SUMMARYSpores 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.

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A gas-vacuolate strain of Methanosarcina barkeri formed protoplasts in substrate-depleted cultures and gas vesicles were isolated from the protoplasts. Vacuolate protoplasts were separated from unvacuolate ones by flotation and the protoplast membrane was removed by Tween 20, liberating the gas vesicles. The gas vesicles were purified by flotation after initial passage through a 0.45 pm filter to remove contaminating material. Gas vesicle membranes were purified by isopycnic gradient centrifugation and were shown by electron microscopy to have a rib spacing of 4.8 nm.

Exosporia surrounding spores of a pigmented Clostridium showed a hexagonal periodicity when negatively stained and were composed of up to 10 lamellae, each 2.6 nm thick, with a centre-to-centre spacing of 5.2 nm. Optical diffraction spectra revealed that isolated fragments of single lainellae contained hexagonal arrays of subunits with a predominant spacing of 5.2 nm and that negatively stained fragments composed of several lamellae showed interference patterns generated by slightly displaced layers of the same hexagonal lattice.

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