Low temperature and anhydrous electron microscopy techniques to observe the infection process of the bacterial pathogen Xanthomonas fragariae on strawberry leaves.

Preserving the structural arrangement of the components of a bacterial infection process within a plant for microscopy study is a technical challenge because of the different requirements of each component for optimal preservation and visualization. We used low temperature scanning electron microscopy (cryo-SEM), anhydrous fixation at ambient temperature and freeze-substitution for transmission electron microscopy to examine fractured and sectioned strawberry leaves infected with Xanthomonas fragariae. Cryo-SEM images of fractured samples showed the bacterial colonization of mesophyll air spaces in the leaf, limited by the vascular bundles and the orientation and packing of bacteria in extracellular polysaccharide. Transmission electron microscopy of samples fixed using osmium tetroxide dissolved in FC-72 solvent at ambient temperature showed that the entire plant/bacteria/extracellular polysaccharide system was preserved in situ, and showed plasmolysis of mesophyll cells and disruption of organelles. In freeze-substitution samples, osmium tetroxide in FC-72 solvent gave superior preservation of the extracellular polysaccharide as compared to a conventional cocktail. In addition, strands believed to be xanthan were preferentially contrasted to show their density and orientation around the bacterial cells. We conclude that anhydrous fixation using osmium tetroxide in FC-72 at ambient temperature gave the best preservation of the entire system, and freeze-substitution using this same fixative enhanced the visualization of strands in the biofilm.

A solvent-based fixative for electron microscopy to improve retention and visualization of the intestinal mucus blanket for probiotics studies.

Samples of pig small intestine, cecum, and large intestine were prepared for scanning electron microscopy (SEM), concentrating on mucus blanket retention and visualization. Samples were fixed using three aqueous-based fixatives which included a standard glutaraldehyde fixative alone as the control and the standard fixative formulation with either ruthenium red or alcian blue added and using one solvent-based fixative, osmium tetroxide dissolved in FC-72 (a degreasing fluorocarbon solvent produced by 3M Canada, Inc.), which had been successfully used by Sims et al. [(1991) Biotech. Histochem., 66:173-180] to preserve tracheal mucus of nonhuman mammals. Pig intestine samples prepared using the solvent-based fixative retained a contiguous mucus blanket, while the aqueous-based treatments retained only patchy or fibrous remnants to a degree depending on fixative composition and intestinal site. We conclude that preparation of the pig intestinal mucus layer using the solvent-based fixative suggested by Sims et al. (1991) preserves the mucus blanket in its entirety and gives superior results to aqueous-based fixatives containing the standard additives ruthenium red and alcian blue. We recommend that this anhydrous fixation, which requires only a slight modification from standard conditions, be adopted when mucus layer retention and visualization is important, as in the field of probiotics. Overcoming this major technical obstacle will now allow electron microscopy (EM) to once again provide new in situ information in this reemerging field.