Two-Stage Enrichment Procedures for Isolating Listeria monocytogenes from Raw Milk

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Abstract
Recovery of two strains of Listeria monocytogenes (Scott A and V7) inoculated into raw milk, and of strains indigenous to milk was investigated. Isolation of the organisms from the milk was attempted using pre-enrichment broths [nutrient broth no. 2 (NB2) and tryptose broth (TB)] at 1:5 and 1:10 dilutions of milk. The broths were incubated at 4°C for 0, 7, 14, 21, and 28 d. Recoveries were compared by direct plating onto acriflavine-nalidixic acid agar (AN), McBride Listeria agar (MLA) and tryptose agar (TA), and after selective enrichment in thiocyanate-nalidixic acid broth, with and without acriflavine. Favorable recoveries were obtained using a two-stage protocol consisting of cold enrichment of the sample diluted 1:10 in TB, followed by plating 0.1 ml of this pre-enrichment to MLA, and transfer of 1 ml to 9 ml Thio-Nal-Acri broth. Selectively-enriched cultures streaked to MLA and TA yielded optimal isolations after 7–14 of cold enrichment.