Evaluation of Recovery Media for Heated Clostridium sporogenes Spores

Abstract

The efficiency of four culture media for recovery of heat-activated and heated Clostridium sporogenes spores was studied. Yeast extract agar gave the highest spore recovery. The effect of the method of preparing the yeast extract agar on the recovery of heated spores was also evaluated. The results indicate that (a) a significantly lower spore recovery was obtained when the dextrose was omitted completely or when added to the medium before autoclaving, and (b) no significant difference in spore recovery was found between yeast extract agar freshly made or prepared and stored at 4 C up to 11 days before use.

Importance of Considering Injured Microorganisms in Sterilization Validation

“…Injured spore formers have significant sensitivity to sodium chloride (Feehery et al, 1987), nitrate and nitrite (Chumney and Adams, 1980), surface-active agents including antibiotics (Chumney and Adams, 1980), pH (Feehery et al, 1987), water activity and osmolarity (Mattick et al, 2001), gas atmosphere (Feehery et al, 1987), culture media, media brand and their lots (Pflug et al, 1979;Pflug et al, 1981;Sasaki et al, 2000;Shintani and Akers, 2000;, and incubation temperature and period (Feehery et al, 1987;. The type and extent of these sensitivities vary with the species and the type of injury.…”

“…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).…”

“…Treatment of B. megaterium with chlorhexidine resulted in a sensitivity to potassium chloride during recovery in SCDA, but recovered in SCDB (Nadir and Gilbert, 1982). Recovery is influenced not only by the kind and constituents of the culture medium, but also by the condition of the medium (Pflug et al, 1981;Pflug et al, 1979), the quality of the constituents (Johnson and Busta, 1984;Sasaki et al, 2000), and the brand and the lot of the medium (Pflug et al, 1981;Pflug et al, 1979;Sasaki et al, 2000). Different lots of soybean casein digest, even from the same manufacturer, resulted in different rates of recovery of heated G. stearothermophilus spores (Pflug et al, 1981;Sasaki et al, 2000;Shintani and Akers, 2000;Shintani, 2006).…”

New Media for Enumeration and Detection of Clostricfium sporogenes (PA3679) Spores

“…Yeast extract agar (YEA; Pflug et al, 1979) was prepared by adding log of yeast extract, l.Og of soluble starch, 2g of K*HPO4 and 15g of Bacto-agar to 1000 ml of distilled water and autoclaving for 15 min at 121'C. Before pouring plates, the following components were aseptically added to 300 ml of tempered medium: 3.75 ml of a 10% sodium thioglycollate solution, 3.75 ml of a 40% dextrose solution and 7.5 ml of a 4% sodium bicarbonate solution.…”

“…The medium recommended to confirm NFPA's spore counts was Andersen's Pork Pea Infusion (APPI) using the Most Probable Number (MPN) technique (Andersen, 195 1;Bernard, 1983). As alternatives to APPI and the MPN method, several anaerobic plating media have been developed for enumeration of PA3679 spores (Wynne et al, 1955;Polvino and Bernard, 1982;Frank and Campbell, 1955;Hauschild and Hilsheimer, 1977;Pflug et al, 1979;Wheaton and Pratt, ,196l;Andersen, 1951;Odlaug and Pflug, 1977). Improvement in subculture methods and especially improvement in the subculture medium for recovering injured spores from inoculated pack tests would increase the accuracy of establishing a safe process.…”