Ruthenium Red for Enhanced Visualization of Alginate Hydrogels Encapsulating Azotobacter vinelandii Cells

Ruthenium Red for Enhanced Visualization of Alginate Hydrogels Encapsulating Azotobacter vinelandii Cells

Abstrakt

Sustainable agriculture is increasingly seeking strategies that reduce dependence on inorganic fertilizers while maintaining plant productivity. Beneficial soil microorganisms, particularly plant growthpromoting rhizobacteria (PGPR), can support plant development through mechanisms related to nutrient availability, stress tolerance, and other factors [1]. Among them, Azotobacter vinelandii is notable for its ability to synthesize phytohormones and extracellular alginate. This polysaccharide contributes to the stabilization of the rhizosphere environment and improves water retention around plant roots, which can help plants better tolerate environmental stress [2]. In this study, bacterially produced alginate was used to form a hydrogel matrix that encapsulated the cells. The encapsulation was achieved by the addition of CaCl2, inducing cross-linking of alginate chains through Ca2+ ions. Due to the polysaccharide-rich nature of the resulting material, visualization of its structural properties using electron microscopy is challenging because commonly used staining agents have limited affinity for these components. To enhance the contrast of polysaccharide structures, ruthenium red was applied as a selective agent with high affinity for acidic polysaccharides and glycoconjugates, where it can also enhance osmium tetroxide contrast [4,5]. As ruthenium red is poorly soluble in common freeze substitution solvents, such as acetone, methanol, or ethanol, it was added directly to the alginate cross-linking solution prior to high-pressure freezing to ensure its presence during cryofixation. After high-pressure freezing, samples were freeze-substituted in either acetone or methanol, embedded in epoxy resin, and ultrathin sections were prepared. Additional contrast was mostly provided by lead citrate. The samples were analysed using a scanning electron microscope (Helios G4 HP, Thermo Fisher Scientific) equipped with a STEM3+ detector. Imaging was performed in STEM bright field mode at an acceleration voltage of 20 kV and probe current of 13 pA. Ruthenium red significantly improved visualization of polysaccharide-rich structures. Both the capsule surrounding A. vinelandii cysts and the surrounding alginate hydrogel network exhibited enhanced contrast while preserving distinguishable fibrillar features. On the other hand, conventional lead citrate post-staining increased overall electron density but partly obscured fine structural details of the cyst capsule, although it improved visualization of the hydrogel matrix. The freeze-substitution solvent also influenced hydrogel morphology. Samples substituted in methanol showed a more open and less dense fibrillar network than those substituted in acetone, possibly due to partial loss of Ca2+ ions or dissolution of hydrogel components. However, given the biological variability of the samples, local heterogeneity must also be considered when interpreting these differences...

Sustainable agriculture is increasingly seeking strategies that reduce dependence on inorganic fertilizers while maintaining plant productivity. Beneficial soil microorganisms, particularly plant growthpromoting rhizobacteria (PGPR), can support plant development through mechanisms related to nutrient availability, stress tolerance, and other factors [1]. Among them, Azotobacter vinelandii is notable for its ability to synthesize phytohormones and extracellular alginate. This polysaccharide contributes to the stabilization of the rhizosphere environment and improves water retention around plant roots, which can help plants better tolerate environmental stress [2]. In this study, bacterially produced alginate was used to form a hydrogel matrix that encapsulated the cells. The encapsulation was achieved by the addition of CaCl2, inducing cross-linking of alginate chains through Ca2+ ions. Due to the polysaccharide-rich nature of the resulting material, visualization of its structural properties using electron microscopy is challenging because commonly used staining agents have limited affinity for these components. To enhance the contrast of polysaccharide structures, ruthenium red was applied as a selective agent with high affinity for acidic polysaccharides and glycoconjugates, where it can also enhance osmium tetroxide contrast [4,5]. As ruthenium red is poorly soluble in common freeze substitution solvents, such as acetone, methanol, or ethanol, it was added directly to the alginate cross-linking solution prior to high-pressure freezing to ensure its presence during cryofixation. After high-pressure freezing, samples were freeze-substituted in either acetone or methanol, embedded in epoxy resin, and ultrathin sections were prepared. Additional contrast was mostly provided by lead citrate. The samples were analysed using a scanning electron microscope (Helios G4 HP, Thermo Fisher Scientific) equipped with a STEM3+ detector. Imaging was performed in STEM bright field mode at an acceleration voltage of 20 kV and probe current of 13 pA. Ruthenium red significantly improved visualization of polysaccharide-rich structures. Both the capsule surrounding A. vinelandii cysts and the surrounding alginate hydrogel network exhibited enhanced contrast while preserving distinguishable fibrillar features. On the other hand, conventional lead citrate post-staining increased overall electron density but partly obscured fine structural details of the cyst capsule, although it improved visualization of the hydrogel matrix. The freeze-substitution solvent also influenced hydrogel morphology. Samples substituted in methanol showed a more open and less dense fibrillar network than those substituted in acetone, possibly due to partial loss of Ca2+ ions or dissolution of hydrogel components. However, given the biological variability of the samples, local heterogeneity must also be considered when interpreting these differences...

Ruthenium red, low-voltage STEM, Azotobacter vinelandii, alginate

Ruthenium red, low-voltage STEM, Azotobacter vinelandii, alginate

HAVLÍČKOVÁ, A.; MRÁZOVÁ, K.; SÚKENÍK, M.; HRUBANOVÁ, K.; SEDLÁČEK, P.; KRZYŽÁNEK, V.

01.06.2025

Czechoslovak Microscopy Society

Brno

978-80-909216-2-7

Microscopy 2026. Book of abstracts.

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