V. parahaemolyticus was inoculated in 96-well polystyrene plates (SPL life science, South Korea) without shaking at 30°C for 24 h with or without the indoles. The biofilms were stained with 100 μL of pre-warmed PBS containing CFSE stain (carboxyfluorescein diacetate succinimidyl ester) (Invitrogen, Molecular Probes, Inc., Eugene, OR, United States) for 20 min at 37°C (final concentration, 5 μM) and washed three times with PBS. The static biofilm plates were visualized by excitation using Ar 488 nm (emission wavelengths 200–550 nm). The cells were visualized by confocal laser microscopy (Nikon Eclipse Ti, Tokyo, Japan) using a 20× objective. DMSO was used as the control. Color confocal images were visualized using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in three independent cultures were chosen for microscopic analysis (Sethupathy et al., 2020 (link)).
Nis elements c version 3
Manufactured by Nikon
Sourced in Japan
NIS-Elements C version 3.2 is a software package designed for microscope control and image analysis. The core function of this software is to provide a user interface for operating and configuring Nikon microscopes, as well as tools for capturing, processing, and analyzing digital microscope images.
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Lab products found in correlation
6 protocols using nis elements c version 3
1
Biofilm Formation Dynamics of V. parahaemolyticus
2
Quantifying Biofilm Formation by COMSTAT
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Biofilm formation was also visualized by confocal laser microscopy (Nikon Eclipse Ti, Nikon Instruments, Tokyo, Japan) using an Ar laser (excitation 488 nm, emission 500–550 nm) and a 20× objective. Color confocal images were produced using NIS-Elements C version 3.2 (Nikon Instruments). For each experiment, at least 10 random positions in each of the three independent cultures were chosen for microscopic analysis. To quantify biofilm formation in the presence and absence of phorbaketals (100 µg/mL), COMSTAT biofilm software (kindly provided by Arne Heydorn, Søborg, Denmark) was used to determine biomasses (μm3/μm2), mean thicknesses (μm), and substratum coverage (%).
3
Quantifying Biofilm Formation by Confocal Microscopy
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Static biofilms in 96-well plates were visualized by confocal laser microscopy (Nikon Eclipse Ti, Nikon Instruments, Tokyo, Japan) using an Ar laser (excitation 488 nm, emission 500–550 nm) and a 20× objective. Color confocal images were produced using NIS-Elements C version 3.2 (Nikon Instruments, Tokyo, Japan). For each experiment, at least 10 random positions in each of three independent cultures were chosen for microscopic analysis. To quantify biofilm formation in the presence and absence of collismycin C, COMSTAT biofilm software (kindly provided by Arne Heydorn, Søborg, Denmark) was used to determine biomass (μm3/μm2) and mean thickness (μm). At least four positions and 20 planar images per position were analyzed.
4
Confocal Imaging of MSSA and MRSA Biofilms
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CLSM was performed using a Nikon Eclipse Ti (Tokyo, Japan) microscope on MSSA and MRSA biofilms in the presence of CNMA, Si-GNPs, or CNMA-GNPs.30 (link) In addition, biofilm formation by both strains was compared when they were grown in medium alone in 96-well polystyrene plates. Bacteria were stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular probes Inc., Eugene, OR, USA) and visualized at a magnification of 20× using an Ar laser with excitation and emission wavelengths of 488 nm and 500–550 nm, respectively. All confocal images of the same strains were captured under the same conditions. Colored 2D sections were reconstructed into 3D images of biofilms using NIS-Elements C version 3.2 (Nikon Eclipse).
5
Visualizing E. coli Biofilm Formation
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Bacterial cells (E. coli O157:H7/pCM18 tagged with green fluorescent protein) were cultured in glass-bottomed dishes (SPL Life Sciences, Korea) without shaking with or without essential oils or eugenol. Planktonic cells were removed by washing with PBS three times, and biofilms were visualized by excitation using an Ar laser 488 nm (emission wavelengths 500 to 550 nm) under a confocal laser microscope (Nikon eclipse Ti, Tokyo) using a 20 × objective48 (link). Color confocal images were constructed using NIS-Elements C version 3.2 (Nikon eclipse). For each experiment, at least 10 random positions in two independent cultures were chosen for microscopic analysis.
To quantify biofilm formation, color confocal images (20 image stacks) were converted to gray scale using ImageJ. COMSTAT biofilm software49 (link) was used to determine biomasses (μm3 per μm2), mean thicknesses (μm), and substratum coverages (%). Thresholding value was fixed for all image stacks, and at least 4 positions and 20 planar images per position were analyzed.
To quantify biofilm formation, color confocal images (20 image stacks) were converted to gray scale using ImageJ. COMSTAT biofilm software49 (link) was used to determine biomasses (μm3 per μm2), mean thicknesses (μm), and substratum coverages (%). Thresholding value was fixed for all image stacks, and at least 4 positions and 20 planar images per position were analyzed.
6
Quantifying Candida albicans Biofilm Formation
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C. albicans biofilms were developed on 96-well plates from re-suspended cells treated with methylindoles at 0.1 mM. After incubation for 24 h at 37°C, cells were washed with water three times to remove non-adherent cells and stained with carboxyfluorescein diacetate succinimidyl ester (Thermo Fisher Scientific, Invitrogen, MA, United States) (Weston and Parish, 1990 (link)). This method has been used previously (Nweze et al., 2012 (link); Lee et al., 2018 (link)). Biofilms were visualized using an Ar laser (emission wavelength 500–550 nm) under a confocal laser microscope (Nikon Eclipse Ti, Tokyo) equipped with a 20× objective (Kim et al., 2012 (link)). Collected color confocal images (of at least 10 random positions) were constructed in three-dimensions using NIS-Elements C version 3.2 (Nikon eclipse). Assays were carried out using two independent cultures in triplicate. Biofilm formation was quantified by converting color confocal images (20 image stacks) to gray scale using ImageJ, and then using COMSTAT biofilm software (Heydorn et al., 2000 (link)) to calculate biomasses (μm3 μm-2), mean biofilm thicknesses (μm) and substratum coverages (%). Thresholding was fixed for all image stacks, and at least four positions and 20 planar images were analyzed per position.
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