AOAC RI ERP Final Action Recommendations eBook

Collaborative Study Approved Protocol Expert Review Panel Use Only

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this inoculated bulk lot and mixed with an additional 300g of uncontaminated test material to create the 325g test portions used in the analysis.

4.3 For orange juice, the inoculum will be prepared by transferring a pure isolated colony of the Salmonella Thompson from SBA into BHI broth containing 1% glucose and incubating the BHI at 35 ± 2 o C for 18-24 ± hours. The inoculum will be added drop-wise to a bulk quantity of orange juice for each inoculation level. The bulk quantity will be

mixed to achieve equal distribution of analyte throughout.

4.4 Test portions for both matrices will be stored at 2-8°C for 48-72 h prior to processing.

5.0 10 11 Each collaborator will received a complete set of test portions (uncontaminated, low, and high) for each 12 matrix, as indicated in 5.1 and 5.2, and will also receive one known uncontaminated sample for each 13 matrix to determine the aerobic plate count (6). All participating collaborators will initiate sample 14 analysis and aerobic plate count for each matrix on the same day. Analysis and Confirmation

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5.1

Hot Dogs – Paired

Each collaborator will receive one set of 36 – 325g test portions (12 uncontaminated, 12 low, 12 high), blind coded so that the contamination level is unknown to the collaborator. The BAX method will be compared to the USDA-FSIS MLG method for testing hot dogs. Both methods use the same enrichment, buffered peptone water (BPW). Approximately one third to one-half of 2925 ± 58.5 mL of sterile BPW will be added to each portion. Each portion will be homogenized approximately 2 minutes, after which the remainder of the 2925 ml of BPW will be added. Samples will be incubated at 35 ° C for 18-24 hours and tested by PCR according to the BAX® system procedure. After primary enrichment, 0.5 mL aliquots of each portion will be transferred to 10 mL TT (Hajna) broth, and 0.1 mL sample will be added to 10 mL modified Rappaport-Vassiliadis (mRV) broth. All secondary enrichments will be incubated at 42 ± 0.5°C for 22-24 h or in a water bath at 42 ± 0.5°C for 18-24 h. Secondary enrichments will be streaked to brilliant green sulfa (BGS) and either double modified lysine iron agar (DMLIA) or xylose lysine Tergitol TM 4 (XLT4) agar plates and incubated 35 ± 2°C for 18-24 h. Isolated colonies will transferred to triple sugar iron (TSI) agar and lysine iron agar (LIA) slants and incubated 35 ± 2°C for 22-26 h. Salmonella colonies will be confirmed using serological (Somatic O and poly H

agglutination) and biochemical procedures according to MLG Ch. 4.05.

5.2

Orange juice – Unpaired

Each collaborator will receive 2 sets of 36 – 25 mL test portions (12 uncontaminated, 12 low, 12 high). Each set will be blind coded so that the contamination level is unknown to the collaborator. The BAX method will be compared to the FDA BAM method for testing orange juice. One set of test portions will be enriched in BAX® MP broth (39-42°C for 22-26 h), for testing with the BAX system, while the second set of portions will be enriched in Universal

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