upright fluorescence microscope  (Nikon)

Journal: Biofilm

Article Title: Inactivation of Cysteine Synthase CysK-A enhances flocculation, biofilm formation, and sensitivity to oxidative stress in Azospirillum brasilense Sp7

doi: 10.1016/j.bioflm.2025.100335

Figure Lengend Snippet: Three-dimensional (3D) reconstruction of A. brasilense biofilms by CLSM. Biofilms were stained to visualize some matrix components: red fluorescence indicates extracellular DNA (eDNA) stained with propidium iodide, and blue fluorescence indicates exopolysaccharides stained with Calcofluor. Upper panels: composite 3D images captured at 20x magnification, showing the distribution of both eDNA and EPS. Lower panels: Detailed 3D images of exopolysaccharides captured at 60x magnification. White arrows indicate structures resembling cyst-like cells within the biofilm of the cysK -A mutant strain. Scale bar = 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Confocal images were acquired using a Nikon C2+ CLSM (Nikon, Tokyo, Japan) equipped with a CFI Plan Apo Lambda 20 × objective and two helium–neon lasers for the excitation of the mCherry fluorophore at wavelengths of 540 nm and 650 nm and an argon laser for the excitation of radish autofluorescence at 488 nm.

Techniques: Staining, Fluorescence, Mutagenesis

Journal: Biofilm

Article Title: Inactivation of Cysteine Synthase CysK-A enhances flocculation, biofilm formation, and sensitivity to oxidative stress in Azospirillum brasilense Sp7

doi: 10.1016/j.bioflm.2025.100335

Figure Lengend Snippet: A. brasilense AR mutant forms thicker aggregates on radish roots than the wild-type strain. Representative 3D CLSM images of radish roots 7 days post-inoculation. ( A ) Wild-type (WT) A. brasilense Sp7 (harboring pMP2449-5) displays a typical rhizosphere colonization pattern. ( B ) cysK -A mutant ( A. brasilense AR, harboring pMP2449-5) forming visible, thicker, and denser aggregates (white arrows) compared to the WT. For both strains, bacteria are tagged with mCherry (red fluorescence), and the plant root autofluorescence is shown in green. Aggregate thickness was measured from the mCherry channel Z-stacks, revealing a depth of 42.5 μm for the WT and 50 μm for the AR mutant. Images were captured with a 20x objective, 2.0x digital zoom, and steps of 0.5 μm. Scale bar = 25 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Confocal images were acquired using a Nikon C2+ CLSM (Nikon, Tokyo, Japan) equipped with a CFI Plan Apo Lambda 20 × objective and two helium–neon lasers for the excitation of the mCherry fluorophore at wavelengths of 540 nm and 650 nm and an argon laser for the excitation of radish autofluorescence at 488 nm.

Techniques: Mutagenesis, Bacteria, Fluorescence

Journal: Biofilm

Article Title: Inactivation of Cysteine Synthase CysK-A enhances flocculation, biofilm formation, and sensitivity to oxidative stress in Azospirillum brasilense Sp7

doi: 10.1016/j.bioflm.2025.100335

Figure Lengend Snippet: Colony and biofilm calcofluor staining assays. Production of exopolysaccharide (EPS) by the wild-type ( A. brasilense Sp7), the cysK-A mutant ( A. brasilense AR), and the complemented ( A . brasilense AR-pAB- cysK ) strains were visualized by staining with calcofluor, which binds to β-linked polysaccharides. ( A ) Colonies were grown for 5 days at 30 °C on solid LB medium (lacking NaCl) supplemented with Calcofluor (200 μg/mL). Images were captured under UV illumination to visualize fluorescence. Scale bar = 10 mm. ( B ) Confocal micrographs of biofilms grown for 5 days at 30 °C on agar-solidified Nfb∗ medium containing Calcofluor (85 μM). The biofilms were observed with a Nikon Eclipse Ti-E C2+ microscope. Scale bar = 20 μm.

Article Snippet: After five days of static incubation at 30 °C, three-dimensional biofilm structures were observed using an Eclipse Ti-E C2+ confocal laser scanning microscope (Nikon) equipped with CFI Plan Apo VC 20x/1.2 and CFI Plan Apo VC 60x/1.2 WI objective lenses.

Techniques: Staining, Mutagenesis, Fluorescence, Microscopy

Journal: Biofilm

Article Title: Inactivation of Cysteine Synthase CysK-A enhances flocculation, biofilm formation, and sensitivity to oxidative stress in Azospirillum brasilense Sp7

doi: 10.1016/j.bioflm.2025.100335

Figure Lengend Snippet: Colony and biofilm calcofluor staining assays. Production of exopolysaccharide (EPS) by the wild-type ( A. brasilense Sp7), the cysK-A mutant ( A. brasilense AR), and the complemented ( A . brasilense AR-pAB- cysK ) strains were visualized by staining with calcofluor, which binds to β-linked polysaccharides. ( A ) Colonies were grown for 5 days at 30 °C on solid LB medium (lacking NaCl) supplemented with Calcofluor (200 μg/mL). Images were captured under UV illumination to visualize fluorescence. Scale bar = 10 mm. ( B ) Confocal micrographs of biofilms grown for 5 days at 30 °C on agar-solidified Nfb∗ medium containing Calcofluor (85 μM). The biofilms were observed with a Nikon Eclipse Ti-E C2+ microscope. Scale bar = 20 μm.

Article Snippet: The biofilms were observed with a Nikon Eclipse Ti-E C2+ microscope.

Techniques: Staining, Mutagenesis, Fluorescence, Microscopy