AOAC-RI ERP Book Micro Jan 19 2017

For the raw spinach matrix, categorized as ‘Leafy Produce’, the 200 g sample was combined with 450 mL of pre-heated (37°C) mBPWp and placed at 37°C + 1°C for 5 h to incubate. No stomaching or blending was performed. Following incubation, 2 mL of ACV supplement was added (1 mL per 225 mL mBPWp) and incubated at 42°C + 1°C for 18-24 h. For the raw sprout matrix, categorized as ‘Cilantro or P arsley’, the 25 g sample was combined with 225 mL of mBPWp and placed at 37°C + 1°C for 5 h to incubate. No stomaching or blending was performed. Following incubation, 1 mL of ACV supplement was added and incubated at 42°C + 1°C for 18-24 h. Following overnight enrichments, the sample was serially diluted in BPB. Appropriate dilutions were plated, in duplicate, onto Tellurite Cefixime-Sorbitol MacConkey Agar (TC-SMAC, Becton, Dickinson and Company, Franklin Lakes, NJ) along with one mRBA plate. Plates were incubated at 37°C + 1°C for 18-24 h. Typical colony morphology was used to identify probable positive for O157. Typical colonies were picked and tested for latex agglutination (Remel kit) and streaked onto TSA + Yeast Extract (TSAYE) with a ColiComplete disc placed in the heaviest streak area. Plates were incubated at 37°C + 1°C for 18-24 h. Isolates were confirmed with commercial antisera (RIM® E. coli O157:H7 Latex Test or equivalent test) followed by VITEK for E. coli identification. 2.5 Analysis of Candidate Method Sub-Portions After the 48 h stabilization period post-inoculation, the samples were subjected to the following enrichment procedures and testing on the MDA. 3M provided all 3M consumable supplies involved with the MDA test. 3M Buffered Peptone Water (BPW) was pre-heated to 41.5°C + 1°C prior to enriching all matrices. For the raw sprouts, the 25 g sample was added to 225 mL 3M BPW and immediately incubated at 41.5°C + 1°C for 18 h. For the raw spinach, to each 200 g sample, 450 mL of pre-warmed BPW was added and immediately incubated at 41.5°C + 1°C for 18 h. The sprout and spinach samples were not homogenized or stomached during preparation. The enriched samples were then subject to analysis as outlined by the instructions for use for the 3M TM Molecular Detection Assay 2 – E. coli O157 test kit. The sample results obtained after completing the MDA wer e considered the ‘screening stage’ result for the candidate method. Concurrent with the transfer and testing on the MDA, aliquots of the enriched samples were transferred and subjected to the testing outlined in Section 2.4 for cultural confirmation. The results obtained after completing this methodology were considered the ‘confirmation stage’ result for the candidate method. 2.6 Most Probable Number Analysis As mentioned above in Section 2.3, samples for each portion, for each food matrix were inoculated with E. coli O157:H7 (i.e. Portion 1, and Portion 2; sprouts, spinach) and used for performing a five tube-three level MPN analysis to determine the concentration of E. coli O157 inoculated into the portion and matrix. This

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