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CONFIDENTIAL INFORMATION
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cells 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, individual portions of 25 g for
frozen blueberry matrices were inoculated. As outlined in Section 2.1,
E. coli
O157:H7 ATCC 700599 was used for the frozen blueberry matrix. 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). 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. Samples consisting of 425 g (blueberries) were removed from 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 then stored at -20°C for
14 d to stabilize. Once this stabilization period is complete, testing will commence.
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 14 d stabilization period post-inoculation, the samples were subjected to
the corresponding reference method according to the FDA BAM testing procedure
(2). The 25 g sample were combined with 225 mL of mBPWp and placed at 37°C
+ 1°C for 5 h to incubate. No stomaching or blending was performed. The mixing
of the enrichment broth and blueberries was performed gently with efforts to limit
any bursting of blueberries. Following incubation, 1 mL of ACV supplement was
added and incubated at 42°C + 1°C for 18-24 h. Following overnight enrichments,
the sample was serially diluted in BPB. Appropriate dilutions were plated, in
duplicate, onto Tellurite Cefixime-Sorbitol MacConkey Agar (TC-SMAC, Becton,
Dickinson and Company, Franklin Lakes, NJ) along with one Chromogenic
Selective Agar (ChromAgar, R&F Laboratories, Downers Grove, IL). Plates were
incubated at 37°C + 1°C for 18-24 h. Typical colony morphology was used to
identify probable positive for O157. Typical colonies were picked and tested for
latex agglutination (Remel kit) and streaked onto TSA + Yeast Extract (TSAYE)
with a ColiComplete disc placed in the heaviest streak area. Plates were incubated
at 37°C + 1°C for 18-24 h. Isolates were confirmed with commercial antisera
(RIM®
E. coli
O157:H7 Latex Test or equivalent test) followed by VITEK for
E. coli
identification.