4. AOACRIMicroMethods-2018Awards

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the details of the collaborative study packet and answer any questions from the participating laboratories.

The method was awarded PTM Certification No. 111501 on November 3, 2015. http://news.3m.com/press-release/3ms-next- generation-molecular-detection-assay-listeria-receives-aoac- ptm-validation. The purpose of this collaborative study was to compare the reproducibility of the 3M MDA 2– Listeria method to U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Chapter 8.09 “Isolation and identification of Listeria monocytogenes from red meat, poultry and egg products, and environmental samples” (5) for deli turkey (125 g) and raw chicken breast fillet (25 g). In this collaborative study, two matrixes, deli turkey and raw chicken breast fillet, were evaluated. The matrixes were obtained from a local retailer and screened for the presence of Listeria by the USDA/FSIS MLG 8.09 reference method. The raw chicken breast fillet was artificially contaminated with fresh unstressed cells of L. monocytogenes [American Type Culture Collection (ATCC) 7644] and the deli turkey was artificially contaminated with heat-stressed cells (Table 1) of L. monocytogenes (ATCC 19115) at two inoculation levels: a high inoculation level of approximately 2–5 CFU/test portion and a low inoculation level of approximately 0.2–2 CFU/test portion. A set of uninoculated control test portions (0 CFU/test portion) was also included. Twelve replicate samples from each of the three inoculation levels were analyzed by each method. Two sets of samples (72 samples total) were sent to each laboratory for analysis by 3M MDA 2– Listeria and the USDA/FSIS MLG 8.09 reference method due to the different sample enrichment procedures for each method. Additionally, collaborators were sent a 60 g test portion and instructed to conduct a total aerobic plate count (APC) using 3M Petrifilm Rapid Aerobic Count Plate (AOAC Official Method 2015.13 ; 6) on the day the samples were received for the purpose of determining the total aerobic microbial load. A detailed collaborative study packet outlining all necessary information related to the study—including media preparation, test portion preparation, and documentation of results—was sent to each collaborating laboratory prior to the initiation of the study. A conference call was then conducted to discuss Collaborative Study Study Design

Preparation of Inocula and Test Portions

The Listeria cultures used in this evaluation were propagated onto tryptic soy agar (TSA) with 5% sheep blood agar from a Q Laboratories frozen stock culture stored at –70°C. Each organism was incubated for 24 ± 2 h at 35 ± 1°C. Isolated colonies were picked to brain heart infusion broth, 10 mL, and incubated for 18 ± 0.5 h at 35 ± 1°C. Raw chicken breast fillet was inoculated in bulk using the fresh broth culture. Prior to inoculation of the deli turkey, the culture suspension was heat-stressed at 55 ± 1°C in a water bath for 15 ± 0.5 min to obtain 50–80% injury, as determined by plating onto selective modified Oxford agar (MOX) and nonselective TSAwith yeast. The degree of injury was estimated as follows: = number of colonies on selective agar and n nonselect = number of colonies on nonselective agar.Appropriate dilutions of each culture were prepared in Butterfield’s phosphate diluent based on previously established growth curves for both low and high inoculation levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed thoroughly to ensure even distribution of microorganisms. The inoculated raw chicken breast fillet was packaged into separate 30 g test portions in sterile Whirl-Pak bags and shipped to the collaborators. For the analysis of the deli turkey, 25 g inoculated test product were mixed with 100 g uninoculated test product to prepare 125 g test portions that were packaged in sterile Whirl- Pak bags and shipped to the collaborators. To determine the level of Listeria in the matrixes, a five- tube most probable number (MPN) was conducted by the coordinating laboratory on the day of the initiation. To determine the MPN for the deli turkey test portions, the coordinating laboratory analyzed 5 × 250 and 5 × 65 g test portions for each inoculation level. Test portions were enriched using a 1:10 dilution scheme in University of Vermont medium (UVM) and analyzed according to the USDA/FSIS MLG 8.09 reference method. Additionally, all test portions submitted by the collaborating laboratories for low and high levels were included in the MPN analysis. The MPN and 95% confidence intervals were calculated using Least Cost Formulations, Ltd MPN Calculator, Version 1.6, provided by the AOAC Research Institute (7). For the raw chicken breast fillet, the MPN of the high and low inoculated levels was determined by analyzing 5 × 50 g test portions, the reference method test portions from the collaborating laboratories, and 5 × 10 g test portions. All samples were labeled with a randomized, blind-coded, three-digit number affixed to the sample container. Test portions were shipped on a Thursday via overnight delivery according to category B dangerous goods shipment regulations set forth by the International Air Transportations Association. The two matrixes were shipped consecutively, with collaborating laboratories performing the analysis on one matrix at a time. −   1   × 100 select nonselect n n where n select Test Portion Distribution

Table 1. Results of heat-stress injury

Degree of injury d

Test organism a

CFU/MOX b CFU/TSA c 9.1 × 10 8 2.5 × 10 9

Matrix

Deli

L. monocytogenes , ATCC 19115 L. monocytogenes , ATCC 7644

60.8%

turkey

NA e

NA Raw chicken product is not heat-treated, so it was not necessary to injure the cells

Raw

chicken breast fillet

a  ATCC=American Type Culture Collection. b  Selective agar. c  Nonselective agar. d  Cultures were heat-stressed for 10 min at 55°C in a recirculating water bath. e  NA=Not applicable.

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