Biofilm formation examined by CLSM was performed as described previously with minor modifications (Takenaka et al., 2001 (link)). Briefly, overnight culture of PA1, PA1RG, and PA1RG/wzy was diluted 1:100 into fresh LB medium, respectively. Then, 2 ml of aliquot was inoculated into the glass-bottom cell culture dish (15 mm in diameter, Nest, China), and incubated at 37°C for 24 h. The biofilm was washed with phosphate-buffered saline (PBS), and fixed with 2.5% glutaraldehyde for 1.5 h. After washing with PBS, the biofilm was labeled by 5 μM fluorescent nucleic acid stain SYTO 61 (Invitrogen, United States) at room temperature for 30 min, followed by 50 μg/ml FITC-labeled concanavalin A (FITC-ConA, Sigma-Aldrich, United States) at 37°C for 5 min. The biofilm was visualized using the confocal laser scanning microscope LSM800 (Zeiss, Germany) with 561 nm excitation and 640 nm emission wavelengths for SYTO 61, and 488 and 537 nm for FITC-ConA, respectively.
Syto 61
Manufactured by Zeiss
Sourced in Germany
SYTO 61 is a fluorescent nucleic acid stain used in cell biology applications. It is designed to bind to both DNA and RNA, providing a way to label and visualize cellular nucleic acids. The core function of SYTO 61 is to enable the detection and analysis of cellular nucleic acid content through fluorescence microscopy or flow cytometry techniques.
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2 protocols using syto 61
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Biofilm Visualization by CLSM
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Biofilm Analysis of Bacterial Species
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Overnight cultures of S. aureus ATCC25923 and N315, S. epidermidis ATCC35984, and P. aeruginosa PAO1 were diluted 1: 100 in TSB or LB media in the presence of 10 mM NAC, or 20% serum, or a combination of NAC and serum. Bacteria were cultured in 15 mm glass-bottom cell culture dishes (polystyrene) on a rocker platform at 37 °C. After incubation for 24 h, the dishes were washed with water three times and fixed with 4% formaldehyde. Two ml of a 1: 1000 dilution of the nucleic acid stain SYTO 61 (Invitrogen) was added to the dishes, followed by incubation in the dark at room temperature for 30 min. The dishes were then washed and 2 ml of 50 µg/ml FITClabelled concanavalin A type IV (Sigma-Aldrich) was added. After incubation in the dark for 5 min at 37 °C, the dishes were rinsed with water three times and air-dried. Visualization of biofilms was performed using a laser scanning confocal microscope (LSM780, Carl Zeiss, Plan-Apochromat 63×/1.40 Oil M27 objective lens) with 561 nm excitation and 640 nm emission wavelengths for SYTO61, and 488 nm and 537 nm for FITC-ConA. Images were acquired from at least three distinct regions of each cell culture dish and processed using the ZEN image analysis package (Carl Zeiss). Biofilm thickness was measured by Z-stack images [15] . Three independent replicas were conducted for each experiment.
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