Microbiology Methods for ERP Review 3-2020

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Following incubation, a 20 μ L aliquot of each sample was lysed for 5 min at >2000 rpm. Lysed samples were transferred to PCR reaction tubes and assayed on the GENE-UP ® thermocycler. Regardless of presumptive results, all test portions were confirmed following two procedures: a traditional confirmation following the MLG 4.10 reference method beginning with a transfer to secondary enrichments and an alternative confirmation procedure by directly streaking the primary enrichment broth to ASAP and CHROMID Salmonella and incubating at 35 ± 1°C for 24 ± 2 h. Isolated colonies from both the traditional and alternative confirmation procedures were confirmed following the procedures outline in MLG 4.10 with biochemical confirmation conducted using VITEK 2 GN (OMA 2011.17) or API 20E (OMA 978.24). For test portions evaluated by the reference method, 25 g samples were enriched in 75 mL mTSB and incubated for 15–24 h at 42 ± 1°C. After incubation, an aliquot of the mTSB was transferred to secondary enrichments (Tetrathionate broth, Hajna, TT and Rappaport-Vassiliadis medium with soya, RVS). Secondary enrichment tubes were incubated at 42 ± 0.5 o C for 22-24 h in an incubator or for 18-24 h in a circulating water bath. After incubation, secondary enrichments were struck to selective agars (Brilliant green sulfa agar, BGS and double modified lysine iron agar, DMLIA) and incubated at 35 ± 1°C for 18-24 h. Regardless of the presence of typical colonies, all plates were reincubated for an additional 18-24 h. Isolated colonies from the selective agars were transferred to triple sugar iron agar (TSI) and lysine iron agar (LIA) slants and incubated at 35 ± 1°C for 24 ± 2 h. Final biochemical confirmation was conducted using VITEK 2 GN or API 20E.

Statistical Analysis

Data for each contamination level was analyzed using the probability of detection (POD) statistical model (10) and conducted using the AOAC Micro Stats Workbook V1.0. The POD

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