4. AOACRIMicroMethods-2018Awards

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B ird et al .: J ournal of AOAC I nternational V ol . 99, N o . 3, 2016  665

Additional PTM parameters (ruggedness, stability, and lot-to-lot variability) tested in the PTM studies satisfied the requirements for PTM approval. The method was awarded PTM certification No. 121403 on December 29, 2014. The purpose of this collaborative study was to compare the 3M Petrifilm RAC Plate to the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 3 (Aerobic Plate Count; 4) and the Standard Methods for the Examination of Dairy Products (SMEDP) Chapter 6 (Standard Plate Count; 5), using BPD as the diluent for raw easy-peel shrimp, pasteurized skim milk, and instant NFDM. In this collaborative study, three matrixes—raw easy-peel shrimp, pasteurized skim milk, and instant NFDM—were evaluated. The matrixes were obtained from local retailers and screened for the presence of naturally occurring aerobic organisms by BAM or the SMEDP reference methods. Three separate levels of contamination were targeted for the evaluation of each matrix using naturally occurring aerobic microflora. The target levels for the naturally contaminated matrixes were low (10–100 CFU/g), medium (100–1000 CFU/g), and high (1000–10 000 CFU/g). To obtain the required contamination levels, bulk lots of the target matrixes were temperature-abused by heat-stressing to elevate the naturally occurring aerobic bacteria present in the matrixes, or diluted using lots containing low numbers of aerobic bacteria. Two replicate samples from each of the three contamination levels were analyzed by both candidate and reference methods in a paired study design. One set of paired samples (six total) per matrix was sent to each laboratory for analysis by the 3M Petrifilm RAC Plate and BAM or SMEDP reference methods. 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 conducted prior to the initiation of the study to discuss the collaborative study packet and answer any questions from the participating laboratories. For raw easy-peel shrimp and pasteurized skim milk test portions, a single bulk lot of the matrix was evaluated for total aerobic plate count following BAM or SMEDP, respectively, to determine baseline aerobic bacterial counts. For both raw easy- peel shrimp and pasteurized skim milk, two 1000 g portions were removed from the bulk lot and temperature-abused to increase the aerobic bacterial counts. For raw easy-peel shrimp, one set of 1000 g was placed in a 35 ± 1°C incubator for 1 h, and the second set of 1000 g was placed at room temperature (20–25°C) for 4 h. For pasteurized skim milk, one set of 1000 g was placed at room temperature (24 ± 2°C) for 4 h, and the second set of 1000 g was placed at room temperature (24 ± 2°C) for 12 h. Following temperature abuse, five replicate test portions from each lot were evaluated for total aerobic count using the specified reference method. Results from both test matrixes indicated that the temperature abuse had increased aerobic bacterial counts to produce two additional levels of contamination. The raw easy- peel shrimp samples were subsampled into 60 g test portions and Collaborative Study Study Design Preparation of the Test Portions

the pasteurized skim milk samples were subsampled into 15 mL test portions to be sent to collaborators for use in the evaluation. For instant NFDM, several lots of product were evaluated for the presence of aerobic bacteria. Initial testing identified two lots (one that produced aerobic plate counts in the high contamination level and one that produced counts in the low contamination level) to be used in the evaluation. The medium contamination level was prepared by mixing a portion of the high contamination lot with the low contamination lot. The instant NFDM samples were subsampled into 15 g test portions to be used in the evaluation. All samples were labeledwith a randomized, blind-coded three- digit number affixed to the sample container. Test portions were shipped in leak-proof insulated containers via overnight delivery according to the Category B Dangerous Goods Regulations (DGR) as set forth by the International Air Transport Association (56th edition). Raw easy-peel shrimp and pasteurized skim milk samples were packed with cold packs to ensure refrigeration temperature (2–8°C) during shipment. Upon receipt, these test portions were held at refrigeration (2–8°C) until analyses were initiated the following day. Instant NFDM samples were packed and shipped at ambient temperature (24 ± 2°C). Upon receipt, samples were held at room temperature (24 ± 2°C) until analysis was initiated. In addition to each of the test portions, collaborators also received a test portion for each matrix labeled as “temperature control” for all three matrixes. Participants were instructed to record the temperature of this portion upon receipt of the shipment, document results on the Sample Receipt Confirmation form provided, and fax to the study director. Collaborators followed the appropriate preparation and analysis protocol according to the method specified for each matrix. For all three matrixes, each collaborator received six test portions (two high, two medium, and two low). For the analysis of the raw easy-peel shrimp by the 3M Petrifilm RAC Plate, a 50 g test portion was diluted with 450 mL BPD and homogenized by blending for 2 min. For pasteurized skim milk, 11 g test portion was diluted with 99 mL BPD and homogenized by shaking 25 times in a 30 cm arc within 7 s. For instant NFDM, 11 g sample was added to 99 mL tempered BPD (40–45°C) ensuring that the entire sample was visibly dissolved throughout the diluent prior to homogenizing by shaking 25 times in a 30 cm arc within 7 s. Ten-fold serial dilutions of each sample were prepared for each matrix and a 1.0 mL aliquot of each dilution was plated onto 3M Petrifilm RAC Plates. For raw easy-peel shrimp, four replicate 3M Petrifilm RAC Plates were prepared for each dilution, with two plates being incubated at 32 ± 1°C for 24 ± 2 h and two plates being incubated at 35 ± 1°C for 24 ± 2 h. For the evaluation of the pasteurized skim milk, two replicate 3M Petrifilm RAC Plates for each dilution were prepared and incubated at 32 ± 1°C for 24 ± 2 h. For the evaluation of the instant NFDM, two replicate 3M Petrifilm RAC Plates for each dilution were prepared and incubated at 32 ± 1°C for 48 ± 3 h. Seafood test portions were evaluated at two temperatures (32 and 35°C) to allow end users the option to choose either temperature for incubation. After incubation, 3M Petrifilm RAC Plates were removed from the incubator and typical colonies (all colonies regardless of size, color, or intensity) were enumerated using a Test Portion Distribution Test Portion Analysis

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