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.

Effect of process variables on particle size and viability of Bifidobacterium lactis Bb-12 in genipin-gelatin microspheres.

Gelatin microspheres cross-linked with genipin were developed to encapsulate the probiotic Bifidobacterium lactis Bb-12 The effects of different gelatin concentrations (10-19% w/v), bloom strengths (175 and 300), surfactants, stirring rates during emulsion formation and genipin concentrations (0-10 mM) on the microsphere sizes and viability of bacterial cells were investigated. Principal Component Analysis revealed microsphere size distribution differed depending on the presence or absence of surfactants as well as a trend of increasing micropshere size with increasing gelatin concentration and bloom strength. Lower stirring rates resulted in larger microspheres with higher encapsulation yields of bifidobacteria Microsphere size and cell viability were not significantly (p < 0.05) influenced by increasing genipin concentrations up to 10 mM whereas microsphere stability in simulated gastric juice increased with increasing genipin concentration. The encapsulation yields were higher in 175 bloom strength gelatin microspheres than in 300. Cold-stage scanning electron microscopy showed encapsulated bacteria distributed throughout the genipin cross-linked gelatin matrix.

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.

Microstructural manifestations of two unusual phenomena detected in experimental roast pork: A scanning and transmission electron microscopy study.

Loin roasts L. dorsi from both barrows and gilts from a breeding and feeding experiment were cooked, cut, presented to sensory evaluation panelists and the excess meat stored at 4°C in tied plastic bags. An interesting phenomenon observed was that a deposit remained on the cutter blade after slicing the experimental roasts, but no deposit remained in the blade after cutting control roasts. Similarly, slices of experimental roast adhered together after refrigeration, but slices of control roast stored under identical conditions did not stick together. Electron microscopic examination showed that the experimental samples were made up of three zones consisting of fat, collagen and muscle tissue. The thickness of the middle (collagen) zone varied with the sex of the animals and possibly with their genetic background. Where the experimental roast slices adhered, they always did so only at the middle collagen layer. In contrast, control samples were made up of only two layers: fat and muscle, with the collagen layer between them being absent or much reduced in thickness.

Intracellular transport and posttranslational cleavage of oat globulin precursors.

The synthesis, transport, and posttranslational processing of reserve globulin in Avena sativa L. seeds were studied by pulse-chase labeling. Developing oat seeds were labeled with radioactive sulfate and tissue homogenates were used for globulin extraction.Two globulin precursors (58-62 kilodaltons) were labeled after 1 hour pulse. The alpha and beta globulin subunits appeared between 2 and 10 hours later, while simultaneously the 58 to 62 kilodaltons polypeptides gradually disappeared. This confirmed a precursor-product relationship. In a second pulse-chase experiment, the tissue extracts were fractionated on a sucrose gradient. The major portion of radioactivity was initially (1 hour pulse) associated with the endoplasmic reticulum. However, a significant amount of radioactivity shifted from the endoplasmic reticulum to protein bodies after 20 hours chase, suggesting the transport of the newly synthesized proteins. Protein bodies isolated from pulse-chased seeds were analyzed for the arrival of the newly synthesized globulin. Labeled precursors were detected after 2 hours chase and gradually disappeared. The alpha and beta subunits appeared during the same chase period and assembled into a 12S oligomer.The data indicated that oat globulin was synthesized as two large precursors which were transported from endoplasmic reticulum into protein bodies where they were processed to the alpha and beta subunits forming a 12S oligomer.

Nematospora sinecauda sp. nov., a yeast pathogen of mustard seeds.

An undescribed yeast species was recovered from oriental (Brassica juncea) and yellow (B. hirta) mustard seeds. The new species most closely resembled Nematospora coryli but its asci were rarely cylindrical. The asci and ascospores of N. sinecauda were smaller and the spores did not possess a whip-like appendage. During germination a sprout cell formed first on the smooth anterior surface of the spore above the median ridge. The posterior region of the spore was decorated with interrupted concentric ridges. A description of the new species is given.