AOAC-RI ERP Book Micro Jan 19 2017

harvested by centrifuging aliquots of the stationary phase TSB culture at maximum speed for 10 min in a Model 16K Microcentrifuge (Bio-Rad Laboratories, Inc., Hercules, CA). The supernatant of each centrifuged aliquot was removed and the pelleted cells were re- suspended in Butterfield’s Phosphate Buffer (BPB; Made In - House From Various Ingredients). The cells re-suspended in BPB were centrifuged a second time, and the supernatant was removed once again. The pelleted cells were re-suspended once more in BPB. These final solutions of cells re-suspended in BPB were adjusted to a concentration of ~8.00 log 10 CFU/ml based on the transmittance of the suspension as determined using a Spectro nic™ 200 Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA). This suspension served as inoculum for E. coli O157:H7 and the concentrations of cells in each were verified on each day of the experiment by serially diluting in BPB and plating onto Sorbitol MacConkey Agar (SMAC). 2.3 Inoculation of Food Samples After the E. coli O157:H7 culture was prepared, the ground beef was prepared in individual portions of 325 g. Three portion types were prepared for each testing method. Each individual portion was placed into a sterile Whirl-Pak bag. This included a high inoculum level (Portion 1), a low inoculum level (Portion 2) and un- inoculated samples (Portion 3). After inoculation, all samples for each inoculum level were removed from each Whirl-Pak bag, combined and homogenized in one large batch unit and re-aliquoted into the appropriate portion. In ground beef, a 5 and 0.5 CFU/sample was targeted for the high and low inoculum levels, respectively. As stated in Section 2.2, the inoculum concentration was enumerated at the time of inoculation. A minimum volume consisting of 4,060 g of ground beef was also removed from each inoculated portion level (i.e. Portion 1 and Portion 2) in order to perform a five tube-three level Most Probable Number (MPN) analysis on each portion as described in Section 2.6. After inoculation, the food samples were stored at 4°C for 48 h to stabilize. Once this stabilization period was complete, testing commenced. A total of 20 samples were prepared for Portion 2, 5 samples were prepared for Portion 1 and 5 samples were prepared for Portion 3. 2.4 Analysis of Reference Method Sub-Portions After the 48 h stabilization period post-inoculation, the samples were subjected to the corresponding reference method according to USDA FSIS MLG testing procedures (2). The 325 g ground beef sample was combined with 975 mL of pre- heated (42°C) mTSB broth and hand massaged in order to homogenize. The samples were then incubated for 15-24 h at 42 + 1°C. In brief, the following procedure followed the USDA MLG method (2). Following the enrichment, 5 + 1 mL of each enriched culture was passed through a 40 µm cell strainer. At least 1 mL of this filtrate was added to a prepared immunomagnetic bead suspension

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