ERP Micro December 2019

phosphate-buffered diluent water (BPBD) and added to the bulk food matrix, stabilized, then mixed well with additional uninoculated food matrix at an appropriate amount where the low- level inoculated samples would yield 0.2–2 CFU/25g and the high-level inoculated samples would yield 2–10 CFU/25 g. Inoculated food matrix was mixed to ensure homogeneous distribution of the organisms and was held for 48–72 h at 5 ± 3°C to allow for equilibration of the organism as per AOAC guidelines. Frozen finely textured beef was equilibrated for at -20°C for a minimum of 2 weeks. See Table A for inoculating organism and food type. After allowing the microorganisms to stabilize, all 25 g test portions analyzed by the UDSA-FSIS MLG (or FDA BAM) were transferred to sterile filter laboratory blender bags. For the 375 g test portions analyzed by the GDS EHEC method, a 25 g test portion of the inoculated matrix was combined with 350 g of uninoculated matrix. For swabbed carcass cloths, the E. coli O157:H7 inoculum was prepared into BHI broth as indicated previously. Following incubation, the BHI culture was diluted to a target level using BPBD. The test strain was cold-stressed for a minimum of 24 h before inoculation onto the cloths by placing the diluted BPBD tubes at refrigerated (2–8°C) condition. While BPBD-diluted cultures were stored refrigerated, the offsite customer scrubbed the surface of beef trim (FSIS Directive 10,010.1 Rev. 4, (12)) using Fremonta MicroTally™ Manual Sampling Device (MSD) cloths. This scrub adhered typical levels of microflora onto the surface of the MSD cloths. Surface-swabbed cloths were placed individually into sterile Whirl-Pac® bags and shipped at 5 ± 3°C to the testing laboratory by overnight delivery. The diluted chilled test strain was spot inoculated directly onto each cloth at an appropriate amount so that the low-level inoculated samples would yield 0.2–2 CFU/cloth and the high-level inoculated samples would yield 2–10 CFU/cloth. Next, inoculated cloths were held for 12 h at 2–8°C to allow for equilibration of the organism. The level of E. coli O157:H7 for beef trim, FTB and green onions in the low-level inoculum was determined on the day of initiation of analysis by Most Probable Number (MPN) by evaluating 5 x 50 g, 20 x 25 g (reference method test portions), and 5 x 10 g inoculated test samples. The level of E. coli O157:H7 in the high-level inoculum was determined by MPN by evaluating 5 x 25g (reference method test portions), 5 x 50 g, and 5 x 10 g inoculated test samples. To the 50 g portions, 450 mL of the reference method enrichment broth was added; to the 10 g portions, 90 mL of the reference method enrichment broth was added. All 25 g portions were utilized from reference method test portions analyzed following the USDA-FSIS MLG (or FDA BAM) reference method. The number of positives from the 3 test levels was used to calculate the MPN using the LCF MPN calculator (version 1.6) provided by AOAC RI (13). No MPN analysis was performed for carcass cloths.

Table A: Matrices and Inoculating Organisms

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