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per AOAC guidelines. For the raw meat matrixes, 25 g of inoculated meat was mixed with
300 g of uninoculated meat to prepare the 325 g test portions.
The level of E. coli O157:H7 or non-O157H7 STECin the low level inoculum for raw ground
beef and raw beef trim was determined by Most Probable Number (MPN) on the day of
analysis by evaluating 5 x 650 g, 20 x 325 g (reference method test portions), and 5 x 130
g inoculated test samples. The high inoculation level for raw ground beef and raw beef trim
was determined by evaluating 5 x 325 g (reference method test portions), 5 x 130 g, and 5
x 65 g. The level of E. coli O111 in the low level inoculum for fresh spinach was determined
by MPN by evaluating 5 x 400 g, 20 x 200 g (reference method test portions),and 5 x 80 g
inoculated test samples. The high level inoculum for fresh spinach test portions was
determined by MPN evaluating 5 x 200 g (reference method test portions), 5 x 80 g, and 5
x 40 g inoculated test samples. Table 3 presents the MPN test portions for the low and the
high inoculation levels. Each test portion was enriched using the reference method
enrichment broth at the reference method dilution scheme and analyzed by the reference
method procedure. The number of positives from the 3 test levels was used to calculate
the MPN using the LCF MPN calculator (version 1.6) provided by AOAC RI. [9]
Table 3:MPN Test Portion Sizes
Matrix Inoculation Level MPN Test Portions
Raw Ground Beef
and Raw Beef Trim
Low 5 x 650 g 20 x 325 g* 5 x130 g
High 5 x 325 g* 5 x 130 g 5 x 65 g
Fresh Spinach
Low 5 x 400 g 20 x 200 g* 5 x 100 g
High 5 x 200 g* 5 x 80 g 5 x 40 g
* - Test portions from matrix study or reference method
FDA/BAM Chapter 4A: Diarrheagenic Escherichia coli
For fresh spinach, 200 g test portions were enriched in 450 mL of 1 x modified Buffered
Peptone water with pyruvate ( mBPWp). All test portions were incubated statically for 5
hours at 37 ± 1 oC followed by the addition of Acriflavin, Cefsulodin, and Vancomycin
supplements per the FDA/BAM method. Samples were re-incubated for an additional 18-24
hours at 42 ± 1 oC.
Following the primary enrichment of fresh spinach, an optional immunomagnetic separation
(IMS) procedure was conducted due to the high background flora. A 1 mL aliquot was
transferred into a 1.5 mL microcentrifuge tube containing 20 µL of immunomagnetic
separation beads (E. coli O26 IMS Beads, Abraxis,Inc.).The microcentrifuge tubes were
rotated on the Labquakerotator for 10 minutes, removed and placed onto a magnetic stand.
The tubes were inverted three times and allowed to sit on the magnetic stand for 3
minutes. The contents of the microcentrifuge tube were decanted making sure that the
pellet of beads was not disturbed. The microcentrifuge tubes were removed from the
magnetic stand and1 mL of wash buffer was added to each tube ensuring the bead pellet
OMAMAN-36 E/ AOAC PTM Report
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January 2017
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