Validation of NMKL method No. 136--Listeria monocytogenes, detection and enumeration in foods and feed

A collaborative study was organised to define the performance characteristics of the revised NMKL Method No.136 "Listeria monocytogenes. Detection and enumeration in foods". Chromogenic L. monocytogenes specific plating medium, Agar Listeria according to Ottaviani and Agosti (ALOA) was introduced in the revised method in order to improve the sensitivity and specificity of the method, and to shorten the analysis time. Efficacy of ALOA One Day from AES (ready-to-use agar in bottles), Listeria Chromogenic Agar (Agosti and Ottaviani Listeria agar) from Lab M (LCA) (dehydrated powder), Chromogenic Listeria Agar Plates from Oxoid (OCLA) (ready-to-use plates) and L. monocytogenes blood agar medium LMBA from Lab M (dehydrated powder) were tested. Three types of food matrices (vacuum-packed hot-smoked salmon, soft cheese and cooked ham) and one feed matrix (wheat grain) inoculated with two levels of L. monocytogenes with or without L. innocua were used in the study. A total of 24 samples were analysed both in the detection and enumeration part of the study by 18 and 17 Nordic laboratories, respectively. The sensitivities of ALOA, LCA, OCLA and LMBA in the detection of L. monocytogenes in food samples after one-step enrichment (Half-Fraser) were 94.4-96.4% and after two-step enrichment (Half-Fraser followed by Fraser) 97.7-100%. For wheat grain the respective figures were 84.7-88.9% and 90.3-93.1%, respectively. The precision characteristics were generally good for the enumeration of L. monocytogenes in the food samples with high levels of inoculation. Several poor values obtained from the food samples with low levels of inoculation probably reflect high uncertainty of measurement when less than 10 cfu/g was counted. Poor values obtained from the wheat grain samples by any of the media evaluated were due to poor precision for feed samples. According to the study, the revised NMKL Method No.136, 4th ed. showed excellent results in the detection and satisfactory results in the enumeration of L. monocytogenes in foods. The results for the detection of L.monocytogenes in wheat grain were good, but the method cannot be recommended for the enumeration of L. monocytogenes in feed-stuffs. Any one of the media evaluated can interchangeably be used as an obligatory isolation medium for the detection and enumeration of L. monocytogenes in foods, and for the detection in feed-stuffs. The L. monocytogenes specific plating media that were evaluated shorten the time of analysis and significantly reduce the work load. The detection of positive samples mostly after Half-Fraser enrichment, reduces the analysis time further, and makes it possible to skip the secondary enrichment. However, secondary enrichment cannot be totally left out, because samples with low levels of L. monocytogenes, with high levels of competing flora, and with injured L. monocytogenes, do need secondary enrichment.