Distribution ofCalcium andOtherElements inCryosectioned Bacillus cereusT Spores, Determined byHigh-Resolution Scanning Electron ProbeX-RayMicroanalysis

Thedistribution ofa numberofkeyelements inBacillus cereusT sporeswas determined byhigh-resolution scanning electron probeX-raymicroanalysis. To circumvent theredistribution ofsoluble orweakly boundelements, freeze-dried cryosections ofspores,whichhadbeenrapidly frozen in50%aqueous polyvinyl pyrrolidone, were employed. Thesections were examined byusing a modified Philips EM400electron microscope fitted witha field emission gun, scanning transmission electron microscopy attachment, anda computer-linked energy- dispersive X-raymicroanalysis system. X-ray maps forselected elements andthe corresponding electron image were produced simultaneously byscanning the cryosections witha fineelectron beamina raster pattern, using thescanning transmission electron microscopy attachment. Theresults indicated thatalmost allofthecalcium, magnesium, andmanganese, together withmostofthephos- phorus, was located inthecoreregion. Anunexpectedly highconcentration of silicon was foundinthecortex/coat layer. Granules containing highconcentra- tions ofcalcium, manganese,andphosphorus were demonstrated insporescon- taining reduced levels ofdipicolinic acid. Spotmodeanalyses, inwhich astation- ary beamwas located over theregion ofinterest inthespore cryosection, confirmed theresults obtained withthescanning modeandalso provided a more accurate quantitation oftheelemental concentrations on a dryweight basis. Bacterial spores showremarkable physiologi- calproperties, inparticular aresistance toheat, radiation, enzymes, disinfectants, andother nor- mally deleterious agents (30). Furthermore, they havenodetectable metabolism whendormant andyetareabletogerminate inamatterof minutes andultimately formvegetative cells. Spores ofallspecies havethesamegeneral morphology, consisting ofaninner core(proto- plast) surrounded by,inturn, agermcell wall, cortex, coats, and,insomespecies, anexospo- rium. Theymay,however, differ dramatically in their resistance andstability. Spores also have adistinctive composition whichisquite unlike that ofvegetative cells, particularly withrespect tolowconcentrations ofsodiumandchlorine andgreatly elevated concentrations of3-phos- phoglyceric acid, dipicolinic acid, calcium, and manganese (19, 30). Thehighconcentration of divalent cations isaconsequence oftheir rapid uptake instage IVofsporulation (33) andmay ultimately comprise upto3%ofthedryweight ofthespore (22). Thesecations arerapidly re- leased during germination andareassociated withspore dormancy andresistance. Theratio oftheconcentration ofcalcium ionstomagne- siumions andthedipicolinic acidconcentration