Changes in spores of Clostridium bifermentans caused by treatment with hydrogen peroxide and cations

Spores of Clostridium bifermentans were treated with hydrogen peroxide until their peripheries had lost refractility. The centres of such spores only retained refractility at acid pH. Adding monovalent cations or increasing the pH caused the treated spores to lose their remaining refractility and decreased the turbidity of spore suspensions. Divalent cations prevented or reversed this loss of central refractility and decreased the fall in turbidity. Calcium ions also prevented but did not reverse the loss of central refractility which occurred on drying or applying pressure. Electron micrographs of spores treated with hydrogen peroxide showed that the cortex was depleted or absent and that the loss of central refractility was accompanied by protoplast swelling. It is suggested that divalent cations make spores resistant to drying and pressure by cross-linking negatively charged groups within the protoplast, and that together with hydrogen ions they neutralize the negatively charged groups, thus preventing the swelling of the protoplast, loss of refractility and fall in extinction which occur when divalent cations are replaced by monovalent cations.