Microsoft Word - 3M Petrifilm REC Plate OMA Manuscript 09121

ERP Methods Book

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Preparation of the Inocula and Test Portions 1 2 The isolates used in this evaluation were lyophilized prior to inoculation. The cultures were 3 first propagated onto Tryptic Soy Agar with 5% Sheep Blood (SBA) from a Q Laboratories 4 frozen stock culture stored at -70°C. To prepare the culture for lyophilization, a single, well 5 isolated colony from SBA was transferred into brain heart infusion (BHI) broth and incubated at 6 37 ± 2 o C for 18-24 hours. The cultures were diluted in a sterile cryoprotectant, reconstituted 7 10% non-fat dry milk (NFDM), and freeze dried for 48-72 hours. A bulk lot of the test matrix 8 was inoculated with each culture at a high level. An aliquot of the high-level inoculated matrix 9 was further mixed with uninoculated matrix to produce the medium and low level inoculum. 10 After inoculation, the matrix was held for a minimum of 2 weeks at ambient temperature (20 - 11 25 o C). The inoculated test product was packaged into separate 10 g (ISO) and 50 g (BAM) 12 samples in sterile Whirl-Pak ® bags and shipped to the collaborators. 13 Test Portion Distribution 16 sample container. Nine participants from 8 separate locations participated. Test portions were 17 shipped in leak-proof insulated containers via overnight delivery according to the Category B 18 Dangerous Goods shipment regulations set forth by International Air Transport Association 19 (IATA). Test portions were shipped at ambient temperatures (20-25 o C). Upon receipt, samples 20 were held at ambient temperature until analysis was initiated. In addition to each of the test 21 portions, collaborators also received a test portion for the matrix labeled as Aerobic Plate Count 22 (APC), to determine total background count in the matrix using the FDA BAM Chapter 3 23 Aerobic Plate Count reference method [6]. The APC background screen samples were prepared 24 from the bulk lot of test matrix, prior to inoculation. Additionally, a temperature probe was 25 included in the shipment. Participants were instructed to submit the data from the temperature 26 probe upon receipt of the shipment. 29 30 Collaborators followed the appropriate preparation and analysis protocol provided to them in 31 the collaborator instructions (Version 2, August,2018). Each collaborator received 16 test 32 portions (2 high, 2 medium, 2 low and 2 uninoculated for paired analysis with the 3M Petrifilm 33 Rapid E. coli /Coliform Count Plate and ISO methods, and 2 high, 2 medium, 2 low and 2 34 uninoculated for analysis with the 3M Petrifilm and BAM method). 37 38 A 50 g test portion was diluted with 450 mL of BPBD, allowed to sit for 20 minutes to soften 39 the dry dog kibble, and homogenized with a paddle blender for 2 minutes ± 10 sec. Ten-fold 40 serial dilutions of each sample were prepared in BPBD and a 1.0 mL aliquot of each dilution was 41 plated onto a single 3M Petrifilm Rapid E. coli /Coliform Count Plate for each dilution. The plate 42 was incubated at 35 ± 1 o C for 18-24 hours. After incubation, plates were enumerated for total 43 coliform and E. coli . Plates containing greater than 100 CFU were recorded as too numerous to 44 count (TNTC). Final results were determined by multiplying the counts by the dilution factor for 45 that plate. 46 14 15 All samples were labeled with a randomized, blind-coded 3 digit number affixed to the 27 28 Test Portion Analysis 35 36 3M Petrifilm Rapid E. coli/Coliform Count Plate & BAM

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December 6, 2018