AOAC-RI ERP Micro June 2016

OMAMAN-30 A/ Collaborative Study Manuscript OMA ERP June 2016 ERP Use Only

conducted to discuss the details of the collaborative study packet and answer any questions from 1 the participating laboratories. 4 5 The Listeria monocytogenes cultures used in this evaluation were propagated onto Tryptic Soy 6 Agar with 5% Sheep Blood (SBA) from a Q Laboratories frozen stock culture stored at -70°C. 7 Each organism was incubated for 24 ± 2 hours at 35 ±1°C. Isolated colonies were picked to 10 8 mL of Brain Heart Infusion (BHI) broth and incubated for 18 ± 0.5 hours at 35 ±1°C. Raw 9 chicken breast fillet was inoculated in bulk using the fresh, broth culture. Prior to inoculation of 10 the deli turkey, the culture suspension was heat stressed at 55 ± 1 o C in a water bath for 15 11 ±0.5minutes to obtain a percent injury of 50-80% (as determined by plating onto selective 12 Modified Oxford agar (MOX) and non-selective tryptic soy agar with yeast (TSA/ye). The 13 degree of injury was estimated as: 14 16 selective agar. Appropriate dilutions of each culture were prepared in Butterfield’s Phosphate 17 Diluent (BPD) based on previously established growth curves for both low and high inoculation 18 levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed 19 thoroughly to ensure an even distribution of microorganisms. The inoculated raw chicken 20 breastfillet was packaged into separate 30 g test portions in sterile Whirl-Pak ® bags and shipped 21 to the collaborators. For the analysis of the deli turkey, 25 g of inoculated test product was 22 mixed with 100 g of un-inoculated test product to prepare 125 g test portions which were 23 packaged in sterile Whirl-Pak ® bags and shipped to collaborators. 24 To determine the level of Listeria monocytogenes in the matrices, a 5-tube most probable 25 number (MPN) was conducted by the coordinating laboratory on the day of the initiation of 26 analysis using the USDA/FSIS-MLG 8.09 reference method. For deli turkey, the MPN was 27 determined by analyzing 5 x 250 g test portions,thereference method test portions from the 28 collaborating laboratories and 5 x 65 g test portions. For the raw chicken breast fillet, the MPN 29 of the high and low inoculated levels was determined by analyzing 5 x 50 g test portions, the 30 reference method test portions from the collaborating laboratories and 5 x 10 g test portions. The 31 MPN and 95% confidence intervals were calculated using the LCF MPN Calculator, Version 1.6, 32 ( www.lcftld.com/customer/LCFMPNCaclucator.exe ), provided by AOAC Research Institute 33 (RI) [7]. 36 37 All samples were labeled with a randomized, blind-coded 3-digit number affixed to the sample 38 container. Test portions were shipped on a Thursday via overnight delivery according to the 39 Category B Dangerous Goods shipment regulations set forth by the International Air 40 Transportations Association(IATA).The two matrices were shipped consecutively, with 41 collaborating laboratories performing analysis on one matrix at a time. Upon receipt, samples 42 were held by the collaborating laboratory at refrigeration temperature (2-8 °C) until the 43 following Monday when analysis was initiatedafter a total equilibration time of 96 hours.All 44 samples were packed with cold packs to target a temperature of < 7°C during shipment. 45 In addition to each of the test portions and a separate APCsample, collaborators received a test 46 portion for each matrix labeled as ‘temperature control’. Participants were instructed to obtain 47 the temperature of this portion upon receipt of the package, document the results on the Sample 48 100 ) 1( x n n nonselect select − AOAC Research Institute Expert Review Panel Use Only 2 3 Preparation of Inocula and Test Portions 15 where n select = number of colonies on selective agar and n nonselect = number of colonies on non- 34 35 Test Portion Distribution

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