4. AOACRIMicroMethods-2018Awards

B ird et al .: J ournal of aoaC i nternational V ol . 99, n o . 4, 2016 981

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instant nonfat dry milk (25 g), black pepper (25 g), cocoa powder (25 g), pasteurized liquid whole egg (100 g), raw head on shrimp (25 g), raw bagged spinach (25 g), spent sprout irrigation water (375 mL), cooked breaded chicken (325 g), creamy peanut butter (25 and 375 g), dry dog food (25 and 375 g), pasteurized American cheese (25 g), sealed concrete environmental surface [4 × 4 in. area with 3M hydrated sponge stick with Dey/Engeley (D/E)], stainless steel environmental surface (1 × 1 in. area with 3M Enviroswab), and sealed ceramic tile environmental surface (4 × 4 in. area with 3M hydrated sponge stick with D/E). 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 number 091501 on September 21, 2015. The purpose of this collaborative study was to compare the reproducibility of the 3M MDA 2 – Salmonella method to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) Chapter 4.08 “Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Catfish Products and Carcass and Environmental Sponges” reference method (USDA/FSIS MLG; 7) for raw ground beef (73% lean) and to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 “ Salmonella ” reference method (FDA BAM; 8) for creamy peanut butter. In this collaborative study, two matrixes, raw ground beef (73% lean) and creamy peanut butter (50% fat, 22% sugar content), were evaluated. The matrixes were obtained from a local retailer and screened for the presence of Salmonella by the appropriate reference method before analysis. The raw ground beef was artificially contaminated with nonheat-stressed cells of Salmonella Agona, American Type Culture Collection (ATCC) 51957, and the creamy peanut butter was artificially contaminated with heat-stressed cells of Salmonella Muenchen, ATCC BAA- 1594, 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 3M MDA 2 – Salmonella and by either the USDA/FSIS MLG (raw ground beef) or FDA BAM (creamy peanut butter) reference method due to the different sample enrichment procedures for each method. In addition, collaborators were sent a 60 g test portion and instructed to conduct a total aerobic plate count (APC) using 3M Petrifilm™Aerobic Count Plate (AOAC Official Method SM 990.12 ; 9) on the day 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 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. Collaborative Study Study Design

Preparation of Inocula and Test Portions

The Salmonella cultures used in this evaluation were propagated onto tryptic soy agar (TSA) with 5% sheep blood agar from a Q Laboratories, Inc. (Cincinnati, OH) 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. Before inoculation, the culture suspension for the creamy peanut butter was heat-stressed at 55 ± 1°C in a water bath for 15 ± 0.5 min to obtain an injury of 50–80% [as determined by plating onto selective xylose lysine deoxycholate (XLD) agar and nonselective TSA]. The degree of injury was estimated as is the number of colonies onnonselective 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 an even distribution of microorganisms. The inoculated creamy peanut butter was packaged into separate 30 g test portions in sterile Whirl-Pak ® bags and shipped to the collaborators. For the analysis of the raw ground beef, 25 g inoculated test product was mixed with 300 g uninoculated test product to prepare 325 g test portions, which were packaged in sterile Whirl-Pak bags and shipped to the collaborators. To determine the level of Salmonella in the matrixes, a most probable number (MPN) assay was conducted by the coordinating laboratory on the day of the initiation of analysis using the USDA/FSIS MLG reference method for raw ground beef and the FDA BAM reference method for the creamy peanut butter. For raw ground beef, the MPN was determined by analyzing 5 × 650 g test portions, the reference method test portions from the collaborating laboratories, and 5 × 160 g test portions by the USDA/FSIS MLG reference method. For the creamy peanut butter, 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 by the FDA BAM reference method. The MPN and 95% confidence intervals were calculated using the Least Cost Formulations, Ltd MPN Calculator, Version 1.6 (www.lcftld.com/customer/ LCFMPNCalculator.exe), provided by the AOAC Research Institute (10). Confirmation of the samples was conducted according to the USDA/FSIS MLG or the FDA BAM reference method, depending on the matrix. 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 the Category B Dangerous Goods shipment regulations set forth by the International Air Transportations Association. Upon receipt, raw ground beef samples were held by the collaborating laboratory at refrigeration temperature (2–8°C) until the following Monday when analysis was initiated after a total equilibration −   1   × n n 100 select nonselect where n select is the number of colonies on selective agar, and n nonselect Test Portion Distribution

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