Location of Calcium and Phosphorus in Ashed Spores of Bacillus megaterium, Determined by Electron Probe X‐Ray Microanalysis

The important roles played by metal ions in the resistance, dormancy and germination of bacterial spores are well established (1-3, 7, 12, 17). Ca is thought to be particularly important and is present in large quantities, probably in conjunction with dipicolinic acid (DPA), a compound specific to bacterial spores (11, 18). The location of Ca and other elements within the spore is of obvious importance. There have been suggestions that Ca is distributed in the coat, in the cortex, and in the core (4,6, 13, 15), but only recently definite evidence for its location has become available (9, 14). Electron probe X-ray microanalysis has enabled distribution of elements to be determined at high resolution directly in an electron microscope. Early applications of this technique by Scherrer and Gerhardt (13) and Ando (2) did not yield completely clear results because of low resolution. Recently, Stewart and his coworkers (14) analyzed cryosectioned spores of Bacillus cereus and showed that the bulk of Ca is located in the core. We (9) have examined B. megaterium spores which have been ashed and an analysis, in which a spot was placed over three different areas in the spore, also suggested that most of the Ca is located in the core, with some in the coat of strain QM BlSSl. We have now extended this analysis by determining the distribution across ashed spores by using line scans for Ca and P. Spores of B. megaterium QM BlSSl and ATCC 19213 were prepared and ashed in a low temperature asher (Yanagimoto: FDA-lS8), as described previously (9). Throughout this ashing procedure, the loss of P was less than 10% (9). Specimens were then examined at 75 kV in a Hitachi HU-12A electron microscope equipped with a Kevex Si(Li) energy dispersive X-ray spectrometer and a Kevex 7000 multichannel analyzer. Line scans for Ca and P were performed with windows of 140 eV centered on the K alpha lines of these elements. Detailed examination of the morphology of the ashed spores shows that they are surrounded by a light cortex region and an outer denser coat region (Figs. 1 and 2). As reported before (9), the coat of strain QM BlSSl is considerably darker