The Effect of Hydrogen Peroxide on Spores of Clostridium bifermentans

Abstract

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 "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. Preincubation of spores with Cu2+ before treatment with hydrogen peroxide produced a similar increase, but this could be eliminated by washing the spores with dilute acid or ethylenediamine tetraacetate. Hydrogen peroxide removed protein from 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. (1940) first showed that the rate of loss of viability of spores in the presence of hydrogen peroxide depends on temperature and pH. Copper and cobalt ions increase the lysis of spores by hydrogen peroxide (Gould & Hitchins, 1963) but their effect on spore viability and structure has not been examined. We have therefore studied the effect of pH, temperature and the concentration of hydrogen peroxide and metal ions on colony formation, germination and the structure of spores of Clostridium bifermentans. METHODSOrganism, spore preparation and maintenance of culture. The strain of Clostridium b fermentans used and the preparation and storage of spores were as described previously (Waites & Wyatt, 1971) except that spores were produced on a trypticase agar containing (g 1-l): Trypticase (BBL), 30 ; yeast extract (Difco), I '0 ; ammonium sulphate, 10 ; agar, I 2 ; adjusted to pH 7-3 with I M-NaOH. The organism was maintained in the reinforced clostridial medium of Hirsch & Grinsted (1954).Treatment of spores with hydrogen peroxide. Hydrogen peroxide (BDH) at the concentrations described was added to spores (about 0.7 mg dry wt ml-l) in sodium phosphate buffer (100 mM in phosphate at the stated pH) which had been pre-incubated at the required

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

“…We have shown previously that addition of 10 ,LAM-CU~+ caused a fivefold increase in the lethal effect of H202 on spores of Clostridium bifermentans and that addition of other divalent metal ions also slightly increased the kill (Bayliss & Waites, 1976). Preliminary results suggested that such ions do not markedly increase the effect of H20, on spores of other species.…”

“…7b). We have shown that the resistance of spores of C. bifermentans to H20, was only slightly decreased by the presence of Fe2+ (Bayliss & Waites, 1976). It is probable, therefore, that only those metal ions like Cu2+ which bind to protoplasts are able to decrease spore resistance.…”

“…We have shown that Cu2+ markedly increased the lethal effect of H20, (Bayliss & Waites, 1976) and of heat on spores of C. bfermentans but not on those of the other species examined, suggesting that certain sites in spores of C. bfermentans must be especially sensitive and/or accessible to Cu2+. Spores of B. subtih and C. bifermentans took up Cu2+ at about the same rate but examination of unstained sections of spores by electron microscopy suggested that Cu2+ was bound by the protoplasts of spores of C. biferrnentans but not those of spores of B. subtilis.…”

“…Examination of electron micrographs of thin sections of spores of C. b$iwnentuns suggested that Cu2+ did not bind to the cortices. Beveridge & Murray (1976) found that the ability of Fez+ and Cu2+ to stain isolated walls of vegetative cells of B. subtilis was diminished after digestion by lysozyme, suggesting that peptidoglycan in the wall bound both Fe2+ and Cu2+. The peptidoglycan of the spore cortex is structurally similar, although not identical, to that of walls of vegetative cells (Tipper & Gauthier, 1972) so that the cortex might be expected to bind Cu2+.…”

“…bifermentans was measured spectrophotometrically with the 'Ala + ' germination system (Bayliss & Waites, 1976) as described by Waites & Wyatt (1971) or, after treatment with urea/mercaptoethanol, with lysozyme by counting 100 spores using a phase-contrast microscope and scoring dark (germinated) and bright (ungerminated) spores. Outgrowth of C. bifermentans was studied at 37 "C in medium containing casein hydrolysate flushed with H,/CO, (9: 1, v/v) as described by .…”