AOAC-RI ERP Micro June 2016

OMAMAN-29 D/ PTM Validation Report 111501 OMA ERP - June 2016 ERP Use Only

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was inverted so that the inoculated end was on the bottom of the sterile bag. The bag was tied

closed and held for 48-72 hours at 2-8 °C.

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

inoculum.

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 seeligeri 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 ±2 o C). The 3M™ Hydrated Sponge Stick (pre-moistened with Dey-Engley) (stainless steel and sealed concrete) and 3M Tecra Enviroswabs (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 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 high inoculation level, the MPN was determined by examining 5 x 50 g, 5 x 25 g (reference method test portions) and 5 x 10 g . The level of Listeria in the low 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. For the high inoculation level, the MPN was determined by examining 5 x 250 g, 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. See Table A for details. The number of positives from the 3 test levels was used to calculate the MPN using the LCF MPN

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AOAC Research Institute Expert Review Panel Use Only

calculator (version 1.6) provided by AOAC RI. [11] (http://www.lcfltd.com/customer/LCFMPNCalculator.exe)

Table A: MPN Test Portion Sizes

Reference Method Test Portion

Inoculation Level

MPN Test Portions

Low High Low High

5 x 50 g 5 x 50 g 5 x 250 g 5 x 250 g

20 x 25 g* 5 x 25 g* 20 x 125 g* 5 x 125 g*

5 x 10 g 5 x 10 g 5 x 50 g 5 x 50 g

25 g

125 g

38 39 40

* Test portions from reference method