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CONFIDENTIAL INFORMATION
Page 3 of 13
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