Development of a protocol for the isolation of Listeria monocytogenes from sludge

Abstract

Given the high level of background flora in sludge, methods for detecting Listeria monocytogenes are not well established. In this study, two critical parameters for the detection of L. monocytogenes were evaluated: the concentration of Listeria sp. in a modified Fraser broth (first stage of the method) and the proportion of L. monocytogenes on Palcam agar (second stage of the method). Concentrations of Listeria sp. estimated in 118 modified Fraser enrichment broths inoculated with four types of sludge, reached 10(4) bacteria per mL for 83% of the positive enrichment broths. Proportion of L. monocytogenes on Palcam agar, which was estimated by transferring all characteristic colonies of Listeria sp. onto Rapid'L Mono agar, was highly variable regardless of the type of sludge. According to these results, we proposed a protocol that consisted of an enrichment in modified Fraser broth for 48 h at 37 degrees C, followed by plating 0.1 mL of appropriate dilutions of broth onto Palcam agar. After an incubation of 48 h at 37 degrees C, a systematic identification of characteristic colonies of Listeria sp. on Rapid'L Mono agar allowed to enhance the detection of Listeria monocytogenes.