Location of calcium within Bacillus spores by electron probe x-ray microanalysis

Spectroscopic microanalysis of the element-characteristic X rays produced by a scanning electron microprobe was employed to detect calcium and carbon in both intact and thin-sectioned spores of Bacillus cereus T and B. megaterium QM B1551. Linear scan profiles and multilinear scan images of the X-ray emissions for calcium (Ca(Kalpha)) were compared with those for carbon (C(Kalpha)) as an index of mass. Location was accomplished by stereological comparisons with secondary electron images and conventional transmission electron micrographs. Although the elements could be detected at the attogram level theoretically, spatial resolution was limited to approximately 500 to 1,000 nm in an intact spore, e.g., by the primary electron beam diameter, the electron-excited spore microvolume, and the type of specimen support. The resolution was improved to approximately 100 to 200 nm by use of thin-sectioned spores, with precautions to prevent calcium leakage from the specimen during preparations. In both intact and sectioned spores, calcium was distributed throughout the spore, similarly to carbon, and concentrated mainly in a central region corresponding to the spore protoplast.