AOAC-RI ERP Book MICRO.pdf

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CONFIDENTIAL INFORMATION
Page 4 of 13
(Dynal® anti-E. coli O157 antibody-coated paramagnetic beads; Dynal Inc., Lake
Success, NY). The sample tubes were then agitated for 10-15 min at 18-30°C.
The sample-bead suspensions were filtered, processed and washed as described
in the USDA MLG method (2). A 1:10 and 1:100 dilution of each treated bead
suspension was prepared with E-Buffer. Once the dilutions were prepared, 0.1 mL
of each were spread onto modified Rainbow® Agar (mRBA, Biolog Inc., Hayward,
CA) plates and incubated at 35°C + 2°C for 20-24 h. Each sample was also
subjected to acid treatment as outlined and plated onto mRBA as well. Colonies
exhibiting typical colony morphologies were then subject to latex agglutination
assays (RIM® E. coli O157:H7 Latex Test (Remel, Lenexa, KS). Latex positive
colonies were streaked onto TSA with 5% Sheep Blood (SBA) plates and
incubated at 35°C + 2°C for 16-24 h. Colonies with typical morphologies were then
subject to biochemical testing using VITEK® 2 GN cards (bioMerieux Vitek, Inc.,
Hazelwood, MO) along with O157 and H7 antigen confirmations by latex
agglutination testing by RIM® E. coli O157:H7 Latex Test. Positive samples were
designated as such by the serological positive for ‘O157’ and biochemical
identifications.
2.5 Analysis of Candidate Method Sub-Portions
After the 48 h stabilization period post-inoculation, the samples were subjected to
the following enrichment procedures and testing on the MDA. 3M provided all 3M
consumable supplies involved with the MDA test. 3M Buffered Peptone Water
(BPW) was pre-heated to 41.5°C + 1°C prior to enriching all samples. For raw
ground beef, the 325 g sample was added to 975 mL 3M BPW. The sample was
homogenized for 2 min and incubated at 41.5°C + 1°C for 18 h. Aliquots from both
time points were subject to confirmation procedures outlined in Section 2.4. The
enriched samples were then subject to analysis as outlined by the instructions for
use for the 3MTM Molecular Detection Assay 2 – E. coli O157 test kit. The sample
results obtained after completing the MDA were considered the ‘screening stage’
result for the candidate method. Concurrent with the transfer and testing on the
MDA, aliquots of the enriched samples were transferred and subjected to the
testing outlined in Section 2.4 for cultural confirmation. The results obtained after
completing this methodology were considered the ‘confirmation stage’ result for
the candidate method.
2.6 Most Probable Number Analysis
As mentioned above in Section 2.3, samples for each portion were inoculated with
E. coli O157:H7 (i.e. Portion 1, and Portion 2) and used for performing a five tube-
three level MPN analysis to determine the concentration of E. coli O157 inoculated
into the portion. This analysis was setup after the 48 hr stabilization period in order
to mimic all handling and treatment of samples.