AOAC ERP MICRO AUGUST 2018

OMAMAN-30 D/ PTM Validation Report 081501 OMA ERP - June 2016 ERP Use Only

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Where n select

= number of colonies on selective agar and n nonselect

= number of colonies on non-

selective agar. Using BHI broth as the diluent, the culture was diluted to a low level expected to yield fractional positive results (5-15 positive results) and a high level expected to yield all positive results. Following inoculation, a bulk lot of the matrix was homogenized by hand and held for 48-72 hours at refrigerated temperature (2-8 °C) prior to analysis to allow time for the For melons, a single whole melon was placed into a large sterile bag and the blossom end of the melon was inoculated with 100 µL of the diluted Listeria monocytogenes culture. The liquid culture was then allowed to soak into the melon. The melon was then inverted so that the inoculated end was on the bottom of the sterile bag. The bag was tied closed and held for 48-72 For environmental surfaces, inocula were prepared by transferring a pure isolated colony of the specified organism from SBA into BHI broth and incubated at 35± 2 o C for 24 ± 2 hours. Following incubation, serial dilutions were performed in BHI broth to achieve the target level For stainless steel and sealed concrete surfaces, 4” x 4” areas were inoculated with 0.25 mL of diluted Listeria monocytogenes culture. Stainless steel was also inoculated with competitor organism Enterococcus faecium ATCC 19434 at 10x the level of the target organism. For plastic surfaces, 1” x 1” areas were inoculated with 0.10 mL of diluted Listeria monocytogenes culture. Plastic was also inoculated with a competitor organism, Enterococcus faecalis ATCC 29212, at 10x the level of the target organism. For the uninoculated test portions, sterile BHI broth was applied to the test area. Each surface was allowed to dry for 16-24 hours at room temperature (24 The 3M hydrated sampling sponges (pre-moistened with Dey-Engley) (stainless steel and sealed concrete) and 3M™Tecra™ Enviro Swabs (pre-wetted with Letheen) (plastic) were sampled by using horizontal and vertical sweeping motions. The sponges and the swabs were held at room temperature for 2 hours prior to analysis. To determine the inoculation level for the environmental surfaces, aliquots of each inoculum were plated on TSA in triplicate. The level of Listeria monocytogenes in the low level inoculum and high level inoculum was determined by Most Probable Number (MPN) on the day of analysis by evaluating 5 x 50 g, 20 x 25 g (reference method test portions), and 5 x 10 g inoculated test samples for the low inoculation level and by examining 5 x 25 g, 5 x 5 g and 5 x 1 g for the high inoculation level. The level of Listeriamonocytogenes in the low level inoculum and high level inoculum for all 125 g test portions was determined by MPN by evaluating 5 x 250 g, 20 or 5 x 125 g (reference method test portions), and 5 x 50 g inoculated test samples. Each test portion was enriched with 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. [10] organism to equilibrate within the sample. hours at 2-8 °C. inoculum. ±2 o C).

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AOAC Research Institute Expert Review Panel Use Only

(http://www.lcfltd.com/customer/LCFMPNCalculator.exe)

USDA/FSIS MLG 8.09 Reference Method

For the USDA/FSIS MLG 8.09 reference method, 25 g test portions were enriched with 225 ± 5 mL of modified University of Vermont Medium broth (UVM) and 125 g test portions were enriched with 1125 ± 25 mL of UVM. All test portions were mechanically stomached for two minutes. The 25 g test portions were incubated at 30 ± 2 °C for 20-26 hours and the 125 g test