Modified methods for the enumeration of spores of mesophilic Clostridium species in dried foods

Abstract

Clostridia constitute markers of limited though definite importance for the microbiological integrity of particular foods processed for safety, provided their application and the results obtained are meticulously considered and guided by proper ecological awareness. Their selective diagnostic enumeration in food specimens relies on their ability to reduce sulphite in agar media, visualised by the presence of ferrous cations leading to the production of black colonies. The composition of the medium used substantially affects the productivity of the procedure. We established that (1) the sulphite activity and the ferrous ion should be rigorously standardised; (2) tryptose is one of the appropriate nitrogen sources for a limited number of clostridia; (3) the basal medium should be free of added acetate and lactate. Black colonies obtained in the newly elaborated medium, termed Differential clostridial agar (DCA) should be further examined for morphology and metronidazole sensitivity, since some bacilli might mimic clostridia under the conditions of the procedure. An elegant variant of the technique relies on using a bottom-layer of mannitol/egg yolk/polymyxin/bromocresol purple agar, inoculated with macerates of food in buffered cysteine hydrochloride peptone saline, immediately liberally overlayered with freshly prepared DCA. Plates are incubated and read in tightly closed bags of plastic with a low oxygen permeability coefficient, which eliminates the need for using anaerobic jars. Colony identification is relying on assessment of sulphite reduction, egg yolk dissimilation, the mode of attack on mannitol and when required to be supported by classical other physiological traits. The mandatory precautions to be observed in this procedure call for extreme caution when introducing reference ranges (“standards”) for clostridial spores in foods, particularly in the international commerce.