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Foodmatrixes were purchased from local grocery retailers and stored according to the
manufacturers’ guidelines. Prior to inoculation, the matrixes were screened for natural
contamination of the target analytes using the specified reference method in the protocol.
Aerobic plate counts were performed following FDA/BAM Chapter 3 [8] to determine the
microbial load of each food matrix. Table2 below outlines the specific inoculation
schemesfor each food evaluated in the study.
For the method comparison, a total of 30 samples for each of the 3 inoculated food
matrixeswasas analyzed. Each sample set included 5 uninoculated control samples, 20
low-level samples, and 5 high-level samples. The target levels for each strain of
E. coli
used for challenging the food matrixes were as follows: 0 colony forming units (CFU)/test
portion for the uninoculated control samples, 0.2-2 CFU/test portion for low-level
inoculation and 2-5 CFU/test portion for the high-level inoculation. Fractionally positive
results, those in which ether the reference or candidate method yields 5 to 15 positive
results out of the 20 low-level inoculum replicates, were required for each matrix.
Table 2: Test Matrixes and Organisms for Inoculation
Matrix
Organism
Target
Inoculum
Levels
# of
Replicates
Reference
Method
Reference
Method Test
Portion Sizes
mericon
Sample
Prep Method
Fresh
Spinach
E. coli
O111
TW06315
1
0 CFU/Test
Portion
5
FDA/BAM
Chapter 4A
200 g
Manual
&
QIAsymphony
(Automated)
0.2-2
CFU/Test
Portion
20
2-5 CFU/Test
Portion
5
Raw
Ground
Beef (70%
Lean)
E. coli
O157:H7
ATCC
43895
2
0 CFU/Test
Portion
5
USDA/FSIS-
MLG 5.09
325 g
0.2-2
CFU/Test
Portion
20
2-5 CFU/Test
Portion
5
Raw Beef
Trim
E. coli
O26
ATCC BAA-
1653
2
0 CFU/Test
Portion
5
USDA/FSIS-
MLG 5B.05
325 g
0.2-2
CFU/Test
Portion
20
2-5 CFU/Test
Portion
5
1
Michigan State University Culture Collection
2
American Type Culture Collection
Each matrix was inoculated with a different strain of
E. coli
as indicated in Table 2. Each
inoculum was prepared by transferring a pure isolated colony of the specified organism
from Trypticase Soy agar containing 5% sheep’s blood (SBA) into BHIbroth followed by
incubation at 35± 2
o
C for 24 ± 2 h. Post-incubation, the BHI broth culture was diluted to the
target level using BHI broth as the diluent. Non-stressed cultures were used for all three
matrixes.
For each matrix, bulk portions were inoculated and blended in a large stainless steel
container using sterile spatulas to distributethe inoculum evenly.. All food matrixes were
held for 48-72 hours post-inoculation at 2-8
o
C to allow for equilibration of the organism as
OMAMAN-36 E/ AOAC PTM Report
ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only