Micro December 2018

1 The isolates used in this evaluation were lyophilized prior to inoculation. The cultures were 2 first propagated onto Tryptic Soy Agar with 5% Sheep Blood (SBA) from a Q Laboratories 3 frozen stock culture stored at -70°C. To prepare the culture for lyophilization, a single, well 4 isolated colony from SBA was transferred into brain heart infusion (BHI) broth and incubated at 5 37 ± 2 o C for 18-24 hours. The cultures were diluted in a sterile cryoprotectant, reconstituted 6 10% non-fat dry milk (NFDM), and freeze dried for 48-72 hours. A bulk lot of the test matrix 7 was inoculated with each culture at a high level. An aliquot of the high-level inoculated matrix 8 was further mixed with uninoculated matrix to produce the medium and low level inoculum. 9 After inoculation, the matrix was held for a minimum of 2 weeks at ambient temperature (20 - 10 25 o C). The inoculated test product was packaged into separate 10 g (ISO) and 50 g (BAM) 11 samples in sterile Whirl-Pak ® bags and shipped to the collaborators. 12 Test Portion Distribution 15 sample container. Nine participants from 8 separate locations participated. Test portions were 16 shipped in leak-proof insulated containers via overnight delivery according to the Category B 17 Dangerous Goods shipment regulations set forth by International Air Transport Association 18 (IATA). Test portions were shipped at ambient temperatures (20-25 o C). Upon receipt, samples 19 were held at ambient temperature until analysis was initiated. In addition to each of the test 20 portions, collaborators also received a test portion for the matrix labeled as Aerobic Plate Count 21 (APC), to determine total background count in the matrix using the FDA BAM Chapter 3 22 Aerobic Plate Count reference method [6]. The APC background screen samples were prepared 23 from the bulk lot of test matrix, prior to inoculation. Additionally, a temperature probe was 24 included in the shipment. Participants were instructed to submit the data from the temperature 25 probe upon receipt of the shipment. 28 29 Collaborators followed the appropriate preparation and analysis protocol provided to them in 30 the collaborator instructions (Version 2, August,2018). Each collaborator received 16 test 31 portions (2 high, 2 medium, 2 low and 2 uninoculated for paired analysis with the 3M Petrifilm 32 Rapid E. coli /Coliform Count Plate and ISO methods, and 2 high, 2 medium, 2 low and 2 33 uninoculated for analysis with the 3M Petrifilm and BAM method). 36 37 A 50 g test portion was diluted with 450 mL of BPBD, allowed to sit for 20 minutes to soften 38 the dry dog kibble, and homogenized with a paddle blender for 2 minutes ± 10 sec. Ten-fold 39 serial dilutions of each sample were prepared in BPBD and a 1.0 mL aliquot of each dilution was 40 plated onto a single 3M Petrifilm Rapid E. coli /Coliform Count Plate for each dilution. The plate 41 was incubated at 35 ± 1 o C for 18-24 hours. After incubation, plates were enumerated for total 42 coliform and E. coli . Plates containing greater than 100 CFU were recorded as too numerous to 43 count (TNTC). Final results were determined by multiplying the counts by the dilution factor for 44 that plate. 45 Each test portion analyzed by the candidate method was also analyzed using the FDA BAM 46 Chapter 4 reference method in a paired study design. A 1.0 mL aliquot from each sample dilution 47 was plated onto a Petri dish. To each plate, 10 mL of Violet Red Bile (VRB) agar was added and 48 13 14 All samples were labeled with a randomized, blind-coded 3 digit number affixed to the 26 27 Test Portion Analysis 34 35 3M Petrifilm Rapid E. coli/Coliform Count Plate & BAM

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