4. AOACRIMicroMethods-2018Awards

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768  B ird et al . : J ournal of AOAC I nternational V ol . 98, N o . 3, 2015

repeatability was observed between the 3M Petrifilm RYMCount Plate method and the reference methods. For the 3M Petrifilm RYM Count Plate method PTM evaluation, 10 matrixes were evaluated: yogurt (1.5% fat), sour cream, raw almonds, sliced apples, frozen bread dough, ready-made cherry pie, ready-to- eat deli sandwiches, dehydrated chicken noodle soup, fermented salami, and raw frozen ground beef patties (77% lean). All other 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. 121301 on December 18, 2013. The purpose of this collaborative study was to compare the 3M Petrifilm RYM Count Plate method to a harmonized version of the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM) Chapter 18, Yeasts, Molds and Mycotoxins  (1) and the ISO 21527:2008 Microbiology of Food and Animal Feeding Stuffs – Horizontal Method for the Enumeration for Yeast and Molds – Part 1: Colony Count Technique in Products with Water Activity Greater Than 0.95 (4) and Part 2: Colony Count Technique in Products with Water Activity Less Than or Equal to 0.95 (5) methods using 0.1% peptone water as the diluent for raw almonds and raw frozen ground beef patties (77% lean). Study Design In this collaborative study, twomatrixes, rawfrozen ground beef patties (77% lean) and raw almonds, were evaluated. The matrixes were obtained from local retailers and screened for the presence of yeast and molds by the BAM and ISO reference methods. The raw frozen ground beef patties were artificially contaminated with a yeast, Trichosporon mucoides American Type Culture Collection (ATCC, Manassas, VA) 201382, and the almonds with a mold, Aspergillus aculeatus ATCC 56925. Four separate levels of contamination, including an uninoculated control level and three levels of artificial contamination, were evaluated for each matrix. The target for the three levels of artificial contamination was as follows: low, 10–100 CFU/g; medium, 100–1000 CFU/g; high 1000–10000 CFU/g. Two replicate samples from each of the four levels were analyzed by both the candidate and reference methods. One set of paired samples (eight total) per matrix was sent to each laboratory for analysis by the 3M Petrifilm RYM Count Plate method and the BAM and ISO reference methods. For both matrixes, collaborators were sent an additional 60 g test portion and instructed to conduct a total aerobic plate count (APC) using 3M TM Petrifilm TM Aerobic Count Plate (AOAC Official Method 990.12 ; 6) on the day samples were received as a quality measure to verify appropriate shipping conditions. 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 prior to the initiation of the study was Collaborative Study

conducted to discuss the collaborative study packet and answer any questions from the participating laboratories.

Preparation of Inocula and Test Portions

The Trichosporon mucoides used in this evaluation was propagated in 10 mL of Brain Heart Infusion (BHI) broth from a frozen stock culture stored at –70°C at Q Laboratories, Inc. (Cincinnati, OH) The broth was incubated for 48±2 h at 30±1°C. Appropriate dilutions of the cultures were prepared in Butterfields Phosphate Buffer (PBW) based on previously established growth curves to obtain low, medium and high contamination levels. For the raw frozen ground beef patties, a bulk lot of the matrix was thawed, inoculated with the diluted liquid inoculum and mixed thoroughly by hand kneading to ensure an even distribution of microorganisms. The inoculated raw frozen ground beef patties samples were separated into 30 g test portions and held at –20±2°C for 2 weeks when testing was initiated. The Aspergillus aculeatus used in this evaluation was propagated on Potato Dextrose Agar (PDA) in a culture tissue flask from a frozen stock spore suspension stored at –70°C at Q Laboratories, Inc. The culture tissue flask was incubated for 7 days at 25±1°C. The mold spore suspension was prepared by rinsing the culture tissue flask twice with 20 mL of 0.9% saline containing 0.05%Tween 20. Each suspension was centrifuged at 4000 × g for 10±0.5 min to pellet the spores and the supernatant was decanted. The two separate spore pellets were resuspended with 2.5 mL of PBW and combined. The spore suspension was combined with a cryoprotectant, reconstituted non-fat dry milk (NFDM), homogenized by vortex to mix and placed onto a freeze dry system for 72±4 h to lyophilize the culture. After the lyophilization process, appropriate dilutions of the culture were prepared in sterile NFDM powder to produce the low, medium and high contamination levels. A bulk lot of whole raw almonds were inoculated with the lyophilized inoculum and mixed thoroughly by hand mixing to ensure an even distribution of microorganisms. The inoculated almonds were separated into 30 g test portions and held at ambient temperature (24±2°C) so that the organism had equilibrated for 2 weeks when testing was initiated. The shipment conditions and hold times of the inoculated test materials were verified as a quality control measure prior to study initiation. All samples were labeled with a randomized, blind-coded 3 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 International Air Transport Association. Frozen raw ground beef patties samples were packed with cold packs to ensure the samples remained frozen (–20±2°C) during shipment. Upon receipt, raw frozen ground beef patties samples were held at –20±2°C until the following Monday when analysis was initiated. Raw almond samples were packed and shipped at ambient temperature. Upon receipt, samples were held at room temperature (24±2°C) until the following Monday when analysis was initiated. In addition to each of the test portions and the total plate count replicate, collaborators also received Test Portion Distribution

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