412 / 596
High
5 x 50 g
5 x 25 g*
5 x 10 g
125 g
Low
5 x 250 g
20 x 125 g*
5 x 50 g
High
5 x 250 g
5 x 125 g*
5 x 50 g
*
Test portions from reference method
1
2
3
USDA/FSIS MLG 8.09 Reference Method
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5
For the USDA/FSIS MLG 8.09 reference method, 25 g test portions were enriched with 225 ± 5
6
mL of modified University of Vermont Medium broth (UVM) and 125 g test portions were
7
enriched with 1125 ± 25 mL of UVM. All test portions were mechanically stomached for two
8
minutes. The 25 g test portions were incubated at 30 ± 2 °C for 20-26 hours and the 125 g test
9
portions were incubated at 30 ± 2 °C for 23-26 hours. For environmental samples, sponges were
10
enriched with 225 mL of UVM and homogenized by hand, while swabs were enriched with 10
11
mL of UVM and mixed by vortex. Sponges and swabs were incubated for 20-26 hours at 30 ±
12
2°C. After incubation of all test portions, 0.1 ± 0.02 mL of the sample enrichment was
13
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
15
streaked to MOX and incubated at 35 ± 2°C for 26 ± 2 hours.
16
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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
20
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
22
hours.
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MOX agar plates streaked from the primary enrichment or the FB secondary enrichment were
25
examined after 26 ± 2 hours and if no suspect colonies were present, the MOX agar plate was re-
26
incubated for an additional 26 ± 2 hours at 35 ± 2°C for a total of 48 ± 2 hours. If suspect
27
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
29
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
31
motility by preparing a wet mount, analyzed by a catalase test and examined for morphology by
32
preparing a Gram stain. Additionally, purified HBO isolates were identified using the VITEK
®
33
GP Biochemical Identification following AOAC OMA 2013.02. [12]
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35
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
44