W. M. Waites

The Effect of Hydrogen Peroxide on Spores of Clostridium bifermentans

The effect of hydrogen peroxide on the germination, colony formation and structure of spores of Clostridium bifermentans was examined. Treatment with 0.35 M-hydrogen peroxide increased the germination rate at 25 degrees C but increasing the temperature or concentration of hydrogen peroxide decreased both the germination rate and colony formation. The presence of Cu2+ increased the lethal effect of hydrogen peroxide on colony formation as much as 3000-fold. Pre-incubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute spores--apparently from the coat--and treatment with dithiothreitol, which also removes spore-coat protein, increased the lethal effect of hydrogen peroxide 500-fold, suggesting that spore-coat protein has a protective effect against hydrogen peroxide.

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.

The Effect of Transition Metal Ions on the Resistance of Bacterial Spores to Hydrogen Peroxide and to Heat

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.

The Effect of Sporulation Medium on Spores of Clostridium bifermentans

SUMMARY: Spores of Clostridium bifermentans produced on a Trypticase medium (TRY) and on reinforced clostridial medium (RCM) were compared. RCM-spores contained less dipicolinic acid, outgrew more slowly, were less resistant to hydrogen peroxide and to heat and lost more dipicolinic acid on heating. After treatment with urea/mercaptoethanol both TRY - and RCM-spores germinated with lysozyme, although heating RCM- but not TRY-spores before urea/mercaptoethanol treatment decreased their subsequent germination rate. In thin-section electron micrographs, TRY-spores showed thicker cortices and smaller protoplasts with less visible ribosomes than RCM-spores. The sporulation medium evidently has a marked effect on spore properties. Resistance to heat and to hydrogen peroxide appears to be related to spore structure.

The effect of chlorine and heat on spores of Clostridium bifermentans.

Waites et al. (1976) showed that spores of Clostridium bifermentans, which had lost peripheral phase brightness after being killed by incubation with high concentrations of hydrogen peroxide, had also lost most of their cortex, calcium and dipicolinic acid (DPA). Addition of monovalent cations to such spores resulted in a fall in turbidity, loss of the remaining phase brightness and swelling of the protoplasts although these changes could be prevented by the presence of calcium ions. Since monovalent cations are required for the germination of untreated spores and since the loss of calcium, DPA and cortex together with protoplast swelling also occur during germination (for a review see Gould, 1969), we suggested that replacing the calcium within the spore by monovalent cations could be one of the initiating events in germination. It is possible, however, that the changes in spores treated with peroxide might be specific to the action of the peroxide. We have therefore investigated whether other agents including chlorine and heat can produce spores which undergo similar changes. This communication reports the effect of these agents in combination.

Microelectrophoretic Studies of Coat-defective Spores of Bacillus megaterium

SUMMARY: Microelectrophoretic studies of lysozyme-resistant spores of Bacillus megaterium QM B1551 suggested that carboxyl groups were the only ionized species on the spore surface. Spores of B. megaterium NCIB 8291 have defective coats, allowing lysozyme to attack the underlying cortical peptidoglycan and initiate germination-like changes. The surface of such spores was characterized by the presence of ionized carboxyl and amino groups, suggesting that the amino groups were present on the cortical surface. Spores of B. megaterium QM B1551 rendered defective by extraction of coat protein with sodium dodecyl sulphate and dithiothreitol at pH 10 were also lysozyme sensitive and had similar electrophoretic behaviour to the naturally coat-defective spores. Since the electrophoretic behaviour of coat defective spores is qualitatively similar to that of germinated spores, holes or channels may appear in the spore coat during the early stages of germination, exposing the cortical peptidoglycan.