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USDA/FSIS MLG 8.09 Reference Method
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For the USDA/FSIS MLG 8.09 reference method, 25 g test portions were enriched with 225 ± 5
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mL of modified University of Vermont Medium broth (UVM) and 125 g test portions were
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enriched with 1125 ± 25 mL of UVM. All test portions were mechanically stomached for two
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minutes. The 25 g test portions were incubated at 30 ± 2 °C for 20-26 hours and the 125 g test
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portions were incubated at 30 ± 2 °C for 23-26 hours. For environmental samples, sponges were
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enriched with 225 mL of UVM and homogenized by hand, while swabs were enriched with 10
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mL of UVM and mixed by vortex. Sponges and swabs were incubated for 20-26 hours at 30 ±
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2°C. After incubation of all test portions, 0.1 ± 0.02 mL of the sample enrichment was
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transferred to 10 ± 0.5 mL of Fraser Broth (FB) containing 0.1 mL of 5% ferric ammonium
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citrate and incubated at 35 ± 2°C for 26 ± 2 hours. A loopful of the sample enrichment was also
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streaked to MOX and incubated at 35 ± 2°C for 26 ± 2 hours.
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After 26 ± 2 hours, FB was examined for any degree of darkening due to esculin hydrolysis. Any
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FB that displayed darkening was streaked to a MOX plate. If no darkening occurred, FB was re-
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incubated at 35 ± 2°C for a total of 48 ± 2 hours and re-examined for evidence of darkening. If
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darkening occurred, the FB was streaked to a MOX plate, if no darkening occurred, samples
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were considered negative. All FB streaked MOX plates were incubated at 35 ± 2°C for 26 ± 2
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hours.
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MOX agar plates streaked from the primary enrichment or the FB secondary enrichment were
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examined after 26 ± 2 hours and if no suspect colonies were present, the MOX agar plate was re-
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incubated for an additional 26 ± 2 hours at 35 ± 2°C for a total of 48 ± 2 hours. If suspect
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colonies were present on the MOX agar plates, these suspect colonies were streaked to Horse
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Blood Overlay agar (HBO) and incubated at 35 ± 2
o
C for 22 ± 4 hours. HBO plates were
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examined for hemolysis reactions and well isolated colonies were transferred to BHI broth and
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incubated at 25°C for 24 ± 2 hours. Sample isolates from BHI broth were analyzed for tumbling
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motility by preparing a wet mount, analyzed by a catalase test and examined for morphology by
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preparing a Gram stain. Additionally, purified HBO isolates were identified using the VITEK
®
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GP Biochemical Identification following AOAC OMA 2013.02. [12]
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FDA/BAM Chapter 10 Reference Method
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Twenty-five gram test portions were enriched in 225 mL ± 5 mL of Buffered Listeria
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Enrichment Broth (BLEB) homogenized for 2 minutes and incubated at 30 ± 1°C for 4 hours.
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For whole melons, a single whole melon was enriched using BLEB with approximately 1.5 times
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the weight of the melon, and soaked for 1 hour at room temperature. After an hour at room
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temperature, the test portions were incubated at 30 ± 1°C for 4 hours. Following 4 hours of
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incubation, selective supplements acriflavine (10mg/L), sodium nalidixate (40mg/L) and
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cycloheximide (50mg/L) were added to each test portion and incubated for an additional 20
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hours. After 24 hours of total incubation, the enriched samples were streaked to MOX agar plates
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and incubated at 35 ±1 °C for 24-48 hours. The enriched samples were re-incubated for an
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additional 24 hours at 30 ± 1 °C and then streaked to a second MOX agar plate which was
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incubated for 24-48 hours at 35 ± 1
o
C. MOX agar plates were examined for suspect colonies, and
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if present, at least 5 colonies were streaked to TSA containing 0.6% yeast extract (TSA/YE). The
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AOAC Research Institute
Expert Review Pan l Use Only
OMAMAN-29 D/ PTM Validation Report 111501
OMA ERP - June 2016
ERP Use Only