Ruthenium Red and the Bacterial Glycocaly

Abstract

Ruthenium red, a promising cationic reagent for electron microscopy (EM), has long been an important tool in histology. The reagent was initially used by botanists as a semispecific stain for pectic substances, but it has gradually been embraced by investigators in microbiology and the animal sciences as a stain for anionic glycosylated polymeric substances. Luft developed a reliable method and demonstrated that ruthenium red was a useful reagent for visualizing ultrastructural detail. Many investigators, using modifications of Luft's approach, have identified numerous applications for this important reagent. Ruthenium red has been used to show the ultrastructural detail of bacterial glycocalyces. Strong, sharp and consistent observations of this ultrastructural component of the bacterial cell have given a better understanding its fibrous anionic matrix. Any variations in staining owing to artifactual alteration of the fine delicate ultrastructural features have been overcome by incorporation of diamine lysine into ruthenium red methods, thus providing flexible processing times under less than ideal laboratory sampling conditions. Ruthenium red has broad utility in the biological sciences, and in combination with lysine, it is an excellent EM stain for enhanced visualization of bacterial glycocalyx from culture or from clinical specimens.

Salmonella enterica Serovar Typhimurium DT104 Displays a Rugose Phenotype

“…This ES was preserved only in closely packed rugose colonies during preparation for electron microscopy. Because of the shrinkage and detachment of the hydrated ES during normal electron microscopy preparation, the ES was lost, as reported previously (9). However, the ES in cells from the pellicle were preserved.…”

“…This procedure was used to ensure the integrity of the extracellular substances (ES). The agar blocks were fixed with 0.075% ruthenium red, 75 mM lysine monohydrochloride (Sigma), 2% paraformaldehyde (from a 10% stock solution of methanol-free pure formaldehyde), and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 h at room temperature and 18 h at 4°C (9). After a series of washes in the same buffer and postfixation with 1% osmium tetroxide followed by 2% aqueous uranyl acetate, samples were dehydrated with ethanol and embedded in Spurr resin.…”

N-Acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila

“…To investigate the surface architecture of bacterial cells aggregated in biofilm formed by the WT and DahyRI mutant, we performed SEM. Ruthenium red staining is an excellent method for visualization of surface properties of bacteria (Fassel & Edmiston, 1999), and hence we stained bacterial aggregates with ruthenium red. As expected, SEM results revealed that WT bacteria formed a structured biofilm in which cells were well connected by filaments (Fig.…”

Investigating Microbial (Micro)colony Heterogeneity by Vibrational Spectroscopy

“…These exopolysaccharides are mainly composed of homopolysaccharides (cellulose, levans, dextrans, and glucans) and heteropolysaccharides (monosaccharides including a uronic acid) (43). It is thought that the formation of the glycocalyx serves as an integral matrix for a biofilm and that following the adhesion of bacteria to a substrate, the glycocalyx forms a protective milieu for cell division and microcolony formation and growth (7,11). Some studies propose that the glycocalyx either acts as a diffusion barrier or, by complexing antibacterial agents, excludes and/or influences the penetration of antimicrobial agents to the underlying cells (10,13).…”