AOAC RI ERP Final Action Recommendations eBook

Collaborative Study Approved Protocol Expert Review Panel Use Only

0 cfu /25 mL

12 12 12

Random number 200-299; 300-399

FDA BAM Ch. 5

0.2–2 cfu/25 mL

Orange Juice

S . Thompson (DD1336)

2–5 cfu/25 mL

1 2

*DD – DuPont Qualicon proprietary culture collection

4.0 3 4 To prepare the artificially contaminated matrix, primary samples will be inoculated with Salmonella so 5 that on the day of initiation of analysis there is a low level (approx. 0.2-2 cfu/test portion) and a high 6 level (approximately 2-5 cfu/test portion). Note that one level of contamination must result in fractional 7 positive results. One sample will remain uninoculated to serve as the uncontaminated control. The 8 organizing laboratory will prepare and ship the samples. Each sample will be divided into test portions, 9 randomized, and blind-coded for analysis. Test portions will be shipped to each collaborator via 10 overnight courier to arrive on a Friday. Containers will be packed with ice packs to maintain a 11 temperature of <7 ° C during transport. A temperature control sample will be included with each 12 shipment to verify temperature of sample upon receipt. Upon arrival, the test portions will be stored at 13 2-8 ° C until they are analyzed. All participating collaborators will begin processing and analysis on the 14 following Monday. Collaborators will complete and return the data sheets to the study director 15 following confirmation, within 2 weeks of the test initiation date (Appendix 10.2). Each matrix will be 16 analyzed in separate weeks. Sample Preparation and Distribution 4.1 Both matrices will be obtained from a local retail outlet and screened for the presence of Salmonella . Naturally contaminated test portions will be used if available at sufficient levels. Alternatively, the test portions will be artificially inoculated to meet the targets of 0.2-2.0 cfu/test portion for the low and 2 – 5 cfu/test portion for the high with the 4.2 For hot dogs, the inoculum will be prepared by transferring a pure isolated colony of the Salmonella Worthington from Trypticase Soy agar with 5% sheep’s blood (SBA) into Brain Heart Infusion broth (BHI) and incubating the BHI at 35 ± 2 o C for 18-24 ± hours. 4.2.1 Prior to inoculating the test material, the inoculum will be heat stressed in a appropriate concentration of a wet inoculum.

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

55°C water bath for 10-20 minutes to obtain a percent injury of 50-80%. The heat stressed culture will be plated onto a selective agar (Xylose Lysine Desoxycholate, XLD) and a non-selective agar (Tryptic Soy agar, TSA). The

n

x

select

1(

100 )

−

n

degree of injury will be estimated as of colonies on selective agar and n nonselect

, where n select

= number

nonselect

= number of colonies on non-selective

agar.

4.2.2 The heat stressed inoculum will be added drop-wise to a bulk quantity of hot dogs for each inoculation level. The bulk quantity will be mixed to achieve equal distribution of analyte throughout. Twenty-five (25) grams will be pulled from

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