AOAC-RI ERP Micro June 2016

OMAMAN-29 A/ Collaboartive Study Manuscript OMA ERP June 2016 ERP Use Only

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

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