Influence of Media and Media Constituents on Recovery of Bacterial Spores Exposed to Hydrogen Peroxide

Abstract

The effects of different media, length of time of incubation, media constituents and pH on recovery of spores of Bacillus subtilis var. niger exposed to hydrogen peroxide were examined. As the time of exposure of spores to hydrogen peroxide increased, the incubation time req'uired for colony formation increased. A medium consisting of 7.5 g/L of yeast extract, 2.5g of glucose/L, 6.Og of vitamin-free casamino acids/L, 15.Og of agar/L, O.lg of ferrous sulfate/L, and O.lg of manganous sulfate/L gave the best recovery. The presence of glucose and yeast extract in the medium was important Autoclaving of ferrous sulfate and manganous sulfate apart from the rest of the medium improved recovery and allowed for pH adjustment of the medium before autoclaving. Maximal recovery occurred between pH 6.8 and 7.0. The results of these experiments indicate that the sporitidal action of hydrogen peroxide may not be as great as previously observed and that careful attention should be paid to the procedures used to evaluate the safety of food processes that utilize hydrogen peroxide as a bactericide.

Importance of Considering Injured Microorganisms in Sterilization Validation

“…Injury towards bacterial spores is expressed in various forms. There is a need for nonnutrient germination stimulants by the injured spores, modified optimum incubation temperatures for the enumeration of survivors, an increased sensitivity of the survivors to inhibitors and selective agents, and altered nutritional requirements by the survivors (Adams, 1978 (Gould, 1984), ethylene oxide Richardson, 1970a, b, Davis et al, 1978), hydrogen peroxide (Wallen and Walker, 1979), hypochlorite (Foegeding and Busta , 1983a,b), alcohols (Craven and Blankenship, 1985) and/or nitrite (Gould, 1984). Hypochlorite injures bacterial spores such as Clostridium botulinum (C. botulinum) (Foegeding and Busta, 1983a, b).…”

“…Hypochlorite injures bacterial spores such as Clostridium botulinum (C. botulinum) (Foegeding and Busta, 1983a, b). Hydrogen peroxide has injured spores of Bacillus atroaeus (B. atrophaeus) (Wallen and Walker, 1979), B. cereus and C. sporogenes (Neal and Walker, 1977). Ethylene oxide injures bacterial spores (Roberts, 1970).…”

“…The culture medium used to detect injured spore formers will influence their recovery (Johnson and Busta, 1984;Pflug et al, 1979;Wallen and Walker, 1979;Sasaki et al, 2000;Shintani and Akers, 2000;Shintani, 2006). Several media have been used for the recovery of injured spores (Table 4).…”

“…The recovery of hydrogen peroxide-damaged B. atrophaeus spore was improved with yeast extract, glucose, and casamino acids (Johnson and Busta, 1984;Wallen and Walker, 1979). In every case of injury, addition of glucose, pyruvate, alanine and divalent cations (Ca and Mg) will cause significant recovery (Shintani, 2006).…”

“…Thus, the use of cysteine as a superior reducing agent, which is noninhibitory to clostridia, has been recommended (Shoesmith and Worsley, 1984 the incubation period has not been prolonged (Lynt et al, 1983). Longer incubation period has been required for heat, irradiation, and chemically injured spores (Futter and Richardson, 1970a;Chowdhury et al, 1976;Wallen and Walker, 1979;Neal and Walker, 1979). In some instances, repair may be accomplished during storage at refrigeration temperature (Edwards et al, 1965b).…”