Comparison of four routine methods for the confirmation of Clostridium perfringens in food

Abstract

In order to compare methods for the confirmation of C. perfringens 52 isolates from strain collections and food samples were examined using four different techniques including the detection of lactose degradation and sulfite reduction in lactose sulfite medium (European Standard EN 13401), examination of nitrate reduction, motility, lactose degradation and gelatine liquefaction in motility–nitrate and lactose–gelatine medium (EN 13401), the reverse CAMP test (German Standard DIN 10103) and the acid phosphatase reaction according to Ueno et al. [Ueno, K., Fujii, H., Marui, T., Takahshi, J., Sugitani, T., Ushijima, T., Suzuki, S. 1970. Acid phosphatase in Clostridium perfringens — A new rapid and simple identification method. Jpn. J. Microbiol. 14, 171–173]. Pure cultures of the test strains were suspended in sterile physiological saline and decimal dilutions inoculated into modified SC agar according to Hauschild and Hilsheimer [Hauschild, A.H.W., Hilsheimer, R., 1974. Evaluation and modifications of media for enumeration of Clostridium perfringens. Appl. Microbiol. 27, 521–526] and Eisgruber and Reuter [Eisgruber, H., Reuter, G., 1995. A selective medium for the detection and enumeration of mesophilic sulphite-reducing clostridia in food monitoring programs. Food Res. Internat. 28, 219–226] by pour-plating. Five individual colonies per test strain (in total 260 colonies) were examined. Using either the reverse CAMP test or the acid phosphatase reaction 94.2% of the colonies were confirmed as C. perfringens. The detection of nitratase and motility in combination with lactose and gelatine degradation enabled the identification of C. perfringens in 88.8% of the colonies. The lowest percentage of C. perfringens colonies was detected via lactose sulfite medium: only 42.7% of the tested colonies showed typical sulfite reduction with gas formation from lactose.