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.