Microbiology Methods for ERP Review 3-2020

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To determine the level of Salmonella in the test matrix, a 5-tube Most Probable Number (MPN) was conducted on the first day of analysis following the FDA/BAM Chapter 5 reference method. The MPN was determined by analyzing 5 x 50 g test portions, the reference method test portions from the collaborating laboratories and 5 x 10 g test portions. The MPN and 95% confidence intervals were calculated using the LCF MPN Calculator, Version 1.6, (www.lcfltd.com/customer/LCFMPNCalculator.exe), provided by AOAC Research Institute (RI) [7]. All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample container. All international test portions were shipped on a Monday while all domestic test portions were shipped on a Wednesday via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by the International Air Transport Association (IATA). Upon receipt, samples were held by the collaborating laboratory at ambient temperature (20-25 °C) until the following Monday when analysis was initiated after a total equilibration time of 2 weeks. A temperature probe was included in each shipment in order to track the temperature of the package during transit. Participants were instructed to obtain the temperature of their package upon receipt and document the results on the Sample Receipt Confirmation form provided and fax or email it back to the study director. Collaborators were instructed to follow the appropriate preparation and analysis as outlined in the study protocol. Two separate sets of 36 test portions (12 high, 12 low and 12 uninoculated controls for each method) were analyzed due to the unpaired study design. The Solus One test portions (375 g) were enriched with 1125 mL of pre-warmed (37 ± 2°C) Solus BPW (with the Solus selective supplement: 4.44 mL of supplement per Liter), homogenized for 2 ± 0.5 minutes and incubated for 21-22 hours at 41.5 ± 1°C. If the Solus One analysis was conducted manually or via automation (DS2 instrument), the exact same procedures are followed. Both the manual and automated extraction procedures have been included in the PTM evaluations of the Solus One Salmonella assay. Out of the 11 collaborators, 7 participants conducted analysis using the automated Dynex DS2 System and 4 participants conducted analysis following the manual assay procedure. Table 2 presents a summary of the collaborator participation along with extraction method utilized. Regardless of the presumptive result, all test portions were confirmed following the FDA/BAM Chapter 5 reference method beginning with a transfer to secondary enrichments (Tetrathionate broth (TT) and Rappaport Vassiliadis (RV) broth). The RV tubes were incubated at 42 ± 0.2°C for 24 ± 2 hours. Because the instant NFDM aerobic plate count (APC) results were <10 4 CFU/g, the Tetrathionate (TT) tubes were incubated at 35 ± 2 o C for 24 ± 2 hours. After incubation, secondary enrichments were streaked to selective agars Xylose Lysine Deoxycholate (XLD), Hektoen Enteric (HE), and Bismuth Sulfite (BS) and incubated at 35 ± 2 °C for 24 ± 2 hours. If no visible colonies were present after 24 hours of incubation on the BS plates, they were re- incubated for an additional 24 ± 2 hours at 35 ± 2°C. Typical isolated colonies from the selective agars were transferred to Triple Sugar Iron (TSI) agar and Lysine Iron Agar (LIA) and incubated at 35 ± 2 °C for 24 ± 2 hours. Typical Isolates were confirmed positive by serological confirmation (polyvalent O and H) and biochemical confirmation by the API 20 E (AOAC Official Method 978.24), VITEK 2 GN biochemical identification test (AOAC Official Method Test Portion Distribution Test Portion Analysis

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