AOAC-RI ERP Book Micro Jan 19 2017

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 transmittan ce of the suspension as determined using a Spectronic™ 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 two different food matrices were prepared in individual portions of 25 g and 200 g for sprouts and spinach matrices, respectively. Three portion types were prepared for each testing method, for each matrix. 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 sprouts and spinach, a 7.5 and 0.75 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. Minimum volumes consisting of 300 g (sprouts) and 555 g (spinach) were also removed from each food matrix and each inoculated portion (i.e. Portion 1 and Portion 2) in order to perform a five tube-three level Most Probable Number (MPN) analysis on each portion/matrix 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. For each food matrix, 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 the FDA BAM testing procedures (2).

CONFIDENTIAL INFORMATION Page 3 of 17