AOAC-RI ERP Book Micro Jan 19 2017

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

Low High

5 x 650 g 5 x 325 g*

20 x 325 g* 5 x 130 g

5 x 130 g 5 x 65 g

325 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

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