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The inoculating organism was prepared by transferring a single colony from trypticase
soy agar with 5% sheep blood (SBA) into brain heart infusion (BHI) broth and incubated
at 35 ± 2
o
C for 24 ± 2 hours. Following incubation, the culture was diluted to a target
level using BHI broth as the diluent. For the inoculated food matrix, bulk portions were
spiked and homogenized in large sterile stainless steel containers. Sterile spatulas were
used to mix the bulk portions to ensure the inoculum was evenly distributed. Prior to
analysis, the organism was allowed to equilibrate in the matrix for 48 – 72 hours at 4
o
C.
To prepare the test portions, 25 g of inoculated matrix was combined with 300 g of
uninoculated matrix.
The level of
E. coli
in the low level inoculum for the 325 g test portions were 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
level of
E. coli
in the high level inoculum for the 325 g test portions were determined by
MPN on the day of analysis by evaluating 5 x 325 g (reference method test portions),
5 x 130 g, and 5 x 65 g inoculated test samples. The test portion size is presented
below in Table B. Each test portion was enriched with the reference method enrichment
medium and analyzed by the reference method procedure. The number of positives
from the 3 test levels were used to calculate the MPN using the LCF MPN calculator
(version 1.6) provided by AOAC RI. [5]
( http://www.lcfltd.com/customer/LCFMPNCalculator.exe )Table B: MPN Test Portion Sizes
Reference Method
Test Portion
Inoculation
Level
MPN Test Portions
325 g
Low
5 x 650 g
20 x 325 g*
5 x 130 g
High
5 x 325 g*
5 x 130 g
5 x 65 g
*
Test portions from reference method
USDA/FSIS-MLG 5.09: Detection, Isolation and Identification of Escherichia coli
O157:H7 from Meat Products and Carcass and Environmental Sponges
The raw ground beef test portions were prepared as outlined in the study protocol.
Following equilibration of the microorganism in the matrix, test portions consisting of
325 ± 32.5 g were enriched with 975 ± 19.5 mL of pre-warmed modified tryptic soy broth
with the addition of casamino acids (mTSB + CAA), homogenized for 2 minutes and
incubated 15-24 hours at 42 ± 1°C. After incubation, the primary enrichment from each
sample was screened using a USDA/FSIS-MLG 5.09 validated lateral flow device test
system. Regardless of the screening result, all samples were subjected to isolation by
immunomagnetic separation (IMS) by transferring 1.0 mL aliquots of the primary
enrichment to a microcentrifuge tube containing a 50 µL suspension of
E. coli
O157
immunomagnetic (paramagnetic) beads. The solution was placed onto a Labquake
™
agitator and rotated for 10 to 15 minutes at 18-30°C. After rotation the bead and sample
solution was transferred to a MACS
®
large cell separation (ferromagnetic) column and