AOAC-RI ERP Book Micro Jan 19 2017

OMAMAN-35 A : Collaborative Study Manuscript For ERP Use Only January 2017

A detailed collaborative study packet outlining all necessary information related to the study including media 1 preparation, test portion preparation and documentation of results was sent to each collaborating laboratory prior to 2 the initiation of the study. A conference call was then conducted to discuss the details of the collaborative study 3 packet and answer any questions from the participating laboratories. 3M Food Safety Technical Service provided 4 on-site hands on training to 12 of the 15 participating laboratories prior to the start of the collaborative study. 7 8 The E. coli O157:H7cultureATCC43895 used in this evaluation were propagated onto Tryptic Soy Agar (TSA) 9 from a Vanguard Sciences(collaborating laboratory) frozen stock culture stored at -70°C. The organism was 0 incubated for 24 ± 2 hours at 35 ±1°C. Isolated colonies were picked to 10 mL of Brain Heart Infusion (BHI) 1 broth and incubated for 18 ± 0.5 hours at 35 ±1°C. The cultures were stored at 2-8°C for 24 h while the inoculum 2 level was determined. Appropriate dilutions of each culture were prepared in Butterfield’s Phosphate Diluent 3 (BPD) for both low and high inoculation levels. Bulk portions of each matrix were inoculated with the diluted 4 liquid inoculum and mixed thoroughly to ensure an even distribution of microorganisms. For the analysis of the 5 raw ground beef, 25 g of inoculated test product was mixed with 300 g of un-inoculated test product to prepare 325 6 g test portions which were packaged in sterile Smasher ® XLbags and shipped to collaborators. 7 A most probable number (MPN) was conducted by the coordinating laboratory on the day of the initiation of 8 analysis using the USDA/FSIS-MLG 5.09 reference method for raw ground beef.The MPN was determined by 9 analyzing 5 x 650 g test portions,thereference method test portions from the collaborating laboratories and 5 x 160 0 g test portions by the USDA/FSIS-MLG 5.09 reference method for the low level inoculated samples and the 1 reference method test portions from the collaborating laboratories, 5 x 160 g, and 5 x 80 g test portions for the high 2 samples. The MPN and 95% confidence intervals were calculated using the LCF MPN Calculator, Version 1.6, 3 ( www.lcftld.com/customer/LCFMPNCalculator.exe ), provided by AOAC RI [6]. Confirmation of the samples was 4 conducted according to the USDA/FSIS-MLG 5.09 reference method. 7 8 All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample container. Test 9 portions were shipped on a Thursday via overnight delivery according to the Category B Dangerous Goods 0 shipment regulations set forth by the International Air Transportations Association(IATA). All raw ground beef 1 samples were packed with cold packs to target a temperature of < 7°C during shipment.Upon receipt, raw ground 2 beef samples were held by the collaborating laboratory at refrigeration temperature (2-8 °C) until the following 3 Monday when analysis was initiatedafter a total equilibration time of 96 hours. 4 In addition to each of the test portions and a separate APCsample, collaborators received a test portion for each 5 matrix labeled as ‘temperature control’.Participants were instructed to measurethe temperature of this portion upon 6 receipt of the package, record the results on the Sample Receipt Confirmation form provided and send the filled 7 form back to the study directorby fax or email. 0 1 Collaborators were instructed to follow the appropriate preparation and analysis protocol for both the 3M MDA 2 – 2 E. coli O157 (including H7) method and reference methods. Each collaborator received 72 test portions (12 high, 3 12 low and 12 un-inoculated controls) for each method to be performed. For the analysis of the raw ground beef 4 test portions by the 3M MDA 2 – E. coli O157 (including H7) method, a 325 g portion was enriched with 975 mL 5 of pre-warmed (41.5 ±1 o C) BPW ISO, manually homogenized by hand for 2 minutes and incubated for 10 hours at 6 41.5 ±1 o C. 7 Following enrichment, samples were assayed by the 3M MDA 2 – E. coli O157 (including H7) method and, 8 regardless of presumptive result, confirmed following the appropriate reference method. The matrix evaluated by 9 AOAC Research Institute Expert Review Panel Use Only 5 6 Preparation of Inocula and Test Portions 5 6 Test Portion Distribution 8 9 Test Portion Analysis

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