, USA), diluted in DMSO. The staining was done for 10 min in dark conditions. After incubation, the cover slips were further washed extensively with 1× PBS and final rinsing in double distilled water. In order to stain for nonviable S. aureus cells, propidium iodide (ThermoFischer Scientific, USA) was diluted in 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) to a final concentration of 500 nm. The diluted propidium iodide was added to the washed and rinsed Syto9 stained cover slip. The staining was done for 10 min, followed by washing with 1× PBS and final rinsing with double distilled water. The imaging was done by Confocal microscope (Zeiss, Germany) at an excitation/emission of 483/503 nm for Syto9 and 535/617 nm for propidium iodide. The images were acquired on a Rolera Em-C2 camera with a 63× oil immersion objective (Zeiss, Germany). The acquired image was processed by Zen software (Zeiss, Germany).
Syto9
Manufactured by Zeiss
Sourced in Germany
SYTO9 is a fluorescent dye used for nucleic acid staining. It binds to both RNA and DNA, and can be used to visualize and quantify nucleic acids in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Thermo Fisher Scientific (6 mentions)
Syto9, by Thermo Fisher Scientific (3 mentions)
Confocal microscope, by Zeiss (2 mentions)
Propidium iodide, by Zeiss (2 mentions)
Oil immersion objective, by Zeiss (2 mentions)
Zen software, by Zeiss (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Zen blue software, by Zeiss (2 mentions)
Zen lite software, by Zeiss (1 mentions)
Rolera em c2 camera, by Zeiss (1 mentions)
P3566, by Thermo Fisher Scientific (1 mentions)
4 protocols using syto9
1
Live/Dead Staining of S. aureus Biofilm
2
Live-Dead Bacterial Cell Staining
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The live–dead staining of bacterial cells was performed according to the protocol described by Manoharadas et al., 2021 [31 (link)]. Briefly, the staining for viable cells was performed by the addition of 5 μM of SYTO9 (ThermoFischer Scientific, Bedford, MA, USA), diluted in DMSO. The staining was performed for 10 m in dark conditions. After incubation, the cover slips were further washed extensively with 1× PBS and final rinsing in double distilled water. In order to stain for nonviable cells, propidium iodide (ThermoFischer Scientific, Bedford, MA, USA) was diluted in 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) to a final concentration of 500 nm. The diluted propidium iodide was added to the cells. The staining was performed for 10 m, followed by washing with 1× PBS and final rinsing with double distilled water. The image was captured by confocal microscope (Zeiss, Jena, Germany) at an excitation/emission of 483/503 nm for SYTO9 and 535/617 nm for propidium iodide. The images were acquired on a Rolera Em-C2 camera with a 63× oil immersion objective (Zeiss, Jena, Germany). The acquired image was processed by the Zen lite software (Zeiss, Jena, Germany).
3
Evaluating Copper's Impact on Persister Cell Membrane
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To determine whether copper ions damage the persister cell membrane, we performed a Live/Dead assay. Persister cells (5 mL) were resuspended in 0.85% NaCl supplemented with cupric sulfate (160 µg/mL final). The bacterial suspensions were incubated for 30 and 60 min at 30 °C incubator, without shaking, and washed twice in PBS buffer before the addition of propidium iodide (PI) (Invitrogen P3566) and SYTO 9 (Invitrogen, L7012), reaching final concentrations of 60 µM and 20 µM, respectively. After a 15 min-incubation (room temperature, light-protected), cells were observed under a Zeiss fluorescence microscope (approximate excitation/emission for PI is 490/635 nm, and for SYTO9, it is 485/500 nm). Pictures taken with different filters were merged using ZEN software. As a control, heat-treated E. coli cells were used to confirm the PI staining of dead cells.
4
Biofilm Formation on Glass Slides during Fish Impregnation
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Biofilm formation was monitored on 76 × 26 mm glass slides during the impregnation of fish from Batch 2. To do so, slides were immersed in fish tanks during the first 8 weeks of impregnation. Thereafter, the slides were collected and stored at + 4°C for 24 h in tank seawater. For analyses, slides were rinsed twice in sterile seawater, dried and stained with 15 µL of 5 µM SYTO-9 (Invitrogen -Life Science Technology) for 45 min. SYTO-9 fluorescence was measured with an 488 nm excitation filter and a 500 and 550 nm emission filter by confocal laser scanning microscopy, CLSM, (Zeiss, LSM710) using a 63x oil immersion objective. At least three images were acquired for each slide. 3D images were acquired using ZEN software (Zeiss). ZenBlue software (Zeiss, license-free version) was used to process data from the 3D images. The biofilm stacks were analyzed using COMSTAT software [29] to estimate the biovolume (µm 3 /µm 2 ),the average thickness (µm) and the maximum thickness (µm).
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