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was fully reconstituted. The tubes were placed back onto the magnetic stand and inverted
three times and allowed to sit for 3 minutes. This wash process was repeated three more
times for a total of four washes. Following the fourth wash, the microcentrifuge tubes were
removed from the magnetic stand and the bead pellet was reconstituted in 1.5 mL of wash
buffer.
The leafy spinach IMS samples were serial diluted (1:10) in phosphate buffered water
(PBW). A 100 µL aliquot of each serial dilution was plated in duplicate on Levine’s eosin-
methylene blue (L-EMB) agar and R&F non-O157 STEC Chromogenic plating mediain
order to achieve isolated colonies. Both sets of plates were incubated for 18-24 hours at 37
± 1
o
C. After incubation, plates containing typical colonies were screened for the O111
antigen by latex agglutination. Up to 10 isolated colonies that screened positive were
streaked to SBA and tryptic soy agar containing 0.6% yeast extract (TSA/YE) to verify
purity and incubated for 18-24 hours at 37 ± 1
o
C. Following incubation, a ColiComplete
(CC) disc was placed on the densest growtharea on the TSA/YE plates and incubated for
an additional 18-24 hours at 37 ± 1
o
C. The CC discs on the TSA/YE plates were observed
for typical reactions (blue color change with no fluorescence under long wave UV) and a
spot indole test was conducted. After incubation, SBA plates were observed for purity.
Final biochemical confirmations were obtained by VITEK 2 GN Biochemical Identification
following AOAC OMA 2011.17. [10]
USDA/FSIS-MLG 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7
from Meat Products and Carcass and Environmental Sponges
All raw ground beef test portions were prepared as outlined in the study protocol. Following
equilibration of the inoculum in the matrix, 325 ± 32.5 g test portions were enriched in 975
± 19.5 mL of pre-warmed mTSB+CAA, homogenized by stomaching 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(Dynal beads, ThermoFisher Scientific).. The
microcentrifuge tube was then 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
MACSlarge cell separation (ferromagnetic) column and washed four times with E buffer
(pre-warmed to 18-35°C) before the final elute was collected with 1 mL of E buffer in a
sterile tube. Following the IMS procedure, 1:10 and 1:100 dilutions of each IMS
suspension in E Buffer were spread-plated on modified Rainbowagar (mRBA).
A 450 µL aliquot of each remaining IMS suspension was transferred into a microcentrifuge
tube and mixed with 25 µL of1 N HCl. The microcentrifuge tubes were vortexed and placed
onto a Labquake agitator and rotated for 1 hour at 18 - 30°C. After rotating, 475 µL of E
buffer was added to each sample tube. The acid-treated IMS suspension and a 1:10
dilution of this suspension in E Buffer were plated onto mRBA. All mRBA plates were
incubated for 20 - 24 hours at 35 ± 2°C. After incubation, mRBA plates containing typical
colonies were tested for O157 latex agglutination and up to 5 isolated colonies were
streaked to SBA. The SBA plates were incubated for 16 - 24 hours at 35 ± 2°C. After
incubation, SBA plates were observed for purity. Isolated colonies from SBA were
confirmed positive by conducting a H7 latex agglutination test and testing for the presence
OMAMAN-36 E/ AOAC PTM Report
ERP Use Only
January 2017
OAC Research Institute
Expert Review Panel Use Only