Characterization of Ustilago HordeiFimbriae Using Scanning Transmission Electron Microscopy and Immunocytochemistry

Sporidia of Ustilago hordeiproduce surface fimbriae which are important in conjugation and pathogenicity. This work focuses on fimbrial origin and production using transmission electron microscopy (TEM) and immunocytochemistry.Wild type I4A sporidial cells cultured to log phase with rotary shaking in yeast extract glucose (YEG) growth for 48 h. at 21° C, were harvested by centrifugation at 8000 rpm, placed on formvar coated grids, negatively stained with 2% uranyl acetate and photographed in the JEOL 1200 STEM. Some cells were prepared for sectioning by fixation with gluteraldehyde and cacodylate buffer, post fixed in osmium tetroxide, dehydrated and embedded in epoxy and stained with uranyl acetate. The remainder of the cells were sheared in a blender to remove fimbriae. The defimbriated cells and 1 ml. of fimbrial suspension were presented to TEM. The rest of the fimbrial suspension was centrifuged at 30,000 rpm and he fimbrial pellet protein concentration was determined to be 1.345 nm. as assayed by UV adsorption.