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CONFIDENTIAL INFORMATION
Page 4 of 17
For the raw spinach matrix, categorized as ‘Leafy Produce’, the 200 g sample
was
combined with 450 mL of pre-heated (37°C) mBPWp and placed at 37°C + 1°C for
5 h to incubate. No stomaching or blending was performed. Following incubation,
2 mL of ACV supplement was added (1 mL per 225 mL mBPWp) and incubated at
42°C + 1°C for 18-24 h. For
the raw sprout matrix, categorized as ‘Cilantro or
P
arsley’, the 25 g sample
was combined with 225 mL of mBPWp and placed at
37°C + 1°C for 5 h to incubate. No stomaching or blending was performed.
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 mRBA plate. 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.
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 matrices. For the raw
sprouts, the 25 g sample was added to 225 mL 3M BPW and immediately
incubated at 41.5°C + 1°C for 18 h. For the raw spinach, to each 200 g sample,
450 mL of pre-warmed BPW was added and immediately incubated at 41.5°C +
1°C for 18 h. The sprout and spinach samples were not homogenized or
stomached during preparation. The enriched samples were then subject to
analysis as outlined by the instructions for use for the 3M
TM
Molecular Detection
Assay 2
–
E. coli
O157 test kit. The sample results obtained after completing the
MDA wer
e 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, for each food matrix
were inoculated with
E. coli
O157:H7 (i.e. Portion 1, and Portion 2; sprouts,
spinach) and used for performing a five tube-three level MPN analysis to determine
the concentration of
E. coli
O157 inoculated into the portion and matrix. This