4. AOACRIMicroMethods-2018Awards

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B ird et al .: J ournal of AOAC I nternational V ol . 100, N o . 2, 2017  455

Aerobic Count Plate (AOAC Official Method SM 2015.13 ; 6) on the day samples were received for the purpose of determining the total APC. 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 before the initiation of the study. A conference call was then conducted to discuss the details of the collaborative study packet and answer any questions from the participating laboratories. The L. monocytogenes cultures (ATCC 7644 and ATCC 19115) used in this evaluation were propagated onto TSA with 5% sheep blood 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 10 mL brain heart infusion broth and incubated for 18 ± 0.5 h at 35 ± 1°C. Raw chicken breast fillet was inoculated in bulk using the fresh broth culture. Before 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 in duplicate onto selective modified Oxford agar and nonselective TSA with yeast extract. The degree of injury was estimated as =thenumberofcoloniesonnonselectiveagar.Appropriate dilutions of each culture were prepared in Butterfield’s phosphate diluent based on previously established growth curves for both the low and high inoculation levels. Bulk portions of each matrix were inoculated with the diluted liquid inoculum and mixed thoroughly to ensure an even distribution of microorganisms. The inoculated raw chicken breast fillet was packaged into separate 30 g samples in sterile Whirl-Pak ® bags and shipped to the collaborators. For the analysis of the deli turkey, 25 g inoculated test product was mixed with 100 g uninoculated test product to prepare 125 g test portions, which were packaged in sterileWhirl- Pak bags and shipped to collaborators. To determine the level of L. monocytogenes in the matrixes, a five-tube most probable number (MPN) test was conducted by the coordinating laboratory on the day of the initiation of analysis using the USDA/FSIS MLG reference method. For deli turkey, the MPN was determined by analyzing five 250 g test portions, the reference method test portions from the collaborating laboratories, and five 65 g test portions. For the raw chicken breast fillet, the MPN of the high and low inoculated levels was determined by analyzing five 50 g test portions, the reference method test portions from the collaborating laboratories, and five 10 g test portions. The MPN and 95% confidence intervals were calculated using the Least Cost Formulations, Ltd, MPN Calculator–Version 1.6, provided by AOAC Research Institute (7). Preparation of Inocula and Test Portions 1   100 −   × n n select nonselect where n select = the number of colonies on selective agar; and n nonselect

melon (whole), raw chicken leg pieces (25 g); raw chicken breast fillet (25 g), sealed concrete (sponge, 225 and 100 mL), stainless steel (sponge, 225 mL), and plastic (high-density polyethylene; 3M EnviroSwab, 10 mL). Additional PTM parameters (inclusivity, exclusivity, ruggedness, stability, and lot-to-lot variability) tested in the PTM studies satisfied the performance requirements for PTM approval. The method was awarded PTM Certification No. 081501 on August 18, 2015. The purpose of this collaborative study was to compare the reproducibility of the 3M MDA 2 – Listeria monocytogenes method to the U.S. Department of Agriculture (USDA) Food Safety 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” (USDA/FSIS MLG) reference method (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 L. monocytogenes by the USDA/FSIS MLG reference method. The raw chicken breast fillet was artificially contaminated with fresh unstressed cells of L. monocytogenes ,AmericanType Culture Collection (ATCC) 7644, and the deli turkey was artificially contaminated with heat-stressed cells of L. monocytogenes , ATCC 19115 (Table 1), 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 total) were sent to each laboratory for analysis by the 3M MDA 2 – Listeria monocytogenes and the USDA/FSIS MLG reference method due to the different sample enrichment procedures required for each method. In addition, collaborators were sent a 60 g test portion and instructed to conduct a total aerobic plate count (APC) using the 3M Petrifilm™ Rapid Collaborative Study Study Design

Table 1. Heat-stress injury results

CFU/TSA (nonselective agar) b

CFU/MOX (selective agar) a

Degree of injury, %

Matrix

Test organism

L. monocytogenes

Deli

9.1 × 10 8

2.5 × 10 9

60.8

turkey

ATCC 19115

Raw

L. monocytogenes

chicken breast fillet c

Not applicable

Not applicable

— d

ATCC 7644

a  MOX = Modified Oxford agar. b  TSA = Tryptic soy agar. c  Cultures were heat-stressed for 10 min at 55°C in a recirculating water bath. d  — = Raw chicken breast fillet sample is not heat treated, so it is not necessary to injure the cells.

Test Portion Distribution

All samples were labeled with a randomized, blind-coded three-digit number affixed to the sample container. Test portions

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