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All samples were labeled with a randomized, blind-coded 3 digit number affixed to the sample
1
container. Test portions were shipped on a Thursday via overnight delivery according to the
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Category B Dangerous Goods shipment regulations set forth by the International Air
3
Transportation Association(IATA). Upon receipt, samples were held by the collaborating
4
laboratory at refrigeration temperature (3-5 °C) until the following Monday when analysis was
5
initiated a total of 96 hours post-inoculation. All samples were packed with cold packs to target
6
a temperature of < 7°C during shipment. In addition to each of the test portions and the total
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plate count replicate, collaborators also received a test portion for each matrix labeled as
8
‘temperature control’. Participants were instructed to obtain the temperature of this portion upon
9
receipt of the package, document results on the Sample Receipt Confirmation form provided and
10
fax to the study director. The shipment and hold times (through 120 hours) of the inoculated test
11
material had been verified as a quality control measure prior to study initiation.
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13
Test Portion Analysis
14
15
Each collaborator received 72 test portions (12 high inoculum, 12 low inoculum and 12
16
uninoculated controls for each method) of each matrix. Collaborators followed the appropriate
17
preparation and analysis protocol according to the method specified for the matrix.
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For the analysis of the deli turkey test portions by the 3M MDA
Listeria monocytogenes
method,
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a 125 g portion was enriched with 1125 mL of pre-warmed (37 ±1
o
C) DF broth base without
20
FAC, homogenized for 2 ± 0.5 minutes and incubated for 26-30 hours at 37 ±1
o
C. For the full
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fat cottage cheese test portions analyzed by the 3M MDA
Listeria monocytogenes
method, a 25 g
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portion was enriched with 225 mL pre-warmed (37 ±1
o
C)DF broth base without FAC,
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homogenized for 2 ± 0.5 minutes and incubated for 24-28 hours at 37 ±1
o
C. Following
24
enrichment, samples were assayed by the 3M MDA
Listeria monocytogenes
method and
25
confirmed following the standard reference method specified for each matrix.
26
Following enrichment, full fat cottage cheese samples assayed by the 3M MDA
27
Listeriamonocytogenes
method were confirmed by streaking an aliquot of the primary
28
enrichment onto Oxford Agar (OXA). Presumptive positive samples were streaked for isolation
29
on TSA/ye, verified morphologically by Gram stain and biochemically confirmed byhemolysis
30
testing and by VITEK 2 GP Biochemical Identification method (AOAC OMA 2012.02) [8] or
31
API Listeria Identification System biochemical test kits. Laboratories utilizing API Listeria kits
32
were also required to conduct catalase and oxidase tests.
33
For samples analyzed using the AOAC OMA 993.12 reference method, 25 g test portions were
34
enriched in pre-warmed (45
o
C) selective enrichment broth, homogenized for 2 ± 0.5 minutes and
35
incubated at 30 ± 2
o
C for 48 hours. Samples were streaked onto OXA and presumptive positive
36
samples were streaked for isolation onto TSA/ye. Colonies from TSA/ye were verified
37
morphologically by Gram stain and biochemically confirmed by evaluation of a hemolytic
38
reaction on sheep blood agar and biochemically confirmed VITEK 2 GP Biochemical
39
Identification method or API Listeria biochemical test kits. Laboratories utilizing API Listeria
40
kits were also required to conductcatalaseand oxidase tests.
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Following enrichment, deli turkey samples assayed by the 3M MDA
Listeriamonocytogenes
42
method were confirmed by streaking an aliquot of the primary enrichment onto MOX and
43
transferring an aliquot into Fraser Broth (FB). Presumptive positive samples were streaked for
44
isolation on Horse Blood Overlay Agar (HL) and confirmed by evaluation of a hemolytic
45
reaction and biochemically confirmedby the VITEK 2 GP Biochemical Identification methodor
46
API Listeria Identification System biochemical test kits. Laboratories utilizing API Listeria kits
47
were also required to conduct catalase and oxidase tests.
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Candidates for 2016 Method of the Year
150